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Sample records for cdna analysis improves

  1. Quantitative Transcript Analysis in Plants: Improved First-strand cDNA Synthesis

    Institute of Scientific and Technical Information of China (English)

    Nai-Zhong XIAO; Lei BA; Preben Bach HOLM; Xing-Zhi WANG; Steve BOWRA

    2005-01-01

    The quantity and quality of first-strand cDNA directly influence the accuracy of transcriptional analysis and quantification. Using a plant-derived α-tubulin as a model system, the effect of oligo sequence and DTT on the quality and quantity of first-strand cDNA synthesis was assessed via a combination of semi-quantitative PCR and real-time PCR. The results indicated that anchored oligo dT significantly improved the quantity and quality of α-tubulin cDNA compared to the conventional oligo dT. Similarly, omitting DTT from the first-strand cDNA synthesis also enhanced the levels of transcript. This is the first time that a comparative analysis has been undertaken for a plant system and it shows conclusively that small changes to current protocols can have very significant impact on transcript analysis.

  2. Application of an improved cDNA competition technique to identify prostate cancer-associated gene.

    Science.gov (United States)

    Rinaldy, A R; Steiner, M S

    1999-11-01

    A technique to improve cDNA library screening was developed by using mixed probes derived from two closely related cDNA populations of high-metastatic MAT-LyLu and low-metastatic AT-1 Dunning R3227 rat prostate cancer sublines. The technique required the generation of a cDNA library from each subline followed by polymerase chain reaction (PCR) amplification of the cDNA insert population. The PCR products derived from the first library were radiolabeled and mixed with an excess amount of PCR products from the second library. The mixture and an excess amount of both the lambda and pBluescript DNA were used as a probe to screen the first cDNA library. This mixed probe (designated the competition probe) differentially cross-hybridized with the plaque lift of the screened first cDNA library. Weak radioactive signals indicated the cross-hybridization of cDNA sequences common to the competition probe mixture and the first cDNA library, whereas strong signals implied unhybridized unique or abundant cDNA sequences in the first cDNA library. The reproducibility of this technique was confirmed by showing that the full-length cDNA clones were associated with the phenotype of the screened first cell line. The isolated clones were characterized as rat nucleolar protein, rat mitochondrial genes coding for 16S and 12S rRNAs, and rat tRNAs specific for valine and phenyl-alanine. This result is consistent with the fact that the first cell line, MAT-LyLu, is metabolically more active than are AT-1 cells because of higher gene dosage or amplification of nucleolar and mitochondrial RNA and its associated genes. Another clone which had a strong signal represented a novel gene associated with the MAT-LyLu cancer phenotype.

  3. cDNA macroarray for analysis of gene expression profiles in prostate cancer

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Early diagnosis and timely treatment are important for improving therapeutic efficiency of prostate cancer. DNA array is a new bio-technology for disease diagnosis. This study was conducted to diagnose prostate cancer with cDNA macroarray and analysis gene expression profiles of some selective genes in prostate cancer.Methods Total RNA was isolated from patients with prostate cancer and from normal people, and poly(A) RNA was further purified. Then it was analyzed for differentially expressed genes in prostate cancer and normal prostate by cDNA macroarray system.Results There were different expressions in the nine prostate-associated specific genes in prostate cancer as compared with normal prostate, in which, 7 were significantly upregulated and 2 were down-regulated.Conclusion As a diagnostic approach at molecular level, the cDNA macroarray is an effectively diagnostic method for prostate cancer.

  4. Single primer amplification (SPA) of cDNA for microarray expression analysis

    OpenAIRE

    2003-01-01

    The potential of expression analysis using cDNA microarrays to address complex problems in a wide variety of biological contexts is now being realised. A limiting factor in such analyses is often the amount of RNA required, usually tens of micrograms. To address this problem researchers have turned to methods of improving detection sensitivity, either through increasing fluorescent signal output per mRNA molecule or increasing the amount of target available for labelling by use of an amplific...

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

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    Jesionek-Kupnicka Dorota

    2009-08-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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.

  6. cDNA Cloning and Sequence Analysis of Rice Sbel and Sbe3 Genes

    Institute of Scientific and Technical Information of China (English)

    CHENXiu-hua; LIUQiao-quan; WuHsin-kan; WANGZong-yang; GuMing-hong

    2004-01-01

    Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes SheI and Shed encoding SBE Ⅰ and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA libray, derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned SheI and Shed cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of She3 was the same as that of shed (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference,which resulted in changes of two deduced amino acids between the cloned She1 cDNA and the reported she1 (Genbank Accession No. D11082). The cloned SheI and Shed cDNAs make it possible to improve rice starch quality through genetic engineering.

  7. cDNA Cloning and Sequence Analysis of Rice Sbe1 and Sbe3 Genes

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiu-hua; LIU Qiao-quan; WU Hsin-kan; WANG Zong-yang; GU Ming-hong

    2004-01-01

    Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes Sbe1 and Sbe3 encoding SBE I and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA library derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned Sbe1 and Sbe3 cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of Sbe3 was the same as that of sbe3 (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference,which resulted in changes of two deduced amino acids between the cloned Sbe1 cDNA and the reported sbe1 (Genbank Accession No. D11082). The cloned Sbe1 and Sbe3 cDNAs make it possible to improve rice starch quality through genetic engineering

  8. Preparation of microbial community cDNA for metatranscriptomic analysis in marine plankton.

    Science.gov (United States)

    Stewart, Frank J

    2013-01-01

    High-throughput sequencing and analysis of microbial community cDNA (metatranscriptomics) are providing valuable insight into in situ microbial activity and metabolism in the oceans. A critical first step in metatranscriptomic studies is the preparation of high-quality cDNA. At the minimum, preparing cDNA for sequencing involves steps of biomass collection, RNA preservation, total RNA extraction, and cDNA synthesis. Each of these steps may present unique challenges for marine microbial samples, particularly for deep-sea samples whose transcriptional profiles may change between water collection and RNA preservation. Because bacterioplankton community RNA yields may be relatively low (microbiology research.

  9. Analysis of gene expression profile of pancreatic carcinoma using CDNA microarray

    Institute of Scientific and Technical Information of China (English)

    ZhiJun Tan; Xian-Gui Hu; Gui-Song Cao; Yan Tang

    2003-01-01

    AIM: To identify new diagnostic markers and drug targets,the gene expression profiles of pancreatic cancer were compared with that of adjacent normal tissues utilizing cDNA microarray analysis.METHODS: cDNA probes were prepared by labeling mRNA from samples of six pancreatic carcinoma tissues with Cy5dUTP and mRNA from adjacent normal tissues with Cy3dUTP respectively through reverse transcription. The mixed probes of each sample were then hybridized with 12 800cDNA arrays (12 648 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3 000scanner (General Scanning, Inc.). The values of CyS-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Differentially expressed genes were screened according to the criterion that the absolute value of natural logarithm of the ratio of Cy5-dUTP to Cy3-dUTP was greater-than 0.69.RESETS: Among 6 samples investigated, 301 genes, which accounted for 2.38% of genes on the microarry slides,exhibited differentially expression at least in 5. There were 166 over-expressed genes including 136 having been registered in Genebank, and 135 under-expressed genes including 79 in Genebank in cancerous tissues.CONCLUSION: Microarray analysis may provide invaluable information on disease pathology, progression, resistance to treatment, and response to cellular microenvironments of pancreatic carcinoma and ultimately may lead to improving early diagnosis and discovering innovative therapeutic approaches for cancer.

  10. Construction of cDNA representational difference analysis based on two cDNA libraries and identification of garlic inducible expression genes in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Li; Lin Yang; Jian-Tao Cui; Wen-Mei Li; Rui-Fang Guo; You-Yong Lu

    2002-01-01

    AIM: To elucidate molecular mechanism of chemopreventiveefficacies of garlic against human gastric cancer (HGC):METHODS: HGC cell line BGC823 was treated with Allitridi (akind of garlic extract) and Allitridi-treated and parentalBGC823 cDNA librarles were constructed respectively byusing λZAP Ⅱ vector. cDNA Representatinal DifferenceAnalysis (cDNA RDA) was perfonmed using BamH Ⅰ cutting-site and abundant ~DNA messages provided by the Iibrarles.Northern blot analysls was applied to identifythe obtaineddifference prnducts.RESULTS: Two specific cDNA fragments were obtained andcharacterized to be derived from homo sapiens folatereceptorα (FRα) gene and calcyclin gene respectively.Northern blot results showed a 4-fold increase in FRα geneexpression level and 9-fold increase in calcyclin mRNA levelin BGC823 cells after Allilridi treatment for 72 h.CONCLUSION: The method of cDNA RDA based on cDNAlibraries combines the high specificity of cDNA RDA withabundant cDNA messages in cDNA library; this expands theapplication of cDNA library and increases the specificity ofcDNA RDA. Up-regulstion of FRα gene and calcyclin geneexpressions induced by Allitridi provide valuable molecularevidence for theefficacy of garlic in treating HGC as well asother diseases.

  11. Cloning and expression analysis of MBLL cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The mbl (muscleblind) gene of Drosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation of mbl gene will disturb the differentiation of all the Drosophila's photoreceptors. Primers have been designed according to human EST086139, which is highly homologous to mbl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designated MBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology to Drosophila's mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show that MBLL is a widely expressed gene, but the expression amounts differ in these tissues.

  12. RAPID SCREENING OF AN ARRAYED cDNA LIBRARY BY IMPROVED PCR-BASED METHOD

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    杜光伟; 潘美辉; 袁建刚; 周彦; 强伯勤; 梁植权

    1998-01-01

    Tbe present study reports an improved PCR-based technique that allows quick and effecfive screening of eDNA libraries. First, the eDNA library was arrayed as follows: about 3×106 cDNA clones were multiplied as individual plsques on solid medium in 24-well culture dishes at 1 200 plaque forming units per well.The phage suspension of each well was transferred to an individual micrccentrifuge tube in 72-tube box. Then,box pool, row pools and column pools were set up that respectively represent a 72-tube box,rows and columns within the box. To screen a specific target cDNA,primers specific for novel ESTs obtained in our laboratory were eznployed to conduct PCR in a fiierarchy mode. PCR began with the box pools, resulting in the identification of some positive box pools. Then PCR went down to the row and column pools of the positive box. Tbe intersection of the positive row(s) and column(s) revealed the candidate positive tubes. The specificity of PCR products were meanwhile checked hy restriction enzyme digestion. Finally, hybridization was carried out to get single specific eDNA clones-from the positive tlabes. This PCR-hased technique features high specificity, high efficiency and Les-cost in large-scale cDNA library screening. Our initial implementation of the technique resulted in the isolation of three longer different cDNA clones from a hnman fetal brain eDNA library. Thus this improved technique can serve as an alternative to the time-consuming and laborious conventional hybridization-hased metfiod for screening cDNA library.

  13. Cloning and analysis of a cDNA encoding acetohydroxy acid isomeroreductase from G2 pea

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Using cDNA representational difference analysis (cDNA RDA) method, we have successfully isolated a gene fragment whose expression was specifically induced by external GA3 application. Screening a G2 pea cDNA library using this fragment as a probe, we obtained a 2036 bp full-length cDNA. It contains a 1746 bp open reading frame and encodes a protein of 581 amino acids with a theoretical molecular weight of 64 ku. It shares high-level sequence identity with AAIR genes from other plant species. This cDNA was cloned into expression vector and recombinant E. coli DH5α cells with remarkable AAIR enzyme activity were obtained.

  14. IDENTIFICATION OF DIFFERENTIAL GENES IN OVARIAN CANCER USING REPRESENTATIONAL DIFFERENCE ANALYSIS OF cDNA

    Institute of Scientific and Technical Information of China (English)

    Hong Chen; Min Wang; Xin-yan Wang; Shan Gao; Jun Wang; Xiao-ming Guan

    2005-01-01

    Objective To identify differential genes between normal ovarian epithelium tissue and ovarian epithelial cancer using representational difference analysis of cDNA (cDNA-RDA). Methods cDNA-RDA was performed to identify the differentially expressed sequences between cDNAs from cancer tissue and cDNAs from normal ovarian tissue in the same patient who was in the early stage of ovarian serous cystadeno carcinoma. These differentially expressed fragments were cloned and analyzed, then sequenced and compared with known genes.Results Three differentially expressed cDNA fragments were isolated using cDNA from normal ovarian tissue as tester and cDNA from cancer tissue as driver amplicon by cDNA-RDA. DP Ⅲ-1 and DP Ⅲ-2 cDNA clone showed significant ho mology to the cDNA of alpha actin gene; DPⅢ-3 cDNA clone showed significant homology to the cDNA of transgelin gene. Conclusion cDNA-RDA can be used to sensitively identify the differentially expressed genes in ovarian serous cystadenocarcinoma. Ovarian serous cystadenocarcinoma involves alteration of multiple genes.

  15. [Construction and analysis of subtractive cDNA library associated with multidrug resistance of acute leukemia].

    Science.gov (United States)

    Ji, Lei; Zhang, Wang-Gang; Liu, Jie; Liu, Xin-Ping; Yao, Li-Bo

    2004-08-01

    The study was aimed to construct subtractive cDNA library associated with multidrug resistance (MDR) of acute leukemia for screening genes related to MDR in leukemia. The improved PCR-based subtractive hybridization was performed to clone differential genes between HL-60/VCR and HL-60 cell line. The mRNA of HL-60/VCR and HL-60 cell line were isolated. Then the mRNA of HL-60/VCR group was reversely transcribed into cDNA by Cap-Finder method, and the mRNA of HL-60 was reversely transcribed into cDNA by ordinary method to be marked by biotin for the hybridization next with HL-60/VCR cDNA. After hybridizing, filtrating through the sephacryl S-400 column, absorbing by the magnetic beads, and amplifying by PCR method, the fragments were cloned by T-A method and the cDNA library was constructed. Then the quality of cDNA library was identified by dot-blotting hybridization method. The results showed that after constriction, the library demonstrated its good quality. There was a high proportion of large fragments in this library. From small amount of samples a large amount of candidate fragments could be screened rapidly at once by dot-blotting hybridization. It is concluded that a differentially-expressed subtractive cDNA library in MDR of leukemia with high quality and larger fragments can be efficiently constructed by improving subtractive hybridization and selective PCR method.

  16. Analysis of common bean (Phaseolus vulgaris L., genotype BAT93 calmodulin cDNA using computational tools

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    Kassim Amelia

    2015-01-01

    Full Text Available Background: Common bean (Phaseolus vulgaris L. is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day and 20-day old bean-pod-tissues. Altogether, 5972 cDNA clones were isolated to have ESTs. While processing ESTs, we found a transcript for calmodulin (CaM gene. It is an important gene that encodes for a calcium-binding protein and known to express in all eukaryotic cells. Hence, this study was undertaken to analyse and annotate it. Objective: The objective of this study was to analyze and annotate P. vulgaris CaM (PvCaM gene cDNA and its deduced protein (amino acids sequence. Materials and Methods: Both strands of PvCaM cDNA clone were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence. The cDNA sequence and deduced protein sequence were analyzed and annotated using bioinformatics tools available online. The secondary structures and three-dimensional (3D structure of PvCaM protein were predicted using the Phyre automatic fold recognition server. Results: Results showed that PvCaM cDNA is 818 bp in length. The cDNA analysis results showed that it contains an open reading frame that encodes for 149 amino acid residues. The deduced protein sequence analysis results showed the presence of conserved domains required for CaM function. The predicted secondary structures and 3D structure are analogous to the Solanum tuberosum CaM. Conclusions: This study analyzed and annotated PvCaM cDNA and protein. However, in order to obtain a complete understanding of PvCaM protein, further study on its expression, structure and regulation is

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

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    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.

  18. Discovery and analysis of pancreatic adenocarcinoma genes using cDNA microarrays

    Institute of Scientific and Technical Information of China (English)

    Gang Jin; Xian-Gui Hu; Kang Ying; Yan Tang; Rui Liu; Yi-Jie Zhang; Zai-Ping Jing; Yi Xie; Yu-Min Mao

    2005-01-01

    AIM: To study the pathogenetic processes and the role of gene expression by microarray analyses in expediting our understanding of the molecular pathophysiology of pancreatic adenocarcinoma, and to identify the novel cancer-associated genes.METHODS: Nine histologically defined pancreatic head adenocarcinoma specimens associated with clinical data were studied. Total RNA and mRNA were isolated and labeled by reverse transcription reaction with Cy5 and Cy3 for cDNA probe. The cDNA microarrays that represent a set of 4 096 human genes were hybridized with labeled cDNA probe and screened for molecular profiling analyses.RESULTS: Using this methodology, 184 genes were screened out for differences in gene expression level after nine couples of hybridizations. Of the 184 genes,87 were upregulated and 97 downregulated, including 11 novel human genes. In pancreatic adenocarcinoma tissue, several invasion and metastasis related genes showed their high expression levels, suggesting that poor prognosis of pancreatic adenocarcinoma might have a solid molecular biological basis.CONCLUSION: The application of cDNA microarray technique for analysis of gene expression patterns is a powerful strategy to identify novel cancer-associated genes, and to rapidly explore their role in clinical pancreatic adenocarcinoma. Microarray profiles provide us new insights into the carcinogenesis and invasive process of pancreatic adenocarcinoma. Our results suggest that a highly organized and structured process of tumor invasion exists in the pancreas.

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

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    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

  20. Analysis of cDNA sequence, protein structure and expression of parotid secretory protein in pig

    Institute of Scientific and Technical Information of China (English)

    YIN Haifang; FAN Baoliang; ZHAO Zhihui; LIU Zhaoliang; FEI Jing; LI Ning

    2003-01-01

    Parotid secretory protein (PSP) secreted abundantly in saliva, whose function is related with the anti-bacterial effect. The PSP cDNA has been isolated from pig parotid glands by 3′ and 5′ rapid amplification of cDNA end (RACE),based on the conserved signal peptide region among the known mammalian PSP. Theresult of homologous comparison shows that pig PSP and human PSP shares the high identity at the level of the primary, secondary and tertiary protein structure. A search for functionally significant protein motifs revealed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu- X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is important to its function. RT-PCR, Dot blot and Northern blot analysis demonstrated that PSP was strongly expressed in parotid glands, but not in other tissues.

  1. [Construction and analysis of subtractive cDNA library of Phellodendron amurense under drought stress].

    Science.gov (United States)

    Wang, Huimei; Wang, Yanbing; Zu, Yuangang; Sun, Lianhui

    2008-02-01

    With cDNA from Phellodendron amurense seedlings treated with drought stress as tester and cDNA from this plant in normal growth as driver, we construct cDNA subtracted library using suppression subtractive hybridization (SSH). In the library, the rate of recombination was 95%, the size of inserts was 300-800 bp. Two hundred and sixty-five new genes were obtained by DNA sequencing 816 positive clones picked randomly, and partitioned to 16 classes after nucleotide Blast and BlastX homological analysis against NT, NR, SWISSPROT, KEGG database. Forty-four drought stress associated genes, such as heat shock protein cognate 70, dehydration responsive protein 22, universal stress protein, metallothionein II, late embryogenesis abundant protein, were obtained, which made 16.6% of the overall genes. These genes included osmotic regulator, signal component regulatory protein and antioxidant enzyme. The research had established a basis for cloning stress resistance genes and further studying genes expression in P. amurense seedlings under drought stress.

  2. cDNA cloning and function analysis of two novel erythroid differentiation related genes

    Institute of Scientific and Technical Information of China (English)

    WANG; Xin; (王鑫); WANG; Duncheng; (王敦成); CHEN; Xing; (陈兴),; HU; Meiru; (胡美茹); WANG; Jian'an; (王建安); LI; Yan; (黎燕); GUO; Ning; (郭宁); SHEN; Beifen; (沈倍奋)

    2001-01-01

    Our previous studies showed that some nuclear proteins that were expressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen l-gtll human cDNA expression library of fetal liver in order to obtain the rele-vant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' func-tions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigat-ed the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid develop-ment, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

  3. cDNA cloning and function analysis of two novel erythroid differentiation related genes

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Our previous studies showed that some nuclear proteins that wereexpressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen l-gtll human cDNA expression library of fetal liver in order to obtain the rele-vant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' func-tions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigat-ed the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid develop-ment, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

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

    Institute of Scientific and Technical Information of China (English)

    Yu-Juan Xiang; Zhi-Gang Yu; Ming-Ming Guo; Qin-Ye Fu; Zhong-Bing Ma; De-Zong Gao; Qiang Zhang; Yu-Yang Li; Liang Li; Lu Liu; Chun-Miao Ye

    2015-01-01

    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 quanti-tatively 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. Copyright © 2015, Chinese Medical Association Production. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

  5. 3' rapid amplification of cDNA ends (RACE) walking for rapid structural analysis of large transcripts.

    Science.gov (United States)

    Ozawa, Tatsuhiko; Kondo, Masato; Isobe, Masaharu

    2004-01-01

    The 3' rapid amplification of cDNA ends (3' RACE) is widely used to isolate the cDNA of unknown 3' flanking sequences. However, the conventional 3' RACE often fails to amplify cDNA from a large transcript if there is a long distance between the 5' gene-specific primer and poly(A) stretch, since the conventional 3' RACE utilizes 3' oligo-dT-containing primer complementary to the poly(A) tail of mRNA at the first strand cDNA synthesis. To overcome this problem, we have developed an improved 3' RACE method suitable for the isolation of cDNA derived from very large transcripts. By using the oligonucleotide-containing random 9mer together with the GC-rich sequence for the suppression PCR technology at the first strand of cDNA synthesis, we have been able to amplify the cDNA from a very large transcript, such as the microtubule-actin crosslinking factor 1 (MACF1) gene, which codes a transcript of 20 kb in size. When there is no splicing variant, our highly specific amplification allows us to perform the direct sequencing of 3' RACE products without requiring cloning in bacterial hosts. Thus, this stepwise 3' RACE walking will help rapid characterization of the 3' structure of a gene, even when it encodes a very large transcript.

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

    Institute of Scientific and Technical Information of China (English)

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

    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.METHODS: 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 SMARTTM 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.RESULTS: 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.CONCLUSION: 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 gastrocardnoma.

  7. ANALYSIS OF GENES ASSOCIATED WITH LYMPHATIC METASTASIS IN PANCREATIC CARCINOMA USING cDNA MICROARRAY

    Institute of Scientific and Technical Information of China (English)

    谭志军; 胡先贵; 曹贵松; 唐岩

    2003-01-01

    Objective: To identify new markers for prediction of lymph node metastasis. Methods: cDNA probes were prepared by labeling mRNA from samples of four pancreatic carcinoma tissues with Cy5-dUTP and mRNA from adjacent normal tissues with Cy3-dUTP respectively through reverse transcription. The mixed probes of each sample were then hybridized with 4,096 cDNA arrays (4,000 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Genes that differentially expresses in each cancerous tissue were sought out according to the standard that the absolute value of natural logarithm of the ratio of Cy5 to Cy3 is greater than 0.69, i. e., more than 2 times change of gene expression, and the signal value of either Cy3 and Cy5 need to be greater than 600. Then, the genes differently expressed in cancer with and without lymphatic metastasis were screened out for further analysis. Results: Among 2 samples with lymphatic metastasis and 2 samples without metastasis, 56 genes, which accounted for 1.40% of genes on the microarray slides, exhibited differentially expression in cancerous tissues with lymphatic metastasis. There were 32 over-expressed genes including 11 having been registered in Genebank, and 24 under-expressed genes including 3 in Genebank. Conclusion: Microarray analysis may provide invaluable information to identify specific gene expression profile of lymphatic metastasis in pancreatic cancer.

  8. Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration

    Directory of Open Access Journals (Sweden)

    Bryant Susan V

    2009-01-01

    Full Text Available Abstract Background Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR and denervated (DL forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. Results Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa. Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST contigs from the Ambystoma EST database more than doubled (3935 to 9411 the number of non-redundant human-A. mexicanum orthologous sequences. Conclusion Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

  9. 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.

  10. Cloning and bioinformatics analysis of cDNA encoding cattle Smad4 gene

    Institute of Scientific and Technical Information of China (English)

    Xiaohui ZHANG; Shangzhong XU; Xue GAO; Hongyan REN; Jinbao CHEN

    2008-01-01

    The cDNA of cattle Smad4 gene was cloned by RT-PCR, 3' RACE and 5' RACE and got a 3503-bp full-long cDNA sequence. The cloned cattle Smad4 cDNA sequence had been send to GenBank and got an accession number: DQ494856. Cattle Smad4 gene consists of 12 exons and codes 553 amino acids. Cattle Smad4 cDNA shares 99%, 96%, 95%, 91% and 91% similarity in nucleic acid sequences, and 99%, 98%, 98%, 99% and 98% sim-ilarity in amino acid sequences with sheep, pig, human, rat and mouse, respectively. Smad4 cDNA was found in the testes, pancreas, liver, small intestine, ovary, lymph, car-diac muscle, skeleton muscle and thymus gland, which indicated that Smad4 was broadly expressed in cattle.

  11. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    OpenAIRE

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-bindin...

  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 and expression analysis of a mannose-binding lectin from Pinellia pedatisecta

    Indian Academy of Sciences (India)

    Juan Lin; Xuanwei Zhou; Shi Gao; Xiaojun Liu; Weisheng Wu; Xiaofen Sun; Kexuan Tang

    2007-03-01

    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 SMART RACE-PCR technology; it was 1146 bp and contained a 771 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acid residues with a 24 amino acid signal peptide. The PPA precursor contained 3 mannose-binding sites (QXDXNXVXY) and two conserved domains of 43% identity, PPA-DOM1 (polypeptides A) and PPA-DOM2 (polypeptides B). PPA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families such as Araceae, Alliaceae, Iridaceae, Liliaceae, Amaryllidaceae and Bromeliaceae. Southern blot analysis indicated that ppa belonged to a multi-copy gene family. Expression pattern analysis revealed that ppa expressed in most tested tissues, with high expression being found in spadix, spathe and tuber. Cloning of the ppa gene not only provides a basis for further investigation of its structure, expression and regulatory mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.

  14. Construction and analysis of a subtracted cDNA library of Betula platyphylla female inflorescence

    Institute of Scientific and Technical Information of China (English)

    WEIJi-cheng; YANGChuan-ping; WANGChao; JIANGJing

    2005-01-01

    Female inflorescence of Betula platyphylla was sampled at an interval of each two days to analyze the background of gene expression in floral phase. On the basis of SMART strategy, the driver cDNA was obtained from total RNA of the last sample and the tester cDNA was from that of the others by RT-PCR which were subsequently used to construct a subtracted cDNA library. The result of the ESTs (expression sequence tags) blastX showed that the genes in the subtracted cDNA library could be mainly clustered into 5 groups related to metabolism, transportation and signal transduction, cell cycle, stress response, and regulation. The relationship between gene expression and development was also discussed.

  15. Gene expression in retinoic acid-induced neural tube defects A cDNA mieroarray analysis

    Institute of Scientific and Technical Information of China (English)

    Xiaodong Long; Zhong Yang; Yi Zeng; Hongli Li; Yangyun Han; Chao You

    2009-01-01

    the cranium and abnormal changes of the metencephalon and face.cDNA microarray analysis suggested that the changes in expression of seven different genes were similar on both days E10.5 and E11.5. These were downregulation of NekT, Igfbp5, Zw10,Csf3r, Psmc6 and Rbl, and upregulation of Apoa-4. This study also indicated that Cdk5 expression was downregulated in the retinoic acid group on day E11.5. The results of the cDNA microarray analysis were partly confirmed by Northern blotting.CONCLUSION: Cdk5, NekT, Igfbp5, ZwlO, Csf3r, Psmc6, Rb 1 and Apoa-4 may be key factors in retinoic acid-induced neural tube defects.

  16. Massive Analysis of cDNA Ends (MACE for transcript-based marker design in pea (Pisum sativum L.

    Directory of Open Access Journals (Sweden)

    Aleksandr Zhernakov

    2017-03-01

    Full Text Available Aimed at gene-based markers design, we generated and analyzed transcriptome sequencing datasets for six pea (Pisum sativum L. genetic lines that have not previously been massively genotyped. Five cDNA libraries obtained from nodules or nodulated roots of genetic lines Finale, Frisson, Sparkle, Sprint-2 and NGB1238 were sequenced using a versatile 3′-RNA-seq protocol called MACE (Massive Analysis of cDNA Ends. MACE delivers a single next-generation sequence from the 3′-end of each individual cDNA molecule that precisely quantifies the respective transcripts. Since the contig generated from the 3′-end of the cDNA by assembling all sequences encompasses the highly polymorphic 3′-untranslated region (3′-UTR, MACE efficiently detects single nucleotide variants (SNVs. Mapping MACE reads to the reference nodule transcriptome assembly of the pea line SGE (Transcriptome Shotgun Assembly GDTM00000000.1 resulted in characterization of over 34,000 polymorphic sites in more than 9700 contigs. Several of these SNVs were located within recognition sequences of restriction endonucleases which allowed the design of co-dominant CAPS markers for the particular transcript. Cleaned reads of sequenced libraries are available from European Nucleotide Archive (http://www.ebi.ac.uk/ under accessions PRJEB18101, PRJEB18102, PRJEB18103, PRJEB18104, PRJEB17691.

  17. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S. (Istituto Nazionale Neurologico C. Besta, Milan (Italy)); Rocchi, M. (Istituto G. Gaslini, Genoa (Italy))

    1991-01-15

    The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH{sub 2}-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH{sub 2}-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids.

  18. Analysis of gene expression profile of aspermia using cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    杨波; 高晓康; 王禾; 刘贺亮; 陈宝琦; 秦荣良; 康福霞; 邵国兴; 邵晨

    2003-01-01

    Objective: To identify the differential gene expression profiles between the normal and aspermia human testes utilizing cDNA microarray. Methods: cDNA probes were prepared by labeling mRNA of aspermia testes tissues with Cy5-dUTP and mRNA of normal testes tissues with Cy3-dUTP respectively through reverse transcription. The mixed cDNA probes were then hybridized with 4096 cDNA arrays (4096 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Differentially expressed genes were screened according to the criterion that the absolute value of natural logarithm of the ratio of Cy5-dUTP to Cy3-dUTP was greater-than 2.0 or less-than 0.5. A randomly chosen gene RAP1A was studied by in situ hybridization to evaluate the accuracy of the results. Results: 623 differential expressed genes related to aspermia were found. There were 303 up-expressed genes and 320 down-expressed genes. A distinct up-expressed gene RAP1A was confirmed by in situ hybridization. Conclusions: Screening the differential gene expression profiles between the normal and aspermia human testis by cDNA microarray can be used in the study of aspermia-related genes and the further research due to its properties, RAP1A may play some roles in the development and progression of aspermia.

  19. Cloning and Sequence Analysis of cDNA Encoding MRJP3 of Apis cerana cerana

    Institute of Scientific and Technical Information of China (English)

    SU Song-kun; ZHNEG Huo-qing; CHEN Sheng-lu; ZHONG Bo-xiong; Stefan Albert

    2005-01-01

    By screening the worker (Apis cerana cerana) heads cDNA library using a fragment of the mrjp3 gene ofApis cerana as probe, 120 positive clones were obtained. The clone containing A. cerana cerana MRJP3 (AccMRJP3) cDNA was selected. Based on the sequencing of the inserts of the positive clone, a sequence of AccMRJP3 cDNA which is 1 887 bp long including a poly (A) tail was obtained. The AccMRJP3 cDNA encompassed an open-reading frame (ORF) with 1 779 bp encoding 593 amino acids. The un-translated regions (UTR) of the 5' end and 3' end are 46 bp and 160 bp in length,respectively. Similar to AmMRJP3 and AdMRJP3, the putative AccMRJP3 also has a repetitive region. The comparison of the repetitive region of AccMRJP3, AmMRJP3 and AdMRJP3 shows some differences between them.

  20. Analysis of differences of gene expressions in keloid and normal skin with the aid of cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    Chen Wei; Fu Xiaobing; Sun Xiaoqing; Sun Tongzhu; Zhao Zhili; Yang Yinhui; Sheng Zhiyong

    2003-01-01

    Background: Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of the disease. Keloid is a intricate lesion which is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skins with the aid of cDNA microarray in order to explore the molecular mechanism underlying keloid formation. Methods: The PCR products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs was isolated from freshly excised human keloids and normal skin, and then was purified to mRNA by Oligotex. Both the mRNA from keloids and normal skin was reversely transcribed to cDNAs with the incorporations of fluorescent dUTP, for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues. Results: Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250were up-regulated (2.98%) and 152 down-regulated (1.81%). Analyses of collagen, fibronectin, proteoglycan,growth factors and apoptosis related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with published biochemical and clinical observations of keloids. Conclusions: DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help understand the molecular mechanism of keloid formation.

  1. Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

    Directory of Open Access Journals (Sweden)

    Børresen-Dale Anne-Lise

    2002-10-01

    Full Text Available Abstract Background T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Results Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation. Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3–3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. Conclusion T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays.

  2. FY 1999 report on the survey of Research Association for biotechnology development. Trend survey on the structural analysis of full length cDNA; 1999 nendo biotechnology kaihatsu gijutsu kenkyu kumiai chosa hokokusho. Kanzen cho cDNA no kozo kaiseki ni kansuru doko chosa

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    Recognizing that the acquisition and structural analysis of full length cDNA clones are important subjects for connecting genome study and proteomics, survey was made of projects and technical trends in each country. The results of the survey were divided into the following four items: 1) trend of full length cDNA projects overseas; 2) study of full length cDNA in Japan; 3) full length cDNA project in Japan; 4) survey on technical trends of the structural analysis of full length cDNA. In 4), studies were made on the following: trend of technical development on the structural analysis of full length cDNA, trend of patents on the making of full length cDNA library, outline of the technology for the making of full length cDNA library. Countries for survey were the U.S., Japan, Germany, France and the U.K., and patents for survey were Japan open patents, U.S. open patents and WPI patents. For reference, included were seven data on full length cDNA related general remarks in Japanese, full length cDNA library related papers in English, full length cDNA related trend in Japan, etc. (NEDO)

  3. [cDNA cloning and sequence analysis of pluripotency genes in tree shrews (Tupaia belangeri)].

    Science.gov (United States)

    Wang, Cai-Yun; Ma, Yun-Han; He, Da-Jian; Yang, Shi-Hua

    2013-04-01

    In this paper, partial sequences of the tree shrew (Tupaia belangeri) Klf4, Sox2, and c-Myc genes were cloned and sequenced, which were 382, 612, and 485 bp in length and encoded 127, 204, and 161 amino acids, respectively. Whereas, their cDNA sequence identities with those of human were 89%, 98%, and 89%, respectively. Their phylogenetic tree results indicated different topologies and suggested individual evolutional pathways. These results can facilitate further functional studies.

  4. Identification of differentially expressed genes of Xanthomonas axonopodis pv. citri by representational difference analysis of cDNA

    Directory of Open Access Journals (Sweden)

    Angela Mehta

    2005-03-01

    Full Text Available Xanthomonas axonopodis pv. citri is a phytopathogenic bacterium responsible for citrus canker, a serious disease which causes severe losses in citriculture around the world. In this study we report the differential expression of X. axonopodis pv. citri in response to specific treatments by using Representational Difference Analysis of cDNA (cDNA RDA. cDNAs from X. axonopodis pv. citri cultured in the presence of leaf extract of the host plant (Citrus sinensis, in vivo, as well as in the complex medium were hybridized against cDNA of the bacterium grown in the minimal medium. Sequencing of the difference products obtained after the second and third hybridizations revealed a total of 37 distinct genes identified by homology searches in the genome of X. axonopodis pv. citri. These genes were distributed in different functional categories, including genes that encode hypothetical proteins, genes involved in metabolism, cellular processes and pathogenicity, and mobile genetic elements. Most of these genes are likely related to growth and/or acquisition of nutrients in specific treatments whereas others might be important for the bacterium pathogenicity.

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

    Directory of Open Access Journals (Sweden)

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

    2010-01-01

    Full Text Available 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.

  6. Generation and Analysis of Expressed Sequence Tags (ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

    Institute of Scientific and Technical Information of China (English)

    ZHAO Su-mei; LIU Yong-gang; PAN Hong-bing; ZHANG Xi; GE Chang-rong; JIA Jun-jing; GAO Shi-zheng

    2014-01-01

    Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identiifed in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1076 bp, and the cDNA fullness ratio was 86.2%. A total of 1058 unique sequences with 342 contigs (32.3%) and 716 singleton (67.7%) expressed sequence tags (EST) were obtained by clustering and assembling. Meanwhile, 826 (78.1%) ESTs were categorized as known genes, and 232 (21.9%) ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index ofSus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank (accession numbers: EU650784-EU650788, GE843306, GH228978-GH229100). The abundantly expressed genes in porcine muscle tissue were related to muscle ifber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.

  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. Cloning and expression analysis of human reticulon 4c cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    RTNs (reticulons) is a gene family related to the growth and differentiation of neuroendocrine cell. This family is composed of several members such as RTN1, RTN2 and RTN3. RTN1 and RTN2 have been proved to have 3 transcripts with different length. Because the RTN1c cDNA was involved in the sologenesis of small cell lung carcinoma (SCLC), it was selected as a bioinformatic probe to clone novel members of RTN family with the electric hybridization assistant new-gene cloning method (EHAC). A 1677-bp cDNA was identified from human brain cDNA library. The cDNA contains an intact open reading frame (ORF) which encodes a protein of 199 amino acids. This deduced protein is highly homologous to RTN1c, RTN2c and RTN3 with identities of 64.4%, 45.8% and 50.0% respectively. This new gene was named RTN4c (GenBank accession number: AF087901). Northern hybridization showed that the full length of RTN4c transcript is about 1.8 kb. It is hardly expressed in heart, placenta, lung, spleen, thymus, testis, ovary, small intestine and peripheral blood white cells; but it is highly expressed in the tissues of skeletal muscle, brain, liver and kidney, and less expressed in the pancreas, prostate and colon. Furthermore, Northern results also showed that there is a 2.3 kb transcript expressed in 14 tissues except liver and skeletal muscle; while another 5.0 kb transcript in brain, skeletal muscle and testis. By the electric hybridization walking, we obtained two full-length contigs with a length of 4632 and 2235 bp respectively. The former encodes a protein with 1192 amino acids and was defined as RTN4a; the latter encodes another protein with 373 amino acids, and was named RTN4b. The RTN4 gene was mapped to human chromosome 2p14-p13 region by the radiation hybridization (RH).

  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. Rat serum amyloid P component. Analysis of cDNA sequence and gene expression.

    Science.gov (United States)

    Dowton, S B; McGrew, S D

    1990-09-01

    cDNA clones for rat serum amyloid P component (SAP) were isolated, and the derived amino acid sequence for pre-SAP was determined from the complete nucleotide sequence. Rat SAP is encoded by approximately 1 kb of mRNA, and the mature SAP protein is predicted to be 208 amino acids long. An increase in hepatic mRNA levels for rat SAP was found after administration of lipopolysaccharide, and SAP mRNA levels in livers of unstimulated male rats were lower than in hepatic RNA from female rats.

  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. Genome-wide expression profiling of the response to terbinafine in Candida albicans using a cDNA microarray analysis

    Institute of Scientific and Technical Information of China (English)

    ZENG Yue-bin; QIAN Yuan-shu; MA Lian; GU Hong-ni

    2007-01-01

    Background Candida albicans is the most frequently seen opportunistic human fungal pathogen. Terbinafine is an allylamine antifungal agent that has been proven to have high clinical efficacy in the therapy of fungal infections, the mechanism of action of terbinafine involves the specific inhibition of fungal squalene epoxidase, resulting in ergosterol deficiency and accumulation of intracellular squalene. We used cDNA microarray analysis technology to monitor global expression profile changes of Candida albicans genes in response to terbinafine treatment, and we anticipated a panoramic view of the responses of Candida albicans cells to the representatives of allylamine antifungal agents at the molecular level in an effort to identify drug class-specific and mechanism-independent changes in gene expression.Methods Candida albicans strain ATCC 90028 was exposed to either medium alone or terbinafine at a concentration equivalent to the 1/2 minimal inhibitory concentrations (MICs, 4 mg/L) for 90 minutes. RNA was isolated and gene expression profiles were compared to identify the changes in the gene expression profile using a cDNA microarray analysis. Differential expression of 10 select genes detected by cDNA microarray analysis was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).Results A total of 222 genes were found to be responsive to terbinafine, including 121 up-regulated genes and 101 down-regulated genes. These included genes encoding membrane transport proteins belonging to the members of the ATP-binding cassette (ABC) or major facilitator superfamily (MFS; CDR1, AGP2, GAP6, PHO84, HOL3, FCY23, VCX1),genes involved in stress response and detoxification (CDR1, AGP2, HOL3), and gene involved in the ergosterol biosynthesis pathway (ERG12). The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis.Conclusions The up-regulation of the gene encoding the multidrug resistance efflux pump

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

    OpenAIRE

    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.

  14. cDNA cloning, characterization, and expression analysis of the Rac1 gene from Scophthalmus maximus.

    Science.gov (United States)

    Jia, Airong; Zhang, Xiao-Hua

    2009-09-01

    Rac1 is a small GTP-binding protein that belongs to the Rho small GTPases, which are important signaling molecules that regulate the dynamics of the actin cytoskeleton and mediate changes in cell morphology and motility. The EST sequence of Rac1 from turbot (Scophthalmus maximus L.) was obtained from a subtractive cDNA library previously. In this study, the full-length cDNA sequence of turbot Rac1 was obtained, which was 2420 nucleotides (nt) encoding a protein of 192 amino acids, with a putative molecular weight of 21.3 kDa. At the amino-acid level, turbot Rac1 was highly conserved to previously characterized GTPases of Rac sub-family, and was nearly identical to human Rac1 (95.3% identity). Quantitative real-time PCR demonstrated that the Rac1 was constitutively expressed in all tissues examined, but at different levels. Upon challenge with Vibrio harveyi, the expression level of Rac1 fluctuated in the liver at different time points. In the head kidney, its expression level decreased to the lowest at 4 h, and then increased to the background level at 24 h. The remarkable degree of evolutionary conservation observed in turbot Rac1 primary structure together with its changing in expression level upon challenge suggested a functionally important role for this Rho family member in the immune response.

  15. Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    Hai-Tao Wang; Jian-Ping Kong; Fang Ding; Xiu-Qin Wang; Ming-Rong Wang; Lian-Xin Liu; Min Wu; Zhi-Hua Liu

    2003-01-01

    AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1.METHODS: The authors first constructed pcDNA3.1/mychis expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes,classification was performed according to their function and cellular component.RESULTS: Human EMP-1 gene can be stably expressed in ECg706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion.CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved incell signaling, cell communication and adhesion regulators.

  16. Full length cDNA cloning and expression analysis of annexinA2 gene from deer antler tissue

    Institute of Scientific and Technical Information of China (English)

    Li Hao; Xianghong Xiao; Heping Li

    2014-01-01

    ANXA2(AnnexinA2), a calcium-dependent phospholipid bind-ing protein, is involved in various Ca2+-related biological activities. In the present study, full-length cDNA of ANXA2 was isolated from the velvet antler tip tissue of sika deer (Cervus nippon hortulorum);the amino acid sequence and gene expression was analyzed by using bioinformatics and real-time reverse transcriptase polymerase chain reaction (RT-PCR) techniques. Nucleotide sequence analysis reveals that the full-length cDNA of the ANXA2 gene was 1372 bp, of which 1020 bp was in the open-reading frame (ORF) encoding 339 amino acids; its relative mo-lecular weight was 38.3 kDa; and isoelectric point was 6.72. Sequence analysis indicates that the protein includes four conserved tan-dem-duplication ANX domains. The gene-accession nucleotide sequence number in GenBank is JX315571. Expression analysis by RT-PCR re-veals that ANXA2 gene expression has a significant positive correlation with the antler-tissue mineralization process, indicating that this gene may play an important role in the regulation of antler-tissue mineraliza-tion.

  17. Analysis of Gene Expression Profile in Lung Adenosquamous Carcinoma Using cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    YANG Fei; YANG Jiong; JIANG Man; YE Bo; ZHANG Yu-xia; CHEN Hong-lei; XIA Dong; LIU Ming-qiu

    2004-01-01

    Gene expression profile of the lung adenosquamous carcinoma was characterized by using cDNA microarray chip containing 4 096 human genes. Among target genes, 508 differentially expressed genes were identified in adenosquamous carcinoma of the lung, 232 genes were overexpressed and 276 genes were underexpressed. Among them, 92 genes are cell signals transduction genes, 34 genes are proto-oncogenes and tumor suppressor genes or cell cycle related genes or cell apoptosis related genes, 29 genes are cell skeleton genes, 28 genes are DNA synthesis, repair and recombination genes, 12 genes are DNA binding and transcription genes. These genes may be associated with the occurence and development of adenosquamous carinome of the lung.

  18. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    Science.gov (United States)

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-08-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene.

  19. ArrayExplorer, a program in Visual Basic for robust and accurate filter cDNA array analysis.

    Science.gov (United States)

    Patriotis, P C; Querec, T D; Gruver, B N; Brown, T R; Patriotis, C

    2001-10-01

    Determining the dynamics in the global regulation of gene expression holds the promise of bringing a better understanding of the processes that govern physiological cell growth regulation and its disruption during the development of disease. The advent for cDNA arrays has created the possibility for the parallel analysis of expression of thousands of genes in a given cell population, simultaneously. The level of expression of a given set of genes within the studied tissue corresponds to the intensity of a labeled cDNA probe synthesized from the studied tissue RNA and bound specifically to the cDNAs of the genes spotted on the array. The accurate extraction of gene expression intensity values is essential for further data analysis and the interpretation of the obtained results. Here, we describe a new array image-processing software developed in Microsoft Visual Basic, the ArrayExplorer, which provides a user-friendly, multiple-window interface and a number of automatic and manual features that facilitate a reliable, robust, and accurate extraction of gene intensity values from filter-array images.

  20. Analysis of cDNA libraries from developing seeds of guar (Cyamopsis tetragonoloba (L. Taub

    Directory of Open Access Journals (Sweden)

    Dixon Richard A

    2007-11-01

    Full Text Available Abstract Background Guar, Cyamopsis tetragonoloba (L. Taub, is a member of the Leguminosae (Fabaceae family and is economically the most important of the four species in the genus. The endosperm of guar seed is a rich source of mucilage or gum, which forms a viscous gel in cold water, and is used as an emulsifier, thickener and stabilizer in a wide range of foods and industrial applications. Guar gum is a galactomannan, consisting of a linear (1→4-β-linked D-mannan backbone with single-unit, (1→6-linked, α-D-galactopyranosyl side chains. To better understand regulation of guar seed development and galactomannan metabolism we created cDNA libraries and a resulting EST dataset from different developmental stages of guar seeds. Results A database of 16,476 guar seed ESTs was constructed, with 8,163 and 8,313 ESTs derived from cDNA libraries I and II, respectively. Library I was constructed from seeds at an early developmental stage (15–25 days after flowering, DAF, and library II from seeds at 30–40 DAF. Quite different sets of genes were represented in these two libraries. Approximately 27% of the clones were not similar to known sequences, suggesting that these ESTs represent novel genes or may represent non-coding RNA. The high flux of energy into carbohydrate and storage protein synthesis in guar seeds was reflected by a high representation of genes annotated as involved in signal transduction, carbohydrate metabolism, chaperone and proteolytic processes, and translation and ribosome structure. Guar unigenes involved in galactomannan metabolism were identified. Among the seed storage proteins, the most abundant contig represented a conglutin accounting for 3.7% of the total ESTs from both libraries. Conclusion The present EST collection and its annotation provide a resource for understanding guar seed biology and galactomannan metabolism.

  1. Molecular cloning and sequence analysis of hamster CENP-A cDNA

    Science.gov (United States)

    Figueroa, Javier; Pendón, Carlos; Valdivia, Manuel M

    2002-01-01

    Background The centromere is a specialized locus that mediates chromosome movement during mitosis and meiosis. This chromosomal domain comprises a uniquely packaged form of heterochromatin that acts as a nucleus for the assembly of the kinetochore a trilaminar proteinaceous structure on the surface of each chromatid at the primary constriction. Kinetochores mediate interactions with the spindle fibers of the mitotic apparatus. Centromere protein A (CENP-A) is a histone H3-like protein specifically located to the inner plate of kinetochore at active centromeres. CENP-A works as a component of specialized nucleosomes at centromeres bound to arrays of repeat satellite DNA. Results We have cloned the hamster homologue of human and mouse CENP-A. The cDNA isolated was found to contain an open reading frame encoding a polypeptide consisting of 129 amino acid residues with a C-terminal histone fold domain highly homologous to those of CENP-A and H3 sequences previously released. However, significant sequence divergence was found at the N-terminal region of hamster CENP-A that is five and eleven residues shorter than those of mouse and human respectively. Further, a human serine 7 residue, a target site for Aurora B kinase phosphorylation involved in the mechanism of cytokinesis, was not found in the hamster protein. A human autoepitope at the N-terminal region of CENP-A described in autoinmune diseases is not conserved in the hamster protein. Conclusions We have cloned the hamster cDNA for the centromeric protein CENP-A. Significant differences on protein sequence were found at the N-terminal tail of hamster CENP-A in comparison with that of human and mouse. Our results show a high degree of evolutionary divergence of kinetochore CENP-A proteins in mammals. This is related to the high diverse nucleotide repeat sequences found at the centromere DNA among species and support a current centromere model for kinetochore function and structural plasticity. PMID:12019018

  2. cDNA cloning, Phylogenic Analysis and Gene Expression Pattern of Phenylalanine ammonia-lyase in Sugarcane (Saccharum officinarum L.

    Directory of Open Access Journals (Sweden)

    Mahmoud Hashemitabar

    2014-08-01

    Full Text Available The aim of the present study was to clone and characterize a full length cDNA of sugarcane (Saccharum officinarum phenylalanine ammonia-lyase (SoPAL. Differential tissue expression pattern of the SoPAL transcript and its enzyme activity was also analyzed during the tillering stage of growth. The full-length of SoPAL cDNA was 2118 bp long and contained a protein with 706 amino acids, determined by encoding technique. The amino acid sequence and phylogenic analysis of the cloned SoPAL showed high similarity to PAL from other monocotyledonous such as sorghum (96%, maize (93% and Bamboos (87.12%. The highest levels of SoPAL transcript were observed in the root and stem, while its minimal gene expression levels were in the leaves and sheath, respectively. The highest level of SoPAL enzyme activity was in the leaves. These results helped to understanding the characteristics of PAL biosynthesis and its regulation at the molecular level in sugarcane. This information could be critical for the manipulation of phenylpropanoid biosynthesis in the plant using biotechnological processes.

  3. cDNA cloning and analysis of betaine aldehyde dehydrogenase, a salt inducible enzyme in sugar beet

    Energy Technology Data Exchange (ETDEWEB)

    McCue, K.F.; Hanson, A.D. (Michigan State Univ., East Lansing (USA))

    1990-05-01

    Betaine accumulates and serves as a compatible osmolyte in some plants subjected to drought or salinity stress. The last enzyme in the betaine biosynthetic pathway is betaine aldehyde dehydrogenase (BADH). The activity of BADH increases in response to increasing salinity levels. This increase in activity corresponds to an increase in protein detectable by immunoblotting, and to an increase in the translatable BADH mRNA. BADH was cloned from a cDNA library constructed in {lambda}gt10 using poly(A){sup +} RNA from sugar beets salinized to 500 mM NaCl. cDNAs were size selected (>1kb) before ligation into the vector, and the library was screened with a spinach BADH cDNA probe. Three nearly full length clones obtained were confirmed as BADH by their nucleotide and deduced amino acid homology to spinach BADH. Clones averaged 1.8 kb and contained open reading frames of 500 amino acids at 80% identity with spinach BADH. RNA gel blot analysis of poly(A){sup +} RNA indicated that salinization to 500 mM NaCl resulted in a 5-fold increase of BADH mRNA level.

  4. Meat speciation by restriction fragment length polymorphism analysis using an α-actin cDNA probe.

    Science.gov (United States)

    Fairbrother, K S; Hopwood, A J; Lockley, A K; Bardsley, R G

    1998-09-01

    Classical DNA fingerprinting is based on separation of DNA restriction fragments by electrophoresis and hybridisation to nucleic acid probes containing repetitive nucleotide sequences. The use of such mini- or micro-satellite probes tends to yield patterns specific to an individual rather than to a species, hence their value in forensic analysis but general unsuitability for meat speciation. In the present study, a cDNA probe based on conserved sequences contained in members of the actin multigene family has been evaluated for potential application in meat speciation. Genomic DNA was extracted from muscle and digested with BamHI before electrophoresis and hybridisation to a murine α-actin cDNA probe. Beef, pork, lamb, horse, chicken and fish DNA restriction fragments formed characteristic 'fingerprints' which were reproducible and varied sufficiently to allow discrimination even between closely-related species. However no major differences were seen between individuals of the same breed or between different breeds within a species. When DNA obtained from fresh tissue and also from meat heated at 120 °C was analysed, the gel patterns were essentially the same. An attractive feature of this approach is that it employs a single cross-reacting probe and set of conditions, and gives different patterns with all species so far studied. This simplicity suggests applications in meat speciation or related areas of biology.

  5. ISOLATION OF HUMAN TRANSCRIPTS EXPRESSED IN 16HBE CELLS RELATED TO CHLOROPHYLLIN ANTITRANSFORMING ACTIVITY AGAINST ANTI-BPDE BY cDNA REPRESENTATIONAL DIFFERENCE ANALYSIS

    Institute of Scientific and Technical Information of China (English)

    ZHU Li-jin; JIANG Yi-guo

    2005-01-01

    Objective: To analyze the differentially expressed cDNA sequences related to chlorophyllin (CHL) mediated inhibition of malignant transformation of human bronchia1 epithelial cell line (16HBE). Methods: 16HBE cells treated with chlorophyllin and anti-BPDE were conducted as tester, 16HBE cells treated only with anti-BPDE were conducted as driver, and cDNA representational difference analysis (cDNA RDA) was used to compare the differential gene expression between the two kinds of cells. The cDNA fragments were ligated to pGEM-T vector and transformed into JM109 bacteria. The plasmid DNA was sequenced and compared with database in GenBank by BLASTN. Results: Among the 5 cloned cDNA sequences, three were novel and were registered in dbEST database, two showed sequence homology to alpha-enolase and a newly found gene ribosomal protein S18/S6-like. Conclusion: These 5 cDNA sequences might play important roles in antitransforming effect of chlorophyllin.

  6. Cu,Zn superoxide dismutase: cloning and analysis of the Taenia solium gene and Taenia crassiceps cDNA.

    Science.gov (United States)

    Parra-Unda, Ricardo; Vaca-Paniagua, Felipe; Jiménez, Lucia; Landa, Abraham

    2012-01-01

    Cytosolic Cu,Zn superoxide dismutase (Cu,Zn-SOD) catalyzes the dismutation of superoxide (O(2)(-)) to oxygen and hydrogen peroxide (H(2)O(2)) and plays an important role in the establishment and survival of helminthes in their hosts. In this work, we describe the Taenia solium Cu,Zn-SOD gene (TsCu,Zn-SOD) and a Taenia crassiceps (TcCu,Zn-SOD) cDNA. TsCu,Zn-SOD gene that spans 2.841 kb, and has three exons and two introns; the splicing junctions follow the GT-AG rule. Analysis in silico of the gene revealed that the 5'-flanking region has three putative TATA and CCAAT boxes, and transcription factor binding sites for NF1 and AP1. The transcription start site was a C, located at 22 nucleotides upstream of the translation start codon (ATG). Southern blot analysis showed that TcCu,Zn-SOD and TsCu,Zn-SOD genes are encoded by a single copy. The deduced amino acid sequences of TsCu,Zn-SOD gene and TcCu,Zn-SOD cDNA reveal 98.47% of identity, and the characteristic motives, including the catalytic site and β-barrel structure of the Cu,Zn-SOD. Proteomic and immunohistochemical analysis indicated that Cu,Zn-SOD does not have isoforms, is distributed throughout the bladder wall and is concentrated in the tegument of T. solium and T. crassiceps cysticerci. Expression analysis revealed that TcCu,Zn-SOD mRNA and protein expression levels do not change in cysticerci, even upon exposure to O(2)(-) (0-3.8 nmol/min) and H(2)O(2) (0-2mM), suggesting that this gene is constitutively expressed in these parasites.

  7. Cloning and Expression Analysis of Downy Mildew Resistance-Related cDNA Sequences in Melon

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Melon downy mildew caused by Pseudoperonospora cubensis leads to significant losses in melon yields worldwide.Reverse-transcription Polymerase Chain Reaction (RT-PCR) was performed using cDNAs as templates from melonHuangdanzi induced with fungus Pseudoperonospora cubensis, and degenerate primers designed based on the conserved amino acid sequences of known plant disease-resistance genes. A polymorphic cDNA fragment which we named mp-19was cloned and sequenced. The Open Reading Frame (ORF) of this product comprised of 510 base pairs which encodes DNA or RNA-binding protein with 170 amino acids. The putative amino acid sequence of mp-19 appeared highly homologous with those of NBS-type resistant-genes isolated from other plants. Southern blot indicated that the melon genome contained more than 3 copies of mp-19. The obvious expression differences detected by semi-quantitative RTPCR could be observed between resistant-line Huangdanzi and susceptible-line Jiashi after Pseudoperonospora cubensis infection, which implied that mp-19 gene may be related to the resistance of downy mildew in melon.

  8. Construction and analysis of a subtractive cDNA library of early embryonic development in duck.

    Science.gov (United States)

    Liu, Y L; Zhong, L X; Li, J J; Shen, J D; Wang, D Q; Tao, Z R; Shi, F X; Lu, L Z

    2013-07-08

    Several studies have documented the process of early embryonic development in poultry; however, the molecular mechanisms underlying its developmental regulation are poorly understood, particularly in ducks. In this study, we analyzed differential gene expression of embryos 6 and 25 h following oviposition to determine which genes regulate the early developmental stage in ducks. Among 216 randomly selected clones, 39 protein-encoding cDNAs that function in metabolism, transcription, transportation, proliferation/apoptosis, cell cycle, cell adhesion, and methylation were identified. Additionally, the full-length cDNA of the Nanog gene, encoding a 302-amino acid protein, was obtained. Quantitative real-time polymerase chain reaction analyses were performed to detect expression levels of the selected genes during early and late embryonic stages, which revealed that these genes are expressed in a particular spatial and temporal pattern. These results indicate that these genes may play pivotal roles in the process of area pellucida formation through a complex and precise regulatory network during development in duck embryos.

  9. cDNA cloning and sequence analysis of NIb gene of soybean mosaic virus

    Institute of Scientific and Technical Information of China (English)

    刘俊君; 彭学贤; 莽克强

    1995-01-01

    cDNA of soybean mosaic virus (Beijing isolate, SMV-BJ) has been synthesized, using viralgenomic RNA as template and random hexanucleotides as primers. Based on the sequences of SMV-BJ coat protein (CP) gene as well as SMV- and WMV-II-related regions, oligonucleotides were made as primers for polymerase chain reaction (PCR). NIb gene of SMV-BJ was amplified by PCR, and cloned into pBluescript SK. The complete sequence was determined. The comparison of NIb genes between SMV-BJ and WMV-II . (USA) shows that similarities for nucleotide sequence reach 80.3%, and the deduced amino acid sequence. 91 3%. In consideration of the high identities in between the CP gene and the 3’-non-coding region between them, WMV-II might be considered as a watermelon strain of SMV Besides, some unexpected sequences were found in the 3’-region of 2 NIb gene clones. Following modification and splicing, a binary vector of NIb gene has been constructed for its expression in higher plant for the purpose of studying the possible repl

  10. Characterization and phylogenetic analysis of a cnidarian LMP X-like cDNA.

    Science.gov (United States)

    Dishaw, Larry J; Herrera, Manuel L; Bigger, Charles H

    2006-06-01

    Proteasomes are multisubunit protease complexes which are partly responsible for metabolism of intracellular, ubiquitinylated proteins. Vertebrates have adapted a second and specialized structure responsible for the generation of peptides presented to the adaptive immune system and is thus, commonly referred to as the immunoproteasome. This complex is assembled from paralogous copies of subunits belonging to the constitutive, housekeeping proteasome. The immunoproteasome is more efficient in the generation of peptides for display on major histocompatibility complex (MHC) molecules. Important components of this complex are the paralogous members, LMP X and 7; where the latter replaces the former in the assembly of the immunoproteasome of vertebrates. In this report, we describe an LMP X-like cDNA from an endosymbiont-free gorgonian coral, Swiftia exserta. Cnidarians predate the phylogenetic divergence of protostomes and deuterostomes (P-D split), and are becoming an essential model for our comprehension of immune system evolution. Phylogenetic analyses of available sequences indicates that invertebrate LMP X-like sequences are outgroups to vertebrate LMP X and LMP 7, and is in agreement with previous observations that the duplication event giving rise to the two rapidly diverging lineages of proteasomal subunits occurred before jawed fished divergence.

  11. Gene expression profile of esophageal cancer in North East India by cDNA microarray analysis

    Institute of Scientific and Technical Information of China (English)

    Indranil Chattopadhyay; Sujala Kapur; Joydeep Purkayastha; Rupkumar Phukan; Amal Kataki; Jagadish Mahanta; Sunita Saxena

    2007-01-01

    AIM: To identify alterations in genes and molecular functional pathways in esophageal cancer in a high incidence region of India where there is a widespread use of tobacco and betel quid with fermented areca nuts.METHODS: Total RNA was isolated from tumor and matched normal tissue of 16 patients with esophageal squamous cell carcinoma. Pooled tumor tissue RNA was labeled with Cy3-dUTP and pooled normal tissue RNA was labeled with Cy5-dUTP by direct labeling method.The labeled probes were hybridized with human 10K cDNA chip and expression profiles were analyzed by Genespring GX V 7.3 (Silicon Genetics).RESULTS: Nine hundred twenty three genes were differentially expressed. Of these, 611 genes were upregulated and 312 genes were downregulated. Using stringent criteria (P ≤ 0.05 and ≥ 1.5 fold change),127 differentially expressed genes (87 upregulated and 40 downregulated) were identified in tumor tissue. On the basis of Gene Ontology, four different molecular functional pathways (MAPK pathway,G-protein coupled receptor family, ion transport activity,and serine or threonine kinase activity) were most significantly upregulated and six different molecular functional pathways (structural constituent of ribosome,endopeptidase inhibitor activity, structural constituent of cytoskeleton, antioxidant activity, acyl group transferase activity, eukaryotic translation elongation factor activity)were most significantly downregulated.CONCLUSION: Several genes that showed alterations in our study have also been reported from a high incidence area of esophageal cancer in China. This indicates that molecular profiles of esophageal cancer in these two different geographic locations are highly consistent.

  12. CDNA microarray analysis of gene expression patterns in blood mononuclear cells of SLA-DRB1-defined Yorkshire pigs.

    Science.gov (United States)

    Nino-Soto, M I; Jozani, R J; Bridle, B; Mallard, B A

    2008-01-01

    Three lines of commercialYorkshire pigs with defined SLA-DRB1 alleles were developed at the University of Guelph for xenotransplantation and immune response studies. Two of the SLA-DRB1 alleles have been previously reported (SLA-DRB1*0502 and *0701), whereas the third one is a new allele. The influence of defined SLA-DRB1 alleles on transcriptional patterns of immune-related genes in blood mononuclear cells (BMCs) of pigs was explored using cDNA microarray. Microarray analysis showed significant differential expression of inflammatory genes in association with the various SLA-DRB1 alleles. A better understanding of the association between SLA genotypes and gene activity can increase the knowledge of the function of these molecules, as well as define new strategies to control animal health and optimize animal production.

  13. Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis.

    Directory of Open Access Journals (Sweden)

    Amparo Galán

    Full Text Available Blastomere fate and embryonic genome activation (EGA during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM (n = 120, stemness (n = 190 and Trophectoderm (TE (n = 45, were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1, stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT, and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR. The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92 such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2 and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4, as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented.

  14. Fiscal 1999 achievement report on the analysis of the complete sequencing of full-length cDNA; 1999 nendo dai 2 ji hosei yosan kanzencho cDNA kozo kaiseki seika hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    This report accommodates the results of study conducted during the period of April 1, 2000, through March 31, 2001. The study began with the partial sequence determination for cDNA (complementary deoxyribonucleic acid) terminals presented by the cDNA library, novel clones were then selected out of them, and efforts proceeded to sequence all the bases therein. In this study, partial sequences were determined for 519,000 clones, and entire sequences for 7252 clones. The obtained sequence data were subjected to a homological analysis and then converted into an amino acid sequence, and then protein function prediction and the like were performed using the SOSUI program or the like. A prototype system to isolate novel clones out of partial sequences and a system for the graphic display of cDNA-genome links were fabricated. As for expression profile databases, the iAFLP (introduced amplified fragment length polymorphism) method was used to construct a high-throughput system and a function analysis database. (NEDO)

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

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Marcelo Bento (New York, NY); Bonaldo, Maria de Fatima (New York, NY)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Soares, M.B.; Fatima Bonaldo, M. de

    1998-12-08

    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. 25 figs.

  17. Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences.

    Science.gov (United States)

    Schmidt, W M; Mueller, M W

    1996-05-01

    Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase is a polymerase chain reaction (PCR)-mediated technique that was developed to facilitate cloning and direct sequence analysis of complete 5'-terminal unknown coding regions of rare RNA molecules. In contrast with standard tailing protocols using dNTPs as the substrate, ribo-tailing of cDNA ends is easily controllable, self-limited (from two to four rNMP incorporations) and highly efficient (>98%). By virtue of the homopolymeric ribo-tail, the modified cDNA is anchored to the 3' overhang of a double-stranded DNA-adaptor in a T4 DNA ligase-dependent ligation. PCR amplification, mediated by two sequence-specific primers, yields the desired unique product suitable for cloning and dideoxy-sequencing.

  18. Identification and expression analysis of a full-length cDNA encoding a Kandelia candel tonoplast intrinsic protein.

    Science.gov (United States)

    Huang, Wei; Fang, Xiao-Dong; Lin, Qi-Fen; Li, Guan-Yi; Zhao, Wen-Ming

    2003-03-01

    Soil salinity is an important issue, as most crop plants are low in salt tolerance. Salt tolerance, a complex, multifactorial, and multigenic process, has been known to be a quantitative trait. The identification of the salt stress responsive genes or salt tolerance genes is essential for the breeding programs. Most recent efforts have been focused on the products of structural genes (transport proteins, ion channels, enzymes of solute synthesis) while little attention were paid to the regulatory aspects of these proteins. Since the first aquaporin gene from plants was cloned and functionally expressed in 1993, there has been a growing interest in the molecular biology of MIPs (membrane intrinsic proteins) and their bearing on the biophysics of water flow across plant membranes. In the last decades, studies on Mangroves, a special kind of wood plants, grow in high-salt and flooding conditions have been concentrated almost exclusively on their physiological and ecological characteristics. Kandelia candel, one of the dominant species of mangroves along the Chinese coast, lacks salt glands or salt hairs used for removal of excess salt in other mangroves. This makes K. candel a perfect model to study the molecular mechanism of salt tolerance in mangrove plants. Using cDNA RDA, a cDNA-specific modification of genomic representational difference analysis, a series of salt responsive genes of Kandelia candel were cloned. Among these gene fragments, a 183 bp fragment (termed as SRGKC1) encoding a tonoplast intrinsic protein (TIP) in Kandelia candel (KCTIP1) was identified. Based on the sequence of SRGKC1, two gene specific primers were designed, and the 3' and 5' end of the KCTIP1 gene were obtained using the SMART RACE cDNA Amplification Kit. RACE products were purified from low-melting agarose, and sequenced directly with GSPs as the sequencing primers. A 500-bp fragment corresponding to the 3'end of this gene was obtained using the GSP1 primer, and a 690 bp fragment

  19. Transcriptome generation and analysis from spleen of Indian catfish, Clarias batrachus (Linnaeus, 1758) through normalized cDNA library.

    Science.gov (United States)

    Singh, Akanksha; Barman, A S; Sood, Neeraj; Mohindra, Vindhya

    2013-12-01

    Catfishes are commercially important fish for both the fisheries and aquaculture industry. Clarias batrachus, an Indian catfish species is economically important owing to its high demand. A normalized cDNA library was constructed from spleen of the Indian catfish to identify genes associated with immune function. One thousand nine hundred thirty seven ESTs were submitted to the GenBank with an average read length of approximately 700 bp. Clustering analysis of ESTs yielded 1,698 unique sequences, including 184 contigs and 1,514 singletons. Significant homology to known genes was found by homology searches against data in GenBank in 576 (34 %) ESTs, including similarity to functionally annotated unigenes for 158 ESTs. Additionally, 433 ESTs revealed similarity to unigenes and ESTs in the dbEST but the remaining 658 EST sequences (39 %) did not match any sequence in GenBank. Of a total of 1,698 ESTs generated, 65 ESTs were found to be associated with immune functions. Gene Ontology and KEGG pathway analyses of C. batrachus ESTs collectively revealed a preponderance of immune relevant pathways apart from the presence of pathways involved in protein processing, localization, folding and protein degradation. This study constitutes first EST analysis of lymphoid organ in aquaculturally important Indian catfish species and could pave the way for further research of immune-related genes and functional genomics in this catfish.

  20. Generation and analysis of expressed sequence tags from a cDNA library of the fruiting body of Ganoderma lucidum

    Directory of Open Access Journals (Sweden)

    Li Xiwen

    2010-03-01

    Full Text Available Abstract Background Little genomic or trancriptomic information on Ganoderma lucidum (Lingzhi is known. This study aims to discover the transcripts involved in secondary metabolite biosynthesis and developmental regulation of G. lucidum using an expressed sequence tag (EST library. Methods A cDNA library was constructed from the G. lucidum fruiting body. Its high-quality ESTs were assembled into unique sequences with contigs and singletons. The unique sequences were annotated according to sequence similarities to genes or proteins available in public databases. The detection of simple sequence repeats (SSRs was preformed by online analysis. Results A total of 1,023 clones were randomly selected from the G. lucidum library and sequenced, yielding 879 high-quality ESTs. These ESTs showed similarities to a diverse range of genes. The sequences encoding squalene epoxidase (SE and farnesyl-diphosphate synthase (FPS were identified in this EST collection. Several candidate genes, such as hydrophobin, MOB2, profilin and PHO84 were detected for the first time in G. lucidum. Thirteen (13 potential SSR-motif microsatellite loci were also identified. Conclusion The present study demonstrates a successful application of EST analysis in the discovery of transcripts involved in the secondary metabolite biosynthesis and the developmental regulation of G. lucidum.

  1. cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period

    Directory of Open Access Journals (Sweden)

    Tokunaga Tomoyuki

    2004-11-01

    Full Text Available Abstract Background After fertilization, embryo development involves differentiation, as well as development of the fetal body and extra-embryonic tissues until the moment of implantation. During this period various cellular and molecular changes take place with a genetic origin, e.g. the elongation of embryonic tissues, cell-cell contact between the mother and the embryo and placentation. To identify genetic profiles and search for new candidate molecules involved during this period, embryonic gene expression was analyzed with a custom designed utero-placental complementary DNA (cDNA microarray. Methods Bovine embryos on days 7, 14 and 21, extra-embryonic membranes on day 28 and fetuses on days 28 were collected to represent early embryo, elongating embryo, pre-implantation embryo, post-implantation extra-embryonic membrane and fetus, respectively. Gene expression at these different time points was analyzed using our cDNA microarray. Two clustering algorithms such as k-means and hierarchical clustering methods identified the expression patterns of differentially expressed genes across pre-implantation period. Novel candidate genes were confirmed by real-time RT-PCR. Results In total, 1,773 individual genes were analyzed by complete k-means clustering. Comparison of day 7 and day 14 revealed most genes increased during this period, and a small number of genes exhibiting altered expression decreased as gestation progressed. Clustering analysis demonstrated that trophoblast-cell-specific molecules such as placental lactogens (PLs, prolactin-related proteins (PRPs, interferon-tau, and adhesion molecules apparently all play pivotal roles in the preparation needed for implantation, since their expression was remarkably enhanced during the pre-implantation period. The hierarchical clustering analysis and RT-PCR data revealed new functional roles for certain known genes (dickkopf-1, NPM, etc as well as novel candidate genes (AW464053, AW465434, AW

  2. 虹鳟 Ndufb2基因全长 cDNA 序列的克隆与分析%Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss

    Institute of Scientific and Technical Information of China (English)

    王家庆; 边佳; 李代宗; 马爽; 王亮; 那广宁

    2013-01-01

    protein kinase phosphorylation sites were predicted using online software NetPhosK 1 and NetPhos 2. Sequence analysis results showed that the rainbow trout Ndufb2 cDNA was 899 bp in length,5'untranslated region(5'UTR) 1 52 bp,3'untranslated region(3'UTR)441 bp,open reading frame(ORF) 306 bp,encoding 101 amino acids.The protein molecular mass was 1 1.4 ku and isoelectric point was 5.31.This gene sequence had been submitted to the GenBank database (accession number:FJ534641).Mitochondrial target sequence of 50 amino acid motif appeared from the 1st to 50th amino acid site.Two antigenic sites (65-ILWHCWHDPD-74 and 23-QKIVIRK-29) of the Ndufb2 protein were found,and one serine phosphorylation site (aa49),two threonine phosphorylation sites (aa1 5,aa47) and seven kinase phosphorylation sites were also found.Sequence alignment exhibited 98% identity of amino acids between O.mykiss and Salmo salar ,and more than 55% identity of amino acids between O.mykiss and mammal.Phylogenetic tree showed that the Ndufb2 of rainbow trout had the closest relationship with those of Atlantic salmon and zebrafish,then the amphibians,birds,marsupials and mammals. The phylogenetic analysis showed that the constructed Ndufb2 protein phylogenetic tree was consistent with the traditional species classification tree. The above results indicate that the Ndufb2 gene is relatively conservative in the progress of evolution and play an important role in electron transport process of mitochondrial respiratory chain.The cloned full-length cDNA sequence of mitochondrial respiratory chain gene Ndufb2 of rainbow trout lays a theoretical foundation for the structure and function of clarifying fish mitochondrial complex I.%从虹鳟鱼(Oncorhynchus mykiss)的脑组织中提取 RNA,经逆转录聚合酶链反应及 cDNA 末端快速扩增技术克隆出 Ndufb2基因全长 cDNA 序列(GenBank 登录号:FJ534641),并对其序列进行分析。扩增结果表明:Ndufb2基因的 cDNA 序列全长899 bp

  3. Construction and quality analysis of cDNA library from flower buds of spring soybean cultivars%春大豆花芽 cDNA 文库的构建及质量分析

    Institute of Scientific and Technical Information of China (English)

    王楠; 周莹; 姚丹; 尹俊琦; 曲静; 王丕武

    2014-01-01

    [目的]为了解大豆多荚、多粒相关基因的调控机制,构建了大豆花芽cDNA文库,根据要求初步鉴定了所构建文库的质量.[方法]以大豆吉农18突变体的幼嫩花芽为材料提取总RNA,采用SMART技术合成双链cDNA.经蛋白酶K的消化及SfiⅠ酶切后,将所得cDNA克隆到λTriplEx质粒载体中,成功构建了大豆花芽全长cDNA文库.[结果和结论]将初始文库经扩增后保存,检测扩增文库滴度为2.13×108 pfu/mL,重组率接近95.3%,菌落PCR鉴定插入片段主要分布在0.5~2.0 kb .插入片段平均大小在1.0 kb左右.表明本研究所构建的文库既满足了目的基因的分离筛选,又可保证全长cDNA文库的获得,该文库的构建为进一步开展相关基因的克隆及分子生物学研究奠定基础.%[Objective] To understand soybean pods and multigrain gene regulation mechanisms of impro-ving soybean production , a soybean flower bud cDNA library was constructed .The quality of library con-struction was initially identified according to the requirements .[Method]In order to study the novel genes from flower bud mutants of soybean , a full-length cDNA library from flower bud mutants of soybean Jinong 18 was constructed .Total RNA from young flower bud mutants of soybean was extracted .Double strand cDNA was synthesized by SMART method .After proteinase K digestion and SfiⅠ digestion , the ds cDNA fragments were ligated to the λTriplEx vector .A cDNA library of soybean was successfully con-structed .[Result and conclusion]With the unamplified library stored after amplification , the titer of the amplified library was estimated as 2.13 ×108 pfu/mL and the recombination rate was approximately 95.3%.PCR results showed that the inserts varied from 0.5 to 2.0 kb with an average size of 1.0 kb or so.It indicated that this library could be used for full-length genes screening and cloning of low abundance genes.The library will lay a

  4. Targeted rapid amplification of cDNA ends (T-RACE)—an improved RACE reaction through degradation of non-target sequences

    OpenAIRE

    Neil I Bower; Johnston, Ian A

    2010-01-01

    Amplification of the 5' ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3' end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-contain...

  5. Cloning and expression of the cDNA encoding the FXPRL family of peptides and a functional analysis of their effect on breaking pupal diapause in Helicoverpa armigera.

    Science.gov (United States)

    Zhang, Tian-Yi; Sun, Jiu-Song; Zhang, Liu-Bin; Shen, Jin-Liang; Xu, Wei-Hua

    2004-01-01

    Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are encoded by a single mRNA in the suboesophegeal ganglion (SG) and are responsible for induction of embryonic diapause in Bombyx mori and sex pheromone biosynthesis in lepidopteran insects. PBAN cDNA analyses revealed that the DH-like peptide is present in several species that have a pupal diapause. However, the function of the DH-like peptide remains unknown. In the present study, we cloned the cDNA encoding DH-PBAN in Helicoverpa armigera utilizing the rapid amplification of the cDNA ends method. The nucleotide se quence analysis revealed that the longest open reading frame of this cDNA encodes a 194-amino acid precursor protein that con tains a 33-aa PBAN, a 24-aa DH-like peptide, and three other neuropeptides, all of which have a common C-terminal pentapeptide motif FXPR/KL ( X=G, T, S). A homology search showed that H. armigera DH-like and PBAN are highly homologous to those from other insects. Northern blot analysis demonstrated a single message RNA corresponding to the size of Har-DH-PBAN cDNA from pupal SG with significantly higher expression in the SG of nondiapause pupae than diapausing pupae. Western blot analysis showed DH-like peptide expression from SG of both males and females. When DH-like peptide was injected into nondiapause larvae and pupae, it did not induce diapause, but rather efficiently broke pupal diapause in H. armigera. The ED(50) of DH to terminate pupal diapause is 20 pmol/pupae. The other four FXPRLamide neuropeptides from the DH-PBAN polyprotein precursor have cross activity for diapause termination. These observations therefore suggest a potential role for these FXPRL family peptides in promoting continuous development in several noctuid species. The high expression of this gene in pharate adults and adults indicates that the FXPRL family peptides may have multiple physiological functions.

  6. A combined de novo protein sequencing and cDNA library approach to the venomic analysis of Chinese spider Araneus ventricosus.

    Science.gov (United States)

    Duan, Zhigui; Cao, Rui; Jiang, Liping; Liang, Songping

    2013-01-14

    In past years, spider venoms have attracted increasing attention due to their extraordinary chemical and pharmacological diversity. The recently popularized proteomic method highly improved our ability to analyze the proteins in the venom. However, the lack of information about isolated venom proteins sequences dramatically limits the ability to confidently identify venom proteins. In the present paper, the venom from Araneus ventricosus was analyzed using two complementary approaches: 2-DE/Shotgun-LC-MS/MS coupled to MASCOT search and 2-DE/Shotgun-LC-MS/MS coupled to manual de novo sequencing followed by local venom protein database (LVPD) search. The LVPD was constructed with toxin-like protein sequences obtained from the analysis of cDNA library from A. ventricosus venom glands. Our results indicate that a total of 130 toxin-like protein sequences were unambiguously identified by manual de novo sequencing coupled to LVPD search, accounting for 86.67% of all toxin-like proteins in LVPD. Thus manual de novo sequencing coupled to LVPD search was proved an extremely effective approach for the analysis of venom proteins. In addition, the approach displays impeccable advantage in validating mutant positions of isoforms from the same toxin-like family. Intriguingly, methyl esterifcation of glutamic acid was discovered for the first time in animal venom proteins by manual de novo sequencing.

  7. Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis.

    Science.gov (United States)

    Rho, Jaerang; Altmann, Curtis R; Socci, Nicholas D; Merkov, Lubomir; Kim, Nacksung; So, Hongseob; Lee, Okbok; Takami, Masamichi; Brivanlou, Ali H; Choi, Yongwon

    2002-08-01

    Bone homeostasis is maintained by the balanced action of bone-forming osteoblasts and bone-resorbing osteoclasts. Multinucleated, mature osteoclasts develop from hematopoietic stem cells via the monocyte-macrophage lineage, which also give rise to macrophages and dendritic cells. Despite their distinct physiologic roles in bone and the immune system, these cell types share many molecular and biochemical features. To provide insights into how osteoclasts differentiate and function to control bone metabolism, we employed a systematic approach to profile patterns of osteoclast-specific gene expression by combining suppression subtractive hybridization (SSH) and cDNA microarray analysis. Here we examined how gene expression profiles of mature osteoclast differ from macrophage or dendritic cells, how gene expression profiles change during osteoclast differentiation, and how Mitf, a transcription factor critical for osteoclast maturation, affects the gene expression profile. This approach revealed a set of genes coordinately regulated for osteoclast function, some of which have previously been implicated in several bone diseases in humans.

  8. Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

    Energy Technology Data Exchange (ETDEWEB)

    Morin, R D; Chang, E; Petrescu, A; Liao, N; Kirkpatrick, R; Griffith, M; Butterfield, Y; Stott, J; Barber, S; Babakaiff, R; Matsuo, C; Wong, D; Yang, G; Smailus, D; Brown-John, M; Mayo, M; Beland, J; Gibson, S; Olson, T; Tsai, M; Featherstone, R; Chand, S; Siddiqui, A; Jang, W; Lee, E; Klein, S; Prange, C; Myers, R M; Green, E D; Wagner, L; Gerhard, D; Marra, M; Jones, S M; Holt, R

    2005-10-31

    Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence between the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.

  9. Identification of Novel Stress-responsive Transcription Factor Genes in Rice by cDNA Array Analysis

    Institute of Scientific and Technical Information of China (English)

    Cong-Qing Wu; Hong-Hong Hu; Ya Zeng; Da-Cheng Liang; Ka-Bin Xie; Jian-Wei Zhang; Zhao-Hui Chu; Li-Zhong Xiong

    2006-01-01

    Numerous studies have shown that array of transcription factors has a role in regulating plant responses to environmental stresses. Only a small portion of them however, have been identified or characterized.More than 2 300 putative transcription factors were predicted in the rice genome and more than half of them were supported by expressed sequences. With an attempt to identify novel transcription factors involved in the stress responses, a cDNA array containing 753 putative rice transcription factors was generated to analyze the transcript profiles of these genes under drought and salinity stresses and abscisic acid treatment at seedling stage of rice. About 80% of these transcription factors showed detectable levels of transcript in seedling leaves. A total of 18 up-regulated transcription factors and 29 down-regulated transcription factors were detected with the folds of changes from 2.0 to 20.5 in at least one stress treatment.Most of these stress-responsive genes have not been reported and the expression patterns for five genes under stress conditions were further analyzed by RNA gel blot analysis. These novel stress-responsive transcription factors provide new opportunities to study the regulation of gene expression in plants under stress conditions.

  10. 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

    DNA 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......, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha......'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human...

  11. Construction and preliminary analysis of a normalized cDNA library from Locusta migratoria manilensis topically infected with Metarhizium anisopliae var. acridum.

    Science.gov (United States)

    Wang, Jie; Xia, Yuxian

    2010-08-01

    The insect immune response to fungal infection is poorly understood at the molecular level. To explore the molecular basis of this process, a novel method to analyze the gene transcripts of insects in response to pathogenic fungus was established. A normalized cDNA library based on the SMART method combined with DSN (duplex-specific nuclease) treatment was constructed using mRNA extracted from the fat body and hemocytes of Locusta migratoria manilensis 6-24h after being topically infected with Metarhizium anisopliae var. acridum. Analysis of 259 unigenes out of 303 sequenced inserts from the cDNA library revealed that the cDNA library was not contaminated with M. anisopliae transcripts and validated the presence of the immune-related genes characterized here. These results suggest that this method overcame the difficulties of contamination from a fungal source in constructing the host cDNA library from mycosed insects and proved that this method is reliable and feasible for investigation of host genes in response to fungal infection. Further studies of the expressed sequence tags from this library will provide insights into the molecular basis of insect immune response to fungal infection.

  12. Targeted rapid amplification of cDNA ends (T-RACE)--an improved RACE reaction through degradation of non-target sequences.

    Science.gov (United States)

    Bower, Neil I; Johnston, Ian A

    2010-11-01

    Amplification of the 5' ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3' end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-containing primers complimentary to the adapter sequences incorporated in the cDNA ends. The double-stranded cDNA-containing dUTP serves as a universal template for the specific amplification of the 3' or 5' end of any gene. To amplify the ends of cDNA, asymmetric PCR is performed using a single gene-specific primer and standard dNTPs. The asymmetric PCR product is purified and non-target transcripts containing dUTP degraded by Uracil DNA glycosylase, leaving only those transcripts produced during the asymmetric PCR. Subsequent PCR using a nested gene-specific primer and the 3' or 5' T-RACE primer results in specific amplification of cDNA ends. This method can be used to specifically amplify the 3' and 5' ends of numerous cDNAs from a single cDNA synthesis reaction.

  13. Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Two keratin-like proteins of 64 and 55 ku were purified from suspension cells of Daucus carota L.,and their partial amino acid sequences were determined.The homological analysis showed that the sequence from the 64 ku protein was highly homological to b -glucosidase,and that from the 55 ku protein had no significant homologue in GenBank.Using conservative sequence of animal IF proteins as primer,we cloned a cDNA fragment from Daucus carota L.Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves.Homological analysis revealed that it had moderate homology to a variety of a -helical proteins.Our results might shed more light on molecular characterization of IF existence in higher plant.

  14. Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

    Institute of Scientific and Technical Information of China (English)

    赵大中; 陈丹英; 杨橙; 翟中和

    2000-01-01

    Two keratin-like proteins of 64 and 55 ku were purified from suspension cells of Caucus carota L, and their partial amino acid sequences were determined. The homological analysis showed that the sequence from the 64 ku protein was highly homological to p-glucosidase, and that from the 55 ku protein had no significant homologue in GenBank. Using conservative sequence of animal IF proteins as primer, we cloned a cDNA fragment from Daucus carota L. Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves. Homological analysis revealed that it had moderate homology to a variety of a-helical proteins. Our results might shed more light on molecular characterization of IF existence in higher plant.

  15. 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.

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

    Indian Academy of Sciences (India)

    Xiaohua Xia; Jie Zhao; Qiyan Du; Zhongjie Chang

    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.

  17. Identification of variations of gene expression of visceral adipose and renal tissue in type 2 diabetic rats using cDNA representational difference analysis

    Institute of Scientific and Technical Information of China (English)

    杨架林; 李果; 张芳林; 刘优萍; 张迪; 周文中; 许光武; 杨义生; 罗敏

    2003-01-01

    Objectives To identify differences in gene expression in renal and visceral adipose tissue in type 2 diabetic rats using cDNA representational difference analysis (RDA) and to explore the molecular pathogenesis of type 2 diabetes and its chronic vascular complications.Methods A rat model of type 2 diabetes was generated by administration of a high fat and calorie diet combined with a low dose of streptozocin (STZ) injected into the tail vein. The difference bands were generated by cDNA representational difference analysis (cDNA RDA). The final difference products were ligated into the pUC-18 vector and sequenced. A bioformatics analysis was performed on the obtained expressed sequence tags (ESTs), and then the expression levels of known and novel genes were verified by semi-quantitative reverse transcription-PCR (RT-PCR). At the same time, full-length cDNA of a novel gene was cloned in silico.Results The type 2 diabetic rats in this experiment experienced hyperglycemia, lipidemia, lower insulin sensitivity and normal body weight. We obtained 9 novel ESTs and 2 novel genes from renal tissue of rats and 6 novel ESTs and 1 known gene, the rat lipoprotein lipase (LPL) gene from their visceral adipose tissue. The 2 novel genes (RS91 and RS2) from the renal tissue were both very similar to serine (or cysteine) proteinase inhibitor, clade F and eukaryotic translation initiation factor 3 and subunit 5 (EIF-3 epsilon). The expression of both novel genes and the LPL gene were upregulated in renal and visceral adipose tissue of type 2 diabetic and fat-enriched rats. Full-length cDNA of the novel gene RS91 was cloned in silico.Conclusions① The rat model of type 2 diabetes generated in this study was ideal because the disease in the animals closely mimicked type 2 diabetic patients ② cDNA RDA is a flexible, inexpensive, more accurate, sensitive and highly effective technique for identifying differences in gene expression ③ Six novel ESTs and 1 known gene were obtained

  18. 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.

  19. Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases

    NARCIS (Netherlands)

    Lücker, J.; El Tamer, 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 pe

  20. Giardia canis: ultrastructural analysis of G. canis trophozoites transfected with full length G. canis virus cDNA transcripts

    Science.gov (United States)

    Giardia canis virus (GCV) is a double-stranded RNA (dsRNA) virus of the family Totiviridae. In this study, the full-length cDNA of the G. canis virus was constructed in pPoly2/sfinot vector and RNA was transcribed in vitro. Virus-free G. canis trophozoites were transfected with in vitro transcribed ...

  1. 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.

  2. Cloning and Sequence Analysis of Interleukin 10 (IL-10) Full-length cDNA from Cyprinus carpio L.

    Institute of Scientific and Technical Information of China (English)

    Xiangru FENG; Yilong CHEN; Xiao ZHAO; Wendong WANG; Junhui ZHANG; Zhenguo YANG SUN; Shengmei JIA; Qiang LU

    2012-01-01

    Abstract [Objective] This study aimed to obtain IL-IO (interleukin 10) full-length cD- NA of common carp (Cyprinus carpio L.) and conduct the sequence analysis. []~lethod] The differentially expressed cDNA fragment was obtained by DD-RTPCR (differential display RT-PCR). The cDNA library of peripheral blood leukocytes which were separated from common carp and stimulated by mitogen was screened with a probe labeled with DIG (digoxigenin). The IL-IO full-length cDNA was cloned from 0.8x104 pfu of recombinant phages, and the sequence analysis and homology com- parison were carried out. [Result] Sequence analysis indicated that the IL-IO full- length cDNA of common carp was 1 117 bp long, containing a.55 bp 5'-UTR, a 522 bp 3"-UTR, and a 540 bp open reading frame(ORF) encoding 179 amino acids. In addition, there were three mRNA instability motifs (ATTTA) in the 3"-untranslated region. The deduced protein sequence shared typical sequence features of the IL-IO family. Homology comparison indicated that the obtained sequence shared 89.1% homology with the carp IL-IO gene from GenBank. [Conclusion] This study laid foun- dation for further study of the expression manner, functional characteristic and regu- lation mechanism of IL-IO in vivo and the interaction mechanism in the inflammatory reaction and immune response.

  3. Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 (HSC70) in Fenneropenaeus chinensis

    Institute of Scientific and Technical Information of China (English)

    JIAO Chuanzhen; WANG Zaizhao; LI Fuhua; ZHANG Chengsong; XIANG Jianhai

    2004-01-01

    The cDNA encoding hsc70 of Chinese shrimp Fenneropenaeus chinensis was cloned from hepatopancreas by RT-PCR based on its EST sequence. The full length cDNA of 2090 bp contained an open reading frame of 1956 nucleotides and partial 5′- and 3′-untranslated region(5′- and 3′-UTR). PCR amplification and sequencing analysis showed the existence of introns in the region of 1-547 bp, but they did not exist in the region of 548-2090 bp of hsc70 cDNA. When the deduced 652 amino acid sequence of HSC70 was compared with the members of HSP70 family from other organisms, the results showed 85.9% similarity with HSC71 from Oncorhynchus mykiss and HSC70 from Homo sapiens. It also exhibited 85.8% similarity with HSP70 from Mus musculu and 85.4% with HSC70 from Manduca sexta. Expression analysis showed that hsc70 mRNA was espressed constitutively in hepatopancreas, muscle, eyestalks, haemocytes, heart, ovary, intestine and gills in Fenneropenaeus chinensis. No difference could be detected on hsc70 mRNA level in muscle between heat-shocked and control animals.

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

    Science.gov (United States)

    Shi, Shuobo; Ji, Haichuan; Siewers, Verena; Nielsen, Jens

    2016-02-01

    Biological production of fatty acid (FA)-derived products has gained increasing attention to replace petroleum-based fuels and chemicals. FA biosynthesis is highly regulated, and usually it is challenging to design rational engineering strategies. In addition, the conventional 'one sample at a time' method for lipid determination is time consuming and laborious, and it is difficult to screen large numbers of samples. Here, a method for detecting free FAs in viable cells using Nile red staining was developed for use in large-scale screening. Following optimization of the method, it was used 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, malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel 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.

  5. Improved rapid amplification of cDNA ends (RACE) for mapping both the 5' and 3' terminal sequences of paramyxovirus genomes.

    Science.gov (United States)

    Li, Zhuo; Yu, Meng; Zhang, Hong; Wang, Hai-Yan; Wang, Lin-Fa

    2005-12-01

    Rapid amplification of cDNA ends (RACE) is a powerful PCR-based technique for determination of RNA terminal sequences. However, most of the RACE methods reported in the literature are developed specifically for the mapping of eukaryotic transcripts with 3' poly-A tail and 5' cap structure. In this study, an improved RACE strategy was developed which allows both 5' and 3' RACE of paramyxovirus genomic RNA using the same set of common molecular biology reagents without having to rely on expensive RACE kits. Mapping of RNA genome terminal sequences is an essential part of characterizing novel paramyxoviruses since these sequences contain important signals for genome replication and transcription, and are important molecular markers for studying virus evolution. The usefulness of this strategy was demonstrated by rapid characterization of both genome ends for a novel paramyxovirus recently isolated from human kidney primary cells. The RACE strategy described in this paper is simple, cost-effective and can be used to map genome ends of any RNA viruses.

  6. 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.

  7. Analysis of gene expression patterns with cDNA micro-array during late stage of spermatogenesis in mice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The differentiation process of round spermatids to spermatozoa during the late stage of spermatogenesis is called spermiogenesis. To explore spermiogenesis-related genes, cDNA microarray was used to study expression patterns of 1176 genes in pachytene spermatocytes, round spermatids and elongating spermatids of Balb/c mice. The results showed that 208 genes were detected in all the three cell types. Most of them were down-regulated from pachytene spermatocytes to round spermatids and elongating spermatids. However, up-regulation of 7 genes expression in round spermatids and 3 genes in elongating spermatids were found. Expression of 7 differentially expressed genes in cDNA arrays was further confirmed by semi-quantitative RT-PCR study. The RT-PCR results indicated that the expression of 6 genes was consistent with that in cDNA arrays, only one gene did not show differential expression by RT-PCR. These results may provide important clues for studying of expression, regulation, and function of spermiogenesis-related genes.

  8. Immunological responses of turbot (Psetta maxima) to nodavirus infection or polyriboinosinic polyribocytidylic acid (pIC) stimulation, using expressed sequence tags (ESTs) analysis and cDNA microarrays.

    Science.gov (United States)

    Park, Kyoung C; Osborne, Jane A; Montes, Ariana; Dios, Sonia; Nerland, Audun H; Novoa, Beatriz; Figueras, Antonio; Brown, Laura L; Johnson, Stewart C

    2009-01-01

    To investigate the immunological responses of turbot to nodavirus infection or pIC stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirus-infected fish and examined the differential gene expression within turbot kidney in response to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were experimentally infected with nodavirus and samples of each tissue were collected at selected time points post-infection. Using equal amount of total RNA at each sampling time, we made three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173 (98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568 (80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768 unique genes were identified. The two largest groups resulting from the classification of ESTs according to function were the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes). Using these clones, a 1920 element cDNA microarray was constructed and used to investigate the differential gene expression within turbot in response to experimental nodavirus infection or pIC stimulation. Kidney tissue was collected at selected times post-infection (HPI) or stimulation (HPS), and total RNA was isolated for microarray analysis. Of the 1920 genes studied on the microarray, we identified a total of 121 differentially expressed genes in the kidney: 94 genes from nodavirus-infected animals and 79 genes from those stimulated with pIC. Within the nodavirus-infected fish we observed the highest number of differentially expressed genes at 24 HPI. Our results indicate that certain genes in turbot have important roles in immune responses to nodavirus infection and dsRNA stimulation.

  9. Cloning and Analysis of Full-Length cDNA of PumNPR1 Gene from Pyrus ussuriensis Maxim

    Institute of Scientific and Technical Information of China (English)

    CHE Daidi; FAN Jinping; WANG Jingang; XU Ping; YANG Tao; LIU Shenkui

    2008-01-01

    The purpose of this study is to find a new gene resource for the researches of molecular breeding of Rosaceae plants disease-resistance. Pyrus ussuriensis Maxim is used as a starting material to clone the full-length cDNA of NPR1(nonexpressor of pathogenesis- related genes 1) which is a key regulator in SA (salicylic acid)-mediated systemic acquired resistance (SAR) by homologous cloning and RACE techniques. The length of the cDNA sequence was 1 767 bp, the ORF was 1 761 bp, it coded 586 amino acids, pI=5.58, the relative molecular weight was 65.009 ku, contained 19 kinds of amino acids, and had full BTB/POZ and ANK domains. Compared the homology of NPR1 gene in GenBank database, the homology with Pyrus pyrifolia, Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum, Oryza sativa, Helianthus annuus were 98%, 62%, 68%, 65%, 57%, 63%. The homology of functional area were 99%, 78%, 82%, 79%, 74%, 77%. This NPR1 gene was considered as homologic gene of Pyrus ussuriensis Maxim and named PumNPR1.

  10. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    Energy Technology Data Exchange (ETDEWEB)

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-04-01

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone lambdaHB''-1 from a phage lambdagt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambdaHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone lambdaHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambdaHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambdaHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

  11. Cloning and Expression Analysis of an XET cDNA in the Peel and Pulp of Banana Fruit Ripening and Softening

    Institute of Scientific and Technical Information of China (English)

    LUWang-Jin; RyoheiNAKANO; YasutakaKUBO; AkitsuguINABAt; JIANGYue-Ming

    2004-01-01

    Xyloglucan endotransglycosylase (XET) is thought to be involved in fruit softening throughdisassembly of xyloglucan, which is the predominant hemicellulose of cell wall. To study the relationshipbetween fruit softening and XET during banana (Musa acuminata Colla cv. Grand Nain) fruit ripening, a fulllength cDNA (1 095 bp) encoding an XET, MA-XET1, was isolated from ripening banana fruit using RT-PCRand RACE-PCR (rapid amplification of cDNA ends) methods. Sequence analysis showed that the cDNAcontains 5' untranslated region of 66 bp, 3' untranslated region of 189 bp and ORF of 840 bp, encoding apredicted polypeptide of 280 amino acids, including DE|DFEFL motif, which is a presumptive catalyticdomain conserved in XETs. DNA gel blot analysis demonstrated that MA-XET1 is encoded by a multi-copyfamily in the banana genome. RNA gel blot analysis revealed that the level of MA-XET1 transcript in thepulp was undetectable, increased and decreased slightly at the preclimacteric, climacteric and postclimactericstages, respectively. In the peel, accumulation of MA-XET1 transcript was low, increased dramatically andthen decreased rapidly, at preclimacteric, climacteric and postclimacteric stages, respectively. Treatmentof fruit with propylene, an analog of ethylene, decreased the firmness and enhanced the accumulation ofMA-XET1 transcript in the peel and pulp. These results suggest that MA-XET1 is involved in softening ofthe peel and pulp during banana fruit ripening and its expression is regulated by ethylene at transcriptionallevel.

  12. Analysis of Metastatic-Related Gene Expression in Gastric Cancer by Low-Density cDNA Microarrays

    Institute of Scientific and Technical Information of China (English)

    Baojun Huang; Huimian Xu; Yujie Zhao; Zhenning Wang; Shaocheng Wang

    2006-01-01

    OBJECTIVE To screen metastatic-related genes in human gastric cancer by a low-density cDNA microarray technique.METHODS A total of 18 paired gastric cancer and adjacent normal mucosa were examined by a low-density cDNA microarray containing 23genes. RT-PCR was used for further verification.RESULTS The mRNA expression of MMP-7, heparanase, S100A4,hTERT, hRad17 in gastric cancers was higher than that in coupled normal mucosa (P =0.002, 0.00011, 0.000072, 0.002, 0.00016 respectively),whereas nm23H1, and CDH1 were lower (P=0.003, 0.012 respectively).The concordance was verified further by RT-PCR with a correlation coefficient of 0.774. In gastric primary lesions the mRNA expression of MMP-7, heparanase and S100A4 was higher in the serosa involved compared to non-involved (P=0.003, 0.009, 0.012 respectively), whereas nm23H1,CDH1, KAI1 were lower (P=0.001, 0.001, 0.006 respectively). With respect to the area of serosa involvement, MMP-7 and heparanase expressions were higher in an area of more than 20 cm2 compared to an area of less than 20 cm2 (P=0.001, 0.02 respectively), whereas nm23H1,CDH1 and KAI1 were lower (P=0.030, 0.041, 0.031 respectively). MMP-7and hTERT expressions were higher in the heavier lymph node metastatic cases (no less than 7) than in the lighter lymph node metastatic cases(no more than 6, P=0.001, 0.005 respectively).CONCLUSION Expression of MMP-7, S100A4, heparanase, hTERT,KAI1, CDH1 and nm23H1 correlated closely with invasion and metastasis in gastric carcinomas. The low-density cDNA microarrays can be used to examine the expression of many genes simultaneously, parallely and quickly.

  13. A juvenile hormone-repressible transferrin-like protein from the bean bug, Riptortus clavatus: cDNA sequence analysis and protein identification during diapause and vitellogenesis.

    Science.gov (United States)

    Hirai, M; Watanabe, D; Chinzei, Y

    2000-05-01

    We found several juvenile hormone-responsive cDNAs in the bean bug, Riptortus clavatus, by using mRNA differential display (Hirai et al., 1998). One of them, a juvenile hormone-repressible cDNA, JR-3, was cloned, sequenced, characterized and identified as a transferrin (RcTf). RcTf cDNA encoded 652 amino acids with a calculated molecular weight of 71,453 Da. The deduced amino acid sequence showed significant homology with the transferin genes of several insects, Manduca sexta (43% identity), Blaberus discoidalis (43%), Aedes aegypti (43%), Drosophila melanogaster (36%), Sarcophaga peregrina (36%) and the human (25%). Antiserum was prepared by using recombinant RcTf protein expressed in Escherichia coli as an antigen. The antiserum reacted specifically with both the recombinant protein and the native protein from the bugs, with sizes of 70 and 75 kDa, respectively. The 75 kDa protein was partially purified from hemolymph of diapausing female bugs and the first ten amino acids were found to be identical to that of RcTf cDNA, indicating that the 75 kDa protein is RcTf. The tissue distribution of RcTf in the bug was examined by Western blot analysis. In diapausing animals, RcTf was detected in the fat body, hemolymph and ovary but not in the gut. In the post-diapause stage, RcTf was also detected in eggs, in addition to the fat body and ovary. These results indicate that RcTf is incorporated into the oocytes during vitellogenesis, and suggest that it may provide iron for the developing embryos.

  14. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Science.gov (United States)

    Qi, Fei; Guo, Huarong; Wang, Jian

    2008-02-01

    Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  15. hSmad5 gene, a human hSmad family member: its full length cDNA, genomic structure, promoter region and mutation analysis in human tumors.

    Science.gov (United States)

    Gemma, A; Hagiwara, K; Vincent, F; Ke, Y; Hancock, A R; Nagashima, M; Bennett, W P; Harris, C C

    1998-02-19

    hSmad (mothers against decapentaplegic)-related proteins are important messengers within the Transforming Growth Factor-beta1 (TGF-beta1) superfamily signal transduction pathways. To further characterize a member of this family, we obtained a full length cDNA of the human hSmad5 (hSmad5) gene by rapid amplification of cDNA ends (RACE) and then determined the genomic structure of the gene. There are eight exons and two alternative transcripts; the shorter transcript lacks exon 2. We identified the hSmad5 promoter region from a human genomic YAC clone by obtaining the nucleotide sequence extending 1235 base pairs upstream of the 5' end of the cDNA. We found a CpG island consistent with a promoter region, and we demonstrated promoter activity in a 1232 bp fragment located upstream of the transcription initiation site. To investigate the frequency of somatic hSmad5 mutations in human cancers, we designed intron-based primers to examine coding regions by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Neither homozygous deletions or point mutations were found in 40 primary gastric tumors and 51 cell lines derived from diverse types of human cancer including 20 cell lines resistant to the growth inhibitory effects of TGF-beta1. These results suggest that the hSmad5 gene is not commonly mutated and that other genetic alterations mediate the loss of TGF-beta1 responsiveness in human cancers.

  16. 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

  17. Cloning of lea cDNA fragment of carrot (Daucus carota L.) and analysis of its expression features

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Addition of concentrated sucrose to MS culture arrests the development of carrot somatic embryo at the stage of cotyledon embryo and, with the sucrose concentration restored to normal level, the embryo thus arrested is reactivated into post-embryonic development. Using the method of RT-PCR, the cDNA fragment of a new member of the Dc3 family of lea has been obtained from carrot somatic embryo under regulated state. As revealed by Northern blotting, strong expression has been observed in carrot somatic embryo under regulated state but the expression was much reduced 12 h after deregulation, and nearly disappeared 24 h after. Based on this finding as well as results of related studies, it is surmised that changing the sucrose concentration in culture enabled carrot somatic embryo under suspension culture to undergo a specific course of development which is comparable to the dormancy-germination process of seeds.

  18. 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....

  19. Molecular cloning and in silico analysis of the duck (Anas platyrhynchos MEF2A gene cDNA and its expression profile in muscle tissues during fetal development

    Directory of Open Access Journals (Sweden)

    Hehe Liu

    2012-01-01

    Full Text Available The role of myogenic enhancer transcription factor 2a (MEF2A in avian muscle during fetal development is unknown. In this work, we cloned the duck MEF2A cDNA sequence (GenBank accession no. HM460752 and examined its developmental expression profiles in cardiac muscle, non-vascular smooth muscle and skeletal muscle. Duck MEF2A cDNA comprised 1479 bp encoding 492 amino acid residues. In silico analysis showed that MEF2A contained MADS (MCM1, AGAMOUS, DEFICIENS and SRF -serum response factor, MEF2 and mitogen-activated protein kinase (MAPK transcription domains with high homology to related proteins in other species. Modified sites in these domains were conserved among species and several variants were found. Quantitative PCR showed that MEF2A was expressed in all three muscles at each developmental stage examined, with the expression in smooth muscle being higher than in the other muscles. These results indicate that the conserved domains of duck MEF2A, including the MADS and MEF2 domains, are important for MEF2A transcription factor function. The expression of MEF2A in duck smooth muscle and cardiac muscle suggests that MEF2A plays a role in these two tissues.

  20. A cDNA Cloning of a Novel Alpha-Class Tyrosinase of Pinctada fucata: Its Expression Analysis and Characterization of the Expressed Protein

    Directory of Open Access Journals (Sweden)

    Ryousuke Takgi

    2014-01-01

    Full Text Available Tyrosinase plays an important role in the formation of the shell matrix and melanin synthesis in mollusks shells. A cDNA clone encoding a 47 kDa protein was isolated from the pearl oyster Pinctada fucata. The cDNA was 1,957 base pairs long and encodes a 417 residue protein that has extensive sequence identity with tyrosinase (polyphenol oxidase: EC 1.14.18.1. This tyrosinase-like protein, termed PfTy, contains an N-terminal signal sequence and the two copper-binding domain signatures (CuA and CuB, suggesting that PfTy belongs to the α-subclass of type-3 copper proteins. Enzyme activity of PfTy was examined by a spectrophotometric method using the translation product derived from an S30 T7 high-yield protein expression system. Tyrosinase activity was seen in this recombinant product. RT-PCR analysis showed that PfTy mRNA was expressed in the mantle pallial, but not in the mantle edge. Therefore, PfTy may participate in insoluble shell matrix formation of the nacreous layer. PfTy expression was also observed in the foot, liver, and adductor muscle, suggesting that PfTy participates in the synthesis of melanins, which are effective scavengers of free radicals formed in multiple intracellular oxidative processes. This is the first report of a novel α-class tyrosinase from the pearl oyster P. fucata.

  1. cDNA cloning, characterization and expression analysis of DTX2, a human WWE and RING-finger gene, in human embryos.

    Science.gov (United States)

    Yi, Zhengfang; Yi, Tingfang; Wu, Zirong

    2006-06-01

    The WWE domain is a conserved globular domain in several proteins and predicted to mediate specificprotein-protein interactions in ubiquitin and ADP ribose conjugation systems. The RING domain is a conserved and specialized zinc-finger motif with 40-60 residues binding to two zinc atoms, which is also probably involved in mediating protein-protein interactions. Here, from human fetal heart cDNA library, we identified DTX2, a human WWE & RING-finger gene, with high similarity with its homologues. Evaluation of full-length cDNA obtained by RACE indicated it encodes a protein composed of two WWE domains and a RING-finger region. The DTX2 gene located in human chromosome 7q11.23 spanning approximately 44.3 kb on the genome and the deduced protein is 622 amino acids. Northern analysis revealed DTX2 was expressed in the 18-week, 22.5-week human embryo hearts and adult hearts, especially with high levels in the 18-week and adult hearts. Taken together, these results indicate that DTX2 is a gene encoding a WWE-RING-finger protein and involved in regulating heart development and heart functions.

  2. Expressed sequence tags analysis of a liver tissue cDNA library from a highly inbred minipig line

    Institute of Scientific and Technical Information of China (English)

    CHEN You-nan; TAN Wei-dong; LU Yan-rong; QIN Sheng-fang; LI Sheng-fu; ZENG Yang-zhi; BU Hong; LI You-ping; CHENG Jing-qiu

    2007-01-01

    Background Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore,we investigated the liver expression profile of a highly inbred minipig line.Methods A cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme.Results Alignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5' terminal sequences and 140 complete 3' terminal sequences.Conclusion These newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation.

  3. cDNA sequence analysis of ribosomal protein S13 gene in Plutella xylostella (Lepidoptera: Plutellidae)

    Institute of Scientific and Technical Information of China (English)

    SHAO-LIWANG; CHENG-FASHENG; CHUAN-LINGQIAO; MIYATATADASHI

    2005-01-01

    Ribosomal protein S 13 gene has been cloned and analyzed in many organisms,but there are few documents relating to insects. In this communication, the full-length cDNA sequence of ribosomal protein S 13 gene in the diamondback moth, Plutella xylostella(Lepidoptera: Plutellidae), was determined by using PCR amplification technique. The features of the ribosomal protein S 13 gene sequence were analyzed and the deduced amino acids sequence was compared with those from other insects. The results of multi-alignment of the amino acid sequences between the diamondback moth and other insect species revealed that this gene sequence is highly conserved in insects. Based on maximum likelihood method, a phylogenetic tree was constructed from 10 different species using PHYLIP software. It showed that nematode is one separate lineage and the five insect speciesbe long to another lineage, whereas those species higher than insects form the third one. The pattern of this phylogenetic tree evidently represented the evolution of different species.

  4. Cathepsin B from the white shrimp Litopenaeus vannamei: cDNA sequence analysis, tissues-specific expression and biological activity.

    Science.gov (United States)

    Stephens, A; Rojo, L; Araujo-Bernal, S; Garcia-Carreño, F; Muhlia-Almazan, A

    2012-01-01

    Cathepsin B is a cystein proteinase scarcely studied in crustaceans. Its function has not been clearly described in shrimp species belonging to the sub-order Dendrobranchiata, which includes the white shrimp Litopenaeus vannamei and other species from the Penaeidae family. Studies on vertebrates suggest that these lysosomal enzymes intracellularly hydrolize protein, as other cystein proteinases. However, the expression of the gene encoding the shrimp cathepsin B in the midgut gland was affected by starvation in a similar way as other digestive proteinases which extracellularly hydrolyze food protein. In this study the white shrimp L. vannamei cathepsin B (LvCathB) cDNA was sequenced, and characterized. Its gene expression was evaluated in various shrimp tissues, and changes in the mRNA amounts were compared with those observed on other digestive proteinases from the midgut gland during starvation. By using qRT-PCR it was found that LvCathB is expressed in most shrimp tissues except in pleopods and eye stalk. Changes on LvCathB mRNA during starvation suggest that the enzyme participates during intracellular protein hydrolysis but also, after food ingestion, it participates in hydrolyzing food proteins extracellularly as confirmed by the high activity levels we found in the gastric juice and midgut gland of the white shrimp.

  5. 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.

  6. cDNA microarray analysis of disk abalone genes in gills and hemocytes after viral hemorrhagic septicemia virus (VHSV) challenge.

    Science.gov (United States)

    De Zoysa, Mahanama; Nikapitiya, Chamilani; Oh, Chulhong; Whang, Ilson; Shin, Hyun-Jin; Lee, Jehee

    2012-06-01

    A disk abalone Haliotis discus discus 4.2 K cDNA microarray was designed by selecting abalone expressed sequence tags (ESTs). Transcriptional profiles in gills and hemocytes were analyzed upon abalone challenged with viral hemorrhagic septicemia virus (VHSV) in order to select candidates for screening of immune response genes. Among the 4188 genes analyzed, 280 (6.6%) transcripts were changed their expression level in gills and hemocytes against VHSV challenge compared to control animals. Total of 88 and 65 genes were up-regulated in gills and hemocytes, respectively. These genes can be grouped under various immune-functional categories such as transcription factors (Krüppell-like factor; ETS-family transcription factor), inflammatory and apoptosis related genes (TNF superfamily members, Fas ligand), IFN regulatory proteins (IFN-44 like, interferon gamma-inducible lysosomal thiol reductase) and detoxification proteins (glutathione peroxidase). In contrast, 25 and 102 genes were shown down-regulation in gills and hemocytes, respectively. Among the differentially expressed transcripts, considerably higher numbers of ESTs were represented as either hypothetical (unknown) proteins or no GenBank match suggesting those may be novel genes associated with internal defense of abalone.

  7. Construction of a cDNA library and preliminary analysis of expressed sequence tags in Piper hainanense.

    Science.gov (United States)

    Fan, R; Ling, P; Hao, C Y; Li, F P; Huang, L F; Wu, B D; Wu, H S

    2015-10-19

    Black pepper is a perennial climbing vine. It is widely cultivated because its berries can be utilized not only as a spice in food but also for medicinal use. This study aimed to construct a standardized, high-quality cDNA library to facilitated identification of new Piper hainanense transcripts. For this, 262 unigenes were used to generate raw reads. The average length of these 262 unigenes was 774.8 bp. Of these, 94 genes (35.9%) were newly identified, according to the NCBI protein database. Thus, identification of new genes may broaden the molecular knowledge of P. hainanense on the basis of Clusters of Orthologous Groups and Gene Ontology categories. In addition, certain basic genes linked to physiological processes, which can contribute to disease resistance and thereby to the breeding of black pepper. A total of 26 unigenes were found to be SSR markers. Dinucleotide SSR was the main repeat motif, accounting for 61.54%, followed by trinucleotide SSR (23.07%). Eight primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among twenty-one piper germplasm. These results present a novel sequence information of P. hainanense, which can serve as the foundation for further genetic research on this species.

  8. Identification of the dichotomous role of age-related LCK in calorie restriction revealed by integrative analysis of cDNA microarray and interactome.

    Science.gov (United States)

    Park, Daeui; Lee, Eun Kyeong; Jang, Eun Jee; Jeong, Hyoung Oh; Kim, Byoung-Chul; Ha, Young Mi; Hong, Seong Eui; Yu, Byung Pal; Chung, Hae Young

    2013-08-01

    Among the many experimental paradigms used for the investigation of aging, the calorie restriction (CR) model has been proven to be the most useful in gerontological research. Exploration of the mechanisms underlying CR has produced a wealth of data. To identify key molecules controlled by aging and CR, we integrated data from 84 mouse and rat cDNA microarrays with a protein-protein interaction network. On the basis of this integrative analysis, we selected three genes that are upregulated in aging but downregulated by CR and two genes that are downregulated in aging but upregulated by CR. One of these key molecules is lymphocyte-specific protein tyrosine kinase (LCK). To further confirm this result on LCK, we performed a series of experiments in vitro and in vivo using kidneys obtained from aged ad libitum-fed and CR rats. Our major significant findings are as follows: (1) identification of LCK as a key molecule using integrative analysis; (2) confirmation that the age-related increase in LCK was modulated by CR and that protein tyrosine kinase activity was decreased using a LCK-specific inhibitor; and (3) upregulation of LCK leads to NF-κB activation in a ONOO(-) generation-dependent manner, which is modulated by CR. These results indicate that LCK could be considered a target attenuated by the anti-aging effects of CR. Integrative analysis of cDNA microarray and interactome data are powerful tools for identifying target molecules that are involved in the aging process and modulated by CR.

  9. 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

  10. 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.

  11. Cloning and characterization of cDNA probes for the analysis of metallothionein gene expression in the Mediterranean bivalves: Ruditapes decussatus and Cerastoderma glaucum.

    Science.gov (United States)

    Ladhar-Chaabouni, Rim; Mokdad-Gargouri, Raja; Denis, Françoise; Hamza-Chaffai, Amel

    2009-05-01

    cDNA probes have been developed for subsequent use in monitoring the cadmium exposure of the clam Ruditapes decussatus and the cockle Cerastoderma glaucum using metallothionein (MT) gene expression in different tissues of these species. Two partial MT cDNAs were isolated from Ruditapes decussatus and Cerastoderma glaucum. The identification of the nucleotide sequences showed that the cDNAs consist of 480 bp coding 72 amino acid proteins containing 21 cysteine residues organized in Cys-X-Cys motifs as classically described for MTs. The induction of MT gene expression in CdCl(2) treated bivalves was confirmed by dot blot analysis and suggests a potential specific tissue expression rate.

  12. Expressed Sequence Tags Analysis and Design of Simple Sequence Repeats Markers from a Full-Length cDNA Library in Perilla frutescens (L.

    Directory of Open Access Journals (Sweden)

    Eun Soo Seong

    2015-01-01

    Full Text Available Perilla frutescens is valuable as a medicinal plant as well as a natural medicine and functional food. However, comparative genomics analyses of P. frutescens are limited due to a lack of gene annotations and characterization. A full-length cDNA library from P. frutescens leaves was constructed to identify functional gene clusters and probable EST-SSR markers via analysis of 1,056 expressed sequence tags. Unigene assembly was performed using basic local alignment search tool (BLAST homology searches and annotated Gene Ontology (GO. A total of 18 simple sequence repeats (SSRs were designed as primer pairs. This study is the first to report comparative genomics and EST-SSR markers from P. frutescens will help gene discovery and provide an important source for functional genomics and molecular genetic research in this interesting medicinal plant.

  13. Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

    Science.gov (United States)

    Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

    2016-08-01

    Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

  14. A cDNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in Helicoverpa armigera: molecular characterization and expression analysis associated with pupal diapause.

    Science.gov (United States)

    Liu, Ming; Zhang, Tian-Yi; Xu, Wei-Hua

    2005-06-01

    The diazepam binding inhibitor (DBI) or the acyl-CoA-binding protein (ACBP) is a 9-10 kDa highly conserved multifunctional protein that plays important roles in GABA(A) receptor activity regulation, lipid absorption and steroidogenesis in various organisms. To study the functions of DBI/ACBP in insect development or diapause, we cloned the cDNA from Helicoverpa armigera (Har) utilizing rapid amplification of cDNA ends (RACE). By homology search, Har-DBI/ACBP is conserved with the DBI/ACBPs known from other insects. Northern blot analysis showed that DBI/ACBP gene expressed in nonneural and neural tissues. RT-PCR combined Southern blot analysis revealed that DBI/ACBP mRNA in the brain of nondiapause individual was much higher than that in the brain of diapausing insects. At early and middle stages of 6th instar larvae, the level of DBI/ACBP mRNA was higher in the midgut of diapause type than that in nondiapause type and low at late 6th instar larval stage and early pupal stage in both types. In the prothoracic gland (PG), DBI/ACBP expression appeared at a high level at middle and late stages of 6th larval instar in both nondiapause and diapause types, and declined after pupation. In vitro experiments revealed that DBI/ACBP mRNA in PG could be stimulated by synthetic H. armigera diapause hormone (Har-DH), suggesting that Har-DH may stimulate the PG to produce ecdysteroids by the DBI/ACBP signal pathway. By in vitro assay, we also found that FGIN-1-27, which has similar functions to DBI/ACBP in ecdysteroidogenesis, could induce PG ecdysteroidogenesis effectively, suggesting that DBI/ACBP regulates biosynthesis of ecdysteroids in PG. Thus, DBI/ACBP indeed plays a key role in metabolism and development in H. armigera.

  15. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Institute of Scientific and Technical Information of China (English)

    QI Fei; GUO Huarong; WANG Jian

    2008-01-01

    Reversible protein phosphorylation,catalyzed by protein kinases and phosphatases,is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes.Protein phosphatase 1(PP1) is the first and well-characterized member of the protein serine/threoninephosphatase family.In the present study.a full-length cDNA encoding the beta isolorm of the catalytic subunit of protein phosphatase 1(PP1cb).was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus,designated SmPP1cb,by the rapid amplification of cDNA ends (RACE) technique.The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame(ORF),flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region.The ORF encodes a putative 327 amino acid protein.and the N-terminal section of this protein iS highly acidic,Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp.a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B(PP2B).And its calculated molecular mass is 37 193 Da and pI 5.8.Sequence analysis indicated that,SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates.and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXXATGG,which is different from mammalian in two positions A-6 and G-3,indicating the possibility of different initiation of translation in turbot,and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals.especially zebrafish.The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  16. Analysis of immune system gene expression in small rheumatoid arthritis biopsies using a combination of subtractive hybridization and high-density cDNA arrays.

    Science.gov (United States)

    Zanders, E D; Goulden, M G; Kennedy, T C; Kempsell, K E

    2000-01-13

    Subtractive hybridization of cDNAs generated from synovial RNA which had been isolated from patients with rheumatoid arthritis (RA) or normal controls was used in conjunction with high-density array hybridization to identify genes of immunological interest. The method was designed to detect gene expression in small needle biopsy specimens by means of a prior amplification of nanogram amounts of total RNA to full-length cDNA using PCR. The latter was cut with Rsa I, ligated with adapters, hybridized with unmodified driver cDNA, and subjected to suppression subtraction PCR. Differentially expressed products were cloned into E. coli and picked into 384 well plates. Inserts were obtained by PCR across the multiple cloning site, and the products arrayed at high density on nylon filters. The subtracted cDNAs were also labelled by random priming for use as probes for library screening. The libraries chosen were the subtracted one described above and a set of 45,000 ESTs from the I.M. A.G.E consortium. Clones showing positive hybridization were identified by sequence analysis and homology searching. The results showed that the subtracted hybridization approach could identify many gene fragments expressed at different levels, the most abundant being immunoglobulins and HLA-DR. The expression profile was characteristic of macrophage, B cell and plasma cell infiltration with evidence of interferon induction. In addition, a significant number of sequences without matches in the nucleotide databases were obtained, this demonstrates the utility of the method in finding novel gene fragments for further characterisation as potential members of the immune system. Although RA was studied here, the technology is applicable to any disease process even in cases where amounts of tissue may be limited.

  17. MarC-V: a spreadsheet-based tool for analysis, normalization, and visualization of single cDNA microarray experiments.

    Science.gov (United States)

    Schageman, J J; Basit, M; Gallardo, T D; Garner, H R; Shohet, R V

    2002-02-01

    The comprehensive analysis and visualization of data extracted from cDNA microarrays can be a time-consuming and error-prone process that becomes increasingly tedious with increased number of gene elements on a particular microarray. With the increasingly large number of gene elements on today's microarrays, analysis tools must be developed to meet this challenge. Here, we present MarC-V, a Microsoft Excel spreadsheet tool with Visual Basic macros to automate much of the visualization and calculation involved in the analysis process while providing the familiarity and flexibility of Excel. Automated features of this tool include (i) lower-bound thresholding, (ii) data normalization, (iii) generation of ratio frequency distribution plots, (iv) generation of scatter plots color-coded by expression level, (v) ratio scoring based on intensity measurements, (vi) filtering of data based on expression level or specific gene interests, and (vii) exporting data for subsequent multi-array analysis. MarC-V also has an importing function included for GenePix results (GPR) raw data files.

  18. cDNA Cloning and Sequence Analysis of ADH Gene in Delia antiqua%葱蝇ADH基因的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    陈春露; 陈斌; 司风玲; 何正波

    2012-01-01

    [ Objective ] The aim was to clone the ADH gene of Delia antiqun, and carry out a sequence analysis. [ Method ] The cDNA sequence of ADH gene was cloned with the method of RACE, and then studied with homology analysis, comparison of amino acid sequence and phylogenetic analysis. [Result] The full length of cDNA obtained was 1 088 bp, among which there were 771 bp of ORF, encoding a protein of 256 amino acids with a calculated molecular weight of 30.80 kKa and a theoretical isolectric point of 8.22. The deduced amino acid sequence had the highest identity with that of Glossina morsitans based on homological analysis,and a phylogenic tree was inferred with homological ADH sequences from other insects. [ Conclusion ] The study provides a basis for the further research of ADH gene.%[目的]对葱蝇(Delia antiqua)ADH基因进行克隆,并对其进行序列分析.[方法]通过RACE的方法克隆葱蝇ADH基因的cDNA序列,同时对该序列进行同源性分析、氨基酸序列比对和系统发育分析.[结果]试验获得的cDNA全长1 088 bp,其中ORF 771 bp,编码256个氨基酸,推测其相对分子质量为30.80 kDa,等电点为8.22;通过该基因推导的氨基酸序列与其他物种的ADH进行相似性比较和系统发育分析,发现葱蝇与刺舌蝇(Glossina morsuans)氨基酸序列的同源性最高.[结论]该研究为ADH基因的进一步研究提供了基础.

  19. cDNA Cloning and Sequence Analysis of ADH Gene in Delia antiqua%葱蝇ADH基因的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    陈春露; 陈斌; 司风玲; 何正波

    2012-01-01

    【目的】对葱蝇(De如antiqua)ADH基因进行克隆,并对其进行序列分析。【方法】通过RACE的方法克隆葱蝇ADH基因的cDNA序列,同时对该序列进行同源性分析、氨基酸序列比对和系统发育分析。[结果]试验获得的cDNA全长1088bp,其中ORF771bp,编码256个氨基酸,推测其相对分子质量为30.80kDa,等电点为8.22;通过该基因推导的氨基酸序列与其他物种的ADH进行相似性比较和系统发育分析,发现葱蝇与刺舌蝇(Glossina morsitans morsitoas)氨基酸序列的同源性最高。【结论】该研究为ADH基因的进一步研究提供了基础。%[Objective] This study aims to conduct cloning and sequence analysis of ADH gene in D. Antiqua. [Method] Full-length cDNA of ADH gene in D. antiqua was cloned by using RACE technology (GenBank access number: JQ666006). Analysis of the homology, characteristics and functional domains of ADH sequence and the phy- Iogenetic relationship to other dipteran ADH were conducted. [Result] The full length of ADH cDNA is 1 088 bp containing a 771 bp of ORF, encoding 256 amino acids, with a calculated relative molecular weight of 30.80 kDa and a theoretical isoelectric point of 8.22. The deduced amino acid sequence shares the highest homology with Glossina morsitans morsitans based on homological analysis and phylogenetic analysis. [Conclusion] This study provides basis for further research of ADH gene.

  20. Identification of gene profiles of CD4~+ and CD8~+ T lymphocyte in systemic lupus erythematosus by generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification

    Institute of Scientific and Technical Information of China (English)

    王惠琳

    2006-01-01

    Objective To identify LongSAGE Tags in systemic lupus erythematosus (SLE) by generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI). Methods CD4+ and CD8+ T lymphocytes were collected from the PBMCs of 25 patients with SLE and 10 healthy controls. Then the total RNA was extracted and reversely

  1. Analysis of Differential Gene Expression Pattern in Brassica napus Hybrid Huayouza 6 and Its Parents Using Arabidopsis cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    SHEN Jun-ru; WU Jian-yong; ZHANG Jian; LIU Ping-wu; YANG Guang-sheng

    2006-01-01

    Huayouza 6, a new semi-winter Brassica napus variety with high-yield, good quality, prematurity and extensive adaptability, was derived from the cross between the female parent 8086A and male parent 7-5. Two cDNA-based Arabidopisis microarray were used to analyze gene differential expression in bud of an elite B. napus hybrid Huayouza6 and its parents,in which there were 83 over-expression transcripts and 331 under-expression transcripts between Huayouza 6 and its female parent 8086A and 94 over-expression transcripts, and 423 under-expression transcripts were demonstrated betweenHuayouza 6 and its male parent 7-5. Further analysis showed that there were significant number of genes responsible for photosynthesis, and its implication for heterosis was discussed. Northern analysis of phosphoribulokinase coincided with its expression pattern derived from hybridization of Arabidopsis cDNA microarray and B. napus mRNA, this system of heterologous hybridization analysis should be applicable to other close relatives of Arabidopsis thaliana.

  2. Screening of genes related to sulfide metabolism in Urechis unicinctus (Echiura, Urechidae) using suppression subtractive hybridization and cDNA microarray analysis.

    Science.gov (United States)

    Shi, Xiaoli; Shao, Mingyu; Zhang, Litao; Ma, Yubin; Zhang, Zhifeng

    2012-09-01

    Exogenous sulfide can generally induce metabolic injuries in most organisms and even cause death. However, organisms inhabiting intertidal zones, hydrothermal vents, and cold seeps, can tolerate, metabolize, and utilize sulfide. In this study, both suppression subtractive hybridization and cDNA microarray analysis were employed to screen sulfide metabolism-related genes from the body wall in echiuran worm Urechis unicinctus, a marine sediment species. A total of 3456 monoclones were isolated and 82 were identified as differentially expressed genes in worms exposed to 50 μM sulfide for 24 h, compared to controls. The identified genes encoded proteins with multiple processes, including metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. Eight genes, serase, vacuolar protein, src tyrosine kinase, sulfide oxidase-like oxidoreductase, aprataxin, SN-RNP, aminopeptidase, and predicted protein, were selected to verify expression in the worm using qRT-PCR. The agreement of gene expression evaluation was 62.5% between the results of microarray analysis and qRT-PCR. These new data will provide clues for further probing of the molecular mechanism of sulfide metabolism.

  3. Rapid amplification of cDNA ends (RACE) improves the PCR-based isolation of immunoglobulin variable region genes from murine and human lymphoma cells and cell lines.

    Science.gov (United States)

    Doenecke, A; Winnacker, E L; Hallek, M

    1997-10-01

    The isolation of rearranged immunoglobulin (Ig) variable region (V) genes is usually performed by PCR with consensus primers binding to conserved regions within the V sequences. However, the isolation of Ig genes by this method is hampered in 15-35% by technical difficulties, mostly mismatches of oligonucleotide primers to V sequences. In order to obtain DNA sequences from V heavy chain (VH) genes which could not be amplified with consensus primers, we used a modified PCR technique, the rapid amplification of cDNA ends (RACE) PCR in combination with new heavy chain constant region primers for the isolation of human and murine VH genes. In comparison, consensus primer PCR with different sets of previously published oligonucleotide primers was used. Both methods were applied to isolate VH genes from murine B cell lymphoma (A20 and BCL1), myeloma (NS1) and hybridoma (SP6) cell lines and from freshly isolated human chronic lymphocytic leukemia and lymphoma cells. RACE PCR allowed the amplification and subsequent cloning of the complete VH gene in all cases. In contrast, consensus primer PCR failed to isolate the VH sequence of the murine A20 cell line; this was explained by a mismatch of consensus primers with VH sequences. When both PCR methods amplified VH sequences, the DNA sequences obtained were identical. Taken together, RACE PCR represents a reliable and versatile method for the isolation of VH genes from human and murine lymphoma cells, in particular if consensus primer PCR fails.

  4. Identification of genes up-regulated in dedifferentiating Nicotania glauca pith tissue, using an improved method for constructing a subtractive cDNA library.

    Science.gov (United States)

    Cecchini, E; Dominy, P J; Geri, C; Kaiser, K; Sentry, J; Milner, J J

    1993-12-11

    Pith explants of Nicotiana glauca grown in vitro in synthetic medium supplemented with 2,4 dichlorophenoxyacetic acid (2, 4 D), are induced to dedifferentiate. Treatment with actinomycin D within the first 4-8 h of culture (but not later) is lethal and the explants die, implying a requirement for de novo transcription. The genes expressed during the initial period of culture are presumably critical for subsequent cell survival and proliferation, but so far their identity is unknown. We have constructed a subtractive cDNA library, enriched in sequences more abundant in dedifferentiating tissue than in pith. The subtractive library contains approximately seven major species, two of which, NGSUB7 and NGSUB8, are highly abundant. In Northern blots, these two hybridized to mRNA species whose abundance increased significantly but transiently during the first 4 to 8 h of culture. The sequence of NGSUB7 showed no significant homology at a nucleotide or derived amino acid level with any previously reported sequence. NGSUB8 however, showed significant homology over part of the derived amino acid sequence to several yeast and bacterial proteins with DNA binding function. We propose that the two recombinants represent transcripts from two novel genes edeA and edeB, which are expressed early in dedifferentiation.

  5. 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...

  6. The effect of column purification on cDNA indirect labelling for microarrays

    Directory of Open Access Journals (Sweden)

    Kiss John Z

    2007-06-01

    Full Text Available Abstract Background The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. Results We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Conclusion Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive

  7. 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

  8. 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.

  9. [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.

  10. cDNA Cloning, Sequence Analysis of the Porcine LIM and Cysteine-rich Domain 1 Gene

    Institute of Scientific and Technical Information of China (English)

    Jun WANG; Chang-Yan DENG; Yuan-Zhu XIONG; Bo ZUO; Lei XING; Feng-E LI; Ming-Gang LEI; Rong ZHENG; Si-Wen JIANG

    2005-01-01

    LIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton by their interaction with various structural proteins, kinases and transcriptional regulators. Using molecular biology combined with in silico cloning, we have cloned the complete coding sequence of pig LIM and the cysteine-rich domain 1 gene (LMCD1) which encodes a 363 amino acid protein. The estimated molecular weight of the LMCD1 protein is 40,788 Da with a pI of 8.39. It was found to be highly expressed in both skeletal muscle and cardiac muscle. Alignment analysis revealed that the deduced protein sequence shares 86%, 91% and 93% homology with that of its human, mouse and rat counterparts, respectively. The LMCD1 protein was predicted by bioinformatics software to contain a novel cysteine-rich domain in the N-terminal region, two LIM domains in the C-terminal region, nine potential protein kinase C phosphorylation sites, seven casein kinase Ⅱ phosphorylation sites, a tyrosine kinase phosphorylation site, seven N-glycosylation and N-myristoylation sites and a single potential N-glycosylation site, which is similar to the protein's human counterpart. Phylogenetic tree was constructed by aligning the amino acid sequences of the LIM domain from different species. In addition, four base mutations were detected by comparing the sequences of Large White pigs with those of Chinese Meishan pigs. The G294A mutation site was confirmed by polymerase chain reaction-single-strand conformation polymorphism analysis. Its allele frequencies were studied in five pig breeds.

  11. 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)

  12. 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.

  13. [mRNA expression analysis and classification of colonic biopsy samples using oligonucleotide and cDNA microarray techniques].

    Science.gov (United States)

    Galamb, Orsolya

    2008-07-20

    Despite tremendous progress in the past few decades, certain important aspects regarding the diagnosis, therapy, and follow-up of colorectal cancer still remain unsolved. In our work we searched for biomarkers of the development of colorectal carcinoma, and performed gene expression analysis for colorectal disease classification. We have established that the oligonucleotide microarray analyses of biopsy samples wholly fulfil the Affymetrix quality requirements, are highly standard and reproducible and the Taqman microfluidic card system is suitable for high-throughput, quick and cost efficient real-time-PCR validation of gene expression changes. We have shown that the sequential overexpression of osteopontin and osteonectin mRNAs and proteins significantly correlates with the progression of the colorectal adenoma-dysplasia-carcinoma sequence. We have identified and validated ten novel markers with continuously increasing mRNA expression in line with the adenoma-dysplasia-carcinoma transition. We have identified the top 27, 13 and 10 genes associated with adenoma, colorectal cancer, and inflammatory bowel diseases.

  14. Classification of Sensitivity or Resistance of Cervical Cancers to Ionizing Radiation According to Expression Profiles of 62 Genes Selected by cDNA Microarray Analysis

    Directory of Open Access Journals (Sweden)

    Osamu Kitahara

    2002-01-01

    Full Text Available To identify a set of genes related to radiosensitivity of cervical squamous cell carcinomas and to establish a predictive method, we compared expression profiles of 9 radiosensitive and 10 radioresistant tumors obtained by biopsy before treatment, on a cDNA microarray consisting of 23,040 human genes. We identified 121 genes whose expression was significantly greater in radiosensitive cells than in radioresistant cells, and 50 genes that showed higher levels of expression in radioresistant cells than in radiosensitive cells. Some of these genes had already known to be associated with the radiation response, such as aldehyde dehydrogenase 1 (ALDH1 and X-ray repair cross-complementing 5 (XRCC5 (P<.05, Mann-Whitney test. The validity of the total of 171 genes as radiosensitivity related genes were certified by permutation test (P<.05. Furthermore, we selected 62 genes on the basis of a clustering analysis, and confirmed the validity of these genes with cross-validation test. The cross-validation test also indicates the possibility of making prediction of radiosensitivity for discriminating radiation-sensitive from radiation resistant biopsy samples by predicting score (PS values calculated from expression values of 62 genes in 19 samples, because the prediction successfully and unequivocally discriminated the radiosensitive phenotype from the radioresistant phenotype in our test panel of 19 cervical carcinomas. The extensive list of genes identified in these experiments provides a large body of potentially valuable information for studying the mechanism(s of radiosensitivity, and selected 62 genes opens the possibility of providing appropriate and effective radiotherapy to cancer patients.

  15. Molecular cloning of a cDNA encoding human calumenin, expression in Escherichia coli and analysis of its Ca2+-binding activity

    DEFF Research Database (Denmark)

    Vorum, H; Liu, X; Madsen, Peder;

    1998-01-01

    By microsequencing and cDNA cloning we have identified the transformation-sensitive protein No. IEF SSP 9302 as the human homologue of calumenin. The nucleotide sequence predicts a 315 amino acid protein with high identity to murine and rat calumenin. The deduced protein contains a 19 amino acid ...

  16. Analysis of differentially expressed genes in the precocious line of Eimeria maxima and its parent strain using suppression subtractive hybridization and cDNA microarrays.

    Science.gov (United States)

    Dong, Hui; Lin, Jiaojiao; Han, Hongyu; Jiang, Lianlian; Zhao, Qiping; Zhu, Shunhai; Huang, Bing

    2011-04-01

    The precocious line of Eimeria spp., obtained by repeated passages of oocysts initially collected from feces of previously infected chickens, has unique phenotypes and plays an important role in immunizing chickens against coccidiosis. However, the genetic basis of precocious phenotype in Eimeria is still poorly understood. To investigate gene expression changes in sporulated oocysts between the precocious line of E. maxima and its parent strain, subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH). A total of 3,164 cDNA fragments were selected from the SSH cDNA libraries to fabricate cDNA microarrays and further identify the differentially expressed genes. The credibility of the microarray data was verified by real-time PCR. A total of 360 valid expressed sequence tags (ESTs) were obtained, which represented 32 unique sequences. Twenty-one genes were validated as downregulated and 11 genes as upregulated in the precocious line. Homology searching of the public sequence database showed that six genes encoded proteins homologous with previously reported proteins, including rhomboid-like protein and transhydrogenase of E. tenella, serpin, and cation-transporting ATPase of E. acervulina, a heat-shock protein of E. maxima, and a conserved hypothetical protein of Toxoplasma gondii. Thus, the remaining 26 ESTs have not been previously reported. Further characterization of these differentially expressed genes will be useful in understanding the genetic basis for the precocious phenotype in Eimeria spp.

  17. Cloning and Sequence Analysis of the Full-length cDNA of a Novel yp05 Gene Associated With Citrinin Production in Monascus aurantiacus

    Institute of Scientific and Technical Information of China (English)

    YON-GHUA XIONG; YANG XU; WEI-HUA LAI; YAN-PIN LI; HUA WEI

    2007-01-01

    Objective To obtain the full-length cDNA of a novel gene (named yp05) associated with citrinin production-related genes in Monascus aurantiacus. Methods Total RNA was extracted from mycelium, 3' and 5' cDNA end of yp05 gene was amplified using smartTM trace cDNA amplification kit, and the full-length cDNA of a novel gene (named yp05) was obtained from the electronic assembly of 3'-RACE and 5'- RACE products. Results This yp05 gene was 787 bp including a 597 bp open reading frame (ORF) and encoded a deduced protein with 199 amino acid residues, and the amino acid sequence of this protein was found similar with the sequences of many fungal manganese-superoxide dismutases in the GenBank with the aid of BLASTp. The transcription of yp05 gene in Monascus strains was analyzed with the aid of Northern blotting. The transcription of yp05 gene was only detected in Monascus strains, provided that citrinin was produced. Conclusion The transcription of yp05 gene belongs to differential expression genes of citrinin yielded from Monascus and has no correlation with the biosynthesis pathway of red pigments.

  18. 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

  19. 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

  20. [Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper].

    Science.gov (United States)

    Ramazanova, A S; Fil'kin, S Iu; Starkov, V G; Utkin, Iu N

    2011-01-01

    Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.

  1. Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: molecular sequence analysis of its cDNA and biochemical properties.

    Science.gov (United States)

    Vivas-Ruiz, Dan E; Sandoval, Gustavo A; Mendoza, Julio; Inga, Rosalina R; Gontijo, Silea; Richardson, Michael; Eble, Johannes A; Yarleque, Armando; Sanchez, Eladio F

    2013-07-01

    The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.

  2. A cDNA library of the eutardigrade Hypsibius klebelsbergi Mihelčič, 1959 and analysis of the actin gene

    Directory of Open Access Journals (Sweden)

    Hartmut GREVEN

    2007-09-01

    Full Text Available A cDNA library was constructed from the glacier-dwelling eutardigrade Hypsibius klebelsbergi from more than 2000 individuals collected in the Austrian Central Alps. RNA, DNA and proteins were successively isolated by the Trizol®-method. From the RNA preparation a cDNA library was constructed with the cDNA inserted unidirectionally in the phagemid expression vector TriplEx2. The primary gene library had a titre of 107 pfu ml-1 and the final amplified gene library a titre of 6×109 pfu ml-1. The average insert length was about 1.6 kb. The partial sequence of H. klebelsbergi actin (746 bp showed highest similarity to GenBank data of Drosophila melanogaster actin at the nucleic acid level (84.9% and at the amino acid level (98%. Compared with actin fragments of the eutardigrades Ramazzottius oberhaeuseri (450 bp and Macrobiotus sp. (453 bp the identities were 85% - 81% and 100% - 98% with respect to the nucleic/amino acids. Identity with actin fragments (359 bp of Hypsibius dujardini from GenBank was 96% - 100%.

  3. cDNA cloning,sequence analysis,and recombinant expression of akitonin beta,a C-type lectin-like protein from Agkistrodon acutus

    Institute of Scientific and Technical Information of China (English)

    Xiang-dong ZHA; Jing LIU; Kang-sen XU

    2004-01-01

    AIM: To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structurefunction relationships and to achieve its recombinant production. METHODS: PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template.The PCR products were cloned into the plasmid pGEM-T and sequenced. The deduced protein sequence was analyzed with some bioinformatic programs. A recombinant expression plasmid was constructed using pBADTOPO as vector and transformed into E. coli TOP10 competent cells. RESULTS: A novel cDNA sequence encoding akitonin β was found and accepted by GenBank (accession number AF387100). Akitonin β consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins. It was predicted to be a platelet antagonist. Upon induction with arabinose rAkitonin β expressing in E coli was achieved at a high level (superior to 150 mg/L). The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro. CONCLUSION: A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was established for its production.

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

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us Dicty_cDB cDNA library... information Data detail Data name cDNA library information Description of data conten...d - Data analysis method - Number of data entries 14 entries Data item Description cDNA library name Names o...(Slug phase)(S) 4) culminating stages (Morphogenetic phase)(C) 5) sexually fusion-competent KAX3 cells (Gamete phase) (F) cDNA librar...y construction method How to construct cDNA library 1) C

  5. 一个新的枳NAC基因cDNA全长的克隆及其亚细胞定位分析%Cloning and Subcellular Localization Analysis of A New Gene NAC cDNA in Poncirus trifoliata

    Institute of Scientific and Technical Information of China (English)

    韩键; 王化坤; 宋长年; 上官林飞; 冷翔朋

    2012-01-01

    Taking the NACl cDNA sequence in Arabidopsis thaliana as the template, the homologous gene in the EST database of citrus was searched and screened, and the cDNA sequence of NACl gene in citrus was cloned by bioinformatics method. Taking the cDNA sequence in the flower of Poncirus trifoliata ( L. ) Raf. As the template, the specific primers were designed according to the a-bove cDNA sequence, and the 5' - end and 3' - end sequences of NAC1 gene were obtained by using 5'RACE and 3' RACE techniques, respectively. Based on these, the corresponding full length cDNA of NACl in Poncirus trifoliata was acquired through sequence splicing. This complete cDNA, designated as Pt - NACl, was 1351 bp in length, it contained a 1047 bp whole open reading frame (ORF) , its 5' - end included a putative translation start codon ( ATG) at the position of 25 bp, and the length of its 3' - end untranslated region was 280 bp. The deduced amino acid sequence of Pt - NACl was 348 residues, which showed 64. 8% , 57. 0% , 61.3% identical levels with that of Malus x domestica, Arabidopsis thaliana, Petunia xhybrida, respectively. Bioinformatics analysis showed that the cDNA of Pt - NACl had the recognition site of microRNA164, and it also had highly conserved NAC domains. Recom-binant plasmid 35S - GW - GFP - FJ619349 was transferred into onion' s epidermal cells by using the particle bombardment method, and the subcellular localization analysis results indicated that Pt - NACl was all localized at the cell membrane.%以拟南芥的NAC1 cDNA序列作为模板,对柑橘EST数据库进行同源检索筛选,利用生物信息学方法克隆了柑橘NAC1基因的cDNA序列.以枳(Poncirus trifoliata)花的cDNA为模板,根据以上cDNA序列设计特异性引物,利用5′RACE和3′RACE技术,分别获得了NAC1基因的5′和3′末端,序列拼接后获得枳的NAC1 cDNA全长,命名为Pt-NAC1.Pt-NAC1全长为1351 bp,含有1个1047 bp完整的开放读码框(ORF),5′

  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. 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

    Science.gov (United States)

    Boltaña, Sebastian; Castellana, Barbara; Goetz, Giles; Tort, Lluis; Teles, Mariana; Mulero, Victor; Novoa, Beatriz; Figueras, Antonio; Goetz, Frederick W.; Gallardo-Escarate, Cristian; Planas, Josep V.; Mackenzie, Simon

    2017-01-01

    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 of gene

  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. Generation and analysis of large-scale expressed sequence tags (ESTs) from a full-length enriched cDNA library of porcine backfat tissue

    OpenAIRE

    2006-01-01

    Background Genome research in farm animals will expand our basic knowledge of the genetic control of complex traits, and the results will be applied in the livestock industry to improve meat quality and productivity, as well as to reduce the incidence of disease. A combination of quantitative trait locus mapping and microarray analysis is a useful approach to reduce the overall effort needed to identify genes associated with quantitative traits of interest. Results We constructed a full-lengt...

  11. Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response

    Directory of Open Access Journals (Sweden)

    Sakaki Yoshiyuki

    2007-12-01

    Full Text Available Abstract Background Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs. Results The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features. Conclusion The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome.

  12. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein.

    OpenAIRE

    Jiang, H P; Serrero, G

    1992-01-01

    We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA ...

  13. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein

    OpenAIRE

    Jiang, Hui-Ping; Serrero, Ginette

    1992-01-01

    We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA ...

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

    Science.gov (United States)

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

    2014-01-01

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

  15. CLONING AND ANALYSIS OF THE FULL-LENGTH cDNA SEQUENCE OF SEPIELLA MAINDRONI SCD GENE%曼氏无针乌贼(Sepiella maindroni)SCD基因全长cDNA的克隆和序列分析

    Institute of Scientific and Technical Information of China (English)

    马明华; 刘慧慧; 迟长凤; 吴常文

    2013-01-01

    grouped with fish,then fell into a neighboring branch with human,mouse and other mammals.The SCD gene was the main candidate gene for improving the meat quality of S.maindroni,and its cloning and related analysis were of great significance to explore mollusks fatty acid metabolism related genes in the biological and regulation mechanisms.

  16. Analysis of full-length cDNA sequence of FAD2 gene in Vernicia fordii seeds%油桐种子FAD2基因全长cDNA序列分析

    Institute of Scientific and Technical Information of China (English)

    谢禄山; 谭晓风; 张琳; 龙洪旭

    2012-01-01

    Linoleic acid produced in seeds of Verniciafordii is the direct material for synthesize eleostearic acid through catalysis of FAD2, the researches on FAD2 gene in seed from V. fordii has practical significance on improving yield of eleostearic acid. Taking 16 FAD2 clones in cDNA library of nearly mature V. fordii 'Duinian tung' seeds as materials, CAP3 splicing, BLAST alignment and DNAMAN analysis were carried on. The results showed that the cloned gene sequence was FAD2 full-length cDNA sequence, its length was 1 537 bp. The sequence contained a complete coding sequence, length of 1 146 bp (106-1 255 bp), encoding 383 amino acids. The relative molecular mass of the enzyme protein was 44 144.4 u, jsoelectric point was 8.57. The N end of amino acid sequence had a signal peptide sequence of 6 residues, 5 transmembrane domains, 3 strong hydrophilic sequences existed at the N end, C end and intermediate part, respectively, and the activity center of enzyme was 3 conserved histidine clusters. In system evolution, FAD2 gene in V. fordii had a nearest phylogenetic relationship with V. montana, nearer relationship with Euphorbiaceae plants such as Ricinus communis, Triadica sebifrea, Hevea brasiliensis, Jatropha curcas, and far relationship with Olea europaea, Arachis hypogaea, Sesamum indicum, further relationship with Camelia oleifera.%油桐种子中FAD2催化形成的亚油酸是合成桐油酸的直接原料,研究油桐种子中的FAD2基因对提高桐油酸的产量具有实际意义.将油桐对年桐近成熟种子cDNA文库中的16个FAD2克隆子进行CAP3拼接,再进行BLAST比对,并进行DNAMAN分析,结果表明所克隆的基因序列为FAD2全长cDNA序列,其长度为1 537 bp,含有1个完整的编码序列,长度为1 146 bp( 106~1 255 bp),编码383个氨基酸.酶蛋白相对分子质量44 144.4 u,等电点为8.57,氨基酸序列N端有6个残基的信号肽序列,有5个跨膜结构域,N端、C端及中间各有一段表现为强

  17. Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes.

    Science.gov (United States)

    Fu, Glenn K; Wang, Jonathan T; Yang, Junming; Au-Young, Janice; Stuve, Laura L

    2004-07-01

    The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

  18. 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.

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

    Science.gov (United States)

    Rönnberg, Tuomas; Jääskeläinen, Kirsi; Blot, Guillaume; Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

    2012-01-01

    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.

  20. Cloning and Sequence Analysis of PGIP gene and cDNA from Prunus persica%桃PGIP基因及cDNA的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    谌悦; 张军科; 熊帅

    2009-01-01

    通过PCR扩增了桃的PGIP基因及cDNA序列,并进行了序列之间的比对及分析.结果表明,桃的PGIP基因全长1 092 bp,而其cDNA序列只有1 045 bp,PGIP基因中含有一段长147 bp的内含子,且内含子符合GT-AG规律.这与其他核果类植物的PGIP基因构成基本相同.其基因序列与GenBank中已登录的3个桃以及其他核果的序列比对,其同源性达92%~99%;与苹果、梨和大桉的同源性分别为85%、84%和84%.%PGIP gene and cDNA were cloned from Prunus persica and the sequences were compared and analyzed. The result showed that the length of PGIP gene was 1 092 bp, while the length of cDNA was only 1 045 bp. The PGIP gene contained 147 bp length intron, and according to GT-AG law, which basically the same with PGIP gene of other stone fruit plants. its gene sequence and logged 3 peaches in GenBank and other stone fruit were compared the homology can reached to 92%-99% . The homology rates of PGIPs ,compared peach with Malus, Pyrus and Eucalyptus , were 85%, 84% and 84% respectively.

  1. Improved Intermittency Analysis of Single Event Data

    OpenAIRE

    Janik, R. A.; Ziaja, B.

    1998-01-01

    The intermittency analysis of single event data (particle moments) in multiparticle production is improved, taking into account corrections due to the reconstruction of history of a particle cascade. This approach is tested within the framework of the $\\alpha$-model.

  2. 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...

  3. 青枯菌诱导的烟草叶片全长cDNA文库的构建和初步分析%Construction and Primary Analysis of Tobacco Leaf Full-length cDNA Library Induced by Ralstonia Solanacearumr

    Institute of Scientific and Technical Information of China (English)

    张冲; 蔡铁城; 陈华; 曾建斌; 庄伟建

    2013-01-01

    Leaves of high quality flue-cured tobacco variety K326 were inoculated by Ralstonia Solanacearum and harvested at different time point. Total RNA was extracted by CTAB method. Library construction was carried out by Creator SMART cDNA Library Construction Kit (CLONTECH Laboratories) in accordance with the manu- facturer's protocol. Briefly, total RNA was used as starting material to synthesize first-strand cDNA. Then, Double-Stranded cDNA was synthesised by Low-Cycle PCR on a DNA Thermal Cycler. Following double- stranded cDNA synthesis, which was incubated with proteinase K to degrade the thermostable DNA polymerases. After digestion, the cDNA was purified from a low-melt agarose gel to remove small fragments (<750 bp) and was directionally cloned into SfiI A&B-digested vector pDNR-LIB as described. A full-length cDNA library was constructed with the primary library titer of 1.902 ×106 cfu/mL and 2.97 ×109 cfu/mL for the amplyfied titer. Inserts ranged from 0.75~2.0 kb and average insert size of the clones was larger than 1 300 bp, and also with a high recombination rate of 99%. Fivety clones were randomly picked from the libraries for sequencing and functional analysis. The results indicated that the library was of high quality, which serve as invaluable genetic resource for further cloning and screening special genes involved in Ralstonia Solanacearum resistance and tobacco genetic improvement.%以优质高抗青枯病烤烟品种K326为材料,在苗期利用注射法接种烟草青枯菌,分不同时期取叶片,利用CTAB法提取接种和非接种的混合RNA,采用SMART (switching mechanism at 5' end of RNA tra-nscript)技术合成双链cDNA,经SfiⅠ酶切后胶回收纯化双链cDNA,连接到质粒载体pDNR-LIB上,电击转化大肠杆菌DH5α感受态细胞,成功构建了青枯菌诱导的烟草叶片全长cDNA文库。经鉴定,初级文库库容为1.902×106 cfu/mL,重组率达到98%以上,插入片段集中在0.75~2.0 kb

  4. 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…

  5. 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.

  6. Stress responses in alfalfa (Medicago sativa L.) 12. Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants.

    Science.gov (United States)

    Gowri, G; Paiva, N L; Dixon, R A

    1991-09-01

    An expression library containing cDNAs derived from transcripts from fungal elicitor-treated alfalfa cell suspension cultures was screened with an antiserum raised against phenylalanine ammonia-lyase (PAL) from alfalfa. A single immunoreactive clone was isolated which encoded a full-length PAL cDNA (APAL1) consisting of a 2175 bp open reading frame, 96 bp 5'-untranslated leader and 128 bp 3'-non-coding region. The deduced amino acid sequence was 86.5% similar to that of the PAL2 gene of bean, and encoded a polypeptide of Mr 78,865. A second PAL cDNA species was isolated, whose 3'-untranslated region was 86% identical to that of APAL1. Southern blot analysis indicated that PAL is encoded by a small multigene family in alfalfa. PAL transcript levels were rapidly and massively induced, and preceded increased PAL extractable activity, on exposure of alfalfa suspension cells to elicitor from baker's yeast. PAL transcripts were most abundant in roots, stems and petioles during growth and development of alfalfa seedlings. These studies provide the basis for an examination of the developmental and environmental control of a key enzyme of phenylpropanoid synthesis in a plant species which is readily amenable to stable genetic transformation.

  7. 沙田柚无机焦磷酸酶基因的 cDNA 克隆及序列分析%Cloning and sequence analysis of inorganic pyrophosphatase gene from Citrus grandis var.shatianyu

    Institute of Scientific and Technical Information of China (English)

    秦新民; 万珊; 李惠敏; 覃屏生; 张渝

    2015-01-01

    related)and non-self(genetically unrelated)pollen.Inorganic pyrophosphatase(IPPase)play important roles in regu-lating the growth and development in plants.In order to better understand the mechanism of IPP gene in the self-in-compatibility of Citrus grandis var.shatianyu ,the inorganic pyrophosphatase gene of C .grandis var.shatianyu was cloned and physicochemical properties of pyrophosphatase were analyzed.The total RNA was isolated from style of C .grandis var.shatianyu used the total RNA Purification System(Invitrogen)and following the manufacture′s pro-tocols.According to the EST sequence(internal fragment of inorganic pyrophosphatase gene)in suppression subtrac-tive hybridization libraries of C .grandis var.shatianyu style,4 specific primers 5′-GSP1,5′-nGSP1,3′-GSP2 and 3′-nGSP2 were designed for amplifying 3-RACE and 5-RACE of the gene.The full-length sequences of cDNA of inor-ganic pyrophosphatase gene were obtained from suppression subtractive hybridization libraries of C .grandis var.shatianyu style by the SMART-RACE PCR method.A comparison of the similarity of the full-length cDNA se-quence of the inorganic pyrophosphatase gene was performed in the GenBank database used the BLAST program.DNAman software was used for mino acid sequence and homology analysis.Prediction of molecular weight, isoeletric point(pI)and hydrophobicity were performed by using on line software ExpASy and DNAman.A 150 bp band in 3-RACE,and a 900 bp band in 5-RACE were cloned by nested and non nested PCR method.For the full-length cDNA sequences,the middle sequence,3-RACE and 5-RACE sequence of the inorganic pyrophosphatase gene were spliced and formed the full-length cDNA sequences.As a result,the cDNA of inorganic pyrophosphatase was 1 136 bp in length containing an 654 bp open reading frame(ORF),which encoded a protein of 217 amino acids with a 170 bp 5 untranslated region(5 UTR)and a 321 bp 3 UTR.The sequence of the cloned cDNA of the inorganic pyro-phosphatase from C .grandis

  8. Molecular cloning, characterization, and expression analysis of a gonadotropin-releasing hormone-like cDNA in the clam, Ruditapes philippinarum.

    Science.gov (United States)

    Song, Ying; Miao, Jingjing; Cai, Yuefeng; Pan, Luqing

    2015-11-01

    Gonadotropin-releasing hormone (GnRH) is a key neuropeptide regulating reproduction in vertebrates. In the present study, we have cloned and identified the cDNA sequence of the prepro-GnRH (rp-GnRH) in the Manila clam Ruditapes philippinarum. The open reading frame encodes a protein of 94 amino acids, which consists of a signal peptide, a GnRH dodecapeptide, a cleavage site, and a GnRH-associated peptide. These deduced peptides were highly homologous to that reported for other molluscs. We used temperature control to promote gonadal development of the clams. The mRNA expression of rp-GnRH in the visceral ganglia from clams at different reproductive stages was determined by quantitative RT-PCR. The levels of steroids progesterone (P), testosterone (T), and estradiol-17β (E2) in the hemolymph of the corresponding clam were measured by ELISA. The rp-GnRH mRNA was highly expressed at the gonadal early development stage. Concentrations of P, T, and E2 also increased at the early development stage in both sexes. Positive significant correlations between rp-GnRH expression and P content as well as between rp-GnRH expression and T content were observed throughout the gonadal maturation. The results from this study may help to better understand the physiological functions of the native forms of GnRH-like peptides and the actions of GnRH on sex steroids release in bivalve molluscs.

  9. Generation and analysis of expressed sequence tags from a normalized cDNA library of young leaf from Ma bamboo (Dendrocalamus latiflorus Munro).

    Science.gov (United States)

    Gao, Z M; Li, C L; Peng, Z H

    2011-11-01

    Ma bamboo (Dendrocalamus latiflorus Munro) belongs to Dendrocalamus genus, Bambusease tribe, Bambusoideae subfamily, Poaceae family. It is a representative species of clumping bamboo, and a principal commercial species for various construction purposes using mature culms and for human consumption using young shoots. A normalized cDNA library was constructed from young leaves of Ma bamboo and 9,574 high-quality ESTs were generated, from which 5,317 unigenes including 1,502 contigs and 3,815 singletons were assembled. The unigenes were assigned into different gene ontology (GO) categories and summarized into 13 broad biologically functional groups according to similar functional characteristics or cellular roles by BLAST search against public databases. Eight hundred and ninety-one unigenes were assigned by KO identifiers and mapped to six KEGG biochemical pathways. The transcripts involved in biosynthesis of secondary metabolites such as cytochrome 450, flavonol synthase/flavanone 3-hydroxylase, and dihydroflavonol-4-reductase were well represented by 14 unigenes in the unigene set. The candidate genes involved in phytohormone metabolism, signal transduction and encoding cell wall-associated receptor kinases were also identified. Sixty-seven unigenes related to plant resistance (R) genes, including RPP genes, RGAs and RDL/RF genes, were discovered. These results will provide genome-wide knowledge about the molecular physiology of Ma bamboo young leaves and tools for advanced studies of molecular mechanism underlying leaf growth and development.

  10. 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.

  11. cDNA Array Analysis of the Differential Expression Change in Virulence-related Genes During the Development of Resistance in Candida albicans

    Institute of Scientific and Technical Information of China (English)

    Zheng XU; Yuan-Ying JIANG; Yong-Bing CAO; Jun-Dong ZHANG; Ying-Ying CAO; Ping-Hui GAO; De-Jun WANG; Xu-Ping FU; Kang YING; Wan-Sheng CHEN

    2005-01-01

    Candida albicans is the most frequently isolated fungus in immunocompromised patients associated with mucosal and deep-tissue infections. To investigate the correlation between virulence and resistance on a gene expression profile in C. albicans, we examined the changes in virulence-related genes during the development of resistance in C. albicans from bone marrow transplant patients using a constructed cDNA array representing 3096 unigenes. In addition to the genes known to be associated with azole resistance,16 virulence-related genes were identified, whose differential expressions were newly found to be associated with the resistant phenotype. Differential expressions for these genes were confirmed by RT-PCR independently. Furthermore, the up-regulation of EFG1, CPH2, TEC1, CDC24, SAP10, ALS9, SNF1, SPO72 and BDF1, and the down-regulation of RAD32, IPF3636 and UBI4 resulted in stronger virulence and invasiveness in the resistant isolates compared with susceptible ones. These findings provide a link between the expression of virulence genes and development of resistance during C. albicans infection in bone marrow transplant (BMT) patients, where C. albicans induces hyphal formation and expression change in multiple virulence factors.

  12. 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.

  13. Comparative analysis of gene expression at early seedling stage between a rice hybrid and its parents using a cDNA microarray of 9198 uni-sequences

    Institute of Scientific and Technical Information of China (English)

    HUANG; Yi; LI; Lihua; CHEN; Ying; LI; Xianghua; XU; Caiguo; WANG; Shiping; ZHANG; Qifa

    2006-01-01

    Using a cDNA microarray consisting of 9198 expressed sequence tags, we surveyed the gene expression profiles in shoots and roots of a rice hybrid, Liangyoupei 9 and its parents Peiai 64s and 93-11 at 72 h after germination. A total of 8587 sequences had detectable signals in both shoots and roots of the three genotypes. A total of 1571 sequences exhibited significant (P<0.01) expression differences in shoots or roots among the three genotypes, of which 121 showed expression polymorphisms in both shoots and roots, and 870 revealed significant expression differences between the hybrid and one of the parents. The expression polymorphism of the sequences was associated with the functional categories of the sequences. They occurred more frequently in categories of carbohydrate, energy and lipid metabolisms and stress response than expected, while less frequently in categories of amino acid metabolism, transcription and translation regulation, and signal transduction. A total of 214 sequences exhibited significant (P<0.05) mid-parent heterosis in expression, of which 117 had homology to genes with known functions, assigned in the categories of basic metabolism, genetic information processing, cell growth and death, signal transduction, transportation and stress response. The results may provide useful information for exploring the relationship between gene expression polymorphism and phenotypic variation, and for characterizing the molecular mechanism of seedling development and heterosis in rice.

  14. Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends.

    Science.gov (United States)

    Schaefer, B C

    1995-05-20

    Rapid amplification of cDNA ends (RACE) is a polymerase chain reaction (PCR)-based technique which was developed to facilitate the cloning of full-length cDNA 5'- and 3'-ends after a partial cDNA sequence has been obtained by other methods. While RACE can yield complete sequences of cDNA ends in only a few days, the RACE procedure frequently results in the exclusive amplification of truncated cDNA ends, undermining efforts to generate full-length clones. Many investigators have suggested modifications to the RACE protocol to improve the effectiveness of the technique. Based on first-hand experience with RACE, a critical review of numerous published variations of the key steps in the RACE method is presented. Also included is a detailed, effective protocol based on RNA ligase-mediated RACE/reverse ligation-mediated PCR, as well as a demonstration of its utility.

  15. 多头带绦虫脑多头蚴cDNA表达文库的构建及初步分析%Construction and Preliminary Analysis of cDNA Expression Library of Coenurus cerebralis of Taenia multiceps

    Institute of Scientific and Technical Information of China (English)

    李文卉; 王建魁; 盖文燕; 姚菊霞; 曲自刚; 贾万忠; 罗建勋; Radu Blaga; 付宝权

    2011-01-01

    从甘肃景泰羊源脑多头蚴原头节提取总RNA,以Oligo(dT)纤维素柱纯化mRNA,利用Lambda ZAP II XR文库构建试剂盒构建了脑多头蚴cDNA表达文库.从构建的原始文库随机挑选单个噬菌斑进行PCR,确定文库重组率和插入外源基因片段大小,鉴定文库质量.结果表明脑多头蚴cDNA表达文库的原始库容量为1.0×106 pfu,扩增后文库滴度为1.6×109 pfu/mL.随机挑选的165个噬菌斑克隆中,0.25 kb以上的克隆155个,重组率为93.9%,对其中0.5 kb以上的115个克隆测序共获得104个表达序列标签(EST),分析后得到96个单一EST序列(Unique EST),其中20个EST与绦虫基因有同源性,38个EST与吸虫基因有同源性,4个EST与线虫基因有同源性,17个EST与其他物种有同源性,其余17个EST没有同源基因.这些EST编码的氨基酸序列有多头带绦虫六钩蚴Tm16抗原、猪带绦虫副肌球蛋白、猪带绦虫免疫原蛋白Ts76等绦虫抗原的同源蛋白以及烯醇化酶等一些酶类、热休克蛋白、肌动蛋白、核糖体蛋白等同源蛋白.%Total RNA was extracted from protoscolex of Coenurus cerebralis from naturally infected sheep of Jingtai Co. ,Gansu, and mRNA was isolated with Oligo ( dT) cellulose column. The Coenurus cerebralis cDNA expression library was constructed with Lambda ZAP Ⅱ XR vector. The randomly picked clones from primary cDNA library was amplified with PCR to detect the recombinant rate of cDNA lihrary and size of DNA inserts. The results indicated the size of the preliminary cDNA library was 1. 0 × 106 pfu with the titer of the amplified cDNA library of 1. 6 x 109 pfu/mL. From randomly picked 165 clones, 155 were with the size more than 250 bp and the recombinant rate was 93. 9% . Furthermore , 115 clones sized more than 500 bp were sequenced and 104 expressed sequence tags (ESTs) were obtained. After analysis 96 unique ESTs were obtained, of which 20 were homologous with cestode, 38 were homologous with

  16. Improving transient analysis technology for aircraft structures

    Science.gov (United States)

    Melosh, R. J.; Chargin, Mladen

    1989-01-01

    Aircraft dynamic analyses are demanding of computer simulation capabilities. The modeling complexities of semi-monocoque construction, irregular geometry, high-performance materials, and high-accuracy analysis are present. At issue are the safety of the passengers and the integrity of the structure for a wide variety of flight-operating and emergency conditions. The technology which supports engineering of aircraft structures using computer simulation is examined. Available computer support is briefly described and improvement of accuracy and efficiency are recommended. Improved accuracy of simulation will lead to a more economical structure. Improved efficiency will result in lowering development time and expense.

  17. AN IMPROVED ALGORITHM FOR DPIV CORRELATION ANALYSIS

    Institute of Scientific and Technical Information of China (English)

    WU Long-hua

    2007-01-01

    In a Digital Particle Image Velocimetry (DPIV) system, the correlation of digital images is normally used to acquire the displacement information of particles and give estimates of the flow field. The accuracy and robustness of the correlation algorithm directly affect the validity of the analysis result. In this article, an improved algorithm for the correlation analysis was proposed which could be used to optimize the selection/determination of the correlation window, analysis area and search path. This algorithm not only reduces largely the amount of calculation, but also improves effectively the accuracy and reliability of the correlation analysis. The algorithm was demonstrated to be accurate and efficient in the measurement of the velocity field in a flocculation pool.

  18. 佛手GRAS基因的克隆及表达分析%On cDNA cloning and expression analysis of GRAS gene in fingered citron

    Institute of Scientific and Technical Information of China (English)

    石瑞; 曹诣斌; 陈文荣; 郭卫东

    2011-01-01

    A fragment of EST was isolated from fingered citron by suppression subtractive hybridization. It was cloned the full-length cDNA of the EST by RACE. The result showed that this fragment was highly associated with GRAS genes of Arabidopsis and the sequence alignment showed that it had a conserved domain of GRAS. Therefore, the GRAS gene of fingered citron could be named as CmsGRAS ( GenBank accession number: JF440647) , which had an ORF of 1 236 bp and encoded 413 amino acids. Fluorescence quantitative PCR method was used to study the expression characteristics of GRAS gene in fingered citron under low temperature stress, the results showed that the gene expression significantly changed after injured by low temperature.%利用抑制消减杂交法从芸香科柑橘属不耐寒植物佛手中分离得到了一个表达序列标签(EST)片段,结合RACE技术克隆获得该基因的1 669 bp序列,编码区长1 236 bp,编码413个氨基酸.通过Blastn同源序列比对分析,结果显示该基因与拟南芥已知的GRAS基因同源性较高;与GenBank数据库比对分析,表明该基因具有GRAS特有的保守结构域.因此,命名该基因为:CmsGRAS(GenBank登录号:JF440647).用荧光定量聚合酶链式反应(qPCR)的方法研究了该基因在低温胁迫处理下的表达特性,结果显示该基因在低温胁迫后的表达量有明显的变化.

  19. 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.

  20. Preparation of cDNA libraries from vascular cells.

    Science.gov (United States)

    Lieb, M E; Taubman, M B

    1999-01-01

    The vast majority of past and present efforts in the molecular cloning of expressed sequences involve isolation of clones from cDNA libraries constructed in bacteriophage lambda (1,2). As discussed in Chapter 6 , screening these cDNA libraries using labeled probes remains the most straightforward method to isolate full length cDNAs for which some partial sequence information is known. Although the availability of high quality reagents and kits over the past decade has made the process of library construction increasingly straightforward, generation of high-quality libraries is a task that still requires a fair amount of dedicated effort. Because alternative PCR-based cloning strategies have become increasingly popular alternatives to cDNA library screening, it is useful to consider the advantages and disadvantages of each strategy before embarking on a project to construct a cDNA library (Table 1). In our opinion, it is worthwhile to construct a cDNA library when the transcript of interest is not exceedingly rare (i.e., can readily be detected by Northern blot analysis of total RNA), when multiple cDNAs will need to be cloned over a period of time, and in situations where occasional mutations can not be tolerated (for example, if the cDNA is to be expressed in mammalian cells to examine function). In situations where the transcript of interest is expressed at exceedingly low levels, or when only a single cDNA needs to be cloned, a PCR-based strategy should be considered. When the tissue source is precious (such as a unique clinical specimen), successful construction of a phage library provides a resource that can be amplified and used for multiple cloning projects over many years, but runs the risk of consuming the available RNA if the library construction fails. Table 1 Comparison of Relative Advantages of cDNA Cloning from Lambda Phage Libraries by Plaque Hybridization Compared to Newer PCR- Based Strategies Lambda phage cDNA library PCR-based strategy Freedom

  1. Isolation, cDNA sequence analysis and tissue expression profile of a novel swine gene differentially expressed in the Longissimus dorsi muscle tissues from Large White × Meishan cross combination

    Institute of Scientific and Technical Information of China (English)

    LIU Yonggang; LEI Minggang; XIONG Yuanzhu; DENG Changyan

    2005-01-01

    In order to study the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences in gene expression in the Longissimus dorsi muscle tissues from Large White × Meishan cross combination.One novel gene differentially expressed between the hybrids and the purebreds was isolated and subsequently identified using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and its complete cDNA sequence was obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction revealed that the open reading frame of this gene encodes a protein of 188 amino acids that contains the putative conserved domain of the PRA1 family protein and this protein has high homology with the PRA1 family protein 3 of three species-rat (88 % ), human(88 % ), and mouse (87 % ), -so that it can be defined as swine PRA1 family protein 3. The phylogenetic tree analysis revealed that the swine PRA1 family protein 3 has a closer genetic relationship with the human PRA1 family protein 3 than with those of mouse and rat.The tissue expression analysis indicated that swine PRA1family protein 3 gene is highly-expressed in muscle and fat, moderately in spleen,weakly in heart, kidney, ovary, lung, and almost not expressed in small intestine and liver. The function of this gene and the relationship between this gene and heterosis are also discussed.

  2. 岩栖蝮蛇类凝血酶纯化、cDNA克隆和序列分析%Purification, cDNA cloning and sequence analysis of thrombin-like enzyme from Gloydius saxatilis

    Institute of Scientific and Technical Information of China (English)

    孙德军; 杨春伟; 杨同书; 颜炜群; 王伟

    2003-01-01

    Thrombin-like enzyme has great medical application in treating thrombus. A thrombin-like enzyme from Gloydius saxatilis snake venom was isolated and purified to homogeneity by a rapid and effective method using ion-exchange chromatography on DEAE-Sepharose and affinity chromatography on heparin-sepharose.SDS-polyacrylamide electrophoresis under reducing condition revealed that the purified enzyme had a single protein band and its molecular weight was 32000 dalton.Total RNAs were extracted from the venom gland of the G.saxatilis snake.Using degenerate primers,we amplified the cDNA of the thrombin-like enzyme gene in the venom gland of G.saxatilis using the reverse transcription-polymerase chainreaction (RT-PCR) method.The cDNA fragment was inserted into pGEMT vector,cloned and its nucleotide sequence was determined.Its open reading frame is composed of 774 nucleotides and codes a protein prezymogen of 258 amino acids,including a putative secretory signal peptide of 18 amino acids and a proposed pro-peptide of 6 amino acid residues.It contains 12 cysteine residues.The sequence analysis indicates that the deduced amino acid sequence of the cDNA fragment shares high identity with the thrombin-like enzyme genes of other snakes in the gene bank.The query sequence exhibits strong amino acid sequence homology of 88%,88% and 86% to the serine proteas of T.gramineus,thrombin-like defibrase Ⅰ of D.acutus and serine protease catroxase Ⅱ of C.atrox respectively.Based on the amino acid sequences of other thrombin-like enzymes,the catalytic residues and disulfide bridges of this thrombin-like enzyme are deduced as follows:catalytic residues,His65,Asp110,Ser%204;and six disulfide bridges Cys31-Cys163,Cys50-Cys66,Cys98-Cys256,Cys142-Cys210,Cys174-Cys189 and Cys200-Cys225.According to the possible linked glycosylation sites N-X-T (Asn-X-Thr) or N-X-S (Asn-X-Ser),its possible glycosylation sites are N44-S45-T46 and N251-T252-T253 residues [Acta Zoologica Sinica 49(6):878-882,2003].

  3. 光皮桦茎叶cDNA文库构建及部分EST序列SSR分析%Library construction of cDNA and SSR analysis of partial ESTs for stem and leaf of Betula luminifera

    Institute of Scientific and Technical Information of China (English)

    张敏; 黄华宏; 林二培; 周厚君; 王亚辉; 童再康

    2012-01-01

    A cDNA library of stem and leaf from Betula luminifera was constructed.The primary titer of cDNA library was about 1.5×106 pfu/mL,its recombinant efficiency reached 97.3%,and the size of insert DNA fragments ranged from 0.5 to 3.0 kb,with an average of 1.3 kb.The results indicated that it was a higher-quality cDNA library,and could be used in gene cloning and gene expression profile analysis.Distribution and frequency of SSRs were analyzed in 224 non-redundant ESTs from B.luminifera cDNA library,using online searching software.The results showed that 60 SSRs distributed in 47 EST sequences,accounting for 26.80% of all ESTs.Dinucleotide would be the major repeat types,accounting for 70.00% of the total number of acquired SSRs.The tri-nucleotide and tetra-nucleotide repeats accounted for 28.30% and 1.70% respectively.This research might lay the foundation for designing the targeted EST-SSR primers and genetic diversity analysis by mining the information of EST-SSR loci in B.luminifera EST sequence data.%以光皮桦茎叶组织为材料,构建了cDNA文库。初级文库滴度为1.5×106pfu/mL,重组率达97.3%,插入片段大小在0.5~3.0kb之间,平均长度约为1.3kb,表明所构建的文库质量较高,可用于后续基因克隆及基因表达谱的研究。利用微卫星查找软件对获得的224条EST序列进行微卫星位点搜寻及其丰度、分布比较,发现47条序列含微卫星位点60个,占全部EST序列的26.80%;在所有SSRs中二碱基重复最多,为42个,占总数的70.00%,含三、四碱基重复分别占总数的28.30%和1.70%。通过对光皮桦EST序列中微卫星位点信息的发掘分析,为有针对性地设计EST-SSR引物、进行遗传多样性分析奠定了基础。

  4. Cloning, Bioinformatics Analysis of RERG Gene cDNA in Guizhou Black Goat%贵州黑山羊RERG基因cDNA克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    朱冠群; 杨红文; 罗卫星; 韩勇; 刘若余; 陈志

    2013-01-01

    选择与羊同源性较高的牛RERG基因组序列设计特异性引物,提取贵州黑山羊脾脏总RNA,通过RT-PCR技术对RERG基因进行克隆测序及序列分析.结果:首次克隆了贵州黑山羊RERG基因cDNA序列629 bp,GenBank登录号为JN672576,编码199个氨基酸;贵州黑山羊RERG基因蛋白与牛的同源性高达98.5%;聚类分析表明RERG基因编码区适于构建种间系统进化树.%Cow RERG genome sequence with high homology with sheep was selectde and one specific primers had been designed. Total RNA was extracted from the Guizhou black goat spleen and the cDNA encoding RERG was obtained by the reverse transcription PCR (RT-PCR). The purified RT-PCR product was cloned into T vector, and then the sequence was analyzed. The results demonstrated that the 629 bp product was the Guizhou black goat RERG cDNA, and GenBank accession number was JN672576. It encoded 199 amino acids. RERG protein in Guizhou black goat had 98.5% homology with cow. Cluster analysis revealed that probably mammals, birds and amphibians, each of these belonged to different categories separately. The result of phylogenetic clustering was identical to the genetic distance and zoological classification, which indicated that the RERG gene was also fit to construct molecular phylogenetic tree among different species.

  5. Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells.

    OpenAIRE

    1991-01-01

    The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-t...

  6. Exon-specific northern analysis and rapid amplification of cDNA ends (RACE) reveal that the proximal promoter II (PII) is responsible for aromatase cytochrome P450 (CYP19) expression in human ovary.

    Science.gov (United States)

    Jenkins, C; Michael, D; Mahendroo, M; Simpson, E

    1993-11-01

    Estrogens are synthesized from C19 steroids by a unique form of cytochrome P450, aromatase cytochrome P-450 (P-450AROM; the product of the CYP19 gene). We have shown that tissue-specific expression of human P-450AROM is determined, in part, by the use of alternative promoters. Previous methods of analysis for determining the specific 5'-termini of the different transcripts included S1 nuclease protection, primer extension, and Northern analysis. In the present study we have used the RACE procedure (rapid amplification of cDNA ends) to amplify and clone the 5' termini of P-450AROM transcripts expressed in human corpus luteum (CL). Sequencing of the resulting clones supports the results of the previously performed studies. Specifically, the proximal promoter, PII, is the predominant promoter utilized in CL, such that the start of transcription occurs 26 bp downstream of the putative TATA sequence. A minority of the clones possess an alternative 5'-end, namely I.3. Exon-specific Northern analysis confirms that the majority of the P-450AROM transcripts in CL tissue contain sequence specific for promoter II. Similarly, exon-specific Northern analysis indicates that transcripts in human follicles, as well as granulosa cells in culture, contain primarily sequence specific for promoter II.

  7. Cloning and Analysis of a cDNA Encoding psbL and psbJ Gene in Rice Chloroplast Genome%水稻叶绿体基因组中一个编码psbL 和psbJ基因cDNA的克隆与分析

    Institute of Scientific and Technical Information of China (English)

    顾克余; 罗林广; 苏昌潮; 翟虎渠

    2001-01-01

    A 505 bp cDNA was cloned from the leaves of rice (Oryza sativaL.) Shanyou 63 combination. DNA sequence analysis showed that it is a part of rice chloroplast genome. Its homology comparison with those known in GenBank found that it encodes 38 amino acid peptide deduced from psbL gene and 40 amino acid peptide deduced from psbJ gene in rice chloroplast PSⅡ. Northern hybridization showed that the cDNA was differentially displayed in hybrid F1 and its parental lines.

  8. Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene

    Energy Technology Data Exchange (ETDEWEB)

    Tebbs, R.S.; Tucker, J.D.; Hwang, M. [Lawrence Livermore National Lab., CA (United States)] [and others

    1995-07-03

    The mutagen-sensitive CHO line irs1SF was previously isolated on the basis of hypersensitivity to ionizing radiation and was found to be chromosomally unstable as well as cross-sensitive to diverse kinds of DNA-damaging agents. The analysis of somatic cell hybrids formed between irs1SF and human lymphocytes implicated a human gene (defined as XRCC3; x-ray repair cross-complementing), which partially restored mitomycin C resistance to the mutant. A functional cDNA that confers mitomycin C resistance was transferred to irs1SF cells by transforming them with an expression cDNA library and obtaining primary and secondary transformants. Functional cDNA clones were recovered from a cosmid library prepared from a secondary transformant. Transformants also showed partial correction of sensitivity to displatin and {gamma}-rays, efficient correction of chromosomal instability, and substantially improved plating efficiency and growth rate. The XRCC3 cDNA insert is {approx} 2.5 kb and detects an {approx} 3.0-kb mRNA on Northern blots. The cDNA was mapped by fluorescence in situ hybridization to human chromosome 14q32.3, which was consistent with the chromosome concordance data of two independent hybrid clone panels. 30 refs., 5 figs., 2 tabs.

  9. Cloning and bioinformatics analysis of a fragment cDNA encoding growth hormone-releasing hormone in Tibetan sheep%草地藏系绵羊生长激素释放激素基因部分 cDNA 的克隆及生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    王金玲; 王永; 刘鲁蜀; 陶永平

    2015-01-01

    为了克隆草地藏系绵羊生长激素释放激素基因,采用Trizol法从草地藏系绵羊下丘脑组织中提取总RNA,用反转录-聚合酶链式反应( RT-PCR)进行cDNA扩增并克隆测序,获得长度为207 bp的促生长激素释放激素基因( GHRH)的部分cDNA序列。结果表明获得的草地藏系绵羊GHRH部分cDNA序列与GenBank中注册的绵羊GHRH基因编码起始位置(86位)到292位区域高度同源,仅有1个碱基的差异,该cDNA序列编码69个氨基酸残基,其内含有信号肽序列,该氨基酸序列的31~57位具有典型胰高血糖素类似激素特征的GLUCA结构域。%Total RNA was extracted from the hypothalamus tissues of Tibetan sheep using Trizol method and cDNA was amplified by reverse transcription polymerase chain reaction ( RT-PCR) . The cDNA of Tibetan sheep GHRH gene was cloned from the amplified PCR product and sequenced. The cDNA was 207 bp in length and showed 99% homology with that of com-mon sheep registered in GenBank from the starting position (86) to 292 bp. The cDNA sequence encodes 69 amino acid resi-dues and contains a signal peptide sequence which has a GLUCA domain characterizing glucagon-like hormone at amino acid 31 to 57.

  10. Improving information retrieval in functional analysis.

    Science.gov (United States)

    Rodriguez, Juan C; González, Germán A; Fresno, Cristóbal; Llera, Andrea S; Fernández, Elmer A

    2016-12-01

    Transcriptome analysis is essential to understand the mechanisms regulating key biological processes and functions. The first step usually consists of identifying candidate genes; to find out which pathways are affected by those genes, however, functional analysis (FA) is mandatory. The most frequently used strategies for this purpose are Gene Set and Singular Enrichment Analysis (GSEA and SEA) over Gene Ontology. Several statistical methods have been developed and compared in terms of computational efficiency and/or statistical appropriateness. However, whether their results are similar or complementary, the sensitivity to parameter settings, or possible bias in the analyzed terms has not been addressed so far. Here, two GSEA and four SEA methods and their parameter combinations were evaluated in six datasets by comparing two breast cancer subtypes with well-known differences in genetic background and patient outcomes. We show that GSEA and SEA lead to different results depending on the chosen statistic, model and/or parameters. Both approaches provide complementary results from a biological perspective. Hence, an Integrative Functional Analysis (IFA) tool is proposed to improve information retrieval in FA. It provides a common gene expression analytic framework that grants a comprehensive and coherent analysis. Only a minimal user parameter setting is required, since the best SEA/GSEA alternatives are integrated. IFA utility was demonstrated by evaluating four prostate cancer and the TCGA breast cancer microarray datasets, which showed its biological generalization capabilities.

  11. Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

    Institute of Scientific and Technical Information of China (English)

    Wan-Song Ni; Zhi-Yong Lei; Xi Chen; David J. Oliver; Cheng-Bin Xiang

    2007-01-01

    Salt cress (Thellungiella halophila), a close relative of the model plant Arabidopsis thaliana L., is an extremophile that is adapted to harsh saline environments. To mine salt-tolerance genes from this species, we constructed an entry cDNA library from the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombinationassisted cDNA library construction method that is completely free of manipulations involving restriction enzymes and DNA ligase. This cDNA library construction procedure is significantly simplified and the quality of the cDNA library is improved. This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for plant transformation and expression via recombination-assisted cloning. The library is plant transformation ready and is used to transform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.

  12. Cloning and SNPs analysis of CYP51 gene cDNA in sea urchin Strongylocentrotus intermedius%虾夷马粪海胆CYP51基因cDNA的克隆及其SNPs分析

    Institute of Scientific and Technical Information of China (English)

    仇雪梅; 吴领知; 郭晓黎; 张伟杰; 丛玉婷; 常亚青; 宋坚; 刘洋; 王秀利

    2012-01-01

    In this study,CYP51 gene was cloned and its single nucleotide polymorphisms (SNPs) was detected by reverse transcription polymerase chain reaction(RT-PCR) and polymerase chain reaction single-strand conformation polymorphism(PCR-SSCP) in sea urchin Strongylocentrotus intermedius. The results showed that the 1 536 bp sequence including a 1 491 bp open reading frame ( ORF) and encoding 496 amino acids was obtained from CYP51 gene. A SNP A→G was found in the 161st nucleotide of open reading frame in CYP51 cDNA. The analysis of the SNPs showed that there was a significant association (P<0. 05) between the SNP and growth traits such as gonad weight and body weight. The CYP51 cDNA cloning and its SNPs analysis was the first time conducted in sea urchin Strongylocentrotus intermedius. The findings provide basic theory of molecular marker-assisted breeding of the sea urchin.%采用RT-PCR、PCR-SSCP等分子生物学技术和关联分析等方法对虾夷马粪海胆Strongylocentrotus intermedius CYP51基因的cDNA序列进行了克隆和单核苷酸多态性分析.结果表明:虾夷马粪海胆的CYP51基因包括一个1491 bp的开放阅读框,编码496个氨基酸;其开放阅读框的第161个核苷酸处存在1个单核苷酸多态性,即核苷酸发生了A→G突变.关联分析结果表明,该单核苷酸多态性与虾夷马粪海胆的体质量、性腺质量之间存在显著相关性(P<0.05).本研究中首次克隆了虾夷马粪海胆CYP51基因的cDNA序列并进行了SNPs分析,研究结果可为虾夷马粪海胆的分子标记辅助育种提供参考.

  13. Molecular Cloning and Expression Analysis of Toll-like Receptor I cDNA in Japanese Flounder, Paralichthys olivaceus%牙鲆Toll样受体1基因全长cDNA的克隆及特征分析

    Institute of Scientific and Technical Information of China (English)

    吴恋; 孙金生; 耿绪云; 潘宝平; 魏俊利; 王雪惠; 高虹

    2012-01-01

    [Objective] The paper aimed to clone the full length gene of Toll-like recep- tors (TLRs) in Japanese flounder (Paralichthys olivaceus), and analyze their structural features and expression regularity. [Method] The full length cDNA sequence of Toll like receptor 1(TLR1) gene was identified from Japanese flounder head kidney by ho- mologous cloning and rapid amplification cDNA ends (RACE). The bioinformatics and expression model of this gene was analyzed. [Result] The TLR1 cDNA was 2 947 bp, a 2 418 bp open reading frame (ORF), encoding 805 amino acid (aa) residues, including signal peptide, six leucine-rich repeat(LRR) motifs, two transmembrane zones and one TolI/IL 1 receptor (TIR) domain. The molecular weight of the deduced protein was 91.15 KDa, and the isoelectric point was 6.49. The amino acid sequence of Japanese flounder TLR1 possessed 69%-35% identity with the TLRls of other verte- brates, further analysis showed that the TIR domain of Japanese flounder TLR1 shared 84%-62% identities with TIR domains in other vertebrates. Japanese flounder TLR1 protein firstly clustered with TLRls in Epinephelus coioides in the phylogenetic analysis. The transcription of Japanese flounder TLR1 was examined by real-time quantitative PCR, and its mRNA was mainly detected in liver, heart and spleen. [Conclusion] The results lay a foundation for further studying the functions of TLR1 and developing immune potentiator in Japanese flounder.%[目的]克隆牙鲆TLR1(Tolllikereceptors)全长基因,并对其结构特征和表达规律进行分析。[方法]采用同源克隆和快速扩增cDNA末端技术,从牙鲆头肾组织中克隆出TLRl基因cDNA全长序列,并对该基因进行生物信息学和表达模式分析。[结果]牙鲆TLR1基因cDNA全K2947bp,开放阅读框(ORF)2418bp,编码805个氨基酸,包括26个氨基酸组成的信号肽、2个跨膜区、6个富含亮氨酸重复结构域(LRR)和一个TIR结构域(Toll/interleukin(IL).1

  14. Cloning and sequence analysis of para sodium channel cDNA fragment from silkworm, Bombyx mori%家蚕Para钠通道cDNA片段克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    何琳; 刘丽花; 汪洋

    2011-01-01

    Previous studies have revealed that a point mutation of a target gene is related to insecticide resistance to pyrethroids. The para sodium channel in the insect central nervous system is the target of pyrethroid insecticides. We used the RT-PCR method to clone the para sodium ion channel in the silkworm, Bombyx mori L. (GenBank No. EF521818).The full length of this cDNA fragment is 4 882 base pairs and its partial ORF is 3 986 bp translated into 1 328 amino acids. BLAST analysis demonstrated that the cloned cDNA fragment is virtually identical to the para sodium channel a subunit gene amplified from other insects. Amino acid homology of the cloned fragment with para sodium channel a subunit genes from Heliothis virescens Fabricius, Aedes aegypti L. , Blattella germanica L. , Drosophila melanogaster Meigen and Musca domestica L. was 95%, 82%, 80%, 79% and 77% respectively.%昆虫神经系统para型钠离子通道是拟除虫菊酯类杀虫剂的主要靶标,已有的研究表明钠离子通道基因发生点突变与昆虫对菊酯类杀虫剂的抗性密切相关.本文通过RT-PCR方法克隆获得了编码家蚕Bombyx mori L.钠离子通道的cDNA片段(GenBank No.EF521818),该片段全长4 882 bp,部分ORF包含3 986 bp核苷酸,翻译成1 328个氨基酸.蛋白序列分析表明,PCR扩增获得的家蚕钠离子通道eDNA片段所编码的氨基酸与其他昆虫的para型钠离子通道α亚基的氨基酸具有很高的同源相似性,与棉铃虫Heliothis virescens Fabricius、埃及伊蚊Aedes aegypti L.、德国小蠊Blattella germanica L.、果蝇Drosophila melanogaster Meigen和家蝇Musca domestica L.的相似性分别为95%、82%、80%、79%、77%.

  15. cDNA Cloning and Expression Analysis of OsWOX4 in Rice%水稻基因OsWOX4cDNA克隆及表达模式分析

    Institute of Scientific and Technical Information of China (English)

    田夏; 张大兵

    2011-01-01

    WOX (WUSCHEL-related homeobox)是一类含有高度保守同源异型结构域的植物特有转录因子,部分WOX基因家族成员对于植物分生组织的维持发挥着重要的作用.分生组织发育和分化对于植物生长发育十分重要,然而目前人们对于水稻分生组织的维持的分子机制并不清楚.通过RACE(Rapid Amplification cDNA End)的方法我们成功克隆了水稻WOX基因家族成员OsWOX4全长cDNA,该基因编码区(Coding sequence,CDS)长度为711 bp碱基,编码一个236个氨基酸残基的蛋白,5’非翻译区(5'Untranslated region,5'UTR)位于起始密码子上游89碱基处,3'UTR位于终止密码子下游313碱基处.RT-PCR分析表明,该基因主要在茎顶端、花序第3时期的花序原基和成熟花器官中表达,暗示该基因可能参与花序和花分生组织的维持.%WUSCHEL-related homeobox (WOX) transcription factors belong to a large family specifically in plants. Some members of WOX family have been shown to be involved in regulation of meristem activities. Maintenance and differentiation of meristem cells are essential for plant growth and development, but the underlying mechanism of meristem activity control in plants particularly for rice remains largely unknown. In this study, we successfully cloned the full cDNA sequence of OsWOXA of rice by RT-PCR and RACE analyses. The coding sequence of OsWOXA is 711 base pairs in length and encodes a protein with 236 amino acid residues, and the length of 5' UTR (Untranslated Region) OsWOXA is 89 bases and 313 bases for its 3'UTR. Expression analysis indicated that OsWOXA is mainly expressed in rice shoot apical meristem (SAM),inflorescence meristem and flower by qualitative RT-PCR and quantitative RT-PCR,suggesting that OsWOXA may play a role in determining meristem activity in rice

  16. cDNA Clone of Prophenoloxidase for Litopenaeus Stylirostris and Sequence Structure Analysis%细角滨对虾酚氧化酶原cDNA 克隆及序列结构分析

    Institute of Scientific and Technical Information of China (English)

    许尤厚; 胡超群

    2015-01-01

    采用 RT-PCR 原理和长片段扩增技术克隆细角滨对虾酚氧化酶原基因。结果表明,细角滨对虾血淋巴细胞内存在2个 proPO 基因。 proPO gene 1的 cDNA 序列包含有372氨基酸,前190个氨基酸为一个M 家族血蓝蛋白,是一个铜结合位点区域,191-372为一个 C 家族的血蓝蛋白,是一个免疫球蛋白样的区域。proPO gene 2的2个功能位点之间的序列有重叠,proPO gene 2 cDNA 序列的6-935bp 包含了第一个功能位点,928-1464bp 则包含了第二个功能位点。系统进化树比对分析发现2个基因之间的序列差异非常大。细角滨对虾和凡纳滨对虾的 proPO gene 2同处于一个密切相关的群,proPO gene 1则和其他几种对虾的 proPO gene 处于一个群。 proPO gene 2与 proPO gene 1在对虾免疫活动中是否存在不同的功能还有待于进一步的研究。%Prophenoloxidase (proPO) is one of the important factors on humoral immunity of shrimp, so far there are no re-ports for Litopenaeus stylirostris. Depend on techniques of RT-PCR and long fragment amplification cloning, prophenoloxidase gene of L. stylirostris was cloned. The results show that, there are two proPO genes in the lymphocytes of L. stylirostris. ProPO gene 1 cDNA sequence contains 372 amino acids, the first 190 amino acids are a family of M hemocyanin, a copper binding site region, 191-372 is one of the C family of hemocyanin, is an immunoglobulin like region. There are sequence overlap between the 2 functional sites of proPO gene 2, which means that 6-935bp contains the first functional sites, while 928-1464bp contains sec-ond functional sites. The phylogenetic tree alignment analysis showed that sequence structures of two genes is very different. Pro-PO gene 2 of L. stylirostris and L. vannamei was in a closely related group; but proPO gene 1 of L. stylirostris and L. vannamei was in another group with other several shrimp. The function of ProPO gene 2 and proPO gene 1 in shrimp immune

  17. 杜仲HDR基因全长cDNA克隆与序列分析%Cloning and Sequence Analysis of 1-Hydroxy-2-Methyl-2-E-Butenyl-4-Diphosphate Reductase Gene cDNA from Eucommia ulmoides

    Institute of Scientific and Technical Information of China (English)

    刘攀峰; 杜红岩; 乌云塔娜; 杜兰英; 孙志强

    2013-01-01

    以杜仲叶片cDNA为模板,采用反转录RCR及RACE技术分离出HDR基因的cDNA克隆,命名为EuHDR.EuHDR基因cDNA全长1 653 bp,5'端非编码区长82 bp,3'端非编码区长188 bp,编码460个氨基酸,与喜树HDR基因序列相似性最高,达82%;推导EuHDR氨基酸序列中包含转运肽序列(A1-A33)及植物HDR蛋白多个保守的功能位点(A117,A208,A262,A345);EuHDR蛋白二级结构α-螺旋占35.65%,β-折叠占19.78%,螺环结构占44.57%;EuHDR蛋白三级结构为单体形式,呈不规则的三叶草形状;系统进化分析表明EuHDR蛋白与葡萄HDR蛋白的亲缘关系最为接近.%1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) synthesizes IPP and DMAPP in the last step of the plant 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.Homologous HDR gene cDNA was isolated from the leaves of Eucommia ulmoides by the method of reverse transcription polymerase chain reaction (RTPCR) and rapid amplification of cDNA ends (RACE) technique,and named as EuHDR.With the highest gene sequence similarity to Camptotheca acuminata (82%),the full-length cDNA of EuHDR was 1 653 bp including 5'non-coding region of 82 bp and 3' non-coding region of 188 bp and encoded 460 amino acids.The transit peptide sequence (A1-A33) and multiple conserved functional sites(A117,A208,A262,A345)of plant HDR protein were found in the deduced coding sequence of EuHDR.The secondary structure of EuHDR protein was predicted with proportion of α-helix to 35.65%,β-sheet to 19.78% and loop/coil to 44.57%.The calculated protein tertiary structure of EuHDR was formed as monomer,which in space displayed asymmetrical shamrock-like shape.Phylogenetic analysis revealed that the evolutionary relationship of EuHDR protein was the closest to Vitis vinifera HDR protein.

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

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data Description of data contents Phred's quality score. PHD format, one file to a single cDNA data, and co...ription Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive ...

  19. 茶树 HMG-CoA 还原酶基因全长 cDNA 克隆及序列分析%Cloning and sequence analysis of HMG-CoA reductase full-length cDNA from tea (Camellia sinensis)

    Institute of Scientific and Technical Information of China (English)

    韩兴杰; 徐玲玲; 廖亮; 李同建; 邓辉胜; 樊启水; 徐小青

    2015-01-01

    )catalyzes the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)to mevalonate,which is the committed step in the synthesis of isoprenoids via the MVA pathway.To help understand the molecular and genetic mechanisms underlying terpenoid synthesis of tea,a full-length cDNA encoding HMGR was cloned from tea (Camellia sinensis (L.)O.Kuntze)by using the RACE-PCR technique (designated as CsHMGR 1).It comprised 1 979 bp,with a 1 722 bp intact open read-ing frame encoding a 573-amino-acid protein.The deduced protein showed 80% to 82% similarities to homologs from rubber tree (Hevea brasiliensis ),common camptotheca fruit (Camptotheca acuminate ),ginseng (Panax ginseng ), litchis (Litchi chinensis ),American ginseng (Panax quinquefolius ),rooted salvia (Salvia miltiorrhiza ),Momordica grosvenori (Siraltia grosvenorii ),and longan (Dimocarpus longan).The phylogenetic tree,constructed with the cat-alytic domalned of CsHMGR1 and homologs from other species,indicated that CsHMGR1 belonged to the eukaryotic class I HMGR family.CsHMGR1 consisted of two transmembrane domalns,implying that it may be localized to en-doplasmic reticulum (ER)similarly to other eukaryotic homologs.It also contalned two HMG-CoA binding sites,two NADP(H)-binding sites,four conserved catalytic active residues and a phosphorylation site,indicating that phospho-rylation/dephosphorylation is likely a crucial mode of regulation of its biochemical activity.Tissue expression analysis indicated that CsHMGR 1 was expressed comparatively in the leaf buds of C .sinensis cv.Dayelong and in both leaf buds and floral buds of the mother plants.The regulation of expression and physiological activity of CsHMGR1 are likely to impact greatly on tea quality,and CsHMGR1 may provide a basis of the quality evaluation and breeding of tea given that its function is further resolved.

  20. 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......, was determined to be on distal mouse Chromosome (Chr) 9 by analysis of two sets of multilocus crosses....

  1. Improving Software Systems By Flow Control Analysis

    Directory of Open Access Journals (Sweden)

    Piotr Poznanski

    2012-01-01

    Full Text Available Using agile methods during the implementation of the system that meets mission critical requirements can be a real challenge. The change in the system built of dozens or even hundreds of specialized devices with embedded software requires the cooperation of a large group of engineers. This article presents a solution that supports parallel work of groups of system analysts and software developers. Deployment of formal rules to the requirements written in natural language enables using formal analysis of artifacts being a bridge between software and system requirements. Formalism and textual form of requirements allowed the automatic generation of message flow graph for the (sub system, called the “big-picture-model”. Flow diagram analysis helped to avoid a large number of defects whose repair cost in extreme cases could undermine the legitimacy of agile methods in projects of this scale. Retrospectively, a reduction of technical debt was observed. Continuous analysis of the “big picture model” improves the control of the quality parameters of the software architecture. The article also tries to explain why the commercial platform based on UML modeling language may not be sufficient in projects of this complexity.

  2. Characterization and simulation of cDNA microarray spots using a novel mathematical model

    OpenAIRE

    2007-01-01

    Abstract Background The quality of cDNA microarray data is crucial for expanding its application to other research areas, such as the study of gene regulatory networks. Despite the fact that a number of algorithms have been suggested to increase the accuracy of microarray gene expression data, it is necessary to obtain reliable microarray images by improving wet-lab experiments. As the first step of a cDNA microarray experiment, spotting cDNA probes is critical to determining the quality of s...

  3. 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

    of genes between different tissues, in particular brain/spinal cord, and found patterns of correlation between genes that share expression in pairs of libraries. Finally, there was remarkable agreement in expression between specialized tissues according to Gene Ontology categories. CONCLUSION: This EST......BACKGROUND: Knowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from...... approximately 25% have a high confidence match to UniProt. Approximately 6,000 new porcine gene clusters were identified. Expression analysis based on the non-normalized libraries resulted in the following findings. The distribution of cluster sizes is scaling invariant. Brain and testes are among the tissues...

  4. 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, Jakob;

    2007-01-01

    of genes between different tissues, in particular brain/spinal cord, and found patterns of correlation between genes that share expression in pairs of libraries. Finally, there was remarkable agreement in expression between specialized tissues according to Gene Ontology categories. Conclusion: This EST......Background: Knowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from...... approximately 25% have a high confidence match to UniProt. Approximately 6,000 new porcine gene clusters were identified. Expression analysis based on the non-normalized libraries resulted in the following findings. The distribution of cluster sizes is scaling invariant. Brain and testes are among the tissues...

  5. Improved Tiled Bitmap Forensic Analysis Algorithm

    Directory of Open Access Journals (Sweden)

    C. D. Badgujar, G. N. Dhanokar

    2012-12-01

    Full Text Available In Computer network world, the needs for securityand proper systems of control are obvious and findout the intruders who do the modification andmodified data. Nowadays Frauds that occurs incompanies are not only by outsiders but also byinsiders. Insider may perform illegal activity & tryto hide illegal activity. Companies would like to beassured that such illegal activity i.e. tampering hasnot occurred, or if it does, it should be quicklydiscovered. Mechanisms now exist that detecttampering of a database, through the use ofcryptographically-strong hash functions. This papercontains a survey which explores the various beliefsupon database forensics through differentmethodologies using forensic algorithms and toolsfor investigations. Forensic analysis algorithms areused to determine who, when, and what data hadbeen tampered. Tiled Bitmap Algorithm introducesthe notion of a candidate set (all possible locationsof detected tampering(s and provides a completecharacterization of the candidate set and itscardinality. Improved tiled bitmap algorithm willcover come the drawbacks of existing tiled bitmapalgorithm.

  6. De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus densiflora).

    Science.gov (United States)

    Liu, Le; Zhang, Shijie; Lian, Chunlan

    2015-12-04

    Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora.

  7. De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus densiflora

    Directory of Open Access Journals (Sweden)

    Le Liu

    2015-12-01

    Full Text Available Japanese red pine (Pinus densiflora is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98% unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%, Amborella trichopoda (9.83%, and Pinus taeda (4.15%. A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite software, of which the tri-nucleotide repeats were most abundant (50.18% and 629 EST-SSR (expressed sequence tag- simple sequence repeats primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s mapping for pine for P. densiflora.

  8. Cloning and sequence analysis of cDNA encoding aquaporin (AQP) gene from Anopheles sinensis%中华按蚊水通道蛋白(AsAQP)cDNA克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    唐建霞; 张超; 白亮; 李菊林; Liu Kun; 周华云; 曹俊; 高琪

    2012-01-01

    目的 克隆中华按蚊水通道蛋白(AsAQP)基因的cDNA全长序列,分析其基因序列特征,为研究AsAQP的生物学功能提供分子基础.方法 根据已报道的昆虫水通道蛋白(AQP)氨基酸序列的保守区域,采用兼并引物从中华按蚊cDNA中获取AsAQP基因片段,在此基础上利用cDNA末端快速扩增(RACE)技术克隆该基因cDNA全长序列,并用生物信息学方法对获取的序列进行分析.结果 利用兼并引物从中华按蚊成蚊cDNA中分离到AsAQP基因片段,利用RACE技术克隆到该基因的全长cDNA.序列分析表明,该基因cDNA全长762 bp,编码253个氨基酸,蛋白分子量约为63.2 kD.生物信息学分析表明,AsAQP具有典型的6个跨膜区结构和2个天冬酰胺酸-脯氨酸-丙氨酸(NPA)结构,该结构是主要内在蛋白(MIP)家族典型的结构特征.AsAQP与致倦库蚊(Culex quinquefasciatus)AQP及埃及伊蚊(Aedes aegypti AQP蛋白的同源性分别为76%和78%.氨基酸序列聚类分析表明,AsAQP与其他蚊种的水通道蛋白遗传距离较近.结论 利用兼并引物结合RACR技术首次获得了编码AsAQP基因的cDNA全长序列,该基因属于MIP蛋白家族成员,具有典型的功能域,为进一步研究该蛋白的功能奠定了基础.%Objective To clone and analyze the full-length sequence of aquaporin gene of Anopheles sinensis (AsAQP) , so as to provide an insight into its biology functions. Methods The degenerate primers were used to amplify conserved region of AQP from An. Sinensis cDNA. After then, the full-length cDNA of AsAQP was obtained by rapid amplification of cDNA ends (RACE). Concurrently, the bioinformatics methods were applied to analyze the obtained sequence. Results The obtained full-length cD-NA of AsAQP consisted of 762 bp and 253 deduced amino acids with a predicted molecular mass of 63.2 kD. Bioinformatics analysis demonstrated that AsAQP had a typical structure with six membrane-spanning domains and an internal symmetry showing

  9. 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.

  10. The cDNA Cloning and Sequence Analysis of Fragment of CART mRNA in Porcine Hypothalamus%猪下丘脑CART完整CDS区结构域分析

    Institute of Scientific and Technical Information of China (English)

    李鹏飞; 李富禄; 于秀菊; 吕丽华

    2011-01-01

    In order to obtain and analysis the complete CDS and its domain of porcine CART (Cocain and amphetamine- regulated transcript, CART), based on the cDNA sequences of CART in other species published in GenBank, three pairs of specific primers were designed and part mRNA sequence was amplified by RT-PCR. By alignment with other species, the sequences had high similarities, suggesting that the sequence was the porcine CART,and the mRNA expressed in porcine hypothalamus; Analysis of the 3D structure of the CART protein,showed that a CART superfamily (CART Superfamily) existed between amino acids 40 to 116. The superfamily may be as the ligand of iron to achieve a variety of physiological functions in vivo.%为获得猪可卡因苯异丙胺调控转录肽(Cocain and amphetamine-regulated transcript,CART)的完整CDS区并分析其结构城,根据GenBank中公布的其他物种CART的cDNA序列,设计了三对特异性引物,采用RT- PCR技术扩增出猪下丘脑组织CART mRNA的部分序列.经NCBI比对,与其他物种的相似性较高,提示该序列为猪CART的mRNA序列,CART mRNA在猪下丘脑上有表达;对该序列进行3D结构分析,发现猪CART蛋白在第40~116位氨基酸间存在一个CART超家族(CART Superfamily),这个超家族可能通过作为铁离子的配合基,在动物体内实现多种生理功能.

  11. Molecular cloning, sequencing, and expression analysis of cDNA encoding metalloprotein II (MP II) induced by single and combined metals (Cu(II), Cd(II)) in polychaeta Perinereis aibuhitensis.

    Science.gov (United States)

    Yang, Dazuo; Zhou, Yibing; Zhao, Huan; Zhou, Xiaoxiao; Sun, Na; Wang, Bin; Yuan, Xiutang

    2012-11-01

    We amplified and analyzed the complete cDNA of metalloprotein II (MP II) from the somatic muscle of the polychaete Perinereis aibuhitensis, the full length cDNA is 904 bp encoding 119 amino acids. The MP II cDNA sequence was subjected to BLAST searching in NCBI and was found to share high homology with hemerythrin of other worms. MP II expression of P. aibuhitensis exposed to single and combined metals (Cu(II), Cd(II)) was analyzed using real time-PCR. MP II mRNA expression increased at the start of Cu(II) exposure, then decreased and finally return to the normal level. Expression pattern of MP II under Cd(II) exposure was time- and dose-dependent. MP II expression induced by a combination of Cd(II) and Cu(II) was similar to that induced by Cd(II) alone.

  12. Construction of cDNA library of the treated Changliver cell and quality analysis%常氏肝癌细胞cDNA文库的构建及质量分析

    Institute of Scientific and Technical Information of China (English)

    林俊堂; 王聪睿; 张会勇; 李玉昌; 徐存拴

    2004-01-01

    目的利用RNA转录5′末端转换机制(SMART)构建适合免疫筛选的经去分化培养基处理的常氏肝癌细胞cDNA文库.方法通过反转录PCR和长距离PCR获得常氏肝癌细胞的全长cDNA,然后利用SMART cDNA文库构建试剂盒建立经去分化培养基处理的常氏肝癌细胞cDNA文库.结果通过测定,高质量的包含常氏肝癌细胞全长cDNA的cDNA文库得到建立,扩增cDNA文库的滴度高达4.5 × 1010 pfu*ml-1,重组子内平均插入外源基因片段长度在200 bp到4000 bp之间,约1500 bp左右,并且此文库适合免疫筛选.结论结果显示所构建的经去分化培养基处理的常氏肝癌细胞cDNA文库具有很高的质量, 为进一步研究常氏肝癌细胞和筛选相关基因获得cDNA全长奠定了坚实的基础.%Objective To construct cDNA library of the treated Changliver cell by switching mechanism at 5′ end of RNA transcript (SMART) technique and analyze its quality.Methods cDNA of Changliver cell was aquired with reverse transcription polymerase chain reaction (RT-PCR) and long-distance PCR (LD-PCR),then the cDNA library was constructed with SMART cDNA library construction kit.Results Through testing,the high quality cDNA library containing full length cDNA of Changliver cell had been constructed.The titer of the amplified cDNA library was 4.5 × 1010 pfu*ml-1 and the average exogenous inserts of the recombinants was 1.5 kb.Conclusion These results suggest that the Changliver cell cDNA library has a high quality and lays a solid foundation for researching on Changliver cell and screening

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

    OpenAIRE

    1988-01-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex...

  14. Conversion of cDNA differential display results (DDRT-PCR into quantitative transcription profiles

    Directory of Open Access Journals (Sweden)

    Koopmann Birger

    2005-04-01

    Full Text Available Abstract Background Gene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Commonly used protocols for gel-based transcript profiling are cDNA differential display (DDRT-PCR and cDNA-AFLP. Both methods have been used merely as qualitative gene discovery tools so far. Results We developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer and detector signals are converted into virtual gel images suitable for semi-automatic analysis. Data processing consists of four steps: (i cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, (ii intensity of bands is determined by densitometry, (iii densitometric values are normalized, and (iv intensity ratio is calculated for each pair of corresponding bands. Transcription profiles are represented by sets of intensity ratios (control vs. treatment for cDNA fragments defined by primer combination and DNA mobility. We demonstrated the procedure by analyzing DDRT-PCR data on the effect of secondary metabolites of oilseed rape Brassica napus on the transcriptome of the pathogenic fungus Leptosphaeria maculans. Conclusion We developed a data processing procedure for the quantitative analysis of amplified cDNA fragments separated by electrophoresis. The system utilizes common software and provides an open-end alternative to DNA microarray analysis of the transcriptome. It is expected to work equally well with DDRT-PCR and cDNA-AFLP data and be useful particularly in reseach on organisms for which microarray analysis is not available or economical.

  15. RICD: A rice indica cDNA database resource for rice functional genomics

    Directory of Open Access Journals (Sweden)

    Zhang Qifa

    2008-11-01

    Full Text Available Abstract Background The Oryza sativa L. indica subspecies is the most widely cultivated rice. During the last few years, we have collected over 20,000 putative full-length cDNAs and over 40,000 ESTs isolated from various cDNA libraries of two indica varieties Guangluai 4 and Minghui 63. A database of the rice indica cDNAs was therefore built to provide a comprehensive web data source for searching and retrieving the indica cDNA clones. Results Rice Indica cDNA Database (RICD is an online MySQL-PHP driven database with a user-friendly web interface. It allows investigators to query the cDNA clones by keyword, genome position, nucleotide or protein sequence, and putative function. It also provides a series of information, including sequences, protein domain annotations, similarity search results, SNPs and InDels information, and hyperlinks to gene annotation in both The Rice Annotation Project Database (RAP-DB and The TIGR Rice Genome Annotation Resource, expression atlas in RiceGE and variation report in Gramene of each cDNA. Conclusion The online rice indica cDNA database provides cDNA resource with comprehensive information to researchers for functional analysis of indica subspecies and for comparative genomics. The RICD database is available through our website http://www.ncgr.ac.cn/ricd.

  16. 双齿围沙蚕Cu/Zn-SOD cDNA基因的克隆及序列分析%Cloning and sequence analysis of Cu/Zn-SOD cDNA from sandworm Perinereis aibuhitensis

    Institute of Scientific and Technical Information of China (English)

    岳宗豪; 樊鑫; 赵欢; 任洪伟; 张旭峰; 周一兵

    2014-01-01

    A full length cDNA of Cu/Zn-SOD was firstly cloned in sandworm Perinereis aibuhitensis by homology cloning and RACE techniques based on the partial copper-zinc superoxide dismutase ( Cu/Zn-SOD) gene from pol-ychaete Alitta succinea. The full length of the cDNA was found to be 870 bp including a 156 bp 5′untranslated re-gion,a 261 bp 3′untranslated region and 453 bp open reading frame encoding 150 amino acids. There were typical Cu2+ and Zn2+ binding sites as well as two Cu/Zn-SOD protein family tag sequences in the deduced protein which was within the intracellular Cu/Zn-SOD with relative molecular mass of 15 249 900 and the isoelectric point of 5. 66 by bioinformatic analysis. No signal peptide and transmembrane domain were observed in the deduced pro-tein, indicating that it belonged to the hydrophilic protein. Multiple sequences alignment analysis revealed that the deduced amino acids had high homology to the proteins of partial molluscs, fishes and insects. The findings will provide basis for research of dose-response between gene expression and environmental pollutants, and defense mechanism of the sandworm.%根据已知多毛类Alitta succinea铜锌超氧化物歧化酶( Cu/Zn-SOD)基因序列设计引物,利用同源克隆及RACE方法首次从双齿围沙蚕Perinereis aibuhitensis中克隆得到Cu/Zn-SOD基因全长cDNA序列。结果表明:双齿围沙蚕Cu/Zn-SOD基因cDNA全长870 bp,其中包括156 bp的5′端非编码区,261 bp 3′端非翻译区和453 bp 开放阅读框,编码150个氨基酸;该蛋白序列具有典型的Cu2+和Zn2+结合位点,并具有两处Cu/Zn-SOD 蛋白家族标签序列。通过生物信息学分析表明,该蛋白属于胞内Cu/Zn-SOD,理论相对分子质量为15249900,等电点为5.66,无信号肽和跨膜区,推测为亲水性蛋白。同源性分析表明,双齿围沙蚕Cu/Zn-SOD氨基酸序列与部分软体动物、鱼类和昆虫的Cu/Zn-SOD蛋白序列具有很高的相似性。该研究结果为后续研

  17. 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.

  18. 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

  19. On Cloning,Sequence Analysis and Tissue Expression of Ceruloplasmin Gene in Rare Gudgeon%稀有鮈鲫铜蓝蛋白基因 cDNA 克隆及组织表达分析

    Institute of Scientific and Technical Information of China (English)

    景致; 彭作刚; 张耀光

    2014-01-01

    铜蓝蛋白(Ceruloplasmin ,Cp)是一种重要的铜转运蛋白,合成于肝脏并参与生物体铁的代谢,在医学上是各种炎症、感染、中毒及癌症疾病的标志性蛋白.铜蓝蛋白的研究已在多种真骨鱼类中被报道,文中第一次在稀有鮈鲫(Gobiocypris rarus)中报道此基因.采用cDNA末端快速扩增技术(rapid amplification of cDNA ends ,RACE)克隆了稀有鮈鲫铜蓝蛋白基因,使用荧光定量PCR的方法构建了该基因组织表达谱.序列分析表明稀有鮈鲫铜蓝蛋白基因包含3264 bp全长编码序列,该序列编码1087个氨基酸,其核苷酸和氨基酸序列与斑马鱼同源性最高(分别为88.1%和90.3%).理论相对分子质量和等电点分别为124429.1 D和6.41.荧光定量PCR检测表明该基因在肝脏和脾脏中相对表达量最高,在肌肉和鳃中相对表达量最低.使用氨基酸序列进行蛋白结构保守域分析,结果表明铜蓝蛋白基因在脊椎动物中是相对保守的,推测其功能也与其他物种相似.这为进一步研究稀有鮈鲫该基因的功能及其应用奠定了基础.%Ceruloplasmin (Cp) ,which is the major copper-carrying protein synthesized in the liver ,plays a role in iron metabolism .It is a marker protein for inflammation ,infection ,poisoning and cancer .The Cp gene has been reported in several teleosts and here the gene in rare gudgeon (Gobiocy p ris rarus) has been first characterized . In this study , the Cp gene has been cloned by rapid amplification of cDNA ends (RACE) .Real-time PCR has been performed to demonstrate the expression pattern in different tissues . The CDS of Cp gene is 3 264 bp long ,which encodes 1 087 amino acids .BLAST result indicates that the most similar homologue of rare gudgeon Cp is from zebrafish ,with a homology of 88 .1% (DNA ) and 90 .3% (amino acid) .The predicted relative molecular mass of the protein is 124 429.1 D with an estimated PI of 6

  20. Molecular characteristics and expression analysis of ScHsc70 cDNA in agamaki clam (Sinonovacula constricta)%缢蛏ScHsc70cDNA的分子特性和表达分析

    Institute of Scientific and Technical Information of China (English)

    冯冰冰; 牛东红; 钟玉民; 陈慧; 林国文; 李家乐

    2012-01-01

    Heat shock proteins consist of several families of highly conserved proteins that play an essential role in a number of cellular processes. Among the 70 kD family of heat shock proteins, heat shock cognate protein 70 kD (Hsc70) and inducible heat shock protein 70 kD (Hsp70) have been extensively studied in vertebrates and invertebrates. Several cDNAs encoding HSP70 have been described in molluscs, including the oyster (Crassostrea gigas), bay scallop (Argopecten irradians), Zhikong scallop (Chlamys farreri), mussel (Mytilus galloprovincialis), and Asiatic hard clam (Meretrix meretrix). Recent studies examining HSP70 expression in different species of mollusc have recognized the physiological and ecological importance of heat-shock gene expression in response to changing environments. We isolated an EST sequence with high homology with heat shock protein 70 gene from the cDNA library of Sinonovacula constricta. Then, the complete express sequence of this gene was obtained using PCR and 5'RACE. The cDNA of this gene was 2 335 bp, and consisted of a 76 bp 5' untranslated region (UTR), a 1 950 bp open reading frame (ORF) and a 309 bp 3' UTR. The translated protein consisted of 649 amino acids (70.89 kD)and its calculated isoelectric point was 5.28. Sequence analysis of the protein revealed that this gene contained three signature sequences of the heat shock protein 70 family (HSP70 family), two glycosylation sites and one ATP-GTP binding site. Four terapeptides of GGXP and a cytoplasm characteristic motif of EEVD were detected in the carboxyl terminal region of the deduced amino acid sequence. This HSP70 is a member of the HSC70 (constitutive genes) subfamily in the HSP70 family, and is designated as ScHsc70. Phylogenetic analysis suggested that the protein was most similar to those of 5. Constricta, Laternula elliptica, and M. Meretrix. Quantitative reverse transcriptase (qRT-PCR) analyses revealed that ScHsc70 mRNA was expressed constitutively in all the tissues

  1. ASSOCIATION OF DIFFERENTIALLY EXPRESSED cDNA FRAGMENT OF FGG WITH HEPATOCELLULAR CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    范秉琳; 朱武凌; 邹国林; 段芳龄

    2002-01-01

    Objective: To identify a cDNA clone from the subtracted library of human hepatocellular carcinoma (HCC). Methods: Suppression subtractive hybridization was used to isolated a panel of genes that are differentially expressed in hepatocellular carcinoma as compared with cirrhotic liver. T/A cloning method was used to construct a subtracted cDNA library. DNA sequencing analysis and Northern blot analysis were also utilized. Results: The cloned cDNA is 787 nucleotides in length and contains an open reading frame of 230 amino acids, which is a cDNA fragment of reported human fibrinogen, gamma polypeptide (FGG). Northern analysis revealed that this gene was overexpressed in two hepatocellular carcinoma cell lines, SMMC-7721 and HepG2. Conclusion: Sequence identity proved the cDNA clone fragment of as FGG gene. Differential expression of the cDNA fragment in HCC suggested that FGG is related to HCC, indicating a new clue for developing a novel diagnostic and prognostic marker.

  2. 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

  3. Construction and Analysis of a Full-Length cDNA Library of Peanut Embryos at Different Developmental Stages%不同发育时期花生胚混合全长cDNA文库的构建与分析

    Institute of Scientific and Technical Information of China (English)

    陈华; 邓烨; 张冲; 蔡铁城; 郑奕雄; 庄伟建

    2014-01-01

    以及DREB转录因子等。%[Objective] The objective of this study is to understand the molecular mechanism of peanut embryo development and obtain important genes related to peanut embryo development. [Method] Using peanut variety Minhua 6 as the experimental material, embryos on 10, 20, 30, 40, 50, and 60th day after pegging were sampled. Total RNA was extracted by improved CTAB method. Double strand cDNA was synthesized based on SMART technique. The purified dscDNA was ligated to pDNR-LIB vector digested by SfiⅠ and transformed into DH5α by electroporation to construct a full-length cDNA library of peanut embryos at different developmental stages. Bioinformatics analysis was performed following small-scale EST sequencing.[Result]A successful full-length cDNA library of peanut embryos at different development stages was constructed. The titer of unamplified cDNA library was about 3.5×106cfu/mL. The average cDNA inserts were more than 1 000 bp with a recombination frequency of 95.8%. Small-scale plasmid extraction and subsequent sequencing resulted in 60 ESTs, which were used for further analysis. BLASTX analysis showed that 39 sequences (65% of total sequences) had high similarity with reported genes in Glycine max, Arachis hypogaea, Medicago truncatula, etc. on NCBI with 32 sequences having known or putative functions and functions of other 7 sequences were unclear. The other 21 (35%of total sequences) could not find similarity with known genes in NCBI, which may be novel genes for peanut. GO annotation was performed with BLAST2GO software and the results revealed that the ESTs generated in this study mainly included responsive to stresses and defenses, protein synthesis and transport, lipid synthesis and metabolism, transcription and regulation, seed germination, dormancy and embryo development related genes. Besides, some genes were involved in signal transduction and light morphogenesis process. KEGG pathway analysis showed that the ESTs generated by randomly sequencing in this study mainly

  4. 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...

  5. SINFAC - SYSTEMS IMPROVED NUMERICAL FLUIDS ANALYSIS CODE

    Science.gov (United States)

    Costello, F. A.

    1994-01-01

    The Systems Improved Numerical Fluids Analysis Code, SINFAC, consists of additional routines added to the April 1983 revision of SINDA, a general thermal analyzer program. The purpose of the additional routines is to allow for the modeling of active heat transfer loops. The modeler can simulate the steady-state and pseudo-transient operations of 16 different heat transfer loop components including radiators, evaporators, condensers, mechanical pumps, reservoirs and many types of valves and fittings. In addition, the program contains a property analysis routine that can be used to compute the thermodynamic properties of 20 different refrigerants. SINFAC can simulate the response to transient boundary conditions. SINFAC was first developed as a method for computing the steady-state performance of two phase systems. It was then modified using CNFRWD, SINDA's explicit time-integration scheme, to accommodate transient thermal models. However, SINFAC cannot simulate pressure drops due to time-dependent fluid acceleration, transient boil-out, or transient fill-up, except in the accumulator. SINFAC also requires the user to be familiar with SINDA. The solution procedure used by SINFAC is similar to that which an engineer would use to solve a system manually. The solution to a system requires the determination of all of the outlet conditions of each component such as the flow rate, pressure, and enthalpy. To obtain these values, the user first estimates the inlet conditions to the first component of the system, then computes the outlet conditions from the data supplied by the manufacturer of the first component. The user then estimates the temperature at the outlet of the third component and computes the corresponding flow resistance of the second component. With the flow resistance of the second component, the user computes the conditions down stream, namely the inlet conditions of the third. The computations follow for the rest of the system, back to the first component

  6. Novel analysis and improvement of Yahalom protocol

    Institute of Scientific and Technical Information of China (English)

    CHEN Chun-ling; YU Han; L(U) Heng-shan; WANG Ru-chuan

    2009-01-01

    The modified version of Yahalom protocol improved by Burrows, Abradi, and Needham (BAN) still has security drawbacks. This study analyzed such flaws in a detailed way from the point of strand spaces, which is a novel method of analyzing protocol's security. First, a mathematical model of BAN-Yahalom protocol is constructed. Second, penetrators' abilities are restricted with a rigorous and formalized definition. Moreover, to increase the security of this protocol against potential attackers in practice, a further improvement is made to the protocol. Future application of this re-improved protocol is also discussed.

  7. 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.

  8. High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses.

    Science.gov (United States)

    Abd El-Rehim, Dalia M; Ball, Graham; Pinder, Sarah E; Rakha, Emad; Paish, Claire; Robertson, John F R; Macmillan, Douglas; Blamey, Roger W; Ellis, Ian O

    2005-09-01

    Recent studies on gene molecular profiling using cDNA microarray in a relatively small series of breast cancer have identified biologically distinct groups with apparent clinical and prognostic relevance. The validation of such new taxonomies should be confirmed on larger series of cases prior to acceptance in clinical practice. The development of tissue microarray (TMA) technology provides methodology for high-throughput concomitant analyses of multiple proteins on large numbers of archival tumour samples. In our study, we have used immunohistochemistry techniques applied to TMA preparations of 1,076 cases of invasive breast cancer to study the combined protein expression profiles of a large panel of well-characterized commercially available biomarkers related to epithelial cell lineage, differentiation, hormone and growth factor receptors and gene products known to be altered in some forms of breast cancer. Using hierarchical clustering methodology, 5 groups with distinct patterns of protein expression were identified. A sixth group of only 4 cases was also identified but deemed too small for further detailed assessment. Further analysis of these clusters was performed using multiple layer perceptron (MLP)-artificial neural network (ANN) with a back propagation algorithm to identify key biomarkers driving the membership of each group. We have identified 2 large groups by their expression of luminal epithelial cell phenotypic characteristics, hormone receptors positivity, absence of basal epithelial phenotype characteristics and lack of c-erbB-2 protein overexpression. Two additional groups were characterized by high c-erbB-2 positivity and negative or weak hormone receptors expression but showed differences in MUC1 and E-cadherin expression. The final group was characterized by strong basal epithelial characteristics, p53 positivity, absent hormone receptors and weak to low luminal epithelial cytokeratin expression. In addition, we have identified significant

  9. 泌盐植物长叶红砂质膜 Na +/H +逆向转运蛋白基因(RtSOS1)全长 cDNA 的克隆及序列分析%Cloning and Sequence Analysis of the Plasma Membrane Na +/H +Antiporter cDNA in Recretohalophyte Reaumuria trigyna Maxim

    Institute of Scientific and Technical Information of China (English)

    党振华; 郑琳琳; 冯智; 王迎春

    2013-01-01

      Reaumuria trigyna Maxim.is an endangered small shrub with the features of a recretohalophyte .This species is endemic to the Eastern Alxa Western Ordos area and developed distinctive strategies to adapt to the semi -desert and salty soil environment .A full-length cDNA of the plasma Na+/H+antiporter (RtSOS1) was isolated from this species by using RT-PCR and RACE technologies.The 3 829 bp sequence comprised a 3 438 bp open reading frame,encoding an 1 145 amino acids protein with the molecular weight of 126.76 kDa.Bioinformatics analyze re-veals that RtSOS1 composed of 11 transmembrane domains within its N terminal portion ,and a hydrophilic cytoplas-mic tail with the length approximately 700 amino acids in its C-terminal portion.In the C-terminal region,the phos-phorylation domain and the auto -inhibited domain are found.The Homology comparison and phylogenetic analysis showed that RtSOS1 is related to plasma membrane Na+/H+antiporter in other plant species.%  长叶红砂为内蒙古东阿拉善-西鄂尔多斯地区特有珍稀泌盐,强旱生小灌木,对盐渍荒漠环境具有极强适应性。利用 RT-PCR 和 RACE 技术从该植物中分离出质膜 Na+/H+逆向转运蛋白基因(RtSOS1),该 cDNA 全长为3829 bp,开放阅读框为3438 bp,编码一个含1145个氨基酸的蛋白质,推测分子量为126.76 kDa。氨基酸序列的生物信息学分析推测,该蛋白 N 端含有11个跨膜结构域,C 端为一个长约700个氨基酸的亲水性尾,具有磷酸化和自我抑制结构域。同源性比对和系统发育分析证实,RtSOS1与其他植物的质膜 Na+/H+逆向转运蛋白亲缘关系较近。

  10. Construction and analysis of a subtracted cDNA library of Betula platyphylla female inflorescence%白桦雌花序cDNA消减文库的构建及分析

    Institute of Scientific and Technical Information of China (English)

    魏继承; 杨传平; 王超; 姜静

    2005-01-01

    为研究白桦雌花序花期基因表达情况,以两天为间隔对其进行取样.基于SMART策略,通过RT-PCR,将源自最后时期样品的cDNA作为Driver cDNA,源自其他时期样品的cDNA作为Tester cDNA,构建抑制性消减文库.EST序列经blastX分析表明,该文库中的基因大致可以归为五类,分别同代谢、物质运输和信号转导、细胞周期、胁迫反应及调控相关.本文对基因表达同发育的关系做了探讨.%Female inflorescence of Betula platyphylla was sampled at an interval of each two days to analyze the background of gene expression in floral phase. On the basis of SMART strategy, the driver cDNA was obtained from total RNA of the last sample and the tester cDNA was from that of the others by RT-PCR which were subsequently used to construct a subtracted cDNA library. The result of the ESTs (expression sequence tags) blastX showed that the genes in the subtracted cDNA library could be mainly clustered into 5 groups related to metabolism, transportation and signal transduction, cell cycle, stress response, and regulation. The relationship between gene expression and development was also discussed.

  11. Cloning and prokaryotic expression of HGLP cDNA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A novel cDNA, HGLP, which encodes a G- protein coupled receptor (GPCR) like protein, has been isolated and cloned. The coding region of the human HGLP predicts a 7-transmembrane region protein with motifs of rhodopsin-like GPCR superfamily. Northern blot analysis reveals a 3-kb transcript in various human tissues examined. The N- and C-terminal coding regions of HGLP, which are deduced as non-transmembrane regions, have been amplified by PCR and cloned into pET30a+ vector. Then the recombinant proteins are highly expressed in E. coli.

  12. 家蝇防御素基因的cDNA克隆及序列分析%Cloning and sequence analysis of the full-length cDNA encoding defensin, an antimicrobial peptide from the housefly (Musca domestica)

    Institute of Scientific and Technical Information of China (English)

    王来城; 王金星; 王来元; 赵小凡

    2003-01-01

    Defensin is a kind of cationic.inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms.with their broad antimicrobial spectrum and strong pharmaceutical effects.antimicrobial peptides,including defensins,represent a source of novel antibiotic agents.A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDnA library of houseflies(Musca domestica) that had been challenged by E.coli and staphylococcus taincd an NH2-terminal signal sequence(1-22)followed by a propeptide and the mature peptide(53-92),The sequence identity with other insect defensin is between 51% and 73%.The mature peptide,with a predicted molecular weight of 4.0kDa,and pI of 8.69,has 1 negative charged amino acid and 4 positice ones,the putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds,Cys1-Cys4,Cys2-Cys5 and Cys3-Cys6,These results suggest that the novel full-length cDNA of the defensin gene.Denominated Mdde,has been successfully cloned from houseflies.

  13. cDNA2Genome: A tool for mapping and annotating cDNAs

    Directory of Open Access Journals (Sweden)

    Suhai Sandor

    2003-09-01

    Full Text Available Abstract Background In the last years several high-throughput cDNA sequencing projects have been funded worldwide with the aim of identifying and characterizing the structure of complete novel human transcripts. However some of these cDNAs are error prone due to frameshifts and stop codon errors caused by low sequence quality, or to cloning of truncated inserts, among other reasons. Therefore, accurate CDS prediction from these sequences first require the identification of potentially problematic cDNAs in order to speed up the posterior annotation process. Results cDNA2Genome is an application for the automatic high-throughput mapping and characterization of cDNAs. It utilizes current annotation data and the most up to date databases, especially in the case of ESTs and mRNAs in conjunction with a vast number of approaches to gene prediction in order to perform a comprehensive assessment of the cDNA exon-intron structure. The final result of cDNA2Genome is an XML file containing all relevant information obtained in the process. This XML output can easily be used for further analysis such us program pipelines, or the integration of results into databases. The web interface to cDNA2Genome also presents this data in HTML, where the annotation is additionally shown in a graphical form. cDNA2Genome has been implemented under the W3H task framework which allows the combination of bioinformatics tools in tailor-made analysis task flows as well as the sequential or parallel computation of many sequences for large-scale analysis. Conclusions cDNA2Genome represents a new versatile and easily extensible approach to the automated mapping and annotation of human cDNAs. The underlying approach allows sequential or parallel computation of sequences for high-throughput analysis of cDNAs.

  14. Random rapid amplification of cDNA ends (RRACE) allows for cloning of multiple novel human cDNA fragments containing (CAG)n repeats.

    Science.gov (United States)

    Carney, J P; McKnight, C; VanEpps, S; Kelley, M R

    1995-04-03

    We describe a new technique for isolating cDNA fragments in which (i) either a partial sequence of the cDNA is known or (ii) a repeat sequence is utilized. We have used this technique, termed random rapid amplification of cDNA ends (random RACE), to isolate a number of trinucleotide repeat (CAG)n-containing genes. Using the random RACE (RRACE) technique, we have isolated over a hundred (CAG)n-containing genes. The results of our initial analysis of ten clones indicate that three are identical to previously cloned (CAG)n-containing genes. Three of our clones matched with expressed sequence tags, one of which contained a CA repeat. The remaining four clones did not match with any sequence in GenBank. These results indicate that this approach provides a rapid and efficient method for isolating trinucleotide repeat-containing cDNA fragments. Finally, this technique may be used for purposes other than cloning repeat-containing cDNA fragments. If only a partial sequence of a gene is known, our system, described here, provides a rapid and efficient method for isolating a fragment of the gene of interest.

  15. Improved Spectrum Analysis Noise Radar Systems.

    Science.gov (United States)

    and evaluated. A new spectrum analysis system designed to detect moving targets is presented. Comparison is made of the detection capabilities of all four noise radar systems in the presence of extraneous noise. (Author)

  16. Construction and analysis of a cDNA library from queen honeybee spermatheca gland%蜂王受精囊腺cDNA文库的构建与分析

    Institute of Scientific and Technical Information of China (English)

    李江红; 刘振; 欧阳昊; 彭文君; 梁勤

    2011-01-01

    Spermatheca is a tissue in queen honeybee for storing the sperm from the drone honeybee in copulation. The long term sperm storage in spermatheca is related to the secretion of spermathecal gland. Gene expression and regulation of spennathecal gland is a basement for understanding the mechanism of long term sperm storage. In this study, four hundred queen honeybees were reared artificially. The spermathecal gland of queen honeybee was dissected under microscope for isolating the total RNA and mRNA. The double strands cDNA were synthesized using the SMART ( switching mechanism at 5' end of RNA transcript) technology and then used to construct a cDNA library of spennathecal gland. The titre of the cDNA library was about 1.1× 106. The recombination rate of the cDNA library was over 99%. Many clones coding the spermathecal fluid protein were obtained by small scale sequencing and analyzing the cDNA library clones. Among them three clones coding the honeybee venom protein of apamin, secapinand icarapin, two major royal jelly protein clones ( MRJP8 and MRJP9) and testis enhanced gene transcript clone were also detected. These works will be helpful for understanding the mechanism of long term sperm storage in spermatheca.%从400只人工培育的处女蜂王中解剖受精囊及其腺体,利用其mRNA构建了一个cDNA文库.该文库的滴度为1.1×106,重组效率大于99%.进一步对文库克隆进行小规模测序和分析,获得了一批编码蜂王受精囊腺内溶蛋白的基因克隆,同时从中也检测到编码3种蜂毒蛋白(明肽、镇静肽和icarapin)、2种王浆蛋白(MRJP8-9)以及睾丸增强因子等克隆.

  17. 三角帆蚌钙网蛋白基因cDNA的全长克隆与表达分析%FULL-LENGTH cDNA CLONING AND EXPRESSION ANALYSIS OF CALRETICULIN GENE FROM HYRIOPSIS CUMINGII

    Institute of Scientific and Technical Information of China (English)

    舒妙安; 胡杭娇; 陆晶莹; 徐宾朋; 王岩; 刘广绪; 郭晓令

    2013-01-01

    采用同源克隆策略和RACE技术,从三角帆蚌(Hyriopsis cumingii)外套膜组织中成功克隆得到钙网蛋白(Calreticulin, CRT)基因的全长cDNA序列,共1838 bp,开放阅读框为1257 bp,编码418个氨基酸,5′端非编码区为75 bp,3′端非编码区为506 bp,基因序列提交GenBank的登录号为JX416227。生物信息学分析表明,三角帆蚌钙网蛋白基因具有一段信号肽序列、两条典型的钙网蛋白家族标签序列 KHEQNIDCGGGY和IMFGPDICG、三个保守的 N-、P-和 C-端功能域及内质网前导序列 HDEL。NJ 法系统进化分析显示三角帆蚌首先与海洋双壳类紧密聚在一起,且与蚯蚓等环节动物亲缘关系较近,聚为一支,然后依次与虾类、昆虫、鱼类、两栖类、哺乳类聚在一起。经荧光定量 PCR 检测,钙网蛋白基因在三角帆蚌的外套膜、闭壳肌、斧足、鳃、肝脏、性腺、心脏、肠等8个组织中均有表达,其中在外套膜、鳃和斧足等与贝类钙代谢相关的组织中表达量较高预示其可能参与三角帆蚌的钙代谢。不同 Ca2+浓度处理试验的结果表明,随着水体中 Ca2+浓度逐渐升高,三角帆蚌钙网蛋白基因在外套膜中的表达水平呈先上升后下降的趋势,并在60 mg/L 时达到最高峰,表明适宜的 Ca2+浓度可促进钙网蛋白基因表达,而过高的 Ca2+浓度则会抑制其表达。同时在60 mg/L Ca2+浓度条件下,对三角帆蚌外套膜进行不同时间的表达试验,结果表明钙网蛋白基因的表达量随时间推移先上升,并于48h达到最大表达量,而后逐渐下降。上述结果为进一步深入研究钙网蛋白基因的功能及其调控机理奠定基础。%The full-length cDNA sequence of Calreticulin CRT gene was isolated from the mantle of Hyriopsis cumingii by using homology cloning strategy and SMART RACE technique. The entire CRT cDNA was 1838 bp, containing a 1257 bp complete open reading frame which encoding a protein with 418 amino

  18. Liquefaction mathematical analysis for improvement structures stability

    Directory of Open Access Journals (Sweden)

    Azam Khodashenas Pelko

    2010-10-01

    Full Text Available The stability of any structure is possible if foundation is appropriately designed. The Bandar abbas is the largest and most important port of Iran, with high seismicity and occurring strong earthquakes in this territory, the soil mechanical properties of different parts of city have been selected as the subject of current research. The data relating to the design of foundation for improvement of structure at different layer of subsoil have been collected and, accordingly, soil mechanical properties have been evaluated. The results of laboratory experiments can be used for evaluation of geotechnical characteristics of urban area for development a region with high level of structural stability. Ultimately, a new method for calculation of liquefaction force is suggested. It is applicable for improving geotechnical and structure codes and also for reanalysis of structure stability of previously constructed buildings.

  19. Construction of cDNA library and partial ESTs analysis of Strongylocentrotus intermedius%虾夷马粪海胆性腺cDNA文库构建及部分EST序列分析

    Institute of Scientific and Technical Information of China (English)

    丁君; 孙巍; 常亚青

    2011-01-01

    应用Gubler-Hoffman cDNA文库技术.构建虾夷马粪海胆(Strongylocentrotus intermedius)性腺cDNA文库.测试结果表明.其库容量为2.10x 106 PFU/mL.长度范围在0.54.2 kb.插人片段平均长度为1.4 kb.达到建库要求.对虾夷马粪海胆cDNA克隆测序.将获得的819条EST序列与NCB1数据库进行BLAST比对.获得了65个有研究价值的EST序列和cDN^克隆.其EST序列已提交到GenBank.序列号分别为G0448010-GO448016,GR410172-GR410229.虾夷马粪海胆性腺cDNA文库的成功构建.使短期内获得大量调控海胆性腺生长和营养性状的关键基因表达信息成为可能.为进一步开发海胆的生物资源提供参考.%Sea urchin(Strongylocentrotus intermedius) is a species of commercial and ecological significance, and as a economic species, sea urchin has been demanded steadily in Europe and Southeastern area for a long time.Recent studies focused on the development of its genomic resources, which is a key step towards further investigation, identification of gene and gene networks involved in its economic characters. Efforts have been focused on genes that can affect expression of important economic traits, such as growth and nutritional traits related genes. In this study, a cDNA library of sea urchin has been constructed from gonad using Gubler-Hoffman cDNA library technique. Total RNA was extracted from the grounded frozen powder of gonad tissue by using acid-guanidinephenol-chloroform (AGPC). Poly(A)+ RNA was isolated and purified by oligo(dT)18 anchor primer containing Not Ⅰ site. Both ends of the newly generated and polished double-stranded (ds) cDNA were attached by EcoR Ⅰ adaptors and the dscDNA was then restricted by Not Ⅰ. The short cDNA (<400 bp) was removed by Spin Column. The library consisted of 2.10× 106 PFU/mL colony forming units with average insert size of 1.4 kb, ranging from 0.5 kb to 4.2 kb and was quantified to construct a cDNA library. Eight hundred and fourteen ESTs were

  20. Construction of subtracted cDNA libraries of gastrocarcinoma and normal tissue with suppression subtractive hybridization and their quality analysis%人胃癌抑制消减cDNA文库的构建及文库质量分析

    Institute of Scientific and Technical Information of China (English)

    吴岚军; 毛秉智; 王升启

    2001-01-01

    Objective: To construct subtracted cDNA libraries of stomach tumors and normal stomach tissue using suppression subtractive hybridization(SSH).Methods: cDNA Library subtraction was performed using the protocol described in the Clontech PCR-Select cDNA Subtraction Kit. cDNA was synthesized from 2 μg of poly A+RNA from the tumor and normal tissues using AMV reverse transcriptase. The tester and driver cDNAs were digested with RsaⅠ, a four-base-cutting restriction enzyme that yields blunt ends. The tester cDNA was then subdivided into two portions, and each was ligated with different cDNA adaptor. Two hybridizations were performed. In the first, an excess of driver was added to each sample of tester. Hybridization kinetics led to equalization and enrichment of differentially expressed sequences. During the second hybridization, the two primary hybridization samples were mixed together without denaturing and thus the templates were generated from differentially expressed sequences for PCR amplification. Using suppression PCR, only differentially expressed sequences were amplified exponentially and after second PCR amplification the background was reduced and differentially expressed sequences were further enriched. The cDNAs were then directly inserted into a T/A cloning vector to generate a stomach tumor-specific subtracted cDNA library. Results: The amplified library contained 800 positive clones. Plasmid inserts were PCR amplified and showed 250-700 bp inserts. Conclusions: The successfully constructed subtracted cDNA library of gastrocarcinoma and normal tissue enables us to compare two populations of mRNA and obtain clones of genes that expressed in one population but not in the other.The characterization of these genes will allow them to be exploited for their diagnostic and therapeutic potentials.%目的:构建人胃癌抑制消减cDNA文库,为进一步大批量筛选、克隆胃癌特异性表达的基因奠定基础。方法:从胃癌和

  1. 杜仲肉桂醇脱氢酶基因全长cDNA克隆及序列分析%Cloning and Sequence Analysis of the Full-length cDNA of Cinnamyl Alcohol Dehydrogenase Gene from Eucommia ulmoides Olive

    Institute of Scientific and Technical Information of China (English)

    赵丹; 李晓毓; 陈建; 赵德刚

    2012-01-01

    以杜仲(Eucommia ulmoides Olive)4、5月份新长成的杜仲幼嫩叶片为材料,在克隆一段肉桂醇脱氢酶(cinnamyl alcohol dehydrogenase,CAD)基因的基础上,以杜仲cDNA为模板,采用cDNA末端快速扩增法(Rapid amplification of cDNA Ends,RACE)克隆了5'端828 bp和3'端798 bp cDNA序列,经5'RACE产物和3'RACE产物序列拼接,获得全长为1243 bp的杜仲CAD cDNA序列,开放阅读框编码243个氨基酸,命名为EuCAD(GenBank登录号:DQ142643).与GenBank中序列比对分析发现,该cDNA序列与苹果树、桉树、红橡树中的CAD基因序列同源性均为81%,预测编码的氨基酸序列与苹果树、桉树、红橡树的同源性分别为73%、70%和70%,因此认为是杜仲肉桂醇脱氢酶基因.该基因为首次从杜仲中克隆,为探索木质素的合成调控机理奠定基础.%Cinnamyl alcohol dehydrogenase ( CAD) plays an important role in the lignin biosynthesis. Cloning and sequence analysis of this gene ( CAD) from Eucommia ulmoides Olive were carried out by Rapid Amplification of cDNA Ends ( RACE) in the current work. The sequence analysis showed that the full-length cDNA of CAD contained 1243 bp, whose open reading frame ( ORF ) predicted a protein of 243 amino acids. The cDNA blast in GenBank showed 81% homology with Malus domestica, Eucalyptus gunnii, and Quercus suber, and amino acid blast demonstrated 73% , 70% , 70% homology with that of just-mentioned species, respectively , suggesting that full-length cDNA was authentic Eucommia CAD. The Cloning of Eucommia CAD may facilitate to unravel the synthetical mechanism of lignin in plant.

  2. Cloning and Bioinformatics Analysis of Toll-like Receptor 4 cDNA Full Length of Buffalo%水牛TLR4cDNA全长的克隆及其生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    匡文华; 祁超; 王凤阳; 张晓茹; 成鹰; 杜丽; 张冬琳; 郝永昌; 雷明; 焦寒伟; 刘涛

    2011-01-01

    The study aimed to clone Toll-like receptor 4 (TLR4) gene of buffalo and analysis the sequence of this gene. Buffalo TLRA gene was cloned by RT-PCR, the PCR products were sequenced and cloned into pMD20-T vector. Nucleotidesequence of this gene and structure of its encoding protein were predicted by using bioinformatics software. Sequence analysis results showed that the TLRi gene ORF was 2526 bp in length and encoded 841 amino acids, including a signal peptide of 25 amino acids(aa) at the N terminal. Relative molecular weight of the encoding protein was 95. 98 ku, and the isoelectric point (pI) was 6. 37. Homology analysis showed that the nucleotide sequence of cloned gene shared 99. 01% identities with the published sequence of buffalo TLR4 gene (DQ857349) in GenBank. The TLR4 gene sequence had a high homology compared with that of cattle, sheep, pig, horse, human and chimpanzee, which were over 80%, the homology with mouse and dog were 72. 17% and 61. 30% respectively, and that with chicken was only 53. 94%. The protein structure prediction showed that TLR4 was a transmembrane protein composed of extracellularC 1 to 634 aa), transmembrane (635 to 657 aa) and the intracellu-lar rejoins (658 to 841 aa) , which contained twelve LRRdeucine-rich repeats) tandem repeat ectodomains and a TIR (Toll-interleukinl-resistance) cytoplsmic domain. TLR4 of buffalo had been successfully cloned in the study, it lay a foundation for further researching the function of the gene and the protein characteristics.%本研究利用RT-PCR方法分段克隆水牛TLR4 cDNA后拼接成全长,并克隆于pMD20-T载体中,同时运用生物信息学软件对其核苷酸序列及编码蛋白质的结构进行分析和预测.结果表明,克隆的TLR4 cDNA ORF全长2526 bp,共编码841个氨基酸,N端具有由25个氨基酸组成的信号肽,该蛋白预测的分子质量为95.98 ku,等电点为6.37;水牛TLR4与GenBank中登录的水牛TLR4(DQ857349)核苷酸序列同源性达99.01%,与黄

  3. The Use of Item Analysis for Improvement of Biochemical Teaching

    Science.gov (United States)

    Nagata, Ryoichi

    2004-01-01

    Item analysis was used to find out which biochemical explanations need to be improved in biochemical teaching, not which items are to be discarded, improved, or reusable in biochemical examinations. The analysis revealed the basic facts of which less able students had more misunderstanding than able students. Identifying these basic facts helps…

  4. Isolation and Characterization of Phytoene Desaturase cDNA from Stigma of Crocus sativus

    Institute of Scientific and Technical Information of China (English)

    Bai Jie(白洁); Xu Ying; Tang Lin; Zeng Yu; Feng Yun; Wang Shenghua; Chen Fang

    2004-01-01

    Phytoene desaturase (PDS) has recently been identified as an important enzyme in carotenoid biosynthesis pathway. A cDNA clone encoding phytoene desaturase gene is isolated from stigma of saffron (Crocus sativus L.) using RT-PCR technique. Sequence analysis shows 83% similarity to Narcissus pseudonarcissus, 79% to Zea mays, 78% to Arabidopsis thaliana, 77% to Lycopersicon esculentum. A new full-length cDNA is obtained by 5'-RACE and 3' -RACE techniques. The cDNA is 2149bp long with an open reading frame of 1697bp, which encodes a polypeptide of 565 amino acids. Southern analysis shows that the PDS gene is a single copy in saffron. Northern blot analysis shows higher expression level of PDS gene in stigma and anther than in leaves and stem.

  5. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes.

    Science.gov (United States)

    Yang, G P; Ross, D T; Kuang, W W; Brown, P O; Weigel, R J

    1999-03-15

    Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.

  6. Cloning and expression of a cDNA encoding ribosomal protein S4 from Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A cDNA clone, pS4, has been isolated from a cDNA library prepared from rice anthers of about 1.0 mm in length. DNA sequence analysis and database search show that the cDNA encodes a protein which is highly homologous to eukaryotic 80S ribosomal protein subunit 4 (S4). Northern hybridization indicates that this gene expresses in all tissues analyzed although the expression level varies and it cannot be induced by mechanical wounding in leaves. Southern blot analysis demonstrates that this rice S4 gene is from a multigene family.

  7. 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.

  8. 水稻EPSP合酶cDNA克隆、序列分析及其拷贝数测定%Isolation of Rice EPSP Synthase cDNA and Its Sequence Analysis and Copy Number Determination

    Institute of Scientific and Technical Information of China (English)

    徐军望; 魏晓丽; 李旭刚; 陈蕾; 冯德江; 朱祯

    2002-01-01

    根据本室分离的水稻EPSP合酶基因的基因组序列设计一对引物,利用RT-PCR方法首次从水稻(Oryza sativa L. subsp. indica)叶片的RNA中扩增获得了水稻编码EPSP合酶的全长为1 585 bp的cDNA片段,它含有一个完整的开放读码框,编码511个氨基酸,包括444个氨基酸组成的成熟肽序列以及N端的67个氨基酸组成的叶绿体转运肽序列.成熟肽氨基酸序列对比表明,除真菌来源的EPSP合酶变异较大外,其他来源的EPSP合酶同源性较高,均在51%以上.而叶绿体转运肽氨基酸序列同源性较低.Southern杂交表明水稻EPSP合酶基因在水稻基因组中以单拷贝形式存在.RT-PCR分析表明,水稻EPSP合酶基因在根、未成熟种子和叶片中均有转录表达,在叶片中表达量最高.%In order to isolate the total cDNA of rice (Oryza sativa L.) epsps gene, RT-PCR was carried out with template of rice first-strand cDNA and primers designed according to rice EPSP synthase genomic sequence obtained in previous study. A 1 585-bp cDNA fragment was amplified and cloned. The 1 585-bp cDNA contains an open reading frame (ORF) comprising of 1 533 nucleotides (nt) which encodes a 511 residue polypepetides, including 67 amino acids chloroplast transit peptide and 444 amino acids EPSP synthase mature peptide. A comparison between the EPSP synthase of different sources indicates that the mature peptide shows more than 51% identity except for the fungi EPSP synthase and the transit peptide shows considerably less sequence conservation. The copy number of rice epsps gene is estimated to be one copy per haploid rice genome using southern blot. RT-PCR indicated that rice epsps gene is expressed in rice leaves, endosperms and roots and has the highest expression level in leaves.

  9. Molecular Cloning and Sequence Analysis on cDNA of Cystatin Gene from Tea Leaves%茶树巯基蛋白酶抑制剂基因的cDNA克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    王朝霞; 李叶云; 江昌俊; 余有本

    2005-01-01

    对多种已知植物巯基蛋白酶抑制剂(cystatin)基因的氨基酸序列进行比对分析,根据其高度保守的氨基酸序列设计一对简并引物,并从茶树品种龙井43鲜叶中提取总RNA,用RT-PCR法扩增出-204 bp的cDNA特异片段,然后通过3'/5'RACE的方法,分别扩增出3'端和5'端的序列,从而获得茶树巯基蛋白酶抑制剂基因的cDNA全长序列,所得序列全长627 bp,编码101个氨基酸,分子量约11.062 KDa.该基因在推测的氨基酸序列中含有巯基蛋白酶抑制剂家族中高度保守的、与其活性有关的QXVXG结构,且经Blast分析表明,该基因序列与其他植物巯基蛋白酶抑制剂基因的氨基酸序列同源性为54%~77%.%Two degenerate primers were designed according to the conserved region among the known plant cystatins. A cDNA fragment of 204 bp was amplified by RT-PCR (reverse transcription polymerase chain reaction)of total RNA extracted from fresh leaves of Tea plant (Camellia sinensis cv Longjing43). A full-length cDNA of the cystatin gene was obtained by 3'/5'RACE (rapid amplification of cDNA ends). The cDNA sequence of this 627 bp clone contained an open reading frame encoding a polypeptide of 101 amino acid residues with a predicable molecular mass of 11.026 KDa. The deduced amino acid sequence contained the motif QXVXG conserved among most members of the cystatin superfamily. By using the program of Blast on GenBank database, the sequence presented a high match with the cystatin genes from other plants, such as European chestnut, Cassava, Cowpea,Tomato, Soybean et al. All researched out sequences were all cystatins, so we can conclude that the cloned sequence is a member of cystatin gene from Tea plant.

  10. SEREX技术筛选及鉴定食管癌肿瘤抗原%Human Esophageal Carcinoma Antigens Screened by Serologic Analysis of Recombinant cDNA Expression Libraries (SEREX)

    Institute of Scientific and Technical Information of China (English)

    遇珑; 胡海; 冉宇靓; 彭良平; 李江伟; 杨治华

    2007-01-01

    背景与目的:正常细胞向癌细胞转化过程中,突变的基因或各种异常表达的蛋白可以成为肿瘤抗原诱导机体的免疫反应,因此肿瘤患者的血清中存在着与肿瘤相关的自身抗体.重组cDNA表达文库血清学分析法(serological analysis of recombinant cDNA expression libraries,SEREX)是利用肿瘤患者血清中的自身抗体筛选、鉴定肿瘤抗原的技术.本研究拟采用SEREX的方法寻找食管癌自身抗体的相关肿瘤抗原,鉴定与食管癌发生、发展相关的基因和免疫治疗分子靶点,并为食管癌的诊断提供候选血清标志物.方法:用食管癌组织建立库容量达1.6×106 pfu的cDNA表达文库,SEREX筛选获得21个不同cDNA序列的阳性克隆,进一步使用SADA法分析其中4个抗原在10例食管癌及10例正常人血清中的反应.结果:在Homosapiens desmin(DES)等21个阳性克隆中,4个克隆与已知EST序列明显无同源性,另外17个克隆与已知基因高度同源.Ribosomal protein S4等4个抗原与食管癌患者和正常人血清反应阳性率分别为40%和0%、60%和10%、70%和20%、30%和20%.结论:Ribosomal protein S4等4个抗原普遍参与了食管癌患者的体液免疫反应,与食管癌患者血清的反应阳性率明显高于正常人的血清.本研究发现的21个食管癌抗原可作为食管癌治疗的潜在分子靶点和食管癌诊断新的候选血清学标志物.

  11. cDNA library construction and EST analysis of larval salivary glands of Helicoverpa armigera ( Lepidoptera: Noctuidae)%棉铃虫幼虫唾液腺cDNA文库的构建及EST分析

    Institute of Scientific and Technical Information of China (English)

    张帅; 崔金杰; 王春义; 雒珺瑜; 吕丽敏

    2012-01-01

    Helicoverpa armigera ( Hiibner) saliva play important roles in insect-host plant interactions. In this study we constructed a cDNA library of salivary glands of H. armigera larvae where saliva is secreted. We randomly sequenced 1 501 expressed sequence tags (ESTs) , and clustering resulted in a total of 821 unigenes. Blast2 GO program was used to do BLASTx, functional annotation and metabolism analysis. Finally, we classified mRNAs in salivary glands of H. armigera larvae. By annotation of these ESTs, genes encoding 17 enzymes for digestion of fat, 5 enzymes for digestion of carbohydrates, and 20 serine proteases (of which 16 are newly reported) were identified, suggesting that the function of salivary glands is secreting saliva for predigestion. The cuticle protein, odorant-binding protein and chemosensory protein genes were identified in salivary glands of H. armigera larvae for the first time. The results will lay a foundation for studying predigestion system in H. armigera.%棉铃虫Helicoverpa armigera(Hübner)幼虫唾液中的各种酶类及各种生化组分在棉铃虫与植物相瓦作用及协同进化中起到重要作用;唾液腺是棉铃虫唾液成分的合成器官.本研究通过构建棉铃虫幼虫唾液腺全长cDNA文库,测序得到1502条EST序列,聚类分析后获得821个unigenes,为筛选棉铃虫与寄主互作信号因子提供基因信息资源.使用Blast2 GO软件对821个unigenes进行了比对和功能注释,初步获得棉铃虫幼虫唾液腺中Mrna的构成特征.结果显示,在棉铃虫唾液腺ESTs文库中,鉴定得到脂类相关消化酶基因17个,糖类相关消化酶基因5个,半胱氨酸蛋白酶基因1个,丝氨酸蛋白酶基因20个(其中16个为新发现),提示唾液腺的主要功能是分泌消化酶进行预消化;还发现在棉铃虫幼虫唾液腺中存在表皮蛋白、气味结合蛋白和化学感受蛋白基因.结果为研究棉铃虫预消化系统打下基础.

  12. Molecular characteristics and expression analysis of Kazal-type serines proteinase inhibitor (KSPI) gene from Hyriopsis cumingii%三角帆蚌 KSPI cDNA 的分子特征及表达分析

    Institute of Scientific and Technical Information of China (English)

    徐龙威; 王芹; 汪桂玲; 李家乐

    2016-01-01

    Kazal-type serines proteinase inhibitor ( KSPI) is very important in the immune reaction of biological body , but the study of KSPI in Hyriopsis cumingii has been rarely reported .In this study , the full length of cDNA sequence of Kazal-type serines proteinase inhibitor(KSPI) gene (accession number:KT901291) of H.cumingii was cloned.It was 1029 bp in length, containing 5′and 3′-UTRs parts of 61 bp and 206 bp, and an ORF of 762 bp encoding 253 amino acids, and the molecular weight was 27397.5 u.The amino acid sequence contains five Kazal domain structures , and the result of homology analysis showed that it has a few of similarity with other species, suggesting that this gene belongs to the typical KSPI gene.Real-time quantitative PCR showed that KSPI gene expressed in a wide range of tissues including the adductor , foot, liver, blood, mantle, gill and gonad, and the highest expression was in mantle , on the contrary, the lowest in liver.After being infected with Aeromonas hydrophila, the expression of KSPI gene had significant increas-ing in 7 tissues compared with the control group .The results suggested that KSPI gene is very important in the immune reaction of Hyri-opsis cumingii.%Kazal型丝氨酸蛋白酶抑制因子( KSPI)在生物体的免疫应答过程中发挥着重要作用,但在三角帆蚌中尚未进行其相关研究与报道,为研究KSPI对三角帆蚌免疫过程的影响,采用RACE法克隆了三角帆蚌KSPI cDNA全序列(登录号:KT901291),获得了1029 bp的全长,其中包含5′、3′端非翻译区分别为61 bp、206 bp,开放阅读框762 bp,共编码氨基酸253个,分子质量为27397.5u,氨基酸序列包含5个Kazal结构域,与一些已知物种的KSPI编码的氨基酸序列进行同源性分析后发现,与各种物种间具有相似的结构域,属于典型的KSPI。实时荧光定量PCR分析结果表明KSPI在外套膜、血液、肝、腮、闭壳肌、性腺、斧足7个组织中均

  13. Burning analysis on the improved confinement mode

    Energy Technology Data Exchange (ETDEWEB)

    Tateishi, Gonta [Interdisciplinary Graduate School of Engineering Science, Kyushu Univ., Kasuga, Fukuoka (Japan); Yagi, Masatoshi; Itoh, Sanae-I.

    1999-10-01

    1-D transport code is used to examine the ignition of plasma on the improved confinement mode and impact of profile effect on the burning performance. Energy transport, He-ash particle transport and poloidal magnetic field transport equations are solved with a thermal diffusivity of current diffusive ballooning mode model. The ratio of a thermal diffusivity and a He-ash diffusivity is introduced as a parameter and assumed to be constant. For a fixed current profile, the existence of the ignited state is shown. An internal transport barrier is formed autonomously even if parameters lie in the L-mode boundary condition. It is found that the sensitivity of the ignition condition on the density is strong and there is no margin of ignition for the density limit when density profile is flat. However, if a peaked profile of density is chosen, solutions which satisfy the density limit exist. The long time sustainment of ignition is also shown, solving poloidal magnetic field transport simultaneously. It is shown that the ignition is sustained within the time scale of burn-time, however, MHD stability should be considered in the time scale of current diffusion. (author)

  14. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

    Science.gov (United States)

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2014-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

  15. Identification of a Herbicide Safener AD-67 Inducible cDNA in Rice

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A herbicide safener AD-67 inducible cDNA was identified in an indica rice variety 9311 by mRNA differential display. The transcript was increased 6 h after sprayed with the safener solution, and 4 days later, the expression still could be detected. The fragment was recycled from the poly-gel and sequenced, and homologous analysis revealed the cDNA was 100% identical to some ESTs and cDNAs in rice database, and the amino acid sequence was 60-84% homologous to those of the Yippee genes in several eukaryotes. The fragment was extended to the whole long cDNA, and thus a primer pair was designed. RT-PCR analysis for the designed primer supported the induction result.

  16. Cloning and expression pattern analysis of a cDNA of pkc-2 gene in Caenorhabditis elegans%秀丽小杆线虫Caenorhabditis elegans pkc-2基因cDNA的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    钱雨; Laurent SEGALAT

    2009-01-01

    蛋白激酶C在秀丽小杆线虫中具有调节肌细胞渐进性萎缩的功能.为了揭示它的调节机制,本研究克隆了秀丽小杆线虫中蛋白激酶C pkc-2基因的cDNA pkc-2-c,构建了含该pkc-2 基因cDNA亚型的重组质粒pPD 118.20-pKG 63;揭示了该cDNA在秀丽小杆线虫体壁肌细胞中的定位.%Protein kinase C regulates the progressive muscle degeneration in Caenorhabditis elegans. In order to investigate the function of PKC involved in muscle degeneration, this paper cloned a cDNA isoform of pkc-2 gene of C. elegans and constructed the recombinant plasmids pPD118.20-pKG63 containing the isoform. The new isoform was then further studied for gene expression pattern. Immunocytochemistry experiment showed that this cDNA isoform expressed in body-wall muscle cells and located near the dense body.

  17. Cloning and screening of cDNA of Psilgramma menephorn allergen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To construct a cDNA expression library of Psilgramma menephorn to screen its major allergen so as to provide the basis for producing recombinant allergen vaccine of Psilgramma menephorn. Methods Total RNA was extracted from the whole body of Psilgramma menephorn with Trizol and mRNA was purified with Oligo (dT) Spin-Column. And dscDNA was synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoRⅠ adapters. Then the cDNAs were digested by XhoⅠ, and the fragments less than 400 bp were removed by using GHROMA SPIN-400 column. The remaining fragments longer than 400 bp were ligated with Uni-ZAP XR vector. The recombinants were packaged in vitro and a small portion of the packaged phage was used to infect E.coli XL1-Blue MRF′ for titration. The recombinants were examined by color selection. The size of cDNA inserts and the diversity of library were analyzed by PCR. The library was screened using SPT positive sera from patients with Psilgramma menephorn allergy repeatedly. Results The cDNA expression library consisting of a 5×105 recombinant bacteriophages was constructed with the recombinant ratio of 67%. The average length of recombinant exogenous inserts was about 1.49 kb. Five positive cDNA clones were obtained. Conclusion The constructed cDNA expression library shows appropriate contents and size of cDNA fragments and the related genes of Psilgramma menephorn major allergens were harbored successfully, which lays the foundation for the positive clone identification and further analysis.

  18. 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.

  19. Construction of cDNA Library from Populus euphratica

    Institute of Scientific and Technical Information of China (English)

    Yu Guangjun; Wang Yiqin; Shen Xin

    2003-01-01

    In order to isolate and clone salt-tolerance involved genes of Populus euphratica, we constructed a cDNA library from salt-treated leaves of P. euphratica. In the experiment, double strand cDNA were synthesized by a beads-based method. The syntheses of the first strand and the second strand cDNA, adapter ligation and restriction reaction for releasing cDNA were all conducted on the beads. The double strand cDNA were released from magnetic beads by digestion with NotI, and cDNA fragments smaller than 500 bp and residual adapters were removed through cDNA size fractionation columns. Finally, double strand cDNA were directionally cloned intoλExcell vector. The results show that the primary titer of the cDNA library is 7.46×106 pfu per mL and the packaging efficiency reaches 1.47×107 recombinants per μg DNA. λDNA extracted from two clones of plaque were digested by EcoR I and NotI, both of the clones contained inserts larger than 900 bp. These results show that the cDNA library of salt-treated P. euphratica leaves has been successfully constructed.

  20. 猪Mx1基因cDNA的克隆及序列分析%Cloning and Sequence Analysis of Porcine Mx1 Gene cDNA

    Institute of Scientific and Technical Information of China (English)

    李想; 沈学文; 李文贵; 毕峻龙; 杨贵树; 尹革芬

    2011-01-01

    为获得云南本地猪Mx1基因cDNA序列,根据GenBank中猪Mx1基因的参考序列,设计合成3对引物.以云南本地猪外周淋巴细胞为样本,采用RT - PCR方法进行分段扩增,对扩增片段进行克隆,测序分析及序列拼接.结果表明,扩增的云南本地猪Mx1基因cDNA全长2546 bp,开放阅读框1992 bp,编码663个氨基酸,与GenBank中他猪种Mx1基因序列对比,核苷酸序列的同源性为99.4%~99.8%,氨基酸序列的同源性为98.8%~99.5%.成功获得云南本地猪Mx1基因的cDNA序列,为研究云南本地猪Mx1蛋白的抗病毒活性及作用机制奠定了基础.%In order to obtain the cDNA sequence of Mxl gene of Yunnan local pig, primers were designed according to the sequences of porcine Mxl gene in GenBank. Fragments were amplified using RT-PCR from peripheral lymphocytes of Yunnan local pigs, and the fragment sequences were analyzed and spliced. The results demonstrated that the cDNA length of Mxl gene amplified from Yunnan local pigs was 2 546 bp, the open reading frame was 1 992 bp with encoding 663 amino acids. Compared with other Mxl gene sequences in GenBank, the homologies of nucleotide sequences were from 99. 4% to 99. 8% , those of amino acid sequences were from 98. 8% to 99. 5%. The cDNA sequence of Mxl gene in Yunnan local pigs was successfully obtained, which may facilitate the study on the antiviral activity and mechanism of Mxl protein in Yunnan local pigs.

  1. Cloning and sequencing of complete -crystallin cDNA from embryonic lens of Crocodylus palustris

    Indian Academy of Sciences (India)

    Raman Agrawal; Reena Chandrashekhar; Anurag Kumar Mishra; Jetty Ramadevi; Yogendra Sharma; Ramesh K Aggarwal

    2002-06-01

    -Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete -crystallin cDNA from the embryonic lens of Crocodylus palustris and establish it to be identical to the -enolase gene from non-lenticular tissues. Quantitatively, the -crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile -crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5′- and 3′-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete -crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5′-and 3′-ends respectively. Further, it was found to be identical to its putative counterpart enzyme -enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of -crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.

  2. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

    Science.gov (United States)

    Honda, A; Sugimoto, Y; Namba, T; Watabe, A; Irie, A; Negishi, M; Narumiya, S; Ichikawa, A

    1993-04-15

    A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

  3. Cloning and characterization of a cDNA encoding human differentiation antigen 5D4

    Institute of Scientific and Technical Information of China (English)

    马凤蓉; 朱立平; 汪燚; 赵方萄; 史耕先; 李波; 李国燕; 张淑珍; 王讯

    2000-01-01

    A 1 846 bp cDNA is isolated from a human tonsil cell λgt 11 cDNA library (ATCC No. 37546) with mAb 5D4 reactive strongly with human B cell line 3D5, but weakly with human B cell line Daudi and human T cell line Jurkat as a probe. RT-PCR also shows a strong reaction in 3D5 cell and a weak reaction in Daudi and Jurkat cell for 5D4 mRNA. There is an open reading frame from 88 to 1 209 bp in 5D4 cDNA encoding a 374 AA protein. Both the Northern blot analysis and the two consecutive stop codens before start coden demonstrate that the cDNA is a full-length cDNA. Secondary structure prediction suggests that there are a region from 295 to 334 AA in the protein with strong hydrophobicity and a transmembrane helix region with high score from 313 to 334 AA with an orientation from the inside to the outside of the cell.

  4. Molecular cloning and nucleotide sequence of cDNA for human liver arginase

    Energy Technology Data Exchange (ETDEWEB)

    Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

    1987-01-01

    Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated lambda hARG6 and lambda hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying lambda hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes.

  5. Construction of cDNA Library from NPC Tissue and Screening of Antigenic Genes

    Institute of Scientific and Technical Information of China (English)

    Jun Shu; Xiaojuan He; Guancheng Li

    2006-01-01

    To construct cDNA library of nasopharyngeal carcinoma (NPC) and obtain the NPC associated or specific antigens from it, we used a powerful new method to identify the antigens eliciting humoral immune response, which is SEREX (serological identification of antigen by recombinant cDNA expression library). Autologous serum of NPC patient was used to screen the reactive clones in the human NPC tissue cDNA library consisted of 3.64×106 recombinants. The 23 exact positive clones were subcloned to monoclonality and the size of cDNA inserts was identified by PCR. Then the nucleotide sequence of cDNA inserts was determined, and the sequence alignments were performed with BLAST software on GenBank database. They represented 16 different antigens. A detailed sequence analysis showed that 10 of 16 genes were high homologous to genes known in GenBank, such as RPL31,S100 A2, MT2A, etc. However, there were also 6 genes with low homology to genes in GenBank. Furthermore, 3 of 6 genes may be novel genes. The associations of these genes to NPC and the roles that they played in the occurrence and development of NPC should be further revealed.

  6. 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

  7. Cloning Full-Length cDNAs from Vascular Tissues and Cells by Rapid Amplification of cDNA Ends (RACE) and RT-PCR.

    Science.gov (United States)

    Shen

    1999-01-01

    The isolation of full-length cDNAs remains a frequent task undertaken in many laboratories. A full-length cDNA is often desirable for one of the following purposes: 1) to complete the sequence of a partial cDNA cloned by library screenings or the yeast one- or two-hybrid system; 2) to derive the cDNA sequence encoding a protein, based on peptide sequences; 3) to obtain the sequence of a reported cDNA for functional analysis or expression studies; and 4) to define exon/intron boundaries of a cloned gene or determine transcription start site(s) of a promoter.

  8. 诸氏鲻虾虎鱼卵黄蛋白原基因全长cDNA的克隆及表达%Full-length cDNA cloning and expression analysis of vitellogenin gene inMugilogobius chulae

    Institute of Scientific and Technical Information of China (English)

    余露军; 蔡磊; 李舸; 陈小曲; 陈琳; 李建军

    2016-01-01

    To explore the tissue distribution of the vitellogenin (Vg) gene inMugilogobius chulae and the effect of the pollutant 17β-estradiol on maleM. chulae, the full length of vitellogenin (Vg) cDNA in M. chulae was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) tech-niques using total RNA extracted from the liver of female fish. Meanwhile, the expression of Vg mRNA ofM. chu-lae was determined by real-time fluorescent quantitative PCR. The full length ofM. chulae’s Vg cDNA sequence contains 5 067 bp nucleotides, with a complete open reading frame (ORF) of 4 992 bp, which encodes 1 664 amino acid proteins with a deduced protein molecular weight of 186.2 ku. The ORF contains signal peptides and a polyserine domain. The real-time RT-PCR result showed thatM. chulae’s Vg mRNA was primarily detected in the liver. Furthermore, Vg mRNA expression levels at different time points of E2 treatment of male fish were deter-mined.M. chulae’s Vg mRNA expression levels reached the peak on the third day and then the expression levels declined on the seventh day. These results indicated that the full length of Vg cDNA in M. chulae was cloned suc-cessfully. This study suggests that Vg in maleM. chulae can be considered as a valid biomarker for offshore envi-ronmental monitoring of estrogenic substances.%为了探讨诸氏鲻虾虎鱼(Mugilogobius chulae)卵黄蛋白原组织分布及17β-雌二醇(E2)暴露对雄性诸氏鲻虾虎鱼Vg的影响,作者采用RT-PCR、RACE方法克隆并分析了诸氏鲻虾虎鱼卵黄蛋白原(Vg)基因的全长cDNA序列,并对Vg在诸氏鲻虾虎鱼体内的组织表达分布及E2诱导后不同时间表达规律进行了研究。结果表明:获得的Vg cDNA序列全长5067 bp,开放阅读框(ORF)含4992 bp,编码1663个氨基酸,含有信号肽、多丝氨酸区域,推测其编码氨基酸分子量为186.2 ku,等电点为9.31。荧光定量PCR结果显示, Vg在诸氏鲻虾虎鱼

  9. 羊驼垂体催乳素(PRL)基因全长cDNA的克隆及序列分析%Cloning and Sequence Analysis of the Full-length cDNA of PRL Gene from Alpaca Pituitary

    Institute of Scientific and Technical Information of China (English)

    薛霖莉; 董常生; 赫晓燕; 范瑞文; 王海东; 曹靖; 郝欢庆

    2011-01-01

    In order to provide theoretical basis for studying biological function and application of alpaca prolactin (PRL), the alpaca PRL cDNA sequence were cloned and analyzed.According to the known cDNA sequences from mammals, alpaca PRL primers was designed and the full-length cDNA of PRL from alpaca pituitary was cloned by RT-PCR and RACE techniques.The size of full-length cDNA of PRL from alpaca pituitary was 959 bp and it contained an open reading frame (ORF) of 687 bp which encoded PRL precursor protein with 229 AA.PRL precursor protein was a single-chain polypeptide composed of 30-AA signal peptide and 199-AA mature peptide.The spatial structure of alpaca PRL protein was similar to human GH.The result of the sequence alignment showed that the amino acids composition of alpaca PRL was similar to most mammals, but the methionine at 81-AA (51-AA for mature peptide) might lead to different spatial structure which might impact functions of alpaca PRL.A phylogenetic tree constructed basing on the amino acid sequences of alpaca PRL and other organisms showed that the relationships between alpaca PRL and camel PRL were closest and that the evolution speed of alpaca was very slow with no ' episodic' evolution pattern as most mammals such as primates, rodents and ruminant.%获得并分析羊驼PRL基因cDNA全序列结构,为研究羊驼催乳素(PRL)的各种生物学作用和生产应用提供理论依据.根据已知的不同哺乳动物的PRL基因cDNA序列,设计羊驼PRL引物,运用RT-PCR方法和cDNA末端快速扩增(RACE)技术获得羊驼PRL基因cDNA全序列.羊驼PRL基因cDNA序列全长959 bp,编码区为687bp,编码229个氨基酸的PRL碰前体蛋白.预测羊驼PRL蛋白质的空间结构类似人生长激素(GH),但在81位(成熟肽为51位)为蛋氨酸可能导致蛋白空间结构的不同而影响羊驼PRL的功能;序列比对结果表明,羊驼PRL的cDNA序列与大多数哺乳动物相似.构建的基因进化树分析结果显示,羊驼PRL与骆

  10. 红笛鲷头肾消减cDNA文库的构建与分析%Construction and analysis of subtractive cDNA library of head kidney in humphead snapper,Lutjanus sanguineus

    Institute of Scientific and Technical Information of China (English)

    张新中; 吴灶和; 简纪常; 鲁义善

    2011-01-01

    应用抑制性消减杂交技术(SSH)构建红笛鲷(Lutjanus sanguineus)头肾消减cDNA文库,筛选红笛鲷免疫相关基因的EST.以哈氏弧菌(Vibrio harveyi)灭活疫苗体内诱导红笛鲷为实验组,以注射无菌生理盐水的红笛鲷为驱动组,通过SSH技术构建红笛鲷头肾消减.DNA文库.利用PCR技术和斑点杂交对文库进行筛选,从2 424个含插人片段的阳性克隆中筛选了680个克隆在上海生工进行了序列测定.使用BLASTx和BLASTn工具对获得的ESTs与GenBank数据库进行同源性比较并根据相似性序列的名称通过GO法对ESTs进行注释.结果获得了30个与红笛鲷免免疫防御相关基因的EST,如组织相容性抗原复合物基因(MHC I和MHCII),免疫球蛋白基因(IgH和IgL)、热休克蛋白基因(HSP10,HSP70和HSP90)等.本研究构建了哈氏弧菌灭活疫苗免疫后与正常组织差异表达的消减cDNA文库,并获得一批与红笛鲷免疫防御相关的ESTs,旨在为探讨红笛鲷分子免疫防御机制、筛选参与免疫防御调控相关的功能基因,揭示红笛鲷免疫抗病机制、提高机体抗病力、实现遗传改良奠定基础.%A subtracted cDNA library of humphead snapper (Lutjanus sanguineus) was constructed by suppression subtractive hybridization technology (SSH) to screen immune-related EST. The cDNA library has been constructed by the mRNA of the test group and driven by SSH. Differential ESTs from the subtracted cDNA library have been identified by both PCR technology and dot blot hybridization. Six hundred and eighty positive clones were sequenced by Sangon Biological Engineering Technology & Services Co., Ltd. The homology of the sequences was analyzed by BLASTx tool and BLASTn tool in GenBank database. Functional distribution was performed based on the features of ESTs by gene ontology annotation (GO) and 30 immune-related ESTs of L. sanguineus, such as major histocompatibility complex gene (MHC Ⅰ and MHC Ⅱ ), immunoglobulin gene

  11. Cloning, sequence analysis and detection of vitellogenin cDNA from Colisa fasciata%条纹密鲈卵黄蛋白原基因的克隆与检测

    Institute of Scientific and Technical Information of China (English)

    梁岳; 方展强

    2011-01-01

    Using RT-PCR,the partial length vitellogenin( VTG)cDNA sequence and (3-actin cDNA sequence of Colisa fasciata were cloned. VTG cDNA sequence contains 3 464 bp nucleotides and encodes 1 150 amino acids, p-actin cDNA sequence contains 1 253 bp nucleotides and encodes 375 amino acids. In vivo and in vitro methods were employed to investigate VTG mRNA expression under exposure to estradiol (E2), octylphenol (OP), cadmium ( Cd2 + ) and perfluorooctane sulfonates ( PFOS) and evaluate the estrogenic activity. The results showed that E2 and OP could induce VTG mRNA expression in dose-dependent way by in vivo and in vitro test. Cd2+ could induce VTG mRNA expression only in the low dose by in vivo test,but VTG mRNA expression was not observed in PFOS groups by in vivo and in vitro test. The results indicated that the strength of estrogenic effects was in the order E2 > OP > Cd2 +. Cd2 + estrogenic effects in vivo and in vitro results are inconsistent, suggesting that the mechanism of Cd2 + induced effects of estrogen and E2 may be different. The results also indicated that VTG cDNA of C. Fasciata is very sensitive to environmental hormone and very suitable to be a biomarker for monitoring the environmental hormones.%采用RT-PCR方法克隆并分析了条纹密鲈卵黄蛋白原(VTG)和β-肌动蛋白(β-actin)cDNA部分序列.获得的VTG cDNA序列片段长3464 bp,全部处于编码区,编码1150个氨基酸;β-actin cDNA序列片段长1253 bp,编码375个氨基酸.使用活体与离体的实验方法,检测了VTG mRNA转录情况,并以此评价雌二醇(E2)、辛基酚(OP)、镉(Cd2+)和全氟辛烷磺酸类化合物( PFOS)引起的雌激素效应.结果显示,E2和OP在活体和离体实验中均能剂量依赖性地诱导VTG mRNA表达.Cd2+仅在活体实验低剂量组诱导VTG mRNA表达,PFOS在活体和离体实验的各个浓度组均未见显著的VTG mRNA表达.结果表明,所诱导的雌激素效应强弱的排列顺序为E2>OP> Cd2.Cd2的雌激素效应

  12. 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…

  13. 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...

  14. Profiling gene expression patterns of nasopharyngeal carcinoma and normal nasopharynx tissues with cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    5 μg of total RNAs from normal nasopharynx and nasopharyngeal carcinoma tissue have been labeled with α-32P-dCTP during reverse transcription. The synthesized cDNA probes have been hybridized to high-density cDNA microarray containing 5184 genes or expression sequence tags (ESTs). Then image analysis software has been applied to comparing their expression profiles. Results show that 187 ESTs were of density value above 200 in nasopharyngeal carcinoma tissue while there were 307 such ESTs in normal nasopharynx tissue; 38 ESTs were strongly expressed in nasopharynx, but weakly expressed in nasopharyngeal carcinoma; 48 ESTs were strongly expressed in nasopharyngeal carcinoma, but weakly expressed in normal nasopharynx. These results suggest that there may exist some new differentially expressed genes involved in nasopharyngeal carcinoma development. Furthermore, the results strongly indicate that high-density cDNA microarray is a powerful and efficient tool for large-scale screening differentially expressed genes.

  15. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    Science.gov (United States)

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury.

  16. LD-RTPCR:\tA NEW METHOD FOR LABELLING TRACE cDNA MICROARRAY PROBE

    Institute of Scientific and Technical Information of China (English)

    范保星; 孙敬芬; 梁好; 王升启; 周平坤; 吴德昌

    2002-01-01

    Objective: To explore the usefulness of long distance reverse transcript combining linear amplification (LD-RTPCR) in labeling slight trace probe used for cDNA microarray. Methods: Total RNA from BEP2D cells was extracted and labeled by two different methods, LD-RTPCR with Cy3-dCTP as fluorescent dye and traditionally used RNA reverse transcript (RT) with Cy5-dCTP as fluorescent dye. Then, the probes labeled by two methods were mixed equally and hybridized with the cDNA microarray. Results: Scan and analysis of the microarray showed that the two methods labeled probes had consistent results. Conclusion: LD-RTPCR was proved useful for labeling cDNA microarray probe, especially for limited RNA material.

  17. 藏羚羊PGC-1α基因编码区的克隆与分析%Molecular Cloning and Sequence Analysis of PGC-1α cDNA in Tibetan Antelope

    Institute of Scientific and Technical Information of China (English)

    马燕; 常荣; 祁玉娟; 格日力

    2012-01-01

    Total RNAs were extracted from myocardium of Tibetan Antelope {Pantholops hodgsonii) and Tibetan Sheep,both inhabiting on Tibetan Plateau (altitude 4 300 m). PGC-la coding cDNA sequences were cloned with reverse transcription polymerase chain reaction ( RT-PCR) , and the sequences were confirmed by DNA sequencing. The cloning and sequencing results confirmed that the PGC-la gene coding sequences of both Tibetan Antelope and Tibetan sheep showed above 90% identity with other species. In addition, the cloned sequences contained the RNA/DNA binding sites, RRM (RNA recognition motif) , the domains involved in the interaction with NRF-1 and MEF2C , Arg/Ser rich domain, negative regulatory domain, LXXLL motif, as well as conserved sequences like TPPTTPP and DHDYCQ, which are present in all PGC-l family members. Fourteen variable amino acid sites were identified in the functional domains mentioned above. Additionally, analysis of generic phosphorylation sites and kinase specific phosphorylation prediction sites indicated that the 329-threonine amino acid site could be phosphorylated by PKG,which may be unique to Tibetan Antelope. Secondary structures of PGC-la protein from Tibetan Antelope and Tibetan Sheep were also predicted in this study. In summary,the PGC-la gene coding regions from Tibetan antelope and Tibetan Sheep have been successfully cloned,which may provide fundamental data for further investigating high altitude adaptation related to genetics in the future.%以藏羚羊(Pantholops hodgsonii)及同海拔分布的藏系绵羊(Tibetan Sheep)的心肌组织为材料,提取总RNA,利用逆转录聚合酶链反应(RT-PCR)技术扩增出过氧化物酶体增生物激活受体γ辅激活因子-1α(PGC-1α)的基因编码区cDNA片段,与载体连接构建重组质粒,经转化、扩增培养、鉴定后测序.利用生物信息学方法分析显示,藏羚羊和藏系绵羊的PGC-1α基因编码区长度均为2 349 bp,编码797个氨基

  18. Lysosomal {beta}-mannosidase: cDNA cloning and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Leipprandt, J.R.; Traviss, C.E. [Michigan State Univ., East Lansing, MI (United States)] [and others

    1994-09-01

    Lysosomal {beta}-mannosidase is an exoglycosidase that cleaves the single {beta}-linked mannose residue from the non-reducing end of all N-linked glycoprotein oligosaccharides. Deficiency of this enzyme results in {beta}-mannosidosis, a severe neurodegenerative disease in goats and cattle. The human cases described have a milder, highly variable presentation. Study of the molecular pathology of this disease in ruminants and humans and development of the animal model for gene therapy studies required cloning of the gene for {beta}-mannosidase has been cloned. {beta}-Mannosidase cDNA were obtained from a bovine thyroid cDNA library by screening with mixed oligonucleotides derived from peptide sequences resulting from microsequencing of bovine {beta}-mannosidase peptides. A total of six independent positive clones were identified from 5 x 10{sup 5} plaques, covering about 80% of the C-terminal region. The missing 5{prime} region was obtained using 5{prime} RACE. The full-length construct contains 3852-bp nucleotides, encoding 879 amino acids. The initiation codon is followed by 17 amino acids containing the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrated a 4.2 kb single transcript in various tissues from both normal and affected goats and calves. The mRNA level was decreased in affected {beta}-mannosidosis animals. The gene encoding {beta}-mannosidase was localized on human chromosome 4 by Southern analysis of rodent/human somatic cell hybrids. The mutation in bovine {beta}-mannosidosis has been identified. This is the first report of cloning of the {beta}-mannosidase gene.

  19. Cloning and Sequence Analysis of the Full-Length cDNA of H-FABP Gene in Lanzhou Fat-Tailed Sheep%兰州大尾羊心脏型脂肪酸结合蛋白(H-FABP)基因克隆及其同源性比较

    Institute of Scientific and Technical Information of China (English)

    徐红伟; 柏家林; 冯玉兰; 曹忻; 蔡勇; 金方圆; 达小强; 杨具田; 臧荣鑫

    2013-01-01

    Bank Accession Number JQ780322).The open reading frame of sheep H-FABP gene is 402 bp in length,encoding a mature protein H-FABP of 133 amino acids and a resulting Mr=14 761.Phylogenetic analysis showed that H-FABP gene in Lanzhou fat-tailed sheep is more close to goat,Capra hircus.Alignment comparison indicated that nucleotide homology of H-FABP gene in sheep is more similar with mammals.However,the base transition from T to G in sixty-six of nueleotide sequence leading to the change from asparagines (N) to lysine (K) in twenty-second of amino acid sequence,which is different from other species.It is predicted that tertiary structure of H-FABP protein is very similar to H-FABP of C.hircus,having 2 a-helix,10 antiparallel [3-pleated sheets that form barrel.[Conclusion] The full length cDNA of 748 bp H-FABP gene was first to be cloned by RACE.This finding may provide basic data for further studying the role ofH-FABP gene in sheep.

  20. 枣结果枝cDNA文库的构建与部分ESTs分析%Construction and Partial ESTs Analysis of a cDNA Library for Fruit-bearing Shoot in Ziziphus jujuba

    Institute of Scientific and Technical Information of China (English)

    孟玉平; 曹秋芬; 孙海峰; 周慧; 张春芬

    2009-01-01

    By using directional cloning, a cDNA Library in the fruit-bearing shoot of Ziziphus jujuba Mill during the early stages of flower bud differentiation was constructed. In 1 388 positive clones, 557 cDNA inserts were obtained. 469 cDNA inserts, length over 500 bp, were selected and sequenced, 390 useful inserts were obtained at last. In these useful sequences,281 ESTs (among them there were 101 repetitive ESTs) were higher similarity with the known genes of CNBI, and 68 ESTs were unknown protein that its sequence had published, another 41 ESTs were unknown sequence (new gene) . The known genes were classified by the classification way of arabidopsis thaliana genes, the result indicated that there were the basal metabolism gene 46 genes,the protein synthesis and transfer gene including 27 genes,the photosyn-thesis gene including 24 genes, the cytoarchitecture gene including 22 genes, the signal transduction gene including 19 genes,the auxesis gene including 19 genes,the resistance adversity gene including 11 genes,the flower development gene including 6 genes, the membrane traffic gene including 4 genes, and the metal transfer gene including 2 genes. The ex-pression of some genes may be relation to the properties of jujube trees's high resistance to the adverse environment, for example,cold resistance,drought resistance,barrenness tolerance and heavy metal tolerance.%用定向克隆法构建了枣(Ziziphus jujuba Mill)生长初期结果枝的部分cDNA文库,获得1 338个阳性克隆,有557个携带cDNA片段,选取469个长度在500 bp以上的进行测序,得到390个有用序列,其中281个ESTs与NCBI中已知功能基因相似性较高(其中重复性ESTs 101个),有68个ESTs是NCBI中有序列注释的未知蛋白,有41个ESTs是NCBI中没有的未知序列(新基因).将已知基因进行功能分类,其中包含有参与基础代谢的基因46个,蛋白质合成与转运基因27个,光合作用基因24个,细胞结构基因22个,信号转导基因19

  1. Improvements to the CONTINUE feature in transient analysis

    Science.gov (United States)

    Pamidi, P. R.

    1989-01-01

    The CONTINUE feature in transient analysis as implemented in the standard release of COSMIC/NASTRAN has inherent errors associated with it. As a consequence, the results obtained by a CONTINUEd restart run do not, in general, match the results that would be obtained in a single run without the CONTINUE feature. These inherent errors were eliminated by improvements to the restart logic that were developed by RPK Corporation and that are available on all RPK-supported versions of COSMIC/NASTRAN. These improvements ensure that the results of a CONTINUEd transient analysis run are the same as those of a non-CONTINUEd run. In addition, the CONTINUE feature was extended to transient analysis involving uncoupled modal equations. The improvements and enhancement were illustrated by examples.

  2. Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons

    Directory of Open Access Journals (Sweden)

    De Paepe Anne

    2004-02-01

    Full Text Available Abstract Background Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes. Results As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences. Conclusions The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.

  3. 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...... 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...

  4. 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...... 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...

  5. Identification and cloning of the cDNA of a Rb-associated protein RAP140a

    Institute of Scientific and Technical Information of China (English)

    李权; 闻宏; 敖世洲

    2000-01-01

    Rb exerts important physiological functions in cell-cycle control, gene expression, cell differentiation, apoptosis, development and tumorigenesis by interacting with many cellular proteins. Using human partial Rb as bait, we screened a human fetal brain cDNA library through yeast two-hybrid system and obtained six novel cDNA fragments. Among them, one cDNA fragment corresponds to two different transcripts, 7 kb and 9 kb in Northern blot analysis. These two transcripts showed uniform distribution in various human tissues. We cloned the full-length cDNA of a 7.2 kb transcript through three times PCR amplifications. It was named RAP140a and predicted to encode a 1 233 amino acids hydrophilic protein. RAP140a was mapped to chromosome 3p13-p14.1. RAP140a may be functionally related to the intracellular translocation of Rb or other proteins.

  6. Identification and cloning of the cDNA of a Rb-associated protein RAP140a

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Rb exerts important physiological functions in cell-cycle control, gene expression, cell differentiation, apoptosis, development and tumorigenesis by interacting with many cellular proteins. Using human partial Rb as bait, we screened a human fetal brain cDNA library through yeast two-hybrid system and obtained six novel cDNA fragments. Among them, one cDNA fragment corre-sponds to two different transcripts, 7 kb and 9 kb in Northern blot analysis. These two transcripts showed uniform distribution in various human tissues. We cloned the full-length cDNA of a 7.2 kb transcript through three times PCR amplifications. It was named RAP140a and predicted to encode a 1 233 amino acids hydrophilic protein. RAP140a was mapped to chromosome 3p13-p14.1. RAP140a may be functionally related to the intracellular translocation of Rb or other proteins.

  7. Improving Cluster Analysis with Automatic Variable Selection Based on Trees

    Science.gov (United States)

    2014-12-01

    ANALYSIS WITH AUTOMATIC VARIABLE SELECTION BASED ON TREES by Anton D. Orr December 2014 Thesis Advisor: Samuel E. Buttrey Second Reader...DATES COVERED Master’s Thesis 4. TITLE AND SUBTITLE IMPROVING CLUSTER ANALYSIS WITH AUTOMATIC VARIABLE SELECTION BASED ON TREES 5. FUNDING NUMBERS 6...2006 based on classification and regression trees to address problems with determining dissimilarity. Current algorithms do not simultaneously address

  8. 大腹园蛛Avg1 cDNA的克隆和序列分析%Cloning and Sequence Analysis of Araneus ventricousus Avg1 cDNA

    Institute of Scientific and Technical Information of China (English)

    任洪林; 柳增善; 卢士英; 潘风光

    2004-01-01

    Quantity and activity of cathepsin B (CB) are increased during the progress of carcinoma cells' invasion and metastasis. So, CB is being applied to the diagnosis of cancers and inhibiting their diffusion. The complete Avgl eDNA was randomly cloned from the cDNA library of major ampullate gland of Araneus ventricousus. It is 1 253 bp and encode 334 amino acids in its 1 002 bp encoding region. The molecular weight of the protein is 36 953.00.GenBank accession number is AY302573. The 3′ non-coding region is composed of 179 bp with a polyadenylation signal AATAAA sequence appearing at the position 129 nt and the poly(A) tail is at the position 153 nt downstream of stop codon TAA. The signal peptide cleavage site of its deduced protein is between codon 16 and 17. Two glycosylation sites of AsnThrThr and AsnValSer, respectively, appear at codon 23 and 202. The high homology genes are not found in all genes known in NCBI, but the typical conserved domain of peptidase-C1 has been detected in NCBI BLASTp, and high homologies with CB of some kinds of creatures are shown. The objective function of the Avgl has not been studied in the spide silk gland yet.

  9. Molecular cloning and character analysis of cDNA of phosphoenolpyruvate carboxykinase (PEPCK) in Erythroculter ilishaeformis%翘嘴红鲌PEPCK基因的克隆和序列分析

    Institute of Scientific and Technical Information of China (English)

    戈贤平; 俞菊华; 吴婷婷

    2006-01-01

    采用RT-PCR和RACE(Rapid Amplification of cDNA Ends)法,分离和克隆翘嘴红鲌(Erythroculterilishaeformis)磷酸烯醇式丙酮酸羧激酶(PEPCK)基因的全序列,得到2 564bp[不含poly(A)]的全长cDNA,包括111bp5′非翻译区,1 911 bp阅读框以及含Poly(A)信号AATAAA的542 bp3'非翻译区[不包括Poly(A)].阅读框共编码636个氨基酸,计算的分子量为69.65 kD.该序列含有PEPCK特有的结合草酰乙酯的结构域以及与GTP三磷酸链和Mg2+结合的激酶1和2基序.与其他鱼PEPCK的相似性高达83%~96%,和爪蟾、变形虫等其他动物的相似性为50%~69%.[中国水产科学,2006,13(3):389 396

  10. Construction of genome-wide physical BAC contigs using mapped cDNA as probes: Toward an integrated BAC library resource for genome sequencing and analysis. Annual report, July 1995--January 1997

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, S.C.; Bocskai, D.; Cao, Y. [and others

    1997-12-31

    The goal of human genome project is to characterize and sequence entire genomes of human and several model organisms, thus providing complete sets of information on the entire structure of transcribed, regulatory and other functional regions for these organisms. In the past years, a number of useful genetic and physical markers on human and mouse genomes have been made available along with the advent of BAC library resources for these organisms. The advances in technology and resource development made it feasible to efficiently construct genome-wide physical BAC contigs for human and other genomes. Currently, over 30,000 mapped STSs and 27,000 mapped Unigenes are available for human genome mapping. ESTs and cDNAs are excellent resources for building contig maps for two reasons. Firstly, they exist in two alternative forms--as both sequence information for PCR primer pairs, and cDoreen genomic libraries efficiently for large number of DNA probes by combining over 100 cDNA probes in each hybridization. Second, the linkage and order of genes are rather conserved among human, mouse and other model organisms. Therefore, gene markers have advantages over random anonymous STSs in building maps for comparative genomic studies.

  11. Construction and analysis of subtractive cDNA library of recovery body wall in sea cucumber Apostichopus japonicus%仿刺参体壁创伤修复消减文库的构建与分析

    Institute of Scientific and Technical Information of China (English)

    秦艳杰; 李霞; 张慧敏; 王雪

    2013-01-01

    应用抑制性消减杂交技术(SSH),构建了仿刺参Apostichopus japonicus(体质量为65 ~90 g)正常体壁及创伤修复(24、48、72、96、120 h后)体壁的消减cDNA文库,利用PCR和斑点杂交技术对文库进行筛选,随机挑取的768个克隆中发现292个阳性克隆,对其中信号强度较强的224个阳性克隆进行测序,得到208个有效EST序列.经BlastX工具对获得的EST与GenBank数据库进行比对分析,结果有171个EST序列与数据库中的基因同源(e≤0.001,相似性>40%),其中153个为未知基因,18个为已知功能或已命名基因,包括在创伤及修复的体壁中上调表达的β微管蛋白、微管蛋白α-1链、肌动蛋白、肌动蛋白ike 7B类似物、细胞色素c氧化酶亚基Ⅰ、tRNA假尿苷合成酶A、GTP酶、细胞分裂周期2类似蛋白、有丝分裂原活化蛋白激酶、homeobox蛋白、延伸因子1A、核糖体蛋白L30、核糖体蛋白L17、60S酸性核糖体蛋白PO、26S蛋白酶调节亚基、泛素特异性肽酶24、大肠癌血清抗原3、清道夫受体蛋白12等.本研究结果可为探讨刺参体壁再生过程和分子机理,以及筛选刺参体壁创伤修复过程中相关功能基因的研究提供基础依据.%A subtracted cDNA library of sea cucumber Apostichopus japonicus(body weight 65-90 g) was constructed by suppression subtractive hybridization technology (SSH) to screen EST associated with recovery body wall.The cDNA library of the test group has been constructed by the mRNA of the body walls 24,48,72,96 and 120h after the operation,and those with no operation as the control group.Differential EST from the subtracted cDNA library have been identified by both PCR technology and dot blot hybridization.Two hundred and ninety-two positive clones were observed from total 768 clones,and 224 positive ones were sequenced.Two hundred and eight EST were found and analyzed by BlastX tool in GenBank database,in which 171 EST were homologous with sequences

  12. Qualitative de novo analysis of full length cDNA and quantitative analysis of gene expression for common marmoset (Callithrix jacchus) transcriptomes using parallel long-read technology and short-read sequencing.

    Science.gov (United States)

    Shimizu, Makiko; Iwano, Shunsuke; Uno, Yasuhiro; Uehara, Shotaro; Inoue, Takashi; Murayama, Norie; Onodera, Jun; Sasaki, Erika; Yamazaki, Hiroshi

    2014-01-01

    The common marmoset (Callithrix jacchus) is a non-human primate that could prove useful as human pharmacokinetic and biomedical research models. The cytochromes P450 (P450s) are a superfamily of enzymes that have critical roles in drug metabolism and disposition via monooxygenation of a broad range of xenobiotics; however, information on some marmoset P450s is currently limited. Therefore, identification and quantitative analysis of tissue-specific mRNA transcripts, including those of P450s and flavin-containing monooxygenases (FMO, another monooxygenase family), need to be carried out in detail before the marmoset can be used as an animal model in drug development. De novo assembly and expression analysis of marmoset transcripts were conducted with pooled liver, intestine, kidney, and brain samples from three male and three female marmosets. After unique sequences were automatically aligned by assembling software, the mean contig length was 718 bp (with a standard deviation of 457 bp) among a total of 47,883 transcripts. Approximately 30% of the total transcripts were matched to known marmoset sequences. Gene expression in 18 marmoset P450- and 4 FMO-like genes displayed some tissue-specific patterns. Of these, the three most highly expressed in marmoset liver were P450 2D-, 2E-, and 3A-like genes. In extrahepatic tissues, including brain, gene expressions of these monooxygenases were lower than those in liver, although P450 3A4 (previously P450 3A21) in intestine and P450 4A11- and FMO1-like genes in kidney were relatively highly expressed. By means of massive parallel long-read sequencing and short-read technology applied to marmoset liver, intestine, kidney, and brain, the combined next-generation sequencing analyses reported here were able to identify novel marmoset drug-metabolizing P450 transcripts that have until now been little reported. These results provide a foundation for mechanistic studies and pave the way for the use of marmosets as model animals

  13. The National Treatment Improvement Evaluation Study: Retention Analysis.

    Science.gov (United States)

    Orwin, Rob; Williams, Valerie

    This study focuses on programmatic factors that predict retention for individuals in drug and alcohol treatment programs through secondary analysis of data from the National Treatment Improvement Evaluation Study (NTIES). It addresses the relationships between completion rates, lengths of stay, and treatment modality. It examines the effect of…

  14. 扩展莫尼茨绦虫蛋白激酶C相互作用蛋白(PICK1)基因的克隆及序列分析%Cloning and Sequence Analysis of the cDNA Encoding Protein Interacting with C Kinase 1 (PICK1) in Moniezia expansa

    Institute of Scientific and Technical Information of China (English)

    赵文娟; 康立超; 薄新文; 王新华

    2011-01-01

    [目的]分离和鉴定扩展莫尼茨绦虫(Monieziaexpansa)新基因,为进一步研究该基因的功能奠定基础.[方法]构建扩展莫尼茨绦虫成虫cDNA文库,随机挑取重组阳性克隆进行测序,对部分序列进行引物步移法测序,获取其全长cDNA序列;采用生物信息学等分析技术对该cDNA序列进行开放阅读框(ORF)的寻找、编码氨基酸的推导、核苷酸和氨基酸同源性比较及蛋白质二级结构的初步预测.[结果]获得了1个扩展莫尼茨绦虫新基因蛋白激酶C相互作用蛋白,全长1 527 bp,编码447个氨基酸,CDS预测存在明显的BAR,PDZ结构域.编码蛋白的理论分子质量为50.173 3 ku,等电点为5.22.[结论]获得了扩展莫尼茨绦虫蛋白激酶C相互作用蛋白的全长cDNA序列,为该基因功能的试验性鉴定工作奠定基础.%[Objective] The purpose of this program was to clone and identify novel genes from an adult Monieda expansa (M. Expansa) cDNA library, and provide a foundation for further research. [Method]A cDNA library was constructed from M. Expansa adult stage. Clones were selected randomly from the cDNA library and were sequenced by using the method of expression sequence tags (ESTs) . Novel genes were acquired by primer -walking. The cDNA sequence encoding M. Expansa PICK1 protein was analyzed, including searching the ORF, translating the nucleotide to protein sequence, similarity searches and secondary structure predication with bioinformatics analysis. [ Result ] PICK 1 genes, 1527 bp and coding for 447 amino acids, was cloned and sequenced, then the sequence was submitted to GenBank and got an accession number, GH291479. The theoretical pi was 5.22 and molecular weight was SO. 173 3 ku. [Conclusion]The full - length cDNA encoding M. Expansa PICK1 was obtained, which laid the foundation for further functional study of this gene.

  15. 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

  16. 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)

  17. Cloning and sequence analysis of squalene synthase gene and cDNA in Glycyrrhiza uralensis%甘草鲨烯合酶基因及cDNA的克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    荣齐仙; 刘春生; 黄璐琦; 张宁; 南博; 呙未

    2011-01-01

    Objective: To clone and sequence the open reading frame and genomic sequence of squalene synthase (SQS) from Glycyrrhiza uralensis. Method: The primers were designed according to cDNA sequence of SQS from G. glabra reported by Hiroaki HAYASHI, SQS cDNA was cloned with total RNA extracted from roots of G. uralensis. Specific fragments were amplified by RT-PCR and then were cloned and sequenced. SQS DNA was cloned with total DNA extracted from roots of G. uralensis. Specific fragments were amplified by PCR and then were cloned and sequenced. Result: GuSQS1 (GenBank accession number: GQ266154) was 1 242 bp in length encoding proteins with 412 amino acid. NCBI Blast x search results showed GuSQS1 had the highest amino acid similarity to the corresponding proteins from G. uralensis The identities of GuSQS1 with the two proteins were 98. 55% and 88. 62%. SQS ( GenBank accession number: GQ180932) gene with 4 484 bp containing 13 exons and 12 introns was then amplified by PCR with genomic DNA extracted from roots of G. uralensis. Conclusion: These findings of cloning and sequencing the open reading frame and genomic sequence of squalene synthase (SQS) from G. uralensis brought some new clues for the further exploration of SmSQS function in sterol and terpenes biosynthesis.%目的:对甘草鲨烯合酶(SQS)基因的cDNA及DNA进行克隆及序列分析.方法:根据已报道的光果甘草SQS1基因的cDNA序列设计引物,采用RT-PCR的方法,提取甘草根的RNA然后反转录成cDNA,以cDNA为模板,扩增出SQS基因的cDNA序列,以甘草总DNA为模板,扩增SQS的DNA序列.结果:序列分析表明,克隆获得的甘草SQS1的cDNA编码区为1242 bp,编码413个氨基酸残基,命名为GuSQS1,登录号为GQ266154,与卢虹玉等报道的甘草的2个SQS(SQS1和SQS2)的氨基酸序列一致性为98.55%,88.62%,对应DNA序列全长为4484 bp,含有13个外显子,12个内含子,登录号为GQ180932.结论:甘草SQS的cDNA及DNA序列的获得为进一步研究

  18. Construction and analysis of suppression subtractive cDNA libraries of continuous monoculture Rehmannia glutinosa%连作地黄cDNA消减文库的构建及分析

    Institute of Scientific and Technical Information of China (English)

    张重义; 范华敏; 杨艳会; 李明杰; 李娟; 许海霞; 陈军营; 陈新建

    2011-01-01

    目的:通过构建连作地黄cDNA消减文库,探讨地黄连作障碍的分子机制.方法:利用抑制性消减杂交(SSH)技术构建连作地黄的正反消减文库,通过蓝白斑筛选、PCR的方法鉴定出阳性克隆,并对其进行测序和生物信息学分析.结果:连作地黄cDNA消减文库构建成功,正向和反向消减文库均筛选了300个阳性克隆.测序结果表明:正库、反库分别获得232条、214条特异的EST序列;经NCBI数据库分析,正库、反库中分别有200,195条EST序列的基因具有蛋白功能注释;COG基因功能预测结果表明,正库、反库中分别有60,61条EST序列具有相应的的基因功能分类,涉及21个代谢途径.结论:差异表达基因的功能注释表明,连作对地黄体内的基因表达具有深刻的影响.本研究筛选地黄响应连作的关键基因,为揭示地黄连作障碍的分子机制奠定了基础.%Objective: To explore the molecular mechanism of continuous monoculture problem by constructing the eDNA libraries of continuous monoculture Rehmannia glutinosa. Method: 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 sereening and PCR to detect the positive clones which would be sequenced and analyzed by bioinformatics. Result: 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

  19. 油茶CoUXS1基因全长cDNA克隆及生物信息学分析%Full-length cDNA Cloning and Bioinformatic Analysis of CoUXS1 Gene in Camellia oleifera

    Institute of Scientific and Technical Information of China (English)

    曾艳玲; 谭晓风; 王保明; 龙洪旭; 刘凯; 曾晓峰

    2012-01-01

    为了揭示油茶UDP-葡萄糖醛酸脱羧酶基因的序列特点和结构功能,为该基因的深入研究和开发应用奠定基础.根据油茶种子EST文库中调取的UDP-葡萄糖醛酸脱羧酶基因序列,设计特异引物,利用RT-PCR技术获得该基因全长cDNA克隆,并对其序列进行生物信息学分析.结果表明:该基因阅读框为1032bp,编码344个氨基酸,具有典型的UXS家族模体序列.与海岛棉GhUXS3基因亲缘关系最近,定位于细胞质,有较强的亲水性,将该基因命名为CoUXS1,GenBank登录号为JN017094.%In order to reveal sequence features and structural function of the UDP-glucuronate decarboxylase gene in Camellia oleifera, and based for the further research, the specific primers were designed according to the UDP-glucuronate decarboxylase gene sequence from Camellia oleifera seed EST library. Using RT-PCR technology, the gene full-length cDNA clones were obtained. Then the sequence was analyzed by bioinformatic software. The results showed that: the ORF box included 1032 bp and encoded 344 ami no acids. The sequence had typical UXS family motif sequence. The gene had closest phylogenetic relationship with GhUXS3 gene of Island cotton and localized in the cytoplasm with strong hydrophilicity. The gene was named CoUXSl and the GenBank accession number was JN017094.

  20. ISOLATION AND CLONING OF cDNA OF GENE ENCODING FOR METALLOTHIONEIN TYPE 2 FROM MELASTOMA AFFINE

    Directory of Open Access Journals (Sweden)

    UTUT WIDYASTUT

    2009-01-01

    Full Text Available Metallothionein is an important protein for detoxifying heavy metal ions. h is research was conducted to isolate and clone cDNA of gene encoding for metallothionein type 2 from Melastoma affi ne . Total RNA was isolated from young leaves. Total cDNA was synthesized from the total RNA by reverse transcription. h e MaMt2 cDNA was successfully isolated by PCR technique. h e MaMt2 cDNA was inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into Escherichia coli DH5 α . DNA sequencing analysis showed that this cDNA is full length consisting of 246 pb encoding 81 amino acid residues. h is cDNA is identical to mRNA of AtMt2 from Arabidopsis thaliana. It does not contain any restriction sites found in the cloning sites of pGEM-T Easy. h e deduced protein of MaMT2 contains 14 cysteine residues distributed in the Cys-Cys, Cys-X-Cys, and Cys-X-X-Cys motifs

  1. Using Operational Analysis to Improve Access to Pulmonary Function Testing

    Directory of Open Access Journals (Sweden)

    Ada Ip

    2016-01-01

    Full Text Available Background. Timely pulmonary function testing is crucial to improving diagnosis and treatment of pulmonary diseases. Perceptions of poor access at an academic pulmonary function laboratory prompted analysis of system demand and capacity to identify factors contributing to poor access. Methods. Surveys and interviews identified stakeholder perspectives on operational processes and access challenges. Retrospective data on testing demand and resource capacity was analyzed to understand utilization of testing resources. Results. Qualitative analysis demonstrated that stakeholder groups had discrepant views on access and capacity in the laboratory. Mean daily resource utilization was 0.64 (SD 0.15, with monthly average utilization consistently less than 0.75. Reserved testing slots for subspecialty clinics were poorly utilized, leaving many testing slots unfilled. When subspecialty demand exceeded number of reserved slots, there was sufficient capacity in the pulmonary function schedule to accommodate added demand. Findings were shared with stakeholders and influenced scheduling process improvements. Conclusion. This study highlights the importance of operational data to identify causes of poor access, guide system decision-making, and determine effects of improvement initiatives in a variety of healthcare settings. Importantly, simple operational analysis can help to improve efficiency of health systems with little or no added financial investment.

  2. Using Operational Analysis to Improve Access to Pulmonary Function Testing.

    Science.gov (United States)

    Ip, Ada; Asamoah-Barnieh, Raymond; Bischak, Diane P; Davidson, Warren J; Flemons, W Ward; Pendharkar, Sachin R

    2016-01-01

    Background. Timely pulmonary function testing is crucial to improving diagnosis and treatment of pulmonary diseases. Perceptions of poor access at an academic pulmonary function laboratory prompted analysis of system demand and capacity to identify factors contributing to poor access. Methods. Surveys and interviews identified stakeholder perspectives on operational processes and access challenges. Retrospective data on testing demand and resource capacity was analyzed to understand utilization of testing resources. Results. Qualitative analysis demonstrated that stakeholder groups had discrepant views on access and capacity in the laboratory. Mean daily resource utilization was 0.64 (SD 0.15), with monthly average utilization consistently less than 0.75. Reserved testing slots for subspecialty clinics were poorly utilized, leaving many testing slots unfilled. When subspecialty demand exceeded number of reserved slots, there was sufficient capacity in the pulmonary function schedule to accommodate added demand. Findings were shared with stakeholders and influenced scheduling process improvements. Conclusion. This study highlights the importance of operational data to identify causes of poor access, guide system decision-making, and determine effects of improvement initiatives in a variety of healthcare settings. Importantly, simple operational analysis can help to improve efficiency of health systems with little or no added financial investment.

  3. Cloning and Sequence Analysis of Partial cDNA of Male Determinant Factor of Self-incompatibility from Six Brassica oleracea L.%六种甘蓝自交不亲和雄性决定因子部分cDNA的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    罗兵; 朱利泉; 薛丽琰; 孙海燕; 张贺翠; 余浩; 杨昆; 王小佳

    2012-01-01

    Many flowering plants possess a self-incompatibility system to prevent inbreeding. In Brassica oleracea, self-incompatibility is genetically controlled by S-locus cysteine rich protein (SCR) and S-locus receptor kinase (SRK). The SCR is the determinant of pollen S-haplotype specificity. In order to compare the structure of the gene and molecular characterization of the protein among the allelic SCRs, the nested PCR primers were designed on the basis of the conserved amino acids in the signal peptide's cleavage site and the ploy A of mRNA. Here we cloned partial cDNA sequence of SCR from six Brassica oleracea L. Sequence analysis showed that the cDNA sequence of SCR in D3, El, 240, Al, Nl and Gl were 319, 311, 290, 288, 385 and 377 bp, respectively, which all encompassed 3'UTR. Their coding regions predicted a protein of 58, 58, 58, 58, 58 and 55 amino acids, respectively. The protein sequences were identical between SCR-D3 and SCR3. SCR-El, SCR-240, SCR-Al and SCR-El also had the same sequences, and they were all identical to the SCR7. The SCR of Gl was a new S haplotype gene. Although SCR -El, SCR-240, SCR-Al and SCR-El were the same S haplotype, their 3'UTR were different. For example, the length, the polyadenylation signal and the adenine nucleotide's content were different among them. Sequencing and bioinformatic analysis indicated that there were some differences in the secondary structure and the 3-dimentional structure of the six SCRs, suggesting that the interactions of SCR with SRK required strict complementary space. All SCRs had potential phosphorylation sites, but no glycosylation sites. It showed that the phosphorylation of SCR might play roles in signal transduction of self-incompatibility. Furthermore, the amino acid residues interacting with SRK were situated on the surface of the SCR molecule, and most of these amino acid residues were basic amino acid. So, we suggested that the process of SCR interacting with SRK required the participation of the

  4. cDNA cloning and sequencing of ostrich Growth hormone

    Directory of Open Access Journals (Sweden)

    Doosti Abbas

    2012-01-01

    Full Text Available In recent years, industrial breeding of ostrich (Struthio camelus has been widely developed in Iran. Growth hormone (GH is a peptide hormone that stimulates growth and cell reproduction in different animals. The aim of this study was to clone and sequence the ostrich growth hormone gene in E. coli, done for the first time in Iran. The cDNA that encodes ostrich growth hormone was isolated from total mRNA of the pituitary gland and amplified by RT-PCR using GH specific PCR primers. Then GH cDNA was cloned by T/A cloning technique and the construct was transformed into E. coli. Finally, GH cDNA sequence was submitted to the GenBank (Accession number: JN559394. The results of present study showed that GH cDNA was successfully cloned in E. coli. Sequencing confirmed that GH cDNA was cloned and that the length of ostrich GH cDNA was 672 bp; BLAST search showed that the sequence of growth hormone cDNA of the ostrich from Iran has 100% homology with other records existing in GenBank.

  5. Cloning and Sequence Analysis of Mannose-6-phosphate lsomerase cDNA from Metarhizium anisopliae ZJ1109%金龟子绿僵菌甘露糖6-磷酸异构酶基因cDNA序列的克隆及分析

    Institute of Scientific and Technical Information of China (English)

    李亚

    2011-01-01

    The primers were designed according to the conservative sequence of mannose-6-phosphate isomerase gene, and the complete cDNA sequence of Metarhizium anisopliae was amplified by RT-PCR and RACE PCR. The complete cDNA sequence of mpi gene was 1513 bp and the complete ORF length was 1328 bp which encoded a protein with 441 amino acid residues. Blast analysis indicated the deduced amino acid sequence of the mpi gene from M. Anisopliae showed high homology with other fungi respectively. The analysis of protein structure showed MPI protein had the characteristic of the conservative phosphate enzyme, and mainly constructed by a-helix and random coil.%通过绿僵菌属甘露糖6-磷酸异构酶基因保守核苷酸区域设计简并性引物,采用RT-PCR及RACE-PCR技术成功克隆了金龟子绿僵菌mpi基因cDNA序列.该基因cDNA序列全长为1513bp,开放阅读框长度为1328 bp,共编码441氨基酸.BLAST分析发现该基因演绎的氨基酸序列与其它真菌同源性较高,蛋白结构分析表明MPI蛋白是较保守的蛋白磷酸酶结构特征,主要由α螺旋和不规则卷曲构成.

  6. Isolation of 24 novel cDNA fragments from microdis—sected human chromosome band

    Institute of Scientific and Technical Information of China (English)

    ZHANGMIN; LONGYU; 等

    1998-01-01

    The strategy of isolating the band0specific expression fragments from a probe pool generated by human chromosome microdissection was reported.A chromosome 14q 24.3 band-specific single copy DNA pool was constructed based on this probe pool.Using total DNA of the pool as probe to hybridize the human marrow cDNA library,68 primary positive clones were selected from 5×105 cDNA clones.Among these primary clones,32 secondary clones were obtained after second-round screening and designed as cFD14-1-32.Finally,24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization.Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but cann't hybridize to 17q11-12 DNA,Partial sequences of 13 fragments of them were sequenced and idenfified as novel cDNA sequences,and these sequences were proved to have some homology with known genes in NCBI database.Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

  7. Molecular cloning and expression of a novel human cDNA containing CAG repeats.

    Science.gov (United States)

    Takeuchi, T; Chen, B K; Qiu, Y; Sonobe, H; Ohtsuki, Y

    1997-12-19

    A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.

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

    OpenAIRE

    Jia-Shing Sheu; Kai-Chung Teng

    2013-01-01

    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 conte...

  9. 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.

  10. 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.

  11. 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.

  12. 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...

  13. Full length cDNA cloning and sequence characteristics analysis of BSP Ⅱ gene from Sika deer antler tissue%梅花鹿茸角组织BSPⅡ基因全长cDNA的克隆及序列特征分析

    Institute of Scientific and Technical Information of China (English)

    郝丽

    2011-01-01

    从梅花鹿鹿茸尖端组织全长cDNA文库中克隆了与骨形成和骨改建有关的一种新的骨生长因子BSPⅡ基因的全长cDNA序列,并结合生物信息学方法和实时荧光定量RT-PCR技术对该基因的氨基酸序列及其在鹿茸尖端不同组织层的表达情况进行了分析。结果表明,BSPⅡ基因cDNA全长为1576bp,编码311个氨基酸。经生物信息学分析表明,该基因编码的蛋白具有N端信号肽及跨膜区,相对分子质量为34100,理论等电点为4.05,其一级结构中谷氨酸所占比例最高;二级结构元件主要以α-螺旋和无规则卷曲为主;同源序列分析表明,梅花%The full length cDNA of BSP Ⅱ gene from velvet tip tissue full-length cDNA library of Sika deer was cloned,bioinformatics method and Real-time RT-PCR technique were used to analyze the amino acid sequence and expression. The results showed the full-length cDNA of the BSP Ⅱ gene was 1 576 bp,encoded a peptide of 311 amino acid,its relative molecular weight was 34 100, isoelectric point was 4.05, contained a N-terminal signal peptid and transmembrane domain; Glu occupied the highest proportion and secondary structure with a-helix and random coilbased. The results obtained through homologous sequence analysis indicated that BSP Ⅱ of Sika deer was highly sim- ilarity to Bos Taurus(93 %) ;with multiple sequences comparison,the N-end and 68-215,265-308 Glu rich area were highly conservative; the molecular evolution trees displayed that BSP Ⅱ of Sika deer had high genetic relationship with Equus caballus. Real-time PCR results showed that there was a significant positive correlation between its expression and velvet mineralization process showed ,it is presumed that the BSP Ⅱ genes in the process of velvet tissue mineralization plays an important role in regulating.

  14. Cloning and Analysis of Cytosolic Glutamine Synthetase cDNA from Eichharnia crassipes%水葫芦胞质型谷氨酰胺合成酶EcGS1全长cDNA的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    蒋丽花; 傅明辉; 李园枚; 严国花; 郑李军; 陈肖丽; 彭进平

    2014-01-01

    以水葫芦根部总RNA逆转录得到的cDNA为模板,参照其他植物的胞质型谷氨酰胺合成酶(GS1)氨基酸保守序列设计简并引物,进行PCR扩增,以得到的产物为基础,采用RACE技术获得水葫芦胞质型谷氨酰胺合成酶EcGS1全长cDNA。全长为1434 bp,开放阅读框为1071 bp,编码356个氨基酸,分子量为39.3 kD,等电点pI为5.52。序列相似性分析显示,该序列与其他植物的GS1氨基酸序列具有较高的相似性。通过亚细胞定位预测,确定EcGS1为胞质型谷氨酰胺合成酶。%In this study, the template cDNA which was reversely transcribed from the total RNA of Eichharnia crassipes, was subjected to polymerase chain reaction(PCR)with the degenerate primers which was designed according to sequences of the Cytosolic Glutamine Synthetase(GS1)from other plants. The full-length cDNA sequence was achieved with Rapid Amplification of cDNA End method(RACE) from the amplification product. It includes 1 434 bp with the open reading frame of 1 071 bp which encoded 356 coding amino acids with a predicted size of 39.3 kD and a calculated pI of 5.52. The result of sequence homology analysis showed that the deduced protein had relatively high amino acid identity with GS1s from other plants. The prediction of sub-cellular localization showed that EcGS1 is a cytosolic glutamine synthetase.

  15. Cloning and Analysis of Trypsin-like Serine Protease cDNA of Yellow Mealworm ( Tenebrio molitor) Larvae%黄粉虫胰蛋白酶样酶基因TMTLSP全长cDNA的克隆和序列分析

    Institute of Scientific and Technical Information of China (English)

    叶韵; 韩雅莉; 黄明星; 孙建平; 代春迎; 林非凡

    2012-01-01

    以黄粉虫(Tenebrio molitor)幼虫全RNA逆转录得到的cDNA为模板,参照地鳖(Eupolyphaga sinensis)纤溶酶(fibrinolytic enzyme)简并引物,进行温度梯度PCR.以得到的扩增产物为基础,采用RACE得到基因全长cDNA,命名为黄粉虫胰蛋白酶样丝氨酸蛋白酶(Tenebrio molitor trypsin-like serine protease,TMTLSP).TMTLSP全长869 bp(GenBank No.JN662461),开放阅读框为777 bp,编码258个氨基酸,并具有蛋白酶样特有的起始位点、活性中心预计底物结合位点.经过比对分析,该基因编码的氨基酸序列与赤拟谷盗、谷蠹、光亮扁角水虻、美洲大蠊等多种昆虫的胰蛋白酶或丝氨酸蛋白酶有较高的相似性.本研究将为胰蛋白酶样丝氨酸蛋白酶的提取及研究提供更为广泛的材料及研究依据.%In this study, the template cDNA which was reversely transcribed from the total RNA of Tenebrio molitor larvae, was subjected to temperature gradient polymerase chain reaction (PCR) with the degenerate primers of Eupolyphaga sinensis fibrinolytic enzyme. The full-length cDNA, achieved with Rapid Amplification of cDNA End method (RACE) from the amplification product, was named Tenebrio molitor Trypsin-like Serine Protease (TMTLSP). TMTLSP includes 869 bp ( GenBank No. JN6624614) with the open reading frame of 777 bp and 258 coding amino acids. It also has the unique trypsin-like initiation sites and expected binding sites of substrate active sites. The results of comparative analysis illustrated that the gene encoding of amino acid sequence was highly similar to those of a variety of insects. Based on the results, this study could be considered as a valid reference for the further investigation of trypsin-like serine protease extraction and examination.

  16. 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

  17. Cloning and sequence analysis of full-length cDNA ofα-actin gene from Chelonia mydas%绿海龟α-actin基因的cDNA克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    陶翠花; 刘莹莹; 赵丽媛; 许敏; 祝茜

    2014-01-01

    To explore the sequence and characteristic of α-actin gene from Chelonia mydas, the full-length cDNA sequence ofα-actin gene was cloned using RT-PCR and RACE technique, which was consisted of 1347 bp nucleo-tides (GenBank accession number: JX073650), with a putative open reading frame (ORF) of 1134 bp encoding a deduced 377 amino acid protein containing a glycosylation site (from 14 to 17) and an Actin domain (from 7 to 377). The molecular weight of the protein was 42.0 kDa and the isoelectric point (pI) was 5.23. The nucleotide sequence similarity ofα-actin gene between C. mydas and other species was above 85.4%, while the similarity of amino acid sequence was more than 98.9%, suggesting that α-actin gene was highly conserved. This study has enriched the Actin gene database and provided basic data for further studies on expression and function of relevant genes.%为探究绿海龟(Chelonia mydas)α-actin基因序列的相关信息,作者利用RT-PCR和RACE方法从绿海龟肌肉组织中获得了α-actin基因的cDNA全长序列,共1347bp(GenBank登录号为JX073650)。所得序列包含一个1134 bp的开放阅读框,编码由377个氨基酸组成的蛋白,该蛋白7~377位为Actin结构域,14~17位有一个糖基化位点,无信号肽;预测分子量为42.0 kDa,理论等电点为5.23。将编码区序列与 GenBank 上同源序列进行比对发现,核苷酸序列相似性均在85.4%以上,氨基酸序列相似性均在98.9%以上,说明α-actin基因作为编码蛋白是高度保守的。

  18. Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Li; You-Yong Lu

    2002-01-01

    AIM: To develop and optimize cDNA representationaldifference analysis (cDNA RDA) method and to identify andclone garlic up-regulated genes in human gastric cancer(HGC) cells.METHODS: We performed cDNA RDA method by usingabundant double-stranded cDNA messages provided by twoself-constructed cDNA libraries (Allitridi-trested and paternalHGC cell line BGC823 cells cDNA libraries respectively).BamH Ⅰ and Xho I restriction sites harbored in the libraryvector were used to select representations. Northern andSlot blots analyses were employed to identify the obtaineddifference products.RESJLTS: Fragments released from the cDNA library vectorafter restriction endonuclease digestion acted as goodmarker indicating the appropriate digestion degree for libraryDNA. Two novel expressed sequence tags (ESTs) and arecombinant gene were obtained. Slot blots result showed a8-fold increase of gila-derived nexin/protease nexin 1 (GDN/PN1 ) gene expression level and 4-fold increase of hepatitis Bvirus x-interacting protein (XIP) mRNA level in BGC823 cellsafter Allitridi treatment for 72 h.CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAsinduced by Allitridi provide valuable molecular evidence forelucidating the garlic' s efficacies against neurodegenerativeand inflammatory diseases. Isolation of a recombinant geneand two novel ESTs further show cDNA RDA based on cDNAlibraries to be a powerful method with high specificity andreproducibility in cloning differentially expressed genes.

  19. cDNA CLONING AND EXPRESSION ANALYSIS OF Dmrt1 IN CLARIAS FUSCUS%胡子鲇Dmrt1基因全长cDNA克隆及其表达分析

    Institute of Scientific and Technical Information of China (English)

    邓思平; 王静杰; 吴天利; 朱春华; 李广丽

    2012-01-01

    Sex of fish is determined by both genetic substance and environment. The studies on the relationship between the sex and sex-related genes expression can not only reveal the mechanisms of sex determination, but also provide the theoretical basis to control the sex differentiation in artificial propagation in the fish. Catfish (Clarias fuscus) is a common freshwater fish in China. The Dmrtl cDNA sequences were isolated from the testes of C. fuscus by RT-PCR and SMART RACE. Structure and function of the predicted DMRT1 protein were analyzed by bioinformatics. Expressions of Dmrtl were examined using semi-quantitative RT-PCR in ten tissues (testes, ovaries, muscles, intestine, livers, heart, head kidney, gills, brains and eyes) and the testes from stage Ⅱ to stage Ⅴ. Results showed that the Dmrtl complete cDNA was 1417 bp in length, and the 5' un-translated region (5'-UTR) and the 3' un-translated region (3'-UTR) were 35 bp and 516 bp, respectively. The open reading frame (ORF) was 864 bp, and a sequence of predicted 287 amino acids residues was encoded. The C. fuscus DMRT1 shared a higher amino acid identity of 83.3% to 96.1% with three Siluri-formes fishes such as C. gariepinus, C. batrachus and Pelteobagrus fulvidraco. DMRT1 protein coded by Dmrtl gene located mainly in nucleus, and worked as an unstable hydrophobic protein. A highly conserved DM domain was found in the DMRT1 protein, which was a common characteristic in DMRT clusters. The C. fuscus shared a higher DM domain identity of 100% with the above-mentioned Siluriformes fishes, 91.9% to 97.3% with Danio rerio, Oryzias latipes and Oncorhynchus mykiss, and over 80% with chicken, rat, pig and human. In addition, the DM domain of C. fuscus had a typical "C2H2C4" zinc finger structure, which bound with the downstream DNA and control the gene transcription. Expression of Dmrtl was found only in the testes, but not in ovaries, muscles, intestine, liver, heart, head kidney, gills, brain and eyes, which

  20. Noise reduction of cDNA microarray images using complex wavelets.

    Science.gov (United States)

    Howlader, Tamanna; Chaubey, Yogendra P

    2010-08-01

    Noise reduction is an essential step of cDNA microarray image analysis for obtaining better-quality gene expression measurements. Wavelet-based denoising methods have shown significant success in traditional image processing. The complex wavelet transform (CWT) is preferred to the classical discrete wavelet transform for denoising of microarray images due to its improved directional selectivity for better representation of the circular edges of spots and near shift-invariance property. Existing CWT-based denoising methods are not efficient for microarray image processing because they fail to take into account the signal as well as noise correlations that exist between red and green channel images. In this paper, two bivariate estimators are developed for the CWT-based denoising of microarray images using the standard maximum a posteriori and linear minimum mean squared error estimation criteria. The proposed denoising methods are capable of taking into account both the interchannel signal and noise correlations. Significance of the proposed denoising methods is assessed by examining the effect of noise reduction on the estimation of the log-intensity ratio. Extensive experimentations are carried out to show that the proposed methods provide better noise reduction of microarray images leading to more accurate estimation of the log-intensity ratios as compared to the other CWT-based denoising methods.

  1. Cloning, expression and mapping of the full-length cDNA of human CCTβ subunit

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Chaperonins assist the proper folding of target proteins without being a part of the substrates. The eukaryotic cytosolic chaperonin, CCT-Chaperonin Containing TCP-1 (tailless complex polypeptide-1), is mainly involved in the formation of cytoskeletal proteins and is essential for cell viability. Mammalian CCT is commonly a protein complex composed of 7-9 subunit species. We have isolated a novel full-length cDNA from human testis cDNA library. This cDNA of 1935 bp contains a 1605 bp open reading frame (ORF) encoding 535 amino acids (aa). The deduced protein of the cDNA is highly homologous to the CCTβ subunit of saccharomyces cerevisiae, schizosaccharomyces pombe, caenorhabditis elegans and mouse, etc. Especially high homology (97%) is found between the deduced protein and mouse CCTb. On the basis of such high homology, the protein encoded by the new gene was proposed to be a human CCTβ subunit. Northern hybridization showed that human CCTβ gene is expressed as a transcript of about 2.0 kb in various tissues. Overexpression was seen in testis with the expression level 3-24 times of those in other tissues. The CCTβ gene was mapped to human chromosome 12q14 by Radiation Hybrid Mapping. Through homologous search, the 5′-end of the cDNA sequence was found to share intermittent regional homology with the 3′-end of human genomic sequence (U91327). The genomic structure of the 5′-end of CCTβ was also described in detail through comparative analysis.

  2. Cloning and expression of a cDNA encoding human sterol carrier protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III (Univ. of Pennsylvania School of Medicine, Philadelphia (United States)); Billheimer, J.T. (E.I. DuPont de Nemours, Inc., Wilmington, DE (United States))

    1991-01-15

    The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP{sub 2}). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP{sub 2} amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP{sub 2}. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A){sup +} RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP{sub 2} gene in the human genome or that the SCP{sub 2} gene is very large. Coexpression of the SCP{sub 2} cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP{sub 2} plays a role in regulating steroidogenesis, among other possible functions.

  3. 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.

  4. Sequence analysis of cDNA encoding an α-neurotoxin from king cobra(Ophiophagus hannah)with PCR techniques%PCR法分子克隆及序列分析广西产眼镜王蛇蛇毒α-神经毒素基因

    Institute of Scientific and Technical Information of China (English)

    李其斌; 渡庆次贺博; 金城记代彦; 中村真理子; 小杉忠诚

    2004-01-01

    目的为了获得眼镜王蛇(Ophiophagus hannah,Oh)蛇毒α-神经毒素(α-Neurotoxin, α-NT)的基因序列.方法我们通过比较了基因库中已知眼镜蛇科不同种类毒蛇来源的α-NT基因,发现它们有较高的同源性,特别是5'和3'非翻译区及引导肽部分高度保守,据此设计了包括翻译起始点的上游引物,以及为了得到3'端较完整非编码部分而设计了基本上属于d(T)的反意链下游RACE-PCR引物.为了克服引物所带来的模糊扩增,还在蛋白编码部分再设计一对上下游引物,由此组成P1、 P2、 P3和 P4四对引物.采用Nacleospin RNA Kit法分别从3条活Oh蛇毒腺中提取mRNA, 以3'端引物合成cDNA的第一链,并以此作为模板,分别用四对引物进行PCR扩增反应,得到了目的基因不同长度的PCR产物,产物经精制后进行测序,对比分析其结果.结果获得了全长474 bp的Oh.cDNA的基因核苷酸序列,包括5'端60 bp,信号肽伴启动子ATG 63 bp,蛋白质密码部分216 bp和3'端186 bp并含有TGA终止码.经基因库信息计算机分析其信号肽与眼镜蛇树属(Pseudonnaja textilis,Pt.)海蛇(Laticauda semifasciata,Ls)100%同源,96.8%与眼镜蛇南洋亚种(Naja sputatrix,Ns)和银环蛇(Bungarus multicinctus,Bm)同源.蛋白密码部分 83.3%与 Ns,79.2%与 Pt, 76.4%与 Ls和 74.1%与 Bm同源.氨基酸顺序分析信号肽后紧接着的72个氨基酸90.3%与已发现的眼镜王蛇毒长链α-NT Toxin a同源,大约 73.6% Toxin b 、69.7% Oh-4 、66.7% Oh-5 、56.9% Oh-6A 和6B同源,并与α-银环蛇毒素54.2%同源.结论新发现的Oh.cDNA属于长链α-NT的基因.%Objective To obtain the cDNA and sequence analysis of α-neurotoxin (α-NT) from Guangxi king cobra (Ophiophagus hannah,Oh).Methods Comparative analysis of the determined cDNA sequences of α-NT from elapidae snake venoms showed that the nucleotide sequences of 5',3'-non-coding regions and the signal peptide coding region were highly conserved

  5. Differential cDNA cloning by enzymatic degrading subtraction (EDS).

    OpenAIRE

    1994-01-01

    We describe a new method, called enzymatic degrading subtraction (EDS), for the construction of subtractive libraries from PCR amplified cDNA. The novel features of this method are that i) the tester DNA is blocked by thionucleotide incorporation; ii) the rate of hybridization is accelerated by phenol-emulsion reassociation; and iii) the driver cDNA and hybrid molecules are enzymatically removed by digestion with exonucleases III and VII rather than by physical partitioning. We demonstrate th...

  6. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    1994-01-04

    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. GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon.

  7. Isolation of a cDNA Encoding a Protease from Perinereis aibuhitensis Grube

    Institute of Scientific and Technical Information of China (English)

    Rong-Gui LI; Dong-Meng QIAN; Dao-Sen GUO; Gui-Cai DU; Zhi-Yong YAN; Bin WANG

    2006-01-01

    The cDNA encoding a protease of Perinereis aibuhitensis Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMALPPA. pMAL-PPA was introduced into Escherichia coli BL21(DE3) and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-β-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.

  8. 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.

  9. RECOGNITION OF CDNA MICROARRAY IMAGE USING FEEDFORWARD ARTIFICIAL NEURAL NETWORK

    Directory of Open Access Journals (Sweden)

    R. M. Farouk

    2014-09-01

    Full Text Available The complementary DNA (cDNA sequence considered the magic biometric technique for personal identification. Microarray image processing used for the concurrent genes identification. In this paper, we present a new method for cDNA recognition based on the artificial neural network (ANN. We have segmented the location of the spots in a cDNA microarray. Thus, a precise localization and segmenting of a spot are essential to obtain a more exact intensity measurement, leading to a more accurate gene expression measurement. The segmented cDNA microarray image resized and used as an input for the proposed artificial neural network. For matching and recognition, we have trained the artificial neural network. Recognition results are given for the galleries of cDNA sequences . The numerical results show that, the proposed matching technique is an effective in the cDNA sequences process. The experimental results of our matching approach using different databases shows that, the proposed technique is an effective matching performance.

  10. RECOGNITION OF CDNA MICROARRAY IMAGE USING FEEDFORWARD ARTIFICIAL NEURAL NETWORK

    Directory of Open Access Journals (Sweden)

    R. M. Farouk

    2014-07-01

    Full Text Available The complementary DNA (cDNA sequence considered th e magic biometric technique for personal identification. Microarray image processing used fo r the concurrent genes identification. In this pape r, we present a new method for cDNA recognition based on the artificial neural network (ANN. We have segmented the location of the spots in a cDNA micro array. Thus, a precise localization and segmenting of a spot are essential to obtain a more exact intensity measurement, leading to a more accurate gene expression measurement. The segmented cDNA microarr ay image resized and used as an input for the proposed artificial neural network. For matching an d recognition, we have trained the artificial neura l network. Recognition results are given for the gall eries of cDNA sequences . The numerical results sho w that, the proposed matching technique is an effecti ve in the cDNA sequences process. The experimental results of our matching approach using different da tabases shows that, the proposed technique is an effective matching performance.

  11. Molecular cloning of GA-suppressed G2 pea genes by cDNA RDA

    Institute of Scientific and Technical Information of China (English)

    朱玉贤; 张翼凤; 李慧英

    1997-01-01

    GA-treated and non-treated G2 pea cDNAs were compared using a newly developed method called cDNA representational difference analysis (cDNA-RDA), and several GA-suppressed mRNAs were found. After cloning of the larger fragments PGAS1-3 ( pea GA-suppressed cDNA 1-3), they were demonstrated to be expressed only in pea tissue not treated with GA3 through Northern analysis. Compared with subtractive hybridization and differ-ential display techniques, this method not only can be easily manipulated but also has a relatively low rate of false posi-tive and is highly repetitive. It is the major progress in molecular cloning techniques.

  12. 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.

  13. 黄龙病诱导下椪柑SSH文库的构建与分析%Construction and analysis of subtractive cDNA library from ponkan (Citrus reticulate ) leaves following infection with Honglongbin pathogen

    Institute of Scientific and Technical Information of China (English)

    钟云; 姜波; 易干军; 曾继吾; 王辉; 蒋侬辉; 周碧容

    2012-01-01

    A suppression subtractive hybridization library was successfully constructed using eDNA synthesized from RNA extracted from leaves of ponkan (Citrus reticulate Blanco) infected with Huang- longbing bacteria as tester and uninfected as driver. One hundred positive clones were randomly select- ed and sequenced, and 71 ESTs were obtained. A search against NCBI GenBank ,after removing the duplications and low quality sequences, revealed that 41 ESTs shared considerable homology with known genes and that 10 ESTs did not have matches. Functional annotation of the genes showed that they were related to metabolic pathways and physiological and biochemical processes such as stress- tolerance, transportation, energy metabolism, photosynthesis, proteometabolism, signaling, anti-oxi- dation. It was noteworthy that the lectin protein precursor gene that was commonly induced by pathogen was also found in the HLB bacteria-infected Ponkan leaf cDNA library. Q-PCR results showed that two analyzed HLB pathogen induced genes were indeed activated. These indicated that an active anti-infection reaction was initiated in Ponkan leaves during the early stage of HLB bacteria in- fection.%以实生苗和平椪柑(Citrus reticulate Blanco)为材料,采用抑制性差减杂交技术,分别以感染黄龙病与未感染黄龙病的椪柑叶片为检测方(tester)和驱动方(driver),成功构建了黄龙病诱导的差减cDNA文库。挑选了100个阳性克隆并成功测序得到71条EST,经NCBI基因库同源性比对。有41条非冗余高质量EST序列找到了同源序列,另有10条非冗余未搜索到同源序列。同源序列的基因涉及抗逆防御、运输、能量代谢、光合作用、蛋白代谢、信号转导、抗氧化等代谢途径和生理生化过程。值得注意的是文库中有由病原引起的韧皮部相关的凝集素蛋白的前体积累。挑选了2条进行Q—PCR定量分析,结果表明感病1周表达量增强不大,2

  14. 枣抑制消减cDNA文库构建与分析%Construction and analysis of suppression subtractive cDNA library in Chinese date (Ziziphus jujube)

    Institute of Scientific and Technical Information of China (English)

    魏琦琦; 段经华; 冯延芝; 李芳东; 张琳

    2016-01-01

    Z. jujube shows some special characters of flowering and fruit bearing. In order to obtain genes involved in blossom development, two suppression subtractive cDNA libraries were constructed using developing blossoms of‘Jinsi No.4’ as tester and leaves as driver. The recombination rates of the SSH libraries from Z. jujube’s flowers and leaves were 92.05% and 90.26%, with the length of inserted fragments ranged from 200 to 2 000 bp, and mainly focusing on 1 000 bp. A total of 1 000 positive clones were randomly selected from the SSH libraries ofZ. jujube’s flowers and leaves respectively for DNA sequencing and assembled. Consequently, 43 contigs, 53 singletons, 73 ORFs, 96 unigenes and 20 annotated sequences were obtained from SSH library ofZ. jujube’s flowers. Forty-ifve contigs, 41 singletons, 70 ORFs, 86 unigenes and 23 annotated sequences were obtained from SSH library ofZ. jujube’s leaves, and the annotated sequences could be divided into six groups.%枣树具有独特的开花结果特性,为获得枣花发育基因,以金丝4号枣不同发育时期的花蕾和同时期的叶片cDNA互为试验方和驱动方,利用抑制消减杂交技术分别构建了枣花和枣叶抑制消减杂交cDNA文库(SSH文库)。枣花和枣叶SSH文库重组率分别是92.05%和90.26%,插入片段长度均介于200~2000 bp之间,主要集中于1000 bp左右。分别从枣花和枣叶SSH文库中随机挑选1000个阳性克隆进行DNA测序、组装拼接,结果显示枣花SSH文库中获得96条unigene,其中contigs 43个,singletons 53个,96条unigene预测得到73个ORF,经过同源比对分析获得有注释的序列20条,按功能分为7类;枣叶SSH文库中获得86条unigene,其中contigs 45个,singletons 41个,86条unigene预测得到70个ORF,经过同源比对分析获得有23条注释的序列,按功能分为6类。

  15. Improved Methods for the Enrichment and Analysis of Glycated Peptides

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qibin; Schepmoes, Athena A; Brock, Jonathan W; Wu, Si; Moore, Ronald J; Purvine, Samuel O; Baynes, John; Smith, Richard D; Metz, Thomas O

    2008-12-15

    Non-enzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. Herein we report improved methods for the enrichment and analysis of glycated peptides using boronate affinity chromatography and electron transfer dissociation mass spectrometry, respectively. The enrichment of glycated peptides was improved by replacing an off-line desalting step with an on-line wash of column-bound glycated peptides using 50 mM ammonium acetate. The analysis of glycated peptides by MS/MS was improved by considering only higher charged (≥3) precursor-ions during data-dependent acquisition, which increased the number of glycated peptide identifications. Similarly, the use of supplemental collisional activation after electron transfer (ETcaD) resulted in more glycated peptide identifications when the MS survey scan was acquired with enhanced resolution. In general, acquiring ETD-MS/MS data at a normal MS survey scan rate, in conjunction with the rejection of both 1+ and 2+ precursor-ions, increased the number of identified glycated peptides relative to ETcaD or the enhanced MS survey scan rate. Finally, an evaluation of trypsin, Arg-C, and Lys-C showed that tryptic digestion of glycated proteins was comparable to digestion with Lys-C and that both were better than Arg-C in terms of the number glycated peptides identified by LC-MS/MS.

  16. Isolation of novel human cDNA (hGMF-gamma) homologous to Glia Maturation Factor-beta gene.

    Science.gov (United States)

    Asai, K; Fujita, K; Yamamoto, M; Hotta, T; Morikawa, M; Kokubo, M; Moriyama, A; Kato, T

    1998-03-13

    A novel full-length human cDNA homologous to Glia Maturation Factor-beta (GMF-beta) gene was isolated. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein sequence of 142 amino acid residues. The deduced amino acid sequences of its putative product is highly homologous to human GMF-beta (82% identity) and named for GMF-gamma. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-beta, is predominantly expressed in human lung, heart, and placenta.

  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.

  18. 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.

  19. Improved spectrum simulation for validating SEM-EDS analysis

    Science.gov (United States)

    Statham, P.; Penman, C.; Duncumb, P.

    2016-02-01

    X-ray microanalysis by SEM-EDS requires corrections for the many physical processes that affect emitted intensity for elements present in the material. These corrections will only be accurate provided a number of conditions are satisfied and it is essential that the correct elements are identified. As analysis is pushed to achieve results on smaller features and more challenging samples it becomes increasingly difficult to determine if all conditions are upheld and whether the analysis results are valid. If a theoretical simulated spectrum based on the measured analysis result is compared with the measured spectrum, any marked differences will indicate problems with the analysis and can prevent serious mistakes in interpretation. To achieve the necessary accuracy a previous theoretical model has been enhanced to incorporate new line intensity measurements, differential absorption and excitation of emission lines, including the effect of Coster-Kronig transitions and an improved treatment of bremsstrahlung for compounds. The efficiency characteristic has been measured for a large area SDD detector and data acquired from an extensive set of standard materials at both 5 kV and 20 kV. The parameterized model has been adjusted to fit measured characteristic intensities and both background shape and intensity at the same beam current. Examples are given to demonstrate how an overlay of an accurate theoretical simulation can expose some non-obvious mistakes and provide some expert guidance towards a valid analysis result. A new formula for calculating the effective mean atomic number for compounds has also been derived that is appropriate and should help improve accuracy in techniques that calculate the bremsstrahlung or use a bremsstrahlung measurement for calibration.

  20. Improved generalized cell mapping for global analysis of dynamical systems

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Three main parts of generalized cell mapping are improved for global analysis. A simple method, which is not based on the theory of digraphs, is presented to locate complete self-cycling sets that corre- spond to attractors and unstable invariant sets involving saddle, unstable periodic orbit and chaotic saddle. Refinement for complete self-cycling sets is developed to locate attractors and unstable in- variant sets with high degree of accuracy, which can start with a coarse cell structure. A nonuniformly interior-and-boundary sampling technique is used to make the refinement robust. For homeomorphic dissipative dynamical systems, a controlled boundary sampling technique is presented to make gen- eralized cell mapping method with refinement extremely accurate to obtain invariant sets. Recursive laws of group absorption probability and expected absorption time are introduced into generalized cell mapping, and then an optimal order for quantitative analysis of transient cells is established, which leads to the minimal computational work. The improved method is applied to four examples to show its effectiveness in global analysis of dynamical systems.

  1. Improved generalized cell mapping for global analysis of dynamical systems

    Institute of Scientific and Technical Information of China (English)

    ZOU HaiLin; XU JianXue

    2009-01-01

    Three main parts of generalized cell mapping are improved for global analysis. A simple method, whichis not based on the theory of digraphs, is presented to locate complete self-cycling sets that corre-spond to attractors and unstable invariant sets involving saddle, unstable periodic orbit and chaotic saddle. Refinement for complete self-cycling sets is developed to locate attractors and unstable in-variant sets with high degree of accuracy, which can start with a coarse cell structure. A nonuniformly interior-and-boundary sampling technique is used to make the refinement robust. For homeomorphic dissipative dynamical systems, a controlled boundary sampling technique is presented to make gen-eralized cell mapping method with refinement extremely accurate to obtain invariant sets. Recursive laws of group absorption probability and expected absorption time are introduced into generalized cell mapping, and then an optimal order for quantitative analysis of transient cells is established, which leads to the minimal computational work. The improved method is applied to four examples to show its effectiveness in global analysis of dynamical systems.

  2. 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.

  3. Improved Aerodynamic Analysis for Hybrid Wing Body Conceptual Design Optimization

    Science.gov (United States)

    Gern, Frank H.

    2012-01-01

    This paper provides an overview of ongoing efforts to develop, evaluate, and validate different tools for improved aerodynamic modeling and systems analysis of Hybrid Wing Body (HWB) aircraft configurations. Results are being presented for the evaluation of different aerodynamic tools including panel methods, enhanced panel methods with viscous drag prediction, and computational fluid dynamics. Emphasis is placed on proper prediction of aerodynamic loads for structural sizing as well as viscous drag prediction to develop drag polars for HWB conceptual design optimization. Data from transonic wind tunnel tests at the Arnold Engineering Development Center s 16-Foot Transonic Tunnel was used as a reference data set in order to evaluate the accuracy of the aerodynamic tools. Triangularized surface data and Vehicle Sketch Pad (VSP) models of an X-48B 2% scale wind tunnel model were used to generate input and model files for the different analysis tools. In support of ongoing HWB scaling studies within the NASA Environmentally Responsible Aviation (ERA) program, an improved finite element based structural analysis and weight estimation tool for HWB center bodies is currently under development. Aerodynamic results from these analyses are used to provide additional aerodynamic validation data.

  4. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  5. New Framework for Improving Big Data Analysis Using Mobile Agent

    Directory of Open Access Journals (Sweden)

    Youssef M. ESSA

    2014-01-01

    Full Text Available the rising number of applications serving millions of users and dealing with terabytes of data need to a faster processing paradigms. Recently, there is growing enthusiasm for the notion of big data analysis. Big data analysis becomes a very important aspect for growth productivity, reliability and quality of services (QoS. Processing of big data using a powerful machine is not efficient solution. So, companies focused on using Hadoop software for big data analysis. This is because Hadoop designed to support parallel and distributed data processing. Hadoop provides a distributed file processing system that stores and processes a large scale of data. It enables a fault tolerant by replicating data on three or more machines to avoid data loss.Hadoop is based on client server model and used single master machine called NameNode. However, Hadoop has several drawbacks affecting on its performance and reliability against big data analysis. In this paper, a new framework is proposed to improve big data analysis and overcome specified drawbacks of Hadoop. These drawbacks are replication tasks, Centralized node and nodes failure. The proposed framework is called MapReduce Agent Mobility (MRAM. MRAM is developed by using mobile agent and MapReduce paradigm under Java Agent Development Framework (JADE.

  6. Analysis and improvement of the MAC shaping mechanism in RPR

    Science.gov (United States)

    Li, Jing; Xu, Zhanqi; Yu, Shaohua

    2005-11-01

    Resilient Packet Ring (RPR) specified by IEEE 802.17 is a new standard for Metropolitan Area Networks (MANs). One of RPR's characteristics is that it can support three priorities traffic in a single datapath, i.e., class A, class B and class C, ranging from high priority to low priority, respectively. Different entities such as shaping, scheduling, fairness, topology and protection coordinate to guarantee the Quality of Service (QoS) for different services. Various pieces of the datapath in RPR are tied together through logical queues, thus we investigate the datapath from the view of logical queues in this paper. With a detailed analysis of the MAC shaping mechanism in RPR, we propose some improvement to achieve better transport performance for RPR's three priorities traffic. Simulation results show that our improvement is efficient.

  7. Crystal quality analysis and improvement using x-ray topography.

    Energy Technology Data Exchange (ETDEWEB)

    Maj, J.; Goetze, K.; Macrander, A.; Zhong, Y.; Huang, X.; Maj, L.; Univ. of Chicago

    2008-01-01

    The Topography X-ray Laboratory of the Advanced Photon Source (APS) at Argonne National Laboratory operates as a collaborative effort with APS users to produce high performance crystals for APS X-ray beamline experiments. For many years the topography laboratory has worked closely with an on-site optics shop to help ensure the production of crystals with the highest quality, most stress-free surface finish possible. It has been instrumental in evaluating and refining methods used to produce high quality crystals. Topographical analysis has shown to be an effective method to quantify and determine the distribution of stresses, to help identify methods that would mitigate the stresses and improve the Rocking curve, and to create CCD images of the crystal. This paper describes the topography process and offers methods for reducing crystal stresses in order to substantially improve the crystal optics.

  8. 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.

  9. 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.

  10. Skill Gap Analysis for Improved Skills and Quality Deliverables

    Directory of Open Access Journals (Sweden)

    Mallikarjun Koripadu

    2014-10-01

    Full Text Available With a growing pressure in identifying the skilled resources in Clinical Data Management (CDM world of clinical research organizations, to provide the quality deliverables most of the CDM organizations are planning to improve the skills within the organization. In changing CDM landscape the ability to build, manage and leverage the skills of clinical data managers is very critical and important. Within CDM to proactively identify, analyze and address skill gaps for all the roles involved. In addition to domain skills, the evolving role of a clinical data manager demands diverse skill sets such as project management, six sigma, analytical, decision making, communication etc. This article proposes a methodology of skill gap analysis (SGA management as one of the potential solutions to the big skill challenge that CDM is gearing up for bridging the gap of skills. This would in turn strength the CDM capability, scalability, consistency across geographies along with improved productivity and quality of deliverables

  11. KARAKTERISTIK SEKUEN cDNA PENGKODE GEN ANTI VIRUS DARI UDANG WINDU, Penaeus monodon

    Directory of Open Access Journals (Sweden)

    Andi Parenrengi

    2016-11-01

    Full Text Available Transgenesis pada ikan merupakan sebuah teknik modern yang berpotensi besar dalam menghasilkan organisme yang memiliki karakter lebih baik melalui rekombinan DNA gen target termasuk gen anti virus dalam peningkatan resistensi pada udang. Gen anti virus PmAV (Penaeus monodon Anti Viral gene merupakan salah satu gen pengkode anti virus yang berasal dari spesies krustase. Penelitian ini dilakukan untuk mengetahui karakteristik gen anti virus yang diisolasi dari udang windu, Penaeus monodon. Isolasi gen anti virus menggunakan metode Polymerase Chain Reaction (PCR dan selanjutnya dipurifikasi untuk sekuensing. Data yang dihasilkan dianalisis dengan program Genetyx Versi 7 dan basic local alignment search tool (BLAST. Hasil penelitian menunjukkan bahwa gen anti virus PmAV yang berhasil diisolasi dari cDNA udang windu dengan panjang sekuen 520 bp yang mengkodekan 170 asam amino. BLAST-N menunjukkan tingkat similaritas yang sangat tinggi (100% dengan gen anti virus yang ada di GeneBank. Komposisi asam amino penyusun gen anti virus yang paling besar adalah serin (10,00%, sedangkan yang terkecil adalah asam amino prolin dan lisin masing-masing 1,76%. Analisis sekuen gen dan deduksi asam amino (BLAST-P memperlihatkan adanya C-type lectin-like domain (CTLD yang memiliki kemiripan dengan gen C-type lectin yang diisolasi dari beberapa spesies krustase. Transgenic fish technology is a potential modern technique in producing better character organism through DNA recombinant of target genes including anti viral gene for improvement of shrimp immunity. PmAV (Penaeus monodon Anti Viral gene is one of anti viral genes isolated from crustacean species. The research was conducted to analyze the characteristics anti viral gene isolated from tiger prawn, Penaeus monodon. Anti viral gene was isolated using Polymerase Chain Reaction (PCR technique and then purified for sequencing. Data obtained were analyzed using Genetyx Version 7 software and basic local alignment

  12. Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States); Kaur, G.P. [Temple Univ. Medical School, Philadelphia, PA (United States)] [and others

    1997-05-01

    We have isolated and sequenced a cDNA that encodes an apparent human orthologue of a rat sulfotransferase (ST) cDNA that has been referred to as {open_quotes}ST1C1{close_quotes} - although it was recently recommended that sulfotransferase proteins and cDNAs be abbreviated {open_quotes}SULT.{close_quotes} The new human cDNA was cloned from a fetal liver-spleen cDNA library and had an 888-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 62% identical with that encoded by the rat ST1C1 cDNA and included signature sequences that are conserved in all cytosolic SULT enzymes. Dot blot analysis of mRNA from 50 human tissues indicated that the cDNA was expressed in adult human stomach, kidney, and thyroid, as well as fetal kidney and liver. Northern blot analyses demonstrated that the major SULT1C1 mRNA in those same tissues was 1.4 kb in length. We next determined the partial human SULT1C1 gene sequence for a portion of the 5{prime}-terminus of one intron. That sequence was used to design SULT1C1 gene-specific primers that were used to perform the PCR with DNA from human/rodent somatic cell hybrids to demonstrate that the gene was located on chromosome 2. PCR amplifications performed with human chromosome 2/rodent hybrid cell DNA as template sublocalized SULT1C1 to a region between bands 2q11.1 and 2q11.2. 14 refs., 2 figs.

  13. 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.

  14. Modification of pGH cDNA using the first intron and adenovirus-mediated expression in CHO cells

    Institute of Scientific and Technical Information of China (English)

    李秀锦; 仲飞; 齐顺章

    2003-01-01

    Objective This study was conducted to investigate the function of the first intron of porcine growth hormone (pGH) gene in the gene expression.Methods PCR method was used to amplify the first intron from pig genomic DNA. The intron was then inserted into pGH cDNA to construct pGH cDNA-intron (pGH cDNA-in). The recombinant adenoviruses containing pGH cDNA and pGH cDNA-in genes under control of CMV promoter were generated by homologous recombination method in HEK 293 cells respectively. The effect of the first intron on gene expression was evaluated by comparing the expression levels of pGH cDNA-in and pGH cDNA mediated by adenovirus vectors in CHO cells.Results The expression level of pGH cDNA containing the first intron increased by 117%, which was significantly higher than that of pGH cDNA without the intron (P<0.001). Conclusion The first intron of pGH gene has the function to improve pGH gene expression.

  15. An Efficient and Configurable Preprocessing Algorithm to Improve Stability Analysis.

    Science.gov (United States)

    Sesia, Ilaria; Cantoni, Elena; Cernigliaro, Alice; Signorile, Giovanna; Fantino, Gianluca; Tavella, Patrizia

    2016-04-01

    The Allan variance (AVAR) is widely used to measure the stability of experimental time series. Specifically, AVAR is commonly used in space applications such as monitoring the clocks of the global navigation satellite systems (GNSSs). In these applications, the experimental data present some peculiar aspects which are not generally encountered when the measurements are carried out in a laboratory. Space clocks' data can in fact present outliers, jumps, and missing values, which corrupt the clock characterization. Therefore, an efficient preprocessing is fundamental to ensure a proper data analysis and improve the stability estimation performed with the AVAR or other similar variances. In this work, we propose a preprocessing algorithm and its implementation in a robust software code (in MATLAB language) able to deal with time series of experimental data affected by nonstationarities and missing data; our method is properly detecting and removing anomalous behaviors, hence making the subsequent stability analysis more reliable.

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

    OpenAIRE

    2008-01-01

    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 synt...

  17. 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.

  18. Improved environmental multimedia modeling and its sensitivity analysis.

    Science.gov (United States)

    Yuan, Jing; Elektorowicz, Maria; Chen, Zhi

    2011-01-01

    Modeling of multimedia environmental issues is extremely complex due to the intricacy of the systems with the consideration of many factors. In this study, an improved environmental multimedia modeling is developed and a number of testing problems related to it are examined and compared with each other with standard numerical and analytical methodologies. The results indicate the flux output of new model is lesser in the unsaturated zone and groundwater zone compared with the traditional environmental multimedia model. Furthermore, about 90% of the total benzene flux was distributed to the air zone from the landfill sources and only 10% of the total flux emitted into the unsaturated, groundwater zones in non-uniform conditions. This paper also includes functions of model sensitivity analysis to optimize model parameters such as Peclet number (Pe). The analyses results show that the Pe can be considered as deterministic input variables for transport output. The oscillatory behavior is eliminated with the Pe decreased. In addition, the numerical methods are more accurate than analytical methods with the Pe increased. In conclusion, the improved environmental multimedia model system and its sensitivity analysis can be used to address the complex fate and transport of the pollutants in multimedia environments and then help to manage the environmental impacts.

  19. Identification and Expression Analysis of a Full-length cDNA Encoding a Kandelia candel Tonoplast Intrinsic Protein%红树植物秋茄中液泡膜内在蛋白(TIP)全长cDNA的克隆和表达分析

    Institute of Scientific and Technical Information of China (English)

    黄薇; 方孝东; 林栖凤; 李冠一; 赵文明

    2003-01-01

    Soil salinity is an important issue, as most crop plants are low in salt tolerance. Salt tolerance, a complex, multifactorial, and multigenic process, has been known to be a quantitative trait. The identification of the salt stress responsive genes or salt tolerance genes is essential for the breeding programs. Most recent efforts have been focused on the products of structural genes (transport proteins, ion channels, enzymes of solute synthesis) while little attention were paid to the regulatory aspects of these proteins. Since the first aquaporin gene from plants was cloned and functionally expressed in 1993, there has been a growing interest in the molecular biology of MIPs (membrane intrinsic proteins) and their bearing on the biophysics of water flow across plant membranes. In the last decades, studies on Mangroves, a special kind of wood plants, grow in high-salt and flooding conditions have been concentrated almost exclusively on their physiological and ecological characteristics. Kandelia candel, one of the dominant species of mangroves along the Chinese coast, lacks salt glands or salt hairs used for removal of excess salt in other mangroves. This makes K. Candel a perfect model to study the molecular mechanism of salt tolerance in mangrove plants. Using cDNA RDA, a cDNA-specific modification of genomic representational difference analysis, a series of salt responsive genes of Kandelia candel were cloned. Among these gene fragments, a 183 bp fragment (termed as SRGKC1) encoding a tonoplast intrinsic protein (TIP) in Kandelia candel (KCTIP1) was identified. Based on the sequence of SRGKC1, two gene specific primers were designed, and the 3′ and 5′end of the KCTIP1 gene were obtained using the SMARTTM RACE cDNA Amplification Kit. RACE products were purified from low-melting agarose, and sequenced directly with GSPs as the sequencing primers. A 500-bp fragment corresponding to the 3′end of this gene was obtained using the GSP1 primer, and a 690 bp

  20. 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.

  1. Construction of cDNA Library of Pyrocystis lunula(Pyrophyta)

    Institute of Scientific and Technical Information of China (English)

    SUI Zhenghong; Klaus V.Kowallik

    2004-01-01

    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 20 μg g-1 net cells, and the band intensity ratio of 28 S/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.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR......Detection of reverse transcriptase termination sites is important in many different applications, such as structural probing of RNAs, rapid amplification of cDNA 5' ends (5' RACE), cap analysis of gene expression, and detection of RNA modifications and protein-RNA cross-links. The throughput...... that do not require formal bioinformatics training. As an example, we apply the method to detection of transcription start sites in mouse liver cells....

  3. Molecular cloning of a cDNA related to vernalization(verc203) in winter wheat

    Institute of Scientific and Technical Information of China (English)

    种康; 谭克辉; 黄华梁; 梁厚果

    1995-01-01

    A cDNA clone related to the vernalization in winter wheat(verc203)was harvested from the en-riched cold-induced cDNA library of 10~4 pfu with differential screening.The insert of verc203 in λ gt10 vector wassubcloned into the sites between BamH Ⅰ and Hind Ⅲ in pUC19 plasmid after being amplified with PCR.the analysis of the Northern blotting with a probe of verc203 indicated that the verc203 has a negative signalfor the control and the devernalized mRNA and a positive signal for the vernalized winter wheat and non-vernalized spring wheat at about 2.6 kb.

  4. Identification of brassinosteroid responsive genes in Arabidopsis by cDNA array

    Institute of Scientific and Technical Information of China (English)

    胡玉欣; 汪政科; 王永红; 包方; 李凝; 彭镇华; 李家洋

    2001-01-01

    We have systematically monitored brassinosteroid (BR) responsive genes in a BR-deficient mutant det2 suspension culture of Arabidopsis by using a cDNA array approach. Among 13000 cDNA clones arrayed on filters, 53 BR responsive clones were identified and designated BRR1-BRR53. Sequence analysis of 43 clones showed that 19 clones are novel genes, 3 clones are genes involved in the control of cell division, 4 clones are genes related to plant stress responses, 4 clones are transcriptional factor or signal transduction component genes, and 3 clones are genes involved in RNA splicing or structure forming. In addition, we also found that BR regulated the transcription of genes related to many physiological processes, such as photoreaction, ion transportation and some metabolic processes. These findings present molecular evidence that BR plays an essential role in plant growth and development.

  5. 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.

  6. Requirements in screening cDNA libraries for new genes and solutions offered by SBH technology

    Energy Technology Data Exchange (ETDEWEB)

    Drmanac, R.; Drmanac, S.; Labat, I.; Stavropoulos, N.

    1993-12-31

    Under different assumptions about the total number of genes, the number of housekeeping and tissue-specific genes, and the difference in the number of mRNAs per cell for functional and nonfunctional genes, significantly different results can be expected from screening random cDNA clones. We have developed gene expression models as a guide for interpretation of experimental results. For statistical, biological, and technical reasons, the search for 100,000 plus genes and discrimination between nonfunctional, housekeeping, and tissue-specific genes requires the analysis of up to 10 million clones from 20 to 50 tissues. Oligonucleotide hybridization of dense clone blots is an inexpensive and fast way to screen such large clone sets. Our preliminary results on control clones and thousands of cDNA clones from an infant brain library demonstrate the feasibility of the method. We present several models of gene expression and analyze the main factors which can influence the hunt for new genes via the screening of random cDNA libraries. The basic steps in the preparation and use of dense DNA dot arrays are described, and some results that demonstrate the feasibility and efficiency of gene inventorying by oligonucleotide hybridization are presented. Furthermore, partial SBH and single-pass gel sequencing are compared and a gene analysis scheme that combines the two approaches is discussed.

  7. cDNA heterogeneity suggests structural variants related to the high-affinity IgE receptor.

    Science.gov (United States)

    Liu, F T; Albrandt, K; Robertson, M W

    1988-08-01

    The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, we identified a number of cDNA clones from a rat basophilic leukemia cell cDNA library. Nucleotide sequencing established four different forms of cDNA: one is nearly identical to the published cDNA; the second differs from the first in the 5' untranslated sequence; the other two forms use either one or the other of the 5'-end sequences as above and lack 163 base pairs in the region coding for the second extracellular domain. RNase protection analysis with radioactive RNA probes established the heterogeneity of rat basophilic leukemia cell mRNA with regard to both the 5' and the internal sequences. Our results suggest the existence of at least four different protein forms related to the alpha subunit of the high-affinity IgE receptor.

  8. 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.

  9. 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)

  10. 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.

  11. 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.

  12. Improving Credit Scorecard Modeling Through Applying Text Analysis

    Directory of Open Access Journals (Sweden)

    Omar Ghailan

    2016-04-01

    Full Text Available In the credit card scoring and loans management, the prediction of the applicant’s future behavior is an important decision support tool and a key factor in reducing the risk of Loan Default. A lot of data mining and classification approaches have been developed for the credit scoring purpose. For the best of our knowledge, building a credit scorecard by analyzing the textual data in the application form has not been explored so far. This paper proposes a comprehensive credit scorecard model technique that improves credit scorecard modeling though employing textual data analysis. This study uses a sample of loan application forms of a financial institution providing loan services in Yemen, which represents a real-world situation of the credit scoring and loan management. The sample contains a set of Arabic textual data attributes defining the applicants. The credit scoring model based on the text mining pre-processing and logistic regression techniques is proposed and evaluated through a comparison with a group of credit scorecard modeling techniques that use only the numeric attributes in the application form. The results show that adding the textual attributes analysis achieves higher classification effectiveness and outperforms the other traditional numerical data analysis techniques.

  13. Simple and inexpensive three-step rapid amplification of cDNA 5′ ends using 5′ phosphorylated primers

    OpenAIRE

    Dallmeier, Kai; Neyts, Johan

    2013-01-01

    Rapid amplification of cDNA 5′ ends (5′-RACE) is routinely used for the sequence analysis of the upstream noncoding regions of cellular mRNAs; however, it represents a tedious and cost-intensive procedure. By employing 5′ phosphorylated gene-specific primers for first-strand cDNA synthesis, we cut short the previously established reverse ligation and amplification protocol of Mandl and coworkers (BioTechniques, 1991, vol. 10, pp. 484–486) to a streamlined three-step procedure that no longer d...

  14. Construction of Midgut Tissue-Specific cDNA Library of Bombyx mandarina M. and Isolation and Sequence Analysis of Serine Protease Gene Fragment%野桑蚕中肠组织cDNA文库的构建及丝氨酸蛋白酶基因片段的克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    王燕红; 李兵; 王东; 朱莎; 赵华强; 卫正国; 沈卫德

    2008-01-01

    [Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion.

  15. 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.

  16. Rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Yeku, Oladapo; Frohman, Michael A

    2011-01-01

    Rapid Amplification of cDNA ends (RACE) provides an inexpensive and powerful tool to quickly obtain full-length cDNA when the sequence is only partially known. Starting with an mRNA mixture, gene-specific primers generated from the known regions of the gene and non-specific anchors, full-length sequences can be identified in as little as 3 days. RACE can also be used to identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5(') and 3(') cDNA ends using the "new RACE" technique.

  17. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    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.

  19. Improved Analysis for Graphic TSP Approximation via Matchings

    CERN Document Server

    Mucha, Marcin

    2011-01-01

    The Travelling Salesman Problem is one the most fundamental and most studied problems in approximation algorithms. For more than 30 years, the best algorithm known for general metrics has been Christofides's algorithm with approximation factor of 3/2, even though the so-called Held-Karp LP relaxation of the problem is conjectured to have the integrality gap of only 4/3. Very recently, significant progress has been made for the important special case of graphic metrics, first by Oveis Gharan et al., and then by Momke and Svensson. In this paper, we provide an improved analysis for the approach introduced by Momke and Svensson yielding a bound of 35/24 on the approximation factor, as well as a bound of 19/12+epsilon for any epsilon>0 for a more general Travelling Salesman Path Problem in graphic metrics.

  20. Improving knowledge management systems with latent semantic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Sebok, A.; Plott, C. [Alion Science and Technology, MA and D Operation, 4949 Pearl East Circle, Boulder, CO 80301 (United States); LaVoie, N. [Pearson Knowledge Technologies, 4940 Pearl East Circle, Boulder, CO 80301 (United States)

    2006-07-01

    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)

  1. [The analysis of tincture for improvement of blood circulation].

    Science.gov (United States)

    Bernatoniene, Rūta; Bernatoniene, Jurga; Ramanauskiene, Kristina

    2004-01-01

    The article describes qualitative and quantitative analysis of tincture for improvement of blood circulation. Flavonoids have been qualitatively determined by methods of thin-layer chromatography, high-pressure liquid chromatography and color reaction with magnesium powder and concentrated hydrochloric acid. Ferments were identified with ferric ammonium sulphate solution; glycosides--with dimethylaminobenzaldehyde solution in sulphur acid; saponin--with lead subacetate solution; reductive materials - with silver nitrate ammoniacal solution; albumen--with ninhydrin solution; and caffeic acid--by method of thin-layer chromatography. An optimal spectrophotometrical method for determining the quantity of quercetin was applied. The refractive index, relative density, loss of drying and ethanol concentration were determined according to European Pharmacopoeia Requirements.

  2. Improving Semantic Search in Digital Libraries Using Multimedia Analysis

    Directory of Open Access Journals (Sweden)

    Ilianna Kollia

    2012-04-01

    Full Text Available Semantic search of cultural content is of major importance in current digital libraries, such as in Europeana. Content metadata constitute the main features of cultural items that are analysed, mapped and used to interpret users' queries, so that the most appropriate content is selected and presented to the users. Multimedia, especially visual, analysis, has not been a main component in these developments. This paper presents a new semantic search methodology, including a query answering mechanism which meets the semantics of users' queries and enriches the answers by exploiting appropriate visual features, both local and MPEG-7, through an interweaved knowledge and machine learning based approach. An experimental study is presented, using content from the Europeana digital library, and involving both thematic knowledge and extracted visual features from Europeana images, illustrating the improved performance of the proposed semantic search approach.

  3. Benchmarking Of Improved DPAC Transient Deflagration Analysis Code

    Energy Technology Data Exchange (ETDEWEB)

    Laurinat, James E.; Hensel, Steve J.

    2013-03-21

    The transient deflagration code DPAC (Deflagration Pressure Analysis Code) has been upgraded for use in modeling hydrogen deflagration transients. The upgraded code is benchmarked using data from vented hydrogen deflagration tests conducted at the HYDRO-SC Test Facility at the University of Pisa. DPAC originally was written to calculate peak deflagration pressures for deflagrations in radioactive waste storage tanks and process facilities at the Savannah River Site. Upgrades include the addition of a laminar flame speed correlation for hydrogen deflagrations and a mechanistic model for turbulent flame propagation, incorporation of inertial effects during venting, and inclusion of the effect of water vapor condensation on vessel walls. In addition, DPAC has been coupled with CEA, a NASA combustion chemistry code. The deflagration tests are modeled as end-to-end deflagrations. The improved DPAC code successfully predicts both the peak pressures during the deflagration tests and the times at which the pressure peaks.

  4. Analysis and improvement measures of flight delay in China

    Science.gov (United States)

    Zang, Yuhang

    2017-03-01

    Firstly, this paper establishes the principal component regression model to analyze the data quantitatively, based on principal component analysis to get the three principal component factors of flight delays. Then the least square method is used to analyze the factors and obtained the regression equation expression by substitution, and then found that the main reason for flight delays is airlines, followed by weather and traffic. Aiming at the above problems, this paper improves the controllable aspects of traffic flow control. For reasons of traffic flow control, an adaptive genetic queuing model is established for the runway terminal area. This paper, establish optimization method that fifteen planes landed simultaneously on the three runway based on Beijing capital international airport, comparing the results with the existing FCFS algorithm, the superiority of the model is proved.

  5. 大鲵皮肤cDNA文库ESTs分析及Dynll2基因的分离与表达%Construction of cDNA library of Andrias davidianus skin tissue and molecular cloning and expression analysis of Dynll 2 gene

    Institute of Scientific and Technical Information of China (English)

    王立新; 郑尧; 李锋刚; 李雯娟; 刘小林

    2011-01-01

    Dyneins are a group of evolutionarily highly conservative molecular proteins, which can be divided into two groups: cytoplasmic dyneins and axonemal dyneins. Cytoplasmic dynein probably moves along the microtubule essential for transporting cargo in eukaryotes. It is also probably involved in the movement of chromosomes and positioning the mitotic spindles for cell division. Dynein light chain 2, cytoplasmic, is a protein that in humans is encoded by the Dynll 2 gene. To clone dynein light chain, LC8-type 2 (Dynll 2) gene from A. davidianus, and to analyze the characteristics of the functional gene by bioinformatics analysis,the structure of Dynll 2 gene was isolated from skin cDNA library. A cDNA library of the skin tissue was constructed by using the isolated mRNA as the template during reverse transcription. The skin cDNA library of A. davidianus were detected and sequenced by picking clones randomly. It was confirmed that the titer of the skin cDNA library of A. davidianus was 1. 50 x 106 cfu ,the recombination rate was 94.8% ,and the PCR results showed that the average size of inserts segment was about 1 000 bp. A total of 343 ESTs from the library were sequenced and made alignment with sequences in GenBank database. Moreover,at least 214 clones (E-value < 10 -6) derived from these identified clones were categorized into nine categories. Immune-related genes accounted for 31. 3% of the largest distribution, metabolism genes (14.0% ),cytoskeleton genes( 13.0% )and signaling pathway-related genes( 12.6% ) took up a larger gene distribution though. It revealed that skin of A. davidianus made intense activities in the form of secretions and took part in metabolism reactions, according to their physiology function in the area of immunity, breathing, and osmotic pressure equilibrium. In order to investigate the contribution of Dynll 2 to motor protein in A. davidianus, molecular cloning, analyzing cDNA sequence and expression analysis of Dynll 2 gene from A

  6. The NASA aircraft noise prediction program improved propeller analysis system

    Science.gov (United States)

    Nguyen, L. Cathy

    1991-01-01

    The improvements and the modifications of the NASA Aircraft Noise Prediction Program (ANOPP) and the Propeller Analysis System (PAS) are described. Comparisons of the predictions and the test data are included in the case studies for the flat plate model in the Boundary Layer Module, for the effects of applying compressibility corrections to the lift and pressure coefficients, for the use of different weight factors in the Propeller Performance Module, for the use of the improved retarded time equation solution, and for the effect of the number grids in the Transonic Propeller Noise Module. The DNW tunnel test data of a propeller at different angles of attack and the Dowty Rotol data are compared with ANOPP predictions. The effect of the number of grids on the Transonic Propeller Noise Module predictions and the comparison of ANOPP TPN and DFP-ATP codes are studied. In addition to the above impact studies, the transonic propeller noise predictions for the SR-7, the UDF front rotor, and the support of the enroute noise test program are included.

  7. 鸭梨多酚氧化酶基因cDNA全长的克隆和生物信息学分析%Cloning and Bioinformatics Analysis for the Full Length cDNA Sequence of PPO Gene in Yali

    Institute of Scientific and Technical Information of China (English)

    李桂琴; 齐靖; 闫洪波; 高志华

    2012-01-01

    In order to investigate the molecular structures of polyphenol oxidase (PPO) in Yali, the author designed series of primers based on the known cDNA fragment of PPO gene in Yali to clone the unknown 3' and 5' cDNA terminal sequence for this gene by using RACE method. With the total RNA extracted from fruit of Yali as the template, the author finally obtained the full length cDNA sequence of PPO gene in Yali, which was 2126 bp in length, and has a open reading frame between 136-1917 bp, in which 276 predicted amino acid residues were encoded. Research the deduce amino acids of PPO gene from Yali by bioinformatics analysis, it showed that the PPO in Yali was a typical hydrophilic soluble protein which belonged to tyrosinase super family, and it did not contain distinct transmembrane domain in tertiary structure, so the Yali PPO should located in thylakoid lume of plastid. That information laid a good foundation for regulating the enzyme activity of PPO in Yali by biological technology and breeding a new cultivar of pear with browning resistance in the future.%为了从分子水平深入了解鸭梨多酚氧化酶结构特点,基于已知的鸭梨多酚氧化酶基因cDNA片段序列设计引物,以鸭梨果实总mRNA为模板,对该基因cDNA 5’端和3’端未知序列进行了RACE扩增,最终获得鸭梨多酚氧化酶基因cDNA全长序列.该序列长度为2126 bp,开放性读码框位于136~1917 bp之间,可编码593个氨基酸残基.对该基因所翻译的氨基酸序列进行生物信息学分析,结果表明基因编码的多酚氧化酶属酪氨酸酶超级家族成员,三级空间结构为可溶性球状蛋白,不具备跨膜结构,在细胞中的定位应该位于类囊体腔中.这些生物信息的获得将为今后应用生物技术开展鸭梨多酚氧化酶的调控,从而抑制鸭梨果实褐变提供十分重要的参考资料.

  8. 重症肌无力患者胸腺组织抑制性消减cDNA文库的构建及分析%Construction and analysis of suppression subtractive cDNA library of thymus from myasthenia gravis patients

    Institute of Scientific and Technical Information of China (English)

    杨俊红; 崔新征; 方华; 关兵峰; 赵国强; 张清勇; 高峰

    2012-01-01

    Aim:To build suppression subtractive cDNA library of thymus from myasthenia gravis (MG) patients.Methods:The total mRNA was extracted from thymus of normal people and MG patients and synthethized to cDNA by reverse transcription.The differentially expressed genes obtained by using suppression subtractive hybridization were proceeded by T-A cloning and sequence analysis.The expressions of GSTM3 and KPNA5 in thymus of 20 MG patients and 10 normal people were detected by real-time quantitative PCR.Results:A total of 125 positive clones were obtained;27 differentially expressed genes were obtained by Blast homology search.The expression levels of GSTM3, KPNA5 in thymus tissue of MG patients(0.671±0.097,0.712±0.080)were significantly higher than those of the control group (0.582±0.047,0.571±0.018)(tGSTM3=5.458,P<0.001;tKPNA5=2.755,P=0.010),consistent with the result of subtractive library.Conclusion:The establishment of suppression subtractive cDNA library of MG thymus is successful.%目的:构建重症肌无力(MG)患者胸腺组织抑制性消减cDNA文库,分析MG胸腺差异表达基因.方法:分别从6例正常和6例MG患者胸腺组织中分离出mRNA,逆转录合成cDNA,行抑制性消减杂交,将得到的差异表达基因进行T-A克隆,构建cDNA文库,并进行序列分析.应用实时荧光定量PCR检测20例MG患者、10例正常对照胸腺组织中GSTM3和KPNA5 mRNA的表达.结果:共获得125个阳性克隆;Blast同源性检索共得到27个差异表达基因;MG患者GSTM3、KPNA5基因表达量(0.671±0.097和0.712±0.080)高于正常对照胸腺(0.582±0.047和0.571±0.018),差异有统计学意义(tGSTM3=5.458,P<0.001;tKPNA5=2.755,P=0.010),与消减文库结果一致.结论:成功建立了MG胸腺组织抑制性消减cDNA文库.

  9. Full-length cDNA Cloning and Tissue Expression Analysis of IRAK-4 Gene from Humphead Snapper (Lutjanus sanguineus)%红笛鲷IRAK-4基因cDNA全长的克隆及组织表达分析

    Institute of Scientific and Technical Information of China (English)

    黄郁葱; 鲁义善; 简纪常; 吴灶和

    2015-01-01

    Interleukin-1 receptor-associated kinase 4 (IRAK-4) is a key molecular which participates in the innate immune and adaptive immune processes. In this paper, full length cDNA sequence of IRAK-4 gene was amplified by RT-PCR and RACE PCR from head kidney of humphead snapper, Lutjanus sanguineus (GenBank accession number:KF279357). The total cDNA sequence of humphead snapper IRAK-4 was 2 015 bp, including 3' UTR of 421 bp,5' UTR of 205 bp,an open reading frame (ORF) of 1 389 bp encoding 462 amino acids with Molecule Mass of 52.0 ku and pI of 5.19. The deduced amino acid sequence of humphead snapper IRAK-4 shared 54.2%-85.7% identities with other species IRAK-4. Phylogenetic analysis showed that humphead snapper was clustered closely with orange-spotted grouper. In addition, the mRNA expression levels in different tissues were analyzed by real time quantitative PCR. The result showed that humphead snapper IRAK-4 expressed in all examined tissues with highest levels in skin, liver and stomach, moderate levels in thymus, gill, heart, intestine, muscle and spleen, and lowest levels in head kidney, kidney and brain.%白细胞介素-1受体相关激酶4( interleukin-1 receptor-associated kinase 4,IRAK-4)是一种参与机体先天性免疫和适应性免疫反应过程中的关键分子。采用RT-PCR和cDNA末端快速扩增(RACE-PCR)的方法从红笛鲷(Lutjanus sanguineus)头肾中克隆 IRAK-4基因的 cDNA 全序列(登录号:KF279357)。该序列全长2015 bp,包含5′非编码区(5′UTR)205 bp,3′非编码区(3′UTR)421 bp,开放阅读框(ORF)1389 bp,编码462个氨基酸。根据推导的氨基酸序列预测其蛋白分子质量为52.0 ku,理论等电点为5.19。氨基酸序列比对结果显示,红笛鲷IRKA-4基因氨基酸序列与其他物种的同源性为54.2%~85.7%。系统进化分析结果显示,红笛鲷与斜带石斑鱼(Epinephelus coioides)聚为一支,两者有较近的亲

  10. Development and validation of a bovine macrophage specific cDNA microarray

    Directory of Open Access Journals (Sweden)

    Waddington David

    2006-09-01

    Full Text Available Abstract Background The response of macrophages to danger signals is an important early stage in the immune response. Our understanding of this complex event has been furthered by microarray analysis, which allows the simultaneous investigation of the expression of large numbers of genes. However, the microarray resources available to study these events in livestock animals are limited. Results Here we report the development of a bovine macrophage specific (BoMP cDNA microarray. The BoMP microarray contains 5026 sequence elements (printed in duplicate and numerous controls. The majority of the clones incorporated on the microarray were derived from the BoMP cDNA library generated from bovine myeloid cells subjected to various stimuli, including over 900 sequences unique to the library. Additional clones representing immunologically important genes have been included on the BoMP microarray. The microarray was validated by investigating the response of bovine monocytes to stimulation with interferon-γ and lipopolysaccharide using amplified RNA. At 2 and 16 hours post stimulation 695 genes exhibited statistically significant differential expression, including; 26 sequences unique to the BoMP library, interleukin 6, prion protein and toll-like receptor 4. Conclusion A 5 K cDNA microarray has been successfully developed to investigate gene expression in bovine myeloid cells. The BoMP microarray is available from the ARK-Genomics Centre for Functional Genomics in Farm Animals, UK.

  11. cDNA cloning and expression of an apoptosis-related gene, human TFAR15 gene

    Institute of Scientific and Technical Information of China (English)

    王玉刚; 刘洪涛; 张颖妹; 马大龙

    1999-01-01

    By means of cDNA-RDA method. some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these tragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1218 nueleotides and encodes 212 amino acids. The putative protein protein product of TFAR15 is partially homologous to C. elegans protein C14A4. 11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung. and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-(?) cells after GM-CSF withdrawal. In vitro analysis showed that the recombinant TFAR15 protein co(?)ld inhibit the natural cell death of 293 cells, an embryonic kidney cell

  12. Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu.

    Science.gov (United States)

    Wee, Ching Ching; Roslan, Hairul Azman

    2012-01-01

    Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group.

  13. Voxel model in BNCT treatment planning: performance analysis and improvements

    Science.gov (United States)

    González, Sara J.; Carando, Daniel G.; Santa Cruz, Gustavo A.; Zamenhof, Robert G.

    2005-02-01

    In recent years, many efforts have been made to study the performance of treatment planning systems in deriving an accurate dosimetry of the complex radiation fields involved in boron neutron capture therapy (BNCT). The computational model of the patient's anatomy is one of the main factors involved in this subject. This work presents a detailed analysis of the performance of the 1 cm based voxel reconstruction approach. First, a new and improved material assignment algorithm implemented in NCTPlan treatment planning system for BNCT is described. Based on previous works, the performances of the 1 cm based voxel methods used in the MacNCTPlan and NCTPlan treatment planning systems are compared by standard simulation tests. In addition, the NCTPlan voxel model is benchmarked against in-phantom physical dosimetry of the RA-6 reactor of Argentina. This investigation shows the 1 cm resolution to be accurate enough for all reported tests, even in the extreme cases such as a parallelepiped phantom irradiated through one of its sharp edges. This accuracy can be degraded at very shallow depths in which, to improve the estimates, the anatomy images need to be positioned in a suitable way. Rules for this positioning are presented. The skin is considered one of the organs at risk in all BNCT treatments and, in the particular case of cutaneous melanoma of extremities, limits the delivered dose to the patient. Therefore, the performance of the voxel technique is deeply analysed in these shallow regions. A theoretical analysis is carried out to assess the distortion caused by homogenization and material percentage rounding processes. Then, a new strategy for the treatment of surface voxels is proposed and tested using two different irradiation problems. For a parallelepiped phantom perpendicularly irradiated with a 5 keV neutron source, the large thermal neutron fluence deviation present at shallow depths (from 54% at 0 mm depth to 5% at 4 mm depth) is reduced to 2% on average

  14. Identification cDNA Cloning and Sequence Analysis on Maize Dwarf Mosaic Virus(MDMV)In Liaoning Province%辽宁省玉米矮花叶病毒原鉴定及cDNA克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    姜华; 陆敏; 安利佳; 韦石泉; 刘维志

    2001-01-01

    This is the report which treats of the identification,genetic cloning of the virus coat protein and sequence analysis of cDNA in Liaoning Province.Two specimens of Maize dwarf mosaic Diseased leaves has been collected,isolated and purified for the studies.The host ranges of these 2 isolates were all limited to live in the plant of Gramineae,transmited by sap;aphids(cotton aphid and peach aphid)and through seed transmission(only about 3%).The basic virus characterization and storage tests shown that:Dilution end-point(DEP)10-3~10-4;Thermal inactivation point(TIP)55~60℃and longevity in vitro(LIV)1~2days.The virus particles are filamentous form;it ’s size is about 430~750nm×13~15nm.There are conical pinwheels,ringlike inclusion bodies in the plant cytoplasm cells.After the centrifugation;the purified virus sap has tested,the violate absorbaece(A):Max.262nm and Min.245nm.A260/A280=1.2.The antiserum which made fromm these isolates,when tested with Beijing isolate(i.e.CK.virus,MDMV-B),and some other isolates,all shown postive reactions.Using virus PNA as template,through CP genetic sequence of MDMV-B,to synthesize the primer;by reverse transcription to synthesize cDNA.Again by cDNA for template to make PCR,then amplified about 1kb CP genetic fragment;using this fragment;using this fragment cloning to the carrier pUC19,after transformation in E.coli DH5a strain,obtained CP genetic clone.Through sequence analysis of cDNA shown that the homologous percentage was 98.7% to the CP genetic sequence of MDMV-B.Thus inferred by us,that the differences of amino acid ;it’s homologous percentage was 99.4%.According to the biological assay,serological test and cDNA sequence analysis of the above mentioned virus isolate,we suggested that the virus induced maize dwarf mosaic virus disease in Liaoning Province is Maize Dwarf Mosaic

  15. Generation and characterisation of a full-length cDNA encoding murine myotonic dystrophy protein kinase from cardiac tissue

    Energy Technology Data Exchange (ETDEWEB)

    Carey, N.; Tongeren, T. van; Winchester, C. [Charing Cross & Wesminster Medical School, London (United Kingdom)] [and others

    1994-09-01

    The mutation underlying myotonic dystrophy (DM) is a CTG trinucleotide expansion in the 3{prime} untranslated region of a putative protein kinase gene (DMPK). We report the isolation of a full-length cDNA clone of the murine (DMPK) gene from a heart cDNA library. Sequence analysis shows that the clone is a splice isoform which has only previously been identified in brain, suggesting that there may be some flexibility of the splicing pattern in some tissues. We are currently analyzing the library for the presence of other isoforms. The full-length cDNA has been cloned into a bacterial expression system and the expressed protein is being used as an immunogen to generate both polyclonal and monoclonal antisera. These reagents will allow the analysis of the intracellular targets of the DMPK. Subclones of the cDNA have been generated for use as in situ hybridization probes, allowing investigation of the normal patterns of expression of the gene and the differential expression of the protein isoforms. These data will be essential for deciding on a rational use of rare patient material and will provide the necessary baseline for the analysis of transgenic and {open_quotes}knock-out{close_quotes} mice.

  16. 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...

  17. Rescue of mumps virus from cDNA.

    Science.gov (United States)

    Clarke, D K; Sidhu, M S; Johnson, J E; Udem, S A

    2000-05-01

    A complete DNA copy of the genome of a Jeryl Lynn strain of mumps virus (15,384 nucleotides) was assembled from cDNA fragments such that an exact antigenome RNA could be generated following transcription by T7 RNA polymerase and cleavage by hepatitis delta virus ribozyme. The plasmid containing the genome sequence, together with support plasmids which express mumps virus NP, P, and L proteins under control of the T7 RNA polymerase promoter, were transfected into A549 cells previously infected with recombinant vaccinia virus (MVA-T7) that expressed T7 RNA polymerase. Rescue of infectious virus from the genome cDNA was demonstrated by amplification of mumps virus from transfected-cell cultures and by subsequent consensus sequencing of reverse transcription-PCR products generated from infected-cell RNA to verify the presence of specific nucleotide tags introduced into the genome cDNA clone. The only coding change (position 8502, A to G) in the cDNA clone relative to the consensus sequence of the Jeryl Lynn plaque isolate from which it was derived, resulting in a lysine-to-arginine substitution at amino acid 22 of the L protein, did not prevent rescue of mumps virus, even though an amino acid alignment for the L proteins of paramyxoviruses indicates that lysine is highly conserved at that position. This system may provide the basis of a safe and effective virus vector for the in vivo expression of immunologically and biologically active proteins, peptides, and RNAs.

  18. Achieving high throughput sequencing of a cDNA library utilizing an alternative protocol for the bench top next-generation sequencing system.

    Science.gov (United States)

    Wan, Minxi; Faruq, Junaid; Rosenberg, Julian N; Xia, Jinlan; Oyler, George A; Betenbaugh, Michael J

    2013-02-15

    The development of next-generation sequencing (NGS) technologies has provided novel tools for genome analysis and expression profiling. A high throughput cDNA sequencing method using a bench top next-generation sequencing system, GS Junior, is now available. Here, we used an alternative protocol to the standard method for generating the cDNA library. This protocol can decrease the number of processing steps to manipulate RNA when constructing a cDNA library from an RNA sample, and does not require mRNA isolation from total RNA. Thus it can decrease the risk of RNA degradation and the cost for preparing a cDNA library. Also, the efficiency of sequencing data obtained with this approach is comparable to the standard method as verified by sequencing characteristics and expression levels of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

  19. Improved Smoothed Analysis of the k-Means Method

    CERN Document Server

    Manthey, Bodo

    2008-01-01

    The k-means method is a widely used clustering algorithm. One of its distinguished features is its speed in practice. Its worst-case running-time, however, is exponential, leaving a gap between practical and theoretical performance. Arthur and Vassilvitskii (FOCS 2006) aimed at closing this gap, and they proved a bound of $\\poly(n^k, \\sigma^{-1})$ on the smoothed running-time of the k-means method, where n is the number of data points and $\\sigma$ is the standard deviation of the Gaussian perturbation. This bound, though better than the worst-case bound, is still much larger than the running-time observed in practice. We improve the smoothed analysis of the k-means method by showing two upper bounds on the expected running-time of k-means. First, we prove that the expected running-time is bounded by a polynomial in $n^{\\sqrt k}$ and $\\sigma^{-1}$. Second, we prove an upper bound of $k^{kd} \\cdot \\poly(n, \\sigma^{-1})$, where d is the dimension of the data space. The polynomial is independent of k and d, and w...

  20. Improving diagnostic criteria for Propionibacterium acnes osteomyelitis: a retrospective analysis.

    Science.gov (United States)

    Asseray, Nathalie; Papin, Christophe; Touchais, Sophie; Bemer, Pascale; Lambert, Chantal; Boutoille, David; Tequi, Brigitte; Gouin, François; Raffi, François; Passuti, Norbert; Potel, Gilles

    2010-07-01

    The identification of Propionibacterium acnes in cultures of bone and joint samples is always difficult to interpret because of the ubiquity of this microorganism. The aim of this study was to propose a diagnostic strategy to distinguish infections from contaminations. This was a retrospective analysis of all patient charts of those patients with >or=1 deep samples culture-positive for P. acnes. Every criterion was tested for sensitivity, specificity, and positive likelihood ratio, and then the diagnostic probability of combinations of criteria was calculated. Among 65 patients, 52 (80%) were considered truly infected with P. acnes, a diagnosis based on a multidisciplinary process. The most valuable diagnostic criteria were: >or=2 positive deep samples, peri-operative findings (necrosis, hardware loosening, etc.), and >or=2 surgical procedures. However, no single criterion was sufficient to ascertain the diagnosis. The following combinations of criteria had a diagnostic probability of >90%: >or=2 positive cultures + 1 criterion among: peri-operative findings, local signs of infection, >or=2 previous operations, orthopaedic devices; 1 positive culture + 3 criteria among: peri-operative findings, local signs of infection, >or=2 previous surgical operations, orthopaedic devices, inflammatory syndrome. The diagnosis of P. acnes osteomyelitis was greatly improved by combining different criteria, allowing differentiation between infection and contamination.

  1. Measuring number of fluorophores labelling cDNA strands, in solution, with Fluorescence Correlation Spectroscopy and photobleaching

    CERN Document Server

    Delon, Antoine; Lambert, Emeline; Lerbs-Mache, Silva; Mache, Régis; Derouard, Jacques; Motto-Ros, Vincent; Galland, Rémi

    2009-01-01

    We present different approaches that aim at determining, in solution, the brightness and the number of Alexa Fluor 647 molecules labelling the C bases of two sequences of cDNA, corresponding to two transcripts of different sizes, a short and a long transcript (123 and 306 base long, with 45 and 74 dCTP residues, respectively). In each case, the Alexa labeled bases have been incorporated during reverse-transcription. Two kinds of experiments have been performed and combined: photobleaching and Fluorescence Correlation Spectroscopy (together with the factorial cumulant analysis method). As a result, we show that the photobleaching cross-section of labelled cDNA strands is about half that of free Alexa in aqueous solution, while their brightness is about twice. The factorial cumulant analysis put into evidence the fact that the brightness of cDNA strands varies from molecule to molecule, due to the statistical distribution of the number of Alexa fluorophores labelling cDNA. Our measurements are consistent with a...

  2. The Arabian camel Camelus dromedarius heat shock protein 90α: cDNA cloning, characterization and expression.

    Science.gov (United States)

    Saeed, Hesham; Shalaby, Manal; Embaby, Amira; Ismael, Mohammad; Pathan, Akbar; Ataya, Farid; Alanazi, Mohammad; Bassiouny, Khalid

    2015-11-01

    Heat shock protein 90 (Hsp90) is a highly conserved ubiquitous molecular chaperone contributing to assisting folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. In the present study, a heat shock protein 90α full length coding cDNA was isolated and cloned from the Arabian one-humped camel by reverse transcription polymerase chain reaction (RT-PCR). The full length cDNA sequence was submitted to NCBI GeneBank under the accession number KF612338. The sequence analysis of the Arabian camel Hsp90α cDNA showed 2202bp encoding a protein of 733 amino acids with estimated molecular mass of 84.827kDa and theoretical isoelectric point (pI) of 5.31. Blast search analysis revealed that the C. dromedarius Hsp90α shared high similarity with other known Hsp90α. Comparative analyses of camel Hsp90α protein sequence with other mammalian Hsp90s showed high identity (85-94%). Heterologous expression of camel Hsp90α cDNA in E. coli JM109 (DE3) gave a fusion protein band of 86.0kDa after induction with IPTG for 4h.

  3. cDNA Cloning, Sequence Analysis and Tissue Specific Expression of Vasoactive Intestinal Peptide Type 1 Receptor (VIPR-1)in Quails%鹌鹑VIPR-1的克隆、序列特征和组织表达分析

    Institute of Scientific and Technical Information of China (English)

    周敏; 李莹; 沈栩; 徐海平; 张成广; 张细权

    2012-01-01

    [目的]对鹌鹑血管活性肠肽1型受体(vasoacitve intestinal peptide type 1 receptor,VIPR-1)cDNA全长基因进行克隆及分析,为鹌鹑的分子育种提供基础资料.[方法]通过比较基因组学,采用RT-PCR和RACE技术,获得鹌鹑了VIPR-1 cDNA全长序列;通过生物学软件对其核苷酸序列和氨基酸序列进行了比对;采用实时定量PCR方法检测了VIPR-1在8个组织中的表达.[结果]鹌鹑VIPR-1的cDNA全长2 427 bp,包含了1 341 bp的开放性阅读框,编码446个氨基酸;序列分析显示,克隆获得的鹌鹑VIPR-1编码区序列与鸡该编码区序列存在41个碱基的差异,造成4个氨基酸残基的不同;VIPR-1氨基酸序列与鸡、火鸡、斑胸草雀的氨基酸的一致性分别为99.1%、92.2%、88%,与其它物种的一致性在60%-78%;各物种VIPR-1蛋白进化树符合物种进化规律;VIPR-1理化性质表明该蛋白为一偏碱性蛋白,蛋白二级结构主要由α-螺旋、β-折叠和β-转角构成;在N-端存在一个由22个氨基酸残基(MKSARLRVLLPLLGCLLSAASS)组成的信号肽,7个α-螺旋构成的跨膜域和C-端结构域,在跨膜域有胆固醇结合位点;在所检测的8个组织中VIPR-1 mRNA均有表达,在小肠中表达量最高.[结论]成功地克隆了鹌鹑VIPR-1 cDNA全长序列,该基因在小肠组织的表达高于其它组织,在跨膜域存在胆固醇结合位点.%[Objective] Vasoactive intestinal peptide type 1 receptor (VIPR-1) plays an important role in poultry reproduction, but the gene and its analysis have not been reported in quail prior to this study. The objective of this study was to clone the cDNA of quail VIPR-1, analyze its sequence and tissue expression pattern. [Method] Based on comparative genomics, the cDNA sequence of quail VIPR-1 was obtained by RT-PCR and RACE PCR. The nucleotide and amino acid sequences were analyzed and compared with that of other species. The expression levels of VIPR-1 in eight quail tissues were detected by

  4. Molecular Cloning and Bioinformatical Analysis of a cDNA Encoding Mitochondrial 50S Ribosomal Protein L21 from Hevea brasiliensis Muell. Arg.%巴西橡胶树线粒体50S核糖体蛋白L21 cDNA的克隆与分析(英文)

    Institute of Scientific and Technical Information of China (English)

    邹智; 杨礼富

    2012-01-01

    [Objective] "Tapping panel dryness (TPD)", a syndrome known as tapping incision blocked partly or entirely during latex exploiting, has become the most important factor causing great losses for rubber production. Aiming to elucidate the molecular mechanism of tapping panel dryness occurrence, this study carried out molecular cloning and bioinformatical analysis of a mRPL21 cDNA sequence, a gene associated with TPD. [Method] In a preliminary study, an expressed sequence tag (EST) encoding a deduced protein homologous to mitochondrial 50S ribosomal protein L21 (mRPL21), which showed to be down-regulated in the latex of TPD-affected rubber trees, was isolated by suppression subtractive hybridization (SSH). After ESTs assembling and RT-PCR validation, an 853 bp cDNA sequence with an open reading frame (ORF) was cloned, which was named as HbmRPL21 under GenBank accession number of HM230670. [Result] Bioinformatical analysis suggests that HbmRPL21 encodes a deduced polypeptide of 271 amino acids with a theoretical molecular weight (Mw) of 30.52 kDa and isolectric point (pI) of 8.40, and HbmRPL21 is a mitochondrion-targeted protein with a conserved domain of Ribosomal_L21p involving translation. Homology analysis reveals high amino acid sequence identity of mRPL21 from plants, while diversity of that between plant and animal kingdom. [Conclusion] This study laid the basis for further revealing the biological functions of mRPL21 in TPD-affected rubber trees.%[目的]在橡胶生产中,一种叫做"死皮"的生理综合症严重制约了橡胶树(Hevea brasiliensis)单产的提高。为揭示橡胶树"死皮"发生的分子机理,研究对一差异表达的HbmRPL21进行了克隆,并在此基础上对其进行深入的生物信息学分析。[方法]在早期构建的差减文库中,筛选到一条在死皮植株中下调表达的基因片段,该片段编码的蛋白与线粒体50S核糖体蛋白L21(mRPL21)同源。通

  5. Improved analysis of bias in Monte Carlo criticality safety

    Science.gov (United States)

    Haley, Thomas C.

    2000-08-01

    Criticality safety, the prevention of nuclear chain reactions, depends on Monte Carlo computer codes for most commercial applications. One major shortcoming of these codes is the limited accuracy of the atomic and nuclear data files they depend on. In order to apply a code and its data files to a given criticality safety problem, the code must first be benchmarked against similar problems for which the answer is known. The difference between a code prediction and the known solution is termed the "bias" of the code. Traditional calculations of the bias for application to commercial criticality problems are generally full of assumptions and lead to large uncertainties which must be conservatively factored into the bias as statistical tolerances. Recent trends in storing commercial nuclear fuel---narrowed regulatory margins of safety, degradation of neutron absorbers, the desire to use higher enrichment fuel, etc.---push the envelope of criticality safety. They make it desirable to minimize uncertainty in the bias to accommodate these changes, and they make it vital to understand what assumptions are safe to make under what conditions. A set of improved procedures is proposed for (1) developing multivariate regression bias models, and (2) applying multivariate regression bias models. These improved procedures lead to more accurate estimates of the bias and much smaller uncertainties about this estimate, while also generally providing more conservative results. The drawback is that the procedures are not trivial and are highly labor intensive to implement. The payback in savings in margin to criticality and conservatism for calculations near regulatory and safety limits may be worth this cost. To develop these procedures, a bias model using the statistical technique of weighted least squares multivariate regression is developed in detail. Problems that can occur from a weak statistical analysis are highlighted, and a solid statistical method for developing the bias

  6. 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.

  7. Construction of the subtractive cDNA library of injured adult and fetal rabbit skins

    Institute of Scientific and Technical Information of China (English)

    张波; 刘大维; 王正国; 朱佩芳; 周继红; 蒋建新

    2004-01-01

    Objective: Early gestational mammalian fetuses possess the amazing ability to heal cutaneous wounds in a scarless fashion. Over the past years, scientists have been working to decipher the mechanisms underlying this regenerative repair. The remarkable phenotypic differences between fetal and adult healings behoves us to learn their characteristics in genetics, which represents potentially important mechanisms involved in wound repair observed in fetal versus adult tissues. In this sense, it is reasonable to construct subtractive cDNA library for future research.Methods: Middle laparotomy and hysterotomy were performed on pregnant rabbits at 20-day gestation to expose the fetal back, and a longitudinal incision through the skin was made on the back of the fetus. The traumatized fetal skin was harvested 12 hours post-operation, the fetus control and traumatized adult skin specimens were taken at the same time. dscDNA was synthesized from total RNA of skin samples with SMART technology. Taking one of the three samples as Tester respectively and the other two as Drivers, we obtained 1 forward and 2 reverse hybridization products. After being amplified with selective polymerase chain reaction, the products were inserted into a vector, and then transferred into E.coli HB101. The colonies were screened afterwards. Results: The wounded fetuses were alive for a long time even after birth. Every determinant step, such as RNA isolation, cDNA synthesis, Rsa I digestion, adaptor ligation and hybridization, was well-operated. Subtractive efficiency identification demonstrated that the suppression subtractive hybridization (SSH) was successful. Insertion into vector and transferring to E.coli were satisfactory. Conclusions: Instead of classic SSH, an improved SSH with 2 Drivers was applied for the experiment. Results confirmed that the improved program was reasonable and correct in both theory and practice. The subtractive cDNA library we have obtained is going to be used for

  8. 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.

  9. 思茅松HDR基因全长cDNA克隆与序列分析%Cloning and sequence analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase gene cDNA from Pinus kesiya var.langbianensis

    Institute of Scientific and Technical Information of China (English)

    王毅; 周旭; 毕玮; 杨宇明; 李江; 王娟

    2015-01-01

    1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase(HDR)catalyzes the last step of the 2C-meth-yl-D-erythritol-4-phosphate(MEP)pathway,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase plays an important role in regulation of terpenes biosynthesis.To explore the function of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase in Pinus kesiya var.langbianensis,and to study the role of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase in regulation of resin biosynthesis,the transcriptome of bark of Pinus kesiya var.langbianen-sis was sequenced by Next-Generation Sequencing.First,a fragment of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphos-phate reductase gene was obtained from Pinus kesiya var.langbianensis transcriptome after gene assemble and gene function annotation.The special primers were designed according to the fragment of 1-hydroxy-2-methyl-2-(E)-bute-nyl-4-diphosphate reductase.RNA of inj ured bark was extracted by Trizol method.The full length gene of PkHDR was cloned from Pinus kesiya var.langbianensis by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE).Bioinformation analysis showed that the obtained full cDNA se-quence of PkHDR had 1 876 bp.It was consisted of 1 464 bp open reading frame (ORF)which encoded 487 amino acid.Homology analysis indicated that the deduced PkHDR protein shared 99% identities with the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase came from Pinus densiflora.Subcellular localization and structural domain analysis showed that the transit peptide sequence (A1-A61)and multiple conserved functional sites (A143, A234,A288,A371)of plant HDR protein were found in the deduced coding sequence of PKHDR.Phylogenetic anal-ysis revealed that the evolutionary relationship of PkHDR protein was the closest to Pinus densiflora HDR protein. Reverse transcription polymerase chain reaction (RT-PCR)detection showed that PkHDR gene expression was up-regulated by wounding

  10. Analysis and Improvement of Authenticatable Ring Signcryption Scheme

    Institute of Scientific and Technical Information of China (English)

    LI Fa-gen; Shirase Masaaki; Takagi Tsuyoshi

    2008-01-01

    We show that the Zhang-Yang-Zhu-Zhang identity-based authenticatable ring signcryption scheme is not secure against chosen plaintext attacks.Furthermore,we propose an improved scheme that remedies the weakness of the Zhang-Yang-Zhu-Zhang scheme.The improved scheme has shorter ciphertext size than the Zhang-Yang-Zhu-Zhang scheme.We then prove that the improved scheme satisfies confidentiality,unforgeability,anonymity and authenticatability.

  11. Isolation and characterization of a cDNA clone of UDP-galactose: flavonoid 3-O-galactosyltransferase (UF3GaT) expressed in Vigna mungo seedlings.

    Science.gov (United States)

    Mato, M; Ozeki, Y; Itoh, Y; Higeta, D; Yoshitama, K; Teramoto, S; Aida, R; Ishikura, N; Shibata, M

    1998-11-01

    Four cDNA clones were isolated from Vigna mungo seedlings by the screening with cDNA encoding UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) of Antirrhinum majus as a probe; the product of the gene corresponding to one cDNA was more highly expressed in the first simple leaves than in stems. Nucleotide sequence analysis revealed 1,691 bp (including 326 bp non-reading) containing an open reading frame of 455 amino acids. The deduced amino acid sequence showed 42% and 23% identity with those of A. majus UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) and Petunia hybrida UDP-rhamnose:anthocyanidin 3-O-glucoside rhamnosyltransferase (RT), respectively. One region of the cDNA (amino acids 325 to 387) showed similarity to ceramide UDP-galactosyltransferases of mice, rats and humans. A crude extract from Escherichia coli, in which the protein was expressed from the cDNA, showed high UF3GaT activity but low UF3GT activity, and was similar in K(m), optimal pH and substrate specificity to UF3GaT from V. mungo. We conclude that we have obtained UDP-galactose:flavonoid 3-O-galactosyltransferase (UF3GaT) cDNA from V. mungo.

  12. Construction of a full-length cDNA library of Solen grandis dunker and identification of defense- and immune-related genes

    Science.gov (United States)

    Sun, Guohua; Liu, Xiangquan; Ren, Lihua; Yang, Jianmin; Wei, Xiumei; Yang, Jialong

    2013-11-01

    The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the `switching mechanism at the 5'-end of the RNA transcript' (SMART) technique. Total RNA was isolated from the immune-relevant tissues, gills and hemocytes, using the Trizol reagent, and cDNA fragments were digested with Sfi I before being ligated to the pBluescript II SK* vector. The cDNA library had a titer of 1048 cfu μL-1 and a storage capacity of 1.05×106 cfu. Approximately 98% of the clones in the library were recombinants, and the fragment lengths of insert cDNA ranged from 0.8 kb to 3.0 kb. A total of 2038 expressed sequence tags were successfully sequenced and clustered into 965 unigenes. BLASTN analysis showed that 240 sequences were highly similar to the known genes (E-value 80%), accounting for 25% of the total unigenes. According to the Gene Ontology, these unigenes were related to several biological processes, including cell structure, signal transport, protein synthesis, transcription, energy metabolism, and immunity. Fifteen of the identified sequences were related to defense and immunity. The full-length cDNA sequence of HSC70 was obtained. The cDNA library of S. grandis provided a useful resource for future researches of functional genomics related to stress tolerance, immunity, and other physiological activities.

  13. Molecular Cloning of a Novel cDNA From Mus Muscular BALB/c Mice Encoding Glycosyl Hydrolase Family 1: A Homolog of HumanLactase-Phlorizin Hydrolase

    Institute of Scientific and Technical Information of China (English)

    WEI HE; ZHEN-YU JI; CHENG-YU HUANG

    2006-01-01

    Objective To study the mechanism of lactose intolerance (LI) by cloning the mouse lactase cDNA and recombining a vector. Methods Total murine RNA was isolated from the small intestine of a 4-week-old BALB/c mouse (♂).Gene-specific primers were designed and synthesized according to the cDNA sequences of lactase-phlorizin hydrolase (LPH) in human, rat, and rabbit. A coding sequence (CDS) fragment was obtained using RT-PCR, and inserted into a clone vector pNEB-193, then the cDNA was sequenced and analyzed using bioinformatics. Results The cDNA from the BALB/c mouse with 912 bp encoding 303 amino acid residues. Analysis of the deduced amino acid sequence using bioinformatics revealed that this cDNA shared extensive sequence homology with human LPH containing a conserved glycosy1 hydrolase family 1 motif important for regulating lactase intolerance. Conclusion BALB/c mouse LPH cDNA (GenBank accession No: AY751548) provides a necessary foundation for study of the biological function and regulatory mechanism of the lactose intolerance in mice.

  14. Characterization of the porcine carboxypeptidase E cDNA

    DEFF Research Database (Denmark)

    Hreidarsdôttir, G.E.; Cirera, Susanna; Fredholm, Merete

    2007-01-01

    Carboxypeptidase E (CPE) is an important enzyme responsible for the proteolytic processing of prohormone intermediates. A naturally occurring point mutation, leading to an accumulation of many neuroendocrine peptides has been characterized within exon 5 of the CPE gene in mice. In the present study...... the sequence of the cDNA for the porcine CPE gene including all the coding region and the 3'-UTR region was generated. Comparisons with bovine, human, mouse, and rat CPE cDNA sequences showed that the coding regions of the gene are highly conserved both at the nucleotide and at the amino acid level. A very low...... nonsynonymousl/synonymous substitution ratios between the proteins was found indicating that purifying selection os acting on the CPE gene. A nonsynonymous SNP identified at position 1272 in the transcript resulting in a codon change from TCA (Serine) to TTA (Leucine) was genotyped in the Danish pig populations...

  15. cDNA Cloning and Expression Analysis of Glutamate Dehydrogenase in Chinese Shrimp (Fenneropenaeus chinensis) Exposed to Ambient Ammonia%氨氮胁迫下中国明对虾(Fenneropenaeus chinensis)谷氨酸脱氢酶基因的表达分析

    Institute of Scientific and Technical Information of China (English)

    何玉英; 李少飞; 王清印; 李健

    2016-01-01

    Chinese shrimp (Fenneropenaeus chinensis) is an ecologically and economically important shrimp species. During the culture, F. chinensis were exposed to a series of stressors that adversely affect biological activities including growth rate. Ammonia, a product of protein degradation and bacterial activity, is a strong stressor in shrimp aquaculture. Glutamate dehydrogenase (GDH) is an abundant and ubiquitous mitochondrial enzyme that catalyzes reversible amination of glutamate. cDNA of GDH from F. chinensis (FcGDH) was cloned by rapid amplification of cDNA ends (RACE). The FcGDH cDNA was 1779 bp in size, and it included a 1659-bp open reading frame (ORF) that encoded a 522 amino-acid polypeptide of which the isoelectric point (pI) was 6.54 and the molecular mass was 61.3 kDa. Homology analysis revealed that the amino acid sequence of FcGDH was highly conserved with its homologs in other arthropod. The similarities between FcGDH and GDHs of Litopenaeus vannamei and Eriocheir sinensis were 98% and 89% respectively. Phylogenetic analysis showed that FcGDH was in the same branch with that of L. vannamei and then in the same branches with those of E. sinensis, Drosophila melanogaster, and Aedes aegypti in order. The tissue expression analysis showed that FcGDH was detected in all tested tissues including muscle, gill, hepatopancreas, stomach, intestine, lymph, and hemocytes. The highest expression of FcGDH was in the muscle that was an amino acid pool and the major tissue for protein deposition. After exposure to ambient ammonia, the expression of FcGDH gene was up-regulated significantly in muscles compared to the control group (P<0.01). The expression level of FcGDH in hepatopancreas was down-regulated significantly at 3 h (P<0.05), and was then stabilized up to 24 h. The expression of FcGDH was increased significantly after 48 h and reached the maximum at 72 h compared to the control group (P<0.01). These results implied that FcGDH might play an important role in the

  16. 香蕉果实XET cDNA的克隆及其在成熟软化的果实果皮和果肉中的表达分析%Cloning and Expression Analysis of an XET cDNA in the Peel and Pulp of Banana Fruit Ripening and Softening

    Institute of Scientific and Technical Information of China (English)

    陆旺金; 中野隆平; 久保康隆; 稻叶昭次; 蒋跃明

    2004-01-01

    Xyloglucan endotransglycosylase (XET) is thought to be involved in fruit softening through disassembly of xyloglucan, which is the predominant hemicellulose of cell wall. To study the relationship between fruit softening and XET during banana (Musa acuminata Colla cv. Grand Nain) fruit ripening, a full length cDNA (1 095 bp) encoding an XET, MA-XET1, was isolated from ripening banana fruit using RT-PCR and RACE-PCR (rapid amplification of cDNA ends) methods. Sequence analysis showed that the cDNA contains 5' untranslated region of 66 bp, 3' untranslated region of 189 bp and ORF of 840 bp, encoding a predicted polypeptide of 280 amino acids, including DEIDFEFL motif, which is a presumptive catalytic domain conserved in XETs. DNA gel blot analysis demonstrated that MA-XET1 is encoded by a multi-copyfamily in the banana genome. RNA gel blot analysis revealed that the level of MA-XET1 transcript in the pulp was undetectable, increased and decreased slightly at the preclimacteric, climacteric and postclimactericstages, respectively. In the peel, accumulation of MA-XET1 transcript was low, increased dramatically andthen decreased rapidly, at preclimacteric, climacteric and postclimacteric stages, respectively. Treatment of fruit with propylene, an analog of ethylene, decreased the firmness and enhanced the accumulation of MA-XET1 transcript in the peel and pulp. These results suggest that MA-XET1 is involved in softening of the peel and pulp during banana fruit ripening and its expression is regulated by ethylene at transcriptional level.%木葡聚糖内糖基转移酶(Xyloglucan endotransglycosylase,XET)通过分解细胞壁半纤维素多糖的主要成分--木葡聚糖而参与果实软化.为了阐明香蕉(Musa acuminata.Colla cv.GrandNain)果实成熟过程中的软化与细胞壁代谢酶XET基因表达模式的关系,采用RT-PCR和RACE-PCR方法,首次从成熟香蕉果实果肉中分离了编码XT基因的全长cDNA(MA-XET1,全长1 095 bp

  17. cDNA cloning and characterization of a mannose-binding lectin from Zingiber officinale Roscoe (ginger) rhizomes

    Indian Academy of Sciences (India)

    Zhonghai Chen; Guoyin Kai; Xiaojun Liu; Juan Lin; Xiaofen Sun; Kexuan Tang

    2005-03-01

    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 510 bp open reading frame (ORF) encoding a lectin precursor of 169 amino acids with a signal peptide. ZOA was a mannose-binding lectin with three typical mannose-binding sites (QDNY). Semi-quantitative RT-PCR analysis revealed that zoa expressed in all the tested tissues of Z. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed in Escherichia coli with the molecular weight expected. To our knowledge, this is the first mannose-binding lectin cDNA cloned from the family Zingiberaceae. Our results demonstrate that monocot mannose-binding lectins also occur within the family Zingiberaceae.

  18. Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray

    Institute of Scientific and Technical Information of China (English)

    Xiao-Jing Liu; Li Yang; Feng-Ming Luo; Hong-Bin Wu; Qu-Qiang

    2004-01-01

    AIM: To characterize the gene expression profiles associated with activation of mouse hepatic stellate cell (HSC) and provide novel insights into the pathogenesis of hepatic fibrosis.METHODS: Mice HSCs were isolated from BALB/c mice by in situ perfusion of collagenase and pronase and singlestep density Nycodenz gradient. Total RNA and mRNA of quiescent HSC and culture-activated HSC were extracted,quantified and reversely transcripted into cDNA. cDNAs from activated HSC were labeled with Cy5 and cDNAs from the quiescent HSC were labeled with Cy3, which were mixed with equal quantity, then hybridized with cDNA chips containing 4 000 genes. Chips were washed, scanned and analyzed. Increased expression of 4 genes and decreased expression of one gene in activated HSC were confirmed by reverse transcription- polymerase chain reaction (RT-PCR).RESULTS: A total of 835 differentially expressed genes were identified by cDNA chip between activated and quiescent HSC, and 465 genes were highly expressed in activated HSC. The differentially expressed genes included those involved in protein synthesis, cell-cycle regulation,apoptosis, and DNA damage response.CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis and proliferation were up-regulated in activated HSC. cDNA microarray is an effective technique in screening for differentially expressed genes between two different situations of the HSC. Further analysis of the obtained genes will help understand the molecular mechanism of activation of HSC and hepatic fibrosis.

  19. Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase.

    Science.gov (United States)

    Giallongo, A; Feo, S; Moore, R; Croce, C M; Showe, L C

    1986-01-01

    We previously purified a 48-kDa protein (p48) that specifically reacts with an antiserum directed against the 12 carboxyl-terminal amino acids of the c-myc gene product. Using an antiserum directed against the purified p48, we have cloned a cDNA from a human expression library. This cDNA hybrid-selects an mRNA that translates to a 48-kDa protein that specifically reacts with anti-p48 serum. We have isolated a full-length cDNA that encodes p48 and spans 1755 bases. The coding region is 1299 bases long; 94 bases are 5' noncoding and 359 bases are 3' noncoding. The cDNA encodes a 433 amino acid protein that is 67% homologous to yeast enolase and 94% homologous to the rat non-neuronal enolase. The purified protein has been shown to have enolase activity and has been identified to be of the alpha type by isoenzyme analysis. The transcriptional regulation of enolase expression in response to mitogenic stimulation of peripheral blood lymphocytes and in response to heat shock is also discussed. Images PMID:3529090

  20. Application of restriction display PCR technique in the preparation of cDNA microarray probes

    Institute of Scientific and Technical Information of China (English)

    Zhao-Hui Sun; Wen-Li Ma; Bao Zhang; Yi-Fei Peng; Wen-Ling Zheng

    2005-01-01

    AIM: To develop a simplified and efficient method for the preparation of hepatitis C virus (HCV) cDNA microarray probes.METHODS: With the technique of restriction display PCR (RD-PCR), restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs. The products were classified and re-amplified by RD-PCR. We separated the differential genes by polyacrylamide gel electrophoresis and silver staining. Single bands cut out from the polyacrylamide gel were isolated. The third-round PCR was performed using the single bands as PCR template.The RD-PCR fragments were purified and cloned into the pMD18-T vector. The recombinant plasmids were extracted from positive clones, and the target gene fragments were sequenced. The cDNA microarray was prepared by spotting RD-PCR products to the surface of amino-modified glass slides using a robot. We validated the detection of microarray by hybridization and sequence analysis.RESULTS: A total of 24 different cDNA fragments ranging from 200 to 800 bp were isolated and sequenced,which were the specific gene fragments of HCV. These fragments could be further used as probes in microarray preparation. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The results of hybridization and sequence analysis showed that the specificity, sensitivity, accuracy, reproducibility,and linearity in detecting HCV RNA were satisfactory.CONCLUSION: The RD-PCR technique is of great value in obtaining a large number of size-comparable gene probes, which provides a speedy protocol in generating probes for the preparation of microarrays. Microarray prepared as such could be further optimized and applied in the clinical diagnosis of HCV.

  1. Full-length cDNA cloning and structural characterization of preproinsulin in Alligator sinensis.

    Science.gov (United States)

    Zhang, R; Zhang, S Z; Li, E; Wang, C; Wang, C L; Wu, X B

    2014-10-27

    Insulin is an important endocrine hormone that plays a critical physiological role in regulating metabolism and glucostasis in vertebrates. In this study, the complete cDNA of Alligator sinensis preproinsulin gene was cloned for the first time by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods; the amino acid sequence encoded and protein structure were analyzed. The full-length of preproinsulin cDNA sequence consists of 528 base pairs (bp), comprising a 34-bp 5'-untranslated region, a 170-bp 3'-untranslated region and an open reading frame that is 324 bp in length. The open reading frame encodes a 107-amino acid preproinsulin with a molecular weight of approximately 12,153.8 Da, theoretical isoelectric point of 5.68, aliphatic index of 92.06, and grand average of hydropathicity of -0.157, from which a signal peptide, a B-chain, a C-peptide, and an A-chain are derived. Online analysis suggested that the deduced preproinsulin amino acid sequence contains a transmembrane region, and that it has a signal peptide whose cleavage site occurs between alanine 24 and alanine 25. Comparative analysis of preproinsulin amino acid sequences indicated that the A-chain and B-chain sequences of preproinsulins are highly conserved between reptiles and birds, and that the preproinsulin amino acid sequence of Alligator sinensis shares 89% similarity to that of Chelonia mydas, but low similarity of 48-63% to those of mammals and fishes. The phylogenetic tree constructed using the neighbor-joining method revealed that preproinsulin of Alligator sinensis had high homology with reptiles and birds, such as Chelonia mydas, Gallus gallus, and Columba livia.

  2. 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

  3. 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

  4. 桔小实蝇电压门控钠离子通道基因cDNA克隆及其生物信息学分析%Cloning and Bioinformatics Analysis of Voltage-Gated Sodium Channel Gene cDNA in Bactrocera dorsalis (Hendel)

    Institute of Scientific and Technical Information of China (English)

    蒋玄赵; 魏丹丹; 申光茂; 豆威; 王进军

    2012-01-01

    [目的]克隆获得桔小实蝇(Bactrocera dorsalis)电压门控钠离子通道基因cDNA序列,明确其典型特征,为研究桔小实蝇抗性分子机理奠定基础.[方法]采用RT-PCR和PCR技术,克隆桔小实蝇钠离子通道基因cDNA序列,利用相关软件对其序列进行生物信息学分析.[结果]克隆得到1条长为6 446 bp的cDNA序列,包含1个6 405 bp的完整开放阅读框,共编码2 134个氨基酸.同源比对发现,桔小实蝇与果蝇(Drosophila melanogaster,NP_001188635)和家蝇(Musca domestica,AAB47604)钠离子通道基因相似度分别高达91.7%和86.9%,而与人的钠离子通道Nav1.2基因(Homo sapiens,NP_066287)相似度为42.3%.所克隆序列包含昆虫钠离子通道所有典型特征.[结论]成功地从桔小实蝇中克隆得到钠离子通道基因完整开放阅读框.该钠离子通道基因存在丰富的选择性剪接,发现了3个可能与抗性相关的碱基取代.钠离子通道基因有可能发展成为一种昆虫系统发育研究的分子标记.%[Objective] The objective of this study is to clone voltage-gated sodium channel gene (VGSC) from Bactrocera dorsalis (Hendel), and to identify its typical hallmarks. It will provide basic molecular information for clarifying insecticide resistance mechanism of B. dorsalis. [Method] The cDNA sequence was isolated using RT-PCR and PCR methods. Based on the sequencing results, the bioinformatics analysis of nucleic acid and putative amino acid was conducted. [Result] An almost full-length cDNA sequence (6 446 bp) of VGSC was obtained, including a complete open reading frame (ORF) of 6 405 bp, which encoded 2 134 amino acids and included all the typical hallmarks of VGSC. The amino acid shared 91.7%, 86.9% and 42.3% identity with sodium channel genes of Drosophila melanogaster (NP_001188635), Musca domestica (AAB47604), and Homo sapiens Navl.2 (NP066287), respectively. [Conclusion] A complete ORF of VGSC was sequenced with clone strategy from oriental

  5. Cloning of partial cDNA of estrogen receptor α and expression analysis during the reproductive cycle in Navodon septentrionalis%绿鳍马面鲀ERα基因部分cDNA序列克隆及其表达研究

    Institute of Scientific and Technical Information of China (English)

    赵梅琳; 温海深; 张冬茜; 何峰; 李吉方

    2014-01-01

    Estrogens are critical sex steroid hormones involved in reproduction. And they need to bind to estrogen receptors (ER) in target organ for biological activity. To explore the physiological function of estrogen receptor α(ERα) in Navodon septentrionalis and provide some theoretical basis for artificial breeding, a partial cDNA sequence of N. septentrionalis ERαgene was obtained by degenerate primer PCR amplification. The length of the cDNA sequence is 1410 bp, and encodes 470 amino acids. The tissue expression analysis using semi-quantitative RT-PCR showed that it had a more restricted distribution in male fish. In female, pituitary, heart and ovary had high expression levels, and in male, pituitary, liver, kidney and testis had relatively high expression levels. In addition, we analyzed the relative expression level of ERαin reproductive cycle in ovary and testis by quantitative RT-PCR, respectively. It revealed that the highest expression level was in May, and the lowest in July in ovary, while in testis, the lowest was in May, and the highest in September. This study indicates that ERαhas a strong relationship with the reproductive cycle of N. septentrionalis, suggesting ERαshould play a role in gonads development.%雌二醇是与生殖相关的重要的性类固醇激素之一,它与雌激素受体相结合作用于靶器官从而发挥生物活性。为进一步鲀阐明绿鳍马面雌激素受体α(Estrogen receptorα, ERα)的生理功能提供了科学依据,同时也为其人工繁育提供一定的理论基础,作者利用简并引物扩增得到绿鳍马面鲀(Navodon septentrionalis)ERα部分cDNA序列,共1410bp,编码470个氨基酸。通过半定量RT-PCR技术分别检测了其在雌鱼、雄鱼中各组织的表达情况,结果表明 ERα在雌鱼体内的表达较雄鱼更广泛,在雌鱼垂体、心脏和卵巢的表达量最高,在雄鱼的垂体、肝脏、肾脏、精巢也有较高的表达。采用实时定量RT-P C R技术,分析了ER

  6. Cloning, Southern blotting and RT-PCR analysis of a cDNA fragment encoding of 70 kDa heat shock cognate protein(Hsc70) from the oyster(Crassostrea ariakensis)%近江牡蛎Hsc70蛋白基因cDNA片段的克隆及Southern杂交和RT-PCR分析

    Institute of Scientific and Technical Information of China (English)

    张其中; 吴信忠; 高劲松; 潘金培

    2003-01-01

    In order to elucidate the molecular mechanisms of the oyster ( Crassostrea ariakensis) against adverse stimulating factors, we cloned and sequenced a partial cDNA encoding a 70 kDa heat shock cognate protein (Hsc70) from the oyster. The live oysters were obtained from Chengcun, Yangxi County, Guangdong Province, China. Various tissues, including mantle, gills, adductor muscle, heart and blood cells, were respectively collected from 5 untreated live oysters or treated ones at 36℃ for 1.5 hours, and immediately frozen in liquid nitrogen except for the blood cells which were suspended with Trizol Reagent after centrifugation (12 000 r/min for 30 s) and stored at - 20℃ . Total RNA was isolated using Trizol Reagent according to the manufacture's instructions. The first strand cDNA was synthesized using reverse transcriptase Superscript Ⅱ according to the manufacture's instructions. The primers were designed from a conserved region of C. gigas Hsc70 cDNA sequence (GeneBank accession No. AF144646). The polymerase chain reaction (PCR)was performed for 30 cycles with denaturation at 94℃ for 30 s, annealing at 49℃ for 40 s, and elongation at 72℃ for 30 s. The product was cloned to pGEM-T easy vector and sequenced. It is 509 base pairs (bp) and possesses 94 % identity with the cDNA encoding C. gigas Hsc70 using Blastn. This homology was strongly confirmed by amino acid sequence comparison using the Blastx (99 % ). The 509 bp fragment was labeled with α-32pdCTP and a random primer DNA labeling kit and employed as a probe to perform Southern blotting, the result demonstrated that the cDNA came from a partial mRNA transcript of C. ariakensis genomic DNA gene. The polymerase chain reaction (PCR) was carried out to investigate the expression of Hsc70, Using the cDNAs of several tissues, such as gills (heat shocked), mantle, adductor muscle (heat shocked), heart, blood cells (one sample with heat shock for 1.5 hours at 36℃ and another without any stimulus).The PCR

  7. Discrete Packet Analysis for Improved Atmospheric Rejection on Modulated Laser Signals

    Energy Technology Data Exchange (ETDEWEB)

    O& #x27; Neill, M., McKenna, I., DiBenedetto, J., Capelle, G., Trainham, R.

    2012-07-19

    This slide-show discusses how the method of discrete packet analysis improves atmospheric compensation for quasi-CW fluorescence detection methods. This is key to improving remote sensing capabilities.

  8. Analysis of the Improvement Methods for Equipment Maintenance Support

    Institute of Scientific and Technical Information of China (English)

    ZHANG Rui-chang; ZHAO Song-zheng

    2005-01-01

    According to military requirement, and based on the problems of equipment maintenance support methods in high-tech battles, each element supporting equipment maintenance is analyzed, and the methods for improving equipment maintenance are proposed.

  9. 10 Inventions on improving keyboard efficiency: A TRIZ based analysis

    OpenAIRE

    Mishra, Umakant

    2013-01-01

    A keyboard is the most important input device for a computer. With the development of technology a basic keyboard does not want to remain confined within the basic functionalities of a keyboard, rather it wants to go beyond. There are several inventions which attempt to improve the efficiency of a conventional keyboard. This article illustrates 10 inventions from US Patent database all of which have proposed very interesting methods for improving the efficiency of a computer keyboard. Some in...

  10. Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

    Institute of Scientific and Technical Information of China (English)

    周兆斓; 朱祯; 刘春明; 张海涛; 肖桂芳; 李向辉

    1996-01-01

    Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and a poly(A) signal AATAAA at the 3’-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3’ region after poly(A) signal.

  11. 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.

  12. Improving Student Understanding of Geological Rates via Chronotopographic Analysis

    Science.gov (United States)

    Linneman, S. R.; Clark, D. H.; Buly, P.

    2010-12-01

    We are investigating the value of incorporating chronotopographic analysis into undergraduate geology courses using terrestrial laser scanning (TLS) to improve student understanding of the rates and styles of geomorphic processes. Repeat high-resolution TLS surveys can track the evolution of active landscapes, including sites of active faulting, glaciation, landslides, fluvial systems and coastal dynamics. We hypothesize that geology students who collect and analyze such positional data for local active landscapes will develop a better sense of the critical (and non-steady) geomorphic processes affecting landscape change and develop a greater interest in pursuing opportunities for geology field work. We have collected baseline TLS scans of actively evolving landscapes identified in cooperation with land-use agencies. The project team is developing inquiry activities for each site and assessing their impact. For example, our faculty partners at 2-year colleges are interested in rapid retreat of coastal bluffs near their campuses. In this situation, TLS will be part of a laboratory activity in which students compare historic air photos to predict areas of the most active long-term bluff retreat; join their instructor to collect TLS data at the site (replicating the baseline scan); sketch outcrops in the field and suggest areas of the site for higher resolution scanning; and in the following class compare their predictions to the deformation maps that are the output of the repeated TLS scans. A brief two question assessment instrument was developed to address both the content and attitudinal targets. It was given WWU Geomorphology classes in 3 sequential quarters of the 2009/2010 academic year, 2 which did not work with the TLS technology (pre treatment) and one that did participate in the redesigned activities (post treatment). Additionally focus group interviews were conducted with the post students so they could verbalize their experience with the TLS. The content

  13. 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.

  14. Why Economic Analysis of Health System Improvement Interventions Matters

    Science.gov (United States)

    Broughton, Edward Ivor; Marquez, Lani

    2016-01-01

    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 medical errors 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 of 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 resource allocation for improvement activities among other competing demands for health-care resources.

  15. cDNA cloning, prokaryotic expression and purification of rat α-synuclein

    Institute of Scientific and Technical Information of China (English)

    Xin LI; Yao-Hua LI; Jun-Yan HAN; Shun YU; Biao CHEN

    2006-01-01

    Objective To clone the cDNA of rat α-Syn gene, investigate its prokaryotic expression and produce purified recombinant rat α-Syn protein. Methods Rat α-Syn cDNA was amplified from the rat brain total RNA by RT-PCR and was cloned into pGEX-4T-1, a prokaryotie expressing vector. The recombinant plasmid containing rat α-Syn gene was transformed into E. Coli BL21 to express a fusion protein with rat α-Syn protein tagged by glutathione-S-transferase (GST). The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant rat α-Syn protein. The recombinant rat α-Syn protein was further purified using Superdex S200 gel filtration.Results DNA sequencing confirmed that the cloned cDNA contained 420 base pairs encoding 140 amino acids, which was identical to the reported amino acid sequence of rat α-Syn. After transformation, the recombinant plasmid pGEX-raSyn expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 18000, which was identical to the reported molecular size of rat α-Syn.Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against α-Syn. Conclusion The rat α-Syn gene was successfully expressed in prokaryotic expression system and highly purified rat α-Syn recombinant protein was produced.

  16. Rice bicoid-related cDNA sequence and its expression during early embryogenesis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation.To clone and characterize the rice bicoid-related genes,one cDNA clone,Rb24 (EMBL accession number: AJ2771380),was isolated by screening of rice unmature seed cDNA library.Sequence analysis indicates that Rb24 contains a putative amino acid sequence,which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity,75% similarity) and involves a lys-9 in putative helix 3.Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner.The transcript was detected strongly in young panicles,but less in young leaves and roots.This results are further confirmed with paraffin section in situ hybridization.The signal is intensive in rice globular embryo and located at the apical tip of the embryo,then,along with the development of embryo,the signal is getting reduced and transfers into both sides of embryo.The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

  17. Analysis and Improvement of Low Rank Representation for Subspace segmentation

    CERN Document Server

    Siming, Wei

    2011-01-01

    We analyze and improve low rank representation (LRR), the state-of-the-art algorithm for subspace segmentation of data. We prove that for the noiseless case, the optimization model of LRR has a unique solution, which is the shape interaction matrix (SIM) of the data matrix. So in essence LRR is equivalent to factorization methods. We also prove that the minimum value of the optimization model of LRR is equal to the rank of the data matrix. For the noisy case, we show that LRR can be approximated as a factorization method that combines noise removal by column sparse robust PCA. We further propose an improved version of LRR, called Robust Shape Interaction (RSI), which uses the corrected data as the dictionary instead of the noisy data. RSI is more robust than LRR when the corruption in data is heavy. Experiments on both synthetic and real data testify to the improved robustness of RSI.

  18. Distortion Parameters Analysis Method Based on Improved Filtering Algorithm

    Directory of Open Access Journals (Sweden)

    ZHANG Shutuan

    2013-10-01

    Full Text Available In order to realize the accurate distortion parameters test of aircraft power supply system, and satisfy the requirement of corresponding equipment in the aircraft, the novel power parameters test system based on improved filtering algorithm is introduced in this paper. The hardware of the test system has the characters of s portable and high-speed data acquisition and processing, and the software parts utilize the software Labwindows/CVI as exploitation software, and adopt the pre-processing technique and adding filtering algorithm. Compare with the traditional filtering algorithm, the test system adopted improved filtering algorithm can help to increase the test accuracy. The application shows that the test system with improved filtering algorithm can realize the accurate test results, and reach to the design requirements.  

  19. Improved Analysis of Kannan's Shortest Lattice Vector Algorithm

    CERN Document Server

    Hanrot, Guillaume

    2007-01-01

    The security of lattice-based cryptosystems such as NTRU, GGH and Ajtai-Dwork essentially relies upon the intractability of computing a shortest non-zero lattice vector and a closest lattice vector to a given target vector in high dimensions. The best algorithms for these tasks are due to Kannan, and, though remarkably simple, their complexity estimates have not been improved since more than twenty years. Kannan's algorithm for solving the shortest vector problem is in particular crucial in Schnorr's celebrated block reduction algorithm, on which are based the best known attacks against the lattice-based encryption schemes mentioned above. Understanding precisely Kannan's algorithm is of prime importance for providing meaningful key-sizes. In this paper we improve the complexity analyses of Kannan's algorithms and discuss the possibility of improving the underlying enumeration strategy.

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

    Science.gov (United States)

    Xie, Wei-Qi; Chai, Xin-Sheng

    2017-03-21

    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. This article is protected by copyright. All rights reserved.

  1. 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…

  2. Fiscal 1998 achievement report. Industrial technology research and development project. (Strategic human cDNA genome application technology development); 1998 nendo senryakuteki hito cDNA genome oyo gijutsu kaihatsu seika hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    A human genome related project named above was started, and studies were conducted for base sequence determination and function analysis for approximately 10,000 kinds of full-length or long-chain human cDNA clones owned by research organizations in this country. The Institute of Medical Science of University of Tokyo and Helix Research Institute dealt with a full-length human cDNA library constructed by oligo-capping, and determined the base sequences of all specimens in the library. The Kazusa DNA Research Institute determined partial sequences for long-chain clones which are not shorter than 4-5kbp, and determined entire sequences for some bases. The obtained base sequence data were subjected to homology analysis, the base sequences were converted into amino acid sequences, and functions of proteins were predicted. In the analysis of gene functions, ATAC-PCR (adaptor tagged competitive-polymerase chain reaction) was applied to the clones covered by this project, and a database was prepared by use of the results of analyses of frequency-related information. For the preparation of a comprehensive gene expression profile, technologies for cDNA microarray construction were established. (NEDO)

  3. Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library.

    OpenAIRE

    1988-01-01

    We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapie-infected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of alpha-crystallin; the latter two may represent a response to stress.

  4. Oceanic Area System Improvement Study (OASIS). Volume I. Executive Summary and Improvement Alternatives Development and Analysis.

    Science.gov (United States)

    1981-09-01

    h ich is shown in Tabla O2 t ha4v :x outib RLs othyr- improvements) would tJM"PJ sysc r"Oar jeyi - -VMG - 3-s &3V)c ee, 2ari4cularly ina one CAR fli...Al 2.867000 (3) :, 6-.0 Xii ’ / i~s i 2 52.399990 5.0-iK (1)? iscua eio corresponding to disc~yio in(ref. 5,6) \\(2) Parame trs corresponding to

  5. 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.

  6. Analysis of Strategies to Improve Heliostat Tracking at Solar Two

    Energy Technology Data Exchange (ETDEWEB)

    Jones, S.A.; Stone, K.W.

    1999-01-14

    This paper investigates dhlerent strategies that can be used to improve the tracking accuracy of heliostats at Solar Two. The different strategies are analyzed using a geometrical error model to determine their performance over the course of a day. By using the performance of heliostats in representative locations of the field aad on representative days of the year, an estimate of the annual performance of each strategy is presented.

  7. Analysis of strategies to improve heliostat tracking at Solar Two

    Energy Technology Data Exchange (ETDEWEB)

    Jones, S.A.; Stone, K.W.

    1999-07-01

    This paper investigates different strategies that can be used to improve the tracking accuracy of heliostats at Solar Two. The different strategies are analyzed using a geometrical error model to determine their performance over the course of a day. By using the performance of heliostats in representative locations of the field and on representative days of the year, an estimate of the annual performance of each strategy is presented.

  8. Analysis and improvement of SNR using time slicing

    Science.gov (United States)

    Karanam, Srikrishna; Singh, Amarjot; Kumar, Devinder; Choubey, Akash; Bacchuwar, Ketan

    2011-06-01

    Noise is a very important factor which in most cases, plays an antagonistic role in the vast field of image processing. Thus noise needs to be studied in great depth in order to improve the quality of images. The quantity of signal in an image, corrupted by noise is generally described by the term Signal-to-Noise ratio. Capturing multiple photos at different focus settings is a powerful approach for improving SNR. The paper analyses a frame work for optimally balancing the tradeoff's between defocus and sensor noise by experimenting on synthetic as well as real video sequences. The method is first applied to synthetic image where the improvement in SNR is studied by the ability of Hough transform to extract the number of lines with respect to the variation in SNR. The paper further experiments on real time video sequences while the improvement in SNR is analyzed using different edge operators like Sobel, Canny, Prewitt, Roberts and Laplacian. The result obtained is further analyzed using different edge operators. The main aim is to detect the edges at different values of SNR which will be a prominent measure of the signal strength as well as clarity of an image. The paper also explains in depth the modeling of noise leading to better understanding of SNR. The results obtain from both synthetic image and real time video sequences elaborate the increase in SNR with the increment in the total number of time slices in a fixed budget leading to clear pictures. This technique can be very effectively applied to capture high quality images from long distances.

  9. Senior Project: Analysis and Improvement of Existing Apparel Technology

    Science.gov (United States)

    1991-09-10

    and improve the quality of the seam , the major cost centers - work in process, operator handlinq, and production control were left far behind. Was it...skilled operation when performed manually; the curve must be stitched without puckering or irregular stitches and the line must remain parallel to the...equipped with side seam expanders. In this plant the Ajax Presses had been modified with a spring I steel clamp on the back of the buck to secure the box

  10. Joint regression analysis and AMMI model applied to oat improvement

    Science.gov (United States)

    Oliveira, A.; Oliveira, T. A.; Mejza, S.

    2012-09-01

    In our work we present an application of some biometrical methods useful in genotype stab