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Sample records for cdna analysis improves

  1. MLPA and cDNA analysis improves COL4A5 mutation detection in X-linked Alport syndrome

    DEFF Research Database (Denmark)

    Hertz, JM; Juncker, I; Marcussen, N

    2008-01-01

    for 10-15% of mutations. We have established a method for mutation analysis of COL4A5 based on reverse transcriptase-polymerase chain reaction analysis of mRNA from cultured skin fibroblasts and multiplex ligation-dependent probe amplification (MLPA) on genomic DNA. One advantage of using skin biopsies...

  2. 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

  3. 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 ...

  4. 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 ...

  5. 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.

  6. 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

  7. [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.

  8. 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

  9. 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

  10. 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.

  11. 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 ...

  12. 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

  13. 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

  14. 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

    or filtered by AFST. Conclusions cDNA terminal pattern analysis, as implemented in the AFST software tool, can be utilized to reveal wet-lab errors such as restriction enzyme cutting abnormities and chimeric EST sequences, detect various data abnormalities embedded in existing Sanger EST datasets, improve the accuracy of identifying and extracting bona fide cDNA inserts from raw ESTs, and therefore greatly benefit downstream EST-based applications.

  15. 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 ...

  16. 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 ...

  17. An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library

    Directory of Open Access Journals (Sweden)

    Wallis James G

    2007-07-01

    Full Text Available Abstract Background Castor seeds are a major source for ricinoleate, an important industrial raw material. Genomics studies of castor plant will provide critical information for understanding seed metabolism, for effectively engineering ricinoleate production in transgenic oilseeds, or for genetically improving castor plants by eliminating toxic and allergic proteins in seeds. Results Full-length cDNAs are useful resources in annotating genes and in providing functional analysis of genes and their products. We constructed a full-length cDNA library from developing castor endosperm, and obtained 4,720 ESTs from 5'-ends of the cDNA clones representing 1,908 unique sequences. The most abundant transcripts are genes encoding storage proteins, ricin, agglutinin and oleosins. Several other sequences are also very numerous, including two acidic triacylglycerol lipases, and the oleate hydroxylase (FAH12 gene that is responsible for ricinoleate biosynthesis. The role(s of the lipases in developing castor seeds are not clear, and co-expressing of a lipase and the FAH12 did not result in significant changes in hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Only one oleate desaturase (FAD2 gene was identified in our cDNA sequences. Sequence and functional analyses of the castor FAD2 were carried out since it had not been characterized previously. Overexpression of castor FAD2 in a FAH12-expressing Arabidopsis line resulted in decreased accumulation of hydroxy fatty acids in transgenic seeds. Conclusion Our results suggest that transcriptional regulation of FAD2 and FAH12 genes maybe one of the mechanisms that contribute to a high level of ricinoleate accumulation in castor endosperm. The full-length cDNA library will be used to search for additional genes that affect ricinoleate accumulation in seed oils. Our EST sequences will also be useful to annotate the castor genome, which whole sequence is being generated by shotgun sequencing at

  18. 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

  19. 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.

  20. 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...

  1. 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.

  2. 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...

  3. A novel method of differential gene expression analysis using multiple cDNA libraries applied to the identification of tumour endothelial genes.

    Science.gov (United States)

    Herbert, John M J; Stekel, Dov; Sanderson, Sharon; Heath, Victoria L; Bicknell, Roy

    2008-04-07

    In this study, differential gene expression analysis using complementary DNA (cDNA) libraries has been improved. Firstly by the introduction of an accurate method of assigning Expressed Sequence Tags (ESTs) to genes and secondly, by using a novel likelihood ratio statistical scoring of differential gene expression between two pools of cDNA libraries. These methods were applied to the latest available cell line and bulk tissue cDNA libraries in a two-step screen to predict novel tumour endothelial markers. Initially, endothelial cell lines were in silico subtracted from non-endothelial cell lines to identify endothelial genes. Subsequently, a second bulk tumour versus normal tissue subtraction was employed to predict tumour endothelial markers. From an endothelial cDNA library analysis, 431 genes were significantly up regulated in endothelial cells with a False Discovery Rate adjusted q-value of 0.01 or less and 104 of these were expressed only in endothelial cells. Combining the cDNA library data with the latest Serial Analysis of Gene Expression (SAGE) library data derived a complete list of 459 genes preferentially expressed in endothelium. 27 genes were predicted tumour endothelial markers in multiple tissues based on the second bulk tissue screen. This approach represents a significant advance on earlier work in its ability to accurately assign an EST to a gene, statistically measure differential expression between two pools of cDNA libraries and predict putative tumour endothelial markers before entering the laboratory. These methods are of value and available http://www.compbio.ox.ac.uk/data/diffex.html to researchers that are interested in the analysis of transcriptomic data.

  4. [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.

  5. Construction and analysis of cotton (Gossypium arboreum L. drought-related cDNA library

    Directory of Open Access Journals (Sweden)

    Zhang Chao-Jun

    2009-07-01

    Full Text Available Abstract Background Drought is one of the most important environmental factors causing water stress for cotton, and it greatly limits cotton growth and crop productivity. So far only a few drought-tolerance genes have been functionally characterized in details, and most efforts on this topic have been made in model organisms. Therefore, to identify more drought-related genes in cotton plays a crucial role in elucidating the underlying mechanisms of drought tolerance as well as utilizing bioengineering techniques to improve the tolerance in this organism. Findings Here we constructed a subtractive drought-tolerance cDNA library using suppressive subtractive hybridization (SSH. Through differential screening and bioinformatics analysis, we identified 392 positive clones with differential expression, corresponding 265 unique genes. By BLAST search against Genbank, we found that more than half of these EST sequences were homologous to those previously known drought-related genes and that there were 57 sequences with unknown functions, suggesting that many more genes are involved in this complex trait. Moreover, using RT-PCR, we examined the expression of nine representative candidate genes and confirmed that their expression levels were increased at different levels under drought stress. Conclusion Our results show that drought tolerance is a complex trait in cotton, which involves the coordination of many genes and multiple metabolism pathways. The candidate EST sequences we identified here would facilitate further functional studies of drought-related genes and provide important insights into the molecular mechanisms of drought-stress tolerance and genetic breeding in cotton.

  6. 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.

  7. Primary analysis of the expressed sequence tags in a pentastomid nymph cDNA library.

    Directory of Open Access Journals (Sweden)

    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.

  8. 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.

  9. 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.

  10. 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: ...

  11. [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.

  12. 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.

  13. cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

    DEFF Research Database (Denmark)

    Young, M F; Kerr, J M; Termine, J D

    1990-01-01

    A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells is...

  14. (+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.

    Science.gov (United States)

    Prosser, Ian; Phillips, Andy L; Gittings, Simon; Lewis, Mervyn J; Hooper, Antony M; Pickett, John A; Beale, Michael H

    2002-08-01

    Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

  15. 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 ...

  16. 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 ...

  17. 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- ...

  18. 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.

  19. Construction of a cDNA library from the ephemeral plant Olimarabidopsis pumila and preliminary analysis of expressed sequence tags.

    Science.gov (United States)

    Zhao, Yun-Xia; Wei, Yan-Ling; Zhao, Ping; Xiang, Cheng-Bin; Xu, Fang; Li, Chao; Huang, Xian-Zhong

    2013-01-01

    Olimarabidopsis pumila is a close relative of the model plant Arabidopsis thaliana but, unlike A. thaliana, it is a salt-tolerant ephemeral plant that is widely distributed in semi-arid and semi-salinized regions of the Xinjiang region of China, thus providing an ideal candidate plant system for salt tolerance gene mining. A good-quality cDNA library was constructed using cap antibody to enrich full-length cDNA with the gateway technology allowing library construction without traditional methods of cloning by use of restriction enzymes. A preliminary analysis of expressed sequence tags (ESTs) was carried out. The titers of the primary and the normalized cDNA library were 1.6 x 10(6) cfu/mL and 6.7 x 10(6) cfu/mL, respectively. A total of 1093 clones were randomly selected from the normalized library for EST sequencing. By sequence analysis, 894 high-quality ESTs were generated and assembled into 736 unique sequences consisting of 72 contigs and 664 singletons. The resulting unigenes were categorized according to the gene ontology (GO) hierarchy. The potential roles of gene products associated with stress-related ESTs are discussed. The 736 unigenes were similar to A. thaliana, A. lyrata, or Thellungiella salsuginea. This research provides an overview of the mRNA expression profile and first-hand information of gene sequence expressed in young leaves of O. pumila.

  20. An improved K-means clustering method for cDNA microarray image segmentation.

    Science.gov (United States)

    Wang, T N; Li, T J; Shao, G F; Wu, S X

    2015-07-14

    Microarray technology is a powerful tool for human genetic research and other biomedical applications. Numerous improvements to the standard K-means algorithm have been carried out to complete the image segmentation step. However, most of the previous studies classify the image into two clusters. In this paper, we propose a novel K-means algorithm, which first classifies the image into three clusters, and then one of the three clusters is divided as the background region and the other two clusters, as the foreground region. The proposed method was evaluated on six different data sets. The analyses of accuracy, efficiency, expression values, special gene spots, and noise images demonstrate the effectiveness of our method in improving the segmentation quality.

  1. 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 ...

  2. 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.

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

    Science.gov (United States)

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

    2010-01-15

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

  4. [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.

  5. 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.

  6. 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

  7. Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

    Directory of Open Access Journals (Sweden)

    Bendahmane Abdelhafid

    2011-05-01

    Full Text Available Abstract Background Melon (Cucumis melo, an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs and 3,073 single nucleotide polymorphisms (SNPs in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but

  8. 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.

  9. 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.

  10. 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.

  11. Isolation and analysis of water stress induced genes in maize seedlings by subtractive PCR and cDNA macroarray.

    Science.gov (United States)

    Zheng, Jun; Zhao, Jinfeng; Tao, Yazhong; Wang, Jianhua; Liu, Yunjun; Fu, Junjie; Jin, Ying; Gao, Peng; Zhang, Jinpeng; Bai, Yunfeng; Wang, Guoying

    2004-08-01

    In order to identify genes induced during the water stress response in maize (Zea mays) seedlings, suppression subtractive hybridization (SSH) was performed using mixed cDNAs prepared from maize seedlings treated with 20% PEG as testers and cDNAs from unstressed maize seedlings as drivers. A forward subtractive cDNA library was constructed, from which 960 recombinant colonies were picked and amplified. Through differential screening of the subtractive cDNA library, 533 clones were identified as water stress induced. After sequencing, 190 unique expressed sequence tags (ESTs) were obtained by clustering and blast analysis, which included transcripts that had previously been reported as responsive to stress as well as some functionally unknown transcripts. The ESTs with significant protein homology were sorted into 13 functional categories. A cDNA marcoarray containing the 190 unique ESTs was used to analyze their expression profiles in maize seedling during both PEG treatment and natural drought. The results indicated that 67 ESTs in leaves and 113 ESTs in roots were significantly up-regulated by PEG-stress. 123 ESTs were found to be up-regulated for at least one time-course point in either maize leaves or roots. Correspondingly, 163 ESTs were significantly up-regulated by drought stress. Results from the hierarchical cluster analysis suggest that the leaves and roots of maize seedlings had different expression profiles after PEG treatment and that there was a lot of overlap between PEG- and drought-stress induced up-regulated transcripts. A set of transcripts has been identified, which have significantly increased expression and probably involved in water stress signaling pathway based on data analysis.

  12. Bioinformatic analysis of barcoded cDNA libraries for small RNA profiling by next-generation sequencing.

    Science.gov (United States)

    Farazi, Thalia A; Brown, Miguel; Morozov, Pavel; Ten Hoeve, Jelle J; Ben-Dov, Iddo Z; Hovestadt, Volker; Hafner, Markus; Renwick, Neil; Mihailović, Aleksandra; Wessels, Lodewyk F A; Tuschl, Thomas

    2012-10-01

    The characterization of post-transcriptional gene regulation by small regulatory RNAs of 20-30 nt length, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for the characterization of small RNAs is their identification and quantification across different developmental stages, normal and diseased tissues, as well as model cell lines. Here we present a step-by-step protocol for the bioinformatic analysis of barcoded cDNA libraries for small RNA profiling generated by Illumina sequencing, thereby facilitating miRNA and other small RNA profiling of large sample collections. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. cDNA cloning and transcriptional controlling of a novel low dose radiation-induced gene and its function analysis

    International Nuclear Information System (INIS)

    Zhou Pingkun; Sui Jianli

    2002-01-01

    Objective: To clone a novel low dose radiation-induced gene (LRIGx) and study its function as well as its transcriptional changes after irradiation. Methods: Its cDNA was obtained by DDRT-PCR and RACE techniques. Northern blot hybridization was used to investigate the gene transcription. Bioinformatics was employed to analysis structure and function of this gene. Results: LRIGx cDNA was cloned. The sequence of LRIGx was identical to a DNA clone located in human chromosome 20 q 11.2-12 Bioinformatics analysis predicted an encoded protein with a conserved helicase domain. Northern analysis revealed a ∼8.5 kb transcript which was induced after 0.2 Gy as well as 0.02 Gy irradiation, and the transcript level was increased 5 times at 4 h after 0.2 Gy irradiation. The induced level of LRIGx transcript by 2.0 Gy high dose was lower than by 0.2 Gy. Conclusion: A novel low dose radiation-induced gene has been cloned. It encodes a protein with a conserved helicase domain that could involve in DNA metabolism in the cellular process of radiation response

  14. An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L

    Directory of Open Access Journals (Sweden)

    Kaur Jatinder

    2010-05-01

    Full Text Available Abstract Background RNA quality and quantity is sometimes unsuitable for cDNA library construction, from plant seeds rich in oil, polysaccharides and other secondary metabolites. Seeds of jatropha (Jatropha curcas L. are rich in fatty acids/lipids, storage proteins, polysaccharides, and a number of other secondary metabolites that could either bind and/or co-precipitate with RNA, making it unsuitable for downstream applications. Existing RNA isolation methods and commercial kits often fail to deliver high-quality total RNA from immature jatropha seeds for poly(A+ RNA purification and cDNA synthesis. Findings A protocol has been developed for isolating good quality total RNA from immature jatropha seeds, whereby a combination of the CTAB based RNA extraction method and a silica column of a commercial plant RNA extraction kit is used. The extraction time was reduced from two days to about 3 hours and the RNA was suitable for poly(A+ RNA purification, cDNA synthesis, cDNA library construction, RT-PCR, and Northern hybridization. Based on sequence information from selected clones and amplified PCR product, the cDNA library seems to be a good source of full-length jatropha genes. The method was equally effective for isolating RNA from mustard and rice seeds. Conclusions This is a simple CTAB + silica column method to extract high quality RNA from oil rich immature jatropha seeds that is suitable for several downstream applications. This method takes less time for RNA extraction and is equally effective for other tissues where the quality and quantity of RNA is highly interfered by the presence of fatty acids, polysaccharides and polyphenols.

  15. 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.

  16. Nucleotide sequence of a cDNA for branched chain acyltransferase with analysis of the deduced protein structure

    International Nuclear Information System (INIS)

    Hummel, K.B.; Litwer, S.; Bradford, A.P.; Aitken, A.; Danner, D.J.; Yeaman, S.J.

    1988-01-01

    Nucleotide sequence was determined for a 1.6-kilobase human cDNA putative for the branched chain acyltransferase protein of the branched chain α-ketoacid dehydrogenase complex. Translation of the sequence reveals an open reading frame encoding a 315-amino acid protein of molecular weight 35,759 followed by 560 bases of 3'-untranslated sequence. Three repeats of the polyadenylation signal hexamer ATTAAA are present prior to the polyadenylate tail. Within the open reading frame is a 10-amino acid fragment which matches exactly the amino acid sequence around the lipoate-lysine residue in bovine kidney branched chain acyltransferase, thus confirming the identity of the cDNA. Analysis of the deduced protein structure for the human branched chain acyltransferase revealed an organization into domains similar to that reported for the acyltransferase proteins of the pyruvate and α-ketoglutarate dehydrogenase complexes. This similarity in organization suggests that a more detailed analysis of the proteins will be required to explain the individual substrate and multienzyme complex specificity shown by these acyltransferases

  17. 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.

  18. Characterization and phylogenetic analysis of lectin gene cDNA isolated from sea cucumber ( Apostichopus japonicus) body wall

    Science.gov (United States)

    Xue, Zhuang; Li, Hui; Liu, Yang; Zhou, Wei; Sun, Jing; Wang, Xiuli

    2017-12-01

    As a `living fossil' of species origin and `rich treasure' of food and nutrition development, sea cucumber has received a lot of attentions from researchers. The cDNA library construction and EST sequencing of blood had been conducted previously in our lab. The bioinformatic analysis provided a gene fragment which is highly homologous with the genes of lectin family, named AjL ( Apostichopus japonicus lectin). To characterize and determine the phylogeny of AjL genes in early evolution, we isolated a full-length cDNA of lectin gene from the body wall of A. japonicus. The open reading frame of this gene contained 489 bp and encoded a 163 amino acids secretory protein being homologous to lectins of mammals and aquatic organisms. The deduced protein included a lectin-like domain. SDS-PAGE analysis showed that AjL migrated as a specific band (about 36.09 kDa under reducing), and agglutinated against rabbit red blood cells. AjL was similar to chain A of CEL-IV in space structure. We predicted that AjL may play the same role of CEL-IV. Our results suggested that more than one lectin gene functioned in sea cucumber and most of other species, which was fused by uncertain sequences during the evolution and encoded different proteins with diverse functions. Our findings provided the insights into the function and characteristics of lectin genes invertebrates. The results will also be helpful for the identification and structural, functional, and evolutionary analyses of lectin genes.

  19. In vitro and in silico cloning of Xenopus laevis SOD2 cDNA and its phylogenetic analysis.

    Science.gov (United States)

    Purrello, Michele; Di Pietro, Cinzia; Ragusa, Marco; Pulvirenti, Alfredo; Giugno, Rosalba; Di Pietro, Valentina; Emmanuele, Giovanni; Travali, Salvo; Scalia, Marina; Shasha, Dennis; Ferro, Alfredo

    2005-02-01

    By using the methodology of both wet and dry biology (i.e., RT-PCR and cycle sequencing, and biocomputational technology, respectively) and the data obtained through the Genome Projects, we have cloned Xenopus laevis SOD2 (MnSOD) cDNA and determined its nucleotide sequence. These data and the deduced protein primary structure were compared with all the other SOD2 nucleotide and amino acid sequences from eukaryotes and prokaryotes, published in public databases. The analysis was performed by using both Clustal W, a well known and widely used program for sequence analysis, and AntiClustAl, a new algorithm recently created and implemented by our group. Our results demonstrate a very high conservation of the enzyme amino acid sequence during evolution, which proves a close structure-function relationship. This is to be expected for very ancient molecules endowed with critical biological functions, performed through a specific structural organization. The nucleotide sequence conservation is less pronounced: this too was foreseeable, due to neutral mutations and to the species-specific codon usage. The data obtained by using AntiClustAl are comparable with those produced with Clustal W, which validates this algorithm as an important new tool for biocomputational analysis. Finally, it is noteworthy that evolutionary trees, drawn by using all the available data on SOD2 nucleotide sequences and amino acid and either Clustal W or AntiClustAl, are comparable to those obtained through phylogenetic analysis based on fossil records.

  20. 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...

  1. 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.

  2. cDNA sequence and tissue expression analysis of glucokinase from ...

    African Journals Online (AJOL)

    Yomi

    2012-01-10

    Jan 10, 2012 ... (Rattus norvegicus) were 98.1, 96.8, 80.3 and 79.8%, respectively. Phylogenetic analysis based on GK amino acid sequences. Phylogenetic analysis among eight fish species, eleven endothermic species and one amphibian species based on glucokinase amino acid sequences is shown in Figure. 3.

  3. 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.

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

    Indian Academy of Sciences (India)

    PRAKASH

    Phaseolus vulgaris erythroagglutinin (PHA-E) (Sun et al. 1995). The nature of the lectin correlates with its mannose- binding active site. 3.3 Southern blot analysis. The presence of multiple copies of the mannose-binding lectin gene in the genome has been reported in many plant species, particularly those belonging to the ...

  5. Construction of a full-length cDNA library and analysis of expressed ...

    African Journals Online (AJOL)

    sunny t

    2015-06-10

    Jun 10, 2015 ... projects provide a very useful and quick means of accessing gene sequence and expression information. (Manickavelu et al., 2012). Some reports have proven that projects based on ESTs are powerful tools for both the analysis of gene ..... genes controlling many important traits of agronomic importance ...

  6. Construction of a full-length cDNA library and analysis of expressed ...

    African Journals Online (AJOL)

    ... in the GenBank databases. Cluster analysis allowed the identification of 61 unique sequences. These genes were classified into six types by Gene Ontology (GO) annotation. The results also indicated that unigenes of C. capsularis have higher homology to Populus trichocarpa, Ricinus communis and Corchorus olitorius.

  7. Identification and functional analysis of a new glyphosate resistance gene from a fungus cDNA library.

    Science.gov (United States)

    Tao, Bo; Shao, Bai-Hui; Qiao, Yu-Xin; Wang, Xiao-Qin; Chang, Shu-Jun; Qiu, Li-Juan

    2017-08-01

    Glyphosate is a widely used broad spectrum herbicide; however, this limits its use once crops are planted. If glyphosate-resistant crops are grown, glyphosate can be used for weed control in crops. While several glyphosate resistance genes are used in commercial glyphosate tolerant crops, there is interest in identifying additional genes for glyphosate tolerance. This research constructed a high-quality cDNA library form the glyphosate-resistant fungus Aspergillus oryzae RIB40 to identify genes that may confer resistance to glyphosate. Using a medium containing glyphosate (120mM), we screened several clones from the library. Based on a nucleotide sequence analysis, we identified a gene of unknown function (GenBank accession number: XM_001826835.2) that encoded a hypothetical 344-amino acid protein. The gene was named MFS40. Its ORF was amplified to construct an expression vector, pGEX-4T-1-MFS40, to express the protein in Escherichia coli BL21. The gene conferred glyphosate tolerance to E. coli ER2799 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. CONSTRUCTION OF SILKWORM MIDGUT cDNA LIBRARY FOR SCREEN AND SEQUENCE ANALYSIS OF PERITROPHIC MEMBRANE PROTEIN GENES.

    Science.gov (United States)

    Zhou, Yi-Jun; Xue, Bin; Li, Yang-Yang; Li, Fan-Chi; Ni, Min; Shen, Wei-De; Gu, Zhi-Ya; Li, Bing; Shen, Wei-De; Gu, Zhi-Ya; Li, Bing

    2016-01-01

    Silkworm is an important economic insect and the model species for Lepidoptera. The midgut of silkworm is an important physiological barrier, as its peritrophic membrane (PM) can resist pathogen invasion. In this study, a silkworm midgut cDNA library was constructed in order to identify silkworm PM genes. The capacity of the initial library was 6.92 × 10(6) pfu/ml, along with a recombination rate of 92.14% and a postamplification titer of 4.10 × 10(9) pfu/ml. Three silkworm PM protein genes were obtained by immunoscreening, two of which were chitin-binding protein (CBP) genes and one of which was a chitin deacetylase (CDA) gene as revealed by sequence analysis. Three genes were named BmCBP02, BmCBP13, and BmCDA17, and their ORF sizes are 678, 1,029, and 645 bp, respectively; all of them contain sequences of chitin-binding domains. Phylogenetic analysis indicated that BmCBP02 has the highest consensus with Mamestra configurata CBP at 61.0%; BmCBP13 has the highest consensus with Loxostege sticticalis PM CBP at 53.35%; BmCDA17 has the highest consensus with Helicoverpa armigera CDA5a at 70.83%. Tissue transcriptional analysis revealed that all three genes were specifically expressed in the midgut, and during the developmental process of fifth-instar silkworms, the transcription of all the genes showed an upward trend. This study laid a foundation for further studies on the functions of silkworm PM genes. © 2015 Wiley Periodicals, Inc.

  9. cDNA Microarray Analysis of Serially Sampled Cervical Cancer Specimens From Patients Treated With Thermochemoradiotherapy

    International Nuclear Information System (INIS)

    Borkamo, Erling Dahl; Schem, Baard-Christian; Fluge, Oystein; Bruland, Ove; Dahl, Olav; Mella, Olav

    2009-01-01

    Purpose: To elucidate changes in gene expression after treatment with regional thermochemoradiotherapy in locally advanced squamous cell cervical cancer. Methods and Materials: Tru-Cut biopsy specimens were serially collected from 16 patients. Microarray gene expression levels before and 24 h after the first and second trimodality treatment sessions were compared. Pathway and network analyses were conducted by use of Ingenuity Pathways Analysis (IPA; Ingenuity Systems, Redwood City, CA). Single gene expressions were analyzed by quantitative real-time reverse transcription-polymerase chain reaction. Results: We detected 53 annotated genes that were differentially expressed after trimodality treatment. Central in the three top networks detected by IPA were interferon alfa, interferon beta, and interferon gamma receptor; nuclear factor κB; and tumor necrosis factor, respectively. These genes encode proteins that are important in regulation cell signaling, proliferation, gene expression, and immune stimulation. Biological processes over-represented among the 53 genes were fibrosis, tumorigenesis, and immune response. Conclusions: Microarrays showed minor changes in gene expression after thermochemoradiotherapy in locally advanced cervical cancer. We detected 53 differentially expressed genes, mainly involved in fibrosis, tumorigenesis, and immune response. A limitation with the use of serial biopsy specimens was low quality of ribonucleic acid from tumors that respond to highly effective therapy. Another 'key limitation' is timing of the post-treatment biopsy, because 24 h may be too late to adequately assess the impact of hyperthermia on gene expression.

  10. EST analysis and annotation of transcripts derived from a trichome-specific cDNA library from Salvia fruticosa.

    Science.gov (United States)

    Chatzopoulou, Fani M; Makris, Antonios M; Argiriou, Anagnostis; Degenhardt, Jörg; Kanellis, Angelos K

    2010-05-01

    Greek sage (Salvia fruticosa Mill., Syn. Salvia triloba L.) is appreciated for its essential oil which is used as an aromatic spice and active against a wide range of microorganisms and viruses. The essential oil is dominated by terpenoids and flavonoids which are produced and stored in glandular trichomes on the plant surface. The present study aims to give insights into the metabolic activities of S. fruticosa trichomes on a transcriptome level. A total of 2,304 clones were sequenced from a cDNA library from leaves' trichomes of S. fruticosa. Exclusion of sequences shorter than 100 bp resulted in 1,615 high-quality ESTs with a mean length of 592 bp. Cluster analysis indicated the presence of 197 contigs (908 clones) and 707 singletons, generating a total of 904 unique sequences. Of the 904 unique ESTs, 628 (69.5%) had significant hits in the non-redundant protein database and were annotated. A total of 517 (82.3%) sequences were functionally classified using the gene ontologies (GO) and established pathway associations to 220 (24.3%) sequences in Kyoto encyclopedia of genes and genomes (KEGG). In addition, 52 (5.8%) of the unique ESTs revealed a GO biological term with relation to terpenoid (78 ESTs), phenylpropanoid (43 ESTs), flavonoid (18 ESTs) or alkaloid (10 ESTs) biosynthesis or to P450s (26 ESTs). Expression analysis of a selected set of genes known to be involved in the pathways of secondary metabolite synthesis showed higher expression levels in trichomes, validating the tissue specificity of the analyzed glandular trichome library.

  11. Monitoring expression profiles of rice (Oryza sativa L.) genes under abiotic stresses using cDNA Microarray Analysis (abstract)

    International Nuclear Information System (INIS)

    Rabbani, M.A.

    2005-01-01

    Transcript regulation in response to cold, drought, high salinity and ABA application was investigated in rice (Oryza sativa L., Nipponbare) with microarray analysis including approx. 1700 independent DNA elements derived from three cDNA libraries constructed from 15-day old rice seedlings stressed with drought, cold and high salinity. A total of 141 non-redundant genes were identified, whose expression ratios were more than three-fold compared with the control genes for at least one of stress treatments in microarray analysis. However, after RNA gel blot analysis, a total of 73 genes were identified, among them the transcripts of 36, 62, 57 and 43 genes were found increased after cold, drought, high salinity and ABA application, respectively. Sixteen of these identified genes have been reported previously to be stress inducible in rice, while 57 of which are novel that have not been reported earlier as stress responsive in rice. We observed a strong association in the expression patterns of stress responsive genes and found 15 stress inducible genes that responded to all four treatments. Based on Venn diagram analysis, 56 genes were induced by both drought and high salinity, whereas 22 genes were upregulated by both cold and high salinity stress. Similarly 43 genes were induced by both drought stress and ABA application, while only 17 genes were identified as cold and ABA inducible genes. These results indicated the existence of greater cross talk between drought, ABA and high salinity stress signaling processes than those between cold and ABA, and cold and high salinity stress signaling pathways. The cold, drought, high salinity and ABA inducible genes were classified into four gene groups from their expression profiles. Analysis of data enabled us to identify a number of promoters and possible cis-acting DNA elements of several genes induced by a variety of abiotic stresses by combining expression data with genomic sequence data of rice. Comparative analysis of

  12. 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.

  13. cDNA cloning and expression analysis of two distinct Sox8 genes in Paramisgurnus dabryanus (Cypriniformes).

    Science.gov (United States)

    Xia, Xiaohua; Zhao, Jie; Du, Qiyan; Chang, Zhongjie

    2010-08-01

    The Sox9 gene attracts a lot of attention because of its connection with gonadal development and differentiation. However, Sox8, belonging to the same subgroup SoxE, has rarely been studied. To investigate the function as well as the evolutionary origin of SOXE subgroup, we amplified the genomic DNA of Paramisgurnus dabryanu using a pair of degenerate primers. Using rapid amplification of the cDNA ends (RACE), it was discovered that P. dabryanu has two duplicates: Sox8a and Sox8b. Each has an intron of different length in the conserved HMG-box region. The overall sequence similarity of the deduced amino acid of PdSox8a and PdSox8b was 46.26%, and only two amino acids changed in the HMG-box. This is the first evidence showing that there are two distinct duplications of Sox8 genes in Cypriniformes. Southern blot analysis showed only one hybrid band, with lengths 7.4 or 9.2 kb. Both semi-quantitative RT-PCR and real-time quantitative PCR assay displayed that both PdSox8a and PdSox8b are downregulated during early embryonic development. In adult tissues, the two Sox8 genes expressed ubiquitously, and expression levels are particularly high in the gonads and brain. In gonads, both PdSox8a and PdSox8b are expressed at a higher level in the tesis than in the ovary. PdSox8a and PdSox8b may have functional overlaps and are essential for the neuronal development and differentiation of gonads.

  14. Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Y. Cui

    2008-05-01

    Full Text Available Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1 allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+, expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG, 267-277 (NYHAVNIVGYG and 284-303 (YWIVRNSWDTTWGDSGYGYF. Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%, an extended strand (17.13%, a ß turn (5.61%, and a random coil (43.30%. A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

  15. 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

  16. 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.

  17. Genomic Profiles in Stage I Primary Non Small Cell Lung Cancer Using Comparative Genomic Hybridization Analysis of cDNA Microarrays

    Directory of Open Access Journals (Sweden)

    Feng Jiang

    2004-09-01

    Full Text Available To investigate the genomic aberrations that are involved in lung tumorigenesis and therefore may be developed as biomarkers for lung cancer diagnosis, we characterized the genomic copy number changes associated with individual genes in 14 tumors from patients with primary non small cell lung cancer (NSCLC. Six squamous cell carcinomas (SQCAs and eight adenocarcinomas (ADCAs were examined by high-resolution comparative genomic hybridization (CGH analysis of cDNA microarray. The SQCAs and ADCAs shared common frequency distributions of recurrent genomic gains of 63 genes and losses of 72 genes. Cluster analysis using 57 genes defined the genomic differences between these two major histologic types of NSCLC. Genomic aberrations from a set of 18 genes showed distinct difference of primary ADCAs from their paired normal lung tissues. The genomic copy number of four genes was validated by fluorescence in situ hybridization of 32 primary NSCLC tumors, including those used for cDNA microarray CGH analysis; a strong correlation with cDNA microarray CGH data emerged. The identified genomic aberrations may be involved in the initiation and progression of lung tumorigenesis and, most importantly, may be developed as new biomarkers for the early detection and classification of lung cancer.

  18. Construction of cDNA library and preliminary analysis of expressed sequence tags from tea plant [Camellia sinensis (L) O. Kuntze].

    Science.gov (United States)

    Phukon, Munmi; Namdev, Richa; Deka, Diganta; Modi, Mahendra K; Sen, Priyabrata

    2012-09-10

    Tea is the most popular non-alcoholic and healthy beverage across the world. The understanding of the genetic organization and molecular biology of tea plant, which is very poorly understood at present, is required for quantum increase in productivity and efficient use of germplasm for either cultivation or breeding program. Single-pass sequencing of randomly selected cDNA clones is the most widely accepted technique for gene identification and cloning. In the present study, a good quality cDNA library was constructed and preliminary analysis of ESTs was carried out. The titers of unamplified and amplified libraries were 1.4 × 10(6)pfu/ml and 5.27 × 10(8)pfu/ml respectively. A total of 210 cDNA clones from the constructed cDNA library were sequenced and analyzed. A total of 84 high quality Expressed Sequence Tags (ESTs) were generated, among which 71 ESTs had significant homology with sequences in NCBI non-redundant protein database by BLAST X analysis. About 80% ESTs had poly (A) tail at 3' end indicating that the cDNAs were full length. The database-matched ESTs were classified into putative cellular roles, viz. energy-related category (corresponding to 20% of total BLAST X matched ESTs), Transcription (14.2%), protein synthesis (14.2%) cell growth and division (8.6%), cell structure (5.7%), signal transduction (5.7%), transporters (2.9%), disease and defenses (2.9%), secondary metabolism (2.9%) and gene regulation (2.9%). This study provides an overview of the mRNA expression profile and first hand information of gene sequence expressed in tender leaves and apical buds of tea plant. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. 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

  20. 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.

  1. [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.

  2. Cloning and structural analysis of alpha-latroinsectotoxin cDNA. Abundance of ankyrin-like repeats.

    Science.gov (United States)

    Kiyatkin, N; Dulubova, I; Grishin, E

    1993-04-01

    alpha-Latroinsectotoxin (alpha-LIT), purified from venom glands of the black widow spider Latrodectus mactans tredecimguttatus, is a presynaptic neurotoxin selective only for insects. A cDNA encoding the putative alpha-LIT precursor was isolated from a spider venom gland cDNA library. The cDNA contains a 4236-base-pair open reading frame corresponding to a 157826-Da protein composed of 1411 amino acids. The mature alpha-LIT, with molecular mass approximately 130 kDa, is probably derived from double processing in the N-terminal and C-terminal regions of the primary translation product. The structure region, extending over residues 464-1176, is composed almost entirely of ankyrin-like repeats which represent a motif also found in the alpha-latrotoxin (alpha-LTX), which has selective action on vertebrates. Total alignment of the alpha-LIT and alpha-LTX amino acid sequences reveals an overall similarity of 34.1%. Strong sequence divergence is observed in analogous cysteine-rich regions situated within the ankyrin-repeat domains of both alpha-LIT and alpha-LTX.

  3. Cloning of canine myocilin cDNA and molecular analysis of the myocilin gene in Shiba Inu dogs.

    Science.gov (United States)

    Kato, Kumiko; Sasaki, Nobuo; Matsunaga, Satoru; Nishimura, Ryohei; Ogawa, Hiroyuki

    2007-01-01

    To identify canine myocilin cDNA and compare its sequence in glaucomatous and nonglaucomatous Shiba Inu dogs with closed and open iridocorneal angles (ICAs). Total RNA was extracted from the ciliary body of the eyes of a healthy Beagle, and the canine myocilin gene was cloned and sequenced. Of the Shiba Inu dogs tested, five were glaucomatous with closed ICA, three were nonglaucomatous with open ICA, and two were nonglaucomatous with closed ICA. The genomic DNA of these dogs was extracted from peripheral blood leukocytes. The exons of the canine myocilin gene were amplified using the polymerase chain reaction (PCR), and sequenced. The frequency of mutation in canine myocilin DNA was verified in these dogs by using the myocilin cDNA of a Beagle. The canine myocilin cDNA was 1452 bp long and contained the entire open reading frame encoding 483 amino acids. A leucine zipper-like motif and olfactomedin-like domain were conserved in the amino acid residues. The presence of sequence variants in the genomic DNA of Shiba Inu dogs was independent of the occurrence of glaucoma and ICA grading. Myocilin RNA was detected in the ciliary body and trabecular meshwork (TM) of a Beagle. The myocilin sequence of Shiba Inu dogs suggests that myocilin mutations are unlikely to play a significant role in the pathogenesis of primary closed-angle glaucoma in this breed. However, several mutations in the myocilin gene in exon 1 of Shiba Inu dogs may predispose them to an obstruction in the anterior aqueous outflow.

  4. Identification of testis-relevant genes using in silico analysis from testis ESTs and cDNA microarray in the black tiger shrimp (Penaeus monodon

    Directory of Open Access Journals (Sweden)

    Wongsurawat Thidathip

    2010-08-01

    Full Text Available Abstract Background Poor reproductive maturation of the black tiger shrimp (Penaeus monodon in captivity is one of the serious threats to sustainability of the shrimp farming industry. Understanding molecular mechanisms governing reproductive maturation processes requires the fundamental knowledge of integrated expression profiles in gonads of this economically important species. In P. monodon, a non-model species for which the genome sequence is not available, expressed sequence tag (EST and cDNA microarray analyses can help reveal important transcripts relevant to reproduction and facilitate functional characterization of transcripts with important roles in male reproductive development and maturation. Results In this study, a conventional testis EST library was exploited to reveal novel transcripts. A total of 4,803 ESTs were unidirectionally sequenced and analyzed in silico using a customizable data analysis package, ESTplus. After sequence assembly, 2,702 unique sequences comprised of 424 contigs and 2,278 singletons were identified; of these, 1,133 sequences are homologous to genes with known functions. The sequences were further characterized according to gene ontology categories (41% biological process, 24% molecular function, 35% cellular component. Through comparison with EST libraries of other tissues of P. monodon, 1,579 transcripts found only in the testis cDNA library were identified. A total of 621 ESTs have not been identified in penaeid shrimp. Furthermore, cDNA microarray analysis revealed several ESTs homologous to testis-relevant genes were more preferentially expressed in testis than in ovary. Representatives of these transcripts, homologs of saposin (PmSap and Dmc1 (PmDmc1, were further characterized by RACE-PCR. The more abundant expression levels in testis than ovary of PmSap and PmDmc1 were verified by quantitative real-time PCR in juveniles and wild broodstock of P. monodon. Conclusions Without a genome sequence, a

  5. Transcriptomic identification of candidate genes involved in sunflower responses to chilling and salt stresses based on cDNA microarray analysis.

    Science.gov (United States)

    Fernandez, Paula; Di Rienzo, Julio; Fernandez, Luis; Hopp, H Esteban; Paniego, Norma; Heinz, Ruth A

    2008-01-26

    Considering that sunflower production is expanding to arid regions, tolerance to abiotic stresses as drought, low temperatures and salinity arises as one of the main constrains nowadays. Differential organ-specific sunflower ESTs (expressed sequence tags) were previously generated by a subtractive hybridization method that included a considerable number of putative abiotic stress associated sequences. The objective of this work is to analyze concerted gene expression profiles of organ-specific ESTs by fluorescence microarray assay, in response to high sodium chloride concentration and chilling treatments with the aim to identify and follow up candidate genes for early responses to abiotic stress in sunflower. Abiotic-related expressed genes were the target of this characterization through a gene expression analysis using an organ-specific cDNA fluorescence microarray approach in response to high salinity and low temperatures. The experiment included three independent replicates from leaf samples. We analyzed 317 unigenes previously isolated from differential organ-specific cDNA libraries from leaf, stem and flower at R1 and R4 developmental stage. A statistical analysis based on mean comparison by ANOVA and ordination by Principal Component Analysis allowed the detection of 80 candidate genes for either salinity and/or chilling stresses. Out of them, 50 genes were up or down regulated under both stresses, supporting common regulatory mechanisms and general responses to chilling and salinity. Interestingly 15 and 12 sequences were up regulated or down regulated specifically in one stress but not in the other, respectively. These genes are potentially involved in different regulatory mechanisms including transcription/translation/protein degradation/protein folding/ROS production or ROS-scavenging. Differential gene expression patterns were confirmed by qRT-PCR for 12.5% of the microarray candidate sequences. Eighty genes isolated from organ-specific cDNA libraries

  6. Transcriptomic identification of candidate genes involved in sunflower responses to chilling and salt stresses based on cDNA microarray analysis

    Directory of Open Access Journals (Sweden)

    Paniego Norma

    2008-01-01

    Full Text Available Abstract Background Considering that sunflower production is expanding to arid regions, tolerance to abiotic stresses as drought, low temperatures and salinity arises as one of the main constrains nowadays. Differential organ-specific sunflower ESTs (expressed sequence tags were previously generated by a subtractive hybridization method that included a considerable number of putative abiotic stress associated sequences. The objective of this work is to analyze concerted gene expression profiles of organ-specific ESTs by fluorescence microarray assay, in response to high sodium chloride concentration and chilling treatments with the aim to identify and follow up candidate genes for early responses to abiotic stress in sunflower. Results Abiotic-related expressed genes were the target of this characterization through a gene expression analysis using an organ-specific cDNA fluorescence microarray approach in response to high salinity and low temperatures. The experiment included three independent replicates from leaf samples. We analyzed 317 unigenes previously isolated from differential organ-specific cDNA libraries from leaf, stem and flower at R1 and R4 developmental stage. A statistical analysis based on mean comparison by ANOVA and ordination by Principal Component Analysis allowed the detection of 80 candidate genes for either salinity and/or chilling stresses. Out of them, 50 genes were up or down regulated under both stresses, supporting common regulatory mechanisms and general responses to chilling and salinity. Interestingly 15 and 12 sequences were up regulated or down regulated specifically in one stress but not in the other, respectively. These genes are potentially involved in different regulatory mechanisms including transcription/translation/protein degradation/protein folding/ROS production or ROS-scavenging. Differential gene expression patterns were confirmed by qRT-PCR for 12.5% of the microarray candidate sequences. Conclusion

  7. Yeast two-hybrid analysis of a human trabecular meshwork cDNA library identified EFEMP2 as a novel PITX2 interacting protein.

    Science.gov (United States)

    Acharya, Moulinath; Sharp, Michael W; Mirzayans, Farideh; Footz, Tim; Huang, Lijia; Birdi, Chanchal; Walter, Michael A

    2012-01-01

    Mutations in the homeobox transcription factor paired-like homeodomain transcription factor 2 (PITX2) cause Axenfeld-Reiger syndrome (ARS), which is associated with anterior segment dysgenesis (ASD) and glaucoma. To understand ARS pathogenesis, it is essential to know the normal functions of PITX2 and the proteins with which PITX2 interacts in the eye. Therefore, we used a unique cDNA library that we created from human trabecular meshwork (TM) primary cells to discover PITX2-interacting proteins (PIPs). A human TM cDNA library was created from primary cells in the ProQuest Two-Hybrid prey vector: pEXP-AD502. Human PITX2A and PITX2C isoforms were used independently as "bait" to identify novel PIPs. A total of 1.25×10⁶ clones were screened by yeast two-hybrid (Y2H) analyses. PIPs obtained from each Y2H experiment were confirmed by yeast retransformation and mammalian co-immunoprecipitation assays. EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) was identified by both PITX2A and PITX2C isoforms as a novel PIP from Y2H analyses. EFEMP2 is 443 amino acids long with six epidermal growth factor (EGF)-like modules and one fibulin-like module. The PITX2-interaction domain in EFEMP2 lies between the second EGF-like module and the COOH-terminal fibulin-like module. Co-immunoprecipitation assays in COS-7 cells confirmed the interaction between PITX2 and EFEMP2. We discovered EFEMP2 as a novel PITX2-interacting protein. Further, our cDNA library made from human TM primary cells is a unique and effective resource to identify novel interacting proteins for glaucoma and ASD candidates. This resource could be used both for discovery and validation of interactomes identified from in silico analysis.

  8. The construction of a cDNA library enriched for immune genes and the analysis of 7535 ESTs from Chinese mitten crab Eriocheir sinensis.

    Science.gov (United States)

    Gai, Yunchao; Wang, Lingling; Zhao, Jianmin; Qiu, Limei; Song, Linsheng; Li, Ling; Mu, Changkao; Wang, Wan; Wang, Mengqiang; Zhang, Ying; Yao, Xuemei; Yang, Jialong

    2009-12-01

    Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from hemocytes of E. sinensis challenged with the mixture of Listonella anguillarum and Staphylococcus aureus, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. Single-pass 5' sequencing of 10368 clones yielded 7535 high quality ESTs (Expressed Sequence Tags) and these ESTs were assembled into 2943 unigenes. BLAST analysis revealed that 1706 unigenes (58.0% of the total) or 4593 ESTs (61.0% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest 1237 unigenes (42.0% of the total) were closely matched to the known genes or sequences deposited in public databases, which could be classed into 20 or 23 classifications according to "molecular function" or "biological process" respectively based on the Gene Ontology (GO). And 221 unigenes (7.5% of all 2943 unigenes, 17.9% of matched unigenes) or 969 ESTs (12.9% of all 7535 ESTs, 32.9% of matched ESTs) were identified to be immune genes. The relative higher proportion of immune-related genes in the present cDNA library than that in the normal library of E. sinensis and other crustaceans libraries, and the differences and changes in percentage and quantity of some key immune-related genes especially the immune inducible genes between two E. sinensis cDNA libraries may derive from the bacteria challenge to the Chinese mitten crab. The results provided a well-characterized EST resource for the genomics community, gene discovery especially for the identification of host-defense genes and pathways in crabs as well as other crustaceans.

  9. Analysis of expression sequence tags from a full-length-enriched cDNA library of developing sesame seeds (Sesamum indicum).

    Science.gov (United States)

    Ke, Tao; Dong, Caihua; Mao, Han; Zhao, Yingzhong; Chen, Hong; Liu, Hongyan; Dong, Xuyan; Tong, Chaobo; Liu, Shengyi

    2011-12-24

    Sesame (Sesamum indicum) is one of the most important oilseed crops with high oil contents and rich nutrient value. However, genetic improvement efforts in sesame could not get benefit from molecular biology technology due to poor DNA and RNA sequence resources. In this study, we carried out a large scale of expressed sequence tags (ESTs) sequencing from developing sesame seeds and further conducted analysis on seed storage products-related genes. A normalized and full-length enriched cDNA library from 5 ~ 30 days old immature seeds was constructed and randomly sequenced, leading to generation of 41,248 expressed sequence tags (ESTs) which then formed 4,713 contigs and 27,708 singletons with 44.9% uniESTs being putative full-length open reading frames. Approximately 26,091 of all these uniESTs have significant matches to the counterparts in Nr database of GenBank, and 21,628 of them were assigned to one or more Gene ontology (GO) terms. Homologous genes involved in oil biosynthesis were identified including some conservative transcription factors regulating oil biosynthesis such as LEAFY COTYLEDON1 (LEC1), PICKLE (PKL), WRINKLED1 (WRI1) and majority of them were found for the first time in sesame seeds. One hundred and 17 ESTs were identified possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin. In total, 9,347 putative functional genes from developing seeds were identified, which accounts for one third of total genes in the sesame genome. Further analysis of the uniESTs identified 1,949 non-redundant simple sequence repeats (SSRs). This study has provided an overview of genes expressed during sesame seed development. This collection of sesame full-length cDNAs covered a wide variety of genes in seeds, in particular, candidate genes involved in biosynthesis of sesame oils and lignans. These EST sequences enriched with full length will contribute to comparative genomic studies on sesame and other oilseed plants and serve as an abundant

  10. Analysis of expression sequence tags from a full-length-enriched cDNA library of developing sesame seeds (Sesamum indicum

    Directory of Open Access Journals (Sweden)

    Ke Tao

    2011-12-01

    Full Text Available Abstract Background Sesame (Sesamum indicum is one of the most important oilseed crops with high oil contents and rich nutrient value. However, genetic improvement efforts in sesame could not get benefit from molecular biology technology due to poor DNA and RNA sequence resources. In this study, we carried out a large scale of expressed sequence tags (ESTs sequencing from developing sesame seeds and further conducted analysis on seed storage products-related genes. Results A normalized and full-length enriched cDNA library from 5 ~ 30 days old immature seeds was constructed and randomly sequenced, leading to generation of 41,248 expressed sequence tags (ESTs which then formed 4,713 contigs and 27,708 singletons with 44.9% uniESTs being putative full-length open reading frames. Approximately 26,091 of all these uniESTs have significant matches to the counterparts in Nr database of GenBank, and 21,628 of them were assigned to one or more Gene ontology (GO terms. Homologous genes involved in oil biosynthesis were identified including some conservative transcription factors regulating oil biosynthesis such as LEAFY COTYLEDON1 (LEC1, PICKLE (PKL, WRINKLED1 (WRI1 and majority of them were found for the first time in sesame seeds. One hundred and 17 ESTs were identified possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin. In total, 9,347 putative functional genes from developing seeds were identified, which accounts for one third of total genes in the sesame genome. Further analysis of the uniESTs identified 1,949 non-redundant simple sequence repeats (SSRs. Conclusions This study has provided an overview of genes expressed during sesame seed development. This collection of sesame full-length cDNAs covered a wide variety of genes in seeds, in particular, candidate genes involved in biosynthesis of sesame oils and lignans. These EST sequences enriched with full length will contribute to comparative genomic studies on sesame and

  11. A simple and novel method for RNA-seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase.

    Science.gov (United States)

    Brouilette, Scott; Kuersten, Scott; Mein, Charles; Bozek, Monika; Terry, Anna; Dias, Kerith-Rae; Bhaw-Rosun, Leena; Shintani, Yasunori; Coppen, Steven; Ikebe, Chiho; Sawhney, Vinit; Campbell, Niall; Kaneko, Masahiro; Tano, Nobuko; Ishida, Hidekazu; Suzuki, Ken; Yashiro, Kenta

    2012-10-01

    Deep sequencing of single cell-derived cDNAs offers novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-seq analysis requires multiple steps with consequent sample loss and stochastic variation at each step significantly affecting output. Thus, a simpler and better protocol is desirable. The recently developed hyperactive Tn5-mediated library preparation, which brings high quality libraries, is likely one of the solutions. Here, we tested the applicability of hyperactive Tn5-mediated library preparation to deep sequencing of single cell cDNA, optimized the protocol, and compared it with the conventional method based on sonication. This new technique does not require any expensive or special equipment, which secures wider availability. A library was constructed from only 100 ng of cDNA, which enables the saving of precious specimens. Only a few steps of robust enzymatic reaction resulted in saved time, enabling more specimens to be prepared at once, and with a more reproducible size distribution among the different specimens. The obtained RNA-seq results were comparable to the conventional method. Thus, this Tn5-mediated preparation is applicable for anyone who aims to carry out deep sequencing for single cell cDNAs. Copyright © 2012 Wiley Periodicals, Inc.

  12. Construction of Geobacillus thermoglucosidasius cDNA library and analysis of genes expressed in response to heat stress.

    Science.gov (United States)

    Tripathy, S; Maiti, N K

    2014-03-01

    Thermophiles exhibit various kinds of molecular mechanisms to survive in extreme environment, but their behavioral responses to long duration stress is poorly understood until date. In the present study, we have prospected for the genes differentially expressed in response to long duration heat stress in thermophilic bacteria. A cDNA library was constructed from Geobacillus thermoglucosidasius grown with a temperature upshift of 10 °C from optimum growth temperature of 45 °C for 16 h. A total of 451 clones from the library were sequenced with accurate base calling that generated 257 high quality sequences with an average read length of 350 bp. We queried our collection of single pass sequences against the NCBI non-redundant database using the BLASTX algorithm and obtained sequences that showed significant similarity (>60%) with heat shock proteins, metabolic proteins and hypothetical proteins. The expressed sequence tags (ESTs) expressed in response to heat stress were annotated that further commuted a strong interaction network among one another. The ESTs based on the best hits were validated by RT-PCR. Di- and tri-nucleotide repeat motifs were also found to be associated with 17 genes involved in heat shock response, metabolism, transport and transcriptional regulation. The present results provide the novel identification of the putative genes responsible for imparting tolerance to bacteria under heat stress and unveil their role for survival of life in environmental extremes.

  13. Identification of differentially-expressed genes potentially implicated in drought response in pitaya (Hylocereus undatus) by suppression subtractive hybridization and cDNA microarray analysis.

    Science.gov (United States)

    Fan, Qing-Jie; Yan, Feng-Xia; Qiao, Guang; Zhang, Bing-Xue; Wen, Xiao-Peng

    2014-01-01

    Drought is one of the most severe threats to the growth, development and yield of plant. In order to unravel the molecular basis underlying the high tolerance of pitaya (Hylocereus undatus) to drought stress, suppression subtractive hybridization (SSH) and cDNA microarray approaches were firstly combined to identify the potential important or novel genes involved in the plant responses to drought stress. The forward (drought over drought-free) and reverse (drought-free over drought) suppression subtractive cDNA libraries were constructed using in vitro shoots of cultivar 'Zihonglong' exposed to drought stress and drought-free (control). A total of 2112 clones, among which half were from either forward or reverse SSH library, were randomly picked up to construct a pitaya cDNA microarray. Microarray analysis was carried out to verify the expression fluctuations of this set of clones upon drought treatment compared with the controls. A total of 309 expressed sequence tags (ESTs), 153 from forward library and 156 from reverse library, were obtained, and 138 unique ESTs were identified after sequencing by clustering and blast analyses, which included genes that had been previously reported as responsive to water stress as well as some functionally unknown genes. Thirty six genes were mapped to 47 KEGG pathways, including carbohydrate metabolism, lipid metabolism, energy metabolism, nucleotide metabolism, and amino acid metabolism of pitaya. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction (RT-PCR) corroborated the results of differential screening. Moreover, time-course expression patterns of these selected ESTs further confirmed that they were closely responsive to drought treatment. Among the differentially expressed genes (DEGs), many are related to stress tolerances including drought tolerance. Thereby, the mechanism of drought tolerance of this pitaya genotype is a very complex physiological and biochemical process, in

  14. The venom gland transcriptome of Latrodectus tredecimguttatus revealed by deep sequencing and cDNA library analysis.

    Directory of Open Access Journals (Sweden)

    Quanze He

    Full Text Available Latrodectus tredecimguttatus, commonly known as black widow spider, is well known for its dangerous bite. Although its venom has been characterized extensively, some fundamental questions about its molecular composition remain unanswered. The limited transcriptome and genome data available prevent further understanding of spider venom at the molecular level. In the present study, we combined next-generation sequencing and conventional DNA sequencing to construct a venom gland transcriptome of the spider L. tredecimguttatus, which resulted in the identification of 9,666 and 480 high-confidence proteins among 34,334 de novo sequences and 1,024 cDNA sequences, respectively, by assembly, translation, filtering, quantification and annotation. Extensive functional analyses of these proteins indicated that mRNAs involved in RNA transport and spliceosome, protein translation, processing and transport were highly enriched in the venom gland, which is consistent with the specific function of venom glands, namely the production of toxins. Furthermore, we identified 146 toxin-like proteins forming 12 families, including 6 new families in this spider in which α-LTX-Lt1a family2 is firstly identified as a subfamily of α-LTX-Lt1a family. The toxins were classified according to their bioactivities into five categories that functioned in a coordinate way. Few ion channels were expressed in venom gland cells, suggesting a possible mechanism of protection from the attack of their own toxins. The present study provides a gland transcriptome profile and extends our understanding of the toxinome of spiders and coordination mechanism for toxin production in protein expression quantity.

  15. Isolation and analysis of genes mainly expressed in adult mouse heart using subtractive hybridization cDNA library.

    Science.gov (United States)

    Komurcu-Bayrak, Evrim; Ozsait, Bilge; Erginel-Unaltuna, Nihan

    2012-08-01

    Subtractive hybridization cDNA library (SHL) is one of the powerful approaches for isolating differentially expressed genes. Using this technique between mouse heart and skeletal muscle (skm) tissues, we aimed to construct a cDNA-library that was specific to heart tissue and to identify the potential candidate genes that might be responsible for the development of cardiac diseases or related pathophysiological conditions. In the first step of the study, we created a cDNA-library between mouse heart and skm tissues. The homologies of the randomly selected 215 clones were analyzed and then classified by function. A total of 146 genes were analyzed for their expression profiles in the heart and skm tissues in published mouse microarray dataset. In the second step, we analyzed the expression patterns of the selected genes by Northern blot and RNA in situ hybridization (RISH). In Northern blot analyses, the expression levels of Myl3, Myl2, Mfn2, Dcn, Pdlim4, mt-Co3, mt-Co1, Atpase6 and Tsc22d1 genes were higher in heart than skm. For first time with this study, expression patterns of Pdlim4 and Tsc22d1 genes in mouse heart and skm were shown by RISH. In the last step, 43 genes in this library were identified to have relationships mostly with cardiac diseases and/or related phenotypes. This is the first study reporting differentially expressed genes in healthy mouse heart using SHL technique. This study confirms our hypothesis that tissue-specific genes are most likely to have a disease association, if they possess mutations.

  16. Ferritin from the Pacific abalone Haliotis discus hannai: Analysis of cDNA sequence, expression, and activity.

    Science.gov (United States)

    Qiu, Reng; Kan, Yunchao; Li, Dandan

    2016-02-01

    Ferritin plays an important role in iron homeostasis due to its ability to bind and sequester large amounts of iron. In this study, the gene encoding a ferritin (HdhFer2) was cloned from Pacific abalone (Haliotis discus hannai). The full-length cDNA of HdhFer2 contains a 5'-UTR of 121 bp, an ORF of 516 bp, and a 3'-UTR of 252 bp with a polyadenylation signal sequence of AATAAA and a poly(A) tail. It also contains a 31 bp iron-responsive element (IRE) in the 5'-UTR position, which is conserved in many ferritins. HdhFer2 consists of 171 amino acid residues with a predicted molecular weight (MW) ∼19.8 kDa and a theoretical isoelectric point (PI) of 4.84. The deduced amino acid sequence of HdhFer2 contains two ferritin iron-binding region signatures (IBRSs). HdhFer2 mRNA was detected in a wide range of tissues and was dominantly expressed in the gill. Infection with the bacterial pathogen Vibrio anguillarum significantly upregulated HdhFer2 expression in a time-dependent manner. Recombinant HdhFer2 (rHdhFer2) purified from Escherichia coli was able to bind ferrous iron in a concentration-dependent manner. In summary, these results suggest that HdhFer2 is a crucial protein in the iron-withholding defense system, and plays an important role in the innate immune response of abalone. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Improved production of fatty acids by Saccharomyces cerevisiae through screening a cDNA library from the oleaginous yeast Yarrowia lipolytica

    DEFF Research Database (Denmark)

    Shi, Shuobo; Ji, Haichuan; Siewers, Verena

    2016-01-01

    for screening a cDNA library from the oleaginous yeast Yarrowia lipolytica for identification of genes/enzymes that were able to enhance free FA accumulation in Saccharomyces cerevisiae. Several novel enzymes resulting in increasing FA accumulation were discovered. These targets include a GPI anchor protein...... method for high-throughput evaluation of the content of free FAs, but also give new insight into how enzymes from Y. lipolytica may increase the production of fatty acids in S. cerevisiae....

  18. Characterization of the venom from the Australian scorpion Urodacus yaschenkoi: Molecular mass analysis of components, cDNA sequences and peptides with antimicrobial activity.

    Science.gov (United States)

    Luna-Ramírez, Karen; Quintero-Hernández, Veronica; Vargas-Jaimes, Leonel; Batista, Cesar V F; Winkel, Kenneth D; Possani, Lourival D

    2013-03-01

    The Urodacidae scorpions are the most widely distributed of the four families in Australia and represent half of the species in the continent, yet their venoms remain largely unstudied. This communication reports the first results of a proteome analysis of the venom of the scorpion Urodacus yaschenkoi performed by mass fingerprinting, after high performance liquid chromatography (HPLC) separation. A total of 74 fractions were obtained by HPLC separation allowing the identification of approximately 274 different molecular masses with molecular weights varying from 287 to 43,437 Da. The most abundant peptides were those from 1 K Da and 4-5 K Da representing antimicrobial peptides and putative potassium channel toxins, respectively. Three such peptides were chemically synthesized and tested against Gram-positive and Gram-negative bacteria showing minimum inhibitory concentration in the low micromolar range, but with moderate hemolytic activity. It also reports a transcriptome analysis of the venom glands of the same scorpion species, undertaken by constructing a cDNA library and conducting random sequencing screening of the transcripts. From the resultant cDNA library 172 expressed sequence tags (ESTs) were analyzed. These transcripts were further clustered into 120 unique sequences (23 contigs and 97 singlets). The identified putative proteins can be assorted in several groups, such as those implicated in common cellular processes, putative neurotoxins and antimicrobial peptides. The scorpion U. yaschenkoi is not known to be dangerous to humans and its venom contains peptides similar to those of Opisthacanthus cayaporum (antibacterial), Scorpio maurus palmatus (maurocalcin), Opistophthalmus carinatus (opistoporines) and Hadrurus gerstchi (scorpine-like molecules), amongst others. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon.

    Science.gov (United States)

    Kang, Hong-Gyu; Jun, Sung-Hoon; Kim, Joonyul; Kawaide, Hiroshi; Kamiya, Yuji; An, Gynheung

    2002-02-01

    We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development.

  20. Full-length transcriptome analysis using a bias-free cDNA library prepared with the vector-capping method.

    Science.gov (United States)

    Kato, Seishi; Oshikawa, Mio; Ohtoko, Kuniyo

    2011-01-01

    Full-length complementary DNAs (cDNAs) are an essential resource for functional genomics. Recently, we have developed a simple and efficient method for preparing a full-length cDNA library from a small amount of total RNA, named the "vector-capping" method. The biggest advantage of this method is that the intactness of the cDNA can be assured by the presence of dG at the 5' end of the full-length cDNA. Furthermore, the cDNA library represents the mRNA population in the cell owing to a bias-free procedure. In this chapter, we describe not only the protocol for preparing the library but also the points for analyzing the 5'-end sequence of the obtained cDNA.

  1. Construction and analysis of cDNA libraries from the antennae of Batocera horsfieldi and expression pattern of putative odorant binding proteins.

    Science.gov (United States)

    Li, Hui; Zhang, Aijun; Chen, Li-Zhen; Zhang, Guoan; Wang, Man-Qun

    2014-04-19

    A high-quality cDNA library was constructed from female and male antenna of the longhorned beetle, Batocera horsfieldi (Hope) (Coleoptera: Cerambycidae), a serious pest of Populus (Salicales: Salicaceae). The titer was approximately 2.37 × 106 pfu/mL, and this complies with the test requirement. From the libraries, 692 clones were selected randomly, sequenced, and further analyzed, and the recombinational efficiency reached 93.85%. By alignment and cluster analysis, we identified four odorant binding proteins, two pheromone-binding proteins (have the characteristic six conserved cysteine residues), four Minus-C odorant binding proteins (lost two conserved cysteines), and three chemosensory proteins. In this study, we describe the identification and characterization of four new cDNAs that encode Minus-C odorant binding proteins (Minus-C OBPs) from B. horsfieldi antennal cDNA libraries. Our investigation focused on the expression pattern of the Minus-C OBP genes in various tissues in both sexes at different developmental stages, using reverse transcription PCR (RT-PCR) and realtime PCR (qPCR) strategies. Minus-C OBP1, 2, and 3 were expressed in all tested tissues, with the exception of the head (without antenna, labial palps, and maxillary palps). Minus-C OBP4 was expressed in the antenna, legs, and abdomen, but not in the labial palps, maxillary palps, or head. The qPCR results revealed MinusC OBPs were expressed in the antenna throughout the adult life, and that the transcript levels of these genes depended on the sex, age, and mating status of adults. This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed.

  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. [Construction of suppression subtractive hybridization cDNA library of half-blood males of Dermacentor silvarum and analysis of differentially expressed genes].

    Science.gov (United States)

    Liu, Qi; Wang, Wei-lin; Meng, Qing-feng; Xu, Zhan; Cui, Jie; Liu, Xin-xin; Wang, Wei-li

    2014-08-01

    To construct a suppression subtractive hybridization (SSH) cDNA library of half-blood males of Dermacentor silvarum, and analyze the differentially expressed genes. Total RNA was extracted from the half-blood males and unfed males of D. silvarum. cDNA was synthesized following the protocol of SMARTER cDNA synthesis kit. After Rsa I digestion, cDNA was ligated to adaptors. The cDNA from the half-blood males was used as the tester, and unfed males as the driver. The SSH library was constructed using TaKaRa PCR-select cDNA subtraction kit. Differentially expressed cDNAs were amplified by nested PCR, cloned into PMD-18T vector, transformed into E. coli DH5alpha, and the white-blue plaque selection was used to get the positive clones. The titer of SSH library and the recombination efficiency were calculated. Individual colonies were randomly selected from library. Subtractive efficiency of the subtracted cDNA library was examined by reverse Northern blotting and RT-PCR. Positive clones with differentially expressed genes were sequenced. Homology comparison and function prediction were performed by Blastn and Blastx. The bands of double-stranded cDNAs from half-blood males and unfed males of D. silvarum were dispersed and longer than 500 bp. After Rsa I digestion, the ds cDNA-fragments were 100-1000 bp. The ligation reaction efficiency of adaptor was more than 25%. Nested PCR showed that the bands of subtracted ds cDNA were gathered, ranging from 250 to 500 bp. The titer of SSH library was 700,000 pfu/ml, and the recombination efficiency was 88.5% (239/270). Reverse Northern hybridization revealed that the clones showed stronger signals in half-blood males cDNA probes than in unfed males cDNA probes. RT-PCR showed that among the eight random selected positive clones, 5 clones were up-expressed under half-blood condition. A total of 87 differentially expressed sequence tags (ESTs, 200-800 bp) were obtained from 115 positive clones. Among the 87 ESTs, 53 ESTs showed

  4. cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

    DEFF Research Database (Denmark)

    Young, M F; Kerr, J M; Termine, J D

    1990-01-01

    on a region of 4q that is near the centromere. A high-frequency restriction fragment length polymorphism was evident in the DNA from 29 unrelated individuals using the enzyme BglII. Analysis of total genomic DNA by digestion with several restriction enzymes, Southern blotting, and hybridization with the human...

  5. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes*

    Science.gov (United States)

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-01-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. PMID:26642182

  6. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes.

    Science.gov (United States)

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-12-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.

  7. [Construction and analysis of a forward and reverse subtractive cDNA library from leaves and stem of Polygonum sibiricum Laxm. under salt stress].

    Science.gov (United States)

    Liu, Guan-Jun; Liu, Ming-Kun; Xu, Zhi-Ru; Yan, Xiu-Feng; Wei, Zhi-Gang; Yang, Chuan-Ping

    2009-04-01

    Using cDNAs prepared from the leaves and stems of Polygonum sibiricum Laxm. treated with NaHCO3 stress for 48 h as testers and cDNAs from unstressed P. sibiricum leaves and stems as drivers library, suppression subtractive hybridization (SSH) was employed to construct a cDNA subtracted library, which contained 2 282 valid sequences including 598 ESTs in the stems forward SSH library and 490 ESTs in the stem reverse SSH library, 627 ESTs in the leaf forward SSH library and 567 in the leaf reverse SSH library. According to the functional catalogue of MIPs and the comparison of the reverse and forward SSH libraries of the stem and leaf, the responses to NaHCO3 stress were different between leaf and stem, except for the same trend in cell rescue defense and transport facilitation. The trend in the metabolism, energy, photosynthesis, protein synthesis, transcription, and signal transduction was opposite. RT-PCR analysis demonstrated that the expression of 12 putative stress related genes in the NaHCO3-treated leaves and stems was different from that in the untreated leaves and stems. This indicated that different mechanisms might be responsible for reactions of leaf and stem in P. sibiricum. The results from this study are useful in understanding the molecular mechanism of saline-alkali tolerance in P. sibiricum.

  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. Identification of late O{sub 3}-responsive genes in Arabidopsis thaliana by cDNA microarray analysis

    Energy Technology Data Exchange (ETDEWEB)

    D' Haese, D. [Univ. of Antwerp, Dept. of Biology, Antwerp (BE) and Univ. of Newcastle, School of Biology and Psychology, Div. of Biology, Newcastle-Upon-Tyne (United Kingdom); Horemans, N.; Coen, W. De; Guisez, Y. [Univ. of Antwerp, Dept. of Biology, Antwerp (Belgium)

    2006-09-15

    To better understand the response of a plant to 0{sub 3} stress, an integrated microarray analysis was performed on Arabidopsis plants exposed during 2 days to purified air or 150 nl l{sup -1} O{sub 3}, 8 h day-l. Agilent Arabidopsis 2 Oligo Microarrays were used of which the reliability was confirmed by quantitative real-time PCR of nine randomly selected genes. We confirmed the O{sub 3} responsiveness of heat shock proteins (HSPs), glutathione-S-tranferases and genes involved in cell wall stiffening and microbial defence. Whereas, a previous study revealed that during an early stage of the O{sub 3} stress response, gene expression was strongly dependent on jasmonic acid and ethylene, we report that at a later stage (48 h) synthesis of jasrnonic acid and ethylene was downregulated. In addition, we observed the simultaneous induction of salicylic acid synthesis and genes involved in programmed cell death and senescence. Also typically, the later stage of the response to O{sub 3} appeared to be the induction of the complete pathway leading to the biosynthesis of anthocyanin diglucosides and the induction of thioredoxin-based redox control. Surprisingly absent in the list of induced genes were genes involved in ASC-dependent antioxidation, few of which were found to be induced after 12 h of 0{sub 3} exposure in another study. We discuss these and other particular results of the microarray analysis and provide a map depicting significantly affected genes and their pathways highlighting their interrelationships and subcellular localization. (au)

  10. Analysis of the effects of sex hormone background on the rat choroid plexus transcriptome by cDNA microarrays.

    Directory of Open Access Journals (Sweden)

    Telma Quintela

    Full Text Available The choroid plexus (CP are highly vascularized branched structures that protrude into the ventricles of the brain, and form a unique interface between the blood and the cerebrospinal fluid (CSF, the blood-CSF barrier, that are the main site of production and secretion of CSF. Sex hormones are widely recognized as neuroprotective agents against several neurodegenerative diseases, and the presence of sex hormones cognate receptors suggest that it may be a target for these hormones. In an effort to provide further insight into the neuroprotective mechanisms triggered by sex hormones we analyzed gene expression differences in the CP of female and male rats subjected to gonadectomy, using microarray technology. In gonadectomized female and male animals, 3045 genes were differentially expressed by 1.5-fold change, compared to sham controls. Analysis of the CP transcriptome showed that the top-five pathways significantly regulated by the sex hormone background are olfactory transduction, taste transduction, metabolism, steroid hormone biosynthesis and circadian rhythm pathways. These results represent the first overview of global expression changes in CP of female and male rats induced by gonadectomy and suggest that sex hormones are implicated in pathways with central roles in CP functions and CSF homeostasis.

  11. Identification and expression analysis of cDNA encoding chitinase-like protein (CLP) gene in Japanese scallop Mizuhopecten yessoensis.

    Science.gov (United States)

    Gao, L; Xu, G J; Su, H; Gao, X G; Li, Y F; Bao, X B; Liu, W D; He, C B

    2014-12-18

    Chitinase-like proteins (CLP) are important members of the glycoside hydrolase family 18 (GH18) and are involved in growth control and remodeling processes. In this study, a CLP transcript was isolated and sequenced from the Japanese scallop (Mizuhopecten yessoensis) after screening expressed sequence tags. The full-length complementary DNA of M. yessoensis CLP (My-Clp1) was 1555 bp in length, consisting of a 75-bp 5'-untranslated region (UTR), a 160-bp 3'-UTR, and a 1320-bp open reading frame bearing characteristics of the GH18 family. The My-Clp1 protein was well conserved, with similar domain structures and architecture across species (e.g., from mollusks to mammals). Expression analysis in healthy tissues and across developmental stages revealed a strong preference for expression; My-Clp1 was abundantly expressed in the mantle and throughout metamorphosis, which suggests the involvement of My-Clp1 in the synthesis of extracellular components, and tissue degeneration and remodeling. My-Clp1 expression was induced after infection with a bacterial pathogen, Vibrio anguillarum, suggesting its involvement in immunity against this intracellular pathogen.

  12. Improving Loop Dependence Analysis

    DEFF Research Database (Denmark)

    Jensen, Nicklas Bo; Karlsson, Sven

    2017-01-01

    Programmers can no longer depend on new processors to have significantly improved single-thread performance. Instead, gains have to come from other sources such as the compiler and its optimization passes. Advanced passes make use of information on the dependencies related to loops. We improve th...

  13. Differential gene expression in a DNA double-strand-break repair mutant XRS-5 defective in Ku80. Analysis by cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Chan, John Y.H.; Chen, Lung-Kun; Chang, Jui-Feng [National Yang Ming Univ., Taipei, Taiwan (China). Inst. of Radiological Sciences] (and others)

    2001-12-01

    The ability of cells to rejoin DNA double-strand breaks (DSBs) usually correlates with their radiosensitivity. This correlation has been demonstrated in radiosensitive cells, including the Chinese hamster ovary mutant XRS-5. XRS-5 is defective in a DNA end-binding protein, Ku80, which is a component of a DNA-dependent protein kinase complex used for joining strand breaks. However, Ku80-deficient cells are known to be retarded in cell proliferation and growth as well as other yet to be identified defects. Using custom-made 600-gene cDNA microarray filters, we found differential gene expressions between the wild-type and XRS-5 cells. Defective Ku80 apparently affects the expression of several repair genes, including topoisomerase-I and -IIA, ERCC5, MLH1, and ATM. In contrast, other DNA repair-associated genes, such as GADD45A, EGR1 MDM2 and p53, were not affected. In addition, for large numbers of growth-associated genes, such as cyclins and clks, the growth factors and cytokines were also affected. Down-regulated expression was also found in several categories of seemingly unrelated genes, including apoptosis, angiogenesis, kinase and signaling, phosphatase, stress protein, proto-oncogenes and tumor suppressors, transcription and translation factors. A RT-PCR analysis confirmed that the XRS-5 cells used were defective in Ku80 expression. The diversified groups of genes being affected could mean that Ku80, a multi-functional DNA-binding protein, not only affects DNA repair, but is also involved in transcription regulation. Our data, taken together, indicate that there are specific genes being modulated in Ku80- deficient cells, and that some of the DNA repair pathways and other biological functions are apparently linked, suggesting that a defect in one gene could have global effects on many other processes. (author)

  14. Gene expression analysis of the rat testis after treatment with di(2-ethylhexyl) phthalate using cDNA microarray and real-time RT-PCR

    International Nuclear Information System (INIS)

    Kijima, Kazuyasu; Toyosawa, Kaoru; Yasuba, Masashi; Matsuoka, Nobuo; Adachi, Tetsuya; Komiyama, Masatoshi; Mori, Chisato

    2004-01-01

    To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment

  15. Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jacob

    2007-01-01

    with the greatest number of different expressed genes, whereas tissues with more specialized function, such as developing liver, have fewer expressed genes. There are at least 65 high confidence housekeeping gene candidates and 876 cDNA library-specific gene candidates. We identified differential expression...

  16. Sequencing and comparative genomics analysis inSenecio scandensBuch.-Ham. Ex D. Don, based on full-length cDNA library.

    Science.gov (United States)

    Qian, Gang; Ping, Junjiao; Zhang, Zhen; Xu, Delin

    2014-09-03

    Senecio scandens Buch.-Ham. ex D. Don, an important antibacterial source of Chinese traditional medicine, has a widespread distribution in a few ecological habitats of China. We generated a full-length complementary DNA (cDNA) library from a sample of elite individuals with superior antibacterial properties, with satisfactory parameters such as library storage (4.30 × 10 6 CFU), efficiency of titre (1.30 × 10 6 CFU/mL), transformation efficiency (96.35%), full-length ratio (64.00%) and redundancy ratio (3.28%). The BLASTN search revealed the facile formation of counterparts between the experimental sample and Arabidopsis thaliana in view of high-homology cDNA sequence (90.79%) with e -values cDNA clones consist of the major of functional genes identified by a large set of microarray data from the present experimental material. For other Compositae species, a large set of full-length cDNA clones reported in the present article will serve as a useful resource to facilitate further research on the transferability of expressed sequence tag-derived simple sequence repeats (EST-SSR) development, comparative genomics and novel transcript profiles.

  17. Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jacob

    2007-01-01

    public databases. The Sino-Danish ESTs were generated from one normalized and 97 non-normalized cDNA libraries representing 35 different tissues and three developmental stages. RESULTS: Using the Distiller package, the ESTs were assembled to roughly 48,000 contigs and 73,000 singletons, of which...

  18. 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

  19. 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.

  20. 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.

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    Czosnek Henryk

    2006-04-01

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

  3. Analysis of cellular responses to aflatoxin B{sub 1} in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Guo Yingying [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Breeden, Linda L. [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Fan, Wenhong [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zhao Lueping [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Eaton, David L. [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zarbl, Helmut [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States) and Fred Hutchinson Cancer Research Center, Seattle, WA (United States)]. E-mail: hzarbl@fhcrc.org

    2006-01-29

    Aflatoxin B1 (AFB{sub 1}) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB{sub 1} is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N{sup 7}-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB{sub 1}, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB{sub 1} that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB{sub 1} treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB{sub 1}-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific

  4. [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.

  5. 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.

  6. Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs

    Directory of Open Access Journals (Sweden)

    Sebastian Boltaña

    2017-02-01

    Full Text Available This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS and peptidoglycan (PGN. Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs, carrier proteins/membrane transport (approximately 15%, effectors/modulators and cell communication (approximately 11%, nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5% and intracellular transducers/signal transduction (approximately 5%. Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ that provides a platform enriched for the study

  7. 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.

  8. 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

  9. Analysis of 4,664 high-quality sequence-finished poplar full-length cDNA clones and their utility for the discovery of genes responding to insect feeding

    Directory of Open Access Journals (Sweden)

    Douglas Carl J

    2008-01-01

    Full Text Available Abstract Background The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. Results As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa × P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones

  10. Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

    Directory of Open Access Journals (Sweden)

    Tuomas Rönnberg

    Full Text Available Hantaviruses (Bunyaviridae are negative-strand RNA viruses with a tripartite genome. The small (S segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs. The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

  11. [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).

  12. 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.

  13. The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies

    Directory of Open Access Journals (Sweden)

    Elnaiem Dia-Eldin

    2008-01-01

    Full Text Available Abstract Background In the life cycle of Leishmania within the alimentary canal of sand flies the parasites have to survive the hostile environment of blood meal digestion, escape the blood bolus and attach to the midgut epithelium before differentiating into the infective metacyclic stages. The molecular interactions between the Leishmania parasites and the gut of the sand fly are poorly understood. In the present work we sequenced five cDNA libraries constructed from midgut tissue from the sand fly Lutzomyia longipalpis and analyzed the transcripts present following sugar feeding, blood feeding and after the blood meal has been processed and excreted, both in the presence and absence of Leishmania infantum chagasi. Results Comparative analysis of the transcripts from sugar-fed and blood-fed cDNA libraries resulted in the identification of transcripts differentially expressed during blood feeding. This included upregulated transcripts such as four distinct microvillar-like proteins (LuloMVP1, 2, 4 and 5, two peritrophin like proteins, a trypsin like protein (Lltryp1, two chymotrypsin like proteins (LuloChym1A and 2 and an unknown protein. Downregulated transcripts by blood feeding were a microvillar-like protein (LuloMVP3, a trypsin like protein (Lltryp2 and an astacin-like metalloprotease (LuloAstacin. Furthermore, a comparative analysis between blood-fed and Leishmania infected midgut cDNA libraries resulted in the identification of the transcripts that were differentially expressed due to the presence of Leishmania in the gut of the sand fly. This included down regulated transcripts such as four microvillar-like proteins (LuloMVP1,2, 4 and 5, a Chymotrypsin (LuloChym1A and a carboxypeptidase (LuloCpepA1, among others. Upregulated midgut transcripts in the presence of Leishmania were a peritrophin like protein (LuloPer1, a trypsin-like protein (Lltryp2 and an unknown protein. Conclusion This transcriptome analysis represents the largest set

  14. 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.

  15. Laser capture microdissection and cDNA array analysis of endometrium identify CCL16 and CCL21 as epithelial-derived inflammatory mediators associated with endometriosis

    Directory of Open Access Journals (Sweden)

    Jones Rebecca L

    2007-05-01

    Full Text Available Abstract Background Understanding the pathophysiology of chemokine secretion in endometriosis may offer a novel area of therapeutic intervention. This study aimed to identify chemokines differentially expressed in epithelial glands in eutopic endometrium from normal women and those with endometriosis, and to establish the expression profiles of key chemokines in endometriotic lesions. Methods Laser capture microdissection isolated epithelial glands from endometrial eutopic tissue from women with and without endometriosis in the mid-secretory phase of their menstrual cycles. Gene profiling of the excised glands used a human chemokine and receptor cDNA array. Selected chemokines were further examined using real-time PCR and immunohistochemistry. Results 22 chemokine/receptor genes were upregulated and two downregulated in pooled endometrial epithelium of women with endometriosis compared with controls. CCL16 and CCL21 mRNA was confirmed as elevated in some women with endometriosis compared to controls on individual samples. Immunoreactive CCL16 and CCL21 were predominantly confined to glands in eutopic and ectopic endometrium: leukocytes also stained. Immunoreactive CCL16 was overall higher in glands in ectopic vs. eutopic endometrium from the same woman (P Conclusion This study provides novel candidate molecules and suggests a potential local role for CCL16 and CCL21 as mediators contributing to the inflammatory events associated with endometriosis.

  16. Identification and expression analysis of peroxisome proliferator-activated receptors cDNA in a reptile, the leopard gecko (Eublepharis macularius).

    Science.gov (United States)

    Kato, Keisuke; Oka, Yoshitaka; Park, Min Kyun

    2008-05-01

    Despite the physiological and evolutionary significance of lipid metabolism in amniotes, the molecular mechanisms involved have been unclear in reptiles. To elucidate this, we investigated peroxisome proliferators-activated receptors (PPARs) in the leopard gecko (Eublepharis macularius). PPARs belong to a nuclear hormone-receptor family mainly involved in lipid metabolism. Although PPARs have been widely studied in mammals, little information about them is yet available from reptiles. We identified in the leopard gecko partial cDNA sequences of PPARalpha and beta, and full sequences of two isoforms of PPARgamma. This is the first report of reptilian PPARgamma mRNA isoforms. We also evaluated the organ distribution of expression of these genes by using RT-PCR and competitive PCR. The expression level of PPARalpha mRNA was highest in the large intestine, and moderate in the liver and kidney. The expression level of PPARbeta mRNA was highest in the kidney and large intestine, and moderate in the liver. Similarly to the expression of human PPARgamma isoforms, PPARgammaa was expressed ubiquitously, whereas the expression of PPARgammab was restricted. The highest levels of their expression, however, were observed in the large intestine, rather than in the adipose tissue as in mammals. Taken together, these results showed that the profile of PPARbeta mRNA expression in the leopard gecko is similar to that in mammals, and that those of PPAR alpha and gamma are species specific. This may reflect adaptation to annual changes in lipid storage due to seasonal food availability.

  17. Construction and analysis of an SSH cDNA library of early heat-induced genes of Vigna aconitifolia variety RMO-40.

    Science.gov (United States)

    Rampuria, Sakshi; Joshi, Uma; Palit, Paramita; Deokar, Amit A; Meghwal, Raju R; Mohapatra, T; Srinivasan, R; Bhatt, K V; Sharma, Ramavtar

    2012-11-01

    Moth bean ( Vigna aconitifolia (Jacq.) Marechal) is an important grain legume crop grown in rain fed areas of hot desert regions of Thar, India, under scorching sun rays with very little supplementation of water. An SSH cDNA library was generated from leaf tissues of V. aconitifolia var. RMO-40 exposed to an elevated temperature of 42 °C for 5 min to identify early-induced genes. A total of 488 unigenes (114 contigs and 374 singletons) were derived by cluster assembly and sequence alignment of 738 ESTs; out of 206 ESTs (28%) of unknown proteins, 160 ESTs (14%) were found to be novel to moth bean. Only 578 ESTs (78%) showed significant BLASTX similarity (<1 × 10(-6)) in the NCBI non-redundant database. Gene ontology functional classification terms were retrieved for 479 (65%) sequences, and 339 sequences were annotated with 165 EC codes and mapped to 68 different KEGG pathways. Four hundred and fifty-two ESTs were further annotated with InterProScan (IPS), and no IPS was assigned to 153 ESTs. In addition, the expression level of 27 ESTs in response to heat stress was evaluated through semiquantitative RT-PCR assay. Approximately 20 different signaling genes and 16 different transcription factors have been shown to be associated with heat stress in moth bean for the first time.

  18. 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

  19. 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.

  20. Gene structure, cDNA characterization and RNAi-based functional analysis of a myeloid differentiation factor 88 homolog in Tenebrio molitor larvae exposed to Staphylococcus aureus infection.

    Science.gov (United States)

    Patnaik, Bharat Bhusan; Patnaik, Hongray Howrelia; Seo, Gi Won; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Han, Yeon Soo

    2014-10-01

    Myeloid differentiation factor 88 (MyD88), an intracellular adaptor protein involved in Toll/Toll-like receptor (TLR) signal processing, triggers activation of nuclear factor-kappaB (NF-κB) transcription factors. In the present study, we analyzed the gene structure and biological function of MyD88 in a coleopteran insect, Tenebrio molitor (TmMyD88). The TmMyD88 gene was 1380 bp in length and consisted of five exons and four introns. The 5'-flanking sequence revealed several putative transcription factor binding sites, such as STAT-4, AP-1, cJun, cfos, NF-1 and many heat shock factor binding elements. The cDNA contained a typical death domain, a conservative Toll-like interleukin-1 receptor (TIR) domain, and a C-terminal extension (CTE). The TmMyD88 TIR domain showed three significantly conserved motifs for interacting with the TIR domain of TLRs. TmMyD88 was grouped within the invertebrate cluster of the phylogenetic tree and shared 75% sequence identity with the TIR domain of Tribolium castaneum MyD88. Homology modeling of the TmMyD88 TIR domain revealed five parallel β-strands surrounded by five α-helices that adopted loop conformations to function as an adaptor. TmMyD88 expression was upregulated 7.3- and 4.79-fold after 12 and 6h, respectively, of challenge with Staphylococcus aureus and fungal β-1,3 glucan. Silencing of the TmMyD88 transcript by RNA interference led to reduced resistance of the host to infection by S. aureus. These results indicate that TmMyD88 is required for survival against Staphylococcus infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Analysis of expressed sequence tags (ESTs) from a normalized cDNA library and isolation of EST simple sequence repeats from the invasive cotton mealybug Phenacoccus solenopsis.

    Science.gov (United States)

    Li, Hui; Lang, Kun-Ling; Fu, Hai-Bin; Shen, Chang-Peng; Wan, Fang-Hao; Chu, Dong

    2015-12-01

    The cotton mealybug, Phenacoccus solenopsis Tinsley, is a serious and invasive pest. At present, genetic resources for studying P. solenopsis are limited, and this negatively affects genetic research on the organism and, consequently, translational work to improve management of this pest. In the present study, expressed sequence tags (ESTs) were analyzed from a normalized complementary DNA library of P. solenopsis. In addition, EST-derived microsatellite loci (also known as simple sequence repeats or SSRs) were isolated and characterized. A total of 1107 high-quality ESTs were acquired from the library. Clustering and assembly analysis resulted in 785 unigenes, which were classified functionally into 23 categories according to the Gene Ontology database. Seven EST-based SSR markers were developed in this study and are expected to be useful in characterizing how this invasive species was introduced, as well as providing insights into its genetic microevolution. © 2014 Institute of Zoology, Chinese Academy of Sciences.

  2. 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

  3. 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...

  4. Differential Gene Expression in Response to Papaya ringspot virus Infection in Cucumis metuliferus Using cDNA- Amplified Fragment Length Polymorphism Analysis

    Science.gov (United States)

    Lin, Chia-Wei; Chung, Chien-Hung; Chen, Jo-Chu; Yeh, Shy-Dong; Ku, Hsin-Mei

    2013-01-01

    A better understanding of virus resistance mechanisms can offer more effective strategies to control virus diseases. Papaya ringspot virus (PRSV), Potyviridae, causes severe economical losses in papaya and cucurbit production worldwide. However, no resistance gene against PRSV has been identified to date. This study aimed to identify candidate PRSV resistance genes using cDNA-AFLP analysis and offered an open architecture and transcriptomic method to study those transcripts differentially expressed after virus inoculation. The whole genome expression profile of Cucumis metuliferus inoculated with PRSV was generated using cDNA-amplified fragment length polymorphism (cDNA-AFLP) method. Transcript derived fragments (TDFs) identified from the resistant line PI 292190 may represent genes involved in the mechanism of PRSV resistance. C. metuliferus susceptible Acc. 2459 and resistant PI 292190 lines were inoculated with PRSV and subsequently total RNA was isolated for cDNA-AFLP analysis. More than 400 TDFs were expressed specifically in resistant line PI 292190. A total of 116 TDFs were cloned and their expression patterns and putative functions in the PRSV-resistance mechanism were further characterized. Subsequently, 28 out of 116 candidates which showed two-fold higher expression levels in resistant PI 292190 than those in susceptible Acc. 2459 after virus inoculation were selected from the reverse northern blot and bioinformatic analysis. Furthermore, the time point expression profiles of these candidates by northern blot analysis suggested that they might play roles in resistance against PRSV and could potentially provide valuable information for controlling PRSV disease in the future. PMID:23874746

  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. 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.

  7. 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 ...

  8. 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...

  9. [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).

  10. Generation and analysis of a large-scale expressed sequence Tag database from a full-length enriched cDNA library of developing leaves of Gossypium hirsutum L.

    Directory of Open Access Journals (Sweden)

    Min Lin

    Full Text Available BACKGROUND: Cotton (Gossypium hirsutum L. is one of the world's most economically-important crops. However, its entire genome has not been sequenced, and limited resources are available in GenBank for understanding the molecular mechanisms underlying leaf development and senescence. METHODOLOGY/PRINCIPAL FINDINGS: In this study, 9,874 high-quality ESTs were generated from a normalized, full-length cDNA library derived from pooled RNA isolated from throughout leaf development during the plant blooming stage. After clustering and assembly of these ESTs, 5,191 unique sequences, representative 1,652 contigs and 3,539 singletons, were obtained. The average unique sequence length was 682 bp. Annotation of these unique sequences revealed that 84.4% showed significant homology to sequences in the NCBI non-redundant protein database, and 57.3% had significant hits to known proteins in the Swiss-Prot database. Comparative analysis indicated that our library added 2,400 ESTs and 991 unique sequences to those known for cotton. The unigenes were functionally characterized by gene ontology annotation. We identified 1,339 and 200 unigenes as potential leaf senescence-related genes and transcription factors, respectively. Moreover, nine genes related to leaf senescence and eleven MYB transcription factors were randomly selected for quantitative real-time PCR (qRT-PCR, which revealed that these genes were regulated differentially during senescence. The qRT-PCR for three GhYLSs revealed that these genes express express preferentially in senescent leaves. CONCLUSIONS/SIGNIFICANCE: These EST resources will provide valuable sequence information for gene expression profiling analyses and functional genomics studies to elucidate their roles, as well as for studying the mechanisms of leaf development and senescence in cotton and discovering candidate genes related to important agronomic traits of cotton. These data will also facilitate future whole-genome sequence

  11. PEX12, the pathogenic gene of group III Zellweger syndrome: cDNA cloning by functional complementation on a CHO cell mutant, patient analysis, and characterization of PEX12p

    NARCIS (Netherlands)

    Okumoto, K.; Shimozawa, N.; Kawai, A.; Tamura, S.; Tsukamoto, T.; Osumi, T.; Moser, H.; Wanders, R. J.; Suzuki, Y.; Kondo, N.; Fujiki, Y.

    1998-01-01

    Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11-20, 1997), using a transient transfection assay and

  12. Improving the production of transgenic fish germlines: in vivo evaluation of mosaicism in zebrafish (Danio rerio using a green fluorescent protein (GFP and growth hormone cDNA transgene co-injection strategy

    Directory of Open Access Journals (Sweden)

    Márcio de Azevedo Figueiredo

    2007-01-01

    Full Text Available In fish, microinjection is the method most frequently used for gene transfer. However, due to delayed transgene integration this technique almost invariably produces mosaic individuals and if the gene is not integrated into germ cells its transmission to descendants is difficult or impossible. We evaluated the degree of in vivo mosaicism using a strategy where a reporter transgene is co-injected with a transgene of interest so that potential germline founders can be easily identified. Transgenic zebrafish (Danio rerio were produced using two transgenes, both comprised of the carp beta-actin promoter driving the expression of either the green fluorescent protein (GFP reporter gene or the growth hormone cDNA from the marine silverside fish Odonthestes argentinensis. The methodology applied allowed a rapid identification of G0 transgenic fish and also detected which fish were transmitting transgenes to the next generation. This strategy also allowed inferences to be made about genomic transgene integration events in the six lineages produced and allowed the identification of one lineage transmitting both transgenes linked on the same chromosome. These results represent a significant advance in the reduction of the effort invested in producing a stable genetically modified fish lineage.

  13. 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.

  14. Analysis and Improvement of Fireworks Algorithm

    OpenAIRE

    Xi-Guang Li; Shou-Fei Han; Chang-Qing Gong

    2017-01-01

    The Fireworks Algorithm is a recently developed swarm intelligence algorithm to simulate the explosion process of fireworks. Based on the analysis of each operator of Fireworks Algorithm (FWA), this paper improves the FWA and proves that the improved algorithm converges to the global optimal solution with probability 1. The proposed algorithm improves the goal of further boosting performance and achieving global optimization where mainly include the following strategies. Firstly using the opp...

  15. Software Process Improvement Using Force Field Analysis ...

    African Journals Online (AJOL)

    An improvement plan is then drawn and implemented. This paper studied the state of Nigerian software development organizations based on selected attributes. Force field analysis is used to partition the factors obtained into driving and restraining forces. An attempt was made to improve the software development process ...

  16. Improved security analysis of Fugue-256 (poster)

    DEFF Research Database (Denmark)

    Gauravaram, Praveen; Knudsen, Lars Ramkilde; Bagheri, Nasoor

    2011-01-01

    We present some improved analytical results as part of the ongoing work on the analysis of Fugue-256 hash function, a second round candidate in the NIST's SHA3 competition. First we improve Aumasson and Phans' integral distinguisher on the 5.5 rounds of the final transformation of Fugue-256 to 16...

  17. 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.

  18. 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

  19. Conducting a SWOT Analysis for Program Improvement

    Science.gov (United States)

    Orr, Betsy

    2013-01-01

    A SWOT (strengths, weaknesses, opportunities, and threats) analysis of a teacher education program, or any program, can be the driving force for implementing change. A SWOT analysis is used to assist faculty in initiating meaningful change in a program and to use the data for program improvement. This tool is useful in any undergraduate or degree…

  20. Improvement of electrophoresis performance by spectral analysis ...

    African Journals Online (AJOL)

    This paper describes a new design of standard agarose gel electrophoresis procedure for nucleic acids analysis. The electrophoresis was improved by using the real-time spectral analysis of the samples to increase its performance. A laser beam illuminated the analysed sample at wavelength with the highest absorption of ...

  1. 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 ...

  2. 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

  3. Expression cloning screening of a unique and full-length set of cDNA clones is an efficient method for identifying genes involved in Xenopus neurogenesis.

    Science.gov (United States)

    Voigt, Jana; Chen, Jun-An; Gilchrist, Mike; Amaya, Enrique; Papalopulu, Nancy

    2005-03-01

    Functional screens, where a large numbers of cDNA clones are assayed for certain biological activity, are a useful tool in elucidating gene function. In Xenopus, gain of function screens are performed by pool screening, whereby RNA transcribed in vitro from groups of cDNA clones, ranging from thousands to a hundred, are injected into early embryos. Once an activity is detected in a pool, the active clone is identified by sib-selection. Such screens are intrinsically biased towards potent genes, whose RNA is active at low quantities. To improve the sensitivity and efficiency of a gain of function screen we have bioinformatically processed an arrayed and EST sequenced set of 100,000 gastrula and neurula cDNA clones, to create a unique and full-length set of approximately 2500 clones. Reducing the redundancy and excluding truncated clones from the starting clone set reduced the total number of clones to be screened, in turn allowing us to reduce the pool size to just eight clones per pool. We report that the efficiency of screening this clone set is five-fold higher compared to a redundant set derived from the same libraries. We have screened 960 cDNA clones from this set, for genes that are involved in neurogenesis. We describe the overexpression phenotypes of 18 single clones, the majority of which show a previously uncharacterised phenotype and some of which are completely novel. In situ hybridisation analysis shows that a large number of these genes are specifically expressed in neural tissue. These results demonstrate the effectiveness of a unique full-length set of cDNA clones for uncovering players in a developmental pathway.

  4. 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.

  5. Improved security analysis of Fugue-256 (poster)

    DEFF Research Database (Denmark)

    Gauravaram, Praveen; Knudsen, Lars Ramkilde; Bagheri, Nasoor

    2011-01-01

    We present some improved analytical results as part of the ongoing work on the analysis of Fugue-256 hash function, a second round candidate in the NIST's SHA3 competition. First we improve Aumasson and Phans' integral distinguisher on the 5.5 rounds of the final transformation of Fugue-256 to 16.......5 rounds. Next we improve the designers' meet-in-the-middle preimage attack on Fugue-256 from 2480 time and memory to 2416. Finally, we comment on possible methods to obtain free-start distinguishers and free-start collisions for Fugue-256. © 2011 Springer-Verlag....

  6. [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.

  7. Studies on the isolation, structural analysis and tissue localization of fetal antigen 1 and its relation to a human adrenal-specific cDNA, pG2

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Teisner, Børge; Højrup, Peter

    1993-01-01

    sequence was back-translated into the appropriate degenerate sequence of nucleic acids, fetal antigen 1 could be partially aligned to a 'human adrenal-specific mRNA, pG2'. The indirect immunoperoxidase technique demonstrated fetal antigen 1 in fetal hepatocytes, glandular cells of fetal pancreas...... is encoded by the mRNA defined by the cDNA clone pG2, but definitive sequencing and expression studies of this mRNA have not been achieved. Udgivelsesdato: 1993-Apr...

  8. Assessment and improvement of the Plasmodium yoelii yoelii genome annotation through comparative analysis.

    Science.gov (United States)

    Vaughan, Ashley; Chiu, Sum-Ying; Ramasamy, Gowthaman; Li, Ling; Gardner, Malcolm J; Tarun, Alice S; Kappe, Stefan H I; Peng, Xinxia

    2008-07-01

    The sequencing of the Plasmodium yoelii genome, a model rodent malaria parasite, has greatly facilitated research for the development of new drug and vaccine candidates against malaria. Unfortunately, only preliminary gene models were annotated on the partially sequenced genome, mostly by in silico gene prediction, and there has been no major improvement of the annotation since 2002. Here we report on a systematic assessment of the accuracy of the genome annotation based on a detailed analysis of a comprehensive set of cDNA sequences and proteomics data. We found that the coverage of the current annotation tends to be biased toward genes expressed in the blood stages of the parasite life cycle. Based on our proteomic analysis, we estimate that about 15% of the liver stage proteome data we have generated is absent from the current annotation. Through comparative analysis we identified and manually curated a further 510 P. yoelii genes which have clear orthologs in the P. falciparum genome, but were not present or incorrectly annotated in the current annotation. This study suggests that improvements of the current P. yoelii genome annotation should focus on genes expressed in stages other than blood stages. Comparative analysis will be critically helpful for this re-annotation. The addition of newly annotated genes will facilitate the use of P. yoelii as a model system for studying human malaria. Supplementary data are available at Bioinformatics online.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Mining the bitter melon (momordica charantia l. seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes

    Directory of Open Access Journals (Sweden)

    Shipp Matthew J

    2010-11-01

    Full Text Available Abstract Background Seeds of Momordica charantia (bitter melon produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological transfer of conjugated fatty acid production to existing oilseed crops. It is expected that these studies will also provide basic information regarding the metabolism of other high-value novel fatty acids. Results Deep sequencing using 454 technology with non-normalized and normalized cDNA libraries prepared from bitter melon seeds at 18 DAP resulted in the identification of transcripts for the vast majority of known genes involved in fatty acid and triacylglycerol biosynthesis. The non-normalized library provided a transcriptome profile of the early stage in seed development that highlighted the abundance of transcripts for genes encoding seed storage proteins as well as for a number of genes for lipid metabolism-associated polypeptides, including Δ12 oleic acid desaturases and fatty acid conjugases, class 3 lipases, acyl-carrier protein, and acyl-CoA binding protein. Normalization of cDNA by use of a duplex-specific nuclease method not only increased the overall discovery of genes from developing bitter melon seeds, but also resulted in the identification of 345 contigs with homology to 189 known lipid genes in Arabidopsis. These included candidate genes for eleostearic acid metabolism such as diacylglycerol acyltransferase 1 and 2, and a phospholipid:diacylglycerol acyltransferase 1-related enzyme. Transcripts were also identified for a novel FAD2 gene encoding a functional Δ12 oleic acid desaturase with potential implications for eleostearic acid biosynthesis. Conclusions 454 deep sequencing, particularly with normalized cDNA populations, was an effective method for mining of genes associated with eleostearic acid metabolism in

  15. In-depth cDNA library sequencing provides quantitative gene expression profiling in cancer biomarker discovery.

    Science.gov (United States)

    Yang, Wanling; Ying, Dingge; Lau, Yu-Lung

    2009-06-01

    Quantitative gene expression analysis plays an important role in identifying differentially expressed genes in various pathological states, gene expression regulation and co-regulation, shedding light on gene functions. Although microarray is widely used as a powerful tool in this regard, it is suboptimal quantitatively and unable to detect unknown gene variants. Here we demonstrated effective detection of differential expression and co-regulation of certain genes by expressed sequence tag analysis using a selected subset of cDNA libraries. We discussed the issues of sequencing depth and library preparation, and propose that increased sequencing depth and improved preparation procedures may allow detection of many expression features for less abundant gene variants. With the reduction of sequencing cost and the emerging of new generation sequencing technology, in-depth sequencing of cDNA pools or libraries may represent a better and powerful tool in gene expression profiling and cancer biomarker detection. We also propose using sequence-specific subtraction to remove hundreds of the most abundant housekeeping genes to increase sequencing depth without affecting relative expression ratio of other genes, as transcripts from as few as 300 most abundantly expressed genes constitute about 20% of the total transcriptome. In-depth sequencing also represents a unique advantage of detecting unknown forms of transcripts, such as alternative splicing variants, fusion genes, and regulatory RNAs, as well as detecting mutations and polymorphisms that may play important roles in disease pathogenesis.

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Alamar Santiago

    2009-09-01

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

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

    Science.gov (United States)

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

    2009-09-11

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

  19. Thorough in silico and in vitro cDNA analysis of 21 putative BRCA1 and BRCA2 splice variants and a complex tandem duplication in BRCA2 allowing the identification of activated cryptic splice donor sites in BRCA2 exon 11.

    Science.gov (United States)

    Baert, Annelot; Machackova, Eva; Coene, Ilse; Cremin, Carol; Turner, Kristin; Portigal-Todd, Cheryl; Asrat, Marie Jill; Nuk, Jennifer; Mindlin, Allison; Young, Sean; MacMillan, Andree; Van Maerken, Tom; Trbusek, Martin; McKinnon, Wendy; Wood, Marie E; Foulkes, William D; Santamariña, Marta; de la Hoya, Miguel; Foretova, Lenka; Poppe, Bruce; Vral, Anne; Rosseel, Toon; De Leeneer, Kim; Vega, Ana; Claes, Kathleen B M

    2018-04-01

    For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies. © 2017 Wiley Periodicals, Inc.

  20. 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...

  1. 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

  2. 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.

  3. 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 ...

  4. 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 ...

  5. Economic Analysis of Improved Alkaline Water Electrolysis

    International Nuclear Information System (INIS)

    Kuckshinrichs, Wilhelm; Ketelaer, Thomas; Koj, Jan Christian

    2017-01-01

    Alkaline water electrolysis (AWE) is a mature hydrogen production technology and there exists a range of economic assessments for available technologies. For advanced AWEs, which may be based on novel polymer-based membrane concepts, it is of prime importance that development comes along with new configurations and technical and economic key process parameters for AWE that might be of interest for further economic assessments. This paper presents an advanced AWE technology referring to three different sites in Europe (Germany, Austria, and Spain). The focus is on financial metrics, the projection of key performance parameters of advanced AWEs, and further financial and tax parameters. For financial analysis from an investor’s (business) perspective, a comprehensive assessment of a technology not only comprises cost analysis but also further financial analysis quantifying attractiveness and supply/market flexibility. Therefore, based on cash flow (CF) analysis, a comprehensible set of metrics may comprise levelised cost of energy or, respectively, levelized cost of hydrogen (LCH) for cost assessment, net present value (NPV) for attractiveness analysis, and variable cost (VC) for analysis of market flexibility. The German AWE site turns out to perform best in all three financial metrics (LCH, NPV, and VC). Though there are slight differences in investment cost and operation and maintenance cost projections for the three sites, the major cost impact is due to the electricity cost. Although investment cost is slightly lower and labor cost is significantly lower in Spain, the difference can not outweigh the higher electricity cost compared to Germany. Given the assumption that the electrolysis operators are customers directly and actively participating in power markets, and based on the regulatory framework in the three countries, in this special case electricity cost in Germany is lowest. However, as electricity cost is profoundly influenced by political decisions as

  6. Analysis and Improvement of Fireworks Algorithm

    Directory of Open Access Journals (Sweden)

    Xi-Guang Li

    2017-02-01

    Full Text Available The Fireworks Algorithm is a recently developed swarm intelligence algorithm to simulate the explosion process of fireworks. Based on the analysis of each operator of Fireworks Algorithm (FWA, this paper improves the FWA and proves that the improved algorithm converges to the global optimal solution with probability 1. The proposed algorithm improves the goal of further boosting performance and achieving global optimization where mainly include the following strategies. Firstly using the opposition-based learning initialization population. Secondly a new explosion amplitude mechanism for the optimal firework is proposed. In addition, the adaptive t-distribution mutation for non-optimal individuals and elite opposition-based learning for the optimal individual are used. Finally, a new selection strategy, namely Disruptive Selection, is proposed to reduce the running time of the algorithm compared with FWA. In our simulation, we apply the CEC2013 standard functions and compare the proposed algorithm (IFWA with SPSO2011, FWA, EFWA and dynFWA. The results show that the proposed algorithm has better overall performance on the test functions.

  7. 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...

  8. 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

  9. [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.

  10. [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.

  11. Methodological considerations for improving Western blot analysis.

    Science.gov (United States)

    MacPhee, Daniel J

    2010-01-01

    The need for a technique that could allow the determination of antigen specificity of antisera led to the development of a method that allowed the production of a replica of proteins, which had been separated electrophoretically on polyacrylamide gels, on to a nitrocellulose membrane. This method was coined Western blotting and is very useful to study the presence, relative abundance, relative molecular mass, post-translational modification, and interaction of specific proteins. As a result it is utilized routinely in many fields of scientific research such as chemistry, biology and biomedical sciences. This review serves to touch on some of the methodological conditions that should be considered to improve Western blot analysis, particularly as a guide for graduate students but also scientists who wish to continue adapting this now fundamental research tool. Copyright 2009 Elsevier Inc. All rights reserved.

  12. 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

  13. Cross-platform analysis of cancer microarray data improves gene expression based classification of phenotypes

    Directory of Open Access Journals (Sweden)

    Eils Roland

    2005-11-01

    Full Text Available Abstract Background The extensive use of DNA microarray technology in the characterization of the cell transcriptome is leading to an ever increasing amount of microarray data from cancer studies. Although similar questions for the same type of cancer are addressed in these different studies, a comparative analysis of their results is hampered by the use of heterogeneous microarray platforms and analysis methods. Results In contrast to a meta-analysis approach where results of different studies are combined on an interpretative level, we investigate here how to directly integrate raw microarray data from different studies for the purpose of supervised classification analysis. We use median rank scores and quantile discretization to derive numerically comparable measures of gene expression from different platforms. These transformed data are then used for training of classifiers based on support vector machines. We apply this approach to six publicly available cancer microarray gene expression data sets, which consist of three pairs of studies, each examining the same type of cancer, i.e. breast cancer, prostate cancer or acute myeloid leukemia. For each pair, one study was performed by means of cDNA microarrays and the other by means of oligonucleotide microarrays. In each pair, high classification accuracies (> 85% were achieved with training and testing on data instances randomly chosen from both data sets in a cross-validation analysis. To exemplify the potential of this cross-platform classification analysis, we use two leukemia microarray data sets to show that important genes with regard to the biology of leukemia are selected in an integrated analysis, which are missed in either single-set analysis. Conclusion Cross-platform classification of multiple cancer microarray data sets yields discriminative gene expression signatures that are found and validated on a large number of microarray samples, generated by different laboratories and

  14. 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.

  15. [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.

  16. 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.

  17. 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...

  18. 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.

  19. Continuous improvement projects: an authorship bibliometric analysis.

    Science.gov (United States)

    Gonzalez Aleu, Fernando; Van Aken, Eileen M

    2017-06-12

    Purpose The purpose of this paper is to describe the current research on hospital continuous improvement projects (CIPs) from an author characteristics' perspective. This work addresses the following questions: who are the predominant research authors in hospital CIPs? To what extent are the research communities collaborating in distinct research groups? How internationalized has hospital CIPs research become with respect to author location? Design/methodology/approach A systematic literature review was conducted, identifying 302 academic publications related to hospital CIPs. Publications were analyzed using: author, quantity, diversity, collaboration, and impact. Findings Hospital CIPs are increasingly attracting new scholars each year. Based on the authors' analysis, authors publishing in this area can be described as a relatively new international community given the countries represented. Originality/value This paper describes the current hospital CIP research by assessing author characteristics. Future work should examine additional attributes to characterize maturity such as how new knowledge is being created and to what extent new knowledge is being disseminated to practitioners.

  20. SPORTS ORGANIZATIONS MANAGEMENT IMPROVEMENT: A SURVEY ANALYSIS

    Directory of Open Access Journals (Sweden)

    Alin Molcut

    2015-07-01

    Full Text Available Sport organizations exist to perform tasks that can only be executed through cooperative effort, and sport management is responsible for the performance and success of these organizations. The main of the paper is to analyze several issues of management sports organizations in order to asses their quality management. In this respect a questionnaire has been desingned for performing a survey analysis through a statistical approach. Investigation was conducted over a period of 3 months, and have been questioned a number of managers and coaches of football, all while pursuing an activity in football clubs in the counties of Timis and Arad, the level of training for children and juniors. The results suggest that there is a significant interest for the improvement of management across teams of children and under 21 clubs, emphasis on players' participation and rewarding performance. Furthermore, we can state that in the sports clubs there is established a vision and a mission as well as the objectives of the club's general refers to both sporting performance, and financial performance.

  1. 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.

  2. 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.

  3. [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.

  4. 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.

  5. 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.

  6. 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

  7. Comparative genomic mapping of the bovine Fragile Histidine Triad (FHIT tumour suppressor gene: characterization of a 2 Mb BAC contig covering the locus, complete annotation of the gene, analysis of cDNA and of physiological expression profiles

    Directory of Open Access Journals (Sweden)

    Boussaha Mekki

    2006-05-01

    Full Text Available Abstract Background The Fragile Histidine Triad gene (FHIT is an oncosuppressor implicated in many human cancers, including vesical tumors. FHIT is frequently hit by deletions caused by fragility at FRA3B, the most active of human common fragile sites, where FHIT lays. Vesical tumors affect also cattle, including animals grazing in the wild on bracken fern; compounds released by the fern are known to induce chromosome fragility and may trigger cancer with the interplay of latent Papilloma virus. Results The bovine FHIT was characterized by assembling a contig of 78 BACs. Sequence tags were designed on human exons and introns and used directly to select bovine BACs, or compared with sequence data in the bovine genome database or in the trace archive of the bovine genome sequencing project, and adapted before use. FHIT is split in ten exons like in man, with exons 5 to 9 coding for a 149 amino acids protein. VISTA global alignments between bovine genomic contigs retrieved from the bovine genome database and the human FHIT region were performed. Conservation was extremely high over a 2 Mb region spanning the whole FHIT locus, including the size of introns. Thus, the bovine FHIT covers about 1.6 Mb compared to 1.5 Mb in man. Expression was analyzed by RT-PCR and Northern blot, and was found to be ubiquitous. Four cDNA isoforms were isolated and sequenced, that originate from an alternative usage of three variants of exon 4, revealing a size very close to the major human FHIT cDNAs. Conclusion A comparative genomic approach allowed to assemble a contig of 78 BACs and to completely annotate a 1.6 Mb region spanning the bovine FHIT gene. The findings confirmed the very high level of conservation between human and bovine genomes and the importance of comparative mapping to speed the annotation process of the recently sequenced bovine genome. The detailed knowledge of the genomic FHIT region will allow to study the role of FHIT in bovine cancerogenesis

  8. Improved security analysis of Fugue-256

    DEFF Research Database (Denmark)

    Gauravaram, Praveen; Bagheri, Nasour; Knudsen, Lars Ramkilde

    2011-01-01

    in the G transform. Next we improve the designers’ meet-in-the-middle preimage attack on Fugue-256 from 2480 time and memory to 2416. Next we study the security of Fugue-256 against free-start distinguishers and free-start collisions. In this direction, we use an improved variant of the differential...... transform is mapped with a transform to a 256-bit digest. In this paper, we present some improved as well as new analytical results of Fugue-256 (with lengthpadding). First we improve Aumasson and Phans’ integral distinguisher on the 5.5 rounds of the G transform to 16.5 rounds, thus showing weak diffusion...

  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 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.

  11. 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.

  12. Construction of cDNA libraries from Pseudocercospora fijiensis Morelet infected leaves of the cultivars Calcutta 4 and Niyarma Yik

    Directory of Open Access Journals (Sweden)

    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.

  13. Occupational Analysis: A Continuous Improvement Approach

    National Research Council Canada - National Science Library

    Duffy, Tom

    1998-01-01

    .... In doing so, the Air Force has implemented "Quality Air Force (QAF)" (AF Handbook 90-502). QAF is a leadership commitment that inspires trust, teamwork, and continuous improvement everywhere in the Air Force...

  14. 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.

  15. 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

  16. Improving Public Perception of Behavior Analysis.

    Science.gov (United States)

    Freedman, David H

    2016-05-01

    The potential impact of behavior analysis is limited by the public's dim awareness of the field. The mass media rarely cover behavior analysis, other than to echo inaccurate negative stereotypes about control and punishment. The media instead play up appealing but less-evidence-based approaches to problems, a key example being the touting of dubious diets over behavioral approaches to losing excess weight. These sorts of claims distort or skirt scientific evidence, undercutting the fidelity of behavior analysis to scientific rigor. Strategies for better connecting behavior analysis with the public might include reframing the field's techniques and principles in friendlier, more resonant form; pushing direct outcome comparisons between behavior analysis and its rivals in simple terms; and playing up the "warm and fuzzy" side of behavior analysis.

  17. Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.).

    Science.gov (United States)

    Gao, Lingchao; Sun, Ruhao; Liang, Yuanxue; Zhang, Mengdan; Zheng, Yusheng; Li, Dongdong

    2014-10-01

    Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176 bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC-MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. The construction of cDNA library and the screening of related antigen of ascitic tumor cells of ovarian cancer.

    Science.gov (United States)

    Hou, Q; Chen, K; Shan, Z

    2015-01-01

    To construct the cDNA library of the ascites tumor cells of ovarian cancer, which can be used to screen the related antigen for the early diagnosis of ovarian cancer and therapeutic targets of immune treatment. Four cases of ovarian serous cystadenocarcinoma, two cases of ovarian mucinous cystadenocarcinoma, and two cases of ovarian endometrial carcinoma in patients with ascitic tumor cells which were used to construct the cDNA library. To screen the ovarian cancer antigen gene, evaluate the enzyme, and analyze nucleotide sequence, serological analysis of recombinant tumor cDNA expression libraries (SEREX) and suppression subtractive hybridization technique (SSH) techniques were utilized. The detection method of recombinant expression-based serological mini-arrays (SMARTA) was used to detect the ovarian cancer antigen and the positive reaction of 105 cases of ovarian cancer patients and 105 normal women's autoantibodies correspondingly in serum. After two rounds of serologic screening and glycosides sequencing analysis, 59 candidates of ovarian cancer antigen gene fragments were finally identified, which corresponded to 50 genes. They were then divided into six categories: (1) the homologous genes which related to the known ovarian cancer genes, such as BARD 1 gene, etc; (2) the homologous genes which were associated with other tumors, such as TM4SFI gene, etc; (3) the genes which were expressed in a special organization, such as ILF3, FXR1 gene, etc; (4) the genes which were the same with some protein genes of special function, such as TIZ, ClD gene; (5) the homologous genes which possessed the same source with embryonic genes, such as PKHD1 gene, etc; (6) the remaining genes were the unknown genes without the homologous sequence in the gene pool, such as OV-189 genes. SEREX technology combined with SSH method is an effective research strategy which can filter tumor antigen with high specific character; the corresponding autoantibodies of TM4SFl, ClD, TIZ, BARDI

  19. 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 ...

  20. [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.

  1. 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.

  2. cDNA library construction of two human Demodexspecies.

    Science.gov (United States)

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

    2017-06-01

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

  3. 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

  4. 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...

  5. 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.

  6. Helping agencies improve their planning analysis techniques.

    Science.gov (United States)

    2011-11-18

    This report summarizes the results of a peer review of the AZTDM. The peer review was : supported by the Travel Model Improvement Program (TMIP), which is sponsored by FHWA. : The peer review of a travel model can serve multiple purposes, including i...

  7. Analysis of improvement ways of creative accounting

    Directory of Open Access Journals (Sweden)

    I.A. Yuhimenko-Nazaruk

    2017-02-01

    Full Text Available The necessity of carrying out the research in the direction of finding out the ways to improve creative accounting is grounded. The existing approaches of researchers to eliminate the negative consequences of creative accounting are analyzed. Four main groups of researchers' approaches to the improvement of creative accounting are singled out and analyzed. The general and distinctive features of the researchers’ proposals on the improvement of creative accounting are examined. The reasons for the impossibility of using the ethical approach to the improvement of creative accounting in Ukraine in modern conditions are grounded. The necessity of procedural aspects perfection of the creative accounting on the basis of the concept of true and fair view is proved. The classification of the approaches to the construction of accounting methodology in the context of the use of creative accounting is developed. The main regulations of the concept of true and fair view are studied, their use provides an adequate reflection of the company's economic reality in financial reporting.

  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. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Tissue-wide expression profiling using cDNA subtraction and microarrays to identify tumor-specific genes.

    Science.gov (United States)

    Amatschek, Stefan; Koenig, Ulrich; Auer, Herbert; Steinlein, Peter; Pacher, Margit; Gruenfelder, Agnes; Dekan, Gerhard; Vogl, Sonja; Kubista, Ernst; Heider, Karl-Heinz; Stratowa, Christian; Schreiber, Martin; Sommergruber, Wolfgang

    2004-02-01

    With the objective of discovering novel putative intervention sites for anticancer therapy, we compared transcriptional profiles of breast cancer, lung squamous cell cancer (LSCC), lung adenocarcinoma (LAC), and renal cell cancer (RCC). Each of these tumor types still needs improvement in medical treatment. Our intention was to search for genes not only highly expressed in the majority of patient samples but which also exhibit very low or even absence of expression in a comprehensive panel of 16 critical (vital) normal tissues. To achieve this goal, we combined two powerful technologies, PCR-based cDNA subtraction and cDNA microarrays. Seven subtractive libraries consisting of approximately 9250 clones were established and enriched for tumor-specific transcripts. These clones, together with approximately 1750 additional tumor-relevant genes, were used for cDNA microarray preparation. Hybridizations were performed using a pool of 16 critical normal tissues as a reference in all experiments. In total, we analyzed 20 samples of breast cancer, 11 of LSCC, 11 of LAC, and 8 of RCC. To select for genes with low or even no expression in normal tissues, expression profiles of 22 different normal tissues were additionally analyzed. Importantly, this tissue-wide expression profiling allowed us to eliminate genes, which exhibit also high expression in normal tissues. Similarly, expression signatures of genes, which are derived from infiltrating cells of the immune system, were eliminated as well. Cluster analysis resulted in the identification of 527 expressed sequence tags specifically up-regulated in these tumors. Gene-wise hierarchical clustering of these clones clearly separated the different tumor types with RCC exhibiting the most homogeneous and LAC the most diverse expression profile. In addition to already known tumor-associated genes, the majority of identified genes have not yet been brought into context with tumorigenesis such as genes involved in bone matrix

  15. 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

  16. International Space Station Future Correlation Analysis Improvements

    Science.gov (United States)

    Laible, Michael R.; Pinnamaneni, Murthy; Sugavanam, Sujatha; Grygier, Michael

    2018-01-01

    Ongoing modal analyses and model correlation are performed on different configurations of the International Space Station (ISS). These analyses utilize on-orbit dynamic measurements collected using four main ISS instrumentation systems: External Wireless Instrumentation System (EWIS), Internal Wireless Instrumentation System (IWIS), Space Acceleration Measurement System (SAMS), and Structural Dynamic Measurement System (SDMS). Remote Sensor Units (RSUs) are network relay stations that acquire flight data from sensors. Measured data is stored in the Remote Sensor Unit (RSU) until it receives a command to download data via RF to the Network Control Unit (NCU). Since each RSU has its own clock, it is necessary to synchronize measurements before analysis. Imprecise synchronization impacts analysis results. A study was performed to evaluate three different synchronization techniques: (i) measurements visually aligned to analytical time-response data using model comparison, (ii) Frequency Domain Decomposition (FDD), and (iii) lag from cross-correlation to align measurements. This paper presents the results of this study.

  17. Planning, Conducting, and Documenting Data Analysis for Program Improvement

    Science.gov (United States)

    Winer, Abby; Taylor, Cornelia; Derrington, Taletha; Lucas, Anne

    2015-01-01

    This 2015 document was developed to help technical assistance (TA) providers and state staff define and limit the scope of data analysis for program improvement efforts, including the State Systemic Improvement Plan (SSIP); develop a plan for data analysis; document alternative hypotheses and additional analyses as they are generated; and…

  18. 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.

  19. 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.

  20. 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...

  1. Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel.

    Science.gov (United States)

    Ravanfar, Seyed Ali; Aziz, Maheran Abdul; Saud, Halimi Mohd; Abdullah, Janna Ong

    2015-11-01

    An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1 mg dm(-3) TDZ. TDZ at 0.1 mg dm(-3) produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

  2. Recent improvements in Thomson scattering data analysis

    International Nuclear Information System (INIS)

    Tillack, M.S.; Lazarus, E.A.

    1980-04-01

    A new profile analysis package for use with the Thomson scattering data on ISX-B has recently been implemented. The primary feature of this package is a weighted least squares fitting of temperature and density data to generate a representative curve, as opposed to the previous hand-fitting technique. The changes will automate the manner in which data are transmitted and manipulated, without affecting the calculational techniques previously used. The computer programs have also been used to estimate the sensitivity of various plasma quantities to the accuracy of the Thomson scattering data

  3. Landsat analysis for improving bamboo forest mapping

    Science.gov (United States)

    Yamamoto, Y.; Suzuoki, Y.; Tsuboi, T.; Iinuma, T.; Iwashita, K.; Nishikawa, H.

    Using satellite data and field data collected periodically over the past years, the vegetation area and the underlying substrate have been mapped. Numerous methods for detecting "vegetation changes" with the aid of digital satellite data have been utilized. Among those methods , the vegetation indices such as RVI, NDVI, or SVI are the most suitable methods to estimate "the change". Vegetation indices are mathematical transformations designed to assess the spectral contribution of vegetation to multispectral observations. Bamboo grove as primary subject in this study is welknown as rapid growing plant, and, on the other hand, the expansion of bamboo grove have been discussed as a regional environmental issue. Change detection of bamboo covered area would be useful to help the preventive countermeasures for bamboo expansion in unwanted areas. As a result, to detect the accurate bamboo covered area, the optimal vegetation indices and band combinations were established through continuous Landsat data based statistical analysis.

  4. Improvement of product design process by knowledge value analysis

    OpenAIRE

    XU, Yang; BERNARD, Alain; PERRY, Nicolas; LAROCHE, Florent

    2013-01-01

    Nowadays, design activities remain the core issue for global product development. As knowledge is more and more integrated, effective analysis of knowledge value becomes very useful for the improvement of product design processes. This paper aims at proposing a framework of knowledge value analysis in the context of product design process. By theoretical analysis and case study, the paper illustrates how knowledge value can be calculated and how the results can help the improvement of product...

  5. 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 ...

  6. 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.

  7. 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 ...

  8. 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.

  9. 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...

  10. Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification.

    Science.gov (United States)

    Tougan, Takahiro; Okuzaki, Daisuke; Nojima, Hiroshi

    2008-09-01

    Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6 x 10(5) cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (K(m)) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate.

  11. A Combinational Clustering Based Method for cDNA Microarray Image Segmentation.

    Directory of Open Access Journals (Sweden)

    Guifang Shao

    Full Text Available Microarray technology plays an important role in drawing useful biological conclusions by analyzing thousands of gene expressions simultaneously. Especially, image analysis is a key step in microarray analysis and its accuracy strongly depends on segmentation. The pioneering works of clustering based segmentation have shown that k-means clustering algorithm and moving k-means clustering algorithm are two commonly used methods in microarray image processing. However, they usually face unsatisfactory results because the real microarray image contains noise, artifacts and spots that vary in size, shape and contrast. To improve the segmentation accuracy, in this article we present a combination clustering based segmentation approach that may be more reliable and able to segment spots automatically. First, this new method starts with a very simple but effective contrast enhancement operation to improve the image quality. Then, an automatic gridding based on the maximum between-class variance is applied to separate the spots into independent areas. Next, among each spot region, the moving k-means clustering is first conducted to separate the spot from background and then the k-means clustering algorithms are combined for those spots failing to obtain the entire boundary. Finally, a refinement step is used to replace the false segmentation and the inseparable ones of missing spots. In addition, quantitative comparisons between the improved method and the other four segmentation algorithms--edge detection, thresholding, k-means clustering and moving k-means clustering--are carried out on cDNA microarray images from six different data sets. Experiments on six different data sets, 1 Stanford Microarray Database (SMD, 2 Gene Expression Omnibus (GEO, 3 Baylor College of Medicine (BCM, 4 Swiss Institute of Bioinformatics (SIB, 5 Joe DeRisi's individual tiff files (DeRisi, and 6 University of California, San Francisco (UCSF, indicate that the improved

  12. A Combinational Clustering Based Method for cDNA Microarray Image Segmentation.

    Science.gov (United States)

    Shao, Guifang; Li, Tiejun; Zuo, Wangda; Wu, Shunxiang; Liu, Tundong

    2015-01-01

    Microarray technology plays an important role in drawing useful biological conclusions by analyzing thousands of gene expressions simultaneously. Especially, image analysis is a key step in microarray analysis and its accuracy strongly depends on segmentation. The pioneering works of clustering based segmentation have shown that k-means clustering algorithm and moving k-means clustering algorithm are two commonly used methods in microarray image processing. However, they usually face unsatisfactory results because the real microarray image contains noise, artifacts and spots that vary in size, shape and contrast. To improve the segmentation accuracy, in this article we present a combination clustering based segmentation approach that may be more reliable and able to segment spots automatically. First, this new method starts with a very simple but effective contrast enhancement operation to improve the image quality. Then, an automatic gridding based on the maximum between-class variance is applied to separate the spots into independent areas. Next, among each spot region, the moving k-means clustering is first conducted to separate the spot from background and then the k-means clustering algorithms are combined for those spots failing to obtain the entire boundary. Finally, a refinement step is used to replace the false segmentation and the inseparable ones of missing spots. In addition, quantitative comparisons between the improved method and the other four segmentation algorithms--edge detection, thresholding, k-means clustering and moving k-means clustering--are carried out on cDNA microarray images from six different data sets. Experiments on six different data sets, 1) Stanford Microarray Database (SMD), 2) Gene Expression Omnibus (GEO), 3) Baylor College of Medicine (BCM), 4) Swiss Institute of Bioinformatics (SIB), 5) Joe DeRisi's individual tiff files (DeRisi), and 6) University of California, San Francisco (UCSF), indicate that the improved approach is

  13. Analysis of event data recorder data for vehicle safety improvement

    Science.gov (United States)

    2008-04-01

    The Volpe Center performed a comprehensive engineering analysis of Event Data Recorder (EDR) data supplied by the National Highway Traffic Safety Administration (NHTSA) to assess its accuracy and usefulness in crash reconstruction and improvement of ...

  14. 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.

  15. Improving Department of Defense Global Distribution Performance Through Network Analysis

    Science.gov (United States)

    2016-06-01

    maximum time allowed by SDDB business rules, 365 days, and run the improvement algorithm again. Using the budget of 20 improvement days for both the...OF DEFENSE GLOBAL DISTRIBUTION PERFORMANCE THROUGH NETWORK ANALYSIS by Justin A. Thompson June 2016 Thesis Advisor: Samuel E. Buttrey...REPORT DATE June 2016 3. REPORT TYPE AND DATES COVERED Master’s thesis 4. TITLE AND SUBTITLE IMPROVING DEPARTMENT OF DEFENSE GLOBAL DISTRIBUTION

  16. Improving Quality Using Architecture Fault Analysis with Confidence Arguments

    Science.gov (United States)

    2015-03-01

    Improving Quality Using Architecture Fault Analysis with Confidence Arguments Peter H. Feiler Charles B. Weinstock John B. Goodenough ...argument are represented explicitly. As reasons for doubt, called defeaters, are removed, confidence in system claims increases [ Goodenough 2013, Weinstock...Peter, Goodenough , John, Gurfinkel, Arie, Weinstock, Charles, & Wrage, Lutz. Reliability Improvement and Validation Framework (CMU/SEI-2012-SR-013

  17. 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 ...

  18. 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 ...

  19. 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% ...

  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

    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. 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

  3. A linear concatenation strategy to construct 5'-enriched amplified cDNA libraries using multiple displacement amplification.

    Science.gov (United States)

    Gadkar, Vijay J; Filion, Martin

    2013-06-01

    In various experimental systems, limiting available amounts of RNA may prevent a researcher from performing large-scale analyses of gene transcripts. One way to circumvent this is to 'pre-amplify' the starting RNA/cDNA, so that sufficient amounts are available for any downstream analysis. In the present study, we report the development of a novel protocol for constructing amplified cDNA libraries using the Phi29 DNA polymerase based multiple displacement amplification (MDA) system. Using as little as 200 ng of total RNA, we developed a linear concatenation strategy to make the single-stranded cDNA template amenable for MDA. The concatenation, made possible by the template switching property of the reverse transcriptase enzyme, resulted in the amplified cDNA library with intact 5' ends. MDA generated micrograms of template, allowing large-scale polymerase chain reaction analyses or other large-scale downstream applications. As the amplified cDNA library contains intact 5' ends, it is also compatible with 5' RACE analyses of specific gene transcripts. Empirical validation of this protocol is demonstrated on a highly characterized (tomato) and an uncharacterized (corn gromwell) experimental system.

  4. [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.

  5. Adapting Job Analysis Methodology to Improve Evaluation Practice

    Science.gov (United States)

    Jenkins, Susan M.; Curtin, Patrick

    2006-01-01

    This article describes how job analysis, a method commonly used in personnel research and organizational psychology, provides a systematic method for documenting program staffing and service delivery that can improve evaluators' knowledge about program operations. Job analysis data can be used to increase evaluators' insight into how staffs…

  6. Improved time complexity analysis of the Simple Genetic Algorithm

    DEFF Research Database (Denmark)

    Oliveto, Pietro S.; Witt, Carsten

    2015-01-01

    A runtime analysis of the Simple Genetic Algorithm (SGA) for the OneMax problem has recently been presented proving that the algorithm with population size μ≤n1/8−ε requires exponential time with overwhelming probability. This paper presents an improved analysis which overcomes some limitations...

  7. an improved structural model for seismic analysis of tall frames

    African Journals Online (AJOL)

    Dr Obe

    ABSTRACT. This paper proposed and examined an improved structural model that overcomes the deficiencies of the shear frame model by considering the effects of flexible horizontal members and column axial loads in seismic analysis of multi-storey frames. Matrix displacement method of analysis is used on the basis of ...

  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 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.

  10. 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.

  11. 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.

  12. 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.

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

    Indian Academy of Sciences (India)

    THOMAS NAICY

    IGF1 gene might play important roles in almost all body functions especially growth, reproduction and meat quality traits in goats. Further studies are required to establish the role of the detected SNP in the IGF1 gene expression and association with economic traits in goats. The results of the present study suggests that IGF1 ...

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

    Indian Academy of Sciences (India)

    The present study was conducted to clone and sequence the full-length coding sequence of the caprine IGF1 gene from Attappady Black and Malabari breeds, two indigenous goat breeds of south India, to analyse its structure, and to ascertainthe relative abundance of IGF1 mRNA in different tissues. The caprine IGF1 ...

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

    Indian Academy of Sciences (India)

    THOMAS NAICY

    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 full-length coding sequence of the caprine IGF1 gene from. Attappady Black and Malabari breeds, two indigenous goat breeds of ...

  16. Genome-Wide Screening of Genes Showing Altered Expression in Liver Metastases of Human Colorectal Cancers by cDNA Microarray

    Directory of Open Access Journals (Sweden)

    Rempei Yanagawa

    2001-01-01

    Full Text Available In spite of intensive and increasingly successful attempts to determine the multiple steps involved in colorectal carcinogenesis, the mechanisms responsible for metastasis of colorectal tumors to the liver remain to be clarified. To identify genes that are candidates for involvement in the metastatic process, we analyzed genome-wide expression profiles of 10 primary colorectal cancers and their corresponding metastatic lesions by means of a cDNA microarray consisting of 9121 human genes. This analysis identified 40 genes whose expression was commonly upregulated in metastatic lesions, and 7 that were commonly downregulated. The upregulated genes encoded proteins involved in cell adhesion, or remodeling of the actin cytoskeleton. Investigation of the functions of more of the altered genes should improve our understanding of metastasis and may identify diagnostic markers and/or novel molecular targets for prevention or therapy of metastatic lesions.

  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. 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

  19. Improvement of software for analysis of visual meteor data

    Science.gov (United States)

    Veljković, K.; Ivanović, I.

    2015-01-01

    In this paper, we present improvements made on our software for the analysis of visual meteor data. R package MetFns received major updates. Selection filters and algorithms for calculation of zenithal hourly rate and population index, as well as accompanying graphics, are corrected and their performance is improved. Web application MetRApp contains a completely remade user interface and some new features. Also, calculation performances are optimized.

  20. Microarray analysis of gene expression on herbal glycoside recipes improving deficient ability of spatial learning memory in ischemic mice.

    Science.gov (United States)

    Wang, Zhong; Du, Qingyou; Wang, Fusheng; Liu, Zhongrong; Li, Baigang; Wang, Anmin; Wang, Yongyan

    2004-03-01

    In order to reveal the mechanism of herbal glycoside recipes retrieving deficient ability of spatial learning memory in mice suffering from cerebral ischemia/reperfusion, a microarray system was used to analyze gene expression in those groups with increasing ability of spatial learning memory who were different from ischemic mice. In this work, we reported a comprehensive characterization of gene expression profiles of mouse hippocampus by the use of cDNA microarray system containing 1176 known genes in middle cerebral artery occlusion (MCAO) ischemic mice after treating with different dosage recipes of glycoside herbs (30, 90, and 270 mg/kg). The ability of spatial learning memory in ischemic mice was found to be decreased. The pathological process in ischemic mouse brain showed that a complex related to 100 genes' expression yielded 1.8-fold. Dose-dependent effects showed an improvement in the deficient ability and reduction in infarct volume when treated with glycoside recipes. Many genes (38-46) in expression were found greater than 1.8-fold in those effective recipes groups, including genes in cell cycle regulation, signal transduction, nerve system transcription factors, DNA binding protein, etc. Nine genes related to retrieving deficient ability of spatial learning memory treated with glycoside recipes were also found in this study. These results suggest that microarray analysis of gene expression might be useful for elucidating the mechanisms of pharmacological function of recipes.

  1. [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.

  2. Improving Automatic Text Classification by Integrated Feature Analysis

    Science.gov (United States)

    Busagala, Lazaro S. P.; Ohyama, Wataru; Wakabayashi, Tetsushi; Kimura, Fumitaka

    Feature transformation in automatic text classification (ATC) can lead to better classification performance. Furthermore dimensionality reduction is important in ATC. Hence, feature transformation and dimensionality reduction are performed to obtain lower computational costs with improved classification performance. However, feature transformation and dimension reduction techniques have been conventionally considered in isolation. In such cases classification performance can be lower than when integrated. Therefore, we propose an integrated feature analysis approach which improves the classification performance at lower dimensionality. Moreover, we propose a multiple feature integration technique which also improves classification effectiveness.

  3. Improvements in analysis techniques for segmented mirror arrays

    Science.gov (United States)

    Michels, Gregory J.; Genberg, Victor L.; Bisson, Gary R.

    2016-08-01

    The employment of actively controlled segmented mirror architectures has become increasingly common in the development of current astronomical telescopes. Optomechanical analysis of such hardware presents unique issues compared to that of monolithic mirror designs. The work presented here is a review of current capabilities and improvements in the methodology of the analysis of mechanically induced surface deformation of such systems. The recent improvements include capability to differentiate surface deformation at the array and segment level. This differentiation allowing surface deformation analysis at each individual segment level offers useful insight into the mechanical behavior of the segments that is unavailable by analysis solely at the parent array level. In addition, capability to characterize the full displacement vector deformation of collections of points allows analysis of mechanical disturbance predictions of assembly interfaces relative to other assembly interfaces. This capability, called racking analysis, allows engineers to develop designs for segment-to-segment phasing performance in assembly integration, 0g release, and thermal stability of operation. The performance predicted by racking has the advantage of being comparable to the measurements used in assembly of hardware. Approaches to all of the above issues are presented and demonstrated by example with SigFit, a commercially available tool integrating mechanical analysis with optical analysis.

  4. 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).

  5. 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

  6. Can an understanding of transactional analysis improve postgraduate clinical supervision?

    Science.gov (United States)

    Sivan, Manoj; McKimm, Judy; Held, Sam

    2011-01-01

    Clinical supervision in postgraduate medical training is vital in producing competent and safe health-care practitioners. Effective communication between supervisors and trainees at an interpersonal and professional level determines the quality of the supervision process. Transactional analysis, a theory of personality, can be used to enhance understanding of interpersonal interactions and improve the outcomes of clinical training.

  7. Costs and returns analysis of improved and alternative cassava ...

    African Journals Online (AJOL)

    The specific objectives of the study was an analysis of the costs and returns of improved and alternative technologies available in the study area by farmers and their level of adoption of the new technologies. Data were collected from a random sample of 250 farmers and 30 extension Staff in the three (3) agricultural zones ...

  8. Sensitivity analysis and its application for dynamic improvement

    Indian Academy of Sciences (India)

    Keywords. Sensitivity analysis; dynamic improvement structural modoficaton; laser beam printer; motorbike; disc drive; mechatronics; automobile engine. Abstract. In order to determine appropriate points where natural frequency or mode shape under consideration can be effectively modified by structural modification, the ...

  9. 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...

  10. Improved Runtime Analysis of the Simple Genetic Algorithm

    DEFF Research Database (Denmark)

    Oliveto, Pietro S.; Witt, Carsten

    2013-01-01

    A runtime analysis of the Simple Genetic Algorithm (SGA) for the OneMax problem has recently been presented proving that the algorithm requires exponential time with overwhelming probability. This paper presents an improved analysis which overcomes some limitations of our previous one. Firstly......, the new result holds for population sizes up to mu = n1/4-epsilon which is an improvement up to a power of 2 larger. Secondly, we present a technique to bound the diversity of the population that does not require a bound on its bandwidth. Apart from allowing a stronger result, we believe this is a major...... improvement towards the reusability of the techniques in future systematic analyses of GAs. Finally, we consider the more natural SGA using selection with replacement rather than without replacement although the results hold for both algorithmic versions. Experiments are presented to explore the limits...

  11. Thermal hydraulic analysis of the JMTR improved LEU-core

    Energy Technology Data Exchange (ETDEWEB)

    Tabata, Toshio; Nagao, Yoshiharu; Komukai, Bunsaku; Naka, Michihiro; Fujiki, Kazuo [Japan Atomic Energy Research Inst., Oarai, Ibaraki (Japan). Oarai Research Establishment; Takeda, Takashi [Radioactive Waste Management and Nuclear Facility Decommissioning Technology Center, Tokai, Ibaraki (Japan)

    2003-01-01

    After the investigation of the new core arrangement for the JMTR reactor in order to enhance the fuel burn-up and consequently extend the operation period, the ''improved LEU core'' that utilized 2 additional fuel elements instead of formerly installed reflector elements, was adopted. This report describes the results of the thermal-hydraulic analysis of the improved LEU core as a part of safety analysis for the licensing. The analysis covers steady state, abnormal operational transients and accidents, which were described in the annexes of the licensing documents as design bases events. Calculation conditions for the computer codes were conservatively determined based on the neutronic analysis results and others. The results of the analysis, that revealed the safety criteria were satisfied on the fuel temperature, DNBR and primary coolant temperature, were used in the licensing. The operation license of the JMTR with the improved LEU core was granted in March 2001, and the reactor operation with new core started in November 2001 as 142nd operation cycle. (author)

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Improving preconception health and care: a situation analysis.

    Science.gov (United States)

    Goodfellow, Ashley; Frank, John; McAteer, John; Rankin, Jean

    2017-08-23

    The purpose of this situation analysis was to explore the views of health and non-health professionals working with women of childbearing age on current and future delivery of preconception care in one National Health Service (NHS) Board area in Scotland. The situation analysis was undertaken using a mixed methods approach. Six focus groups were conducted organised by profession - general practitioners (GPs), practice nurses, health visitors, family nurses, guidance teachers and youth workers. Existing evidence of effective preconception care interventions informed focus group guides. A survey was undertaken with community pharmacists which provided qualitative data for analysis. Focus group transcripts were analysed by two researchers using a thematic analysis approach. There was lack of awareness of preconception health and its importance amongst the target group. Levels of unplanned pregnancy hampered efforts to deliver interventions. Professional knowledge, capacity and consistency of practice were viewed as challenges, as was individual compliance with preconception care advice. Improvement requires multifaceted action, including ensuring the school curriculum adequately prepares adolescents for future parenthood, increasing awareness through communication and marketing, supporting professional knowledge and practice and capitalising on existing opportunities for preconception care, and ensuring services are equitable and targeted to need. Delivery of preconception care needs to be improved both before and between pregnancies to improve outcomes for women and infants. Action is required at individual, organisational and community levels to ensure this important issue is at the forefront of preventative care and preventative spending.

  19. Domain analysis and modeling to improve comparability of health statistics.

    Science.gov (United States)

    Okada, M; Hashimoto, H; Ohida, T

    2001-01-01

    Health statistics is an essential element to improve the ability of managers of health institutions, healthcare researchers, policy makers, and health professionals to formulate appropriate course of reactions and to make decisions based on evidence. To ensure adequate health statistics, standards are of critical importance. A study on healthcare statistics domain analysis is underway in an effort to improve usability and comparability of health statistics. The ongoing study focuses on structuring the domain knowledge and making the knowledge explicit with a data element dictionary being the core. Supplemental to the dictionary are a domain term list, a terminology dictionary, and a data model to help organize the concepts constituting the health statistics domain.

  20. Process Correlation Analysis Model for Process Improvement Identification

    Directory of Open Access Journals (Sweden)

    Su-jin Choi

    2014-01-01

    software development process. However, in the current practice, correlations of process elements are often overlooked in the development of an improvement plan, which diminishes the efficiency of the plan. This is mainly attributed to significant efforts and the lack of required expertise. In this paper, we present a process correlation analysis model that helps identify correlations of process elements from the results of process assessment. This model is defined based on CMMI and empirical data of improvement practices. We evaluate the model using industrial data.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-09-01

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

  3. Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp. M128.

    Science.gov (United States)

    Yaoi, Katsuro; Mitsuishi, Yasushi

    2004-02-27

    A novel xyloglucan-specific endo-beta-1,4-glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3-1,4-beta-glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (-2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full-length cDNA encoding XEG was cloned and sequenced. It consists of a 2436-bp open reading frame encoding a 776-amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained.

  4. 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.

  5. Process correlation analysis model for process improvement identification.

    Science.gov (United States)

    Choi, Su-jin; Kim, Dae-Kyoo; Park, Sooyong

    2014-01-01

    Software process improvement aims at improving the development process of software systems. It is initiated by process assessment identifying strengths and weaknesses and based on the findings, improvement plans are developed. In general, a process reference model (e.g., CMMI) is used throughout the process of software process improvement as the base. CMMI defines a set of process areas involved in software development and what to be carried out in process areas in terms of goals and practices. Process areas and their elements (goals and practices) are often correlated due to the iterative nature of software development process. However, in the current practice, correlations of process elements are often overlooked in the development of an improvement plan, which diminishes the efficiency of the plan. This is mainly attributed to significant efforts and the lack of required expertise. In this paper, we present a process correlation analysis model that helps identify correlations of process elements from the results of process assessment. This model is defined based on CMMI and empirical data of improvement practices. We evaluate the model using industrial data.

  6. 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...

  7. 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...

  8. cDNA analyses of CAPN3 enhance mutation detection and reveal a low prevalence of LGMD2A patients in Denmark

    DEFF Research Database (Denmark)

    Duno, M.; Sveen, M.L.; Schwartz, M.

    2008-01-01

    suspected to have LGMD2A, based on western blot results. Four of these patients were shown to have LGMD2I upon molecular analysis, whereas 16 of the remaining 42 patients harbored mutations in CAPN3 by both direct genomic sequencing and cDNA analyses. In 10 patients, we identified both mutant alleles....... In three other, only one heterozygous mutation could be identified on the genomic level; however, CAPN3 cDNA analyses demonstrated homozygosity for the mutant allele, indicating the presence of an unidentified allele that somehow compromise correct CAPN3 RNA processing. In the three remaining patients......, only a single heterozygous mutation could be identified both at the genomic level and on full-length CAPN3 cDNA. All three patients exhibited a highly abnormal western blot for calpain-3 and clinical characteristics of LGMD2A. Only three of the genetically confirmed LGMD2A patients were of Danish...

  9. Molecular cloning of cDNA for lysenin, a novel protein in the earthworm Eisenia foetida that causes contraction of rat vascular smooth muscle.

    Science.gov (United States)

    Sekizawa, Y; Kubo, T; Kobayashi, H; Nakajima, T; Natori, S

    1997-05-20

    Lysenin, which causes contraction of rat vascular smooth muscle, is a protein that was isolated from the earthworm Eisenia foetida. A cDNA encoding lysenin was isolated by use of a partial cDNA probe that had been generated by the PCR with a primer designed by reference to an internal peptide sequence of lysenin. This clone had an ORF encoding 297 amino acid residues. The amino acid sequence deduced from the cDNA revealed the absence of any significant homology to those of previously characterized vasoactive substances. The recombinant lysenin was produced in Escherichia coli. This protein and native lysenin isolated from the earthworm had similar contractive activities when tested on rat aorta. Northern blot analysis of the RNA from various tissues of the earthworm indicated that lysenin is produced by the coelomocytes.

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

    OpenAIRE

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

    1988-01-01

    Lysozyme is a major secretory product of human and rodent macrophages and a useful marker for myelomonocytic cells. Based on the known human lysozyme amino acid sequence, oligonucleotides were synthesized and used as probes to screen a phorbol 12-myristate 13-acetate-treated U937 cDNA library. A full-length human lysozyme cDNA clone, pHL-2, was obtained and characterized. Sequence analysis shows that human lysozyme, like chicken lysozyme, has an 18-amino-acid-long signal peptide, but unlike t...

  11. 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...

  12. Training needs analysis for MSMEs: how to improve training effectiveness

    Science.gov (United States)

    Rohayati, Y.; Wulandari, S.

    2017-12-01

    The study aims to analyze training needs for MSMEs in the area of Kabupaten Bandung by selecting the case of MSMEs joined in Association for Agricultural Product Process, focusing on marketing as the main topic of the training. The needs analysis was required to improve training participation and effectiveness. Both aspects are important to notice since making MSMEs participate in training is not an easy task. Similarly, the needs analysis was carried out to anticipate participants’ thoughts that the training does not give any benefits for them or is ineffective because it does not meet their needs although it was actually to help MSMEs improve their marketing knowledge expected to lead to their success. This research involved 100 MSMEs with business ages starting from less than five years to more than 15 years. Those involved MSMEs were dominated by MSMEs targeting local marketing areas. The data were collected by survey and judgmental sampling technique. By conducting a descriptive analysis, it can be concluded that the needs of SMEs on marketing training materials should focus on improving marketing skills such as product development, sales, and use of marketing media as well as discussing legal aspects such as the need for certification and product brand. The results of the study also concluded that there is a need for training that is supplemented by making visits to more successful SMEs as well as practices with on the job training methods.

  13. Improvement of human reliability analysis method for PRA

    International Nuclear Information System (INIS)

    Tanji, Junichi; Fujimoto, Haruo

    2013-09-01

    It is required to refine human reliability analysis (HRA) method by, for example, incorporating consideration for the cognitive process of operator into the evaluation of diagnosis errors and decision-making errors, as a part of the development and improvement of methods used in probabilistic risk assessments (PRAs). JNES has been developed a HRA method based on ATHENA which is suitable to handle the structured relationship among diagnosis errors, decision-making errors and operator cognition process. This report summarizes outcomes obtained from the improvement of HRA method, in which enhancement to evaluate how the plant degraded condition affects operator cognitive process and to evaluate human error probabilities (HEPs) which correspond to the contents of operator tasks is made. In addition, this report describes the results of case studies on the representative accident sequences to investigate the applicability of HRA method developed. HEPs of the same accident sequences are also estimated using THERP method, which is most popularly used HRA method, and comparisons of the results obtained using these two methods are made to depict the differences of these methods and issues to be solved. Important conclusions obtained are as follows: (1) Improvement of HRA method using operator cognitive action model. Clarification of factors to be considered in the evaluation of human errors, incorporation of degraded plant safety condition into HRA and investigation of HEPs which are affected by the contents of operator tasks were made to improve the HRA method which can integrate operator cognitive action model into ATHENA method. In addition, the detail of procedures of the improved method was delineated in the form of flowchart. (2) Case studies and comparison with the results evaluated by THERP method. Four operator actions modeled in the PRAs of representative BWR5 and 4-loop PWR plants were selected and evaluated as case studies. These cases were also evaluated using

  14. 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.

  15. Transcriptome Analysis of Potato Tubers - Effects of Different Agricultural Practices

    NARCIS (Netherlands)

    Dijk, van J.P.; Cankar, K.; Scheffer, S.J.; Beenen, H.G.; Shepherd, L.V.T.; Stewart, D.; Davies, H.V.; Wilkockson, S.J.; Leifert, C.; Gruden, K.; Kok, E.J.

    2009-01-01

    The use of profiling techniques such as transcriptomics, proteomics, and metabolomics has been proposed to improve the detection of side effects of plant breeding processes. This paper describes the construction of a food safety-oriented potato cDNA microarray (FSPM). Microarray analysis was

  16. Transcriptome analysis of potato tubers: Effects of different agricultural practices

    NARCIS (Netherlands)

    Dijk, van Jeroen; Kok, Esther; Cankar, Katarina

    2009-01-01

    The use of profiling techniques such as transcriptomics, proteomics, and metabolomics has been proposed to improve the detection of side effects of plant breeding processes. This paper describes the construction of a food safety-oriented potato cDNA microarray (FSPM). Microarray analysis was

  17. Requirements Analysis for Future Satellite Gravity Mission Improved-GRACE

    Science.gov (United States)

    Zheng, Wei; Hsu, Houtse; Zhong, Min; Yun, Meijuan

    2015-01-01

    The Earth's gravitational field from the Next-Generation Gravimetry Mission (NGGM) and the Improved-Gravity Recovery and Climate Experiment (Improved-GRACE) complete up to degree and order 120 is recovered by a closed-loop numerical simulation using different orbital altitudes of 325 and 300 km, different orbital inclinations of 96.78° and 89° and different inter-satellite ranges of 10 and 50 km. The preferred orbit parameters of the future twin Improved-GRACE satellites are proposed based on the results of the simulations in this study. The research results show: (1) In order to achieve the scientific objectives, which require that the accuracy of the next-generation Earth gravity field models is at least one order of magnitude better than that of the current gravity models, the orbit design at an altitude of 300 ± 50 km is recommended for the future Improved-GRACE mission. This altitude is determined by a trade-off analysis between the recovery accuracy of the gravity field and the operational lifetime of the satellite system. (2) Because the accuracy of the Earth's gravitational field from NGGM with an orbital inclination of 96.78° will be decreased due to a lack of the observation data in the polar areas, we propose that a near-polar orbit (inclination of 89° ± 2°) is a preferable selection for the future twin Improved-GRACE satellites. (3) The future Improved-GRACE mission has to adopt an inter-satellite range of 50 ± 10 km, because the common signals of the Earth's gravitational field between the twin NGGM satellites will be substantially eliminated with a shorter inter-satellite range of 10 km. With these orbit design parameters, the Earth's gravitational field from the Improved-GRACE mission is precisely recovered complete up to degree and order 120 with a cumulative geoid height error of about 0.7 mm.

  18. Spiral analysis-improved clinical utility with center detection.

    Science.gov (United States)

    Wang, Hongzhi; Yu, Qiping; Kurtis, Mónica M; Floyd, Alicia G; Smith, Whitney A; Pullman, Seth L

    2008-06-30

    Spiral analysis is a computerized method that measures human motor performance from handwritten Archimedean spirals. It quantifies normal motor activity, and detects early disease as well as dysfunction in patients with movement disorders. The clinical utility of spiral analysis is based on kinematic and dynamic indices derived from the original spiral trace, which must be detected and transformed into mathematical expressions with great precision. Accurately determining the center of the spiral and reducing spurious low frequency noise caused by center selection error is important to the analysis. Handwritten spirals do not all start at the same point, even when marked on paper, and drawing artifacts are not easily filtered without distortion of the spiral data and corruption of the performance indices. In this report, we describe a method for detecting the optimal spiral center and reducing the unwanted drawing artifacts. To demonstrate overall improvement to spiral analysis, we study the impact of the optimal spiral center detection in different frequency domains separately and find that it notably improves the clinical spiral measurement accuracy in low frequency domains.

  19. Factor analysis improves the selection of prescribing indicators

    DEFF Research Database (Denmark)

    Rasmussen, Hanne Marie Skyggedal; Søndergaard, Jens; Sokolowski, Ineta

    2006-01-01

    OBJECTIVE: To test a method for improving the selection of indicators of general practitioners' prescribing. METHODS: We conducted a prescription database study including all 180 general practices in the County of Funen, Denmark, approximately 472,000 inhabitants. Principal factor analysis was used...... to model correlation between 19 register-based indicators for the quality of non-steroidal anti-inflammatory drug (NSAID) prescribing. RESULTS: The correlation between indicators ranged widely from 0 to 0.93. Factor analysis revealed three dimensions of quality: (1) "Coxib preference", comprising...... appropriate and inappropriate prescribing, as revealed by the correlation of the indicators in the first factor. CONCLUSION: Correlation and factor analysis is a feasible method that assists the selection of indicators and gives better insight into prescribing patterns....

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

    Directory of Open Access Journals (Sweden)

    Reginaldo M Kuroshu

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

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

    Science.gov (United States)

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

    2010-05-07

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

  2. Systematic Isolation and Characterization of Cadmium Tolerant Genes in Tobacco: A cDNA Library Construction and Screening Approach.

    Directory of Open Access Journals (Sweden)

    Mei Zhang

    Full Text Available Heavy metal pollution is a major limiting factor that severely affects plant growth worldwide, and the accumulation of heavy metal in the plant may be hazardous to human health. To identify the processes involved in cadmium detoxification, we constructed a cDNA library of tobacco roots acclimated to cadmium (Cd stress. According to the results of functional screening cDNA library with a yeast Cd-sensitive mutant, ycf1Δ, we obtained a series of candidate genes that were involved in Cd response. Sequence analysis and yeast functional complementation of 24 positive cDNA clones revealed that, in addition to antioxidant genes, genes implicated in abiotic and biotic stress defenses, cellular metabolism, and signal transduction showed Cd detoxification effects in yeast. The real time RT-PCR analyses revealed that some Cd tolerance/ detoxification genes may be able to anticipate in other stresses such as biotic defense and water balance in tobacco. Taken together, our data suggest that plants' acclimation to Cd stress is a highly complex process associated with broad gene functions. Moreover, our results provide insights into the Cd detoxification mechanisms along with the antioxidant system, defense gene induction, and calcium signal pathway.

  3. Systematic Isolation and Characterization of Cadmium Tolerant Genes in Tobacco: A cDNA Library Construction and Screening Approach.

    Science.gov (United States)

    Zhang, Mei; Mo, Hui; Sun, Wen; Guo, Yan; Li, Jing

    2016-01-01

    Heavy metal pollution is a major limiting factor that severely affects plant growth worldwide, and the accumulation of heavy metal in the plant may be hazardous to human health. To identify the processes involved in cadmium detoxification, we constructed a cDNA library of tobacco roots acclimated to cadmium (Cd) stress. According to the results of functional screening cDNA library with a yeast Cd-sensitive mutant, ycf1Δ, we obtained a series of candidate genes that were involved in Cd response. Sequence analysis and yeast functional complementation of 24 positive cDNA clones revealed that, in addition to antioxidant genes, genes implicated in abiotic and biotic stress defenses, cellular metabolism, and signal transduction showed Cd detoxification effects in yeast. The real time RT-PCR analyses revealed that some Cd tolerance/ detoxification genes may be able to anticipate in other stresses such as biotic defense and water balance in tobacco. Taken together, our data suggest that plants' acclimation to Cd stress is a highly complex process associated with broad gene functions. Moreover, our results provide insights into the Cd detoxification mechanisms along with the antioxidant system, defense gene induction, and calcium signal pathway.

  4. Characterization of MMP-9 gene from a normalized cDNA library of kidney tissue of yellow catfish (Pelteobagrus fulvidraco).

    Science.gov (United States)

    Ke, Fei; Wang, Yun; Hong, Jun; Xu, Chen; Chen, Huan; Zhou, Shuai-Bang

    2015-08-01

    Matrix metalloproteinase-9 (MMP-9), one of members of the MMP family, is important for the cleaving of structural extracellular matrix (ECM) molecules and involved in inflammatory processes. In this study, MMP-9 cDNA was isolated and characterized from a normalized cDNA library of kidney tissue of yellow catfish (designated as YcMMP-9). The complete sequence of YcMMP-9 cDNA consisted of 2561 nucleotides. The open reading frame potentially encoded a protein of 685 amino acids with a calculated molecular mass of approximately 77.182 kDa. Amino acid sequence of YcMMP-9 have typical characteristics of MMP-9 family and showed highest identity (85.3%) to channel catfish MMP-9. The YcMMP-9 genomic DNA contains 13 exons and 12 introns. Quantitative RT-PCR (qRT-PCR) analysis showed that YcMMP-9 mRNA was constitutively expressed in all examined tissues in normal fish with high expression in head kidney, trunk kidney, blood, and spleen. However, expression of YcMMP-9 mRNA was induced by Aeromonas hydrophila stimulation, especially in these four tissues mentioned above. It indicated that YcMMP-9 was involved in innate immune responses against bacterial infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Cloning a Chymotrypsin-Like 1 (CTRL-1 Protease cDNA from the Jellyfish Nemopilema nomurai

    Directory of Open Access Journals (Sweden)

    Yunwi Heo

    2016-07-01

    Full Text Available An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His69, Asp117, and Ser216. The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita or Hydrozoan (Hydra vulgaris. The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5′ donor splice (GT and 3′ acceptor splice sequences (AG are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.

  6. Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai

    Science.gov (United States)

    Heo, Yunwi; Kwon, Young Chul; Bae, Seong Kyeong; Hwang, Duhyeon; Yang, Hye Ryeon; Choudhary, Indu; Lee, Hyunkyoung; Yum, Seungshic; Shin, Kyoungsoon; Yoon, Won Duk; Kang, Changkeun; Kim, Euikyung

    2016-01-01

    An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His69, Asp117, and Ser216. The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5′ donor splice (GT) and 3′ acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai. PMID:27399771

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

    Science.gov (United States)

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

    1992-04-01

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

  8. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. (Vanderbilt Univ., Nashville, TN (United States))

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.

  9. Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, T.G.; Cohn, D.V.; Gorr, S.U.; Ornstein, D.L.; Kashdan, M.A.; Levine, M.A.

    1987-07-01

    Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. The authors have isolated cDNA clones from a bovine pituitary lambdagt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysognes demonstrated specific antibody binding to an SP4B/..beta..-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lystates with SP-I antiserum yielded parallel displacement curves of /sup 125/I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene.

  10. Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

    International Nuclear Information System (INIS)

    Ahn, T.G.; Cohn, D.V.; Gorr, S.U.; Ornstein, D.L.; Kashdan, M.A.; Levine, M.A.

    1987-01-01

    Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. The authors have isolated cDNA clones from a bovine pituitary λgt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysognes demonstrated specific antibody binding to an SP4B/β-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lystates with SP-I antiserum yielded parallel displacement curves of 125 I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene

  11. Improvement of QR Code Recognition Based on Pillbox Filter Analysis

    Directory of Open Access Journals (Sweden)

    Jia-Shing Sheu

    2013-04-01

    Full Text Available The objective of this paper is to perform the innovation design for improving the recognition of a captured QR code image with blur through the Pillbox filter analysis. QR code images can be captured by digital video cameras. Many factors contribute to QR code decoding failure, such as the low quality of the image. Focus is an important factor that affects the quality of the image. This study discusses the out-of-focus QR code image and aims to improve the recognition of the contents in the QR code image. Many studies have used the pillbox filter (circular averaging filter method to simulate an out-of-focus image. This method is also used in this investigation to improve the recognition of a captured QR code image. A blurred QR code image is separated into nine levels. In the experiment, four different quantitative approaches are used to reconstruct and decode an out-of-focus QR code image. These nine reconstructed QR code images using methods are then compared. The final experimental results indicate improvements in identification.

  12. Security analysis and improvements to the PsychoPass method.

    Science.gov (United States)

    Brumen, Bostjan; Heričko, Marjan; Rozman, Ivan; Hölbl, Marko

    2013-08-13

    In a recent paper, Pietro Cipresso et al proposed the PsychoPass method, a simple way to create strong passwords that are easy to remember. However, the method has some security issues that need to be addressed. To perform a security analysis on the PsychoPass method and outline the limitations of and possible improvements to the method. We used the brute force analysis and dictionary attack analysis of the PsychoPass method to outline its weaknesses. The first issue with the Psychopass method is that it requires the password reproduction on the same keyboard layout as was used to generate the password. The second issue is a security weakness: although the produced password is 24 characters long, the password is still weak. We elaborate on the weakness and propose a solution that produces strong passwords. The proposed version first requires the use of the SHIFT and ALT-GR keys in combination with other keys, and second, the keys need to be 1-2 distances apart. The proposed improved PsychoPass method yields passwords that can be broken only in hundreds of years based on current computing powers. The proposed PsychoPass method requires 10 keys, as opposed to 20 keys in the original method, for comparable password strength.

  13. Micromechanical analysis of polyacrylamide-modified concrete for improving strengths

    International Nuclear Information System (INIS)

    Sun Zengzhi; Xu Qinwu

    2008-01-01

    This paper studies how polyacrylamide (PAM) alters the physicochemical and mechanical properties of concrete. The microstructure of PAM-modified concrete and the physicochemical reaction between PAM and concrete were studied through scanning electron microscope (SEM), differential thermal analysis (DTA), thermal gravimetric analysis (TGA), and infrared spectrum analysis. Meanwhile, the workability and strengths of cement paste and concrete were tested. PAM's modification mechanism was also discussed. Results indicate that PAM reacts with the Ca 2+ and Al 3+ cations produced by concrete hydration to form the ionic compounds and reduce the crystallization of Ca(OH) 2 , acting as a flexible filler and reinforcement in the porosity of concrete and, therefore, improving concrete's engineering properties. PAM also significantly alters the microstructure at the aggregate-cement interfacial transition zone. Mechanical testing results indicate that the fluidity of cement paste decreases initially, then increases, and decreases again with increasing PAM content. PAM can effectively improve the flexural strength, bonding strength, dynamic impact resistance, and fatigue life of concrete, though it reduces the compressive strength to some extent

  14. Modified paraffin wax for improvement of histological analysis efficiency.

    Science.gov (United States)

    Lim, Jin Ik; Lim, Kook-Jin; Choi, Jin-Young; Lee, Yong-Keun

    2010-08-01

    Paraffin wax is usually used as an embedding medium for histological analysis of natural tissue. However, it is not easy to obtain enough numbers of satisfactory sectioned slices because of the difference in mechanical properties between the paraffin and embedded tissue. We describe a modified paraffin wax that can improve the histological analysis efficiency of natural tissue, composed of paraffin and ethylene vinyl acetate (EVA) resin (0, 3, 5, and 10 wt %). Softening temperature of the paraffin/EVA media was similar to that of paraffin (50-60 degrees C). The paraffin/EVA media dissolved completely in xylene after 30 min at 50 degrees C. Physical properties such as the amount of load under the same compressive displacement, elastic recovery, and crystal intensity increased with increased EVA content. EVA medium (5 wt %) was regarded as an optimal composition, based on the sectioning efficiency measured by the numbers of unimpaired sectioned slices, amount of load under the same compressive displacement, and elastic recovery test. Based on the staining test of sectioned slices embedded in a 5 wt % EVA medium by hematoxylin and eosin (H&E), Masson trichrome (MT), and other staining tests, it was concluded that the modified paraffin wax can improve the histological analysis efficiency with various natural tissues. (c) 2010 Wiley-Liss, Inc.

  15. Toward improved analysis of concentration data: Embracing nondetects.

    Science.gov (United States)

    Shoari, Niloofar; Dubé, Jean-Sébastien

    2018-03-01

    Various statistical tests on concentration data serve to support decision-making regarding characterization and monitoring of contaminated media, assessing exposure to a chemical, and quantifying the associated risks. However, the routine statistical protocols cannot be directly applied because of challenges arising from nondetects or left-censored observations, which are concentration measurements below the detection limit of measuring instruments. Despite the existence of techniques based on survival analysis that can adjust for nondetects, these are seldom taken into account properly. A comprehensive review of the literature showed that managing policies regarding analysis of censored data do not always agree and that guidance from regulatory agencies may be outdated. Therefore, researchers and practitioners commonly resort to the most convenient way of tackling the censored data problem by substituting nondetects with arbitrary constants prior to data analysis, although this is generally regarded as a bias-prone approach. Hoping to improve the interpretation of concentration data, the present article aims to familiarize researchers in different disciplines with the significance of left-censored observations and provides theoretical and computational recommendations (under both frequentist and Bayesian frameworks) for adequate analysis of censored data. In particular, the present article synthesizes key findings from previous research with respect to 3 noteworthy aspects of inferential statistics: estimation of descriptive statistics, hypothesis testing, and regression analysis. Environ Toxicol Chem 2018;37:643-656. © 2017 SETAC. © 2017 SETAC.

  16. Gap Analysis Approach for Construction Safety Program Improvement

    Directory of Open Access Journals (Sweden)

    Thanet Aksorn

    2007-06-01

    Full Text Available To improve construction site safety, emphasis has been placed on the implementation of safety programs. In order to successfully gain from safety programs, factors that affect their improvement need to be studied. Sixteen critical success factors of safety programs were identified from safety literature, and these were validated by safety experts. This study was undertaken by surveying 70 respondents from medium- and large-scale construction projects. It explored the importance and the actual status of critical success factors (CSFs. Gap analysis was used to examine the differences between the importance of these CSFs and their actual status. This study found that the most critical problems characterized by the largest gaps were management support, appropriate supervision, sufficient resource allocation, teamwork, and effective enforcement. Raising these priority factors to satisfactory levels would lead to successful safety programs, thereby minimizing accidents.

  17. Road Network Vulnerability Analysis Based on Improved Ant Colony Algorithm

    Directory of Open Access Journals (Sweden)

    Yunpeng Wang

    2014-01-01

    Full Text Available We present an improved ant colony algorithm-based approach to assess the vulnerability of a road network and identify the critical infrastructures. This approach improves computational efficiency and allows for its applications in large-scale road networks. This research involves defining the vulnerability conception, modeling the traffic utility index and the vulnerability of the road network, and identifying the critical infrastructures of the road network. We apply the approach to a simple test road network and a real road network to verify the methodology. The results show that vulnerability is directly related to traffic demand and increases significantly when the demand approaches capacity. The proposed approach reduces the computational burden and may be applied in large-scale road network analysis. It can be used as a decision-supporting tool for identifying critical infrastructures in transportation planning and management.

  18. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

    International Nuclear Information System (INIS)

    Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

    1988-01-01

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16 + natural killer cells and CD3 + , CD16 - T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

  19. An improved convergence analysis of smoothed aggregation algebraic multigrid

    Energy Technology Data Exchange (ETDEWEB)

    Brezina, Marian [Univ. of Colorado, Boulder, CO (United States). Dept. of Applied Mathematics; Vaněk, Petr [University of West Bohemia (Czech Republic). Dept. of Mathematics; Vassilevski, Panayot S. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Center for Applied Scientific Computing

    2011-03-02

    We present an improved analysis of the smoothed aggregation (SA) alge- braic multigrid method (AMG) extending the original proof in [SA] and its modification in [Va08]. The new result imposes fewer restrictions on the aggregates that makes it eas- ier to verify in practice. Also, we extend a result in [Van] that allows us to use aggressive coarsening at all levels due to the special properties of the polynomial smoother, that we use and analyze, and thus provide a multilevel convergence estimate with bounds independent of the coarsening ratio.

  20. Improved method and apparatus for chromatographic quantitative analysis

    Science.gov (United States)

    Fritz, J.S.; Gjerde, D.T.; Schmuckler, G.

    An improved apparatus and method are described for the quantitative analysis of a solution containing a plurality of anion species by ion exchange chromatography which utilizes a single element and a single ion exchange bed which does not require periodic regeneration. The solution containing the anions is added to an anion exchange resin bed which is a low capacity macroreticular polystyrene-divinylbenzene resin containing quarternary ammonium functional groups, and is eluted therefrom with a dilute solution of a low electrical conductance organic acid salt. As each anion species is eluted from the bed, it is quantitatively sensed by conventional detection means such as a conductivity cell.

  1. MITG post-test analysis and design improvements

    International Nuclear Information System (INIS)

    Schock, A.

    1983-01-01

    The design, performance analysis, and key attributes of the Modular Isotopic Thermoelectric Generator (MITG) were described in a 1981 IECEC paper; and the design, fabrication, and testing of prototypical MITG test assemblies were described in preceding papers in these proceedings. Each test assembly simulated a typical modular slice of the flight generator. The present paper describes a detailed thermal-stress analysis, which identified the causes of stress-related problems observed during the tests. It then describes how additional analyses were used to evaluate design changes to alleviate those problems. Additional design improvements are discussed in the next paper in these proceedings, which also describes revised fabrication procedures and updated performance estimates for the generator

  2. Missile placement analysis based on improved SURF feature matching algorithm

    Science.gov (United States)

    Yang, Kaida; Zhao, Wenjie; Li, Dejun; Gong, Xiran; Sheng, Qian

    2015-03-01

    The precious battle damage assessment by use of video images to analysis missile placement is a new study area. The article proposed an improved speeded up robust features algorithm named restricted speeded up robust features, which combined the combat application of TV-command-guided missiles and the characteristics of video image. Its restrictions mainly reflected in two aspects, one is to restrict extraction area of feature point; the second is to restrict the number of feature points. The process of missile placement analysis based on video image was designed and a video splicing process and random sample consensus purification were achieved. The RSURF algorithm is proved that has good realtime performance on the basis of guarantee the accuracy.

  3. 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 ...

  4. [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.

  5. 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 ...

  6. 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

  7. 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 ...

  8. 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

  9. [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.

  10. Improvements and experience in the analysis of reprocessing samples

    International Nuclear Information System (INIS)

    Koch, L.; Cricchio, A.; Meester, R. de; Romkowski, M.; Wilhelmi, M.; Arenz, H.J.; Stijl, E. van der; Baeckmann, A. von

    1976-01-01

    Improvements in the analysis of input samples for reprocessing were obtained. To cope with the decomposition of reprocessing input solutions owling to the high radioactivity, an aluminium capsule technique was developed. A known amount of the dissolver solution was weighed into an aluminium can, dried, and the capsule was sealed. In this form, the sample could be stored over a long period and could be redissolved later for the analysis. The isotope correlation technique offers an attractive alternative for measuring the plutonium isotopic content in the dissolver solution. Moreover, this technique allows for consistency checks of analytical results. For this purpose, a data bank of correlated isotopic data is in use. To improve the efficiency of analytical work, four automatic instruments have been developed. The conditioning of samples for the U-Pu isotopic measurement was achieved by an automatic ion exchanger. A mass spectrometer, to which a high vacuum lock is connected, allows the automatic measurement of U-Pu samples. A process-computer controls the heating, focusing and scanning processes during the measurement and evaluates the data. To ease the data handling, alpha-spectrometry as well as a balance have been automated. (author)

  11. Multispectral fingerprinting for improved in vivo cell dynamics analysis

    Directory of Open Access Journals (Sweden)

    Cooper Cameron HJ

    2010-09-01

    Full Text Available Abstract Background Tracing cell dynamics in the embryo becomes tremendously difficult when cell trajectories cross in space and time and tissue density obscure individual cell borders. Here, we used the chick neural crest (NC as a model to test multicolor cell labeling and multispectral confocal imaging strategies to overcome these roadblocks. Results We found that multicolor nuclear cell labeling and multispectral imaging led to improved resolution of in vivo NC cell identification by providing a unique spectral identity for each cell. NC cell spectral identity allowed for more accurate cell tracking and was consistent during short term time-lapse imaging sessions. Computer model simulations predicted significantly better object counting for increasing cell densities in 3-color compared to 1-color nuclear cell labeling. To better resolve cell contacts, we show that a combination of 2-color membrane and 1-color nuclear cell labeling dramatically improved the semi-automated analysis of NC cell interactions, yet preserved the ability to track cell movements. We also found channel versus lambda scanning of multicolor labeled embryos significantly reduced the time and effort of image acquisition and analysis of large 3D volume data sets. Conclusions Our results reveal that multicolor cell labeling and multispectral imaging provide a cellular fingerprint that may uniquely determine a cell's position within the embryo. Together, these methods offer a spectral toolbox to resolve in vivo cell dynamics in unprecedented detail.

  12. Group learning improves case analysis in veterinary medicine.

    Science.gov (United States)

    Pickrell, John A; Boyer, John; Oehme, Frederick W; Clegg, Victoria L; Sells, Nikki

    2002-01-01

    Group learning has become important to professional students in the healing sciences. Groups share factual and procedural resources to enhance their performances. We investigated the extent to which students analyzing case-based evaluations as teams acquired an immediate performance advantage relative to those analyzing them as individuals and the extent to which group work on one problem led to better performance by individual students on related problems. We blinded written evaluations by randomly assigning numbers to groups of students and using removable tracers. Differences between groups and individuals were evaluated using Student's t statistic. Similar comparisons were evaluated by meta-analysis to determine overall trends. Students who analyzed evaluations as a group had an 8.5% performance advantage over those who analyzed them as individuals. When evaluations were divided into those asking questions related to treatment, differential diagnosis, and prognosis, specific performance advantages for groups relative to individuals were 8.9%, 5.9%, and 6.1% respectively. Students who had previously been trained by group evaluations had a 1.5% advantage relative to those who received their training as individuals. Answers by students analyzing evaluations as groups suggested a deeper understanding, in large part because of their improved ability to explain treatment and to conduct differential diagnosis. These improvements suggested limited abilities to use previous experience to improve present performance.

  13. Skill analysis part 3: improving a practice skill.

    Science.gov (United States)

    Price, Bob

    In this, the third and final article in a series on practice skill analysis, attention is given to imaginative ways of improving a practice skill. Having analysed and evaluated a chosen skill in the previous two articles, it is time to look at new ways to proceed. Creative people are able to be analytical and imaginative. The process of careful reasoning involved in analysing and evaluating a skill will not necessarily be used to improve it. To advance a skill, there is a need to engage in more imaginative, free-thinking processes that allow the nurse to think afresh about his or her chosen skill. Suggestions shared in this article are not exhaustive, but the material presented does illustrate measures that in the author's experience seem to have potential. Consideration is given to how the improved skill might be envisaged (an ideal skill in use). The article is illustrated using the case study of empathetic listening, which has been used throughout this series.

  14. ECONOMIC AND ENERGETICAL ANALYSIS OF IMPROVED WASTE UTILIZATION PLASMA TECHNOLOGY

    Directory of Open Access Journals (Sweden)

    Serghei VAMBOL

    2015-07-01

    Full Text Available Purpose. Energy and economic evaluation of the improved plasma waste utilization technological process, as well as an expediency substantiation of the use of improved plasma technology by comparing its energy consumption with other thermal methods of utilization. Methodology. Analysis of existing modern and advanced methods of waste management and its impact on environmental safety. Considering of energy and monetary costs to implement two different waste management technologies. Results. Studies have shown regular gasification ensure greater heating value due to differences, a significant amount of nitrogen than for plasma gasification. From the point of view of minimizing energy and monetary costs and environmental safety more promising is to offer advanced technology for plasma waste. To carry out the energy assessment of the appropriateness of the considered technologies-comparative calculation was carried out at the standard conditions. This is because in the processing of waste produced useful products, such as liquefied methane, synthetic gas (94% methane and a fuel gas for heating, suitable for sale that provides cost-effectiveness of this technology. Originality. Shown and evaluated ecological and economic efficiency of proposed improved plasma waste utilization technology compared with other thermal techniques. Practical value. Considered and grounded of energy and monetary costs to implement two different waste management technologies, namely ordinary gasification and using plasma generators. Proposed plasma waste utilization technology allows to obtain useful products, such as liquefied methane, synthetic gas and a fuel gas for heating, which are suitable for sale. Plant for improved plasma waste utilization technological process allows to compensate the daily and seasonal electricity and heat consumption fluctuations by allowing the storage of obtained fuel products.

  15. Does plyometric training improve strength performance? A meta-analysis.

    Science.gov (United States)

    Sáez-Sáez de Villarreal, Eduardo; Requena, Bernardo; Newton, Robert U

    2010-09-01

    Majority of the research suggests plyometric training (PT) improves maximal strength performance as measured by 1RM, isometric MVC or slow velocity isokinetic testing. However, the effectiveness of PT depends upon various factors. A meta-analysis of 15 studies with a total of 31 effect sizes (ES) was carried out to analyse the role of various factors on the effects of PT on strength performance. The inclusion criteria for the analysis were: (a) studies using PT programs for lower limb muscles; (b) studies employing true experimental design and valid and reliable measurements; (c) studies including sufficient data to calculate ES. When subjects can adequately follow plyometric exercises, the training gains are independent of fitness level. Subjects in either good or poor physical condition, benefit equally from plyometric work, also men obtain similar strength results to women following PT. In relation to the variables of program design, training volume of less than 10 weeks and with more than 15 sessions, as well as the implementation of high-intensity programs, with more than 40 jumps per session, were the strategies that seem to maximize the probability to obtain significantly greater improvements in performance (p<0.05). In order to optimise strength enhancement, the combination of different types of plyometrics with weight-training would be recommended, rather than utilizing only one form (p<0.05). The responses identified in this analysis are essential and should be considered by the strength and conditioning professional with regard to the most appropriate dose-response trends for PT to optimise strength gains.

  16. Improving patient safety in radiotherapy through error reporting and analysis

    International Nuclear Information System (INIS)

    Findlay, Ú.; Best, H.; Ottrey, M.

    2016-01-01

    Aim: To improve patient safety in radiotherapy (RT) through the analysis and publication of radiotherapy errors and near misses (RTE). Materials and methods: RTE are submitted on a voluntary basis by NHS RT departments throughout the UK to the National Reporting and Learning System (NRLS) or directly to Public Health England (PHE). RTE are analysed by PHE staff using frequency trend analysis based on the classification and pathway coding from Towards Safer Radiotherapy (TSRT). PHE in conjunction with the Patient Safety in Radiotherapy Steering Group publish learning from these events, on a triannual and summarised on a biennial basis, so their occurrence might be mitigated. Results: Since the introduction of this initiative in 2010, over 30,000 (RTE) reports have been submitted. The number of RTE reported in each biennial cycle has grown, ranging from 680 (2010) to 12,691 (2016) RTE. The vast majority of the RTE reported are lower level events, thus not affecting the outcome of patient care. Of the level 1 and 2 incidents reported, it is known the majority of them affected only one fraction of a course of treatment. This means that corrective action could be taken over the remaining treatment fractions so the incident did not have a significant impact on the patient or the outcome of their treatment. Analysis of the RTE reports demonstrates that generation of error is not confined to one professional group or to any particular point in the pathway. It also indicates that the pattern of errors is replicated across service providers in the UK. Conclusion: Use of the terminology, classification and coding of TSRT, together with implementation of the national voluntary reporting system described within this report, allows clinical departments to compare their local analysis to the national picture. Further opportunities to improve learning from this dataset must be exploited through development of the analysis and development of proactive risk management strategies

  17. Response surface analysis to improve dispersed crude oil biodegradation

    Energy Technology Data Exchange (ETDEWEB)

    Zahed, Mohammad A.; Aziz, Hamidi A.; Mohajeri, Leila [School of Civil Engineering, Universiti Sains Malaysia, Nibong Tebal, Penang (Malaysia); Isa, Mohamed H. [Civil Engineering Department, Universiti Teknologi PETRONAS, Tronoh, Perak (Malaysia)

    2012-03-15

    In this research, the bioremediation of dispersed crude oil, based on the amount of nitrogen and phosphorus supplementation in the closed system, was optimized by the application of response surface methodology and central composite design. Correlation analysis of the mathematical-regression model demonstrated that a quadratic polynomial model could be used to optimize the hydrocarbon bioremediation (R{sup 2} = 0.9256). Statistical significance was checked by analysis of variance and residual analysis. Natural attenuation was removed by 22.1% of crude oil in 28 days. The highest removal on un-optimized condition of 68.1% were observed by using nitrogen of 20.00 mg/L and phosphorus of 2.00 mg/L in 28 days while optimization process exhibited a crude oil removal of 69.5% via nitrogen of 16.05 mg/L and phosphorus 1.34 mg/L in 27 days therefore optimization can improve biodegradation in shorter time with less nutrient consumption. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  18. TENDENCY OF IMPROVEMENT ANALYSIS OF VENTURE ACTIVITY FOR MANAGEMENT DECISIONS

    Directory of Open Access Journals (Sweden)

    G.Yu. Iakovetс

    2015-03-01

    Full Text Available The questions concerning the definition of current trends and prospects of venture financing new innovative enterprises as one of the most effective and alternative, but with a high degree of risk financing sources of the entity. The features of venture financing that is different from other sources of business financing, as well as income from investments of venture capital can greatly exceed the volume of investments, but at the same time such financing risks are significant, so it all makes it necessary to build an effective system of venture capital investments in the workplace. In the course of the study also revealed problems of analysis and minimization of risks in the performance of venture financing of innovative enterprises. Defining characteristics analysis and risk assessment of venture financing helps to find ways to minimize and systematization, avoidance and prevention of risks in the performance of venture capital. The study also identified the major areas of improvement analysis of venture capital for management decisions.

  19. Full-motion video analysis for improved gender classification

    Science.gov (United States)

    Flora, Jeffrey B.; Lochtefeld, Darrell F.; Iftekharuddin, Khan M.

    2014-06-01

    The ability of computer systems to perform gender classification using the dynamic motion of the human subject has important applications in medicine, human factors, and human-computer interface systems. Previous works in motion analysis have used data from sensors (including gyroscopes, accelerometers, and force plates), radar signatures, and video. However, full-motion video, motion capture, range data provides a higher resolution time and spatial dataset for the analysis of dynamic motion. Works using motion capture data have been limited by small datasets in a controlled environment. In this paper, we explore machine learning techniques to a new dataset that has a larger number of subjects. Additionally, these subjects move unrestricted through a capture volume, representing a more realistic, less controlled environment. We conclude that existing linear classification methods are insufficient for the gender classification for larger dataset captured in relatively uncontrolled environment. A method based on a nonlinear support vector machine classifier is proposed to obtain gender classification for the larger dataset. In experimental testing with a dataset consisting of 98 trials (49 subjects, 2 trials per subject), classification rates using leave-one-out cross-validation are improved from 73% using linear discriminant analysis to 88% using the nonlinear support vector machine classifier.

  20. Improved nowcasting of precipitation based on convective analysis fields

    Directory of Open Access Journals (Sweden)

    T. Haiden

    2007-04-01

    Full Text Available The high-resolution analysis and nowcasting system INCA (Integrated Nowcasting through Comprehensive Analysis developed at the Austrian national weather service provides three-dimensional fields of temperature, humidity, and wind on an hourly basis, and two-dimensional fields of precipitation rate in 15 min intervals. The system operates on a horizontal resolution of 1 km and a vertical resolution of 100–200 m. It combines surface station data, remote sensing data (radar, satellite, forecast fields of the numerical weather prediction model ALADIN, and high-resolution topographic data. An important application of the INCA system is nowcasting of convective precipitation. Based on fine-scale temperature, humidity, and wind analyses a number of convective analysis fields are routinely generated. These fields include convective boundary layer (CBL flow convergence and specific humidity, lifted condensation level (LCL, convective available potential energy (CAPE, convective inhibition (CIN, and various convective stability indices. Based on the verification of areal precipitation nowcasts it is shown that the pure translational forecast of convective cells can be improved by using a decision algorithm which is based on a subset of the above fields, combined with satellite products.

  1. 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.

  2. Improving knowledge management systems with latent semantic analysis

    International Nuclear Information System (INIS)

    Sebok, A.; Plott, C.; LaVoie, N.

    2006-01-01

    Latent Semantic Analysis (LSA) offers a technique for improving lessons learned and knowledge management systems. These systems are expected to become more widely used in the nuclear industry, as experienced personnel leave and are replaced by younger, less-experienced workers. LSA is a machine learning technology that allows searching of text based on meaning rather than predefined keywords or categories. Users can enter and retrieve data using their own words, rather than relying on constrained language lists or navigating an artificially structured database. LSA-based tools can greatly enhance the usability and usefulness of knowledge management systems and thus provide a valuable tool to assist nuclear industry personnel in gathering and transferring worker expertise. (authors)

  3. A improved method for the analysis of alpha spectra

    International Nuclear Information System (INIS)

    Equillor, Hugo E.

    2004-01-01

    In this work we describe a methodology, developed in the last years, for the analysis of alpha emitters spectra, obtained with implanted ion detectors, that tend to solve some of the problems that shows this type of spectra. This is an improved methodology respect to that described in a previous publication. The method is based on the application of a mathematical function that allows to model the tail of an alpha peak, to evaluate the part of the peak that is not seen in the cases of partial superposition with another peak. Also, a calculation program that works in a semiautomatic way, with the possibility of interactive intervention of the analyst, has been developed simultaneously and is described in detail. (author)

  4. Improving assessment of personality disorder traits through social network analysis.

    Science.gov (United States)

    Clifton, Allan; Turkheimer, Eric; Oltmanns, Thomas F

    2007-10-01

    When assessing personality disorder traits, not all judges make equally valid judgments of all targets. The present study uses social network analysis to investigate factors associated with reliability and validity in peer assessment. Participants were groups of military recruits (N=809) who acted as both targets and judges in a round-robin design. Participants completed self- and informant versions of the Multisource Assessment of Personality Pathology. Social network matrices were constructed based on reported acquaintance, and cohesive subgroups were identified. Judges who shared a mutual subgroup were more reliable and had higher self-peer agreement than those who did not. Partitioning networks into two subgroups achieved more consistent improvements than multiple subgroups. We discuss implications for multiple informant assessments.

  5. Plant improvements through the use of benchmarking analysis

    International Nuclear Information System (INIS)

    Messmer, J.R.

    1993-01-01

    As utilities approach the turn of the century, customer and shareholder satisfaction is threatened by rising costs. Environmental compliance expenditures, coupled with low load growth and aging plant assets are forcing utilities to operate existing resources in a more efficient and productive manner. PSI Energy set out in the spring of 1992 on a benchmarking mission to compare four major coal fired plants against others of similar size and makeup, with the goal of finding the best operations in the country. Following extensive analysis of the 'Best in Class' operation, detailed goals and objectives were established for each plant in seven critical areas. Three critical processes requiring rework were identified and required an integrated effort from all plants. The Plant Improvement process has already resulted in higher operation productivity, increased emphasis on planning, and lower costs due to effective material management. While every company seeks improvement, goals are often set in an ambiguous manner. Benchmarking aids in setting realistic goals based on others' actual accomplishments. This paper describes how the utility's short term goals will move them toward being a lower cost producer

  6. Generalization in the XCSF classifier system: analysis, improvement, and extension.

    Science.gov (United States)

    Lanzi, Pier Luca; Loiacono, Daniele; Wilson, Stewart W; Goldberg, David E

    2007-01-01

    We analyze generalization in XCSF and introduce three improvements. We begin by showing that the types of generalizations evolved by XCSF can be influenced by the input range. To explain these results we present a theoretical analysis of the convergence of classifier weights in XCSF which highlights a broader issue. In XCSF, because of the mathematical properties of the Widrow-Hoff update, the convergence of classifier weights in a given subspace can be slow when the spread of the eigenvalues of the autocorrelation matrix associated with each classifier is large. As a major consequence, the system's accuracy pressure may act before classifier weights are adequately updated, so that XCSF may evolve piecewise constant approximations, instead of the intended, and more efficient, piecewise linear ones. We propose three different ways to update classifier weights in XCSF so as to increase the generalization capabilities of XCSF: one based on a condition-based normalization of the inputs, one based on linear least squares, and one based on the recursive version of linear least squares. Through a series of experiments we show that while all three approaches significantly improve XCSF, least squares approaches appear to be best performing and most robust. Finally we show how XCSF can be extended to include polynomial approximations.

  7. 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.

  8. An efficiency improvement in warehouse operation using simulation analysis

    Science.gov (United States)

    Samattapapong, N.

    2017-11-01

    In general, industry requires an efficient system for warehouse operation. There are many important factors that must be considered when designing an efficient warehouse system. The most important is an effective warehouse operation system that can help transfer raw material, reduce costs and support transportation. By all these factors, researchers are interested in studying about work systems and warehouse distribution. We start by collecting the important data for storage, such as the information on products, information on size and location, information on data collection and information on production, and all this information to build simulation model in Flexsim® simulation software. The result for simulation analysis found that the conveyor belt was a bottleneck in the warehouse operation. Therefore, many scenarios to improve that problem were generated and testing through simulation analysis process. The result showed that an average queuing time was reduced from 89.8% to 48.7% and the ability in transporting the product increased from 10.2% to 50.9%. Thus, it can be stated that this is the best method for increasing efficiency in the warehouse operation.

  9. Sensitivity analysis for improving nanomechanical photonic transducers biosensors

    International Nuclear Information System (INIS)

    Fariña, D; Álvarez, M; Márquez, S; Lechuga, L M; Dominguez, C

    2015-01-01

    The achievement of high sensitivity and highly integrated transducers is one of the main challenges in the development of high-throughput biosensors. The aim of this study is to improve the final sensitivity of an opto-mechanical device to be used as a reliable biosensor. We report the analysis of the mechanical and optical properties of optical waveguide microcantilever transducers, and their dependency on device design and dimensions. The selected layout (geometry) based on two butt-coupled misaligned waveguides displays better sensitivities than an aligned one. With this configuration, we find that an optimal microcantilever thickness range between 150 nm and 400 nm would increase both microcantilever bending during the biorecognition process and increase optical sensitivity to 4.8   ×   10 −2  nm −1 , an order of magnitude higher than other similar opto-mechanical devices. Moreover, the analysis shows that a single mode behaviour of the propagating radiation is required to avoid modal interference that could misinterpret the readout signal. (paper)

  10. Improvements in biamperometric method for remote analysis of uranium

    International Nuclear Information System (INIS)

    Palamalai, A.; Thankachan, T.S.; Balasubramanian, G.R.

    1979-01-01

    One of the titrimetric methods most suitable for remote operations with Master Slave Manipulators inside hot cells is the biamperometric method. The biamperometric method for the analysis of uranium reported in the literature is found to give rise to a significant bias, especially with low aliquots of uranium and the waste volume is also considerable which is not desirable from the point of view of radioactive waste disposal. In the present method, the bias as well as waste volume are reduced. Also addition of vanadyl sulphate is found necessary to provide a sharp end point in the titration curve. The role of vanadyl sulphate in improving the titration method has been investigated by spectrophotometry and electrometry. A new mechanism for the role of vanadyl sulphate which is in conformity with the observations made in coulometric titration of uranium, is proposed. Interference from deliberate additions of high concentrations of stable species of fission product elements is found negligible. Hence this method is considered highly suitable for remote analysis of uranium in intensely radioactive reprocessing solutions for control purposes, provided radioactivity does not pose new problems. (auth.)

  11. Improved inhomogeneous finite elements for fabric reinforced composite mechanics analysis

    Science.gov (United States)

    Foye, R. L.

    1992-01-01

    There is a need to do routine stress/failure analysis of fabric reinforced composite microstructures to provide additional confidence in critical applications and guide materials development. Conventional methods of 3-D stress analysis are time consuming to set up, run and interpret. A need exists for simpler methods of modeling these structures and analyzing the models. The principal difficulty is the discrete element mesh generation problem. Inhomogeneous finite elements are worth investigating for application to these problems because they eliminate the mesh generation problem. However, there are penalties associated with these elements. Their convergence rates can be slow compared to homogeneous elements. Also, there is no accepted method for obtaining detailed stresses in the constituent materials of each element. This paper shows that the convergence rate can be significantly improved by a simple device which substitutes homogeneous elements for the inhomogeneous ones. The device is shown to work well in simple one and two dimensional problems. However, demonstration of the application to more complex two and three dimensional problems remains to be done. Work is also progressing toward more realistic fabric microstructural geometries.

  12. [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.

  13. [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.

  14. Characterization of cDNA for PMT: a Partial Nicotine Biosynthesis-Related Gene Isolated from Indonesian Local Tobacco (Nicotiana tabacum cv. Sindoro1

    Directory of Open Access Journals (Sweden)

    SESANTI BASUKI

    2013-12-01

    Full Text Available Nicotine is the major alkaloid compound in cultivated tobacco (Nicotiana tabacum that could potentially be converted into carcinogenic compound (nor-nicotine. The PMT gene encoding putrescine N-methyltransferase (PMT is one of the two key genes that play a prominent role in nicotine biosynthesis. The aimed of this study was to isolate and characterize the cDNA sequence originated from Indonesian local tobacco cv. Sindoro1 (Ntpmt_Sindoro1. The results showed that the Ntpmt_Sindoro1 was 1124 bp in length. This cDNA fragment encodes for 374 amino acid residues. The predicted polypeptide from the cDNA is a hidrophilic protein, and has a predicted molecular weight of 40.95 kDa. The predicted amino acids sequence also showed high similarity to the PMT gene product Nicotiana sp. available in the GenBank data base. The amino acid sequences also exert conserved residues specifically exhibited only by PMT gene originated from N. tabacum. Clustering analysis revealed that Ntpmt_Sindoro1 belongs to the same clade as the PMT3 gene, a member of the N. tabacum PMT gene family. The Ntpmt_Sindoro1 cDNA sequence covering exon1-exon8 of the PMT gene fragment has been registered in the GenBank data base, under the accession number JX978277.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  16. 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)

  17. 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.

  18. 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

  19. 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...

  20. 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...

  1. 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.

  2. 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 ...

  3. Molecular characterization of a cDNA encoding copper/zinc superoxide dismutase from cultured cells of Manihot esculenta.

    Science.gov (United States)

    Shin, Seung-Yong; Lee, Haeng-Soon; Kwon, Suk-Yoon; Kwon, Soon-Tae; Kwak, Sang-Soo

    2005-01-01

    Superoxide dismutase (SOD) cDNA, mSOD2, encoding cytosolic copper/zinc SOD (CuZnSOD) cDNA was isolated from suspension-cultured cells of cassava (Manihot esculenta Crantz) by cDNA library screening, and its expression was investigated in relation to environmental stress. mSOD2 is 774 bp in length with an open reading frame (ORF) of 152 amino acids, corresponding to a protein of predicted molecular mass 15 kDa and a pI of 5.22. One copy of the mSOD2 gene was found to be present in the cassava genome by Southern analysis using an mSOD2 cDNA-specific probe. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed diverse expression patterns for the mSOD2 gene in various tissues of intact cassava plants, at various stages of the growth in suspension cultures, and in the leaf tissues exposed to different stresses. The mSOD2 gene was highly expressed in suspension-cultured cells and in the stems of intact plants. However, it was expressed at low levels in leaves and roots. During suspension cell growth, the mSOD2 transcript progressively increased during culture. Moreover, the mSOD2 gene in excised cassava leaves responded to various stresses in different ways. In particular, it was highly induced in leaf tissue by several abiotic stresses, including high temperature (37 degrees C), chilling (4 degrees C), methyl viologen (MV) exposure, and wounding treatment. These results indicate that the mSOD2 gene is involved in the antioxidative process triggered by oxidative stress induced by environmental change.

  4. Development and characterization of a high temperature stress responsive subtractive cDNA library in Pearl Millet Pennisetum glaucum (L.) R.Br.

    Science.gov (United States)

    James, Donald; Tarafdar, Avijit; Biswas, Koushik; Sathyavathi, Tara C; Padaria, Jasdeep Chatrath; Kumar, P Ananda

    2015-08-01

    Pearl millet (Pennisetum glaucum L. R. Br.) is an important cereal crop grown mainly in the arid and semi-arid regions of India known to possess the natural ability to withstand thermal stress. To elucidate the molecular basis of high temperature response in pearl millet, 12 days old seedlings of P. glaucum cv. 841A were subjected to heat stress at 46 degrees C for different time durations ( 30 min, 2, 4, 8, 12 and 24 h) and a forward subtractive cDNA library was constructed from pooled RNA of heat stressed seedlings. A total of 331 high quality Expressed Sequence Tags (ESTs) were obtained from randomly selected 1050 clones. Sequences were assembled into 103 unique sequences consisting of 37 contigs and 66 singletons. Of these, 92 unique sequences were submitted to NCBI dbEST database. Gene Ontology through RGAP data base and BLASTx analysis revealed that about 18% of the ESTs showed homology to genes for "response to abiotic and biotic stimulus". About 2% of the ESTs showed no homology with genes in dbEST, indicating the presence of uncharacterized candidate genes involved in heat stress response in P. glaucum. Differential expression of selected genes (hsp101 and CRT) from the SSH library were validated by qRT-PCR analysis. The ESTs thus generated are a rich source of heat stress responsive genes, which can be utilized in improving thermotolerance of other food crops.

  5. Assessing the utility of the Oxford Nanopore MinION for snake venom gland cDNA sequencing

    Directory of Open Access Journals (Sweden)

    Adam D. Hargreaves

    2015-11-01

    Full Text Available Portable DNA sequencers such as the Oxford Nanopore MinION device have the potential to be truly disruptive technologies, facilitating new approaches and analyses and, in some cases, taking sequencing out of the lab and into the field. However, the capabilities of these technologies are still being revealed. Here we show that single-molecule cDNA sequencing using the MinION accurately characterises venom toxin-encoding genes in the painted saw-scaled viper, Echis coloratus. We find the raw sequencing error rate to be around 12%, improved to 0–2% with hybrid error correction and 3% with de novo error correction. Our corrected data provides full coding sequences and 5′ and 3′ UTRs for 29 of 33 candidate venom toxins detected, far superior to Illumina data (13/40 complete and Sanger-based ESTs (15/29. We suggest that, should the current pace of improvement continue, the MinION will become the default approach for cDNA sequencing in a variety of species.

  6. Receiver operating characteristic analysis improves diagnosis by radionuclide ventriculography

    International Nuclear Information System (INIS)

    Dickinson, C.Z.; Forman, M.B.; Vaugh, W.K.; Sandler, M.P.; Kronenberg, M.W.

    1985-01-01

    Receiver operating characteristic analysis (ROC) evaluates continuous variables to define diagnostic criteria for the optimal sensitivity (SENS) and specificity (SPEC) of a test. The authors studied exercise-induced chest pain (CP), ST-changes on electrocardiography (ECG) and rest-exercise gated radionuclide ventriculography (RVG) using ROC to clarify the optimal criteria for detecting myocardial ischemia due to coronary artherosclerosis (CAD). The data of 95 consecutive patients studied with coronary angiography, rest-exercise RVG and ECG were reviewed. 77 patients had ''significant'' CAD (≥50% lesions). Exercise-induced CP, ECG abnormalities (ST-T shifts) and RVG abnormalities (change in ejection fraction, 2-view regional wall motion change and relative end-systolic volume) were evaluated to define optimal SENS/SPEC of each and for the combined data. ROC curves were constructed by multiple logistic regression (MLR). By MLR, RVG alone was superior to ECG and CP. The combination of all three produced the best ROC curve for the entire group and for clinical subsets based on the number of diseased vessels and the presence or absence of prior myocardial infarction. When CP, ECG and RVG were combined, the optimal SENS/SPEC for detection of single vessel disease was 88/86. The SENS/SPEC for 3 vessel disease was 93/95. Thus, the application of RVG for the diagnosis of myocardial ischemia is improved with the inclusion of ECG and CP data by the use of a multiple logistic regression model. ROC analysis allows clinical application of multiple data for diagnosing CAD at desired SENS/SPEC rather than by arbitrary single-standard criteria

  7. 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.

  8. 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.

  9. 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 ...

  10. An integrated sampling and analysis approach for improved biodiversity monitoring.

    Science.gov (United States)

    DeWan, Amielle A; Zipkin, Elise F

    2010-05-01

    Successful biodiversity conservation requires high quality monitoring data and analyses to ensure scientifically defensible policy, legislation, and management. Although monitoring is a critical component in assessing population status and trends, many governmental and non-governmental organizations struggle to develop and implement effective sampling protocols and statistical analyses because of the magnitude and diversity of species in conservation concern. In this article we describe a practical and sophisticated data collection and analysis framework for developing a comprehensive wildlife monitoring program that includes multi-species inventory techniques and community-level hierarchical modeling. Compared to monitoring many species individually, the multi-species approach allows for improved estimates of individual species occurrences, including rare species, and an increased understanding of the aggregated response of a community to landscape and habitat heterogeneity. We demonstrate the benefits and practicality of this approach to address challenges associated with monitoring in the context of US state agencies that are legislatively required to monitor and protect species in greatest conservation need. We believe this approach will be useful to regional, national, and international organizations interested in assessing the status of both common and rare species.

  11. Improving CMD Areal Density Analysis: Algorithms and Strategies

    Directory of Open Access Journals (Sweden)

    R. E. Wilson

    2014-06-01

    Full Text Available Essential ideas, successes, and difficulties of Areal Density Analysis (ADA for color-magnitude diagrams (CMD’s of resolved stellar populations are examined, with explanation of various algorithms and strategies for optimal performance. A CMDgeneration program computes theoretical datasets with simulated observational error and a solution program inverts the problem by the method of Differential Corrections (DC so as to compute parameter values from observed magnitudes and colors, with standard error estimates and correlation coefficients. ADA promises not only impersonal results, but also significant saving of labor, especially where a given dataset is analyzed with several evolution models. Observational errors and multiple star systems, along with various single star characteristics and phenomena, are modeled directly via the Functional Statistics Algorithm (FSA. Unlike Monte Carlo, FSA is not dependent on a random number generator. Discussions include difficulties and overall requirements, such as need for fast evolutionary computation and realization of goals within machine memory limits. Degradation of results due to influence of pixelization on derivatives, Initial Mass Function (IMF quantization, IMF steepness, low Areal Densities (A, and large variation in A are reduced or eliminated through a variety of schemes that are explained sufficiently for general application. The Levenberg-Marquardt and MMS algorithms for improvement of solution convergence are contained within the DC program. An example of convergence, which typically is very good, is shown in tabular form. A number of theoretical and practical solution issues are discussed, as are prospects for further development.

  12. Improving CMD Areal Density Analysis: Algorithms and Strategies

    Science.gov (United States)

    Wilson, R. E.

    2014-06-01

    Essential ideas, successes, and difficulties of Areal Density Analysis (ADA) for color-magnitude diagrams (CMD¡¯s) of resolved stellar populations are examined, with explanation of various algorithms and strategies for optimal performance. A CMDgeneration program computes theoretical datasets with simulated observational error and a solution program inverts the problem by the method of Differential Corrections (DC) so as to compute parameter values from observed magnitudes and colors, with standard error estimates and correlation coefficients. ADA promises not only impersonal results, but also significant saving of labor, especially where a given dataset is analyzed with several evolution models. Observational errors and multiple star systems, along with various single star characteristics and phenomena, are modeled directly via the Functional Statistics Algorithm (FSA). Unlike Monte Carlo, FSA is not dependent on a random number generator. Discussions include difficulties and overall requirements, such as need for fast evolutionary computation and realization of goals within machine memory limits. Degradation of results due to influence of pixelization on derivatives, Initial Mass Function (IMF) quantization, IMF steepness, low Areal Densities (A ), and large variation in A are reduced or eliminated through a variety of schemes that are explained sufficiently for general application. The Levenberg-Marquardt and MMS algorithms for improvement of solution convergence are contained within the DC program. An example of convergence, which typically is very good, is shown in tabular form. A number of theoretical and practical solution issues are discussed, as are prospects for further development.

  13. An integrated sampling and analysis approach for improved biodiversity monitoring

    Science.gov (United States)

    DeWan, Amielle A.; Zipkin, Elise F.

    2010-01-01

    Successful biodiversity conservation requires high quality monitoring data and analyses to ensure scientifically defensible policy, legislation, and management. Although monitoring is a critical component in assessing population status and trends, many governmental and non-governmental organizations struggle to develop and implement effective sampling protocols and statistical analyses because of the magnitude and diversity of species in conservation concern. In this article we describe a practical and sophisticated data collection and analysis framework for developing a comprehensive wildlife monitoring program that includes multi-species inventory techniques and community-level hierarchical modeling. Compared to monitoring many species individually, the multi-species approach allows for improved estimates of individual species occurrences, including rare species, and an increased understanding of the aggregated response of a community to landscape and habitat heterogeneity. We demonstrate the benefits and practicality of this approach to address challenges associated with monitoring in the context of US state agencies that are legislatively required to monitor and protect species in greatest conservation need. We believe this approach will be useful to regional, national, and international organizations interested in assessing the status of both common and rare species.

  14. Improvements of Defect Analysis by Space-Charge Wave Spectroscopy

    Science.gov (United States)

    Voit, Kay-Michael; Hilling, Burkhard; Schmidt, Heinz-Jürgen; Imlau, Mirco

    2011-03-01

    We report on our recent advancements in space-charge wave spectroscopy, which can be used to investigate defect structures in classical high-resistive semiconductors and insulators. It permits to estimate the effective trap concentrations as well as the effective donor density Neff and the product μτ of electron mobility and life-time in the conduction band. We present a novel method of space-charge wave excitation, using a superposition of a running and a static sinusoidal illumination pattern. Thus we acquire -- in contrast to the former oscillating pattern -- a distinct direction of movement. The proposed new technique can be regarded as an effective amelioration, as the theoretical analysis is no longer limited by numerous presumptions like low modulation depth or small oscillation amplitudes. It not only overcomes these limits of the experimental configuration improving accuracy of SCW spectroscopy, but it also provides additional information, such as the sign of the charge carriers. Financial support by the DFG within the graduate college 695 ``Nonlinearities of Optical Materials'' and the project IM 37/5-1 is gratefully acknowledged.

  15. Improved sampling and analysis of images in corneal confocal microscopy.

    Science.gov (United States)

    Schaldemose, E L; Fontain, F I; Karlsson, P; Nyengaard, J R

    2017-10-01

    Corneal confocal microscopy (CCM) is a noninvasive clinical method to analyse and quantify corneal nerve fibres in vivo. Although the CCM technique is in constant progress, there are methodological limitations in terms of sampling of images and objectivity of the nerve quantification. The aim of this study was to present a randomized sampling method of the CCM images and to develop an adjusted area-dependent image analysis. Furthermore, a manual nerve fibre analysis method was compared to a fully automated method. 23 idiopathic small-fibre neuropathy patients were investigated using CCM. Corneal nerve fibre length density (CNFL) and corneal nerve fibre branch density (CNBD) were determined in both a manual and automatic manner. Differences in CNFL and CNBD between (1) the randomized and the most common sampling method, (2) the adjusted and the unadjusted area and (3) the manual and automated quantification method were investigated. The CNFL values were significantly lower when using the randomized sampling method compared to the most common method (p = 0.01). There was not a statistical significant difference in the CNBD values between the randomized and the most common sampling method (p = 0.85). CNFL and CNBD values were increased when using the adjusted area compared to the standard area. Additionally, the study found a significant increase in the CNFL and CNBD values when using the manual method compared to the automatic method (p ≤ 0.001). The study demonstrated a significant difference in the CNFL values between the randomized and common sampling method indicating the importance of clear guidelines for the image sampling. The increase in CNFL and CNBD values when using the adjusted cornea area is not surprising. The observed increases in both CNFL and CNBD values when using the manual method of nerve quantification compared to the automatic method are consistent with earlier findings. This study underlines the importance of improving the analysis of the

  16. [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.

  17. 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

  18. 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

  19. 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.

  20. [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.

  1. 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...

  2. 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.

  3. 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...

  4. 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 ...

  5. 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 ...

  6. Construction and characterization of Japanese medaka (Oryzias latipes) hepatic cDNA library and its implementation to biomarker screening in aquatic toxicology.

    Science.gov (United States)

    Pham, Chi Hoa; Park, Kyung Seo; Kim, Byoung Chan; Kim, Han Na; Gu, Man Bock

    2011-10-01

    To strengthen the toxicogenomic study, we constructed a library of hepatic cDNA from Japanese medaka under influence of specific chemical mediated stress responses. Gene expression profile analysis of the cDNA microarrays followed by real time RT-PCR assay were conducted to screen particular biomarkers for 17-beta estradiol (E2), nonylphenol (NP) and 2-chlorophenol (2CP). Information of 1509 high-quality ESTs including 260 new ESTs was added onto GenBank and dbEST. The ESTs were clustered and assembled into 159 contigs and 372 singletons. Among them, 128 contigs and 163 singletons (54.8%) were functionally characterized and 13 UniESTs (2.5%) were hypothetical proteins. Ontology analysis resulting in 282 UniESTs which involved with 2102 GOs and 93 sequences associated with 116 enzyme codes. For each test chemical, two specific biomarkers were selected from the gene expression profiling of microarrays. The expression patterns of the marker genes in real time PCR analysis were consistent with the regulated gene expression patterns in microarrays. The tentative biomarkers showed unique gene expression patterns depending on chemical concentration(s) and exposure duration in real time RT-PCR analysis. The analysis accomplished of the hepatic cDNA library and its information added to genetic and genomic resources could be sufficiently valuable specifically for aquatic toxicity studies. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. cDNA Cloning, expression and characterization of an allergenic 60s ribosomal protein of almond (prunus dulcis).

    Science.gov (United States)

    Abolhassani, Mohsen; Roux, Kenneth H

    2009-06-01

    Tree nuts, including almond (prunus dulcis) are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP) was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control) possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

  8. Characterization of ovine hepatic gene expression profiles in response to Escherichia coli lipopolysaccharide using a bovine cDNA microarray

    Directory of Open Access Journals (Sweden)

    Boermans Herman J

    2006-11-01

    Full Text Available Abstract Background During systemic gram-negative bacterial infections, lipopolysaccharide (LPS ligation to the hepatic Toll-like receptor-4 complex induces the production of hepatic acute phase proteins that are involved in the host response to infection and limit the associated inflammatory process. Identifying the genes that regulate this hepatic response to LPS in ruminants may provide insight into the pathogenesis of bacterial diseases and eventually facilitate breeding of more disease resistant animals. The objective of this research was to profile the expression of ovine hepatic genes in response to Escherichia coli LPS challenge (0, 200, 400 ng/kg using a bovine cDNA microarray and quantitative real-time PCR (qRT-PCR. Results Twelve yearling ewes were challenged iv with E. coli LPS (0, 200, 400 ng/kg and liver biopsies were collected 4–5 hours post-challenge to assess hepatic gene expression profiles by bovine cDNA microarray and qRT-PCR analyses. The expression of CD14, C3, IL12R, NRAMP1, SOD and IGFBP3 genes was down regulated, whereas the expression of ACTHR, IFNαR, CD1, MCP-1 and GH was increased during LPS challenge. With the exception of C3, qRT-PCR analysis of 7 of these genes confirmed the microarray results and demonstrated that GAPDH is not a suitable housekeeping gene in LPS challenged sheep. Conclusion We have identified several potentially important genes by bovine cDNA microarray and qRT-PCR analyses that are differentially expressed during the ovine hepatic response to systemic LPS challenge. Their potential role in regulating the inflammatory response to LPS warrants further investigation.

  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 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.

  11. [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.

  12. [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.

  13. 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.

  14. Characterization of porcine ENO3: genomic and cDNA structure, polymorphism and expression

    Directory of Open Access Journals (Sweden)

    Xiong Yuanzhu

    2008-09-01

    Full Text Available Abstract In this study, a full-length cDNA of the porcine ENO3 gene encoding a 434 amino acid protein was isolated. It contains 12 exons over approximately 5.4 kb. Differential splicing in the 5'-untranslated sequence generates two forms of mRNA that differ from each other in the presence or absence of a 142-nucleotide fragment. Expression analysis showed that transcript 1 of ENO3 is highly expressed in liver and lung, while transcript 2 is highly expressed in skeletal muscle and heart. We provide the first evidence that in skeletal muscle expression of ENO3 is different between Yorkshire and Meishan pig breeds. Furthermore, real-time polymerase chain reaction revealed that, in Yorkshire pigs, skeletal muscle expression of transcript 1 is identical at postnatal day-1 and at other stages while that of transcript 2 is higher. Moreover, expression of transcript 1 is lower in skeletal muscle and all other tissue samples than that of transcript 2, with the exception of liver and kidney. Statistical analysis showed the existence of a polymorphism in the ENO3 gene between Chinese indigenous and introduced commercial western pig breeds and that it is associated with fat percentage, average backfat thickness, meat marbling and intramuscular fat in two different populations.

  15. Comparative analysis of metagenomes of Italian top soil improvers

    International Nuclear Information System (INIS)

    Gigliucci, Federica; Brambilla, Gianfranco; Tozzoli, Rosangela; Michelacci, Valeria; Morabito, Stefano

    2017-01-01

    Biosolids originating from Municipal Waste Water Treatment Plants are proposed as top soil improvers (TSI) for their beneficial input of organic carbon on agriculture lands. Their use to amend soil is controversial, as it may lead to the presence of emerging hazards of anthropogenic or animal origin in the environment devoted to food production. In this study, we used a shotgun metagenomics sequencing as a tool to perform a characterization of the hazards related with the TSIs. The samples showed the presence of many virulence genes associated to different diarrheagenic E. coli pathotypes as well as of different antimicrobial resistance-associated genes. The genes conferring resistance to Fluoroquinolones was the most relevant class of antimicrobial resistance genes observed in all the samples tested. To a lesser extent traits associated with the resistance to Methicillin in Staphylococci and genes conferring resistance to Streptothricin, Fosfomycin and Vancomycin were also identified. The most represented metal resistance genes were cobalt-zinc-cadmium related, accounting for 15–50% of the sequence reads in the different metagenomes out of the total number of those mapping on the class of resistance to compounds determinants. Moreover the taxonomic analysis performed by comparing compost-based samples and biosolids derived from municipal sewage-sludges treatments divided the samples into separate populations, based on the microbiota composition. The results confirm that the metagenomics is efficient to detect genomic traits associated with pathogens and antimicrobial resistance in complex matrices and this approach can be efficiently used for the traceability of TSI samples using the microorganisms’ profiles as indicators of their origin. - Highlights: • Sludge- and green- based biosolids analysed by metagenomics. • Biosolids may introduce microbial hazards in the food chain. • Metagenomics enables tracking biosolids’ sources.

  16. Ethical analysis to improve decision-making on health technologies.

    Science.gov (United States)

    Saarni, Samuli I; Hofmann, Bjørn; Lampe, Kristian; Lühmann, Dagmar; Mäkelä, Marjukka; Velasco-Garrido, Marcial; Autti-Rämö, Ilona

    2008-08-01

    Health technology assessment (HTA) is the multidisciplinary study of the implications of the development, diffusion and use of health technologies. It supports health-policy decisions by providing a joint knowledge base for decision-makers. To increase its policy relevance, HTA tries to extend beyond effectiveness and costs to also considering the social, organizational and ethical implications of technologies. However, a commonly accepted method for analysing the ethical aspects of health technologies is lacking. This paper describes a model for ethical analysis of health technology that is easy and flexible to use in different organizational settings and cultures. The model is part of the EUnetHTA project, which focuses on the transferability of HTAs between countries. The EUnetHTA ethics model is based on the insight that the whole HTA process is value laden. It is not sufficient to only analyse the ethical consequences of a technology, but also the ethical issues of the whole HTA process must be considered. Selection of assessment topics, methods and outcomes is essentially a value-laden decision. Health technologies may challenge moral or cultural values and beliefs, and their implementation may also have significant impact on people other than the patient. These are essential considerations for health policy. The ethics model is structured around key ethical questions rather than philosophical theories, to be applicable to different cultures and usable by non-philosophers. Integrating ethical considerations into HTA can improve the relevance of technology assessments for health care and health policy in both developed and developing countries.

  17. Comparative analysis of metagenomes of Italian top soil improvers.

    Science.gov (United States)

    Gigliucci, Federica; Brambilla, Gianfranco; Tozzoli, Rosangela; Michelacci, Valeria; Morabito, Stefano

    2017-05-01

    Biosolids originating from Municipal Waste Water Treatment Plants are proposed as top soil improvers (TSI) for their beneficial input of organic carbon on agriculture lands. Their use to amend soil is controversial, as it may lead to the presence of emerging hazards of anthropogenic or animal origin in the environment devoted to food production. In this study, we used a shotgun metagenomics sequencing as a tool to perform a characterization of the hazards related with the TSIs. The samples showed the presence of many virulence genes associated to different diarrheagenic E. coli pathotypes as well as of different antimicrobial resistance-associated genes. The genes conferring resistance to Fluoroquinolones was the most relevant class of antimicrobial resistance genes observed in all the samples tested. To a lesser extent traits associated with the resistance to Methicillin in Staphylococci and genes conferring resistance to Streptothricin, Fosfomycin and Vancomycin were also identified. The most represented metal resistance genes were cobalt-zinc-cadmium related, accounting for 15-50% of the sequence reads in the different metagenomes out of the total number of those mapping on the class of resistance to compounds determinants. Moreover the taxonomic analysis performed by comparing compost-based samples and biosolids derived from municipal sewage-sludges treatments divided the samples into separate populations, based on the microbiota composition. The results confirm that the metagenomics is efficient to detect genomic traits associated with pathogens and antimicrobial resistance in complex matrices and this approach can be efficiently used for the traceability of TSI samples using the microorganisms' profiles as indicators of their origin. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Cloning of a nitrate reductase inactivator (NRI) cDNA from Spinacia oleracea L. and expression of mRNA and protein of NRI in cultured spinach cells.

    Science.gov (United States)

    Sonoda, Masatoshi; Ide, Hiroaki; Nakayama, Shinya; Sasaki, Asako; Kitazaki, Shinei; Sato, Takahide; Nakagawa, Hiroki

    2003-04-01

    The spinach ( Spinacia oleracea L. (cv. Hoyo) nitrate reductase inactivator (NRI) is a novel protein that irreversibly inactivates NR. Using degenerate primers based on an N-terminal amino acid sequence of NRI purified from spinach leaves and a cDNA library, we isolated a full-length NRI cDNA from spinach that contains an open reading frame encoding 479 amino acid residues. This protein shares 67.4% and 51.1-68.3% amino acid sequence similarities with a nucleotide pyrophosphatase (EC 3.6.1.9) from rice and three types of the nucleotide pyrophosphatase-like protein from Arabidopsis thaliana, respectively. Immunoblot analysis revealed that NRI was constitutively expressed in suspension-cultured spinach cells; however, its expression level is quite low in 1-day-subcultured cells. Moreover, northern blot analysis indicated that this expression was regulated at the mRNA level. These results suggest that NRI functions in mature cells.

  19. Cloning of a cDNA that encodes farnesyl diphosphate synthase and the blue-light-induced expression of the corresponding gene in the leaves of rice plants.

    Science.gov (United States)

    Sanmiya, K; Iwasaki, T; Matsuoka, M; Miyao, M; Yamamoto, N

    1997-02-28

    A cDNA encoding farnesyl diphosphate synthase (FPPS), a key enzyme in isoprenoid biosynthesis, was isolated from a cDNA library constructed from mRNA that had been prepared from etiolated rice (Oriza sativa L. variety Nipponbare) seedlings after three hours of illumination by a subtraction method. The putative polypeptide deduced from the 1289 bp nucleotide sequence consisted of 353 amino acids and had a molecular mass of 40 676 Da. The predicted amino acid sequence exhibited high homology to those of FPPS from Arabidopsis (73% to type 1, 72% to type 2) and white lupin (74%). Southern blot analysis showed that the rice genome might contain only one gene for FPPS. The highest level of expression of the gene was demonstrated in leaves by RNA blot analysis. Moreover, light, in particular blue light, effectively enhanced expression of the gene.

  20. Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.).

    Science.gov (United States)

    Blair, Matthew W; Fernandez, Andrea C; Ishitani, Manabu; Moreta, Danilo; Seki, Motoaki; Ayling, Sarah; Shinozaki, Kazuo

    2011-11-25

    Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean. Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes. The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the library has a large number of

  1. Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.

    Directory of Open Access Journals (Sweden)

    Blair Matthew W

    2011-11-01

    Full Text Available Abstract Background Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean. Results Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes. Conclusions The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole

  2. 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

  3. Durable Expression of Minicircle DNA-Liposome-Delivered Androgen Receptor cDNA in Mice with Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Tian-You Chang

    2014-01-01

    Full Text Available Background. The most common gene-based cancer therapies involve the suppression of oncogenic molecules and enhancement of the expression of tumor-suppressor genes. Studies in noncancer disease animal models have shown that minicircle (MC DNA vectors are easy to deliver and that the proteins from said MC-carrying DNA vectors are expressed over a long period of time. However, delivery of therapeutic genes via a liposome-mediated, MC DNA complex has never been tested in vascular-rich hepatocellular carcinoma (HCC. Liposome-mediated DNA delivery exhibits high in vivo transfection efficiency and minimal systemic immune response, thereby allowing for repetitive interventions. In this study, we evaluated the efficacy of delivering an MC-liposome vector containing a 3.2 kb androgen receptor (AR; HCC metastasis suppressor cDNA into Hepatitis B Virus- (HBV- induced HCC mouse livers. Results. Protein expression and promoter luciferase assays revealed that liposome-encapsulated MC-AR resulted in abundant functional expression of AR protein (100 kD for up to two weeks. The AR cDNA was also successfully delivered into normal livers and diseased livers, where it was persistently expressed. In both normal livers and livers with tumors, the expression of AR was detectable for up to 60 days. Conclusion. Our results show that an MC/liposome delivery system might improve the efficacy of gene therapy in patients with HCC.

  4. Improvements to Integrated Tradespace Analysis of Communications Architectures (ITACA) Network Loading Analysis Tool

    Science.gov (United States)

    Lee, Nathaniel; Welch, Bryan W.

    2018-01-01

    NASA's SCENIC project aims to simplify and reduce the cost of space mission planning by replicating the analysis capabilities of commercially licensed software which are integrated with relevant analysis parameters specific to SCaN assets and SCaN supported user missions. SCENIC differs from current tools that perform similar analyses in that it 1) does not require any licensing fees, 2) will provide an all-in-one package for various analysis capabilities that normally requires add-ons or multiple tools to complete. As part of SCENIC's capabilities, the ITACA network loading analysis tool will be responsible for assessing the loading on a given network architecture and generating a network service schedule. ITACA will allow users to evaluate the quality of service of a given network architecture and determine whether or not the architecture will satisfy the mission's requirements. ITACA is currently under development, and the following improvements were made during the fall of 2017: optimization of runtime, augmentation of network asset pre-service configuration time, augmentation of Brent's method of root finding, augmentation of network asset FOV restrictions, augmentation of mission lifetimes, and the integration of a SCaN link budget calculation tool. The improvements resulted in (a) 25% reduction in runtime, (b) more accurate contact window predictions when compared to STK(Registered Trademark) contact window predictions, and (c) increased fidelity through the use of specific SCaN asset parameters.

  5. [Construction of the female subtractive cDNA library and screening of the specific expressing genes].

    Science.gov (United States)

    Wang, Yan-hai; Peng, Hong-juan; Chen, Xiao-guang; Shen, Shu-man

    2006-02-28

    To screen the Schistosoma japonicum female specific expressing genes. S. japonicum adult worms were collected from the rabbits' vein after six-week infection by affusing method. The adult worms were stabilized by RNA-later liquid, the male and female worms were carefully separated with nipper. The high quality total RNA was extracted and mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Female subtractive (female as tester, male as driver) and male subtractive (male as tester, female as driver) cDNA libraries were constructed. The differentially expressed genes were further screened by dot-blot hybridization. The clones were selected and sequenced, which showed apparently higher signals when hybridizing with the female subtracting male probes, than those signals when hybridizing with the male subtracting female probes. The homology of these sequences was searched with BLAST program. The semi-quantitative PCR was applied to test the differential gene expression in female and male adult worms. Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. After dot-blot hybridization, 50 clones were tested to be the potential female differentially expressed genes and were sequenced. 42 expressing sequence tags (ESTs) were received. After bioinformatics analysis, 17 fragments (about 40.5%) showed high identity with the S. japonicum egg-shell protein genes, 17 sequences (about 40.5%) were highly homologous to unknown S. japonicum genes and partly homologous to female specific 800 protein. 8 fragments (about 19.0%) showed high identity with other S. japonicum unknown genes. The fragments in clones of 577, 579, 668, 695, 720, and 708 were tested by RT-PCR to be the differentially expressed genes in female adult worms using S. japonicum actin gene as the internal standard. These fragments were highly homologous to S. japonicum egg shell protein gene AY222885, AY222895, AB

  6. A multivariate analysis of factors affecting adoption of improved ...

    African Journals Online (AJOL)

    This paper analyzes the synergies/tradeoffs involved in the adoption of improved varieties of multiple crops in the mixed crop-livestock production systems of the highlands of Ethiopia A multivariate probit (MVP) model involving a system of four equations for the adoption decision of improved varieties of barley, potatoes, ...

  7. A Multivariate Analysis of Factors Affecting Adoption of Improved ...

    African Journals Online (AJOL)

    and significant effects on the likelihood of adopting improved varieties of barley and wheat and vice versa - indicating synergistic effects among the adoption decisions of the two groups of crops. On the other hand, the area share of improved varieties of wheat negatively and significantly effects the chances of using ...

  8. A novel joint analysis framework improves identification of differentially expressed genes in cross disease transcriptomic analysis

    Directory of Open Access Journals (Sweden)

    Wenyi Qin

    2018-02-01

    Full Text Available Abstract Motivation Detecting differentially expressed (DE genes between disease and normal control group is one of the most common analyses in genome-wide transcriptomic data. Since most studies don’t have a lot of samples, researchers have used meta-analysis to group different datasets for the same disease. Even then, in many cases the statistical power is still not enough. Taking into account the fact that many diseases share the same disease genes, it is desirable to design a statistical framework that can identify diseases’ common and specific DE genes simultaneously to improve the identification power. Results We developed a novel empirical Bayes based mixture model to identify DE genes in specific study by leveraging the shared information across multiple different disease expression data sets. The effectiveness of joint analysis was demonstrated through comprehensive simulation studies and two real data applications. The simulation results showed that our method consistently outperformed single data set analysis and two other meta-analysis methods in identification power. In real data analysis, overall our method demonstrated better identification power in detecting DE genes and prioritized more disease related genes and disease related pathways than single data set analysis. Over 150% more disease related genes are identified by our method in application to Huntington’s disease. We expect that our method would provide researchers a new way of utilizing available data sets from different diseases when sample size of the focused disease is limited.

  9. Analysis and improvement of security of energy smart grids

    International Nuclear Information System (INIS)

    Halimi, Halim

    2014-01-01

    The Smart grid is the next generation power grid, which is a new self-healing, self-activating form of electricity network, and integrates power-flow control, increased quality of electricity, and energy reliability, energy efficiency and energy security using information and communication technologies. Communication networks play a critical role in smart grid, as the intelligence of smart grid is built based on information exchange across the power grid. Its two-way communication and electricity flow enable to monitor, predict and manage the energy usage. To upgrade an existing power grid into a smart grid, it requires an intelligent and secure communication infrastructure. Because of that, the main goal of this dissertation is to propose new architecture and implementation of algorithms for analysis and improvement of the security and reliability in smart grid. In power transmission segments of smart grid, wired communications are usually adopted to ensure robustness of the backbone power network. In contrast, for a power distribution grid, wireless communications provide many benefits such as low cost high speed links, easy setup of connections among different devices/appliances, and so on. Wireless communications are usually more vulnerable to security attacks than wired ones. Developing appropriate wireless communication architecture and its security measures is extremely important for a smart grid system. This research addresses physical layer security in a Wireless Smart Grid. Hence a defense Quorum- based algorithm is proposed to ensure physical security in wireless communication. The new security architecture for smart grid that supports privacy-preserving, data aggregation and access control is defined. This architecture consists of two parts. In the first part we propose to use an efficient and privacy-preserving aggregation scheme (EPPA), which aggregates real-time data of consumers by Local Gateway. During aggregation the privacy of consumers is

  10. [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.

  11. A comparison of parametric and nonparametric methods for normalising cDNA microarray data.

    Science.gov (United States)

    Khondoker, Mizanur R; Glasbey, Chris A; Worton, Bruce J

    2007-12-01

    Normalisation is an essential first step in the analysis of most cDNA microarray data, to correct for effects arising from imperfections in the technology. Loess smoothing is commonly used to correct for trends in log-ratio data. However, parametric models, such as the additive plus multiplicative variance model, have been preferred for scale normalisation, though the variance structure of microarray data may be of a more complex nature than can be accommodated by a parametric model. We propose a new nonparametric approach that incorporates location and scale normalisation simultaneously using a Generalised Additive Model for Location, Scale and Shape (GAMLSS, Rigby and Stasinopoulos, 2005, Applied Statistics, 54, 507-554). We compare its performance in inferring differential expression with Huber et al.'s (2002, Bioinformatics, 18, 96-104) arsinh variance stabilising transformation (AVST) using real and simulated data. We show GAMLSS to be as powerful as AVST when the parametric model is correct, and more powerful when the model is wrong. (c) 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  12. cDNA cloning and mRNA expression of cat and dog Cdkal1

    Directory of Open Access Journals (Sweden)

    Sako T

    2012-08-01

    Full Text Available Ichiro Yamamoto, Shingo Ishikawa, Li Gebin, Hiroshi Takemitsu, Megumi Fujiwara, Nobuko Mori, Yutaka Hatano, Tomoko Suzuki, Akihiro Mori, Nobuhiro Nakao, Koh Kawasumi, Toshinori Sako, Toshiro AraiLaboratory of Veterinary Biochemistry, Nippon Veterinary and Life Science University, Tokyo, JapanAbstract: The cyclin-dependent kinase 5 regulatory subunit–associated protein 1–like 1 (CDKAL1 gene encodes methylthiotransferase, and the gene contains risk variants for type 2 diabetes in humans. In this study, we performed complementary DNA cloning for Cdkal1 in the cat and dog and characterized the tissue expression profiles of its messenger RNA. Cat and dog Cdkal1 complementary DNA encoded 576 and 578 amino acids, showing very high sequence homology to mammalian CDKAL1 (>88.4%. Real-time polymerase chain reaction analyses revealed that Cdkal1 messenger RNA is highly expressed in smooth muscle and that tissue distribution of Cdkal1 is similar in cats and dogs. Genotyping analysis of single-nucleotide polymorphism for cat Cdkal1 revealed that obese cats had different tendencies from normal cats. These findings suggest that the cat and dog Cdkal1 gene is highly conserved among mammals and that cat Cdkal1 may be a candidate marker for genetic diagnosis of obesity.Keywords: cat, dog, Cdkal1, obese, cDNA cloning, Q-PCR

  13. Identification of some unknown transcripts from SSH cDNA library of buffalo follicular oocytes.

    Science.gov (United States)

    Rajput, S K; Kumar, P; Roy, B; Verma, A; Pandey, H P; Singh, D; De, S; Datta, T K

    2013-03-01

    A buffalo oocyte-specific subtracted cDNA library was constructed to identify exclusively or preferentially oocyte-expressed genes. The library represented an enriched population of transcripts obtained from oocytes of diverse ovarian follicular origin and at different stages of in vitro maturation. A total of 1173 high-quality sequences of oocyte-specific genes were clustered into 645 unique sequences, out of which 65.76% were represented as singlets and 34.26% as contig expressed sequence tags (ESTs; clusters). Analysis of sequences revealed that 498 of these sequences were identified as a known sequence in mammalian species including buffalo, 103 as uncharacterized ESTs and 44 unknown sequences including 1 novel EST, so far not reported in any species. Gene ontology annotation classified these sequences into functional categories of cellular events and biological processes associated with oocyte competence. Expression status of the isolated unknown ESTs confirmed that many of these are expressed in oocytes exclusively and in others preferentially, some in excess of 80-fold greater in comparison with a variety of somatic tissues. The isolated novel EST was detected to be expressed exclusively in oocytes and testicular cells only. To our knowledge, this is the first report giving a detailed transcriptome account of oocyte-expressed genes in buffalo. This study will provide important information on the physiological control of oocyte development, as well as many questions yet to be addressed on the reproductive process of buffalo.

  14. Nucleotide sequence of Phaseolus vulgaris L. alcohol dehydrogenase encoding cDNA and three-dimensional structure prediction of the deduced protein.

    Science.gov (United States)

    Amelia, Kassim; Khor, Chin Yin; Shah, Farida Habib; Bhore, Subhash J

    2015-01-01

    Common beans (Phaseolus vulgaris L.) are widely consumed as a source of proteins and natural products. However, its yield needs to be increased. In line with the agenda of Phaseomics (an international consortium), work of expressed sequence tags (ESTs) generation from bean pods was initiated. Altogether, 5972 ESTs have been isolated. Alcohol dehydrogenase (AD) encoding gene cDNA was a noticeable transcript among the generated ESTs. This AD is an important enzyme; therefore, to understand more about it this study was undertaken. The objective of this study was to elucidate P. vulgaris L. AD (PvAD) gene cDNA sequence and to predict the three-dimensional (3D) structure of deduced protein. positive and negative strands of the PvAD cDNA clone were sequenced using M13 forward and M13 reverse primers to elucidate the nucleotide sequence. Deduced PvAD cDNA and protein sequence was analyzed for their basic features using online bioinformatics tools. Sequence comparison was carried out using bl2seq program, and tree-view program was used to construct a phylogenetic tree. The secondary structures and 3D structure of PvAD protein were predicted by using the PHYRE automatic fold recognition server. The sequencing results analysis showed that PvAD cDNA is 1294 bp in length. It's open reading frame encodes for a protein that contains 371 amino acids. Deduced protein sequence analysis showed the presence of putative substrate binding, catalytic Zn binding, and NAD binding sites. Results indicate that the predicted 3D structure of PvAD protein is analogous to the experimentally determined crystal structure of s-nitrosoglutathione reductase from an Arabidopsis species. The 1294 bp long PvAD cDNA encodes for 371 amino acid long protein that contains conserved domains required for biological functions of AD. The predicted deduced PvAD protein's 3D structure reflects the analogy with the crystal structure of Arabidopsis thaliana s-nitrosoglutathione reductase. Further study is required

  15. Molecular cloning of the cDNA encoding aspartate aminotransferase from bean root nodules and determination of its role in nodule nitrogen metabolism.

    Science.gov (United States)

    Silvente, Sonia; Camas, Alberto; Lara, Miguel

    2003-06-01

    A cDNA clone encoding aspartate aminotransferase (PVAAT-2) (EC 2.6.1.1) was isolated from the common bean Phaseolus vulgaris nodule cDNA library. The nucleotide sequence analysis of the full-length cDNA allowed its identification by comparison with sequence databases. The amino acid sequence of the bean PvAAT-2 showed high similarity with the AAT-2 isoforms described in other leguminous plants. The amino-terminal region of the PvAAT-2 contains a sequence, which shares common features of plastid transit peptides. Southern blot analysis showed that the PvAAT-2 clone is encoded by a single gene in the P. vulgaris genome. Analysis of the PvAAT-2 mRNA levels suggests that the expression of this gene is nodule enhanced. The PvAAT-2 transcript is more abundant in nodules with increased synthesis of amides and is down-regulated in conditions where ureides accumulate. When plants were supplemented with ureides or with amides, PvAAT-2 expression was reduced, while it was not affected when plants were treated with allopurinol, an inhibitor of ureide synthesis. On the other hand, the expression of asparagine synthetase (another enzyme involved in the synthesis of amides) is not affected either by ureides or amides. These data suggest a role for AAT-2 in the mechanism involved in the synthesis of nitrogen compounds in bean nodules.

  16. Improved Extreme Learning Machine based on the Sensitivity Analysis

    Science.gov (United States)

    Cui, Licheng; Zhai, Huawei; Wang, Benchao; Qu, Zengtang

    2018-03-01

    Extreme learning machine and its improved ones is weak in some points, such as computing complex, learning error and so on. After deeply analyzing, referencing the importance of hidden nodes in SVM, an novel analyzing method of the sensitivity is proposed which meets people’s cognitive habits. Based on these, an improved ELM is proposed, it could remove hidden nodes before meeting the learning error, and it can efficiently manage the number of hidden nodes, so as to improve the its performance. After comparing tests, it is better in learning time, accuracy and so on.

  17. Does ownership of improved dairy cow breeds improve child nutrition? A pathway analysis for Uganda.

    Science.gov (United States)

    Kabunga, Nassul S; Ghosh, Shibani; Webb, Patrick

    2017-01-01

    The promotion of livestock production is widely believed to support enhanced diet quality and child nutrition, but the empirical evidence for this causal linkage remains narrow and ambiguous. This study examines whether adoption of improved dairy cow breeds is linked to farm-level outcomes that translate into household-level benefits including improved child nutrition outcomes in Uganda. Using nationwide data from Uganda's National Panel Survey, propensity score matching is used to create an unbiased counterfactual, based on observed characteristics, to assess the net impacts of improved dairy cow adoption. All estimates were tested for robustness and sensitivity to variations in observable and unobservable confounders. Results based on the matched samples showed that households adopting improved dairy cows significantly increased milk yield-by over 200% on average. This resulted in higher milk sales and milk intakes, demonstrating the potential of this agricultural technology to both integrate households into modern value chains and increase households' access to animal source foods. Use of improved dairy cows increased household food expenditures by about 16%. Although undernutrition was widely prevalent in the study sample and in matched households, the adoption of improved dairy cows was associated with lower child stunting in adopter household. In scale terms, results also showed that holding larger farms tends to support adoption, but that this also stimulates the household's ability to achieve gains from adoption, which can translate into enhanced nutrition.

  18. Does ownership of improved dairy cow breeds improve child nutrition? A pathway analysis for Uganda.

    Directory of Open Access Journals (Sweden)

    Nassul S Kabunga

    Full Text Available The promotion of livestock production is widely believed to support enhanced diet quality and child nutrition, but the empirical evidence for this causal linkage remains narrow and ambiguous. This study examines whether adoption of improved dairy cow breeds is linked to farm-level outcomes that translate into household-level benefits including improved child nutrition outcomes in Uganda. Using nationwide data from Uganda's National Panel Survey, propensity score matching is used to create an unbiased counterfactual, based on observed characteristics, to assess the net impacts of improved dairy cow adoption. All estimates were tested for robustness and sensitivity to variations in observable and unobservable confounders. Results based on the matched samples showed that households adopting improved dairy cows significantly increased milk yield-by over 200% on average. This resulted in higher milk sales and milk intakes, demonstrating the potential of this agricultural technology to both integrate households into modern value chains and increase households' access to animal source foods. Use of improved dairy cows increased household food expenditures by about 16%. Although undernutrition was widely prevalent in the study sample and in matched households, the adoption of improved dairy cows was associated with lower child stunting in adopter household. In scale terms, results also showed that holding larger farms tends to support adoption, but that this also stimulates the household's ability to achieve gains from adoption, which can translate into enhanced nutrition.

  19. Full-length cDNA cloning of Toll-like receptor 4 in dogs and cats.

    Science.gov (United States)

    Asahina, Yuka; Yoshioka, Noriyuki; Kano, Rui; Moritomo, Tadaaki; Hasegawa, Atsuhiko

    2003-12-15

    In the present study, full length of canine and feline Toll-like receptor 4 (TLR4) cDNAs were sequenced, and the expression of canine and feline TLR4 mRNAs in dog and cat tissues were investigated. The full-length cDNA of TLR4 of dog and cat was 2709 bp encoding 637 amino acids and 3113 bp encoding 833 amino acids, respectively. The similarity of canine and feline TLR4 were 83.6% at the nucleotide sequence level and 77.6% at the amino acid sequence level. At the amino acid sequence level, canine and feline TLR4 showed sequence similarities of approximately 62-78% with those of Homo sapiens, Mus musculus, Bos taurus and Equus caballus, respectively. Southern hybridization analyses with TLR4 cDNA probes gave one distinct band in BamHI, EcoRI and HindIII digests of genomic DNA from dogs and cats, respectively, indicating the likely presence of a single TLR4 gene in each species. By RT-PCR analysis, mRNA of canine TLR4 was expressed highly in peripheral blood leukocytes (PBL), moderately in spleen, stomach and small intestine, at low levels in liver, with no expression in kidney, large intestine and skin. On the other hand, mRNA of feline TLR4 was expressed highly in lung, bladder and PBL, moderately in kidney, liver, spleen and large intestine and at low levels in pancreas and small intestine.

  20. A method for diagnosis of plant environmental stresses by gene expression profiling using a cDNA macroarray

    International Nuclear Information System (INIS)

    Tamaoki, Masanori; Matsuyama, Takashi; Nakajima, Nobuyoshi; Aono, Mitsuko; Kubo, Akihiro; Saji, Hikaru

    2004-01-01

    Plants in the field are subjected to numerous environmental stresses. Lengthy continuation of such environmental stresses or a rapid increase in their intensity is harmful to vegetation. Assessments of the phytotoxicity of various stresses have been performed in many countries, although they have largely been based on estimates of leaf injury. We developed a novel method of detecting plant stresses that is more sensitive and specific than those previously available. This method is based on the detection of mRNA expression changes in 205 ozone-responsive Arabidopsis expressed sequence tags (ESTs) by cDNA macroarray analysis. By using this method, we illustrated shifts in gene expression in response to stressors such as drought, salinity, UV-B, low temperature, high temperature, and acid rain, as distinct from those in response to ozone. We also made a mini-scale macroarray with 12 ESTs for diagnosis of the above environmental stresses in plants. These results illustrate the potential of our cDNA macroarray for diagnosis of various stresses in plants

  1. BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA from disk abalone Haliotis discus discus.

    Science.gov (United States)

    Kim, Yucheol; De Zoysa, Mahanama; Lee, Youngdeuk; Whang, Ilson; Lee, Jehee

    2010-11-01

    A BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA was cloned from the disk abalone (Haliotis discus discus) and designated as AbLECT-1. A full-length (705 bp) of AbLECT-1 cDNA was composed of a 576 bp open reading frame that translates into a putative peptide of 192 amino acids. Deduced amino acid sequence of AbLECT-1 had 15.5- and 27.8% identity and similarity to human LECT-1, respectively. Quantitative real-time PCR analysis results showed that the mRNA of AbLECT-1 was constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas in a tissue-specific manner. Moreover, the AbLECT-1 transcription level was induced in hemocytes after challenge with Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes suggesting that it may be involved in immune response reactions in abalone. Copyright 2010 Elsevier Ltd. All rights reserved.

  2. A plant class V chitinase from a cycad (Cycas revoluta): biochemical characterization, cDNA isolation, and posttranslational modification.

    Science.gov (United States)

    Taira, Toki; Hayashi, Hiroko; Tajiri, Yoshiko; Onaga, Shoko; Uechi, Gen-ichiro; Iwasaki, Hironori; Ohnuma, Takayuki; Fukamizo, Tamo

    2009-12-01

    Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)(3) from the substrate (GlcNAc)(6) through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.

  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. cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata.

    Science.gov (United States)

    Yang, Yanzhen; Cun, Shujian; Peng, Lisheng; Xie, Xiaojin; Wei, Jianwen; Yang, Wenli; Xu, Anlong

    2003-10-01

    Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser(97)-Trp(98), which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately M(r) = 12,800 displayed an obvious inhibition of papain (K(i) value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at >50 degrees C.

  5. Alternative Frameworks for Improving Government Organizational Performance: A Comparative Analysis

    National Research Council Canada - National Science Library

    Simon, Cary

    1997-01-01

    .... Six major frameworks emerging in the U.S. since 1980, applicable to the public sector, and designed to enhance organizational change toward improved performance are reviewed and analyzed: Total Quality; 'Excellence...

  6. Using external data sources to improve audit trail analysis.

    OpenAIRE

    Herting, R. L.; Asaro, P. V.; Roth, A. C.; Barnes, M. R.

    1999-01-01

    Audit trail analysis is the primary means of detection of inappropriate use of the medical record. While audit logs contain large amounts of information, the information required to determine useful user-patient relationships is often not present. Adequate information isn't present because most audit trail analysis systems rely on the limited information available within the medical record system. We report a feature of the STAR (System for Text Archive and Retrieval) audit analysis system wh...

  7. Improved Methods for Pitch Synchronous Linear Prediction Analysis of Speech

    OpenAIRE

    劉, 麗清

    2015-01-01

    Linear prediction (LP) analysis has been applied to speech system over the last few decades. LP technique is well-suited for speech analysis due to its ability to model speech production process approximately. Hence LP analysis has been widely used for speech enhancement, low-bit-rate speech coding in cellular telephony, speech recognition, characteristic parameter extraction (vocal tract resonances frequencies, fundamental frequency called pitch) and so on. However, the performance of the co...

  8. Improving the flash flood frequency analysis applying dendrogeomorphological evidences

    Science.gov (United States)

    Ruiz-Villanueva, V.; Ballesteros, J. A.; Bodoque, J. M.; Stoffel, M.; Bollschweiler, M.; Díez-Herrero, A.

    2009-09-01

    Flash floods are one of the natural hazards that cause major damages worldwide. Especially in Mediterranean areas they provoke high economic losses every year. In mountain areas with high stream gradients, floods events are characterized by extremely high flow and debris transport rates. Flash flood analysis in mountain areas presents specific scientific challenges. On one hand, there is a lack of information on precipitation and discharge due to a lack of spatially well distributed gauge stations with long records. On the other hand, gauge stations may not record correctly during extreme events when they are damaged or the discharge exceeds the recordable level. In this case, no systematic data allows improvement of the understanding of the spatial and temporal occurrence of the process. Since historic documentation is normally scarce or even completely missing in mountain areas, tree-ring analysis can provide an alternative approach. Flash floods may influence trees in different ways: (1) tilting of the stem through the unilateral pressure of the flowing mass or individual boulders; (2) root exposure through erosion of the banks; (3) injuries and scars caused by boulders and wood transported in the flow; (4) decapitation of the stem and resulting candelabra growth through the severe impact of boulders; (5) stem burial through deposition of material. The trees react to these disturbances with specific growth changes such as abrupt change of the yearly increment and anatomical changes like reaction wood or callus tissue. In this study, we sampled 90 cross sections and 265 increment cores of trees heavily affected by past flash floods in order to date past events and to reconstruct recurrence intervals in two torrent channels located in the Spanish Central System. The first study site is located along the Pelayo River, a torrent in natural conditions. Based on the external disturbances of trees and their geomorphological position, 114 Pinus pinaster (Ait

  9. 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.

  10. 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.

  11. [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.

  12. [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.

  13. 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.

  14. [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.

  15. 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.

  16. Why economic analysis of health system improvement interventions matters

    Directory of Open Access Journals (Sweden)

    Edward Ivor Broughton

    2016-10-01

    Full Text Available There is little evidence to direct health systems toward providing efficient interventions to address medical errors, defined as an unintended act of omission or commission or one not executed as intended that may or may not cause harm to the patient but does not achieve its intended outcome. We believe that lack of guidance on what is the most efficient way to reduce adverse events and improve the quality of health care limits the scale-up of health system improvement interventions. Challenges to economic evaluation of these interventions include defining and implementing improvement interventions in different settings with high fidelity, capturing all of the positive and negative effects of the intervention, using process measures of effectiveness rather than health outcomes, and determining the full cost of the intervention and all economic consequences its effects. However, health system improvement interventions should be treated similarly to individual medical interventions and undergo rigorous economic evaluation to provide actionable evidence to guide policy-makers in decisions of resources allocation for improvement activities among other competing demands for health care resources.

  17. Nucleotide sequence of a cDNA coding for the barley seed protein CMa: an inhibitor of insect α-amylase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Johansson, A.

    1992-01-01

    The primary structure of the insect alpha-amylase inhibitor CMa of barley seeds was deduced from a full-length cDNA clone pc43F6. Analysis of RNA from barley endosperm shows high levels 15 and 20 days after flowering. The cDNA predicts an amino acid sequence of 119 residues preceded by a signal...... peptide of 25 amino acids. Ala and Leu account for 55% of the signal peptide. CMa is 60-85% identical with alpha-amylase inhibitors of wheat, but shows less than 50% identity to trypsin inhibitors of barley and wheat. The 10 Cys residues are located in identical positions compared to the cereal inhibitor...

  18. Microarrays in ecological research: A case study of a cDNA microarray for plant-herbivore interactions

    Directory of Open Access Journals (Sweden)

    Gase Klaus

    2004-09-01

    Full Text Available Abstract Background Microarray technology allows researchers to simultaneously monitor changes in the expression ratios (ERs of hundreds of genes and has thereby revolutionized most of biology. Although this technique has the potential of elucidating early stages in an organism's phenotypic response to complex ecological interactions, to date, it has not been fully incorporated into ecological research. This is partially due to a lack of simple procedures of handling and analyzing the expression ratio (ER data produced from microarrays. Results We describe an analysis of the sources of variation in ERs from 73 hybridized cDNA microarrays, each with 234 herbivory-elicited genes from the model ecological expression system, Nicotiana attenuata, using procedures that are commonly used in ecologic research. Each gene is represented by two independently labeled PCR products and each product was arrayed in quadruplicate. We present a robust method of normalizing and analyzing ERs based on arbitrary thresholds and statistical criteria, and characterize a "norm of reaction" of ERs for 6 genes (4 of known function, 2 of unknown with different ERs as determined across all analyzed arrays to provide a biologically-informed alternative to the use of arbitrary expression ratios in determining significance of expression. These gene-specific ERs and their variance (gene CV were used to calculate array-based variances (array CV, which, in turn, were used to study the effects of array age, probe cDNA quantity and quality, and quality of spotted PCR products as estimates of technical variation. Cluster analysis and a Principal Component Analysis (PCA were used to reveal associations among the transcriptional "imprints" of arrays hybridized with cDNA probes derived from mRNA from N. attenuata plants variously elicited and attacked by different herbivore species and from three congeners: N. quadrivalis, N. longiflora and N. clevelandii. Additionally, the PCA

  19. Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression.

    Science.gov (United States)

    Phiriyangkul, Pharima; Utarabhand, Prapaporn

    2006-04-01

    In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp. Copyright 2006 Wiley-Liss, Inc.

  20. Molecular cloning of a cDNA and chromosomal localization of a human theta-class glutathione S-transferase gene (GSTT2) to chromosome 22

    Energy Technology Data Exchange (ETDEWEB)

    Tan, K.L.; Baker, R.T.; Board, P.G. [Australian National Univ., Canberra (Australia)] [and others

    1995-01-20

    Until recently the Theta-class glutathione S-transferases (GSTs) were largely overlooked due to their low activity with the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) and their failure to bind to immobilized glutathione affinity matrices. Little is known about the number of genes in this class. Recently, Pemble et al. reported the cDNA cloning of a human Theta-class GST, termed GSTT1. In this study, we describe the molecular cloning of a cDNA encoding a second human Theta-class GST (GSTT2) from a {lambda}gt11 human liver 5{prime}-stretch cDNA library. The encoded protein contains 244 amino acids and has 78.3% sequence identity with the rat subunit 12 and only 55.0% identity with human GSTT1. GSTT2 has been mapped to chromosome 22 by somatic cell hybrid analysis. The precise position of the gene was localized to subband 22q11.2 by in situ hybridization. The absence of other regions of hybridization suggests that there are no closely related sequences (e.g., reverse transcribed pseudogenes) scattered throughout the genome and that if there are closely related genes, they must be clustered near GSTT2. Southern blot analysis of human DNA digested with BamHI shows that the size of the GSTT2 gene is relatively small, as the coding sequence falls within a 3.6-kb BamHI fragment. 35 refs., 6 figs.

  1. Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

    Science.gov (United States)

    Lardizabal, K D; Metz, J G; Sakamoto, T; Hutton, W C; Pollard, M R; Lassner, M W

    2000-03-01

    Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

  2. Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy

    International Nuclear Information System (INIS)

    Mangin, M.; Webb, A.C.; Dreyer, B.E.

    1988-01-01

    Humoral hypercalcemia of malignancy is a common paraneoplastic syndrome that appears to be mediated in many instances by a parathyroid hormone-like peptide. Poly(A) + RNA from a human renal carcinoma associated with this syndrome was enriched by preparative electrophoresis and used to construct an enriched cDNA library in phage λgt10. The library was screened with a codon-preference oligonucleotide synthesized on the basis of a partial N-terminal amino acid sequence from a human tumor-derived peptide, and a 2.0 kilo-base cDNA was identified. The cDNA encodes a 177 amino acid protein consisting of a 36 amino acid leader sequence and a 141 amino acid mature peptide. The first 13 amino acids of the deduced sequence of the mature peptide display strong homology to human PTH, with complete divergence thereafter. RNA blot-hybridization analysis revealed multiple transcripts in mRNA from tumors associated with the humor syndrome and also in mRNA from normal human keratinocytes. Southern blot analysis of genomic DNA from humans and rodents revealed a simple pattern compatible with a single-copy gene. The gene has been mapped to chromosome 12

  3. Improving prehospital trauma care in Rwanda through continuous quality improvement: an interrupted time series analysis.

    Science.gov (United States)

    Scott, John W; Nyinawankusi, Jeanne D'Arc; Enumah, Samuel; Maine, Rebecca; Uwitonze, Eric; Hu, Yihan; Kabagema, Ignace; Byiringiro, Jean Claude; Riviello, Robert; Jayaraman, Sudha

    2017-07-01

    Injury is a major cause of premature death and disability in East Africa, and high-quality pre-hospital care is essential for optimal trauma outcomes. The Rwandan pre-hospital emergency care service (SAMU) uses an electronic database to evaluate and optimize pre-hospital care through a continuous quality improvement programme (CQIP), beginning March 2014. The SAMU database was used to assess pre-hospital quality metrics including supplementary oxygen for hypoxia (O2), intravenous fluids for hypotension (IVF), cervical collar placement for head injuries (c-collar), and either splinting (splint) or administration of pain medications (pain) for long bone fractures. Targets of >90% were set for each metric and daily team meetings and monthly feedback sessions were implemented to address opportunities for improvement. These five pre-hospital quality metrics were assessed monthly before and after implementation of the CQIP. Met and unmet needs for O2, IVF, and c-collar were combined into a summative monthly SAMU Trauma Quality Scores (STQ score). An interrupted time series linear regression model compared the STQ score during 14 months before the CQIP implementation to the first 14 months after. During the 29-month study period 3,822 patients met study criteria. 1,028 patients needed one or more of the five studied interventions during the study period. All five endpoints had a significant increase between the pre-CQI and post-CQI periods (pimprovement of +6.1% (p=0.017) and sustained monthly improvements in care delivery-improving at a rate of 0.7% per month (p=0.028). The SAMU experience demonstrates the utility of a responsive, data-driven quality improvement programme to yield significant immediate and sustained improvements in pre-hospital care for trauma in Rwanda. This programme may be used as an example for additional efforts engaging frontline staff with real-time data feedback in order to rapidly translate data collection efforts into improved care for the

  4. Method for improving accuracy in full evaporation headspace analysis.

    Science.gov (United States)

    Xie, Wei-Qi; Chai, Xin-Sheng

    2017-05-01

    We report a new headspace analytical method in which multiple headspace extraction is incorporated with the full evaporation technique. The pressure uncertainty caused by the solid content change in the samples has a great impact to the measurement accuracy in the conventional full evaporation headspace analysis. The results (using ethanol solution as the model sample) showed that the present technique is effective to minimize such a problem. The proposed full evaporation multiple headspace extraction analysis technique is also automated and practical, and which could greatly broaden the applications of the full-evaporation-based headspace analysis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. A Note on Improved Homotopy Analysis Method for Solving the Jeffery-Hamel Flow

    OpenAIRE

    Motsa, Sandile Sydney; Sibanda, Precious; Marewo, Gerald T.; Shateyi, Stanford

    2010-01-01

    This paper presents the solution of the nonlinear equation that governs the flow of a viscous, incompressible fluid between two converging-diverging rigid walls using an improved homotopy analysis method. The results obtained by this new technique show that the improved homotopy analysis method converges much faster than both the homotopy analysis method and the optimal homotopy asymptotic method. This improved technique is observed to be much more accurate than these traditional ...

  6. Error analysis to improve the speech recognition accuracy on ...

    Indian Academy of Sciences (India)

    Telugu language is one of the most widely spoken south Indian languages. In the proposed Telugu speech recognition system, errors obtained from decoder are analysed to improve the performance of the speech recognition system. Static pronunciation dictionary plays a key role in the speech recognition accuracy.

  7. An Analysis of Appropriateness of Improved Rice Technology for ...

    African Journals Online (AJOL)

    The study was carried out in selected states of South Western Nigeria in order to investigate the appropriateness of improved rice technology for women farmers. A purposive sampling technique was used to select 320 women farmers from the study area. Data collection was carried out using structured interview schedule.

  8. Improved omit set displacement recoveries in dynamic analysis

    Science.gov (United States)

    Allen, Tom; Cook, Greg; Walls, Bill

    1993-09-01

    Two related methods for improving the dependent (OMIT set) displacements after performing a Guyan reduction are presented. The theoretical bases for the methods are derived. The NASTRAN DMAP ALTERs used to implement the methods in a NASTRAN execution are described. Data are presented that verify the methods and the NASTRAN DMAP ALTERs.

  9. Does Competition Improve Public School Efficiency? A Spatial Analysis

    Science.gov (United States)

    Misra, Kaustav; Grimes, Paul W.; Rogers, Kevin E.

    2012-01-01

    Advocates for educational reform frequently call for policies to increase competition between schools because it is argued that market forces naturally lead to greater efficiencies, including improved student learning, when schools face competition. Researchers examining this issue are confronted with difficulties in defining reasonable measures…

  10. Improving Capabilities for Irregular Warfare. Volume 2. Capabilities Analysis

    Science.gov (United States)

    2007-08-01

    and Iraq were limited to six orbits at any one time. Only six operator stations were available at Nellis AFB in Nevada , where Predators were...improve this system will need to hit the ground running with a well thought-out plan. The cost of “learn as you go” will be high: hunger and patience

  11. A Multivariate Analysis of Factors Affecting Adoption of Improved ...

    African Journals Online (AJOL)

    Internal factors that affect the adoption and use of agricultural technologies include farmers' attitude ... Single probit and logit models are often employed to model discrete choices such as adoption of improved ..... suggests that there are unobservable factors affecting both choices and reveals an association after controlling ...

  12. Rapid economic analysis of northern hardwood stand improvement options

    Science.gov (United States)

    William B. Leak

    1980-01-01

    Data and methodology are provided for projecting basal area, diameter, volumes, and values by product for northern hardwood stands, and for determining the rate of return on stand improvement investments. The method is rapid, requires a minimum amount of information, and should prove useful for on-the-ground economic analyses.

  13. Error analysis to improve the speech recognition accuracy on ...

    Indian Academy of Sciences (India)

    measures, error-rate and Word Error Rate (WER) by application of the proposed method. Keywords. Speech recognition; pronunciation dictionary modification method; error analysis; F-measure. 1. Introduction. Speech is one of the easiest modes of ...

  14. Models, Web-Based Simulations, and Integrated Analysis Techniques for Improved Logistical Performance

    National Research Council Canada - National Science Library

    Hill, Raymond

    2001-01-01

    ... Laboratory, Logistics Research Division, Logistics Readiness Branch to propose a research agenda entitled, "Models, Web-based Simulations, and Integrated Analysis Techniques for Improved Logistical Performance...

  15. Optimizing Bus Passenger Complaint Service through Big Data Analysis: Systematized Analysis for Improved Public Sector Management

    Directory of Open Access Journals (Sweden)

    Weng-Kun Liu

    2016-12-01

    Full Text Available With the advances in industry and commerce, passengers have become more accepting of environmental sustainability issues; thus, more people now choose to travel by bus. Government administration constitutes an important part of bus transportation services as the government gives the right-of-way to transportation companies allowing them to provide services. When these services are of poor quality, passengers may lodge complaints. The increase in consumer awareness and developments in wireless communication technologies have made it possible for passengers to easily and immediately submit complaints about transportation companies to government institutions, which has brought drastic changes to the supply–demand chain comprised of the public sector, transportation companies, and passengers. This study proposed the use of big data analysis technology including systematized case assignment and data visualization to improve management processes in the public sector and optimize customer complaint services. Taichung City, Taiwan, was selected as the research area. There, the customer complaint management process in public sector was improved, effectively solving such issues as station-skipping, allowing the public sector to fully grasp the service level of transportation companies, improving the sustainability of bus operations, and supporting the sustainable development of the public sector–transportation company–passenger supply chain.

  16. [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.

  17. [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.

  18. Analysis and improvement of face detection based on surf cascade

    Science.gov (United States)

    Hu, Siquan; Zhang, Caihong; Liu, Lei

    2017-08-01

    This paper aims to study limitations of the commonly employed boosting cascade framework. We focus on the factors like data, feature, weak classifier and stages. A set of novel experiments were done to show the relationship. The model contains three key points: SURF feature, weak classifier based on logistic regression and AUC-based cascade learning algorithm. This paper adds cross validation in logistic regression creatively which improves accuracy and speeds up convergence greatly. Eventually only five stages and about 100 weak classifiers are needed. The frontal face detector improves reject rate to 99% for the first three stages, decreases number of false positive greatly and achieves comparable performance among non-CNN techniques on FDDB dataset.

  19. 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.

  20. [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.

  1. 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

  2. Mass Spectrometry Vapor Analysis for Improving Explosives Detection Canine Proficiency

    Science.gov (United States)

    2017-02-10

    ionization (SESI), 8,19-21 dielectric barrier discharge ionization (DBDI), 21,22 selected-ion-flow-tube ( SIFT ), 23,24 and proton transfer reaction...sensors in addition to as service teams, will improve training efficiency and opera- tional performance. In support of canine training, the Department of...for both materials, but transfer efficiency into the source may be poor. Instrument response also saturat- ed when analyzing cyclohexanone. These

  3. Improving the usefulness of accounting data in financial analysis

    Directory of Open Access Journals (Sweden)

    A Saville

    2004-04-01

    Full Text Available Accounting practices are flawed.  As a consequence, the accounting data generated by firms are generally open to interpretation, often misleading and sometimes patently false.  Yet, financial analysts place tremendous confidence in accounting data when appraising investments and investment strategies.  The implications of financial analysis based on questionable information are numerous, and range from inexact analysis to acute investment error.  To rectify this situation, this paper identifies a set of simple, yet highly effective corrective measures, which have the capacity to move accounting practice into a realm wherein accounting starts to ‘count what counts’.  The net result would be delivery of accounting data that more accurately reflect firms’ economic realities and, as such, are more useful in the task of financial analysis.

  4. Using external data sources to improve audit trail analysis.

    Science.gov (United States)

    Herting, R L; Asaro, P V; Roth, A C; Barnes, M R

    1999-01-01

    Audit trail analysis is the primary means of detection of inappropriate use of the medical record. While audit logs contain large amounts of information, the information required to determine useful user-patient relationships is often not present. Adequate information isn't present because most audit trail analysis systems rely on the limited information available within the medical record system. We report a feature of the STAR (System for Text Archive and Retrieval) audit analysis system where information available in the medical record is augmented with external information sources such as: database sources, Light-weight Directory Access Protocol (LDAP) server sources, and World Wide Web (WWW) database sources. We discuss several issues that arise when combining the information from each of these disparate information sources. Furthermore, we explain how the enhanced person specific information obtained can be used to determine user-patient relationships that might signify a motive for inappropriately accessing a patient's medical record.

  5. The Subjectivity Problem: Improving Triangulation Approaches in Metaphor Analysis Studies

    Directory of Open Access Journals (Sweden)

    Sonya L. Armstrong

    2011-06-01

    Full Text Available Metaphor analysis procedures for uncovering participant conceptualizations have been well-established in qualitative research settings since the early 1980s; however, one common criticism of metaphor analysis is the trustworthiness of the findings. Namely, accurate determination of the conceptual metaphors held by participants based on the investigation of linguistic metaphors has been identified as a methodological issue because of the subjectivity involved in the interpretation; that is, because they are necessarily situated in specific social and cultural milieus, meanings of particular metaphors are not universally constructed nor understood. In light of these critiques, this article provides examples of two different triangulation methods that can be employed to supplement the trustworthiness of the findings when metaphor analysis methodologies are used.

  6. A quality improvement study using fishbone analysis and an electronic medical records intervention to improve care for children with asthma.

    Science.gov (United States)

    Gold, Jonathan; Reyes-Gastelum, David; Turner, Jane; Davies, H Dele

    2014-01-01

    Despite expert guidelines, gaps persist in quality of care for children with asthma. This study sought to identify barriers and potential interventions to improve compliance to national asthma prevention guidelines at a single academic pediatric primary care clinic. Using the plan-do-check-act (PDCA) quality improvement framework and fishbone analysis, several barriers to consistent asthma processes and possible interventions were identified by a group of key stakeholders. Two interventions were implemented using the electronic medical record (EMR). Physician documentation of asthma quality measures were analyzed before intervention and during 2 subsequent time points over 16 months. Documentation of asthma action plans (core group P PDCA and fishbone analysis in conjunction with embedded EMR tools can improve asthma care in a pediatric primary care setting.

  7. 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.

  8. Rasch Analysis for Psychometric Improvement of Science Attitude Rating Scales

    Science.gov (United States)

    Oon, Pey-Tee; Fan, Xitao

    2017-01-01

    Students' attitude towards science (SAS) is often a subject of investigation in science education research. Survey of rating scale is commonly used in the study of SAS. The present study illustrates how Rasch analysis can be used to provide psychometric information of SAS rating scales. The analyses were conducted on a 20-item SAS scale used in an…

  9. Improving the Computational Morphological Analysis of a Swahili ...

    African Journals Online (AJOL)

    approach to the morphological analysis of Swahili. We particularly focus our discussion on its ability to retrieve lemmas for word forms and evaluate it as a tool for corpus-based dictionary compilation. Keywords: LEXICOGRAPHY, MORPHOLOGY, CORPUS ANNOTATION, LEMMATIZATION, MACHINE LEARNING, SWAHILI ...

  10. Temporal Land Cover Analysis for Net Ecosystem Improvement

    Energy Technology Data Exchange (ETDEWEB)

    Ke, Yinghai; Coleman, Andre M.; Diefenderfer, Heida L.

    2013-04-09

    We delineated 8 watersheds contributing to previously defined river reaches within the 1,468-km2 historical floodplain of the tidally influenced lower Columbia River and estuary. We assessed land-cover change at the watershed, reach, and restoration site scales by reclassifying remote-sensing data from the National Oceanic and Atmospheric Administration Coastal Change Analysis Program’s land cover/land change product into forest, wetland, and urban categories. The analysis showed a 198.3 km2 loss of forest cover during the first 6 years of the Columbia Estuary Ecosystem Restoration Program, 2001–2006. Total measured urbanization in the contributing watersheds of the estuary during the full 1996-2006 change analysis period was 48.4 km2. Trends in forest gain/loss and urbanization differed between watersheds. Wetland gains and losses were within the margin of error of the satellite imagery analysis. No significant land cover change was measured at restoration sites, although it was visible in aerial imagery, therefore, the 30-m land-cover product may not be appropriate for assessment of early-stage wetland restoration. These findings suggest that floodplain restoration sites in reaches downstream of watersheds with decreasing forest cover will be subject to increased sediment loads, and those downstream of urbanization will experience effects of increased impervious surfaces on hydrologic processes.

  11. An improved quantitative analysis method for plant cortical microtubules.

    Science.gov (United States)

    Lu, Yi; Huang, Chenyang; Wang, Jia; Shang, Peng

    2014-01-01

    The arrangement of plant cortical microtubules can reflect the physiological state of cells. However, little attention has been paid to the image quantitative analysis of plant cortical microtubules so far. In this paper, Bidimensional Empirical Mode Decomposition (BEMD) algorithm was applied in the image preprocessing of the original microtubule image. And then Intrinsic Mode Function 1 (IMF1) image obtained by decomposition was selected to do the texture analysis based on Grey-Level Cooccurrence Matrix (GLCM) algorithm. Meanwhile, in order to further verify its reliability, the proposed texture analysis method was utilized to distinguish different images of Arabidopsis microtubules. The results showed that the effect of BEMD algorithm on edge preserving accompanied with noise reduction was positive, and the geometrical characteristic of the texture was obvious. Four texture parameters extracted by GLCM perfectly reflected the different arrangements between the two images of cortical microtubules. In summary, the results indicate that this method is feasible and effective for the image quantitative analysis of plant cortical microtubules. It not only provides a new quantitative approach for the comprehensive study of the role played by microtubules in cell life activities but also supplies references for other similar studies.

  12. Improving Family Forest Knowledge Transfer through Social Network Analysis

    Science.gov (United States)

    Gorczyca, Erika L.; Lyons, Patrick W.; Leahy, Jessica E.; Johnson, Teresa R.; Straub, Crista L.

    2012-01-01

    To better engage Maine's family forest landowners our study used social network analysis: a computational social science method for identifying stakeholders, evaluating models of engagement, and targeting areas for enhanced partnerships. Interviews with researchers associated with a research center were conducted to identify how social network…

  13. Stiffness Analysis and Improvement of Bolt-Plate Contact Assemblies

    DEFF Research Database (Denmark)

    Pedersen, Niels Leergaard; Pedersen, Pauli

    2008-01-01

    and the plate is extended by the possibility of designing a gap, that is, a nonuniform distance between the bolt and plate before prestressing. Designing the gap function generates the possibility for a better stress field by which the stiffness of the bolt is lowered, and at the same time the stiffness...... of the members is increased. Both of these changes have a positive influence on the lifetime of the connections. From designing a varying gap size distribution, it is found that the stiffness become a function of the loading. It is shown that similar improvements in the stiffness ratio between the bolt...

  14. Analysis and improvement of vehicle information sharing networks

    Science.gov (United States)

    Gong, Hang; He, Kun; Qu, Yingchun; Wang, Pu

    2016-06-01

    Based on large-scale mobile phone data, mobility demand was estimated and locations of vehicles were inferred in the Boston area. Using the spatial distribution of vehicles, we analyze the vehicle information sharing network generated by the vehicle-to-vehicle (V2V) communications. Although a giant vehicle cluster is observed, the coverage and the efficiency of the information sharing network remain limited. Consequently, we propose a method to extend the information sharing network's coverage by adding long-range connections between targeted vehicle clusters. Furthermore, we employ the optimal design strategy discovered in square lattice to improve the efficiency of the vehicle information sharing network.

  15. cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

    Directory of Open Access Journals (Sweden)

    F.H.R. Fagundes

    2010-03-01

    Full Text Available To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR, we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49, from the venom of Crotalus durissus collilineatus (Cdc PLA2. The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC. The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.

  16. 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

  17. 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.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  19. Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells

    Science.gov (United States)

    Balcer-Kubiczek, E. K.; Meltzer, S. J.; Han, L. H.; Zhang, X. F.; Shi, Z. M.; Harrison, G. H.; Abraham, J. M.

    1997-01-01

    A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

  20. Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer's disease: Identification as the microtubule-associated protein tau

    International Nuclear Information System (INIS)

    Goedert, M.; Wischik, C.M.; Crowther, R.A.; Walker, J.E.; Klug, A.

    1988-01-01

    Screening of cDNA libraries prepared from the frontal cortex of an Alzheimer's disease patient and from fetal human brain has led to isolation of the cDNA for a core protein of the paired helical filament of Alzheimer's disease. The partial amino acid sequence of this core protein was used to design synthetic oligonucleotide probes. The cDNA encodes a protein of 352 amino acids that contains a characteristic amino acid repeat in its carboxyl-terminal half. This protein is highly homologous to the sequence of the mouse microtubule-associated protein tau and thus constitutes the human equivalent of mouse tau. RNA blot analysis indicates the presence of two major transcripts, 6 and 2 kilobases long, with a wide distribution in normal human brain. Tau protein mRNAs were found in normal amounts in the frontal cortex from patients with Alzheimer's disease. The proof that at least part of tau protein forms a component of the paired helical filament core opens the way to understanding the mode of formation of paired helical filaments and thus, ultimately, the pathogenesis of Alzheimer's disease