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Sample records for cdna library construction

  1. 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.

  2. Construction and characterization of a normalized whole-life-cycle cDNA library of rice

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A cDNA library with genomic complete coverage is a powerful tool for functional genomic studies. For studying the functions of rice genes on a large scale, a normalized whole-life-cycle cDNA library is constructed based on the strategy of saturation hybridization with genomic DNA using rice cultivar Minghui 63, an elite restorer line for a number of rice hybrids that are widely cultivated in China. This library consists of cDNA from 15 directionally cloned cDNA libraries constructed with different tissues from 9 developmental stages. For normalization, the denatured plasmids purified from the 15 directionally cloned libraries are mixed and hybridized with saturated genomic DNA labeled with magnetic beads in two complementary systems. Well-matched plasmids are captured from the hybridized genomic DNA and electroporated into competent DH10B E. coli for construction of the normalized whole-life-cycle cDNA library. This library consists of 62000 clones with an average insert length about 1.4 kb. Inverse Northern blotting shows that this cDNA library included many rarely expressed genes and tissue-specific genes. Sequencing of 10750 cDNA clones of this library reveals 6399 unique ESTs (expressed sequence tags), indicating that the non-redundancy of the library is about 59.5%. This library has been used to make cDNA microarrays for functional genomic studies.

  3. [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.

  4. 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.

  5. [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.

  6. Construction of full length cDNA expression library of hepatopancreas of Penaeus monodon

    Institute of Scientific and Technical Information of China (English)

    罗田; 徐洵

    2002-01-01

    --mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyATtract System 1000 Kit. By using mRNA as template, double- strand cDNA with EcoR I/Xho I ends was synthesized by using a ZAP Express cDNA Synthesis Kit. The cDNA was inserted into the lambda ZAP Express vector predigested with EcoR I/Xho I, and the recombinant DNA was in vitro packaged into larnbda phage with GigapackⅢ Gold packaging extracts. These recombinant phages were then used to transfect E. coli XLl - Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2 × 105pfu in capacity and its recombination ratio is higher than 99%. The size of the inserted cDNAs was determined by EcoR I/Xho I digestion of 9 phagemids prepared by in vivo excision of plaques selected randomly from amplified cDNA library . The longest inserted cDNA is about 1.6 kb in length. The complete sequence (about 1.2 kb) of actin cDNA was amplified from the library by PCR reveals that this library contains full-length cDNAs of Penaeus mod on hepatopancreas and is available for screening and expression of shrimp genes.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. A NEW METHOD TO CONSTRUCT A FULL-LENGTH cDNA LIBRARY OF HUMAN NORMAL BLADDER TISSUE

    Institute of Scientific and Technical Information of China (English)

    成瑜; 李旭; 陈葳; 杨玉琮; 赵乐

    2003-01-01

    Objective Using template-switch mechanism at the 5'-end of mRNA technique (SMART) to construct a full-length cDNA library of human normal bladder tissue. Methods The novel procedures used the template-switching activity of powerscript reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, 5 cycles of PCR were performed using a modified oligo(dT) primer and an anchor primer to enrich the full-length cDNA population with 1.0 g human normal bladder poly(A)+RNA, then double-strand cDNA was synthesized. After digestion with sfiI and size-fractionation by CHROMA SPIN-400 columns, double-strand cDNA was ligated into λTripIEx2 vector and was packaged. We determined the titer of the primary library and the percentage of recombinant clones and finally amplified the library. Results The titer of the cDNA library constructed was 2.1×106 pfu*mL-1, and the amplified cDNA library was 6×1011 pfu*mL-1, the percentage of recombination clones was 99%. Conclusion Using SMART technique helps us to construct full-length cDNA library with high efficiency and high capacity which lays solid foundation for screening target genes of bladder diseases with probes and antibodies.

  15. Construction of human and mouse brain cDNA libraries and isolation of full-length cDNAs

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    cDNA libraries from aborted human 3-month fetal brain,adult rat and mouse brain were constructed by using a yZAP express cDNA library construction kin.Low molecular weight fragments of the second strand cDNASA were removed by flowing through the Sepharose CL-4B column and the frractionated long,Middle,Short fragments and the combined fragments weire respectively inserted into clone vectors to construct the cDNA libraries of the brain of human 3-month fetus.The 5'ends of 1200 clones from each of human fetal brain cDNA libraries were sequenced.A total of 894 ESTs were obtained and some full-length clones were squenced.By andalyaing the se-quences,12 novel full-length cDNAs were obtained.

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

    Science.gov (United States)

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

    2007-11-01

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

  17. [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.

  18. 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.

  19. 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

  20. Construction of Hypericin Gland-Specific cDNA Library via Suppression Subtractive Hybridization.

    Science.gov (United States)

    Singh, Rupesh Kumar; Hou, Weina; Franklin, Gregory

    2016-01-01

    Hypericin, an important determinant of the pharmacological properties of the genus Hypericum, is considered as a major molecule for drug development. However, biosynthesis and accumulation of hypericin is not well understood. Identification of genes differentially expressed in tissues with and without hypericin accumulation is a useful strategy to elucidate the mechanisms underlying the development of the dark glands and hypericin biosynthesis. Suppression Subtractive Hybridization (SSH) is a unique method for PCR-based amplification of specific cDNA fragments that differ between a control (driver) and experimental (tester) transcriptome. This technique relies on the removal of dsDNA formed by hybridization between a control and test sample, thus eliminating cDNAs of similar abundance, and retaining differentially expressed or variable in sequence cDNAs. In our laboratory we applied this method to identify the genes involved in the development of dark glands and accumulation of hypericin in Hypericum perforatum. Here we describe the complete procedure for the construction of hypericin gland-specific subtracted cDNA library.

  1. Construction and characterization of a full-lengh cDNA library from non-fresh Giardia lamblia

    Institute of Scientific and Technical Information of China (English)

    Jun-Li Guo; Jie Jiang; Wen-Yu Zheng; Ming-Luan Li; Xi-Feng Tian; Xian-Min Feng; Yue-Hua Wang; Xiao-Hong Ju; Yue-Qiong Kong

    2012-01-01

    Objective: To construct rapidly a full-length cDNA library from nanogram amounts total RNA of Giardia lamblia (G. lamblia) trophozoites stocked in RNA stabilization reagent. Methods: Total RNA of Giardia was extracted using Trizol reagent. A full-length cDNA library of G. lamblia trophozoites was constructed by a long-distance PCR (LD-PCR) method. The recombinant rate and the coverage rate of full-length clones of the library were evaluated. The inserted fragments were identified and sequenced by PCR amplification. Results: The titer of cDNA library was 3.85 ×107 pfu/mL. The length of inserted fragments ranged from 0.4 to 2.5 kb, and the recombination efficiency accounted for 100% (20/20). The coverage rate of full-length clones is high (17/20). Conclusions: The RNA stabilization reagent may be used to fix the cells and prevent the RNA in cells even though delivered under normal atmospheric temperature. The long-distance PCR can be used to construct a full-length cDNA library rapidly and it needs less RNA than the traditional method from mRNA.

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

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiao-hong; CHEN Zhi; YAO Hang-ping; CHEN Feng; ZHU Hai-hong; ZHOU Hong-juan

    2005-01-01

    Objective: To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. Methods: The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript(SMART) technique and CDS Ⅲ/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction(LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to λTriplEx2 vector. Then λ phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. Results: The titers of unamplifed and amplified libraries were 1.94×106 pfu/ml and1.49×109 pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%.Conclusion: A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

  3. 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.

  4. 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.

  5. Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization

    Institute of Scientific and Technical Information of China (English)

    Li Liang; Yan-Qing Ding; Xin Li; Guang-Zhi Yang; Jun Xiao; Li-Chun Lu; Jin-Hua Zhang

    2004-01-01

    AIM: To construct a differentially-expressed gene subtracted cDNA library from two colorectal carcinoma (CRC) cell lines with different metastatic phenotypes by suppression subtractive hybridization.METHODS: Two cell lines of human CRC from the same patient were used. SW620 cell line showing highly metastatic potential was regarded as tester in the forward subtractive hybridization, while SW480 cell line with lowly metastatic potential was treated as tester in the reverse hybridization. Suppression subtractive hybridization (SSH)was employed to obtain cDNA fragments of differentially expressed genes for the metastasis of CRC. These fragments were ligated with T vectors, screened through the bluewhite screening system to establish cDNA library.RESULTS: After the blue-white screening, 235 white clones were picked out from the positive-going hybridization and 232 from the reverse. PCR results showed that 200-700 bp inserts were seen in 98% and 91% clones from the forward and reverse hybridizations, respectively.CONCLUSIONS: A subtractive cDNA library of differentially expressed genes specific for metastasis of CRC can be constructed with SSH and T/A cloning techniques.

  6. 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.

  7. Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica*

    OpenAIRE

    2005-01-01

    To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosoma japonicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes. Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Ampli...

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

    OpenAIRE

    Soliman, Hatem Mohamed Touhan

    2005-01-01

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

  9. Construction and Characterization of a cDNA Expression Library for the North American Ginseng Root Tissues

    Institute of Scientific and Technical Information of China (English)

    WANG Ying; WANG Kun; BAO Yong-li; WU Yin; MENG Xiang-ying; LI Yu-xin

    2007-01-01

    The root of Panax ginseng plant undergoes a specific developmental process to become a biosynthesis and accumulation tissue for ginsenosides. To identify and analyze genes involved in the biosynthesis of ginsenoside, we constructed and characterized a full-length cDNA library for 6-year-old North American ginseng. The titer of primary cDNA library is 1.2×106 pfu/mL, the titer of amplified library is 2.6×1010 pfu/mL and the rate of recombinant is above 86%. The insert size ranges from 0.3 to 2.0 kb. Sequencing results show that 18 of 58 genes are high homologous to the genes(GBR5, GBR3 and GBR1) known in GenBank, which are involved in biosynthesis of ginsenoside in North American ginseng plant; 16 of 58 genes are novel genes. The full-length cDNA library of North American ginseng root tissues is essential for the cloning of genes known and it is also an initial key for the screening and cloning of new genes.

  10. Construction of SMART cDNA Library of Sheep Ovary and Identification of Candidate Gene by Homologous Cloning

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The cDNA library of an ovary from Small Tail Han sheep before estrus was constructed by switching mechanism at 5' end of RNA transcript (SMART) approach. This library had a plaque titer of 1 × 109pfu mL-1 and a 96% recombinant ratio of which the fragment length of inserted average eDNA sequences was 1.0 kb. Based on bioinformatics analysis of the sequences, we obtained 338 expressed sequence tags (ESTs) from 380 cDNA clones which indicated 191 contigs. These contigs consist of 89 unmatched ESTs, 9 homologous known genes in sheep, and 93 homologous sequences in species of mouse, bovine, and human beings, including 19 sequences expressed in the ovary or follicle and 14 unknown sequences.Several candidate genes associated with sheep reproduction trait such as epidermal growth factor (EGF), estrogen receptor (ESR), Inhibin, follicle stimulating hormone receptor (FSHR), prostaglandin (PG), and transforming growth factor-β (TGF-β) were identified and the homologous were cloned from this library, which will contribute to compile expression profies and find the major genes of prolificacy of Small Tail Han sheep.

  11. Construction and Characterization of a cDNA Library from the Pulp of Coconut (Cocos nucifera L.)

    Institute of Scientific and Technical Information of China (English)

    LI Dong-dong; FAN Yong-mei

    2008-01-01

    To investigate the gene expression profile of endosperm development,a cDNA library was constructed and characterized from the pulp of coconut at different developmental stages.The constructed cDNA library incorporated approximately 1 × 107 clones in total,and the size of the insertion fragments ranged from 800 to 2000 bp.Sequencing results of 100 randomly picked clones showed that the recombination rate was 96%.In subsequent sequence analysis,41 clones (41%)were homologous to known function proteins,and 23 clones showed high amino acid identity (more than 80%) with the corresponding genes of different plants.Semi-quantitative RT-PCR indicated that oleosin and globulin genes are pulpspecific expression,and have differential expression level in different developmental stage.Clone 29,recognized as homologous to KIAA1239 protein (Homo sapiens),was observed to occur nine times,indicating that this gene may be over-expressed during the endosperm development stage.However,the homologous protein was found only in mammals,and the detailed function is still unknown.Elucidation of the functional characterization of these genes will be carried out immediately.

  12. Construction of cDNA subtractive library from pearl oyster (Pinctada fucata Gould) with red color shell by SSH

    Institute of Scientific and Technical Information of China (English)

    GUAN Yunyan; HUANG Liangmin; HE Maoxian

    2011-01-01

    The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization (SSH) to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively.Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6,shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COI. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and COI so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.

  13. Construction of cDNA subtractive library from pearl oyster ( Pinctada fucata Gould) with red color shell by SSH

    Science.gov (United States)

    Guan, Yunyan; Huang, Liangmin; He, Maoxian

    2011-05-01

    The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization (SSH) to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively. Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6, shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COI. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and COI so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.

  14. Construction of the Subtracted cDNA Library of Striatal Neurons Treated with Long-term Morphine

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

    Objective To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization (SSH).Methods Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons (10-5 mol/L for 72 hours). To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein (CREB) mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons (62.85±1.98) compared with normal striatal neurons (28.43±1.46, P<0.01). Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis. And the 36 clones showed homology with 19 known genes and 2 novel genes. The expression of 2 novel genes, mitochondrial carrier homolog 1 (Mtch1; 96.81±2.04 vs. 44.20±1.31, P<0.01) and thymoma viral proto-oncogene 1 (Akt1; 122.10±2.17 vs. 50.11 ±2.01, P<0.01), showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons. Conclusions A reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed. Mtchl and Akt1 might be the candidate genes for the development of morphine tolerance.

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

    Directory of Open Access Journals (Sweden)

    Zhou Sun

    2012-05-01

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

  16. Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica

    Institute of Scientific and Technical Information of China (English)

    Zheng Min; Wu Yi-jun; Cai Wei-min; Weng Hong-lei; Liu Rong-hua

    2005-01-01

    To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosomajaponicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes.Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library.Amplification of the library was carried out with transformation of DH5α. The amplified library contained more than 400 positive bacterial clones, which were then hybridized with forward and backward subtracted probes for differential screening. One hundred positive bacterial clones were randomly selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. The subtracted cDNA library of differentially expressed genes of HSCs was constructed successfully,the library is efficient and lays foundation for screening and cloning new and specific genes of schistosomiasis.

  17. 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.

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

    Science.gov (United States)

    He, Tao; Jia, Jing Fen

    2008-12-01

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

  19. 微量RNA的cDNA PCR文库的构建%The Construction of cDNA PCR Library from a Small Amount of RNA

    Institute of Scientific and Technical Information of China (English)

    李晶泉; 袁晓东; 汤敏谦

    2001-01-01

    By the method of PCR (Polymerase Chain Reaction),we have constructed the cDNA PCR library from mRNA.The cDNA PCR library can amplify the original cDNA up to hundreds of times.With the total RNA of human K562 cultured cell,the cDNA of β-Actin has been obtained by the methods of cDNA PCR library and reverse transcription respectively.As contrast,the amount of β-Actin′s cDNA from the cDNA PCR library is much higher than from reverse transcription.75pg total RNA of human K562 Cultured cell is employed to construct 50μl cDNA PCR library,and the cDNA of β-Actin can even be detected by using 1μl of the library as template to perform the PCR.Therefore cDNA PCR library can greatly enlarge the amount of information.%使用PCR(polymerase chain reaction)技术,调制了mRNA的cDNA PCR文库,实验证明,cDNA PCR文库能使原cDNA的量放大数百倍。同时,使用人体K562培养细胞的总RNA,对cDNA PCR文库法和反转录中的β-Actin的cDNA量进行了比较,cDNA PCR文库法中的β-Actin的cDNA量大大高于反转录中的β-Actin的cDNA量。使用75pg的人体K562培养细胞的总RNA,调制成50μl的cDNA PCR文库,使用1μl的cDNA PCR文库进行PCR反应时,可对文库中的β-Actin的cDNA进行PCR检测。因此,cDNA PCR文库显示了良好的信息放大性能。

  20. 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.

  1. Construction and Characterization of a cDNA Library from the Pulp of Cara Cara Navel Orange (Citrus sinensis Osbeck)

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A cDNA library was constructed and characterized from the pulp of Cara Cara navel orange (Citrus sinensis Osbeck) at different stages of ripening. Tittering results revealed that approximately 5.086x105 independent clones were included in this library. Electrophoresis gel results of 15 randomly selected clones revealed that the size of the insertion fragments ranged from 400 bp to 2 kb, with an average size of 900 bp. Sequencing results of 150 randomly picked clones showed that the recombination rate was 94%. During subsequent sequence analysis, 41 of 139 clones failed to be identified and the amino sequence of 71 clones shared less than 30% identity with related plants in GenBank. Of 27 clones whose amino sequences shared more than 60% identity with other related plants in GenBank, 17 clones showed an 80% identity with the corresponding candidate genes of citrus. The clone recognized as the type Ⅲ metallothionein-like (MT) gene was observed to occur 13 times, indicating that the protein may play an important role in fruit development and ripening.

  2. Construction of a cDNA library for sea cucumber Acaudina leucoprocta and differential expression of ferritin peptide

    Science.gov (United States)

    Zhou, Jun; Hou, Fujing; Li, Ye; Su, Xiurong; Li, Taiwu; Jin, Chunhua

    2016-07-01

    Acaudina leucoprocta is an edible sea cucumber of economic interest that is widely distributed in China. Little information is available concerning the molecular genetics of this species although such knowledge would contribute to a better understanding of the optimal conditions for its aquaculture and its mechanisms of defense against disease. Therefore, we constructed a cDNA library and, based on bioinformatics analysis of the sequences, the functions of 75% of the cDNAs were identified, including those involved in cell structure, energy metabolism, mitochondrial function, and signal transduction pathways. Approximately 25% of genes in the library were unmatched. The gene for A. leucoprocta ferritin was also cloned. The predicted amino-acid sequence of ferritin displayed significant homology with other sea-cucumber counterparts but indicated that it was a new member of the ferritin family. Semiquantitative real-time RT-PCR indicated the highest levels of ferritin mRNA expression in the intestine. A polyclonal antibody of ferritin was also produced. These data provide a set of molecular tools essential for further studies of the functions of ferritin protein in A. leucoprocta.

  3. 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.

  4. 人胎儿骨骼和关节RACE cDNA文库的构建%The construction of rapid amplification of cDNA ends cDNA libraries from human fetal bone and joint

    Institute of Scientific and Technical Information of China (English)

    梁晓媛; 龚瑶琴; 刘奇迹; 李江夏; 陈丙玺; 郭辰虹

    2001-01-01

    目的 建立人胎儿骨骼和关节快速扩增cDNA末端(rapid amplification of cDNA ends,RACE cDNA)文库,为分离骨骼和关节发育相关基因奠定基础。方法 采用改进的异硫氰酸胍-酚-氯仿-异戊醇一步法提取骨骼和关节总RNA,用TaKaRa公司生产的cDNA合成试剂盒合成平末端的双链cDNA,然后与衔接子连接。再用位于双链cDNA末端的通用引物扩增全部cDNA。结果 建立了从骨骼和关节构建RACE cDNA文库的方法,并用该方法成功地构建了人胎儿骨骼和关节RACE cDNA文库。结论 所构建的的利用少量总RNA构建RACE cDNA文库方法切实可行,所构建的文库适用于用RACE方法从中分离骨骼和关节发育相关基因。%Objective To construct rapid amplification cDNA ends(RACE) cDNA libraries from human fetal bone and joint and provide resources for isolation of bone- and joint- specific development-related genes.Methods Total RNA of bone and joint were extracted with the modified single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. The double-stranded end-blunted cDNA were synthesized using TaKaRa's cDNA synthesis kit and ligated to cassette adaptors. All of the cDNA molecules were amplified by a pair of common primers.Results A protocol for RACE cDNA library construction from bone and joint was established and two RACE cDNA libraries from human fetal bone and joint were successfully constructed.Conclusion The protocol of RACE cDNA library construction from limited materials proved to be simple and efficient and the library was suitable for RACE to isolate tissue-specific genes.

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

    Directory of Open Access Journals (Sweden)

    Chen Xianming

    2007-06-01

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

  6. 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.

  7. [Construction of suppression subtractive hybridization (SSH) library of copepod Pseudodiaptomous annandalei and its ferritin cDNA cloning and differential expression under nickel stress].

    Science.gov (United States)

    Jiang, Jie-Lan; Wang, Gui-Zhong; Wu, Li-Sheng; Li, Shao-Jing

    2012-07-01

    To study the molecular response mechanisms of copepod to nickel stress, a suppression subtractive hybridization (SSH) cDNA library of Pseudodiaptomous annandalei under nickel stress was constructed by using SSH technique, and a total of 140 clones were randomly picked from the growing colonies and identified by PCR. The recombinant rate of the library was 98.6%, and the volume of the library was 1.12 x 10(6) cfu. After the recombinant plasmids were sequenced, a partial cDNA fragment of ferritin was recognized based on BLAST searches in NCBI, with a size of 859 bp and continuously encoding 170 amino acid residues. The semi-quantitative PCR results showed that the ferritin cDNA under 24 h nickel stress was distinctly up-regulated. The successful construction of the SSH library and the obtaining of ferritin cDNA fragment would supply basis for the further study of the molecular response mechanisms of copepod to nickel stress.

  8. 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.

  9. Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes

    Institute of Scientific and Technical Information of China (English)

    Xing Zhong; Yu-Liang Ran; Jin-Ning Lou; Dong Hu; Long Yu; Yu-Shan Zhang; Zhuan Zhou; Zhi-Hua Yang

    2004-01-01

    AIM: To gain tumor endothelium associated antigen genes from human liver cancer vascular endothelial cells (HLCVECs)cDNA expression library, so as to find some new possible targets for the diagnosis and therapy of liver tumor.METHODS: HLCVECs were isolated and purified from a fresh hepatocellular carcinoma tissue sample, and were cultured and proliferated in vitro. A cDNA expression library was constructed with the mRNA extracted from HLCVECs.Anti-sera were prepared from immunized BALB/c mice through subcutaneous injection with high dose of fixed HLCVECs, and were then tested for their specificity against HLCVECs and angiogenic effectsin vitro, such as inhibiting proliferation and inducing apoptosis of tumor endothelial cells, using immunocytochemistry, immunofiuorescence,cell cycle analysis and MTT assays, etc. The identified xenogeneic sera from immunized mice were employed to screen the library of HLCVECs by modified serological analyses of recombinant cDNA expression libraries (SEREX).The positive clones were sequenced and analyzed by bioinformatics.RESULTS: The primary cDNA library consisted of 2x106recombinants. Thirty-six positive clones were obtained from6×10s independent clones by immunoscreening. Bio-informatics analysis of cDNA sequences indicated that 36 positive clones represented 18 different genes. Among them, 3 were new genes previously unreported, 2 of which were hypothetical genes. The other L5 were already known ones. Series analysis of gene expression (SAGE) database showed that ERP70,GRP58, GAPDH, SSB, S100A6, BMP-6, DVS27, HSP70 and NAC alpha in these genes were associated with endothelium and angiogenesis, but their effects on HLCVECs were still unclear. GAPDH, S100A6, BMP-6 and hsp70 were identified by SEREX in other tumor cDNA expression libraries.CONCLUSION: By screening of HLCVECs cDNA expression library using sera from immunized mice with HLCVECs,the functional genes associated with tumor endothelium or angiogenesis were identified. The

  10. Optimization of RNA isolation from Brittle Leaf Disease affected date palm leaves and construction of a subtractive cDNA library.

    Science.gov (United States)

    Saïdi, Mohammed Najib; Gargouri-Bouzid, Radhia; Rayanni, Mariem; Drira, Noureddine

    2009-01-01

    A simple and efficient method was described here for the isolation of high-quality RNA from date palm leaves affected with Brittle Leaf Disease (BLD) and containing high amount of phenolic compounds. The procedure was based on the use of a non-ionic detergent Nonidet-P40 (NP-40), Polyvinylpyrrolidone (PVP), and beta-mercaptoethanol in the extraction buffer in order to isolate cytoplasmic RNA and to prevent the oxidation of phenolic compounds. This method allowed the isolation of intact RNA, suitable for cDNA synthesis and library construction. Differential screening of the subtractive cDNA library from affected leaf RNA led to the identification of some BLD-induced genes.

  11. 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.

  12. Construction of Yeast Two-hybrid cDNA Library of Verticillium dahliae%大丽轮枝菌酵母双杂交 cDNA 文库的构建

    Institute of Scientific and Technical Information of China (English)

    康静敏; 刘坤; 刘严; 张怡; 李成伟; 谭光轩

    2015-01-01

    为了分离克隆与植物互作的大丽轮枝菌致病相关蛋白基因,利用 SMART 技术构建了大丽轮枝菌酵母双杂交 cDNA 文库。结果表明,构建的文库滴度为5.2×107 cfu/mL,库容为3.9×107 cfu;文库 cDNA 插入片段长度主要分布在0.5~2.0 kb,平均为1 kb;文库重组率为96%。表明文库质量较好,为筛选与植物互作的大丽轮枝菌致病蛋白基因奠定了基础。%To seek out the V. dahliae proteins involved in the interaction of plant with V. dahliae,a yeast two-hybrid library of V. dahliae was constructed using SMART technique. Results showed that the titer of cDNA library was 5. 2 × 107 cfu/mL,the library contained 3. 9 × 107 cfu independent clones,the size distribution of insert frag-ments ranged from 0. 5 -2. 0 kb,and the recombination rate was about 96%. These data demonstrated that the li-brary could meet the requirements of standard cDNA library,which laid a foundation for screening the interaction proteins from V. dahliae.

  13. [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.

  14. 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

  15. 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

  16. 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

  17. Construction and identification of directional cDNA library from Chinese giant salamander Andrias davidianus liver%大鲵肝脏组织定向cDNA文库的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    杨芳; 贺智敏; 詹显全; 陈主初; 严斌; 黄宏科; 李廷宝

    2004-01-01

    To construct a directional cDNA library from Chinese giant salamander Andrias davidianus liver by SMART (switching mechanism at 5' end of RNA transcript) technique, we purified the mRNA from Andrias davidianus liver and the first-strand cDNA was synthesized through reverse transcription by using a modified oligo (dT) primer (contained sfi Ⅰ B site). We used the SMART oligonucleotide (contained sfi Ⅰ A site) as a template so that the first-strand cDNA could be extended over the 5' end of mRNA. The double-strand cDNA was amplified by LD-PCR (long-distancePCR) with the above two primers and then digested by sfi Ⅰ ( Ⅰ A and Ⅰ B) restriction enzyme. After cDNA fractionation through CHROMA SPIN column, the double-strand cDNA was ligated into the sfi Ⅰ -digested λtripIEx2 vector and then the recombinant DNA was packaged in vitro. The content of the unamplified Andrias davidianus liver cDNA library is 1.5 × 106 in which the percentage of recombinant clones is about 98.9 %. The titer of the amplified cDNA library is 1.0 × 1010 pfu/ml and the average exogenous inserts of the recombinants is 1.25 kb. These results show that the Andrias davidianus liver cDNA library has excellent quality [Acta Zoologica Sinica 50 (3): 475 -478, 2004].

  18. Construction of a full-length cDNA library for Senecio scandens%千里光全长cDNA文库的构建及分析

    Institute of Scientific and Technical Information of China (English)

    平军娇; 张珍; 蔡振锋; 汤贤春; 钱刚

    2012-01-01

    目的 构建千里光全长cDNA文库,以期研究千里光的功能基因组学信息,为克隆药理学性状相关的功能基因提供数据资源.方法 Trizol法提取千里光叶片总RNA,通过SMART(switching mechanism at 5’end of RNA transcript)构建全长cDNA文库,随机挑取600个单克隆测序分析文库滴度、全长率及冗余率,得到的EST序列进行Blast分析(NR、NT、Swiss-Prot、KEGG)及COG功能分类.结果 文库的库容为4.3×106 cfu/mL,插入片段大小平均1.7 kb,文库重组率96.35%,全长率58.24%,冗余率10.88%;获得524条全长EST序列,含有467条独立基因(unigenes),其中5条序列与千里光次生代谢产物的合成、运输与代谢有关.结论 经检测,SMART技术成功构建了千里光全长cDNA文库,该文库可用于千里光功能基因组鉴定、新基因筛选及次生代谢产物生物合成的表达调控研究.%Objective In the present study, our information from Senecio scandens full-length cDNA clones will serve as a useful resource for elucidating functional genes and will also aid a precise annotation of genomics in Compositae plants. Methods The total RNA was extracted from S. Scandens using Trizol method. SMART (switching mechanism at 5' end of RNA transcript) was applied to constructing the full-length cDNA library. Titer of the library, full-length ratio, and redundancy rate for 600 monoclone randomly selected sequencing library were evaluated by PCR amplification. NCBI and COG database was used to compare those sequences. Results Parameters of the the quality of cDNA library were as follows: the capacity of the library (4.3* 106 cfu/mL), the average size of the inserted fragment (1.7 kb), the recombination rate (96.35%), the full-length rate (58.24%), and the redundancy rate (10.88%). EST sequences for 524 full-length were obtained in this study, involving 467 unigenes, among which five sequences associated with synthesis, transport, and metabolism of S. Scandens secondary

  19. 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.

  20. 中华蜜蜂工蜂 cDNA 文库的构建及ESTs 测序分析%Construction of cDNA libraries and ESTs sequencing of Apis cerana cerana workers

    Institute of Scientific and Technical Information of China (English)

    张卫星; 郗学鹏; 秦明; 王帅; 刘春蕾; 王红芳; 胥保华

    2016-01-01

    Objectives] To build a cDNA library to improve understanding of how honey bee workers respond to adverse conditions and analyze the quality of the resultant library. [Methods] A cDNA library of Apis cerana cerana was constructed using the SMART technique. [Results] The library’s capacity was 3.6×106 cfu/mL, the recombination rate was 97% and the average length of inserts was approximately 1 000 bp. 306 ESTs were generated by ESTs sequencing. Additionally, 234 non-repetitive sequences were formed, including 207 singletons and 27 contigs after initial assembly. Using Blastx to query, compare and annotate these sequences with those in GenBank, revealed that 141 sequences could be assigned putative functions because they were homologous to known genes. Other sequences had no obvious homology, which suggests there is potential for the discovery of new functional genes. [Conclusion] The construction of a cDNA library has important benefits for cloning, screening and gene function research in Apis cerana cerana.%【目的】为了解中华蜜蜂 Apis cerana cerana 工蜂的抗逆性,构建了中华蜜蜂工蜂的 cDNA 文库,并对文库质量进行分析。【方法】本研究利用 SMART 技术构建了中华蜜蜂工蜂的全长 cDNA 文库。【结果】文库库容为3.6×106 cfu/mL,文库重组率为97%,插入片段长度多数分布在1000 bp 左右。挑取 cDNA克隆进行 EST 测序,共进行了306个成功反应,软件拼接共得到234个单基因簇(Unigene),其中包括207个单拷贝(Singletons)序列及27个重叠群(Contigs)。使用 Blastx 将这些序列同 GenBank 等数据库进行查询、比对和注释,结果显示141条序列有相关同源性,其他序列没有明显的同源性,这也为我们发现新功能基因提供了可靠依据。【结论】此文库的构建在中华蜜蜂功能基因的分离、克隆、筛选以及基因功能研究等方面具有重要作用。

  1. Construction of a cDNA expression library of Suaeda salsa%碱蓬cDNA表达文库的构建

    Institute of Scientific and Technical Information of China (English)

    马秀灵; 王丽萍; 张慧

    2002-01-01

    以盐生植物盐地碱蓬(Suaeda salsa)地上部分为材料,提取植物总RNA,纯化出mRNA,合成cDNA第一链,得到双链cDNA后,连接入植物表达载体,构建cDNA表达文库.该文库重组子约10-6.将该文库转化农杆菌GV3101,可直接用以转化拟南芥.%In order to find new genes related to salt stress,we constructed a Suaeda salsa aerial part tissue cDNA expression library.Halophyte S.salsa was used as plant material,the first strands of cDNAs were synthesized from mRNA.Ligated the cDNAs into the expression vector,then transformed Escherichia.coli.The library titer is about 10-6.Plasmids were extracted from amplified library and were transferred intoAgrobacterium,which subsequently was used to transform Arabidopsis thaliana.

  2. Construction and characterization of normalized cDNA libraries by 454 pyrosequencing and estimation of DNA methylation levels in three distantly related termite species.

    Science.gov (United States)

    Hayashi, Yoshinobu; Shigenobu, Shuji; Watanabe, Dai; Toga, Kouhei; Saiki, Ryota; Shimada, Keisuke; Bourguignon, Thomas; Lo, Nathan; Hojo, Masaru; Maekawa, Kiyoto; Miura, Toru

    2013-01-01

    In termites, division of labor among castes, categories of individuals that perform specialized tasks, increases colony-level productivity and is the key to their ecological success. Although molecular studies on caste polymorphism have been performed in termites, we are far from a comprehensive understanding of the molecular basis of this phenomenon. To facilitate future molecular studies, we aimed to construct expressed sequence tag (EST) libraries covering wide ranges of gene repertoires in three representative termite species, Hodotermopsis sjostedti, Reticulitermes speratus and Nasutitermes takasagoensis. We generated normalized cDNA libraries from whole bodies, except for guts containing microbes, of almost all castes, sexes and developmental stages and sequenced them with the 454 GS FLX titanium system. We obtained >1.2 million quality-filtered reads yielding >400 million bases for each of the three species. Isotigs, which are analogous to individual transcripts, and singletons were produced by assembling the reads and annotated using public databases. Genes related to juvenile hormone, which plays crucial roles in caste differentiation of termites, were identified from the EST libraries by BLAST search. To explore the potential for DNA methylation, which plays an important role in caste differentiation of honeybees, tBLASTn searches for DNA methyltransferases (dnmt1, dnmt2 and dnmt3) and methyl-CpG binding domain (mbd) were performed against the EST libraries. All four of these genes were found in the H. sjostedti library, while all except dnmt3 were found in R. speratus and N. takasagoensis. The ratio of the observed to the expected CpG content (CpG O/E), which is a proxy for DNA methylation level, was calculated for the coding sequences predicted from the isotigs and singletons. In all of the three species, the majority of coding sequences showed depletion of CpG O/E (less than 1), and the distributions of CpG O/E were bimodal, suggesting the presence of

  3. Construction and characterization of normalized cDNA libraries by 454 pyrosequencing and estimation of DNA methylation levels in three distantly related termite species.

    Directory of Open Access Journals (Sweden)

    Yoshinobu Hayashi

    Full Text Available In termites, division of labor among castes, categories of individuals that perform specialized tasks, increases colony-level productivity and is the key to their ecological success. Although molecular studies on caste polymorphism have been performed in termites, we are far from a comprehensive understanding of the molecular basis of this phenomenon. To facilitate future molecular studies, we aimed to construct expressed sequence tag (EST libraries covering wide ranges of gene repertoires in three representative termite species, Hodotermopsis sjostedti, Reticulitermes speratus and Nasutitermes takasagoensis. We generated normalized cDNA libraries from whole bodies, except for guts containing microbes, of almost all castes, sexes and developmental stages and sequenced them with the 454 GS FLX titanium system. We obtained >1.2 million quality-filtered reads yielding >400 million bases for each of the three species. Isotigs, which are analogous to individual transcripts, and singletons were produced by assembling the reads and annotated using public databases. Genes related to juvenile hormone, which plays crucial roles in caste differentiation of termites, were identified from the EST libraries by BLAST search. To explore the potential for DNA methylation, which plays an important role in caste differentiation of honeybees, tBLASTn searches for DNA methyltransferases (dnmt1, dnmt2 and dnmt3 and methyl-CpG binding domain (mbd were performed against the EST libraries. All four of these genes were found in the H. sjostedti library, while all except dnmt3 were found in R. speratus and N. takasagoensis. The ratio of the observed to the expected CpG content (CpG O/E, which is a proxy for DNA methylation level, was calculated for the coding sequences predicted from the isotigs and singletons. In all of the three species, the majority of coding sequences showed depletion of CpG O/E (less than 1, and the distributions of CpG O/E were bimodal, suggesting

  4. 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.

  5. 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.

  6. 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

  7. Construct breast carcinoma T7 phage display cDNA library%乳腺癌T7噬菌体展示cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    张涛; 施宝民; 王洪; 陈鹊汀; 季堃; 余松林

    2015-01-01

    目的 构建乳腺癌组织T7噬菌体展示cDNA文库,为下一步筛选差异蛋白打下基础.方法 利用乳腺癌新鲜标本,提取总RNA,分离mRNA并进行纯化,然后合成cDNA,连接体外包装获得T7噬菌体展示cDNA文库.结果 总RNA经检测,A260/A280=1.87,纯化的mRNA产量为4.0μg,A260/A280=1.91,合成的cDNA大小在200~6 000 bp之间,原始文库的容量为2×107pfu,文库重组率为90%,插入片段长度在300~2 000 bp之间.结论 噬菌体展示技术是进行蛋白质功能研究的高效方法,构建高质量的乳腺癌噬菌体展示cDNA文库,可用于肿瘤标志物的筛选、肿瘤疫苗的研制、多肽药物的开发、靶向治疗的研究等众多领域.%Objective To construct the breast carcinoma T7 phage display cDNA library,so the foundation for the screening of differentially expressed proteins was laid. Methods Firstly,fresh specimens of breast carcinoma was used to extract total RNA,separating and purifying of mRNA. Then synthesis cDNA was done. At last,the packaging was connected in vitro and T7 phage display cDNA library was obtained. Results The total RNA was tested,the result is A260/A280=1.87. The purified mRNA is 4.0μg,A260/A280=1.91. The size of cDNA is 200-6 000 bp. The primary library capacity is 2 × 107pfu. Recombination rate of the library is 90%. The length of inserted fragments is 300-2 000 bp. Conclusions Phage display is an efficient method of protein function research. We constructed a high quality breast cancer phage display cDNA Library. The library can be used for screening tumor markers,tumor vaccine,polypeptide drug development,targeted research,and so on.

  8. Isolation, Identification and cDNA Library Construction of Glyphosate-Resistant Fungus ( Candida palmioleophila )%抗草甘膦真菌(Candida palmioleophila)分离鉴定及其cDNA文库构建

    Institute of Scientific and Technical Information of China (English)

    于海涛; 金龙国; 蒋凌雪; 韩玉军; 陶波; 邱丽娟

    2012-01-01

    A glyphosate-resistant fungus strain was isolated from sludge of glyphosate factory outfall by the flask-foster enrichment technology. Based on its morphological, physiological and biochemical properties as well as the 18S rRNA sequence analysis results,the strain was tentatively identified as Candida palmioleophila. A cDNA library driven by the total RNA extracted from the strain was constructed by SMARTer technology of Clontech company. The library quality was evaluated, and the results showed that the titer of primary cDNA library and amplified cDNA library were 2. 58 x 106 cfu/mL and 3. 42 x 109 cfu/mL, respectively, with the library volume of about 2. 58 x 106 cfu and the recombination rate of 97. 6%. The inserted fragments were distributed from 500bp to 3kb,and the most cDNA fragments were distributed around lkb. These results indicated that the strain TY-JM cDNA library was constructed successfully.%利用瓶培养富集技术从生产草甘膦工厂排污口的污泥中筛选抗草甘膦菌株,通过菌株形态学鉴定、生理生化特性测定及18S rRNA序列分析,确定其为假丝酵母菌(Candida palmioleophila).以供试的抗草甘膦菌株总RNA为起始模板,利用Clontech公司的SMARTer技术构建cDNA文库.文库质量鉴定表明:原始文库滴度为2.58×106cfu/mL,扩增后文库滴度为3.42×109cfu/mL,文库库容为2.58×106cfu,重组率为97.6%,插入片段范围为500bp ~3kb,主要集中在1kb附近.表明构建的TY-JM的cDNA文库质量符合要求.

  9. Construction of an adult barnacle (Balanus amphitrite cDNA library and selection of reference genes for quantitative RT-PCR studies

    Directory of Open Access Journals (Sweden)

    Burgess J Grant

    2009-06-01

    Full Text Available Abstract Background Balanus amphitrite is a barnacle commonly used in biofouling research. Although many aspects of its biology have been elucidated, the lack of genetic information is impeding a molecular understanding of its life cycle. As part of a wider multidisciplinary approach to reveal the biogenic cues influencing barnacle settlement and metamorphosis, we have sequenced and annotated the first cDNA library for B. amphitrite. We also present a systematic validation of potential reference genes for normalization of quantitative real-time PCR (qRT-PCR data obtained from different developmental stages of this animal. Results We generated a cDNA library containing expressed sequence tags (ESTs from adult B. amphitrite. A total of 609 unique sequences (comprising 79 assembled clusters and 530 singlets were derived from 905 reliable unidirectionally sequenced ESTs. Bioinformatics tools such as BLAST, HMMer and InterPro were employed to allow functional annotation of the ESTs. Based on these analyses, we selected 11 genes to study their ability to normalize qRT-PCR data. Total RNA extracted from 7 developmental stages was reverse transcribed and the expression stability of the selected genes was compared using geNorm, BestKeeper and NormFinder. These software programs produced highly comparable results, with the most stable gene being mt-cyb, while tuba, tubb and cp1 were clearly unsuitable for data normalization. Conclusion The collection of B. amphitrite ESTs and their annotation has been made publically available representing an important resource for both basic and applied research on this species. We developed a qRT-PCR assay to determine the most reliable reference genes. Transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, although other genes also performed well and could prove useful to normalize gene expression studies.

  10. 玉米弯孢叶斑病菌酵母双杂交cDNA文库的构建及评价%Construction and characterization of yeast two hybrid cDNA library of Curvularia lunata

    Institute of Scientific and Technical Information of China (English)

    荆晶; 刘铜; 陈捷

    2012-01-01

    由新月弯孢[Curvularia lunata (Wakker)Boed]引起的玉米弯孢叶斑病是我国玉米生产区的一种重要性叶斑病,曾在20世纪90年代给我国玉米生产造成较大损失.目前对该病原菌致病类型鉴定与致病相关基因、病害发生规律、综合防治等方面均有较好的研究基础[1],并且在前期的研究中发现该病菌调控毒素形成的相关基因与色素合成相关基因在表达上存在某种关联性[2],由于这2个基因均为致病相关基因,因此研究两基因表达的蛋白的关联性对于全面揭示病菌致病机理和调控网络具有重要意义.%BD SMART technique and LD-PCR were applied to synthesize double strand cDNA(ds cDNA). The ds cDNA and pGADT7-Rec were transfered into competent yeast cell to construct yeast two-hybrid cDNA library of Curvularia lunata by homologous recombination method. The results showed that library capacitance was 2.46×105 and transformation rate was 2.13×105/μg pGADT7-Rec. The plaque titer of the library was 2.5×108 pfu/mL and the recombination frequency was 88. 24%. The length of inserted cDNA fragment was ranged from 0.4 kb to 3 kb in average. This is the first time to use BD SMART technique to construct yeast two-hybrid cDNA library of C. lunata by homologous recombination.

  11. Construction and evaluation of yeast two-hybrid cDNA library of Diaphorina citri (Hemiptera: Psyllidae)%柑橘木虱酵母双杂交cDNA文库的构建及评价

    Institute of Scientific and Technical Information of China (English)

    马晓芳; 陈国庆; 张学潮; 徐海君

    2013-01-01

    In order to study the interacting proteins between the Asian citrus psyllid ( Diaphorina citri) and Candidatus Liberibacter asiaticus ( CLas) which is the pathogenic bacterium causing Huanglongbing, yeast two-hybrid cDNA library of D. citri was constructed using the Switching Mechanism at 5' End of the RNA Transcript (SMART) technique. The total RNA was isolated from the citrus psyllid adults bred in the laboratory and subjected to reverse transcription, and the double-strand cDNAs ( ds cDNAs) were synthesized. The ds cDNAs were ligated with homologous adapter and purified by the chromatography column. By using homologous recombination reaction, the ds cDNAs were transformed into the Y187 competent cell with the library plasmid pGADT7-Rec to construct yeast two-hybrid cDNA library. Detection of the library indicated that it contained more than 106 independent clones, the titer of the amplified library was 2. 23 × 10 cfu/mL, and the average size of inserts was above 750 bp in the cDNA library. These results demonstrate that the library meets the requirements of the standard cDNA library. Moreover, two membrane proteins, ORF420 and ORF3420, from ( CLas) were used as bait proteins to screen the interacting proteins in the library, but no positive clone was screened in the tests. The yeast two-hybrid cDNA library of D. citri will be useful for the research on the interaction between insect vectors and C. Liberibacter asiaticus in the future.%为了探索研究柑橘木虱Diaphorina citri与柑橘黄龙病(Huanglongbing,HLB)病原菌的相互作用蛋白,本研究运用RNA转录中5 '末端转换机制(Switching Mechanism at 5'End of the RNA Transcript,SMART)技术构建了柑橘木虱的酵母双杂交cDNA文库.以实验室饲养的柑橘木虱为材料,提取总RNA,经反转录后合成ds cDNA,两端添加同源重组序列,并用层析柱纯化;ds cDNA与文库质粒pGADT7-Rec在酵母Y187感受态细胞内发生同源重组,柑橘木虱cDNA重组到文库质粒

  12. 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.

  13. Rapid construction of directional cDNA library from human nasopharynx%鼻咽上皮组织定向cDNA文库的快速构建

    Institute of Scientific and Technical Information of China (English)

    张必成; 余鹰; 邱元正; 钱骏; 周鸣; 李忠花; 张小慧; 向娟娟; 朱诗国; 李桂源

    2001-01-01

    Objective To construct a directional cDNA library from human adult nasopharynx by SMART (switching mechanism at 5′ end of RNA transcript) technique. Methods The total RNA was separated from human adult nasopharynx epithelial fissue and the frist-strand cDNA was synthesized through reverse transcription by a modified oligo(dT) primer(contained sfi IB site) while the SMART oligonudeotide(contained sfi IA site) was utilized as a template so that the frist-strand cDNA could be extended over the 5′end of mRNA. The double-strand cDNA was amplified by LD-PCR(long-distance PCR) with the above two primers and then digested by sfi I (IA & IB) restriction enzyme.After cDNA size fractionation through CHROMA SPIN column,the double-strand cDNA was ligated into the sfi I-digested λTripIEx2 vector and then the recombinant DNA was packaged in vitro. Results The unamplified human adult nasopharynx cDNA library consists of 1.5×106 independent clones in which the percentage of recombinant clones is about 100%.The titer of the amplified cDNA library is 3.8×109 pfu/ml and the average exogenous inserts of the recombinants is 1.5 kb. Conclusion These results shows that the human adult nasopharynx cDNA library has an excellent quality and lays solid foundation for screening and cloning new tumor suppressor genes of nasopharyngeal carcinoma(NPC) and tissue-specific genes of human nasopharynx.%目的采用SMART (switching mechanism at 5′ end of RNA transcript)技术,快速构建了高质量的成人鼻咽上皮组织定向cDNA文库。方法从成人正常鼻咽上皮组织分离总RNA,利用经修饰的oligo(dT)引物(含sfi IB酶切位点)合成cDNA第一链,同时根据真核生物mRNA 5′端帽子结构特点,利用SMART核苷酸(含sfi IA酶切位点)作为cDNA第二链在mRNA 5′端延伸出去的模板,进而以此序列为引物利用LD-PCR(Long-distance-PCR)合成双链cDNA,双链cDNA经sfi I(IA & IB)酶切和过柱分

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Construction and Identification ofa cDNA Library of Human Rheumatoid Arthritis Synovial Tissue%人类风湿性关节炎滑膜组织cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    闫永毅; 任蕾; 高飞; 卢秀敏; 刘彦虹

    2012-01-01

    Objective:To construct a cDNA library of human synovial tissue of RA and indentify the quality of the library. Methods: Total RNA was extracted and mRNA was purified. mRNA was reversed to first-strand cDNA which was amplified to double-strand cD-NA by long distance PCR. PCR products were digested by proteinase K and Sfi I, and were fractionated by CHROMA SPIN-400 column. The cDN A of length longer than 0.4kb were collected and ligated toλ TriplEx2 vector. The λ phage packaging reaction for the ligated DNA was performed to produce an unamplified library. Thereafter, the unamplified library was titered and the percentage of recombinant clones were detected. In the end, fourty plaques were randomly selected and amplified by PCR using universal primers from vector in order to test the qualify of the obtained library. Results: The titers of unamplifed and amplified libraries were 6.89x106 pfu/mL and 2.63x 109 pfu/mL respectively. The rate of recombinant was 93%. The insert size range from 300 to 1800 bp. Conclusions: A high quality cD-NA library from human synovial tissue of RA was constructed successfully, and it lays solid foundation not only for screening and cloning new special genes associated with the occurrence of RA, but also for gene therapy of it.%目的:构建人类风湿关节炎(RA)滑膜组织cDNA文库.筛查与RA相关的特异基因,为探讨RA的发病机制及基因治疗奠定基础.方法:提取人类风湿关节炎滑膜组织RNA并使用Trizol法纯化mRNA;运用mRNA5'末端的模板转换方法以powerscriptTM逆转录酶进行转录,使用COS Ⅲ/3'PCR引物合成第1链cDNAs;长距离聚合酶链反应(LD-PCR)合成双链cDNA; PCR产物经蛋白酶K水解并纯化后,经SfiI酶切、柱层析洗脱,重组于TripIEx2载体并包装后,测定滴度、重组率、扩增文库,随机挑取40个噬菌斑,用载体克隆位点两端的通用引物进行PCR扩增,以检测所构建cDNA文库的质量.结果:未扩增文库的滴度为6.89× 106pfu/m

  3. 莫氏巴贝斯虫裂殖子cDNA表达文库的构建及免疫学筛选%Construction and Immunoscreening of a Merozoites cDNA Expression Library of Babesia motasi

    Institute of Scientific and Technical Information of China (English)

    王锦明; 罗建勋; 殷宏; 关贵全; 刘军龙; 刘爱红; 马米玲; 牛庆丽; 任巧云; 杨吉飞; 刘志杰; 李有全

    2012-01-01

    为了构建莫氏巴贝斯虫(Babesia motasi)裂殖子cDNA表达文库,从中筛选和鉴定功能基因,利用差速离心法从莫氏巴贝斯虫感染的红细胞中纯化裂殖子,提取总RNA并纯化mRNA.在合成的双链cDNA两端加上含EcoRⅠ和Hind Ⅲ定向接头后连接到λ SCREEN载体上.通过体外包装形成完整的噬菌体颗粒,并用之转染ER1647,构建莫氏巴贝斯虫裂殖子的cDNA表达文库.用莫氏巴贝斯虫阳性血清筛选cDNA文库,获得的阳性克隆,经过测序和Blast软件分析鉴定,然后利用末端快速扩增技术(RACE)对筛选到的功能基因片段进行全长扩增.结果表明构建的cDNA表达文库其初级库容量约为1.0×106 PFU,扩增文库为3.5×109 PFU;通过免疫学筛选,获得50个阳性克隆.测序和Blast分析后,鉴定出10个莫氏巴贝斯虫基因片段,RACE扩增获得了其中8个基因的全长.本试验构建的莫氏巴贝斯虫裂殖子的cDNA表达文库和筛选到的10个结构和功能基因,为研究巴贝斯虫生物学特性、筛选疫苗与诊断用抗原及药物靶位奠定了基础.%The objective of this study was to obtain functional genes of Babesia motasi, a cDNA expression library of the merozoites was constructed and immunoscreened with positive sera from sheep infected with B. motasi. The merozoites of B. motasi were purified from red blood cell with differential centrifugation. The mRNA was purified from extracted total RNA. Synthetized double-strand cDNA was added directional EcoR I /Hind H linkers and ligated to the EcoR I / Hind III arms of X screen vector. To produce a primary cDNA library of B. motasi, the phages DNA was packaged in vitro and transfected into ER1647. The positive clones were obtained by immunoscreening with the positive sera against B. motasi and amplified with rapid amplificationof cDNA ends (RACE). The titers of the primary and amplified cDNA expression library were 1. 0X106 PFU and 3. 5X1O9PFU ·mL-1, respectively. The results

  4. Construction of Full-length cDNA Library for Antler Tip Tissue of Sika Deer%东北梅花鹿鹿茸尖端组织全长cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    郝丽; 李和平; 严厉

    2009-01-01

    为克隆出与鹿茸生长发育相关基因的全长序列,采用SMART技术构建了东北梅花鹿鹿茸尖端组织的全长cDNA文库.用SV Total RNA Isolation System试剂盒提取总RNA,以逆转录酶PowerScriptTM 反转录合成第一链cDNA,然后通过LD-PCR合成并扩增ds cDNA.扩增产物经纯化、SfiⅠ酶切、过CHROMA SPIN-400柱去除小片段后,连接到SfiⅠ消化过的pDNR-LIB质粒载体中,最后用电转化法将重组质粒转化到E. coli DH5α内得到原始文库.经测定,构建的原始文库约含有2.56×10~6个重组子,插入片段多在0.5~2kb之间,平均插入片段长度约1.1kb,重组效率接近100%.结果表明,东北梅花鹿鹿茸尖端组织的全长cDNA文库已构建成功.%A study was conducted to construct full-length cDNA library from antler tip tissues of Sika Deer (Cenna nippon hortu-lonun) by SMART technique in order to clone new special genes for development of antler. The total RNA was extracted u-sing SV Total RNA Isolation System. Single-stranded cDNA was synthesized using PowerScripiTM reverse transcriptase,and double-stranded cDNA was synthesized and amplified by long-distance PCR. The PCR products were digested by pro-teinase K and purified. After digestion with Sfi I and size fractionation using CHROMA SPIN -400TM Columns, SMART cDNA was ligated to the Sfi I-digested, dephosphorylated pDNR-LIB vector, and the ligation mixture was transformed into E. call DH5a by electroporation. The primary cDNA library contained 2.56×10~6 independent clones with DNA inserts of 0.5~2. 0 kb, the average size of inserted cDNAs was 1.1 kb, and the recombination percentage was about 100%. Results showed that the full-length cDNA library from antler tip tissues of Sika Deer was successfully constructed.

  5. 龙须菜四分孢子体cDNA文库的构建%Construction of a cDNA Library from the Tetrasporophyte of Gracilaria lemaneiformis (Rhodophyceae)

    Institute of Scientific and Technical Information of China (English)

    孙雪; 张学成; 隋正红; 茅云翔

    2003-01-01

    为了研究不同世代和性别基因表达谱的差异,本文构建了龙须菜(Gracilaria lemaneiformis)四分孢子体的cDNA文库.总RNA提取使用RNeasy Plant Mini Kit(Qiagen),cDNA文库构建使用SMART cDNA Library Construction Kit(Clontech),包装蛋白购自Promega公司.该cDNA文库含有1.28×106个克隆,扩增文库的滴度是1.98×109 pfu/mL,重组频率是85.0%,插入片段几乎全部集中在500~1500 bp之间.

  6. 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

  7. Construction of a full-length cDNA Library from Chinese oak silkworm pupa and identification of a KK-42-binding protein gene in relation to pupa-diapause termination

    Directory of Open Access Journals (Sweden)

    Yu-Ping Li, Run-Xi Xia, Huan Wang, Xi-Sheng Li, Yan-Qun Liu, Zhao-Jun Wei, Cheng Lu, Zhong-Huai Xiang

    2009-01-01

    Full Text Available In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 × 105 cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4% were known genes but only five from A. pernyi, 37 (21.2% were known ESTs without function annotation, and 41 (23.4% were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151 was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination.

  8. 凡纳滨对虾肌肉组织cDNA全长文库的构建%Construction of the full length cDNA library from muscular tissue of Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    熊建华; 高永华; 马宁; 盛小伟; 陈晓汉

    2011-01-01

    [目的]为了在短期内获得大量凡纳滨对虾肌肉组织的功能基因表达信息,为深入了解功能基因在凡纳滨对虾肌肉组织中的表达提供分子生物学依据.[方法]通过构建凡纳滨对虾肌肉组织的全长cDNA文库,并进行EST测序分析.[结果]文库质量分析表明,初始文库库容约8.50×106 CFU,重组率在95%左右,插入片断大小为0.54~4.0 kb,多数在1.0 kb以上.随机测序72条cDNA,可得到有功能注释的37条全长cDNA和18条编码未知蛋白的基因序列.通过Gene Ontology功能分类可将有功能注释的37个基因分为蛋白质合成、细胞骨架、细胞信号传导、代谢、转运、能量、转录、抗病及防御、生殖发育和未知功能基因等10类,其中蛋白质合成类基因最多(27.03%).与细胞骨架(13.51%)、细胞信号传导(13.51%)及代谢类基因(13.51%)共占67.56%.[结论]构建凡纳滨对虾肌肉组织的全长cDNA文库,可实现短期内获得大量凡纳滨对虾肌肉组织的功能基因表达信息.%[Objective]The studies had been undertaken in order to understand the biological basis of expression of functional genes in muscular tissue of Litopenaeus vannamei. [Method]The full length cDNA library from muscular tissue of Litopenaeus vannmei was constructed and expressed Sequence Tags (ESTs) were sequenced. [Result]The constructed library was 8.50×106 CFU in capacity with 95% recombinant coefficient. The PCR results showed that the inserts ranged from 0.5 to 4.0 kb and most of them were larger than 1.0 kb. 72 clones were randomly selected and sequenced for full length. Of which, 37 cDNA sequences were identified with known functions, and 18 cDNA sequences remained as unidentiffed. Using gene ontology function classification, 37 cDNA sequences with known function were classified into groups of protein synthesis, cytoskeleton, signal transduction, metabolism, transporter, energy, transcription factors, response to disease

  9. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    Science.gov (United States)

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

  10. 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. 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

  12. 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.

  13. 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.

  14. CDNA library from the Latex of Hevea brasiliensis

    Directory of Open Access Journals (Sweden)

    Wilaiwan Chotigeat

    2010-12-01

    Full Text Available Latex from Hevea brasiliensis contains 30-50% (w/w of natural rubber (cis-1,4-polyisoprene, the important rawmaterial for many rubber industries. We have constructed a cDNA library from the latex of H. brasiliensis to investigate theexpressed genes and molecular events in the latex. We analyzed 412 expressed sequence tags (ESTs. More than 90% of theEST clones showed homology to previously described sequences in public databases. Functional classification of the ESTsshowed that the largest category were proteins of unknown function (30.1%, 11.4% of ESTs encoded for rubber synthesisrelatedproteins (RS and 8.5% for defense or stress related proteins (DS. Those with no significant homology to knownsequences (NSH accounted for 8.7%, primary metabolism (PM and gene expression and RNA metabolism were 7.8% and6.6%, respectively. Other categories included, protein synthesis-related proteins (6.6%, chromatin and DNA metabolism(CDM 3.9%, energy metabolism (EM 3.4%, cellular transport (CT 3.2%, cell structure (CS 3.2%, signal transduction (ST2.2%, secondary metabolism (SM 1.7%, protein fate (PF 2.2%, and reproductive proteins (RP 0.7%.

  15. 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。

  16. 申克孢子丝菌酵母相和菌丝相cDNA消减文库的构建%Construction of cDNA subtractive library for the yeast and mycelium phases of Sporothrix Schenckii

    Institute of Scientific and Technical Information of China (English)

    周汛; 杨致邦; 郭丽媛; 肖异珠

    2011-01-01

    Objective To explore molecular mechanism of dimorphic transition of Sporothrix schenckii, differential gene expression in dimorphic transition of Sporothrix schenckii was screened. Methods cDNA subtractive library for the yeast ( Y ) and mycelium ( M ) phases was constructed by suppression subtractive hybridization ( SSH ) . Bioinformatics analysis was performed to profile the relationship between the differently expressed genes and dimorphic transition. Results 751 ESTs were obtained in M + Y library , with the average length of 690. 98bp. Meanwhile . 875 ESTs were obtained in Y + M library, with the average length of 575. 9bp. After splicing of ESTs,101 unigenes were obtained in M + Y library and 249 unigenes in Y + M library. Some structure genes and function-unknown genes in these differentially expressed genes appeared to be related to dimorphic transition as compared with BLASTN. Conclusion It is evident that the subtracted cDNA library for the dimorphic transition of Sporothrix Schenckii was successfully constructed, thereby providing solid foundation for screening the genes involved the dimorphic transition of Sporothrix schenckii.%目的 筛选与申克孢子丝菌酵母相与菌丝相双相转换相关的差异表达基因,为探讨其双相转换的分子的机制奠定基础.方法 应用抑制性消减杂交技术,构建高特异性的申克孢子丝菌菌丝相(mycelium,M)和酵母相(yeast,Y)的正反cDNA消减文库,并对其差异表达的基因进行生物信息学分析.结果 M+Y文库获得751条表达序列标签,平均长度为690.98 bp,经拼接后获得101条非冗余序列.Y+M文库获得875条表达序列标签,平均长度为575.9 bp,拼接获得249条非冗余序列.经BLASTN比对,这些差异表达基因中,某些结构基因和功能不明的细胞分子类基因可能与双相转换有关.结论 成功构建了高特异性的申克孢子丝菌菌丝相和酵母相的正反cDNA 消减文库,为进一步筛选申克孢子丝菌的双相转换基因奠定了基础.

  17. Construction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr.) Different Developmental Stages

    Science.gov (United States)

    Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng

    2012-01-01

    To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing. PMID:23202944

  18. Construction and evaluation of normalized cDNA libraries enriched with full-length sequences for rapid discovery of new genes from Sisal (Agave sisalana Perr.) different developmental stages.

    Science.gov (United States)

    Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng

    2012-10-12

    To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing.

  19. 黄瓜幼果cDNA文库构建与EST测序分析%Construction of a Young Fruit cDNA Library and EST Sequencing in Cucumis sativus

    Institute of Scientific and Technical Information of China (English)

    潘宇; 蒲志群; 肖雅文; 赵名琛; 郑浴; 石士涛; 胡小燕; 张兴国

    2013-01-01

    将黄瓜授粉前后多个发育阶段的幼果组织等量混合后提取总RNA和mRNA,以λTriplEx2为栽体、XL1-Blue为宿主茵,构建了1个黄瓜幼果cDNA文库;其滴度为1.165×106pfu/mL,重组率在94.4%左右.测序获得116条EST,92.2%的长度在400 bp以上,19%为重叠序列.在GenBank中进行BLAST分析后确认与已知功能基因相似的EST序列有71条,有相似序列而功能未知的基因和没有相似序列的EST序列各占19.83%和18.97%.从对文库的检验结果看,构建的cDNA文库重组率较高,库容达到预期要求.%The growth and development of cucumber (Cucumis sativus L.) fruit is closely related to its yield and quality.To gain the gene expression pattern of the young fruit just before and after pollination is important to exploring the molecular mechanisms of parthenocarpy and fruit growth initiation.In this study,tissues of young fruit of cucumber at different development stages before and after pollination were mixed and total RNA and mRNA were extracted.Then,a cDNA library of cucumber young fruit with a titer of 1.165 × 106 pfu/mL and a recombinant frequency of 94.4% was constructed,using λTriplEx2 as a vector and XL1-Blue as the host strain.One hundred and sixteen EST sequences were obtained,of which 92.2% were over 400 bp in size and 19% were contigs.BLAST analysis in GenBank revealed that 71 of the 116 ESTs were homologous to genes of known function,19.83% were related to genes with unknown functions and 18.97 % were novel.The cDNA library sufficed the criteria with high recombinant efficiency and wide representativeness.The results will facilitate the cloning of development-related genes from cucumber fruit.

  20. 致农民肺嗜热吸水链霉菌cDNA文库的构建及体外表达%Construction of cDNA library and antigen expression of Streptomyces Thermohydroscopicus in vitro

    Institute of Scientific and Technical Information of China (English)

    凌媛; 王玲玲; 刘朔; 马列; 王群; 张丽娇; 何晓雨; 赵明静; 王笑歌

    2013-01-01

    目的 构建致农民肺嗜热吸水链霉菌cDNA文库并进行EST测序,通过生物信息学软件分析EST序列同源性及生物学功能,筛选重组阳性基因克隆进行体外表达.方法 利用SMART方法构建致农民肺嗜热吸水链霉菌的cDNA文库,随机挑选重组阳性克隆进行EST测序.挑选有关目的基因连接入pET-28a载体并转化大肠埃希菌BL2 (DE3),IPTG诱导体外表达.结果 该文库的重组率为96.3%,是一个较高质量的文库.从文库中随即挑选的1 020个重组阳性的克隆,获得978个有义序列,平均长度为495 bp,含有347个单一基因,同源性比较发现部分序列与猪胸膜肺炎放线菌外膜蛋白(omla),转铁蛋白B(TbpB)具有较高的同源性,分别为51%和42%.将重组阳性的基因克隆至载体中,IPTG诱导得到表达产物的相对分子质量分别为63×10 3和30× 103.结论 所构建的致农民肺嗜热吸水链霉菌cDNA文库包含全部EST序列,可能包含嗜热吸水链霉菌相关疾病巨大部分毒力分子,这些数据的积累将有助于农民肺特异性毒力分子的进一步筛选,同时为研究农民肺的发病机制奠定基础,如果能准确筛选出相关毒力基因,将为农民肺血清学诊断以及特异性疫苗的研发奠定基础.%Objective To construct the cDNA library of Streptomyces Thermohydroscopicus and to express some of the specific antigens in vitro.Methods The cDNA library of Streptomyces Thermohydroscopicus was constructed by switching mechanism at 5'end of RNA transcript approach.The recombinant positive clones were selected randomly for EST sequencing and two candidate genes were sub-cloned into expression vector pET-28a.The recombinants were then transformed into BL2 (DE3) and proteins were expressed by the induction of IPTG.Results A high-quality cDNA library of Streptomyces Thermohydroscopicus was constructed with the recombination rate of 96.3%.A set of 978 valid sequences were obtained out of total 1020

  1. 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

  2. Transposase mediated construction of RNA-seq libraries.

    Science.gov (United States)

    Gertz, Jason; Varley, Katherine E; Davis, Nicholas S; Baas, Bradley J; Goryshin, Igor Y; Vaidyanathan, Ramesh; Kuersten, Scott; Myers, Richard M

    2012-01-01

    RNA-seq has been widely adopted as a gene-expression measurement tool due to the detail, resolution, and sensitivity of transcript characterization that the technique provides. Here we present two transposon-based methods that efficiently construct high-quality RNA-seq libraries. We first describe a method that creates RNA-seq libraries for Illumina sequencing from double-stranded cDNA with only two enzymatic reactions. We generated high-quality RNA-seq libraries from as little as 10 pg of mRNA (∼1 ng of total RNA) with this approach. We also present a strand-specific RNA-seq library construction protocol that combines transposon-based library construction with uracil DNA glycosylase and endonuclease VIII to specifically degrade the second strand constructed during cDNA synthesis. The directional RNA-seq libraries maintain the same quality as the nondirectional libraries, while showing a high degree of strand specificity, such that 99.5% of reads map to the expected genomic strand. Each transposon-based library construction method performed well when compared with standard RNA-seq library construction methods with regard to complexity of the libraries, correlation between biological replicates, and the percentage of reads that align to the genome as well as exons. Our results show that high-quality RNA-seq libraries can be constructed efficiently and in an automatable fashion using transposition technology.

  3. 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。

  4. 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

  5. Cassava cDNA Library Construction: One Tool for Biotechnological Development of the Crop CONSTRUCCIÓN DE UNA LIBRERÍA DE ADNc EN YUCA: UNA HERRAMIENTA PARA EL DESARROLLO BIOTECNOLÓGICO DEL CULTIVO

    Directory of Open Access Journals (Sweden)

    CAROLINA GONZÁLEZ ALMARIO

    el estudio de su función a través de la identificación y la interacción entre proteínas.Cassava is one of the most important crops for global food security and provides food and livelihood for 600 million people in the developing world. It is also good source of starch, with levels between 73.7 y 84.9% of total dry weight in roots (FAO, 2007. Cassava starch can be used in a wide range of industries (textile, cosmetic, nourishing, etc and it has a high potential for the production of biofuel. Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam, is one of the most important diseases that affects cassava. This disease can compromise the starch supply not only for bioetanol production but also affect global food security. The long reproductive cycle, high heterozigosity and tetraploid character of cassava are characteristics that have complicated the genetic breeding for this crop. For these reasons new alternatives based on biotechnology are necessary to accelerate its improvement. In the postgenomic era many experiments rely on the availability of transcript sequences for cloning. As these clones usually originate from cDNA libraries, the quality of these libraries is crucial. In this article we report the construction of the first cassava cDNA library employing the Gateway® system. For this, in vitro grown plants were inoculated with the Xam strain CIO151. The expression library shows a high titer of 1 x 10(7 cfu/ml, with inserts ranging between 600 and 1500 bp. The sequence analyses from 14 random clones confirmed that these are expressed genes and showed similarity with previously cloned genes from species related to cassava. This library is an excellent resource for the identification of novel genes and for functional studies through the identification of their interactions with other proteins.

  6. 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.

  7. SMARTer技术构建辣椒黄绿苗突变体叶片全长cDNA文库%Construction of full-length cDNA library of yellow bud mutant leaves in Capsicum annuum L.using SMARTer technique

    Institute of Scientific and Technical Information of China (English)

    马志虎; 孙国胜; 张昌伟; 杨玉霞; 潘跃平

    2013-01-01

    本研究以辣椒黄绿苗嫩叶为材料,提取总RNA,采用LD-PCR技术合成First-strand cDNA和ds cDNA.将分级纯化后的ds cDNA连接到载体pSMART2IFD上,用电穿孔法将重组子转化到大肠杆菌感受态细胞DH5α中,构建辣椒全长cDNA文库.文库质量检测结果显示:原始文库滴度为1.76×106 PFU/ml,重组率为94%,插入片段长度为500~2 000 bp,平均长度为1 170 bp,表明构建的辣椒叶片cDNA文库较为理想,可用于目的基因筛选.%Total RNA was extracted from yellow bud mutant leaves of Capsicum annuum L. , and first-strand cDNA and ds cDNA were synthesized by LD-PCR technology. The purified ds cDNA was connected to vector pSMART2IFD, and the recombinant vectors were transformed into competent Escherichia coli cells DH5a by electroporation to construct full-length cDNA library of Capsicum annuum L_ The library quality test results showed the titer of original library was 1.76× 106PFU/ml, the recombination rate was 94% , and the inserted fragment length was 500-2 000 bp, indicating that the library was ideal for target genes selection.

  8. 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

  9. A method for generating subtractive cDNA libraries retaining clones containing repetitive elements.

    OpenAIRE

    1997-01-01

    Here we describe a two-stepped photobiotin-based procedure to enrich a target (canine retinal) cDNA library for tissue specific clones without removing those containing repetitive ( SINE ) elements, despite the presence of these elements in the driver population. In a first hybridization excess SINE elements were hybridized to a driver (canine cerebellar) cDNA. In a second hybridization target cDNA was added to this reaction. The resulting cDNA library was enriched for retinal specific clones...

  10. 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.

  11. Sequence characterization of a human embryonic craniofacial cDNA library

    Energy Technology Data Exchange (ETDEWEB)

    Padanilam, B.J.; Barsel, S.; Solursh, M. [and others

    1994-09-01

    Broad-based sequencing approaches for the characterization of human cDNA libraries have proven successful in identifying large numbers of novel genes of specific tissue or developmental stages. To pursue our interests in human craniofacial development, stages. To pursue our interests in human craniofacial development, we have made use of both subtracted and unsubtracted cDNA libraries constructed from embryonic craniofacial tissue obtained from pooled samples at 42-54 days gestation. Single-pass sequencing was carried out using an ABI automated sequencer and T3 or T7 primers. Sequences were characterized using BLAST and GRAIL, and the identified homologous sequences grouped according to gene class and family. Four genes have been mapped using repeat sequence elements identified in the clones. Using primers developed from sequence data, other genes are being mapped using a panel of somatic cell hybrids. To date, a total of 786 sequences have been returned with 35% identifying no homologies, and 35% with strong homologies to previously identified genes. A number of genes previously identified to play a role in human embryonic development have been returned from the sequence comparisons providing evidence that the library is representative of this tissue and stage of development. Previous characterization of the library has also identified a number of novel embryonically expressed human homeobox genes. Genes felt to be of special relevance based on their homology to characterized genes known to play a role in development or that are members of novel classes but with high scores on GRAIL searches are being characterized using whole mount in situ hybridization with mouse embryos. Characterization of the library with respect to chromosomal mapping, gene types and make-up, and embryonic expression patterns will be presented.

  12. 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.

  13. Construction of cDNA Libraries and Identification of Head Kidney from Scomberomorus niphonius%蓝点马鲛头肾cDNA文库的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    王丹妮; 刘军

    2015-01-01

    研究运用SMART技术对蓝点马鲛头肾构建了cDNA文库,该文库原始文库滴度为2.26×106pfu/mL,文库容量为1.36×106pfu,符合文库构建的要求。从文库中随机挑选24个单克隆菌,以载体的通用引物进行菌落检测PCR,结果表明:重组率为95.8%,插入片段大小平均为1000bp。该文库的构建满足了基因的分离与筛选,为进一步研究其相关基因克隆及分子生物学研究奠定基础。%cDNA library of Scomberomorus niphonius head kidney was constracted by SMART technology. The original library titer is 2.26×106pfu/mL,and library capacity is 1.36×106pfu,meeting the requirements of library construction. RandomLy selected 24 monoclonal bacteria from the library,subjected to colony PCR detected by using universal primers. The results showed that recombination rate was 95.8%,an average in⁃sert size was 1 000bp. Construction of the library meet the isolation and screening of gene,and lay the foundation for further study of melecμlar biology and gene cloning.

  14. Construction and characterization of a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B%类泛素FAT10高表达肝癌细胞株Hep3B酵母双杂交cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    余新; 刘天; 德洪波; 李国惠; 邵江华

    2011-01-01

    AIM: To construct a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B.METHODS: Total RNA was prepared from Hep3B cells and used to purify poly (A) mRNA.Double-stranded cDNA was synthesized from the purified mRNA, ligated to EcoR Ⅰ adaptor,digested with EcoR Ⅰ/Xho Ⅰ enzymes, and then cloned into the pGADT7 vector.The recombinant vector was transformed into E.coli DH10B to obtain a primary cDNA library.The primary library was amplified and used to determine the size of cDNA inserts through enzyme digestion.RESULTS: The primary cDNA library contained 1.03 × 106independent clones.The titer of the cDNA library was estimated to be 2.50 × 106 cfu/mL, and that of the amplified library was 3.60 × 109 cfu/mL.The size of the inserts varied from 0.5 to 3.5 kb, with an average value of about 2.0 kb.CONCLUSION: A yeast two-hybrid cDNA library has been successfully generated from FAT10-over-expressing Hep3B ceils and can be used for future screening of proteins interacting with FAT10.%目的:构建类泛素FAT10高表达肝癌细胞株Hep3B的酵母双杂交用cDNA文库.方法:从肝癌细胞Hep3B中提取总RNA,分离mRNA.利用反转录酶M-MLV与Oligo(dT)AnchorPrimer合成1 Strand cDNA,用E.coli DNAPolymerase与E.coli DNA Ligase将RNA链置换成DNA链,合成2 Strand cDNA.将双链cDNA与EcoR Ⅰ Adaptor连接,然后用EcoR Ⅰ /Xho Ⅰ进行酶切.使用Spin Column除去短链cDNA与pGADT7载体连接,转化入E.coli DH10B,建成原始文库.然后对其进行扩增并随机挑取单菌落,酶切鉴定重组子插入片段大小.结果:提取的总RNA降解少且分子完整;RNA纯度高,相对分子质量为400-5000 bp;成功合成双链cDNA,均符合建库要求:库容量达到1.03×10克隆,原始文库滴度为2.50×10 cfu/L,扩增后的文库滴度为3.60×10 cfu/L.插入片段大小分布为0.5-3.5 kb,平均长度约为2.0 kb.结论:所构建文库的各项指标均达到要求,为筛选FAT10作用蛋白奠定了重要基础.

  15. 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

  16. Construction of a smart cDNA library of Asian yellow pond turtle stimulated with Serratia marcescens and identification of related genes%黏质沙雷氏菌诱导的黄喉拟水龟SMART cDNA文库构建及相关基因的鉴定

    Institute of Scientific and Technical Information of China (English)

    赵密; 朱新平; 史燕; 高明英

    2011-01-01

    以致病黏质沙雷氏菌人工感染的黄喉拟水龟肝组织为材料,应用SMART(switching mechanism at5'end of RNA transcript)技术,构建了黄喉拟水龟的全长cDNA文库.首先用SMARTTM PCR cDNA systhesis kit合成全长的双链cDNA,通过琼脂糖凝胶分级分离技术切除小片段的cDNA,将大于500 bp的cDNA连接到pGEM-T载体中,电转化到JM109感受态细胞.在构建好的文库中,经测定,文库约含有1.8×105个重组子,重组效率达90%,插入片段多在0.5~3.0 kb之间.对库中长度约为1 000 bp的80个基因进行了测序,结果显示大部分首次在龟类发现.测序鉴定的基因包括免疫相关基因9个、信号传导基因6个、催化酶类基因8个、糖代谢相关基因2个、转运相关基因1个、结构基因2个.%To understand anti-infectious response to bacteria in the Asian yellow pond turtle (Mauremys mutica), a full length cDNA library was constructed for it by SMART technique experimentally infected with Serratia marcescens. Firstly, the double-strand cDNA was synthesized using SMARTTM PCR cDNA systhesis kit. Second, the ds cDNA was separated into two parts based on the size distribution of amplified ds cDNA by agarose gel size fractionation. The part shorter than 500 bp was discarded and the other one longer than 500 bp was ligated to the pGEM-T vector. The ligation mixture was transformed into E. coli JM109 by electroporation. The cDNA library contained 1.8 × 105 independent clones with DNA inserts of 0. 5-3. 0 kb. The recombination rate was 90. 30%. We sequenced 80 cDNA clones about 1 kb and most of the genes were found the first time in reptiles. We classified these clones in functions with 9 in immunity, 6 in cell signaling, 8 in catalytic activity, 2 in sugar/glycolysis metabolism, 1 in transport metabolism, and 2 in cell structure. The successfully constructed cDNA library will be essential for rapid isolation of differentially expressed genes related to Serratia marcescens

  17. 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.

  18. 人牙周膜成纤维细胞与牙龈成纤维细胞差异表达基因扣除文库的构建%Subtractive cDNA library construction of genes differentially xpressed in human periodontalligament fibroblast in comparison with gingival fibroblasts

    Institute of Scientific and Technical Information of China (English)

    郭希民; 吴补领; 肖明振; 蒲勤; 柴玉波; 朱峰; 赵忠良

    2000-01-01

    AIM: To construct a subtractive cDNA library of genes differentially expressed in cultured primary human PDLF in comparison with GF. METHODS: mRNAs were isolated fiom primary cultures of human PDLF and GF. Subtractive eDNA library of PDLF was constructed with a recently developed gene cloning technique that is based on PCR and subtractive hybridization. Part of randomly selected clones were identified by restriction analysis and reverse Southern dotblot. RESULTS: A subtbtetive cDNA library of PDLF was constructed with a capieity of 4×l02 clones. CONCLUSION:The gene cloning method we used in this study is both simple and effective in cloning differentially expressed gene.%目的:建立人牙周膜成纤维细胞(periodontal ligament fibroblast, PDLF) 与牙龈成纤维细胞(gin-gival fibroblast, GF)差异表达基因的扣除文库。方法:体外培养人PDLC和GF,分别提取mRNA,采用基于PCR和消减杂交的基因克隆技术构建人PDLC与GF差异表达基因的扣除文库。结果:成功构建了人PDLF与GF细胞差异表达基因的扣除文库,文库容量为4×l02。结论:基于PCR和消减杂交的基因克隆技术是构建细胞间差异表达基因扣除文库的较为简洁而有效的方法。

  19. 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).

  20. Construction and Identification of Full-length cDNA Library of the Peripheral Blood Leucocytes in Oreochromis niloticus after Vaccination%尼罗罗非鱼免疫后外周血白细胞全长cDNA文库的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    陈明; 梁万文; 李超; 王瑞; 李莉萍; 甘西; 余晓丽; 雷爱莹; 黄婷; 陈福艳

    2011-01-01

    为大规模快速克隆罗非鱼细胞免疫功能基因,采用SMART技术,构建了尼罗罗非鱼链球菌疫苗免疫后外周血白细胞全长cDNA文库.提取免疫后第3、5和7天外周血白细胞总RNA,用Powerscrip TM反转录酶逆转录合成第一链cDNA,LD-PCR扩增获得双链cDNA,经sfiⅠ酶切和CHROMA SPIN-400TM柱分级分离,收集500 bp以上的片段重组于改造的pBluescriptⅡ SK载体并转化大肠杆菌DH5α,测定文库滴度、重组率及库容量.结果表明,构建的cDNA文库原始库容量1.021×106克隆,文库滴度1.078×106pfu/mL,重组率94.71%,插入片段大小0.75-3.0 kb,平均长度约为1.5 kb.随机对24个克隆测序所获得的18条Contigs进行BLASTx分析,发现有15条Contigs有相关同源信忠,其中10条为全长cDNA,全长串为66.7%.说明所构建文库的各项指标均达到要求,可为筛选罗非鱼免疫功能相关基因和进一步研究基因的结构和功能奠定基础.%In order to rapidly clone the cellular immune function genes of the Tiplia on a large scale, a full-length cDNA library of the peripheral blood leucocytes from the Oreochromis niloticus thud vaccinated of S. iniae vaccine by i. p was constructed by using SMART ( switching mechanism at 5′ end of RNA transcript) techniques. The total RNA was extracted from peripheral blood teucocytes after vaccinated 3 , 5 , and 7 days. The PowerscriptTM reverse transcriptase was used to synthesize and anchor the first-strand cDNA, and the long distance PCR ( LD-PCR) method was used to amplify double-strand cDNA based on the SMART techniquea for construction of a full-length cDNA library. The PCR products were digested by proteinase K. After digestion with Sfi Ⅰ and size fractionation using CHROMA SPIN-400TM columns, cDNAa ( > 500 bp ) were ligated to the Sfi Ⅰ digested , dephosphorylated pBluescript Ⅱ SK vector. The ligation mixture was transformed into E. coil DH5a. The recmnbinant vectors were titered and the recombinant rate

  1. Screening a Novel Human Breast Cancer-Associated Antigen from a cDNA Expression Library of Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    Shuhua Yang; Lin Zhang; Ruifang Niu; Defa Wang; Yurong Shi; Xiyin Wei; Yi Yang

    2005-01-01

    OBJECTIVE The aim of this research was to clone and express the antigen of the previously prepared monoclonal antibody named M4G3.METHODS Western blots were used to screen a breast cancer cell line that overexpresses the M4G3-associated antigen. A λ zap cDNA expression library of breast cancer cells was constructed and screened using M4G3 as a probe to clone the antigen. The positive clones were subcloned and identified by homologous comparison using BLAST.RESULTS The λ zap cDNA expression library had 1.0x106 independent clones. Fifteen positive clones were isolated following 3 rounds of immunoscreening and identified as being from Mycoplasma pulmonis.CONCLUSION The specific antigen that matched the monoclonal M4G3 antibody is an unknown protein of M. pulmonis. This work is helpful for the further study of the association of M. pulmonis infection with breast cancer.

  2. Optimization of yeast surface-displayed cDNA library screening for low abundance targets.

    Science.gov (United States)

    Kim, Juh-Yung; Kim, Hyung Kyu; Jang, Hye Jeong; Kim, Eun-Kyung; Kim, Moon Kyu

    2015-04-01

    The yeast surface-displayed cDNA library has been used to identify unknown antigens. However, when unknown target antigens show moderate-to-low abundance, some modifications are needed in the screening process. In this study, a directional random-primed cDNA library was used to increase the number of candidates for the unknown antigen. To avoid the loss of target yeast clones that express proteins at a low frequency in the cDNA library, a comprehensive monitoring system based on magnetic-activated cell sorting, fluorescence-activated cell sorting, and immunofluorescence was established, and a small number of target yeast cells was successfully enriched. These results showed that our optimized method has potential application for identifying rare unknown antigens of the human monoclonal antibody.

  3. 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.

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

    Science.gov (United States)

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

    2013-03-01

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

  5. 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.

  6. Construction of differentially expressed cDNA libraries of aldehyde dehydrogenase with high and low activity from tongue squamous carcinoma Tca8113 cell line%基于舌鳞癌Tca8113细胞醛脱氢酶活性不同构建差异表达基因cDNA文库

    Institute of Scientific and Technical Information of China (English)

    孙守娟; 季平; 邓诚; 李颖; 邹波; 漆小娟

    2012-01-01

    Objective To construct the differentially expressed cDNA libraries of aldehyde dehydro genase with high and low activity (ALDHhigh/ALDHlow) from tongue squamous carcinoma Tca8113 cell line. Methods Expression of stem cell marker ALDH was detected, and ALDHhighand ALDHlow cells were collected by Aldefluor assay combined with flow cytometry. Differentially expressed genes of total RNA that was extracted from the two cell subpopulations by Trizol were screened and amplified by suppressing subtractive hybridization ( SSH) , and the PCR products were connected with pMD18-T vector and then transfected into E. Coli DH5a for amplification. Enzyme digestion, gene sequencing and homology analysis were performed in 24 positive clones that were randomly picked from each library. Results Two subproportions of ALDHhigh and ALDHlowwere " screened out, and ALDHhigh cells in Tca8113 cells accounted for 2. 5%. RNA D(260)/D(280) of ALDHhigh and ALDHlow were 1. 93 and 1. 92, respectively. Two-directional subtractive cDNA libraries of ALDHhigh and ALDHlow were constructed, and each library comprised 500 clones. PCR analysis of 24 clones randomly picked from each library showed that insert-fragments distributed in 200 - 700 bp, and no false positive clones were detected. Gene sequencing result that was analyzed and indexed by PubMed showed that cancer related genes included SLC25A13, KLHL2, NPC1, WAPL, BARD1, Notch2 and EEF2K. Conclusion Two-directional subtractive cDNA libraries of ALDHhigh and ALDHlow cells were successfully constructed.%目的 构建舌鳞癌Tca8113细胞系中高、低醛脱氢酶活性(high/low aldehyde dehydrogenase activity,ALDHhigh/ALDHlow)细胞差异表达基因cDNA文库.方法 用流式细胞仪检测ALDEFLUOR(R)染色的Tca8113细胞中干细胞标志物ALDH的表达,并收集ALDHhigh和ALDHlow细胞;用Trizol分别提取两亚群细胞的总RNA;用抑制性消减杂交(SSH)对2组RNA进行差异基因筛选和扩增,扩增产物与pMD18-T载体

  7. Genomic libraries: I. Construction and screening of fosmid genomic libraries.

    Science.gov (United States)

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Large insert genome libraries have been a core resource required to sequence genomes, analyze haplotypes, and aid gene discovery. While next generation sequencing technologies are revolutionizing the field of genomics, traditional genome libraries will still be required for accurate genome assembly. Their utility is also being extended to functional studies for understanding DNA regulatory elements. Here, we present a detailed method for constructing genomic fosmid libraries, testing for common contaminants, gridding the library to nylon membranes, then hybridizing the library membranes with a radiolabeled probe to identify corresponding genomic clones. While this chapter focuses on fosmid libraries, many of these steps can also be applied to bacterial artificial chromosome libraries.

  8. cDNA library Table: Nnor [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available Nnor NA Nnor NA ovary-derived cell-line NA NA pBluescript SK- EcoR1 for 5' Xho1for 3' sequenced from T3 prim...er (5' -> 3') BY916644-BY916866 E_ET_Nnor_[number]_F_0,E_ET_Nnor_[number]_F_1 BmN normalized library ...

  9. Construction of Subtracted cDNA Library of the Early Growth Plate in Broiler Chickens with Thiram-induced Tibial Dyschondroplasia%福美双诱发肉鸡胫骨软骨发育不良早期生长板消减cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    宁官保; 郭定宗; 田文霞; 王瑞; 覃平; 乔建钢; 李宏全; 李家奎; 毕丁仁; 潘思轶

    2011-01-01

    [Objective] An experiment was conducted to construct subtracted cDNA library for selecting time series genes differentially expressed in the TD growth plate of broiler chickens at the early stage with cDNA microarray. [Method] AVIAN (AV) broiler chicks at 7 days of age were randomly divided into two groups. After fasting overnight, they were fed with regular diet (control) or the same diet containing 100 mg/kg thiram for 48h to induce TD (thiram diet-fed). Forward and reverse-subtracted eDNA libraries were generated by suppression subtractive hybridization technology (SSH). Identification of the inserted cDNA fragments in subtractive library was done using PCR. One hundred clones were randomly selected for further DNA sequencing, blast homology analysis and function prediction. [Result] A total of 2 227 positive clones were obtained and the size of inserts was between 200 bp and 1 000 bp. There were 97 homologous gene sequences shared more than 99% identity with genes known in chicken (Gallus gallus). Non-redundancy of sequenced genes was 68.7%. Meanwhile, 3 clones were found to be novel EST as no functional clues were associated with them by bioinformatic analysis. Most of these genes are involved in matrix formation,endochondral ossification and remodelling, developmental regulation, signal transduction, electron transport in mitochondrial respiratory chain and vascularization. [ Conclusion ] Successfully produced cDNA library would make a good foundation for further printing cDNA microarray, screening differential expression genes of TD growth plates at different stages, and also may provide new insights into understanding the pathogenesis of TD.%[目的]为应用cDNA芯片技术筛选肉鸡胫骨软骨发育不良(TD)时序性差异表达基因,本研究构建了早期生长板消减cDNA文库.[方法]将7日龄AV肉鸡随机分为两组,对照组(control,C)饲以基础日粮,试验组(thiram diet-fed,T)饲以基础日粮添加福美双100 mg·kg(-1),2 d

  10. Isolation of carrot Argonaute1 from subtractive somatic embryogenesis cDNA library.

    Science.gov (United States)

    Takahata, Kiminori

    2008-03-01

    Carrot Argonaute1 (C-Ago1) was isolated from a subtractive cDNA library to obtain somatic embryogenesis related genes. C-Ago1 has three conserved domains, which are found in all other Argonautes. C-Ago1 has specific expression during somatic embryogenesis, which indicates that microRNA gene expression controlling system is required for somatic embryogenesis.

  11. Construction of subtractive cDNA Library of apoptosis-related genes in NB4 cells treated by arsenic trioxide%用抑制性差减杂交构建As_2O_3诱导的NB4细胞凋亡相关基因文库

    Institute of Scientific and Technical Information of China (English)

    狄春红; 顾少华; 谭晓华; 仙玲玲; 吴奇涵; 杨磊

    2009-01-01

    Objective: Construct the gene library of apoptosis related genes in acute promyelocytic leukemia (APL) cell line NB4 cells treated by arsenic trioxide to clarify the apoptotic mechanism of NB4 cells. Method: APL cell line NB4 cells treated with or without arsenic trioxide for 24 hours. Total RNA was extracted and suppress subtractive hybridization (SSH) was conducted according to the manual. With the cDNA of the apoptosis cells as the tester and that of control cells as the driver, forward and reverse hybridization was performed- Differentially expressed genes were linked with pGEM-Teasy cloning vector and transformed into E.coli DH5α. The positive clones were screened by blue and white spot. PCR were used to amplify these genes. Result: The subtractive cDNA libraries related with apoptosis of NB4 cells were successfully constructed. Conclusion: The constructed subtractive libraries are suitable for further study on the functional genes associated with apoptosis of NB4 cells induced by arsenic trioxide.%目的: 构建三氧化二砷(As_2O_3)诱导的急性早幼粒细胞白血病细胞株NB4细胞凋亡相关基因文库.方法:用含4 μmol·L~(-1)As_2O_3和正常培养基培养NB4细胞24 h,抽提总RNA,经逆转录酶合成双链cDNA,分别以砷诱导凋亡组和对照组作为tester和driver,进行双向抑制性差减杂交(supptess-ion sublxactive hybridization,SSH),筛选As_2O_3诱导的NB4细胞凋亡相关基因,将差异表达基因进行PCR扩增并与pGEM-Teasy克隆载体连接,转化DH5 α大肠杆菌,经蓝白斑筛选获得白色阳性克隆,PCR扩增出未知基因片段.结果:成功构建了分别代表在NB4细胞中表达上调和下调的基因文库.结论: 经双向抑制性差减杂交获得了NB4细胞差异表达基因文库,为克隆NB4细胞凋亡相关基因奠定了基础.

  12. Construction and diversity analysis of a murine IgE phage surface display library

    Institute of Scientific and Technical Information of China (English)

    LIZONGDONG; MINGYEH

    1997-01-01

    To make further investigation of the IgE antibody repertoire in Trichosanthin (TCS) allergic responses,a murine IgE phage surface display library was constructed (3.0×105 independent clones).We first constructed the Vε cDNA library (4.6×105 independent clones) and Vκ cDNA library (3.0×105 independent clones).Then,the Vε and Vκgene segments were amplified from both libraries by PCR respectively,and assembled into Fab fragment by SOE PCR.The phage library containing Fabs was thus constructed.The diversity of Vε from this library was analyzed and proved.Fab clones with high specificity to TCS have been screened out.

  13. Construction of cDNA subtractive library between yeast and mycelium phase of Sporothrix schenckii and screening of differently expressed genes about dimorphic transition%申克孢子丝菌菌丝相和酵母相cDNA消减文库的构建及差异表达基因的筛选

    Institute of Scientific and Technical Information of China (English)

    周汛; 杨致邦; 肖异珠

    2011-01-01

    The purpose of this study was to screen the differentially expressed genes about dimorphic transition of Sporothrix schenckii by construction of eDNA subtractive library between yeast and mycelium phase.The eDNA subtractive library between yeast and mycelium phase was constructed by suppression subtractive hybridization(SSH)and bioinformatics analysis was performed to profile the relationship between those differently expressed genes and dimorphic transition.Of 751 and 875 ESTs were obtained in M+Y and Y+M library, separately.After splicing of ESTs, 101 and 249 unigenes were obtained in M +Y and Y+ M libraries.During the construction of cDNA subtractive libraries, the distribution of differently expressed genes varied with dimorphic transition.The over expressed genes with diversity and complexity were divided into structural genes,metabolic enzymes, molecule on cell surface and molecule with indistinct function.Results disclose that construction of cDNA subtractive libraries for the dimorphic transition and bioinformatics analysis for those differently expressed genes lay the foundation for screening the related genes involved in the pathogenesis of Sporothrix schenckii infection.%目的 构建申克孢子丝菌双相cDNA消减文库,筛选与双相转换相关的差异表达基因.方法 运用抑制性消减杂交技术,构建申克孢子丝菌菌丝相(Mycelium,M)和酵母相(Yeast,Y)的正反cDNA消减文库,并对其差异表达的基因进行生物信息学分析.结果 M+Y文库获得751条表达序列标签(Expressed Sequence Tags,ESTs),经拼接后获得101条unigenes;Y+M文库获得875条ESTs,拼接获得249条unigenes.申克孢子丝菌酵母相菌丝相的转换伴随着不同菌相细胞差异基因的高表达,这些高表达的差异基因可分为结构基因类、代谢酶类、细胞表面分子类及功能不明的细胞分子.结论 成功构建了申克孢子丝菌双相转换相关的cDNA消减文库基础上,筛选出部分差异表达基

  14. Characterization of Expressed Sequence Tags From a Gallus gallus Pineal Gland cDNA Library

    OpenAIRE

    2005-01-01

    The pineal gland is the circadian oscillator in the chicken, regulating diverse functions ranging from egg laying to feeding. Here, we describe the isolation and characterization of expressed sequence tags (ESTs) isolated from a chicken pineal gland cDNA library. A total of 192 unique sequences were analysed and submitted to GenBank; 6% of the ESTs matched neither GenBank cDNA sequences nor the newly assembled chicken genomic DNA sequence, three ESTs aligned with sequences designated to be on...

  15. THE CLONING OF HRNT-1 USING A COMBINATION OF cDNA LIBRARY SCREENING WITH BIOTIN-LABELED PROBE AND RAPID AMPLIFICATION OF cDNA ENDS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To clone the human counterpart of rat ZA73, EST cloned from rat tracheal epithelial (RTE) neoplastic transformed cell model induced by (a-particles radiation by using mRNA differential display. Methods: According to the sequence of rat ZA73, a probe was biotin-labeled to screen human cDNA library, and then the gene sequence was extended by RACE (rapid amplification of cDNA ends). Result: Human gene HRNT-1 (GenBank Accession Number: AF223393) is 4.256 kb in length, with an ORF located in the region between 254 and 3013 bp. 5' UTS (untranslated sequences) is 253 bp, 3' UTS is 1243 bp. Conclusion: The combination of cDNA library screening with biotin-labeled probes and RACE is an effective method to clone full-length cDNA, especially for sequences longer than 2 kb.

  16. Construction and Sequence Analysis of SSH cDNA Library form Aphid-resistant Sorghum%高粱广谱抗蚜基因SSH文库的构建及其序列分析

    Institute of Scientific and Technical Information of China (English)

    齐金凤; 孙权; 常金华; KhalidHussain; 林凤

    2012-01-01

    The aim was to study broad-spectrum anti-aphid gene in sorghum and sorghum aphid interactions in the molecular genetic basis and mechanism, and to explore and use the information of aphid resistance genes. In this study, 'Henongl6' with aphid-resistance genes and 'Qian3' susceptible to sorghum aphid were used as the tester and the driver and vice versa to construct SSH library, so that the aphid resistance genes were enriched effectively, then the differential gene sequence was analyzed. 200 positive clones were randomly selected and sequenced. 18 open reading frame (ORF) sequences were found through the NCBI ORF Finder online prediction. Then the 18 ORF sequences were blasted and analyzed using online Blastx. The results showed that the 18 ORF sequences were highly related with putative oxygen-evolving enhancer protein in chloroplast, adenylate cyclase-associated protein, ribosomal protein, putative senescence-associated protein. The contigs without ORF were blasted and analyzed using online Blastx and Blastn, and 66 ESTs had highly homologous to genes sequences with known disease resistance in many plants, such as ribosomal protein,transport membrane protein, NBS-LRR disease resistance protein family-1, zinc finger protein, ATP synthase, NADH dehydrogenase, retrotransposon, Adenylyl cyclase-associated protein, and bZIP transcription factor etc.. These homologous genes involved in secondary metabolism, energy metabolism, membrane transport, signal transduction and transcription regulation and so on. The SSH library of sorghum aphid resistance gene was constructed successfully, which indicated that the SSH technique in the mechanism of aphid resistance of sorghum was a feasibility study. The results laid the foundation for further studying on sorghum aphid-resistance gene function.%为了从分子水平上解析高粱广谱抗蚜基因在高粱与蚜虫互作中的分子遗传基础和作用机理,为发掘并利用抗蚜基因提供可靠的信息支持.

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Directory of Open Access Journals (Sweden)

    Wallis James G

    2007-07-01

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

  19. 败血症鲢肝与肾cDNA文库构建及MHC class Ⅰ的克隆与分析%Construction of a cDNA library and cloning of the MHC class Ⅰ gene in silver carp (Hypophthalmichthys molitrix) infected with bacterial septicemia

    Institute of Scientific and Technical Information of China (English)

    汪登强; 罗晓松; 陈大庆

    2011-01-01

    Silver carp (Hypophthalmichthys molitrix) is one of the most commonly cultured freshwater species in China. However, the development of silver carp aquaculture is threatened by bacterial septicemia. To isolate and study genes relevant to the disease, we constructed a cDNA library from silver carp liver and kidney tissue using a CloneMiner? cDNA kit. The primary cDNA library titer was 1.34× 107 cfu/mL yielding 2.68×l07cfu recombi-nants with 97.5% positive clones. The exogenous inserts of the recombinants ranged in size from 0.8 to 3.5 kb. We attempted to sequence 80 positive clones from both terminals to test the completeness of the coding sequence. We successfully sequenced 74 clones, of which 49 contained the complete coding sequence. Among the clones successfully sequenced, 57 sequences were identified to 49 known genes in GenBank. The cDNA library was subsequently screened by PCR yielding a single clone containing the complete coding sequence for MHC class I. The H. Molitrix MHC class I was 1 026 bp long and encoded a 341 amino acid (aa) protein that included a leader peptide, a\\, a2, a3, and transmembrane and cytosolic domains of 16, 88, 90, 87, and 60 aa, respectively. In addition, we identified sites that were highly conserved among vertebrate MHC class I. Phylogenetic comparison of the complete coding sequences and the a3 domain ofhH, molitrix MHC class I with other vertebrate species revealed different topology, suggesting a different evolutionary history for different domains of MHC class I and the occurrence of gene recombination among cyprinidae.%采用CloneMinerTM cDNA文库构建试剂盒构建了患败血症的鲢(Hypophthalmichthys molitrix)肝和肾的cDNA文库.经检验,文库的滴度为1.34×107 cfu/mL,总库容为2.68×107 cfu,阳性克隆率为97.5%,平均插入片段大于1.2 kb.对80个随机挑选的克隆进行两端测序,结果显示有74个测序成功.经在GenBank上BLAST比对,其中57个为已知功能基因,属于45

  20. 黑曲霉H1的cDNA文库构建及其溶磷相关基因的筛选%Construction of cDNA library of Aspergillus niger H1 and screening of phosphate-dissolving related gene

    Institute of Scientific and Technical Information of China (English)

    唐超西; 龚明波; 李顺鹏; 朱昌雄

    2012-01-01

    [目的]构建溶磷黑曲霉H1的cDNA文库,并从中筛选溶磷相关基因.[方法]利用SMART技术合成黑曲霉H1的双链cDNA并将其连接于pBluescript Ⅱ SK(+)载体上,将重组质粒转化E.coli HST08,得到黑曲霉的初级cDNA文库.利用难溶磷培养基筛选具有溶磷能力的克隆子,测序并利用Blast分析基因序列.在难溶磷液体培养基中,进行克隆子对溶液pH值、可溶磷含量的影响和产有机酸实验.[结果]成功构建了黑曲霉H1的cDNA文库,其初级库容量约为5.65×106cfu/mL,重组率约为99.15%;通过难溶磷固体培养基筛选,得到具有溶磷圈的克隆子61个,其中克隆子H-54的cDNA序列长839 bp,基因编码氨基酸残基序列长179n.t.克隆子E.coli HST08 H-54在液体难溶磷培养基中培养,提高了有机酸的表达量,并增加了有机酸的种类,在培养12h后,溶液中开始产生甲酸和乙酸,在24 h后,溶液中产生苹果酸和α-酮戊二酸,培养36 h,溶液pH值由6.32降到3.93,可溶磷含量达到0.105 mg/mL.[结论]从黑曲霉H1中获得1个溶磷相关基因,将其命名为psgA.%[ Objective]To obtain phosphate-dissolving genes from cDNA library of Aspergillus niger HI. [Methods] The double-stranded cDNA was synthesized using switching mechanism at 5'end of RNA transcript technique and ligated to the vector pBluescript II SK ( + ). We transformed recombinant plasmid into E. Coli HST08, resulting in a primary cDNA library. We screened clones with phosphate-dissolving activities on the insoluble phosphate medium and blasted the sequence in National Center for Biotechnology Information (NCBI). To study the phosphate dissolving mechanisms of the cloned gene, we analyzed the changes of the pH value, the soluble phosphate content and the production of organic acids in the insoluble phosphate liquid medium inoculated with the clones harboring the phosphate-dissolving gene. [Results] A cDNA library of A. Niger HI was successfully constructed. Titer tests

  1. Construction and ESTs Analysis of a Normalized Full-Length cDNA Library of Brown Prunus salicina%(木奈)褐变果实均一化全长cDNA文库的构建及部分ESTs序列分析

    Institute of Scientific and Technical Information of China (English)

    姜翠翠; 陈桂信; 潘东明; 赖燕; 郑鸿昌

    2013-01-01

    以发生褐变初期的(木奈)果肉总RNA为材料,对SMARTTM cDNA合成方法进行改良,并将长距离PCR、DSN处理和定向克隆相结合,成功构建(木奈)褐变果实均一化全长cDNA文库.经检测,该文库的初始滴度为2.0×106 pfu/mL,重组率为99%,插入片段大小平均为1.8 kb,文库质量良好.随机挑取720个阳性克隆进行5'EST测序,共获得684条有效的ESTs序列;并将684条有效序列拼接后,58条被组装成21个重叠群(contigs),单拷贝(singlets)基因有626条,共得到647个单基因簇(unigenes),文库冗余率为8.4%.用Blast 2 go在线软件对测序结果进行功能注释和归类分析,结果显示,已知功能基因与能量代谢、蛋白质合成与降解、次生代谢物质、细胞壁代谢和转录因子有关.该文库的构建有利于从分子水平上研究(木奈)果实褐变发生的机制.%Total RNA was extracted from the fruit of "Oil Nai" with beginning of browning. The full-length normalized cDNA library was constructed by the combination of long distance PCR, DSN (Duplex -Specific Nuclease) treatment and directed cloning. The titer of primary library was about 2.0 ×106 pfu/mL and 99% clones were recombinant, and the length of insert cDNAs was about 1.8 kb. The cDNA library was well normalized with 8.4% redundancy. A total of 720 random clones in this cDNA library were then sequenced by 5EST. A total of valid 684 ESTs were attained by fragment assembly, removing the contaminated sequences and frameshift mutation. Then 647 unigenes were attained, which included 21 contigs and 626 singlets. Annotation by the online Blast 2 go search against Genbank database on NCBI web server revealed that most of the genes with predicted function were related to energy metabolism, protein synthesis, degradation of secondary metabolites, cell wall metabolism and transcription factors. These results would lead to further research of mechanism of browning fruit in molecular level and develop good

  2. 中国美利奴超细型与哈萨克羊毛囊兴盛期皮肤组织消减cDNA文库的构建%Construction of Subtractive cDNA Library of Skin Tissue in Follicle Anagen Between Chinese Merino and Kazakh Sheep

    Institute of Scientific and Technical Information of China (English)

    杨剑波; 甘尚权; 李晶; 高磊; 杨井泉; 张科; 杨永林; 沈敏; 石国庆

    2012-01-01

    [Objective] The aim of the study is to screen important candidate genes influencing wool traits in sheep, and to lay a theoretical foundation for exploring the molecular mechanism of wool traits formation. [Method] Using suppression subtractive hybridzation (SSH) technique, positive and reverse subtracted cDNA library between skin tissue in follicle anagen of Chinese Merino (superfine type) and Kazakh sheep was constructed, respectively. Genes of differential expression profiles were sequenced for bioinformatics analysis. Semi-quantitative RT-PCR and real-time fluorescent quantitative RT-PCR methods were used to assess partial genes of differential expression profiles. [Result] A total of 153 and 143 ESTs were obtained from the forward and reverse subtracted cDNA library, respectively. Among these ESTs, 5 from positive library and 4 from reverse library of unknown function were identified and speculated as novel genes. The GO cluster, pathway and protein-protein interaction analysis of the partial ESTs of known function showed that there were certain differences between coarse wool and fine wool sheep. The three differential genes from the constrcted cDNA libraries, KRTAP8-2, Trichohyalin and KRTAP3-2 genes were all specifically expressed at high level in the skin. The abundance of KRTAP8-2 and Trichohyalin genes mRNA in skin of Chinese Merino was 3.45 and 2.07 times than that of Kazakh sheep, respectively, while the abundance of KRTAP3-2 gene mRNA in skin of Kazakh sheep was 1.87 times than that of Chinese Merino. [Conclusion] The subtracted cDNA library of skin tissue between Chinese Merino and Kazakh sheep was successfully constructed, a batch of ESTs with differential expression profiles which possibly influence woo! Traits were initially screened.%[目的]筛选影响绵羊羊毛性状的重要候选基因,为研究羊毛性状形成的分子机制奠定基础.[方法]利用抑制性消减杂交技术构建中国美利奴超细型和哈萨克羊兴盛期皮

  3. 干旱胁迫的水稻根高效酵母双杂交体系建立%Construction and High-efficient Screenings of a Yeast Two-Hybrid cDNA Library from the Drought-stressed Roots of Rice

    Institute of Scientific and Technical Information of China (English)

    付坚; Linkun Gu; 郭怡卿; Liyuan ZHANG; 王玲仙; 李定琴; 王波; Jeff Qingxi SHEN; 程在全

    2013-01-01

    采用SMART技术在酵母菌株AH109中构建了干旱胁迫下水稻根部全长cDNA文库,采用改进的滤膜杂交方法建立了高效酵母杂交体系,以抗旱相关水稻转录调控因子OsWRKy71基因作为诱饵对该方法进行检验.实验获得的酵母杂交文库容量为4.9×10 6,平均插入片段为800 bp,达到了基因分离和克隆等后续研究的标准.同时采用滤膜杂交法将文库的杂交效率提高到了6.9%,是液体杂交法的6倍以上.该方法不需要特殊的设备,且具有低消耗和高效率的特点,可用于高通量酵母双杂交分析.%By using the SMART technique, a yeast two-hybrid cDNA library of rice roots was constructed and utilized to study protein-protein interactions induced by drought. An optimized high efficiency membrane mating method, substituting for liquid mating method, was developed for library screenings. As results, there are about 4. 9 × 106 clones in the library with the averaged size of the insert fragments to be about 800 base pairs. To verify the efficiency of the membrane mating method, OsWRKY71 , a well-studied gene encoding a transcriptional repressor of gibberellin signaling, was used as a bait to screen this library. The mating efficiency was shown to be as high as 6. 9% , which was six times higher than that of the liquid mating method. This high efficient mating method is simple, cost-effective and suitable for large scale yeast hybrid analyses.

  4. Production of Arrayed and Rearrayed cDNA Libraries for Public Use

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, K

    2005-08-29

    Researchers studying genes and their protein products need an easily available source for that gene. The I.M.A.G.E. Consortium at Lawrence Livermore National Laboratory is an important source of such genes in the form of arrayed cDNA libraries. The arrayed clones and associated data are available to the public, free of restriction. Libraries are transformed and titered into 384-well master plates, from which 2-8 copies are made. One copy plate is stored by LLNL while others are sent to sequencing groups, plate distributors, and to the group which contributed the library. Clones found to be unique and/or full-length are rearrayed and also made publicly available. Bioinformatics tools supporting the use of I.M.A.G.E. clones are accessible via the World Wide Web.

  5. 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.

  6. 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%.

  7. Screening cDNA Libraries Using Partial Probes to Isolate Full-Length cDNAs from Vascular Cells.

    Science.gov (United States)

    Csortos, C; Lazar, V; Garcia, J G

    1999-01-01

    The purpose of screening cDNA libraries is to isolate a particular cDNA clone encoding a mRNA and by implication, a protein, of interest. The screening is based on identification of the desired clone among a large number of recombinant clones within the library selected (1,2). As an example of both the utility and power of library screening, we will relate our own library screening efforts utilized to isolate the nonmuscle high molecular weight myosin light chain kinase isoform from a human umbilical vein endothelial cell cDNA library (3). This unique nonmuscle myosin light chain kinase isoform phosphorylates myosin light chains, thereby playing an essential role in agonist-mediated endothelial cell contraction, paracellular gap formation and increased vascular permeability. We are hopeful that this step-by-step approach will help the reader to understand the discussed methods.

  8. 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

  9. 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

  10. 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

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

    Science.gov (United States)

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

    2015-04-01

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

  12. 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.

  13. [Development of new SSR markers from EST of SSH cDNA libraries on rose fragrance].

    Science.gov (United States)

    Yan, Hui-Jun; Zhang, Hao; Xie, Ji-Rong; Li, Shu-Fa; Jian, Hong-Ying; Qiu, Xian-Qin; Wang, Qi-Gang; Wang, Ji-Hua; Tang, Kai-Xue

    2009-09-01

    The new SSR markers of rose related fragrance were developed based on the SSH cDNA libraries of rose floral scent mutant. In this study, 10 EST-SSRs (2.6%) from 391 ESTs in the libraries were identified. Six EST-SSRs primers were designed to sequence flanking SSRs. The primer pairs designed were screened on the wild-type Jinyindao, which has flowers full of pleasant scent, and the mutant-type Wangriqinghuai without perceivable floral scent. Five primer pairs were amplified effectively in Jinyindao and Wangriqinghuai, and 3 were polymorphic between Jinyindao and Wangriqinghuai. Eighteen rose cultivars including fragrant roses and nonfragrant roses were identified by the five prime pairs. These results proved that EST-SSR markers are effective markers to identify the polymorphism of the rose.

  14. Test: Construction of genomic libraries.

    Science.gov (United States)

    Szeberényi, Jozséf

    2005-03-01

    Terms to be familiar with before you start to solve the test: genomic library, gel filtration, restriction endonuclease, plasmid, sticky ends, blunt ends, ligation, recombinant DNA, bacterium transformation, denaturation and renaturation of DNA, satellite DNA, telomere, centromere, unique and repetitive sequences.

  15. Discovery of Phytophthora infestans genes expressed in planta through mining of cDNA libraries.

    Directory of Open Access Journals (Sweden)

    Roberto Sierra

    Full Text Available BACKGROUND: Phytophthora infestans (Mont. de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora--Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta. METHODOLOGY/PRINCIPAL FINDINGS: We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family. CONCLUSIONS/SIGNIFICANCE: We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant--oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta.

  16. Studies of the hyperthermophile Thermotoga maritima by random sequencing of cDNA and genomic libraries. Identification and sequencing of the trpEG (D) operon.

    Science.gov (United States)

    Kim, C W; Markiewicz, P; Lee, J J; Schierle, C F; Miller, J H

    1993-06-20

    Random sequencing of cDNA and genomic libraries has been used to study the genome of the hyperthermophile Thermotoga maritima. To date, 175 unique clones have been analyzed by comparing short sequence tags with known proteins in the PIR and GenBank databases. We find that a significant proportion of sequences can be matched to previously identified protein from non-Thermotoga sources. A high match rate was obtained from an oligo(dT)-primed cDNA library, where one-third of all unique sequences analyzed (21/65) shared high amino acid sequence similarity with proteins in the PIR and GenBank databases. Also, approximately one-third of the unique sequences from a second cDNA library (28/89), constructed with random oligo primers, could be matched to sequences in PIR and GenBank. Identification of genes from the oligo(dT)-primed cDNA library indicates that some Thermotoga mRNAs are polyadenylated. Genes have also been identified from a 1 to 2 kb genomic DNA library. Here, (3/21) of genomic sequences analyzed could be matched to protein in PIR and GenBank. One of the genomic clones had high sequence similarity to the tryptophan synthesis gene anthranilate synthase component I (trpE). Using this sequence tag, the Thermotoga trp operon was isolated and sequenced. The Thermotoga maritima trp operon is arranged with trpE forming an overlapping transcript with a second protein consisting of a fusion of anthranilate synthase component II (trpG) and anthranilate phosphoribosyltransferse (trpD). With regard to the fusion, the operon organization is similar to Escherichia coli and Salmonella typhimurium, but lacks the classic attenuation system of enteric bacteria. Amino acid sequence comparison with 19 trpE, 18 trpG and 14 trpD genes from other organisms suggest that the Thermotoga trp genes resemble corresponding genes from other thermophiles more closely than expected.

  17. 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.

  18. Targeting a complex transcriptome: the construction of the mouse full-length cDNA encyclopedia.

    Science.gov (United States)

    Carninci, Piero; Waki, Kazunori; Shiraki, Toshiyuki; Konno, Hideaki; Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Arakawa, Takahiro; Ishii, Yoshiyuki; Sasaki, Daisuke; Bono, Hidemasa; Kondo, Shinji; Sugahara, Yuichi; Saito, Rintaro; Osato, Naoki; Fukuda, Shiro; Sato, Kenjiro; Watahiki, Akira; Hirozane-Kishikawa, Tomoko; Nakamura, Mari; Shibata, Yuko; Yasunishi, Ayako; Kikuchi, Noriko; Yoshiki, Atsushi; Kusakabe, Moriaki; Gustincich, Stefano; Beisel, Kirk; Pavan, William; Aidinis, Vassilis; Nakagawara, Akira; Held, William A; Iwata, Hiroo; Kono, Tomohiro; Nakauchi, Hiromitsu; Lyons, Paul; Wells, Christine; Hume, David A; Fagiolini, Michela; Hensch, Takao K; Brinkmeier, Michelle; Camper, Sally; Hirota, Junji; Mombaerts, Peter; Muramatsu, Masami; Okazaki, Yasushi; Kawai, Jun; Hayashizaki, Yoshihide

    2003-06-01

    We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

  19. Construction of subtracted cDNA library of diferentially expressed genes of multidrug resistant tubercle bacillus by suppression subtracted hybridization%应用抑制消减杂交技术构建耐多药结核杆菌差异表达基因消减cDNA文库

    Institute of Scientific and Technical Information of China (English)

    张运玲; 郑改焕; 刘芮汐; 彭哲; 李奇志; 幸琳琳; 朱朝敏

    2012-01-01

    Objective: To build the subtracted cDNA library of differentially expressed genes of multidrug-resistant tubercle bacillus (MDR-TB)and to further discuss the molecular mechanism of MDR-TB. Methods:Tester was MDR-TB and Driver was sensitive tuberculosis. Suppression subtractive hybridization (SSH) and T/A cloning technology were done to build the subtracted cDNA library of differentially expressed genes of MDR-TB. Results:The subtracted cDNA library of differentially expressed genes of MDR-TB was successfully built and 113 differentially expressed cDNA fragements of MDR-TB were obtained. Sequencing and homology analysis showed that 5 of them were novel cDNA sequences and 5 sequenced genes were reported to be related with MDR in TB. Conclusions: SSH is an effective method for screening new function genes. Many genes both known and unknown are in correlation with MDR in TB. Discovery of these genes provides a solid foundation for the explanation of MDR mechanism in TB.%目的:构建结核杆菌耐多药株与敏感株的差异表达消减cDNA文库,进一步探索结核杆菌耐多药的分子机制.方法:以耐多药菌株cDNA为实验组(Tester),敏感株cDNA为驱动组(Driver)应用抑制消减杂交(Suppression subtractive hybridization,SSH)技术结合T/A克隆技术构建结核杆菌耐多药株与敏感株的差异表达消减cDNA文库.结果:成功构建了耐多药结核菌株差异表达消减cDNA文库,获得113个差异表达cDNA片段.结论:研究表明SSH技术是筛选新功能基因的有效方法;多种已知或未知基因均参与了结核杆菌耐多药的调节,大规模筛选与克隆这些基因为进一步研究结核杆菌耐多药机制的产生奠定了必要的理论基础.

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  1. Mouse protein arrays from a TH1 cell cDNA library for antibody screening and serum profiling

    OpenAIRE

    2005-01-01

    The mouse is the premier genetic model organism for the study of disease and development. We describe the establishment of a mouse T helper cell type 1 (TH1) protein expression library that provides direct access to thousands of recombinant mouse proteins, in particular those associated with immune responses. The advantage of a system based on the combination of large cDNA expression libraries with microarray technology is the direct connection of the DNA sequence information from a particula...

  2. 巴西橡胶树胶乳均一化酵母双杂交cDNA文库构建%Construction of a normalized yeast two-hybrid cDNA library of the latex of rubber tree (Hevea brasiliensis Müll. Arg.)

    Institute of Scientific and Technical Information of China (English)

    余海洋; 张宇; 王萌; 覃碧

    2016-01-01

    In this study, the latex of rubber tree (Hevea brasiliensis) clone ‘Reyan 7-33-97’ was used as plant material. SMART® cDNA synthesis technology was used to generate the ifrst strand cDNA, and then long distance PCR (LD-PCR) was used to amplify double-strand cDNA (ds-cDNA). The ds-cDNA was normalized by duplex-speciifc nuclease (DSN). The normalized cDNA was puriifed by running CHROMA SPIN+TE-400 column to remove short cDNA fragments. The puriifed cDNA and linear vector pGADT7-Rec were co-trans-formed into competent Y187 yeast cell to generate a normalized yeast two-hybrid cDNA library. The results show that the cDNA of the latex of rubber tree was wide range of fragment sizes and with uneven abundance before normalization. After normalized by DSN and puriifed using CHROMA SPIN+TE-400 column, cDNA below 500 bp had been removed efficiently, and high abundance of cDNA had been reduced significantly. Moreover, RT-PCR revealed that the transcripts of two housekeeping genes18S rRNA andβ-actinwere decreased signiifcantly after normalization. The harvested library had 1.26×106 independent clones. The titer of the library was up to 3.23×107 cfu·mL-1, the recombination rate was 87%, and the average insert size was more than 1.0 kb. In this study, a normalized yeast two-hybrid cDNA library from the latex of rubber tree has been successfully established, which provides a reference for studying natural rubber biosynthetic pathway and its molecular regulation mechanism in rubber tree.%以巴西橡胶树无性系‘热研7-33-97’胶乳为材料,采用SMART® cDNA合成技术反转录合成cDNA第一链,并通过LD-PCR合成双链cDNA (ds-cDNA),采用双链特异性核酸酶(DSN)对ds-cDNA进行均一化处理,并经过CHROMA SPIN+TE-400柱子去除短片段的cDNA,纯化后的cDNA和线性化载体pGADT7-Rec共转化酵母Y187感受态细胞构建均一化酵母双杂交cDNA文库。结果显示:均一化之前橡胶树胶乳cDNA片段分布较

  3. 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

  4. 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

  5. Preparation of a subtractive cDNA library enriched in cDNAs which expressed at a high level in cultured senescent human fibroblasts.

    Science.gov (United States)

    Tahara, H; Hara, E; Tsuyama, N; Oda, K; Ide, T

    1994-03-30

    Subtracted cDNA library was prepared by subtracting [cDNA from young growing SV40-transformed human fibroblasts] from [cDNA from growing SV40-transformed fibroblasts in extended lifespan]. Isolated cDNA clones which expressed at high level in life-extended transformed cells also expressed at high level in normal senescent fibroblasts but did at low level in growing and growth-arrested young cells. Neither fibronectin nor procollagen cDNA was isolated. This cDNA library is useful for isolation of senescent-specific cDNA species which express at high level in normal senescent cells but at low level in growing and growth-arrested young cells, avoiding growth-arrest-specific cDNAs.

  6. 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.

  7. 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.

  8. A subtractive cDNA library from an identified regenerating neuron is enriched in sequences up-regulated during nerve regeneration.

    Science.gov (United States)

    Korneev, S; Fedorov, A; Collins, R; Blackshaw, S E; Davies, J A

    1997-01-01

    We have constructed a subtractive cDNA library from regenerating Retzius cells of the leech, Hirudo medicinalis. It is highly enriched in sequences up-regulated during nerve regeneration. Sequence analysis of selected recombinants has identified both novel sequences and sequences homologous to molecules characterised in other species. Homologies include alpha-tubulin, a calmodulin-like protein, CAAT/enhancer-binding protein (C/EBP), protein 4.1 and synapsin. These types of proteins are exactly those predicted to be associated with axonal growth and their identification confirms the quality of the library. Most interesting, however, is the isolation of 5 previously uncharacterised cDNAs which appear to be up-regulated during regeneration. Their analysis is likely to provide new information on the molecular mechanisms of neuronal regeneration.

  9. Human BAC library: construction and rapid screening.

    Science.gov (United States)

    Asakawa, S; Abe, I; Kudoh, Y; Kishi, N; Wang, Y; Kubota, R; Kudoh, J; Kawasaki, K; Minoshima, S; Shimizu, N

    1997-05-20

    We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.

  10. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

    OpenAIRE

    1996-01-01

    A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model sys...

  11. Construction of Differential-Methylation Subtractive Library

    Directory of Open Access Journals (Sweden)

    Wei Hu

    2014-01-01

    Full Text Available Stress-induced ROS changes DNA methylation patterns. A protocol combining methylation-sensitive restriction endonuclease (MS-RE digestion with suppression subtractive hybridization (SSH to construct the differential-methylation subtractive library was developed for finding genes regulated by methylation mechanism under cold stress. The total efficiency of target fragment detection was 74.64%. DNA methylation analysis demonstrated the methylation status of target fragments changed after low temperature or DNA methyltransferase inhibitor treatment. Transcription level analysis indicated that demethylation of DNA promotes gene expression level. The results proved that our protocol was reliable and efficient to obtain gene fragments in differential-methylation status.

  12. 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.

  13. 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

  14. 孔石莼在干出胁迫下上调表达基因消减cDNA文库的构建及分析%Construction and analysis of subtractive cDNA library of up-regulated genes from Ulva pertusa under emersed stress

    Institute of Scientific and Technical Information of China (English)

    李世国; 杨帆; 吴倩倩; 乔坤; 佟少明; 侯和胜

    2011-01-01

    利用抑制消减杂交技术构建了潮间带绿藻孔石莼(Ulva pertusa)在干出胁迫状态下上调表达基因的消减cDNA文库.获得的阳性克隆经测序得到150条上调表达的EST片段,其中21条为冗余序列(RS),137条非冗余序列(NRS).片段中最长833 bp,最短106 bp,平均长度364 bp.参与比对的EST片段按照其注释功能可分类为:细胞生长与发育、蛋白合成与分解、代谢过程、胁迫应答、光合作用与光诱导、转录调节、信号转导等相关基因.未知功能和无对比结果序列共60条,占非冗余序列的43.80%.比对相似度主要分布在20%~60%之间,主要与已经完成基因组测序的藻类或高等植物的氨基酸序列具有较高的相似性.孔石莼对密码子第三位碱基的使用偏好无显著差别(P>0.05),GC使用总频率为50.66%,而对终止密码子则明显偏好TGA.实时荧光定量结果表明,部分分离的基因在干出胁迫状态下表达量上调.这些不同功能基因的表达构成了复杂的调控网络,能够为揭示孔石莼抵御潮间带不良环境的分子机制提供多方面的切入点.%The subtractive cDNA library of up-regulated genes from Ulva pertusa under emersed stress was constructed by suppression subtractive hybridization (SSH) technology. 150 EST fragments of up-regulated genes were isolated after sequencing the positive clones, in which 21 fragments were redundant (RS) and other 137 fragments were non-redundant sequences (NRS). PCR analysis indicated that the length of the EST inserts were in the range of 100 to 900 bp. The longest was 833 bp, and the shortest was 106 bp and the average length was 364 bp. Based on the functional similarity with homologous genes in algae and higher plants, the EST fragments could be divided into several functions: cell growth and development, protein synthesis and degradation, metabolism, stress response, photosynthesis and photoinduction, transcription regulation and signal transduetion

  15. Construction of BAC libraries from flow-sorted chromosomes

    OpenAIRE

    Šafář, J.; Šimková, H; Doležel, J

    2016-01-01

    Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by construct...

  16. Peroxidases identified in a subtractive cDNA library approach show tissue-specific transcript abundance and enzyme activity during seed germination of Lepidium sativum.

    Science.gov (United States)

    Linkies, Ada; Schuster-Sherpa, Uta; Tintelnot, Stefanie; Leubner-Metzger, Gerhard; Müller, Kerstin

    2010-01-01

    The micropylar endosperm is a major regulator of seed germination in endospermic species, to which the close Brassicaceae relatives Arabidopsis thaliana and Lepidium sativum (cress) belong. Cress seeds are about 20 times larger than the seeds of Arabidopsis. This advantage was used to construct a tissue-specific subtractive cDNA library of transcripts that are up-regulated late in the germination process specifically in the micropylar endosperm of cress seeds. The library showed that a number of transcripts known to be up-regulated late during germination are up-regulated in the micropylar endosperm cap. Detailed germination kinetics of SALK lines carrying insertions in genes present in our library showed that the identified transcripts do indeed play roles during germination. Three peroxidases were present in the library. These peroxidases were identified as orthologues of Arabidopsis AtAPX01, AtPrx16, and AtPrxIIE. The corresponding SALK lines displayed significant germination phenotypes. Their transcripts were quantified in specific cress seed tissues during germination in the presence and absence of ABA and they were found to be regulated in a tissue-specific manner. Peroxidase activity, and particularly its regulation by ABA, also differed between radicles and micropylar endosperm caps. Possible implications of this tissue-specificity are discussed.

  17. Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

    Directory of Open Access Journals (Sweden)

    Halkier Barbara A

    2006-10-01

    Full Text Available Abstract Background We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector. Results Injection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized. Conclusion Expression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified.

  18. Lung fibrosis: drug screening and disease biomarker identification with a lung slice culture model and subtracted cDNA Library.

    Science.gov (United States)

    Guo, Tong; Lok, Ka Yee; Yu, Changhe; Li, Zhuo

    2014-09-01

    Pulmonary fibrosis is a progressive and irreversible disorder with no appropriate cure. A practical and effective experimental model that recapitulates the disease will greatly benefit the research community and, ultimately, patients. In this study, we tested the lung slice culture (LSC) system for its potential use in drug screening and disease biomarker identification. Fibrosis was induced by treating rat lung slices with 1ng/ml TGF-β1 and 2.5μM CdCl2, quantified by measuring the content of hydroxyproline, and confirmed by detecting the expression of collagen type III alpha 1 (Col3α1) and connective tissue growth factor (CTGF) genes. The anti-fibrotic effects of pirfenidone, spironolactone and eplerenone were assessed by their capability to reduce hydroxyproline content. A subtractive hybridisation technique was used to create two cDNA libraries (subtracted and unsubtracted) from lung slices. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed to assess the subtraction efficiency of the subtracted cDNA library. Clones from the two libraries were sequenced and the genes were identified by performing a BLAST search on the NCBI GenBank database. Furthermore, the relevance of the genes to fibrosis formation was verified. The results presented here show that fibrosis was effectively induced in cultured lung slices, which exhibited significantly elevated levels of hydroxyproline and Col3α1/CTGF gene expression. Several inhibitors have demonstrated their anti-fibrotic effects by significantly reducing hydroxyproline content. The subtracted cDNA library, which was enriched for differentially expressed genes, was used to successfully identify genes associated with fibrosis. Collectively, the results indicate that our LSC system is an effective model for the screening of drug candidates and for disease biomarker identification.

  19. 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...

  20. Construction and screening of marine metagenomic libraries.

    Science.gov (United States)

    Weiland, Nancy; Löscher, Carolin; Metzger, Rebekka; Schmitz, Ruth

    2010-01-01

    Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. They harbor an enormous diversity of microbes with still unknown and probably new physiological characteristics. Besides, the surfaces of marine multicellular organisms are typically covered by a consortium of epibiotic bacteria and act as barriers, where diverse interactions between microorganisms and hosts take place. Thus, microbial diversity in the water column of the oceans and the microbial consortia on marine tissues of multicellular organisms are rich sources for isolating novel bioactive compounds and genes. Here we describe the sampling, construction of large-insert metagenomic libraries from marine habitats and exemplarily one function based screen of metagenomic clones.

  1. Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Rat calcineurin (CaN) A α isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemiareperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG-FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared.The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results veri fied that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A α (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

  2. Gene discovery in Eimeria tenella by immunoscreening cDNA expression libraries of sporozoites and schizonts with chicken intestinal antibodies.

    Science.gov (United States)

    Réfega, Susana; Girard-Misguich, Fabienne; Bourdieu, Christiane; Péry, Pierre; Labbé, Marie

    2003-04-01

    Specific antibodies were produced ex vivo from intestinal culture of Eimeria tenella infected chickens. The specificity of these intestinal antibodies was tested against different parasite stages. These antibodies were used to immunoscreen first generation schizont and sporozoite cDNA libraries permitting the identification of new E. tenella antigens. We obtained a total of 119 cDNA clones which were subjected to sequence analysis. The sequences coding for the proteins inducing local immune responses were compared with nucleotide or protein databases and with expressed sequence tags (ESTs) databases. We identified new Eimeria genes coding for heat shock proteins, a ribosomal protein, a pyruvate kinase and a pyridoxine kinase. Specific features of other sequences are discussed.

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

    Science.gov (United States)

    Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

    2016-01-01

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

  4. Differential screening of mitochondrial cDNA libraries from male-fertile and cytoplasmic male-sterile sugar-beet reveals genome rearrangements at atp6 and atpA loci.

    Science.gov (United States)

    Xue, Y; Collin, S; Davies, D R; Thomas, C M

    1994-04-01

    As part of a strategy to define differences in genome organization and expression between cytoplasmic male-sterile (CMS) and male-fertile (MF) sugar-beet mitochondria, cDNA libraries from both mitochondrial genotypes were constructed. Preliminary screening with ribosomal RNA gene probes identified candidate cDNA clones corresponding to structural genes. In addition, reciprocal hybridization experiments were performed using labelled first-strand cDNA to identify uniquely transcribed sequences. One cDNA clone (pYC700) is unique to CMS mitochondria and is located upstream of the F0F1-ATPase subunit 6 gene (atp6). Another cDNA clone (pYC130), when used as a probe in northern hybridization analysis, revealed novel transcript profiles in CMS sugar-beet mitochondria. Sequence analysis of this cDNA showed strong homology with the F0F1-ATPase subunit alpha (atpA) coding sequences from several higher plants. The atp6 and atpA loci from each genotype were cloned and the genomic organization, DNA sequence and transcription of each locus was studied. Differences in the transcript profiles of each gene are a consequence of genomic rearrangements 5' to the coding sequence.

  5. 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

  6. 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.

  7. Identification of a panel of tumor-associated antigens from breast carcinoma cell lines, solid tumors and testis cDNA libraries displayed on lambda phage

    Directory of Open Access Journals (Sweden)

    Cianfriglia Maurizio

    2004-11-01

    Full Text Available Abstract Background Tumor-associated antigens recognized by humoral effectors of the immune system are a very attractive target for human cancer diagnostics and therapy. Recent advances in molecular techniques have led to molecular definition of immunogenic tumor proteins based on their reactivity with autologous patient sera (SEREX. Methods Several high complexity phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines MCF-7, MDA-MB-468 were constructed. The cDNAs were expressed in the libraries as fusion to bacteriophage lambda protein D. Lambda-displayed libraries were efficiently screened with sera from patients with breast cancer. Results A panel of 21 clones representing 18 different antigens, including eight proteins of unknown function, was identified. Three of these antigens (T7-1, T11-3 and T11-9 were found to be overexpressed in tumors as compared to normal breast. A serological analysis of the 21 different antigens revealed a strong cancer-related profile for at least five clones (T6-2, T6-7, T7-1, T9-21 and T9-27. Conclusions Preliminary results indicate that patient serum reactivity against five of the antigens is associated with tumor disease. The novel T7-1 antigen, which is overexpressed in breast tumors and recognized specifically by breast cancer patient sera, is potentially useful in cancer diagnosis.

  8. Survey of a cDNA library from the turkey (Meleagris gallopavo).

    Science.gov (United States)

    Chaves, L D; Rowe, J A; Reed, K M

    2005-02-01

    Genome characterization and analysis is an imperative step in identifying and selectively breeding for improved traits of agriculturally important species. Expressed sequence tags (ESTs) represent a transcribed portion of the genome and are an effective way to identify genes within a species. Downstream applications of EST projects include DNA microarray construction and interspecies comparisons. In this study, 694 ESTs were sequenced and analyzed from a library derived from a 24-day-old turkey embryo. The 437 unique sequences identified were divided into 76 assembled contigs and 361 singletons. The majority of significant comparative matches occurred between the turkey sequences and sequences reported from the chicken. Whole genome sequence from the chicken was used to identify potential exon-intron boundaries for selected turkey clones and intron-amplifying primers were developed for sequence analysis and single nucleotide polymorphism (SNP) discovery. Identified SNPs were genotyped for linkage analysis on two turkey reference populations. This study significantly increases the number of EST sequences available for the turkey.

  9. cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-telomeric Roles of Arabidopsis Telomerase

    Directory of Open Access Journals (Sweden)

    Ladislav eDokládal

    2015-11-01

    Full Text Available Telomerase-reverse transcriptase (TERT plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE TERT domain and identified a nuclear-localized protein that contains a RNA recognition motif (RRM. This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions.

  10. cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-Telomeric Roles of Arabidopsis Telomerase.

    Science.gov (United States)

    Dokládal, Ladislav; Honys, David; Rana, Rajiv; Lee, Lan-Ying; Gelvin, Stanton B; Sýkorová, Eva

    2015-01-01

    Telomerase-reverse transcriptase (TERT) plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE) TERT domain and identified a nuclear-localized protein that contains an RNA recognition motif (RRM). This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions.

  11. Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi

    NARCIS (Netherlands)

    Gemeren, I.A. van; Musters, W.; Hondel, C.A.M.J.J. van den; Verrips, C.T.

    1995-01-01

    A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the subseq

  12. An annotated cDNA library of juvenile Euprymna scolopes with and without colonization by the symbiont Vibrio fischeri

    Directory of Open Access Journals (Sweden)

    Tong Deyan

    2006-06-01

    Full Text Available Abstract Background Biologists are becoming increasingly aware that the interaction of animals, including humans, with their coevolved bacterial partners is essential for health. This growing awareness has been a driving force for the development of models for the study of beneficial animal-bacterial interactions. In the squid-vibrio model, symbiotic Vibrio fischeri induce dramatic developmental changes in the light organ of host Euprymna scolopes over the first hours to days of their partnership. We report here the creation of a juvenile light-organ specific EST database. Results We generated eleven cDNA libraries from the light organ of E. scolopes at developmentally significant time points with and without colonization by V. fischeri. Single pass 3' sequencing efforts generated 42,564 expressed sequence tags (ESTs of which 35,421 passed our quality criteria and were then clustered via the UIcluster program into 13,962 nonredundant sequences. The cDNA clones representing these nonredundant sequences were sequenced from the 5' end of the vector and 58% of these resulting sequences overlapped significantly with the associated 3' sequence to generate 8,067 contigs with an average sequence length of 1,065 bp. All sequences were annotated with BLASTX (E-value Conclusion Both the number of ESTs generated from each library and GO categorizations are reflective of the activity state of the light organ during these early stages of symbiosis. Future analyses of the sequences identified in these libraries promise to provide valuable information not only about pathways involved in colonization and early development of the squid light organ, but also about pathways conserved in response to bacterial colonization across the animal kingdom.

  13. A novel approach to investigation of the pathogenesis of active minimal-change nephrotic syndrome using subtracted cDNA library screening.

    Science.gov (United States)

    Sahali, Djillali; Pawlak, André; Valanciuté, Asta; Grimbert, Philippe; Lang, Philippe; Remy, Philippe; Bensman, Albert; Guellaën, Georges

    2002-05-01

    Clinical and experimental observations suggest that minimal-change nephrotic syndrome (MCNS) results from T cell dysfunction, via unknown mechanisms. For the identification of genes that are potentially involved in MCNS, a subtractive cDNA library was constructed from cDNA from T cell-enriched peripheral blood mononuclear cells obtained from the same patient during relapse versus remission ("relapse minus remission"). This library was screened by differential hybridization with forward ("relapse minus remission") and reverse ("remission minus relapse") subtractive cDNAs probes, as well as unsubtracted probes from relapse and remission, and irrelevant nephrotic syndrome (membranous nephropathy). A total of 84 transcripts were isolated, of which 12 matched proteins of unknown function and 30 were unknown clones. Among the 42 known transcripts, at least 18 are closely involved in the T cell receptor-mediated complex signaling cascade, including genes encoding components of the T cell receptor and proteins associated with the cytoskeletal scaffold, as well as transcription factors. In particular, it was demonstrated that the expression levels of Fyb/Slap, L-plastin, and grancalcin were increased during relapse, suggesting that the integration of proximal signaling after T cell engagement involves the preferential recruitment of these cytoskeleton-associated proteins in MCNS. Because very low levels of interleukin-12 receptor beta2 mRNA were detected in relapse samples, the interleukin-12 signaling pathway might be defective, suggesting that, in MCNS, T cell activation evolves toward a T helper 2 phenotype. Therefore, the combination of subtractive cloning and differential screening constitutes an efficient approach to the identification of genes that are likely to be involved in the pathophysiologic processes of MCNS.

  14. Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene.

    Science.gov (United States)

    Sun, Wei; Li, Da; Su, Rui; Musa, Hassan H; Chen, Ling; Zhou, Hong

    2014-02-01

    RT-PCR, 5'RACE, 3'RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities.

  15. New algebraic constructions for pooling design in DNA library screening.

    Science.gov (United States)

    Li, Zengti; Gao, Suogang; Du, Hongjie; Shi, Yan

    2010-01-01

    Pooling design is an important mathematical tool in DNA library screening. It has been showed that using pooling design, the number of tests in DNA library screening can be greatly reduced. In this paper, we present some new algebraic constructions for pooling design.

  16. Construction and Analysis of N-phosphoryl Peptide Libraries

    Institute of Scientific and Technical Information of China (English)

    Shu Xia CAO; Jian Chen ZHANG; Ming Yu NIU; Kui LU; Xin Cheng LIAO; Yu Fen ZHAO

    2004-01-01

    N-Phosphoryl peptide libraries were constructed by transformation from homo-oligopeptide libraries, which was synthesized by self-assembly of amino acids with the assistance of phosphorus oxychloride. Electrospray ionization mass spectrometry (ESI-MS) was used to monitor the reaction.

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

    Science.gov (United States)

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

    2015-08-01

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

  18. In-depth cDNA Library Sequencing Provides Quantitative Gene Expression Profiling in Cancer Biomarker Discovery

    Institute of Scientific and Technical Information of China (English)

    Wanling Yang; Dingge Ying; Yu-Lung Lau

    2009-01-01

    procedures may allow detection of many expres-sion features for less abundant gene variants. With the reduction of sequencing cost and the emerging of new generation sequencing technology, in-depth sequencing of cDNA pools or libraries may represent a better and powerful tool in gene expression profiling and cancer biomarker detection. We also propose using sequence-specific subtraction to remove hundreds of the most abundant housekeeping genes to in-crease sequencing depth without affecting relative expression ratio of other genes, as transcripts from as few as 300 most abundantly expressed genes constitute about 20% of the total transcriptome. In-depth sequencing also represents a unique ad-vantage of detecting unknown forms of transcripts, such as alternative splicing variants, fusion genes, and regulatory RNAs, as well as detecting mutations and polymorphisms that may play important roles in disease pathogenesis.

  19. Identification and characterization of a new autoimmune protein in membranous nephropathy by immunoscreening of a renal cDNA library.

    Directory of Open Access Journals (Sweden)

    Fabrizio Cavazzini

    Full Text Available Membranous Nephropathy (MN represents a large amount of Nephrotic Syndromes in the adult population and its definitive diagnosis is currently carried out through biopsy. An autoimmune condition has been demonstrated in idiopathic MN (iMN in which some kidney structures are targeted by patient autoantibodies. Some candidate antigens have been described and other likely involved target proteins responsible for the disease are not known yet. In this work our aim is to identify these proteins by screening a lambda-phage library with patients' sera. We enrolled four groups of patients: two MN groups of 12 full iMN patients; one control group of 15 patients suffering from other renal diseases; one control group of 15 healthy individuals. A commercial cDNA phagemide library was screened using the above described sera, in order to detect positive signals due to antigen-antibody bond. We detected one phagemide clone expressing a protein which was shown to be targeted by the antibodies of the iMN sera only. Control sera were negative. The sequence analysis of cDNA matched the Synaptonemal Complex protein 65 (SC65 coding sequence. Further proteomic analyses were carried out to validate our results. We provide evidence of an involvement of SC65 protein as an autoimmune target in iMN. Considering the invasiveness and the resulting risk coming from renal biopsy, our ongoing aim is to set a procedure able to diagnose affected patients through a little- or non-invasive method such as blood sampling rather than biopsy.

  20. 羊种布鲁菌05/43株侵染绵羊肺泡巨噬细胞cDNA文库的构建%Construction of cDNA library of sheep alveolar macrophages infected with Brucella melitensis 05/43 strain

    Institute of Scientific and Technical Information of China (English)

    王远志; 陈创夫; 曹旭东; 盛金良; 张辉; 任艳; 高剑峰; 王端明

    2008-01-01

    目的 构建羊种布鲁菌 05/43 株侵染绵羊肺泡巨噬细胞的cDNA 文库.方法 培养的绵羊肺泡巨噬细胞经羊种布鲁菌 05/43 株侵染后,以 Trizol 试剂提取其总 RNA,用 cDNA 文库构建试剂盒构建巨噬细胞的 cDNA 文库.随机选取 cDNA 文库中的 18 个克隆进行表达序列标签 (EST) 序列测定,测序结果 用 BlastX 和 BlastN 软件在 GenBank 蛋白质库和核酸库中进行序列同源性比对.结果 构建的 cDNA 文库的库容量为1.192×106,重组率为 95.65%.18 个 EST 测序,12 个与 CD 抗原基因、细胞因子基因、蛋白酶基因等已知功能的基因序列相关,6 个为未知功能基因的 EST 序列.结论 成功构建了羊种布鲁菌 05/43 株侵染绵羊肺泡巨噬细胞的 cDNA 文库,这将有助于阐明该菌株的致病机制.

  1. The tetramethylammonium chloride method for screening of cDNA libraries using highly degenerate oligonucleotides obtained by backtranslation of amino-acid sequences

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Leffers, H

    1993-01-01

    We describe a method for screening of cDNA libraries with highly degenerate oligonucleotides using tetramethylammonium chloride (TMAC). This method is a convenient alternative to using probes generated by the polymerase chain reaction (PCR), especially when these cannot easily be made. Nylon filt...

  2. 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.

  3. Identification of expressed genes during compatible interaction between stripe rust (Puccinia striiformis and wheat using a cDNA library

    Directory of Open Access Journals (Sweden)

    Huang Lili

    2009-12-01

    Full Text Available Abstract Background Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst, is one of the most destructive diseases of wheat worldwide. To establish compatibility with the host, Pst forms special infection structures to invade the plant with minimal damage to host cells. Although compatible interaction between wheat and Pst has been studied using various approaches, research on molecular mechanisms of the interaction is limited. The aim of this study was to develop an EST database of wheat infected by Pst in order to determine transcription profiles of genes involved in compatible wheat-Pst interaction. Results Total RNA, extracted from susceptible infected wheat leaves harvested at 3, 5 and 8 days post inoculation (dpi, was used to create a cDNA library, from which 5,793 ESTs with high quality were obtained and clustered into 583 contigs and 2,160 singletons to give a set of 2,743 unisequences (GenBank accessions: GR302385 to GR305127. The BLASTx program was used to search for homologous genes of the unisequences in the GenBank non-redundant protein database. Of the 2,743 unisequences, 52.8% (the largest category were highly homologous to plant genes; 16.3% to fungal genes and 30% of no-hit. The functional classification of all ESTs was established based on the database entry giving the best E-value using the Bevan's classification categories. About 50% of the ESTs were significantly homologous to genes encoding proteins with known functions; 20% were similar to genes encoding proteins with unknown functions and 30% did not have significant homology to any sequence in the database. The quantitative real-time PCR (qRT-PCR analysis determined the transcription profiles and their involvement in the wheat-Pst interaction for seven of the gene. Conclusion The cDNA library is useful for identifying the functional genes involved in the wheat-Pst compatible interaction, and established a new database for studying Pst pathogenesis genes

  4. Screening of genes of proteins interacting with p7 protein of hepatitis C virus from human liver cDNA library by yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    Yan-Ping Huang; Xue-Song Gao; Dong Ji; Shu-Mei Lin; Yan-Wei Zhong; Qing Shao; Shu-Lin Zhang; Jun Cheng; Lin Wang; Jiang Guo; Yan Liu; Yuan Yang; Li-Ying Zhang; Gui-Qin Bai

    2005-01-01

    AIM: To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes.METHODS: We constructed p7 protein bait plasmid by doning the gene of p7 protein into pGBKT7, then transformed it into yeast AH109 (a type). The transformed yeast was mated with yeast Y187 (α type) containing liver cDNA library plasmid, pACT2 in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection and screening. After extracting and sequencing of plasmids from blue colonies, we performed sequence analysis by bioinformatics.RESULTS: Fifty colonies were selected and sequenced.Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nucleoporin 214 ku and two colonies were CLL-associated antigens.CONCLUSION: The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its associated protein.

  5. 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.

  6. Characterization of Leukemia-Inducing Genes Using a Proto-Oncogene/Homeobox Gene Retroviral Human cDNA Library in a Mouse In Vivo Model.

    Directory of Open Access Journals (Sweden)

    Su Hwa Jang

    Full Text Available The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30% had MYC as the only transgene, and seven mice (70% had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.

  7. Characterization of Leukemia-Inducing Genes Using a Proto-Oncogene/Homeobox Gene Retroviral Human cDNA Library in a Mouse In Vivo Model.

    Science.gov (United States)

    Jang, Su Hwa; Lee, Sohyun; Chung, Hee Yong

    2015-01-01

    The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML) using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM) cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30%) had MYC as the only transgene, and seven mice (70%) had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.

  8. Construction and analysis of liver suppression subtractive hybridization library of silver carp (Hypophthalmichthys molitrix) intraperitoneally injected with microcystin-LR.

    Science.gov (United States)

    Qu, Xiancheng; Zhang, Kaiyue; Cui, Zhihui; Zhang, Yong; Jiang, Jiaoyun; Feng, Long; Liu, Qigen

    2011-09-01

    Microcystin-LR (MC-LR) is the most frequently studied cyclic heptapeptide hepatotoxin produced by cyanobacteria. The toxin accumulates rapidly in the liver where it exerts most of its damage, but the molecular mechanisms behind its toxicity remain unclear. Here, suppression subtractive hybridization (SSH) was used to identify alterations in gene transcription of the silver carp (Hypophthalmichthys molitrix) after exposure to MC-LR. After hybridization and cloning, the forward and reverse subtractive cDNA libraries were obtained. At random, 150 positive clones (70 forward and 80 reverse) were selected and sequenced from the subtractive libraries, which gave a total of 88 gene fragment sequences (48 forward and 40 reverse). Sequencing analysis and homology searches showed that these ESTs represented 75 unique genes and 13 duplicates. Of the 75 unique genes, 38 shared high homology with fish genes of known functions, including immune-related genes, transporters and some involved in cell metabolism. Four sequenced genes (Fs59, Fs70, Rs2 and Rs15) were analyzed further using semi-quantitative RT-PCR. The genes from the forward library (Fs59 and Fs70) were found to be transcriptionally upregulated, while the genes from the reverse library (Rs2 and Rs15) were found to be transcriptionally downregulated. These results confirmed the successful construction of the subtractive cDNA library that was enriched for genes that were differentially transcribed in the silver carp liver challenged with MC-LR.

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

    Directory of Open Access Journals (Sweden)

    Czosnek Henryk

    2006-04-01

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

  10. Identification of midgut microvillar proteins from Tenebrio molitor and Spodoptera frugiperda by cDNA library screenings with antibodies.

    Science.gov (United States)

    Ferreira, A H P; Cristofoletti, P T; Lorenzini, D M; Guerra, L O; Paiva, P B; Briones, M R S; Terra, W R; Ferreira, C

    2007-11-01

    The objective of this study was to identify midgut microvillar proteins in insects appearing earlier (Coleoptera) and later (Lepidoptera) in evolution. For this, cytoskeleton-free midgut microvillar membrane from Spodoptera frugiperda (Lepidoptera) and Tenebrio molitor (Coleoptera) were used to raise antibodies. These were used for screening midgut cDNA expression libraries. Positive clones were sequenced, assembled and searched for similarities with gene/protein databases. The predicted midgut microvillar proteins from T. molitor were: cockroach allergens (unknown function), peritrophins (peritrophic membrane proteins), digestive enzymes (aminopeptidase, alpha-mannosidase) and unknown proteins. Predicted S. frugiperda midgut proteins may be grouped into six classes: (a) proteins involved in protection of midgut (thioredoxin peroxidase, aldehyde dehydrogenase, serpin and juvenile hormone epoxide hydrolase); (b) digestive enzymes (astacin, transporter-like amylase, aminopeptidase, and carboxypeptidase); (c) peritrophins; (d) proteins associated with microapocrine secretion (gelsolin, annexin); (e) membrane-tightly bound-cytoskeleton proteins (fimbrin, calmodulin) and (f) unidentified proteins. The novel approach is compared with others and microvillar function is discussed in the light of the predicted proteins.

  11. Modification of the GS LT Paired-end Library Protocol for Constructing Longer Insert Size Libraries

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Ze; Peng, Ze; Hamilton, Matthew; Ting, Sara; Tu, Hank; Goltsman, Eugene; Lapidus, Alla; Lucas, Susan; Cheng, Jan-Fang

    2008-05-22

    Paired-end library sequencing has been proven useful in scaffold construction during de novo assembly of genomic sequences. The ability of generating mate pairs with 8 Kb or greater insert sizes is especially important for genomes containing long repeats. While the current 454 GS LT Paired-end library preparation protocol can successfully construct libraries with 3 Kb insert size, it fails to generate longer insert sizes because the protocol is optimized to purify shorter fragments. We have made several changes in the protocol in order to increase the fragment length. These changes include the use of Promega column to increase the yield of large size DNA fragments, two gel purification steps to remove contaminated short fragments, and a large reaction volume in the circularization step to decrease the formation of chimeras. We have also made additional changes in the protocol to increase the overall quality of the libraries. The quality of the libraries are measured by a set of metrics, which include levels of redundant reads, linker positive, linker negative, half linker reads, and driver DNA contamination, and read length distribution, were used to measure the primary quality of these libraries. We have also assessed the quality of the resulted mate pairs including levels of chimera, distribution of insert sizes, and genome coverage after the assemblies are completed. Our data indicated that all these changes have improved the quality of the longer insert size libraries.

  12. 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....

  13. Cloning of open reading frames and promoters from the Saccharomyces cerevisiae genome: construction of genomic libraries of random small fragments.

    Science.gov (United States)

    Santangelo, G M; Tornow, J; Moldave, K

    1986-01-01

    We have developed a novel efficient method, carrier-facilitated insertion, to insert small (150-600 bp) DNA fragments into plasmid vectors. This method employs a carrier segment of vector DNA to circumvent the difficulties in ligating two fragments together to generate a recombinant circle efficiently. We have used carrier-facilitated insertion to construct three genomic libraries of random (DNase I-generated) fragments from the Saccharomyces cerevisiae genome. One of these was an expression library, and the other two were promoter-cloning libraries. 87-90% of the Escherichia coli colonies in each library contained recombinant plasmids, and less than 3% of the recombinants contained more than one insert. Detection of open reading frames among the inserts in the expression library was accomplished by testing for beta-galactosidase activity. This methodology, unencumbered by the intrinsic disproportionality of cDNA libraries, can be used to identify and clone DNA that codes for a specific antigenic determinant. When used in combination with a method to detect and isolate random constitutive, repressible and inducible yeast promoters, these libraries should permit a comprehensive analysis of the yeast genome and its expression.

  14. Construction and characterization of an expressed sequenced tag library for the mosquito vector Armigeres subalbatus

    Directory of Open Access Journals (Sweden)

    Tsai Shih-Feng

    2007-12-01

    Full Text Available Abstract Background The mosquito, Armigeres subalbatus, mounts a distinctively robust innate immune response when infected with the nematode Brugia malayi, a causative agent of lymphatic filariasis. In order to mine the transcriptome for new insight into the cascade of events that takes place in response to infection in this mosquito, 6 cDNA libraries were generated from tissues of adult female mosquitoes subjected to immune-response activation treatments that lead to well-characterized responses, and from aging, naïve mosquitoes. Expressed sequence tags (ESTs from each library were produced, annotated, and subjected to comparative analyses. Results Six libraries were constructed and used to generate 44,940 expressed sequence tags, of which 38,079 passed quality filters to be included in the annotation project and subsequent analyses. All of these sequences were collapsed into clusters resulting in 8,020 unique sequence clusters or singletons. EST clusters were annotated and curated manually within ASAP (A Systematic Annotation Package for Community Analysis of Genomes web portal according to BLAST results from comparisons to Genbank, and the Anopheles gambiae and Drosophila melanogaster genome projects. Conclusion The resulting dataset is the first of its kind for this mosquito vector and provides a basis for future studies of mosquito vectors regarding the cascade of events that occurs in response to infection, and thereby providing insight into vector competence and innate immunity.

  15. Large-scale Identification of Expressed Sequence Tags (ESTs from Nicotianatabacum by Normalized cDNA Library Sequencing

    Directory of Open Access Journals (Sweden)

    Alvarez S Perez

    2014-12-01

    Full Text Available An expressed sequence tags (EST resource for tobacco plants (Nicotianatabacum was established using high-throughput sequencing of randomly selected clones from one cDNA library representing a range of plant organs (leaf, stem, root and root base. Over 5000 ESTs were generated from the 3’ ends of 8000 clones, analyzed by BLAST searches and categorized functionally. All annotated ESTs were classified into 18 functional categories, unique transcripts involved in energy were the largest group accounting for 831 (32.32% of the annotated ESTs. After excluding 2450 non-significant tentative unique transcripts (TUTs, 100 unique sequences (1.67% of total TUTs were identified from the N. tabacum database. In the array result two genes strongly related to the tobacco mosaic virus (TMV were obtained, one basic form of pathogenesis-related protein 1 precursor (TBT012G08 and ubiquitin (TBT087G01. Both of them were found in the variety Hongda, some other important genes were classified into two groups, one of these implicated in plant development like those genes related to a photosynthetic process (chlorophyll a-b binding protein, photosystem I, ferredoxin I and III, ATP synthase and a further group including genes related to plant stress response (ubiquitin, ubiquitin-like protein SMT3, glycine-rich RNA binding protein, histones and methallothionein. The interesting finding in this study is that two of these genes have never been reported before in N. tabacum (ubiquitin-like protein SMT3 and methallothionein. The array results were confirmed using quantitative PCR.

  16. Development and construction of China’s higher education libraries

    Institute of Scientific and Technical Information of China (English)

    YAN; Jinwei; ZHENG; Lan; SONG; Xue; ZHANG; Yan; SONG; Jiao; DAI; Longji; LI; Dejuan

    2008-01-01

    Libraries in China’s higher education institutions have been developing in keeping pace with the flourishing development of China’s higher education.This article aims to make an introduction to the construction of China’s higher education libraries,especially the recent three decades’achievements since China’s reform and opening-up in 1978.In this article,the authors draw a general picture of the development of libraries in China’s higher education institutions,covering such eight aspects as management,types and positioning,organizational structure and personnel,expenditure and buildings,reader service,building and sharing of resources as well as automation system.

  17. Construction of the Seed-Coat cDNA Microarray and Screening of Differentially Expressed Genes in Barley

    Institute of Scientific and Technical Information of China (English)

    Jin-Song PANG; Meng-Yuan HE; Bao LIU

    2004-01-01

    Some barley mutants can synthesize neither anthocyanins nor proanthocyanidins in the seed coat, which is related to several genes in locus Ant13, but the exact model of action remains unknown. We used the cDNA microarray technology with barley transcription-deficient mutant (ant13-152) that does not synthesize proanthocyanidins as the tester, and its wild type genotype (Triumph) as the driver, to study this question. Six-thousand and forty-eight clones from the wild type Morex testa+pericarp cDNA library were amplified using PCR, and the DNA fragments were spotted on commercial amino-modified glass slide as microarray. The mRNAs from the developing seed coat (8-15 days) of both the mutant and the wild-type barley plants were isolated, and labeled respectively with Cy3-dUTP and Cy5-dUTP when reversely transcribed to cDNAs. The labeled cDNAs were used as probes, mixed at the same molar concentration, and hybridized with the DNA fragments on the slide. Seventy clones exhibiting marked differential expression (ratio>4) were identified from the microarray. All the 25 cDNA clones that showed an over-expression in wild type in comparison to the mutant ant13-152 were sequenced. It was found that most of these overexpressing clones were transcription/translation and hordein-associated genes. These results have laid a solid material basis for further elucidation of the metabolic pathway in proanthocyanidin synthesis in barley and likely other plants.

  18. Construction and characterization of infectious cDNA clones of a chicken strain of hepatitis E virus (HEV), avian HEV.

    Science.gov (United States)

    Huang, F F; Pierson, F W; Toth, T E; Meng, X J

    2005-09-01

    Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important human pathogen. Increasing evidence indicates that hepatitis E is a zoonosis. Avian HEV was recently discovered in chickens with hepatitis-splenomegaly syndrome in the USA. Like swine HEV from pigs, avian HEV is also genetically and antigenically related to human HEV. The objective of this study was to construct and characterize an infectious cDNA clone of avian HEV for future studies of HEV replication and pathogenesis. Three full-length cDNA clones of avian HEV, pT7-aHEV-5, pT7G-aHEV-10 and pT7G-aHEV-6, were constructed and their infectivity was tested by in vitro transfection of leghorn male hepatoma (LMH) chicken liver cells and by direct intrahepatic inoculation of specific-pathogen-free (SPF) chickens with capped RNA transcripts from the three clones. The results showed that the capped RNA transcripts from each of the three clones were replication competent when transfected into LMH cells as demonstrated by detection of viral antigens with avian HEV-specific antibodies. SPF chickens intrahepatically inoculated with the capped RNA transcripts from each of the three clones developed active avian HEV infections as evidenced by seroconversion to avian HEV antibodies, viraemia and faecal virus shedding. The infectivity was further confirmed by successful infection of naïve chickens with the viruses recovered from chickens inoculated with the RNA transcripts. The results indicated that all three cDNA clones of avian HEV are infectious both in vitro and in vivo. The availability of these infectious clones for a chicken strain of HEV now affords an opportunity to study the mechanisms of HEV cross-species infection and tissue tropism by constructing chimeric viruses among human, swine and avian HEVs.

  19. Construction and characterization of genomic libraries from specific human chromosomes.

    Science.gov (United States)

    Krumlauf, R; Jeanpierre, M; Young, B D

    1982-05-01

    Highly purified fractions of human chromosomes 21 and 22 were isolated from a suspension of metaphase chromosomes stained with ethidium bromide by using a fluorescence-activated cell sorter (FACS II). Two recombinant DNA libraries, representing chromosomes 21 and 22, were constructed by complete digestion of DNA from these fractions with EcoRI and insertion into the vector lambda gtWES lambda B. Twenty clones selected at random from the chromosome 22 library hybridized to EcoRI-digested human DNA, and five of these clones hybridized to single bands identical in size to the phage inserts. These five single-copy sequences and a clone coding for an 8S RNA isolated by screening the chromosome 22 library for expressed sequences were characterized in detail. Hybridization of all six clones to a panel of sorted chromosomes and hybrid cell lines confirmed the assignment of the sequences to chromosome 22. The sequences were localized to regions of chromosome 22 by hybridization to translocated chromosomes sorted from a cell line having a balanced translocation t(17;22)(p13;q11) and to hybrid cell lines containing the various portions of another translocation t(X;22)(q13;q112). Five clones reside on the long arm of chromosome 22 between q112 and pter, while one clone and an 18S rRNA gene isolated from the chromosome 22 library reside pter and g112. The construction of chromosome-specific libraries by this method has the advantage of being direct and applicable to nearly all human chromosomes and will be important in molecular analysis of human genetic diseases.

  20. Construction and analysis of gonad suppression subtractive hybridization libraries for the rice field eel, Monopterus albus.

    Science.gov (United States)

    Qu, Xiancheng; Jiang, Jiaoyun; Shang, Xiaoli; Cheng, Cui; Feng, Long; Liu, Qigen

    2014-04-25

    The objective of this study was to investigate gene transcription profiles of the stage IV ovary and the ovotestis of the rice field eel (Monopterus albus) in an attempt to uncover genes involved in sex reversal and gonad development. Suppression subtractive hybridization (SSH) libraries were constructed using mRNA from the stage IV ovary and the ovotestis. In total 100 positive clones from the libraries were selected at random and sequenced, and then expressed sequence tags (ESTs) were used to search against sequences in the GenBank database using the BLASTn and BLASTx search algorithms. High quality SSH cDNA libraries and 90 ESTs were obtained. Of these ESTs, 43 showed high homology with genes of known function and these are associated with energy metabolism, signal transduction, transcription regulation and so on. The remaining 47 ESTs shared no homology with any genes in GenBank and are thus considered to be hypothetical genes. Furthermore, the four genes F11, F63, R11, and R47 from the forward and reverse libraries were analyzed in gonad, brain, heart, spleen, liver, kidney and muscle tissues. The results showed that the transcription of the F11 and F63 genes was significantly increased while the expression of the R11 and R47 genes was significantly decreased from IV or V ovary. In addition, the results also indicated that the four genes' expression was not gonad-tissue specific. This results strongly suggested that they may be involved in the rice field eel gonad development and/or sex reversal.

  1. THE CLONING OF HRNT-1 USING A COMBINATION OF cDNA LIBRARY SCREENING WITH BIOTIN-LABELED PROBE AND RAPID AMPLIFICATION OF cDNA ENDS

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Kai-tai

    2001-01-01

    [1]Tom S, Andrew PR. Human Molecular Genetics [M]. John Wiley & Sons, Inc. United States of America 1996; 335.[2]Zhao Yong-liang, Jin Cui-zhen, Wu De-chang et al. Neoplastic transformation and cytogenetic changes of rat tracheal epithelial cells induced by a-particles irradiation [J]. Chin Med Sci J 1997; 12:202.[3]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[4]Frederick A, Roger B. Current Protocols in Molecular Biology [M]. John Wiley & Sons, Inc. United States of America 1998; 2.1.1.[5]Roux KH. Optimization and troubleshooting in PCR [J]. PCR Methods Appl 1995; 4:5158.[6]Sambrook, J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual [M]. 2nd Ed. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989; 54.[7]Zhang Y, Frohman MA. Using rapid amplification of cDNA ends (RACE) to obtain full-length cDNAs [J]. Methods Mol Biol 1997; 69:61.[8]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[9]Iqbal S, Robinson J, Deere D, et al. Efficiency of the polymerase chain reaction amplification of the uid gene for detection of Escherichia coli in contaminated water [J]. Lett Appl Microbiol 1997; 24:498.[10]Schunck B, Kraft W, Truyen U. A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces [J]. J Virol Methods 1995; 55:427.

  2. 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.

  3. 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...

  4. Yeast surface display for antibody isolation: library construction, library screening, and affinity maturation.

    Science.gov (United States)

    Van Deventer, James A; Wittrup, Karl Dane

    2014-01-01

    Antibodies play key roles as reagents, diagnostics, and therapeutics in numerous biological and biomedical research settings. Although many antibodies are commercially available, oftentimes, specific applications require the development of antibodies with customized properties. Yeast surface display is a robust, versatile, and quantitative method for generating these antibodies and is accessible to single-investigator laboratories. This protocol details the key aspects of yeast surface display library construction and screening.

  5. Construction of gene targeting vectors from lambda KOS genomic libraries.

    Science.gov (United States)

    Wattler, S; Kelly, M; Nehls, M

    1999-06-01

    We describe a highly redundant murine genomic library in a new lambda phage, lambda knockout shuttle (lambda KOS) that facilitates the very rapid construction of replacement-type gene targeting vectors. The library consists of 94 individually amplified subpools, each containing an average of 40,000 independent genomic clones. The subpools are arrayed into a 96-well format that allows a PCR-based efficient recovery of independent genomic clones. The lambda KOS vector backbone permits the CRE-mediated conversion into high-copy number pKOS plasmids, wherein the genomic inserts are automatically flanked by negative-selection cassettes. The lambda KOS vector system exploits the yeast homologous recombination machinery to simplify the construction of replacement-type gene targeting vectors independent of restriction sites within the genomic insert. We outline procedures that allow the generation of simple and more sophisticated conditional gene targeting vectors within 3-4 weeks, beginning with the screening of the lambda KOS genomic library.

  6. Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

    Directory of Open Access Journals (Sweden)

    Motedayen, M.H.

    2015-12-01

    Full Text Available Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthesis and amplification of the heavy (Fd segment and kappa light chains of IgG antibody. After digestion of the heavy chain with SpeI and XhoI and light chain with XbaI and SacI enzymes, inserted successively into the cloning vector pComb3x, and then recombinant vector transformed to TG1 bacteria to construct the Fab library. For determination insertion rate of Fab segment into cloning vector, plasmids of 12 clones of library were extracted and digested with SfiI. Length of amplified Fd and κ chains, were 650 - 750 bp. Primary library size was determined to contain 4.9×105 members out of which half of them contained the same size of Fab fragment. This result is comparable to some researchers and shows that this method could be appropriate tool for the production of human polyclonal Fab fragment antibodies for management of poisonous snake bitted victims.

  7. Construction and characterization of human chromosome 2-specific cosmid, fosmid, and PAC clone libraries

    Energy Technology Data Exchange (ETDEWEB)

    Gingrich, J.C.; Boehrer, D.M.; Garnes, J.A. [Lawrence Livermore National Lab., CA (United States)] [and others

    1996-02-15

    This article discusses the construction and characterization of three human chromosome 2-specific clone libraries. A chromosome 2-specific PAC library was also constructed from a hybrid cell line. The chromosome 2 coverage of each of the three libraries was further determined by PCR screening clone pools with 82 chromosome 2-specific STSs. 47 refs., 3 figs., 1 tab.

  8. A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector.

    Science.gov (United States)

    Junqueira, Bruna Rayane Teodoro; Nicolini, Cícero; Lucinda, Natalia; Orílio, Anelise Franco; Nagata, Tatsuya

    2014-03-01

    Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants.

  9. The construction of genomic libraries of Cowdria ruminantium in an expression vector, lambda gt11.

    Science.gov (United States)

    Ambrosio, R E; Du Plessis, J L; Bezuidenhout, J D

    1987-09-01

    Genomic libraries of the Welgevonden and Kwanyanga isolates of Cowdria ruminantium have been constructed in an expression vector. These libraries contain approximately 4 x 10(5) and 3 x 10(5) recombinants respectively.

  10. Construction of differentially expressed genes library of bighead carp (Aristichthys nobilis) exposed to microcystin-lr using ssh and expression profile of related genes.

    Science.gov (United States)

    Cui, Zhihui; Zhang, Kaiyue; Qu, Xiancheng; Liu, Qigen

    2011-12-01

    Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria (blue-green algae). There are more than 70 MCs variants of which the most common and widely studied is MC-LR. We screened the hepatocellular differentially expressed genes against MC-LR in the bighead carp (Aristichthys nobilis). Suppression subtractive hybridization was used to construct the forward subtracted and reverse subtracted cDNA libraries, and one hundred and thirty two positive clones (seventy one in forward library and sixty one in reverse library) were randomly selected and sequenced. Finally, fifty five reliable sequences from the forward subtracted library were used in a homology search by BLASTn and BLASTx, as were 57 reliable sequences from the reverse subtracted library. Furthermore, eight analyzed sequences from the forward subtracted cDNA library and seven from the reverse subtracted library were found to be non-homologous sequences. The screening identified genes induced by MC-LR in both libraries that are involved in various processes, such as energy metabolism, immunity, and apoptosis. Some are cytoskeleton- and transportation-related genes, while signal transduction-related genes were also found. Significant genes, such as the apoptosis-related gene p53 and the proto-oncogene c-myc, are involved in inhibition of the MC-LR response in the reverse subtracted library. In addition, several immune-related genes, which play an important role in antioxidation and detoxification of MC-LR, were characterized and identified in both of the subtracted libraries. The study provides the basic data to further identify the genes and molecular mechanism of detoxification of microcystins.

  11. 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...

  12. 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

  13. Construction and high expression of retroviral vector with human clotting factor IX cDNA in vitro

    Institute of Scientific and Technical Information of China (English)

    卢大儒; 邱信芳; 郑冰; 邱晓赟; 薛京伦

    1995-01-01

    The construction of the high liter and highly expressed safety retroviral vector carrying human clotting factor IX cDNA is reported. Retroviral vectors LNCTX, LIXSN and LCTXSN, driven by hCMV, LTR and hCMV combined with LTR promoter respectively, were constructed, based on the retroviral vector LNL6, and transferred into packaging cell line PA317 with electroporalion. Human dolling factor IX was delected in the cultured cells transduced with LNCIX and LIXSN but not in the cells transduced with LCIXSN. The viral titer of PA317/LNC1X was 800000 CFU per mL. With ELISA detection, it was found that the cells transduced with this vector can express human clotting factor IX at the level of 3.3μg per 106 cells in 24 h in human fibrosarcoma cells HT-1080 and 2.5μg per 106 cells in 24 h in hemophilia B patients’ skin fibroblast HSF cells, and more than 80% of them were biologically active. The viral liter and expression of human FIX were increased, and the construction of retroviral vector backbone was improved

  14. Representative appressorium stage eDNA library of Magnaporthe grisea

    Institute of Scientific and Technical Information of China (English)

    LU Jian-ping; LIU Tong-bao; YU Xiao-yun; LIN Fu-cheng

    2005-01-01

    A mature appressorium cDNA library of rice blast fungus, Magnaporthe grisea, was constructed in a ?TriplEx2 vector by SMARTTM cDNA library containing 2.37?106 independent clones about 100% of which harbor foreign cDNA inserts with average size of 660 bp. Of 9 randomly selected clones, 2 expressed sequence tags (ESTs) sequences did not have homologous EST sequences of M. grisea in GenBank. The appressorium cDNA library is suitable for gene expression analysis and function analysis of the late stages of appressorium formation and the early stages of penetration of M. grisea.

  15. Seek protein which can interact with hepatitis B virus X protein from human liver cDNA library by yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    Xiao-Zhong Wang; Xiang-Rong Jiang; Xiao-Chun Chen; Zhi-Xing Chen; Dan Li; Jian-Yin Lin; Qi-Min Tao

    2002-01-01

    AIM: To seek the X associated protein (XAP) with theconstructed bait vector pAS2-1X from normal human livercDNA library.METHODS: The X region of the HBV gene was amplied byPCR and cloned into the eukaryotic expression vector pAS2-l.The reconstituted plasmid pAS2-1X was transformed intothe yeast cells and the expression of X protein (pX) wasconfirmed by Western blot analysis. Yeast cells werecotransformed with pAS2-1X and the normal human livercDNA library and were grown in selective SC/-trp-leu-his-ade medium, the second screen was performed with theLacZ report gene. Furthermore, segregation analysis andmating experiment were performed to eliminate the falsepositive and the true positive clones were selected for PCRand sequencing.RESULTS: Reconstituted plasmid pAS2-1X including theanticipated fragment of X gene was proved by auto-sequencing assay. Western blot analysis showed thatreconstituted plasmid pAS2-1X expressed BD: X fusionprotein in yeast cells. Of 5 × 106 transformed coloniesscreened, 65 grew in the selective SC/-trp-leu-his-ademedium, 5 scored positive for β-gal activity, and only 2remaining clones passed through the segregation analysisand mating experiment. Sequence analysis identified thattwo clones contained similar cDNA fragment: GAACFFGCG.CONCLUSION: The short peptide (glutacid-leucine-alanine)is a possible required site for XAP binding to pX. Normalhuman liver cDNA library has difficulties in expressing theintegrated XAP on yeast cells.

  16. Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region-specific markers

    Energy Technology Data Exchange (ETDEWEB)

    Kandpal, R.P.; Kandpal, G.; Weissman, S.M. (Yale Univ. School of Medicine, New Haven, CT (United States))

    1994-01-04

    The authors describe a simple and rapid method for constructing small-insert genomic libraries highly enriched for dimeric, trimeric, and tetrameric nucleotide repeat motifs. The approach involves use of DNA inserts recovered by PCR amplification of a small-insert sonicated genomic phage library or by a single-primer PCR amplification of Mbo I-digested and adaptor-ligated genomic DNA. The genomic DNA inserts are heat denatured and hybridized to a biotinylated oligonucleotde. The biotinylated hybrids are retained on a Vectrex-avidin matrix and eluted specifically. The eluate is PCR amplified and cloned. More than 90% of the clones in a library enriched for (CA)[sub n] microsatellites with this approach contained clones with inserts containing CA repeats. They have also used this protocol for enrichment of (CAG)[sub n] and (AGAT)[sub n] sequence repeats and for Not I jumping clones. They have used the enriched libraries with an adaptation of the cDNA selection method to enrich for repeat motifs encoded in yeast artificial chromosomes.

  17. Exploiting the colloidal nanocrystal library to construct electronic devices

    Science.gov (United States)

    Choi, Ji-Hyuk; Wang, Han; Oh, Soong Ju; Paik, Taejong; Sung, Pil; Sung, Jinwoo; Ye, Xingchen; Zhao, Tianshuo; Diroll, Benjamin T.; Murray, Christopher B.; Kagan, Cherie R.

    2016-04-01

    Synthetic methods produce libraries of colloidal nanocrystals with tunable physical properties by tailoring the nanocrystal size, shape, and composition. Here, we exploit colloidal nanocrystal diversity and design the materials, interfaces, and processes to construct all-nanocrystal electronic devices using solution-based processes. Metallic silver and semiconducting cadmium selenide nanocrystals are deposited to form high-conductivity and high-mobility thin-film electrodes and channel layers of field-effect transistors. Insulating aluminum oxide nanocrystals are assembled layer by layer with polyelectrolytes to form high-dielectric constant gate insulator layers for low-voltage device operation. Metallic indium nanocrystals are codispersed with silver nanocrystals to integrate an indium supply in the deposited electrodes that serves to passivate and dope the cadmium selenide nanocrystal channel layer. We fabricate all-nanocrystal field-effect transistors on flexible plastics with electron mobilities of 21.7 square centimeters per volt-second.

  18. [Construction and identification on enriched microsatellite library from yak genome].

    Science.gov (United States)

    Li, Qi-Fa; Zhao, Xing-Bo; Luo, Xiao-Lin; Yao, Ping; Li, Ning; Tian, Zhi-Hua; Wu, Chang-Xin; Xie, Zhuang

    2004-05-01

    We constructed the first microsatellite-enriched library of yak according to the strong affinity between biotin and streptavidin. The method included ligation of 300 approximately 1 000 bp enzyme-digested fragments and adaptors, affinity capture of microsatellite repeat using biotinylated oligoprobe ((CA)12, (CCG) 8, (CAG)8, (TTTC) 8) attached to streptavidin-coated magnetic beads, PCR amplification using the 21-mer adaptor oligonucleotide as primer to obtain double-stranded targeted fragments, religated into pMD18-T vector and transformed to DH5alpha. The results of sequencing showed that 37 of 48 readable sequences contained microsatellites indicating a high degree of microsatellite enrichment. The new polymorphic microsatellite markers we have identified and characterized will contribute to the yak genetic linkage mapping, molecular evolution and phylogenetic studies, marker assistant selection and QTLs location of yak main economic traits.

  19. Construction and analysis of a suppression subtractive hybridization library of regeneration-related genes in soybean.

    Science.gov (United States)

    Sun, J; Li, J; Liu, M; Zhang, B B; Li, D M; Wang, M; Zhang, C; Li, W B; Su, A Y; Wu, X X

    2015-01-30

    The development of a genetic transformation system is needed to address the problem of the low efficiency associated with soybean regeneration. To contribute to the enhancement of the soybean regenerative capacity, we explored the developmental mechanisms of soybean regeneration at the molecular level using a suppression subtractive hybridization cDNA library constructed from cotyledonary nodes of soybean cultivar DN50. A total of 918 positive clones were identified and screened, with most inserted fragments ranging from 100 to 750 bp. Of these, 411 differentially expressed functional expressed sequence tags were identified and annotated based on their similarity to orthologs and paralogs detected in GenBank using the nucleotide and translated nucleotide Basic Local Alignment Search Tools. Functional analysis revealed that the associated genes were involved in signal transduction, synthesis, and metabolism of macromolecules, glucose and protein synthesis and metabolism, light and leaf morphogenesis, regulation of apoptosis, cell defense, cell wall differentiation, and a variety of hormone and cytokinin-mediated signaling pathways. The information uncovered in our study should serve as a foundation for the establishment of an efficient and stable genetic transformation system for soybean regeneration.

  20. [Construction and analysis of SSH library of Gossypium barbadense upon infection with Verticillium dahliae].

    Science.gov (United States)

    Zhu, Long-Fu; Tu, Li-Li; Zhang, Xian-Long; Nie, Yi-Chun; Guo, Xiao-Ping; Xia, Qi-Zhong

    2005-05-01

    Roots were collected from the seedlings inoculated with pathogen Verticillium dahliae after 2, 4, 8, 12, 24, 48, 72 and 96 hours for total RNA extraction. The cDNAs from the inoculated seedlings were used as the tester and those from the control seedlings as the driver. SSH method was employed to find the differently expressed cDNAs responding to the pathogen. T/A clone library was constructed containing 534 clones. The cDNA inserts were amplified from the bacterial clones directly with M13 primers by PCR. The size of the products ranged 0.2 - 1.2 kb with an average size of 0.5 kb. The SSH products were dotted on nylon filters, and the positive clones were screened by virtual Northern blotting with probes of the two kinds of initiative cDNAs. Totally 78 clones which were up-regulated and putatively involved in the defense response of G. barbadense were identified and sequenced. Sequence similarity searches were performed with the Blastn and Blastx. Most of them showed high or partial homology to genes or ESTs induced by different stresses in Arabidopsis thaliana and other species,such as the pathogenesis-related 10 family of G. hirsumtum and disease resistance-responsive family protein in Arabidopsis thaliana. The results would be helpful to understand the molecular mechanisms of disease response in cotton.

  1. Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin

    Directory of Open Access Journals (Sweden)

    MacFarlane Amanda J

    2009-07-01

    Full Text Available Abstract Background Exposure to dietary wheat proteins in genetically susceptible individuals has been associated with increased risk for the development of Type 1 diabetes (T1D. Recently, a wheat protein encoded by cDNA WP5212 has been shown to be antigenic in mice, rats and humans with autoimmune T1D. To investigate the genomic origin of the identified wheat protein cDNA, a hexaploid wheat genomic library from Glenlea cultivar was screened. Results Three unique wheat globulin genes, Glo-3A, Glo3-B and Glo-3C, were identified. We describe the genomic structure of these genes and their expression pattern in wheat seeds. The Glo-3A gene shared 99% identity with the cDNA of WP5212 at the nucleotide and deduced amino acid level, indicating that we have identified the gene(s encoding wheat protein WP5212. Southern analysis revealed the presence of multiple copies of Glo-3-like sequences in all wheat samples, including hexaploid, tetraploid and diploid species wheat seed. Aleurone and embryo tissue specificity of WP5212 gene expression, suggested by promoter region analysis, which demonstrated an absence of endosperm specific cis elements, was confirmed by immunofluorescence microscopy using anti-WP5212 antibodies. Conclusion Taken together, the results indicate that a diverse group of globulins exists in wheat, some of which could be associated with the pathogenesis of T1D in some susceptible individuals. These data expand our knowledge of specific wheat globulins and will enable further elucidation of their role in wheat biology and human health.

  2. 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.

  3. 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.

  4. A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes.

    Directory of Open Access Journals (Sweden)

    Moritz Eißmann

    Full Text Available Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems.

  5. Construction of BIBAC and BAC libraries from a variety of organisms for advanced genomics research.

    Science.gov (United States)

    Zhang, Hong-Bin; Scheuring, Chantel F; Zhang, Meiping; Zhang, Yang; Wu, Cheng-Cang; Dong, Jennifer J; Li, Yaning

    2012-02-16

    Large-insert BAC (bacterial artificial chromosome) and BIBAC (binary BAC) libraries are essential for modern genomics research for all organisms. We helped pioneer the BAC and BIBAC technologies, and by using them we have constructed hundreds of BAC and BIBAC libraries for different species of plants, animals, marine animals, insects, algae and microbes. These libraries have been used globally for different aspects of genomics research. Here we describe the procedure with the latest improvements that we have made and used for construction of BIBAC libraries. The procedure includes the preparation of BIBAC vectors, the preparation of clonable fragments of the desired size from the source DNA, the construction and transformation of BIBACs and, finally, the characterization and assembly of BIBAC libraries. We also specify the modifications necessary for construction of BAC libraries using the protocol. The entire protocol takes ∼7 d.

  6. [Construction of cDNA infectious clones of EV71 highly-pathogenic and cell-culture-adapted strains].

    Science.gov (United States)

    Zhang, Yong-xin; Li, Xiao-yu; Huang, Yu-ming; Zhou, Yong-dong; Bi, Sheng-li; Cen, Shan

    2014-11-01

    The highly-pathogenic EV71 strain is the primary cause of mortality in hand-foot-and-mouth disease (HFMD) associated with neurological symptoms, for which no clinically effective drugs or vaccines exist. This study aimed to construct infectious cDNA clones of the EV71 highly-pathogenic strain and the cell-culture adapted strain using a reverse genetics approach. The genomic RNAs of EV71 parent strains were used as the templates for RT-PCR amplification, and then the PCR products that overlapped the full-length genome were connected into the pBR322 vector to produce infectious clones of pEV71 (HP) and pEV71 (CCA), respectively. The results showed that the HP strain propagated much more quickly than the CCA strain. The rescued viruses derived from the infectious clones not only maintained their consistency with their parent strains in terms of genomic sequences, but also retained their respective biological phenotypes. This research will contribute to our understanding of EV71 pathogenesis and the development of novel vaccines against HFMD.

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

    Science.gov (United States)

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

    1993-01-01

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

  8. Chemiluminescent detection of sequential DNA hybridizations to high-density, filter-arrayed cDNA libraries: a subtraction method for novel gene discovery.

    Science.gov (United States)

    Guiliano, D; Ganatra, M; Ware, J; Parrot, J; Daub, J; Moran, L; Brennecke, H; Foster, J M; Supali, T; Blaxter, M; Scott, A L; Williams, S A; Slatko, B E

    1999-07-01

    A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.

  9. Construct Primary Education Semantic Ontology Library Based Mind Mapping

    Directory of Open Access Journals (Sweden)

    Hu Dong-Hong

    2016-01-01

    Full Text Available Researches conducted for Mind mapping application in primary education semantic ontology, while considering unique characteristics of primary education, found there were rare widely used ontology libraries and few connections between ontology libraries for information sharing and reuse. In addition, primary semantic ontology library lack precise definitions of the semantics. This paper proposed a solution based on cluster structure derived from mind mapping by providing logical description of the ontologies to precisely define semantics; Meanwhile, tags were adapted to associate different ontologies to form ontology library.

  10. Computational methods for the construction, editing, and error correction of DNA molecules and their libraries.

    Science.gov (United States)

    Raz, Ofir; Ben Yehezkel, Tuval

    2015-01-01

    The field of synthetic biology is fueled by steady advances in our ability to produce designer genetic material on demand. This relatively new technological capability stems from advancements in DNA construction biochemistry as well as supporting computational technologies such as tools for specifying large DNA libraries, as well as planning and optimizing their actual physical construction. In particular, the design, planning, and construction of user specified, combinatorial DNA libraries are of increasing interest. Here we present some of the computational tools we have built over the past decade to support the multidisciplinary task of constructing DNA molecules and their libraries. These technologies encompass computational methods for [1] planning and optimizing the construction of DNA molecules and libraries, [2] the utilization of existing natural or synthetic fragments, [3] identification of shared fragments, [4] planning primers and overlaps, [5] minimizing the number of assembly steps required, and (6) correcting erroneous constructs. Other computational technologies that are important in the overall process of DNA construction, such as [1] computational tools for efficient specification and intuitive visualization of large DNA libraries (which aid in debugging library design pre-construction) and [2] automated liquid handling robotic programming [Linshiz et al., Mol Syst Biol 4:191, 2008; Shabi et al., Syst Synth Biol 4:227-236, 2010], which aid in the construction process itself, have been omitted due to length limitations.

  11. Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

    Science.gov (United States)

    Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2015-12-01

    Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

  12. Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

    Directory of Open Access Journals (Sweden)

    Decai Tuo

    2015-12-01

    Full Text Available Papaya leaf distortion mosaic virus (PLDMV is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV. The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA, was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

  13. Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

    Science.gov (United States)

    Putney, S; Herlihy, W; Royal, N; Pang, H; Aposhian, H V; Pickering, L; Belagaje, R; Biemann, K; Page, D; Kuby, S

    1984-12-10

    A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

  14. Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning.

    Science.gov (United States)

    Sone, Takefumi; Yahata, Kazuhide; Sasaki, Yukari; Hotta, Junko; Kishine, Hiroe; Chesnut, Jonathan D; Imamoto, Fumio

    2008-09-10

    Much attention has been focused on manipulating multiple genes in living cells for analyzing protein function. In order to perform high-throughput generation of multi-gene expression clones, gateway cloning technology (which represents a high-throughput DNA transfer from vector to vector) can be anticipated. In the conventional strategy for gateway cloning, the construction of two or more expression elements into tandem elements on a single plasmid requires the recombination of multiple entry clones with a destination vector in a single reaction mixture. Use of increasing numbers of entry clones in a single reaction is inefficient due to the difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, a "Modular Destination" vector has been devised and constructed, whereby cDNA inserts are sequentially introduced, resulting in a tandem structure with multiple inserts. Whereas the standard destination vector contains only Cm(R) and ccdB genes flanked by two attR signals, this destination vector contains, in addition, one or two cDNA expression elements. Here, we show the rapid construction of expression vectors containing three or four tandemly arrayed cDNA expression elements and their expression in mammalian cells.

  15. 构建图书馆文化的思考%On Construction of Library Culture

    Institute of Scientific and Technical Information of China (English)

    郁丽玲

    2012-01-01

    Library culture is the necessity of library development,and it plays an important role in library development.This essay discusses the library culture to promote library development by strengthening library culture construction,so as to build the library culture with time characteristics.%图书馆文化是图书馆发展的必然产物,它对图书馆发展起着重要作用。本文通过对图书馆文化讨论研究,以加强图书馆文化的建设来促进图书馆事业的发展,着力塑造具有时代特质的图书馆文化。

  16. Preservation Concerns in Construction and Remodeling of Libraries: Planning for Preservation.

    Science.gov (United States)

    Trinkley, Michael

    To help libraries and other holdings institutions better incorporate preservation concerns in construction, renovation, and routine maintenance, various techniques are presented that allow preservation concerns to be integrated. The following topics are considered: (1) site selection; (2) design of the building envelope; (3) the library interior;…

  17. 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...

  18. Carver County Library System, Final Performance Report for Library Services and Construction Act (LSCA) Title VI, Library Literacy Program.

    Science.gov (United States)

    Colhapp, Barbara; Miller, Lana

    The Carver County Library System (Chaska, Minnesota) conducted a project to demonstrate adult educational teamwork between libraries and literacy agents in the Carver-Scott two county area. The project, "National Issues Forums (NIF) for the New Reader," extended educational opportunities that encouraged new adult readers and the public…

  19. Lowell Public Library, Final Performance Report for Library Services and Construction Act (LSCA) Title VI, Library Literacy Program.

    Science.gov (United States)

    Maravilla, Virginia

    The Lowell Public Library (Indiana) Adult Literacy Program expanded literacy efforts of the library and its volunteer tutors by increasing the program enrollment numbers of the functionally illiterate English-speaking, English as a Second Language (ESL), migrant workers, and Basic Math students; assisted students in achieving their stated goals in…

  20. 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.

  1. Screening for Novel Binding Proteins Interacting with Human Papillomavirus Type 18 E6 Oncogene in the Hela cDNA Library by Yeast Two-Hybrid System

    Institute of Scientific and Technical Information of China (English)

    Shuang LI; Ping LIU; Ling XI; Xuefeng JIANG; Jianfeng ZHOU; Shixuan WANG; Li MENG; Yunping LU; Ding Ma

    2008-01-01

    To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH109 was transformed with pGBKT7-HPV18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPVl8 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate im- muno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transeriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.

  2. Functional screening of a cDNA library from the desiccation-tolerant plant Selaginella lepidophylla in yeast mutants identifies trehalose biosynthesis genes of plant and microbial origin.

    Science.gov (United States)

    Pampurova, Suzana; Verschooten, Katrien; Avonce, Nelson; Van Dijck, Patrick

    2014-11-01

    Trehalose is a non-reducing disaccharide that accumulates to large quantities in microbial cells, but in plants it is generally present in very low, barely-detectible levels. A notable exception is the desiccation-tolerant plant Selaginella lepidophylla, which accumulates very high levels of trehalose in both the hydrated and dehydrated state. As trehalose is known to protect membranes, proteins, and whole cells against dehydration stress, we have been interested in the characterization of the trehalose biosynthesis enzymes of S. lepidophylla; they could assist in engineering crop plants towards better stress tolerance. We previously isolated and characterized trehalose-6-phosphate synthases from Arabidopsis thaliana (desiccation sensitive) and S. lepidophylla (desiccation tolerant) and found that they had similar enzymatic characteristics. In this paper, we describe the isolation and characterization of trehalose-6-phosphate phosphatase from S. lepidophylla and show that its catalytic activities are also similar to those of its homolog in A. thaliana. Screening of an S. lepidophylla cDNA library using yeast trehalose biosynthesis mutants resulted in the isolation of a large number of trehalose biosynthesis genes that were of microbial rather than plant origin. Thus, we suggest that the high trehalose levels observed in S. lepidophylla are not the product of the plant but that of endophytes, which are known to be present in this plant. Additionally, the high trehalose levels in S. lepidophylla are unlikely to account for its desiccation tolerance, because its drought-stress-sensitive relative, S. moellendorffii, also accumulated high levels of trehalose.

  3. DNA libraries for the construction of phage libraries: statistical and structural requirements and synthetic methods.

    Science.gov (United States)

    Lindner, Thomas; Kolmar, Harald; Haberkorn, Uwe; Mier, Walter

    2011-02-15

    Peptide-based molecular probes identified by bacteriophage (phage) display technology expand the peptide repertoire for in vivo diagnosis and therapy of cancer. Numerous peptides that bind cancer-associated antigens have been discovered by panning phage libraries. However, until now only few of the peptides selected by phage display have entered clinical applications. The success of phage derived peptides essentially depends on the quality of the library screened. This review summarizes the methods to achieve highly homogenous libraries that cover a maximal sequence space. Biochemical and chemical strategies for the synthesis of DNA libraries and the techniques for their integration into the viral genome are discussed in detail. A focus is set on the methods that enable the exclusion of disturbing sequences. In addition, the parameters that define the variability, the minimal numbers of copies per library and the use of alternating panning cycles to avoid the loss of selected hits are evaluated.

  4. EST sequencing and fosmid library construction in a non-model moth, Mamestra brassicae, for comparative mapping.

    Science.gov (United States)

    Kamimura, Manabu; Tateishi, Ken; Tanaka-Okuyama, Makiko; Okabe, Takuya; Shibata, Fukashi; Sahara, Ken; Yasukochi, Yuji

    2012-11-01

    Genome data are useful for both basic and applied research; however, it is difficult to carry out large-scale genome analyses using species with limited genetic or genomic resources. Here, we describe a cost-effective method to analyze the genome of a non-model species, using the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae). First, we conducted expression sequence tag (EST) analysis. In this analysis, we performed PCR-based prescreening of a non-normalized embryonic cDNA library to eliminate already sequenced cDNAs from further sequencing, which significantly increased the percentage of unique genes. Next, we constructed a fosmid library of M. brassicae and isolated 120 clones containing 119 putative single copy genes by PCR-based screening with primer sets designed from the ESTs. Finally, we showed that the isolated fosmid clones could be used as probes for multicolor fluorescence in situ hybridization (FISH) analysis against an M. brassicae chromosome and confirmed conserved gene order between M. brassicae and the silkworm, Bombyx mori. Thus, we developed new genomic resources for comparative genome analysis in M. brassicae using robust and relatively low cost methods that can be applied to any non-model organism.

  5. Construction of human antibody gene libraries and selection of antibodies by phage display.

    Science.gov (United States)

    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

    2014-01-01

    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  6. Modular system for the construction of zinc-finger libraries and proteins.

    Science.gov (United States)

    Gonzalez, Beatriz; Schwimmer, Lauren J; Fuller, Roberta P; Ye, Yongjun; Asawapornmongkol, Lily; Barbas, Carlos F

    2010-04-01

    Engineered zinc-finger transcription factors (ZF-TF) are powerful tools to modulate the expression of specific genes. Complex libraries of ZF-TF can be delivered into cells to scan the genome for genes responsible for a particular phenotype or to select the most effective ZF-TF to regulate an individual gene. In both cases, the construction of highly representative and unbiased libraries is critical. In this protocol, we describe a user-friendly ZF technology suitable for the creation of complex libraries and the construction of customized ZF-TFs. The new technology described here simplifies the building of ZF libraries, avoids PCR-introduced bias and ensures equal representation of every module. We also describe the construction of a customized ZF-TF that can be transferred to a number of expression vectors. This protocol can be completed in 9-11 d.

  7. Isolation of high molecular weight DNA suitable for the construction of genomic libraries.

    Science.gov (United States)

    Steven, J; McKechnie, D; Graham, A

    1988-01-01

    Recent advances in molecular biology have made it possible to construct complete gene libraries for any organism that uses DNA as its carrier of genetic information. A gene library should contain a large number of cloned DNA fragments that in total contain the entire donor genome. The construction of a genomic library first requires the isolation of DNA from the donor organism. To be of maximum use in the construction of genomic libraries, DNA isolated from the donor organism should fulfill the following criteria. First, the DNA must represent all sequences in the genome to be cloned. Second, it must be of high molecular weight. Third, no contaminants must taint the DNA so that its use as a substrate for restriction endonucleases and other enzymes used in genetic engineering is uninhibited.

  8. 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.

  9. Characterization of the human HOX 7 cDNA and identification of polymorphic markers.

    Science.gov (United States)

    Padanilam, B J; Stadler, H S; Mills, K A; McLeod, L B; Solursh, M; Lee, B; Ramirez, F; Buetow, K H; Murray, J C

    1992-09-01

    cDNA clones for a human HOX 7 gene obtained with homologous clones of Drosophila were used in human gene mapping studies. The human cDNA clone was isolated from a library constructed from human embryonic craniofacial material. The sequence of the cDNA demonstrates significant homology with mouse HOX 7. A search for RFLPs identified MboII and BstEII variants. A CA dinucleotide repeat with 5 alleles was also identified and allowed placement of HOX 7 into a defined linkage map. Evidence for linkage disequilibrium was found with markers tested. These results place the human HOX 7 gene in a defined position on 4p.

  10. Construction of infectious cDNA clones of PRRSV:Separation of coding regions for nonstructural and structural proteins

    Institute of Scientific and Technical Information of China (English)

    YUAN ShiShan; WEI ZuZhang

    2008-01-01

    Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the causative agent of the ongoing"porcine high fever syndrome" in China, is capable of genetic and antigenic mutations at high frequency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great Interest. We developed an infectious cDNA clone of an attenuated strain of Type Ⅱ PRRSV, and further manipulated the infectious cDNA clone by inserting polylinker between ORF1 and ORF2, encoding for nonstructural- or structural-protein, respectively. The cDNA was generated from the cell-attenuated virus strain, APRRS, via RT-PCR, and followed by nucleotide sequencing and molecular cloning. The full-length of the APRRS genomic RNA was determined as 15521 nucleotides in length excluding poly(A) tail, which has a 99.7% nucleotide identity with that of PRRSV Nsp strain, also a vaccine strain. Based on the nucleotide sequencing results, the full-length cDNA clone was assembled in pBlueScript vector, under the control of T7 promoter at the immediate 5' terminus of genome. To discern the rescued viruses from that of parental virus, a Mlu I restriction site was engineered into ORF5 coding region. Upon trensfection of the in vitro transcripts of both the original and MIu I-tagged cDNAs into MA-104 cells, typical PRRSV cytopathic effects were observed. The rescued viruses from the full-length cDNA clones displayed the same virological and molecular properties. Subsequently,PCR-based mutagenesis was conducted to separate the coding regions between PRRSV nonstructural genes, ORF1, and structural proteins, ORF2-ORF7. The synthetic RNA of such mutant clone, pCSA,was infectious and the rescued virus shared similar properties with that of the parental virus. This study provided a valuable tool for development of chimeric PRRSV as vaccine candidate offering crose-protection to various genetically diversified PRRSV strains, and a platform for further development of PRRSV as a gene

  11. Construction of infectious cDNA clones of PRRSV: Separation of coding regions for nonstructural and structural proteins

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the causative agent of the ongoing "porcine high fever syndrome" in China, is capable of genetic and antigenic mutations at high fre- quency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great interest. We developed an infectious cDNA clone of an attenuated strain of Type II PRRSV, and further manipulated the infectious cDNA clone by inserting polylinker between ORF1 and ORF2, en- coding for nonstructural- or structural-protein, respectively. The cDNA was generated from the cell-attenuated virus strain, APRRS, via RT-PCR, and followed by nucleotide sequencing and molecular cloning. The full-length of the APRRS genomic RNA was determined as 15521 nucleotides in length excluding poly(A) tail, which has a 99.7% nucleotide identity with that of PRRSV Nsp strain, also a vac- cine strain. Based on the nucleotide sequencing results, the full-length cDNA clone was assembled in pBlueScript vector, under the control of T7 promoter at the immediate 5′ terminus of genome. To dis- cern the rescued viruses from that of parental virus, a Mlu I restriction site was engineered into ORF5 coding region. Upon transfection of the in vitro transcripts of both the original and Mlu I-tagged cDNAs into MA-104 cells, typical PRRSV cytopathic effects were observed. The rescued viruses from the full-length cDNA clones displayed the same virological and molecular properties. Subsequently, PCR-based mutagenesis was conducted to separate the coding regions between PRRSV nonstructural genes, ORF1, and structural proteins, ORF2-ORF7. The synthetic RNA of such mutant clone, pCSA, was infectious and the rescued virus shared similar properties with that of the parental virus. This study provided a valuable tool for development of chimeric PRRSV as vaccine candidate offering cross-protection to various genetically diversified PRRSV strains, and a platform for further develop- ment of

  12. The characterisation of tapetum-specific cDNAs isolated from a Lilium henryi L. meiocyte subtractive cDNA library.

    Science.gov (United States)

    Crossley, S J; Greenland, A J; Dickinson, H G

    1995-01-01

    Differential screening of a meiocyte subtractive cDNA library from Lilium henryi L. has identified a group of 16 anther-specific partial cDNAs. Three of these sequences, LHM2, LHM6 and LHM7 have been further characterised. Hybridisation in situ with antisense riboprobes of LHM2, LHM6, and LHM7 gives a strong, clear signal which, contrary to expectations, is localised to the tapetal layer surrounding the meiocytes and not the meiocytes themselves. Developmental slot blots demonstrate that mRNAs corresponding to the three LHM cDNAs are transcribed from prophase of meiosis I to the uninucleate microspore stage, while Northern analysis reveals these tapetally expressed cDNAs to correspond with transcripts of some 500 bp. Although LHM2 is less abundant than LHM6 and LHM7, the pattern of developmental expression, and the size range of the transcripts suggests that all three cDNAs may be related. The deduced polypeptide products of LHM6 and LHM7 share 71% identity over a conserved region of 38 residues. Inverse polymerase chain reaction was used to obtain the full sequence for LHM7. Its deduced protein sequence has a signal peptide indicating it may be secreted; the cleaved protein has a molecular weight of 8.9 kDa. Furthermore, the LHM7 protein has significant levels of homology with tapetally expressed proteins from Arabidopsis thaliana, Antirrhinum majus and Lycopersicon esculentum. All contain a highly conserved pattern of cysteine residues present in seed and non-specific lipid transfer proteins. The function of this gene product is discussed in the perspective of current models of another development.

  13. Biomass Scenario Model Scenario Library: Definitions, Construction, and Description

    Energy Technology Data Exchange (ETDEWEB)

    Inman, D.; Vimmerstedt, L.; Bush, B.; Peterson, S.

    2014-04-01

    Understanding the development of the biofuels industry in the United States is important to policymakers and industry. The Biomass Scenario Model (BSM) is a system dynamics model of the biomass-to-biofuels system that can be used to explore policy effects on biofuels development. Because of the complexity of the model, as well as the wide range of possible future conditions that affect biofuels industry development, we have not developed a single reference case but instead developed a set of specific scenarios that provide various contexts for our analyses. The purpose of this report is to describe the scenarios that comprise the BSM scenario library. At present, we have the following policy-focused scenarios in our library: minimal policies, ethanol-focused policies, equal access to policies, output-focused policies, technological diversity focused, and the point-of-production- focused. This report describes each scenario, its policy settings, and general insights gained through use of the scenarios in analytic studies.

  14. Hopkinsville-Christian County Library, Final Performance Report for Library Services and Construction Act (LSCA) Title VI, Library Literacy Program.

    Science.gov (United States)

    Nevels, Vada Germaine

    The Hopkinsville-Christian County Library (Kentucky) conducted a project that involved recruitment, public awareness, basic literacy, collection development, tutoring, computer-assisted, other technology, and intergenerational/family programs. The project served a community of 50,000-100,000 people, and targeted the learning disabled,…

  15. Lee Library Association, Final Performance Report for Library Services and Construction Act (LSCA) Title VI, Library Literacy Program.

    Science.gov (United States)

    Dalheim, Zoe; Mauke, Martha

    The Lee Library Association conducted a project that involved recruitment, public awareness, training, rural oriented, basic literacy, collection development, tutoring, computer assisted services, and English as a Second Language (ESL) programs. The project served a community of 25,000-50,000 people, and targeted the homeless, learning disabled,…

  16. Irvington Public Library, Final Performance Report for Library Services and Construction Act (LSCA) Title VI, Library Literacy Program.

    Science.gov (United States)

    Shader, Holly

    The Irvington Public Library (Irvington, New Jersey) conducted a project that involved recruitment, retention, public awareness, training, basic literacy, collection development, tutoring, and intergenerational/family programs. The project served a community of 25,000-50,000 people, and targeted the learned disabled and intergenerational/families.…

  17. Logan Library, Final Performance Report for Library Services and Construction Act (LSCA) Title VI, Library Literacy Program.

    Science.gov (United States)

    Steinhoff, Nadene; Jenkins, Ronald

    The Bridgerland Literacy program of Logan Library (Logan, Utah) involved recruitment, retention, public awareness, training, basic literacy, collection development, tutoring, computer assisted, employment oriented, intergenerational/family, and English as a Second Language (ESL) programs. The program served a community of 50,000-100,000 people,…

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  19. Construction and Characterization of Grass Carp(Ctenopharyngodon idellus)Fosmid Library

    Institute of Scientific and Technical Information of China (English)

    JIA Zhen-hu; LIN Chang-you; YANG Tian-yao; JIANG Yi-nan; XIA Chun

    2010-01-01

    Grass carp(Ctenopharyngodon idellus)genomic fosmid library cotaining 129014 clones was constructed and characterized from one diploid grass carp.The average insert size of the fosmid library was determined to be 35 kb by pulsed field gel electrophoresis,which is 4.1-fold coverage of the grass carp genome.To demonstrate the probability of picking the functional genes from the library,eleven functional genes were screened by three-dimensional PCR technique.The number of positive clones of these genes was from 1 to 6.So,this library may screen any useful genes from grass carp.This grass carp genome fosmid library will be integrated in the presently ongoing efforts to determine the sequence of the grass carp genome.

  20. Construction and characterization of a bovine BAC library with four genome-equivalent coverage

    Directory of Open Access Journals (Sweden)

    Eilertsen Ken

    2001-09-01

    Full Text Available Abstract A bovine artificial chromosome (BAC library of 105 984 clones has been constructed in the vector pBeloBAC11 and organized in 3-dimension pools and high density membranes for screening by PCR and hybridization. The average insert size, determined after analysis of 388 clones, was estimated at 120 kb corresponding to a four genome coverage. Given the fact that a male was used to construct the library, the probability of finding any given autosomal and X or Y locus is respectively 0.98 and 0.86. The library was screened for 164 microsatellite markers and an average of 3.9 superpools was positive for each PCR system. None of the 50 or so BAC clones analysed by FISH was chimeric. This BAC library increases the international genome coverage for cattle to around 28 genome equivalents and extends the coverage of the ruminant genomes available at the Inra resource center to 15 genome equivalents.

  1. Construction and validation of two metagenomic DNA libraries from Cerrado soil with high clay content.

    Science.gov (United States)

    de Castro, Alinne Pereira; Quirino, Betania Ferraz; Allen, Heather; Williamson, Lynn L; Handelsman, Jo; Krüger, Ricardo Henrique

    2011-11-01

    A challenge of metagenomic studies is in the extraction and purification of DNA from environmental samples. The soils of the Cerrado region of Brazil present several technical difficulties to DNA extraction: high clay content (>55% w/w), low pH (4.7) and high iron levels (146 ppm). Here we describe for the first time the efficient recovery and purification of microbial DNA associated with these unusual soil characteristics and the construction and validation of two metagenomic libraries: a 150,000 clones library with insert size of approximately 8 kb and a 65,000 clones library with insert size of approximately 35 kb. The construction of these metagenomic libraries will allow the biotechnological exploitation of the microbial community present in the soil from this endangered biome.

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  3. Construction of Large Human Single-chain Antibody Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A large human naive single chain antibody (scFv) library is constructed from 60 healthy donors via phage display technique. During the period, some methods are employed to optimize the diversity, such as multi donors, different annealing temperature, half-nest PCR, and assembly by two-way fusion PCR. In this stud y, 78 electroporations resulted in 1010 library, diversity of which is assayed by enzyme fingerprint. The efficiency and diversity are all better than other rese arches.

  4. Construction of a Metagenomic DNA Library of Sponge Symbionts and Screening of Antibacterial Metabolites

    Institute of Scientific and Technical Information of China (English)

    CHEN Juan; ZHU Tianjiao; LI Dehai; CUI Chengbin; FANG Yuchun; LIU Hongbing; LIU Peipei; GU Qianqun; ZHU Weiming

    2006-01-01

    To study the bioactive metabolites produced by sponge-derived uncultured symbionts, a metagenomic DNA library of the symbionts of sponge Gelliodes gracilis was constructed. The average size of DNA inserts in the library was 20 kb. This library was screened for antibiotic activity using paper disc assaying. Two clones displayed the antibacterial activity against Micrococcus tetragenus. The metabolites of these two clones were analyzed through HPLC. The result showed that their metabolites were quite different from those of the host E. coli DH5α and the host containing vector pHZ132. This study may present a new approach to exploring bioactive metabolites of sponge symbionts.

  5. [Construction of SSH library from haemocyte of variously colored abalone challenged with bacteria and differential expression analysis of macrophage expressed protein].

    Science.gov (United States)

    Ren, Hong-Lin; Xu, Dan-Dan; Qiao, Kun; Cai, Ling; Huang, Wei-Bin; Zhang, Nai; Wang, Ke-Jian

    2008-08-01

    Abalones are considered to be the most precious delicacy from the sea, and become very important commercial seafood in aquaculture worldwide. Variously colored abalone (Haliotis diversicolor Reeve, 1846) has been widely cultured on the southeast coast for more than twenty years. However, abalone culture frequently suffers from bacterial infection and mass mortality of reared abalones causes serious economic losses. Unfortunately, knowledge of the defense mechanism in this animal is still lacking. In this study, using suppression subtractive hybridization (SSH) technology, a forward SSH library was constructed from haemocytes of H. diversicolor, with the content of 1.37x10(6) pfu and the recombinant rate of 98.18%. After the recombinant plasmids were sequenced, partial cDNA of macrophage expressed protein (MEP) was recognized based on BLAST searches in NCBI, with the size of 1,551 bp, and continuously encoding 517 amino acids. Semi-quantitative PCR and quantitative real-time PCR results showed that MEP cDNA was distinctly up-regulated in haemocytes of the bacterial-challenged group compared to the unchallenged group. The gene information obtained from this library will provide new insights into the immune mechanism of H. diversicolor and facilitate future study of target genes involved in the response to invading microorganisms.

  6. [Construction and analysis of the SSH library with the resistant wheat near-isogenic line and its susceptible parent infected by Puccinia striiformis Westend. f. sp. tritici].

    Science.gov (United States)

    Shu, Wei; Chen, Xiao-Hong; Niu, Yong-Chun

    2011-09-01

    To analyze the differentially expressed genes between resistant and susceptible wheat near-isogenic lines infected by Puccinia striiformis Westend. f. sp. tritici, a subtractive library containing about 1300 clones was constructed using suppression subtractive hybridization (SSH) in which the cDNA from resistant Yr4/6 × Taichung 29 seedlings inoculated with race CY26 was used as the tester, and the corresponding cDNA from susceptible Taichung 29 as the driver. Six hundred clones from the library were analyzed with reverse Northern blot. The positive clones were further tested by Northern blotting analysis. Twelve clones were verified and showed significant difference. By means of sequencing and BlastX analysis, six function-known differentially expressed sequences were detected, and their putative products were leucine-rich repeat protein, catalase, thioredoxin H-type, RNA binding protein, ascorbate peroxidase, and heat shock protein, respectively. Among them, leucine-rich repeat protein belongs to signal transduction protein, and others belong to defense response protein.

  7. Infectious RNA transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis virus.

    Science.gov (United States)

    Kuhn, R J; Niesters, H G; Hong, Z; Strauss, J H

    1991-06-01

    We have constructed a full-length cDNA clone of the virulent T48 strain of Ross River virus, a member of the alphavirus genus. Infectious RNA can be transcribed from this clone using SP6 or T7 RNA polymerase. The rescued virus has properties indistinguishable from those of the T48 strain of Ross River virus. We have used this clone, together with a full-length cDNA clone of Sindbis virus, to construct chimeric plasmids in which the 5' and the 3' nontranslated regions of the Sindbis and Ross River genomes were exchanged. The nontranslated regions of the two viral genomes differ in both size and sequence although they maintain specific conserved sequence elements. Virus was recovered from all four chimeras. Chimeras containing heterologous 3' nontranslated regions had replicative efficiencies equal to those of the parents. In contrast, the chimeras containing heterologous 5' nontranslated regions were defective in RNA synthesis and virus production, and the severity of the defect was dependent upon the host. Replication of a virus containing a heterologous 5' nontranslated region may be inefficient due to the formation of defective protein-RNA complexes, whereas, the presumptive complexes formed between host or virus proteins and the 3' nontranslated region to promote RNA synthesis appear to function normally in the chimeras.

  8. 酵母双杂交筛选胎肾上腺cDNA文库中HNP-1结合蛋白%SCREENING THE GENE SEQUENCES OF THE INTERACTION PROTEINS OF HNP-1 FROM HUMAN FETAL ADRENAL CDNA LIBRARY BY YEAST TWO HYBRID SYSTEM

    Institute of Scientific and Technical Information of China (English)

    杜润滋; 邓璐霞; 黄宁; 罗朝志

    2011-01-01

    [Objective] To screen proteins binding with α-defensin (HNP-1) mature peptide from placenta cDNA libraty by yeast two hybrid technique. [Methods] The cDNA fragment encoding HNP-1 mature peptide was amplified by polymera-sechain reaction (PCR) and constructed into pGBK-T7 vector as the bait plasmid in yeast two hybrid system 3. Subsequently , the RNA from fetal adrenal gland was obtained and then transformed into cDNA library using SMART technology. The fetal adrenal cDNA library was screened with pGBKT7-HNP-1 as bait plasmid by yeast-two hybrid system Matchmaker Lexa. Finally, the positive clone was obtained by PCR and then identified by sequence. Then the interaction between them was determined by GST pull down in vitro and coimmunoprecipitates experiments in vivo. [Results] Bait and cDNA library have been constructed successfully and transformed into yeast. Then the interaction protein was found-melanocortin 2 receptor (ACTHR), CCAAT-enhancer-binding proteins (C/EBP), Tramembrane trafficking protein (TMP21), low density lipoprotein receptor-related protein 6 (LRP6). Therefore, melanocortin 2 receptor (ACTH-R) was determined into the major subjects. And bands which can demonstrate the relationship between HNP-1 and ACTH-R was obtained in GST pull down and coimmunoprecipitates experiments. [Conclusion] ACTH-R can bind to HNP-1 we obtained from fetal adrenal cDNA library and it may play important roles in the function of HNP-1 mature peptide.%[目的]筛选胎肾上腺cDNA文库中与α防御素HNP-1成熟肽具有相互作用的蛋白分子.[方法]通过聚合酶链反应(PCR)成功获得HNP-1成熟肽基因插入酵母表达载体pGBK-T7中构建诱饵质粒,同时提取胎肾上腺RNA,SMART技术制备人胎肾上腺cDNA文库,并采用Matchmaker LexA酵母双杂交系统从胎肾上腺cDNA文库中筛选与HNP-1成熟肽相互作用的蛋白.最后通过PCR筛选获得阳性克隆并测序,而后经回转实验,GST pull down以及免疫共沉淀再次验证

  9. Construction of Mammalian Genomic Libraries Using λ Replacement Vectors.

    Science.gov (United States)

    Bateson, A N; Pollard, J W

    1988-01-01

    The ideal genomic library should consist of a series of clones containing overlapping sequences that are representative of the entire genome. Such an ideal state can be approached by cutting the DNA randomly and cloning large pieces of this DNA into a suitable vector (1). The DNA can be cleaved either by mechanical shearing, in which case the DNA is fragmented in a truely random fashion, but with the introduction of problems associated with blunt end ligation, or better, by partial digestion with a restriction endonuclease such as Mbol or Sau3A. These enzymes recognize four base sequences that are predicted to occur on average (although in practice this is not the case) every 256 bases, and hence digestion results in cleavage of the DNA in a pseudorandom manner.

  10. 数字化图书馆建设%Digitization Library Construction

    Institute of Scientific and Technical Information of China (English)

    庞丽

    2014-01-01

    With the development of society informatization, digitization library has entered the information era, therefore, China's library has a new opportunity for development at the same time facing unprecedented challenges with the rapid development of science and technology. From the perspective of the construction of library development, digitization library will replace the traditional library is represent the general trend. This paper analyzes influences and changes of network digitization for library, and discusses that digitization library is not only changes the printed, audio and video works of the traditional library into data digitization, but also makes the digitized data upload network and become the online resources, finally puts forward some countermeasures for the digitization library construction.%随着社会信息化进程的加快,数字化图书馆已经进入了信息时代,因此,我国的图书馆随着科技的飞速发展,在拥有新的发展机遇的同时也面临着前所未有的挑战。从图书馆事业发展建设来看,数字化图书馆将代替传统的图书馆已是大势所趋。文章分析了网络数字化对图书馆所产生的影响及所发生的变化,并论述了数字图书馆不仅是将传统图书馆的印刷、音像作品等资料数字化,而且把数字化了的资料上传网络,成为网上资源,提出了数字化图书馆建设的对策。

  11. T cell-based functional cDNA library screening identified SEC14-like 1a carboxy-terminal domain as a negative regulator of human immunodeficiency virus replication.

    Science.gov (United States)

    Urano, Emiko; Ichikawa, Reiko; Morikawa, Yuko; Yoshida, Takeshi; Koyanagi, Yoshio; Komano, Jun

    2010-05-26

    Genome-wide screening of host factors that regulate HIV-1 replication has been attempted using numerous experimental approaches. However, there has been limited success using T cell-based cDNA library screening to identify genes that regulate HIV-1 replication. We have established a genetic screening strategy using the human T cell line MT-4 and a replication-competent HIV-1. With this system, we identified the C-terminal domain (CTD) of SEC14-like 1a (SEC14L1a) as a novel inhibitor of HIV-1 replication. Our T cell-based cDNA screening system provides an alternative tool for identifying novel regulators of HIV-1 replication.

  12. CitEST libraries

    Directory of Open Access Journals (Sweden)

    Maria Luísa P. Natividade Targon

    2007-01-01

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

  13. Exploring the potential of megaprimer PCR in conjunction with orthogonal array design for mutagenesis library construction.

    Science.gov (United States)

    Tang, Lixia; Zheng, Kai; Liu, Yu; Zheng, Huayu; Wang, Hu; Song, Chunlei; Zhou, Hong

    2013-01-01

    Although megaprimer PCR mutagenesis has been used routinely in protein directed evolution, users sometimes encounter technical hurdles, particularly inefficiency during amplification when large fragments are used or the template is difficult to be amplified. Instead of methodology development, here we simply overcome the limitation by optimizing megaprimer PCR conditions via orthogonal array design of the four PCR components in three levels of each: template, primer, Mg(2+) , and dNTPs. For this, only nine PCRs need to be performed. The strategy (termed as OptiMega) was not only successfully applied for the construction of one multiple-site saturation mutagenesis library of halohydrin dehalogenase HheC, which failed to be constructed previously using the standard QuikChange™ protocol, but also expanded the construction of two high-quality random mutagenesis libraries of HheA and HheC. Most importantly, OptiMega offers a quick and simple way of constructing random mutagenesis libraries by eliminating the ligation step. Our results demonstrated that the OptiMega strategy could greatly strengthen the potential of megaprimer PCR mutagenesis for library construction.

  14. Restriction enzyme-free construction of random gene mutagenesis libraries in Escherichia coli.

    Science.gov (United States)

    Pai, Jen C; Entzminger, Kevin C; Maynard, Jennifer A

    2012-02-15

    Directed evolution relies on both random and site-directed mutagenesis of individual genes and regulatory elements to create variants with altered activity profiles for engineering applications. Central to these experiments is the construction of large libraries of related variants. However, a number of technical hurdles continue to limit routine construction of random mutagenesis libraries in Escherichia coli, in particular, inefficiencies during digestion and ligation steps. Here, we report a restriction enzyme-free approach to library generation using megaprimers termed MegAnneal. Target DNA is first exponentially amplified using error-prone polymerase chain reaction (PCR) and then linearly amplified with a single 3' primer to generate long, randomly mutated, single-stranded megaprimers. These are annealed to single-stranded dUTP-containing template plasmid and extended with T7 polymerase to create a complementary strand, and the resulting termini are ligated with T4 DNA ligase. Using this approach, we are able to reliably generate libraries of approximately 10⁷ colony-forming units (cfu)/μg DNA/transformation in a single day. We have created MegAnneal libraries based on three different single-chain antibodies and identified variants with enhanced expression and ligand-binding affinity. The key advantages of this approach include facile amplification, restriction enzyme-free library generation, and a significantly reduced risk of mutations outside the targeted region and wild-type contamination as compared with current methods.

  15. Construction of Human ScFv Phage Display Library against Ovarian Tumor

    Institute of Scientific and Technical Information of China (English)

    XIA Jinsong; BI Hao; YAO Qin; QU Shen; ZONG Yiqiang

    2006-01-01

    In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E.coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 × 109 cfu/μg was obtained. After amplification with helper phage, the titer of antibody library reached 5 × 1012 cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

  16. Preliminary Study of BAC Library Construction in Black Tiger Shrimp, Penaeus monodon

    Directory of Open Access Journals (Sweden)

    Suwit WUTHISUTHIMETHAVEE

    2009-01-01

    Full Text Available Availability of shrimp genome information is necessary for shrimp genetic studies and large-insert DNA clones, bacterial artificial chromosome (BACs serve as valuable tools for obtaining genomic sequences. The construction of a BAC library was achieved from this preliminary study of P. monodon. High molecular weight (HMW genomic DNA was isolated from abdominal muscle and the resulting hemocytes were of high quality and sufficient quantity for a BAC library construction. This BAC library was constructed from hemocyte HMW genomic DNA fragments ranging between 100 and 300 kb ligated to a pBAC-lac vector. The average insert size of the BAC clones was calculated as 100 kb ranging from 40 - 150 kb with over 50 % of the clones containing inserts larger than 100 kb. This BAC library contained approximately 60,000 BAC clones representing a 3-fold coverage of the haploid shrimp genome size. Furthermore, the consistency of BAC clones was also proven as no apparent rearrangements were observed before and after 100 generations in serial growth. From the above result, it is essential to improve the insert size and clone numbers of the BAC library to be a powerful tool in the future.

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

    Science.gov (United States)

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

    2012-01-01

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

  18. Derivation of a mathematical expression useful for the construction of complete genomic libraries.

    Science.gov (United States)

    Zilsel, J; Ma, P H; Beatty, J T

    1992-10-12

    We present and derive a formula that is useful for the design and evaluation of gene cloning experiments in which a complete gene library of the entire genome of an organism is desired. The formula n = ln(1-phi f)/ln(1-f) (in which n is the number of recombinant clones required to ensure a probability, phi, of obtaining at least one of each of all possible gene sequences, and f is the fraction of the genome contained in an average-sized DNA fragment) applies to construction of libraries, in which at least one copy of all the genetic information of a genome is required. The use of this formula for quantitative evaluation of genomic libraries should give greater assurance that a given library would be complete.

  19. Construction of genomic libraries of Cryptosporidium parvum and identification of antigen-encoding genes.

    Science.gov (United States)

    Dykstra, C C; Blagburn, B L; Tidwell, R R

    1991-01-01

    Genomic libraries have been constructed from bovine C. parvum DNA in the lambda ZAP and lambda DASH vectors. Based on an estimated genome size of 2 x 10(4) kilobases (kb), each recombinant library contains greater than 10 genomic equivalents. The average recombinant size for the lambda ZAP library is 2.1 kb and for the lambda DASH library is 14 kb. We have identified genes to major antigens recognized by hyperimmune bovine antiserum. These recombinants are currently being purified and characterized. Limited DNA sequence analysis of random C. parvum clones confirms suggestions that the genome is quite AT-rich. The DNA sequence of random lambda ZAP fusion proteins has identified a potential ATPase, a structural protein and a DNA-binding protein.

  20. Versatile broad-host-range cosmids for construction of high quality metagenomic libraries.

    Science.gov (United States)

    Cheng, Jiujun; Pinnell, Lee; Engel, Katja; Neufeld, Josh D; Charles, Trevor C

    2014-04-01

    We constructed IncP broad-host-range Gateway® entry cosmids pJC8 and pJC24, which replicate in diverse Proteobacteria. We demonstrate the functionality of these vectors by extracting, purifying, and size-selecting metagenomic DNA from agricultural corn and wheat soils, followed by cloning into pJC8. Metagenomic DNA libraries of 8×10(4) (corn soil) and 9×10(6) (wheat soil) clones were generated for functional screening. The DNA cloned in these libraries can be transferred from these recombinant cosmids to Gateway® destination vectors for specialized screening purposes. Those library clones are available from the Canadian MetaMicroBiome Library project (http://www.cm2bl.org/).

  1. Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species

    Directory of Open Access Journals (Sweden)

    Doležel Jaroslav

    2010-02-01

    Full Text Available Abstract Background The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy. Results The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the

  2. Recursive construction of perfect DNA molecules and libraries from imperfect oligonucleotides.

    Science.gov (United States)

    Linshiz, Gregory; Yehezkel, Tuval Ben; Shapiro, Ehud

    2012-01-01

    Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology, and synthetic biology. We developed an error-correcting recursive construction procedure that attempts to address this challenge. Making DNA molecules from synthetic oligonucleotides using the procedure described here surpasses existing methods for de novo DNA synthesis in speed, precision, and amenability to automation. It provides for the first time a unified DNA construction platform for combining synthetic and natural DNA fragments, for constructing designer DNA libraries, and for making the faultless long synthetic DNA building blocks needed for de novo genome construction.

  3. 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.

  4. Rapid DNA Library Construction for Functional Genomic and Metagenomic Screening▿ †

    OpenAIRE

    2007-01-01

    A rapid protocol was developed for constructing plasmid libraries from small quantities of genomic/metagenomic DNA. The technique utilizes linker amplification with topoisomerase cloning and allows for inducible transcription in Escherichia coli. As proof of principle, several anti-Bacillus lysins were cloned from bacteriophage genomes and an aerolysin was cloned from a metagenomic sample.

  5. BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction.

    Directory of Open Access Journals (Sweden)

    Brad Thomas Townsley

    2015-05-01

    Full Text Available Next Generation Sequencing (NGS is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing inherent properties of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE libraries and can easily extend to full transcript coverage shotgun (SHO type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.

  6. Research on the Construction of Subject Branch Library of University Library%高校图书馆学科分馆建设研究

    Institute of Scientific and Technical Information of China (English)

    王晓征

    2012-01-01

    学科分馆建设是高校图书馆创新服务形式、配合学校加强学科建设的重要措施。分析了学科分馆的含义及高校图书馆学科分馆建设现状,重点就学科分馆的信息资源建设和学科服务进行探讨。%The construction of subject branch library is an important measure for library to innovate on service form and strengthen the discipline construction coordinating with university.This paper analyses the meaning and the construction status of subject branch library,emphatically discusses the information resources construction and subject services of subject branch library.

  7. Comparison of Three Cre-LoxP Based Paired-End Library Construction Methods

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Ze; Nath, Nandita; Tritt, Andrew; Liang, Shoudan; Han, James; Pennacchio, Len; Chen, Feng

    2013-03-26

    Paired-end library sequencing has been proven useful in scaffold construction during de novo whole genome shotgun assembly. The ability of generating mate pairs with > 8 Kb insert sizes is especially important for genomes containing long repeats. To make mate paired libraries for next generation sequencing, DNA fragments need to be circularized to bring the ends together. There are several methods that can be used for DNA circulation, namely ligation, hybridization and Cre-LoxP recombination. With higher circularization efficiency with large insert DNA fragments, Cre-LoxP recombination method generally has been used for constructing >8 kb insert size paired-end libraries. Second fragmentation step is also crucial for maintaining high library complexity and uniform genome coverage. Here we will describe the following three fragmentation methods: restriction enzyme digestion, random shearing and nick translation. We will present the comparison results for these three methods. Our data showed that all three methods are able to generate paired-end libraries with greater than 20 kb insert. Advantages and disadvantages of these three methods will be discussed as well.

  8. Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

    Indian Academy of Sciences (India)

    Vikas Anathy; Thayanithy Venugopal; Ramanathan Koteeswaran; Thavamani J Pandian; Sinnakaruppan Mathavan

    2013-03-01

    A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.

  9. Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

    Indian Academy of Sciences (India)

    Vikas Anathy; Thayanithy Venugopal; Ramanathan Koteeswaran; Thavamani J Pandian; Sinnakaruppan Mathavan

    2001-09-01

    A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.

  10. Construction of genomic libraries of mycobacterial origin: identification of recombinants encoding mycobacterial-specific proteins.

    Science.gov (United States)

    Khandekar, P S; Munshi, A; Sinha, S; Sharma, G; Kapoor, A; Gaur, A; Talwar, G P

    1986-09-01

    A complete genomic library from Mycobacterium vaccae (2785 recombinants) and a partial genomic library of M. leprae and BCG (300 and 1750 clones, respectively) were constructed in the plasmid pBR322. Bam HI was selected as the restriction endonuclease for obtaining DNA cleavage products. Evidence was obtained for limited expression of the cloned mycobacterial DNA inserts in Escherichia coli. A recombinant has been identified which codes for antigen immunoreactive with rabbit anti-M. leprae antibody but not with anti-H37Rv antibody.

  11. SMART技术构建的辣椒疫霉菌与辣椒互作AD-cDNA文库%Construction of AD-cDNA Library for Interaction ofPhytophthora capsici and Pepper in SMART Technology

    Institute of Scientific and Technical Information of China (English)

    周志国; 明月梅

    2015-01-01

    为了分离和克隆辣椒中疫霉菌诱导基因,以接种辣椒疫霉菌的叶片为材料,利用SMART技术构建辣椒疫霉菌与辣椒互作的双杂交cDNA文库。结果表明:该文库容量为3.6×106 cfu,重组率88%左右,插入片段集中在300~2000 bp之间,平均长度约为800 bp,表明获得的文库质量较高,该文库将为分子育种提供重要的基因资源。%In order to identify thePhytophthora capsici stress induced genes of pepper, a double-hybrid cDNA library ofPhytophthora capsici infected pepper leaves was constructed using SMART technology. The results showed that the cDNA library contained 3.6×106 independent clones, and the recombination rate was 88%. Insert fragments ranged from 300 bp to 2 000 bp, and the average length of the library was about 800 bp. These data demonstrate that the library is qualified, and able to provide important genetic resources for molecular breeding.

  12. Construction of representative NotI linking libraries specific for the total human genome and for human chromosome 3.

    Science.gov (United States)

    Zabarovsky, E R; Allikmets, R; Kholodnyuk, I; Zabarovska, V I; Paulsson, N; Bannikov, V M; Kashuba, V I; Dean, M; Kisselev, L L; Klein, G

    1994-03-15

    NotI linking clones represent valuable tools for both physical and genetic mapping. Using procedures that we have previously described, several chromosome 3-specific NotI linking libraries have been constructed. Here, we describe the construction of six independent NotI linking libraries specific for the total human genome. These libraries were made using three different vectors and two combinations of restriction enzymes. Altogether, these six libraries contain more than 1 million recombinant phages. Considering that the human genome contains about 3000-5000 NotI sites, it is likely that all clonable NotI sites are present in these libraries. Two of the six libraries were transferred into plasmid form. At the same time, a chromosome 3-specific EcoRI-NotI library (NRL1) was constructed. This library considerably increases the representation of cloned NotI sites in combination with previously constructed libraries that were made using BamHI-NotI digestion. All libraries are available on request.

  13. Construction of representative NotI linking libraries specific for the total human genome and for human chromosome 3

    Energy Technology Data Exchange (ETDEWEB)

    Zabarovsky, E.R.; Kholodnyuk, I.; Zabarovska, V.I.; Paulsson, N.; Bannikov, V.M.; Kashuba, V.I.; Klein, G. (Karolinska Institute, Stockholm (Sweden)); Allikmets, R.; Dean, M. (National Cancer Institute, Frederick, MD (United States)); Kisselev, L.L. (Engelhardt Institute of Molecular Biology, Moscow (Russian Federation))

    1994-03-15

    NotI linking clones represent valuable tools for both physical and genetic mapping. Using procedures previously described, several chromosome 3-specific NotI linking libraries have been constructed. Here, the authors describe the construction of six independent NotI linking libraries specific for the total human genome. These libraries were made using three different vectors and two combinations of restriction enzymes. Altogether, these six libraries contain more than 1 million recombinant phages. Considering that the human genome contains about 3000-5000 NotI sites, it is likely that all clonable NotI sites are present in these libraries. Two of the six libraries were transferred into plasmid form. At the same time, a chromosome 3-specific EcoRI-NotI library (NRL1) was constructed. This library considerably increases the representation of cloned NotI sites in combination with previously constructed libraries that were made using BamHI-NotI digestion. All libraries are available on request. 18 refs., 3 figs., 1 tab.

  14. High yield of functional metagenomic library from mangroves constructed in fosmid vector.

    Science.gov (United States)

    Gonçalves, A C S; dos Santos, A C F; dos Santos, T F; Pessoa, T B A; Dias, J C T; Rezende, R P

    2015-10-02

    In the present study, metagenomic technique and fosmid vectors were used to construct a library of clones for exploring the biotechnological potential of mangrove soils by isolation of functional genes encoding hydrolytic enzymes. The library was built with genomic DNA from the soil samples of mangrove sediments and the functional screening of 1824 clones (~64 Mbp) was performed to detect the hydrolytic activity specific for cellulases, amylases (at acidic, neutral and basic pH), lipases/esterases, proteases, and nitrilases. Significant numbers of clones, positive for the tested enzyme activities were obtained. Our results indicate the importance and biotechnological potential of mangrove soils especially when compared to those obtained using other soil metagenomic libraries.

  15. Construction and characterization of a sugar beet (Beta vulgaris) fosmid library.

    Science.gov (United States)

    Lange, Cornelia; Holtgräwe, Daniela; Schulz, Britta; Weisshaar, Bernd; Himmelbauer, Heinz

    2008-11-01

    A sugar beet (Beta vulgaris) fosmid library from the doubled haploid accession KWS2320 encompassing 115 200 independent clones was constructed and characterized. The average insert size of the fosmid library was determined by pulsed field gel electrophoresis to be 39 kbp on average, thus representing 5.9-fold coverage of the sugar beet genome (758 Mbp). PCR screening of plate pools with primer pairs against nine sugar beet genes supported the insert size estimation. BLAST searches with 2951 fosmid end-sequences originating from 1510 clones (1536 clones attempted) revealed little contamination with organellar DNA (2.1% chloroplast DNA, 0.3% mitochondrial DNA). The sugar beet fosmid library will be integrated in the presently ongoing efforts to determine the sequence of the sugar beet genome. Fosmids will be publicly available in the format of plate pools and individual clones.

  16. NCBI和cDNA文库中栽培花生EST-SSR分子标记的开发及其特点%Development and Characterization of EST-SSR Markers from NCBI and cDNA Library in Cultivated Peanut (Arachis hypogaea L.)

    Institute of Scientific and Technical Information of China (English)

    王金彦; 潘丽娟; 杨庆利; 禹山林

    2009-01-01

    86 132 ESTs downloaded from GenBank in NCBI and 12 501 ESTs from cDNA library constructed by high-oil linoleic acid accession E 12 were analysed. After the preprocession, there were 18 051 singletons and 9 972 contigs in the GenBank of NCBI and cDNA library. Totally 3 104 SSR loci had been screened by MISA software, accounting for 11.08% for these non-redundant ESTs. All SSR loci are divided into di-nucleotide, thi-nucleotide, tetra-nucleotide, penta-nucleotide, hexa-nucleotide and multi-nucleotide etc., and thi-nucleotide motif is the most motifs and the frequency was 43.0% and 56.8% in NCBI and cDNA libraray, respectively. The number of di-and penta-nucleotide motifs were second and third in all motifs. And the hexa-nucleotide was the least mo-tif both in NCBI and cDNA library. In all repeat motifs nucleotide, AG/TC was the most motifs and accounted for 8.65% and 13.42% in NCBI and cDNA library, respectively. Among the tri-nucleotide repeats, CTT/GAA was the most frequent motif, accounting for 6.7% and 13.42%, respectively. The repeat unit number of SSR loci is from 4 to 51.%本研究利用NCBI的GenBank数据库中公布的花生86 132条EST序列以及利用高油酸品种E12所创建的cDNA文库中的12 501条EST序列,对这些序列进行前期处理,总共获得非冗余且拼接较长的singleton 11 260条,contig 9 972条.通过MISA软件分析发现两个EST库中共包含有3 104个SSR位点,占到总共非冗余序列的11.08%.这些SSR位点被分成二核苷酸重复、三核苷酸重复、四核苷酸重复、五核苷酸重复、六核苷酸重复以及混合核苷酸重复等,其中三核苷酸重复占的比例最多,分别占到NCBI和cDNA文库的43.0%和56.8%,二核苷酸和五核苷酸重复占到所有重复位点的第二位和第三位,六核苷酸重复的比例最少.在所有重复基序中,AG/TC重复的数量最多,分别占到NCBI和cDNA文库的8.65%和13.42%.在三核苷酸重复中,CTT/GAA出现的频率最大,分别占到6.7%

  17. BAC Library Construction and Physical Mapping of Bacillus anthracis A16R

    Institute of Scientific and Technical Information of China (English)

    Zhang Da; Zhu Houchu; Huang Liuyu

    2013-01-01

    Bacillus anthracis is an endospore-forming bacterium that causes severe inhalational anthrax, and bacillus anthracis A16R is an attenuated strain derived from Bacillus anthracis A16. The development of bacterial artificial chromosome (BAC) system has allowed the construction of large insert-size DNA libraries, and the bacterial artificial chromosomes (BACs) have become the preferred large insert cloning system for genomic analysis because such libraries are characteristically stable, high in ifdelity and easy to handle. To facilitate genome studies of this bacterium, a bacterial artiifcial chromosome library (BAC) has been established from genome DNA of Bacillus anthracis A16R. This library consisted of 9 600 clones randomly selected from more than 15 000 recombinant clones carrying inserts in the plindigoBAC-5 vectors. The mean insert size was 56 kbp, representing an approximate 12-fold genome coverage, while end sequences were obtained from 700 randomly selected clones. Sequences were compared with Bacillus anthracis Ames and Bacillus cereus ATCC 14579 Genome Project databases using the NCBI BLASTN search project. And most BLASTN results showed high identities and that the sequences’ sites could be used as STSs. To construct this physical map, Excel was used for the array of STSs and some gaps of the map were iflled up by PCR walking. Artemis-V4 was used in the construction of a genome-wide physical map with 93%genome coverage. The A16R BAC library proved to be a vital tool for the generation of a map that would not only allow the subsequent sequencing of defined areas of genome, but also provide immediate access to clones that were stable and convenient for functional genomic researches.

  18. BAC Library Construction and Physical Mapping of Bacillus anthracis A16R

    Directory of Open Access Journals (Sweden)

    Da Zhang

    2013-12-01

    Full Text Available Bacillus anthracis is an endospore-forming bacterium that causes severe inhalational anthrax, and bacillus anthracis A16R is an attenuated strain derived from Bacillus anthracis A16. The development of bacterial artificial chromosome (BAC system has allowed the construction of large insert-size DNA libraries, and the bacterial artificial chromosomes (BACs have become the preferred large insert cloning system for genomic analysis because such libraries are characteristically stable, high in fidelity and easy to handle. To facilitate genome studies of this bacterium, a bacterial artificial chromosome library (BAC has been established from genome DNA of Bacillus anthracis A16R. This library consisted of 9 600 clones randomly selected from more than 15 000 recombinant clones carrying inserts in the plindigoBAC-5 vectors. The mean insert size was 56 kbp, representing an approximate 12-fold genome coverage, while end sequences were obtained from 700 randomly selected clones. Sequences were compared with Bacillus anthracis Ames and Bacillus cereus ATCC 14579 Genome Project databases using the NCBI BLASTN search project. And most BLASTN results showed high identities and that the sequences’ sites could be used as STSs. To construct this physical map, Excel was used for the array of STSs and some gaps of the map were filled up by PCR walking. Artemis-V4 was used in the construction of a genome-wide physical map with 93% genome coverage. The A16R BAC library proved to be a vital tool for the generation of a map that would not only allow the subsequent sequencing of defined areas of genome, but also provide immediate access to clones that were stable and convenient for functional genomic researches.

  19. Accelerating plant DNA barcode reference library construction using herbarium specimens: improved experimental techniques.

    Science.gov (United States)

    Xu, Chao; Dong, Wenpan; Shi, Shuo; Cheng, Tao; Li, Changhao; Liu, Yanlei; Wu, Ping; Wu, Hongkun; Gao, Peng; Zhou, Shiliang

    2015-11-01

    A well-covered reference library is crucial for successful identification of species by DNA barcoding. The biggest difficulty in building such a reference library is the lack of materials of organisms. Herbarium collections are potentially an enormous resource of materials. In this study, we demonstrate that it is likely to build such reference libraries using the reconstructed (self-primed PCR amplified) DNA from the herbarium specimens. We used 179 rosaceous specimens to test the effects of DNA reconstruction, 420 randomly sampled specimens to estimate the usable percentage and another 223 specimens of true cherries (Cerasus, Rosaceae) to test the coverage of usable specimens to the species. The barcode rbcLb (the central four-sevenths of rbcL gene) and matK was each amplified in two halves and sequenced on Roche GS 454 FLX+. DNA from the herbarium specimens was typically shorter than 300 bp. DNA reconstruction enabled amplification fragments of 400-500 bp without bringing or inducing any sequence errors. About one-third of specimens in the national herbarium of China (PE) were proven usable after DNA reconstruction. The specimens in PE cover all Chinese true cherry species and 91.5% of vascular species listed in Flora of China. It is very possible to build well-covered reference libraries for DNA barcoding of vascular species in China. As exemplified in this study, DNA reconstruction and DNA-labelled next-generation sequencing can accelerate the construction of local reference libraries. By putting the local reference libraries together, a global library for DNA barcoding becomes closer to reality.

  20. Korean BAC library construction and characterization of HLA-DRA, HLA-DRB3.

    Science.gov (United States)

    Park, Mi-Hyun; Lee, Hye-Ja; Bok, Jeong; Kim, Cheol-Hwan; Hong, Seong-Tshool; Park, Chan; Kimm, KuChan; Oh, Bermseok; Lee, Jong-Young

    2006-07-31

    A human bacterial artificial chromosome (BAC) library was constructed with high molecular weight DNA extracted from the blood of a male Korean. This Korean BAC library contains 100,224 clones of insert size ranging from 70 to 150 kb, with an average size of 86 kb, corresponding to a 2.9-fold redundancy of the genome. The average insert size was determined from 288 randomly selected BAC clones that were well distributed among all the chromosomes. We developed a pooling system and three-step PCR screen for the Korean BAC library to isolate desired BAC clones, and we confirmed its utility using primer pairs designed for one of the clones. The Korean BAC library and screening pools will allow PCR-based screening of the Korean genome for any gene of interest. We also determined the allele types of HLA-DRA and HLA-DRB3 of clone KB55453, located in the HLA class II region on chromosome 6p21.3. The HLA-DRA and DRB3 genes in this clone were identified as the DRA*010202 and DRB3*01010201 types, respectively. The haplotype found in this library will provide useful information in future human disease studies.

  1. Construction and validation of metagenomic DNA libraries from landfarm soil microorganisms.

    Science.gov (United States)

    Pessoa, T B A; de Souza, S S; Cerqueira, A F; Rezende, R P; Pirovani, C P; Dias, J C T

    2013-06-28

    Landfarming biodegradation is a strategy used by the petrochemical industry to reduce pollutants in petroleum-contaminated soil. We constructed 2 metagenomic libraries from landfarming soil in order to determine the pathway used for mineralization of benzene and to examine protein expression of the bacteria in these soils. The DNA of landfarm soil, collected from Ilhéus, BA, Brazil, was extracted and a metagenomic library was constructed with the Copy Control(TM) Fosmid Library Production Kit, which clones 25-45-kb DNA fragments. The clones were selected for their ability to express enzymes capable of cleaving aromatic compounds. These clones were grown in Luria-Bertani broth plus L-arabinose, benzene, and chloramphenicol as induction substances; they were tested for activity in the catechol cleavage pathway, an intermediate step in benzene degradation. Nine clones were positive for ortho-cleavage and one was positive for meta-cleavage. Protein band patterns determined by SDS-polyacrylamide gel electrophoresis differed in bacteria grown on induced versus non-induced media (Luria-Bertani broth). We concluded that the DNA of landfarm soil is an important source of genes involved in mineralization of xenobiotic compounds, which are common in gasoline and oil spills. Metagenomic library allows identification of non-culturable microorganisms that have potential in the bioremediation of contaminated sites.

  2. Construction and packaging of pseudotype retrovirus containing human N—ras cDNA antisense sequence and its biological effects on human hepatoma cells

    Institute of Scientific and Technical Information of China (English)

    JIALIBIN; WANGXIANG; 等

    1990-01-01

    N-ras is one of the transforming genes in human hepatic cancer cells.It has been found that N-ras was overexpressed at the mRNA and protein level in hepatoma cells.In order to explore the biological roles of N-ras in human hepatic carcinogenesis and the potential application in control of cancer cell growth,a preudotype retrovirus containing antisense sequence of human N-ras was constructed and packaged.A recombinant retrovirus vector containing antisense or sense sequences of N-ras cDNA was constructed by pZIP-NeoSV(X)1.The pseudotype virus was packaged ang rescued by transfection and infection in PA317 and ψ 2 helper cells.It has been demonstrated that the pseudotype retrovirus containing antisense N-ras sequence did inhibit the growth of human PLC/PRF/5 hepatoma cells accompanied with inhibition of p21 expression,while the retrovirus containing sense sequence had none.The pseudotype virus had no effect on human diploid fibroblasts.

  3. 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.

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

    OpenAIRE

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

    2009-01-01

    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 an...

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

    Science.gov (United States)

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

    1997-01-01

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

  6. Impact of Three Illumina Library Construction Methods on GC Bias and HLA Genotype Calling

    Science.gov (United States)

    Lan, James H; Yin, Yuxin; Reed, Elaine F; Moua, Kevin; Thomas, Kimberly; Zhang, Qiuheng

    2016-01-01

    Next-generation sequencing (NGS) is increasingly recognized for its ability to overcome allele ambiguity and deliver high-resolution typing in the human leukocyte antigen (HLA) system. Using this technology, non-uniform read distribution can impede the reliability of variant detection, which renders high-confidence genotype calling particularly difficult to achieve in the polymorphic HLA complex. Recently, library construction has been implicated as the dominant factor in instigating coverage bias. To study the impact of this phenomenon on HLA genotyping, we performed long-range PCR on 12 samples to amplify HLA-A, -B, -C, -DRB1, and -DQB1, and compared the relative contribution of three Illumina library construction methods (TruSeq Nano, Nextera, Nextera XT) in generating downstream bias. Here, we show high GC% to be a good predictor of low sequencing depth. Compared to standard TruSeq Nano, GC bias was more prominent in transposase-based protocols, particularly Nextera XT, likely through a combination of transposase insertion bias being coupled with a high number of PCR enrichment cycles. Importantly, our findings demonstrate non-uniform read depth can have a direct and negative impact on the robustness of HLA genotyping, which has clinical implications for users when choosing a library construction strategy that aims to balance cost and throughput with data quality. PMID:25543015

  7. Construction and characterization of the transformation-competent artificial chromosome (TAC) libraries of Leymus multicaulis

    Institute of Scientific and Technical Information of China (English)

    XU YueYu; ZHOU YuLei; SONG LinLin; ZHANG Yan; ZHAO MaoLin

    2008-01-01

    Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants. In order to clone genes highly tolerant to barley yellow dwarf virus (BYDV), Aphids, drought and salt from Leymus multicaulis, the two TAC genomic libraries Ⅰ and Ⅱ were constructed in vector pYLTAC17 and pYLTAC747H/sacB, which contain about 165000 and 236000 recombinant clones sepa-rately. The genome coverage of the two libraries was totally estimated to be about 3-5 haploid genome equivalents, as size selection of genomic DNA fragments was approximately from 9 to 300 kb. Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so, stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern using a GeneTACTM G3 arraying robot after being transferred manually into three 384-well plates. Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates. Nineteen positive clones were detected by using the probe glutahione reductase gene of L. Multicaulis. TAC libraries constructed here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning. Once the target TAC clones were isolated, they could be immediately transferred into plant genomes with the Agrobacterium system.

  8. Construction and characterization of the transformation-competent artificial chromosome(TAC)libraries of Leymus multicaulis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants.In order to clone genes highly tolerant to barley yellow dwarf virus(BYDV),Aphids,drought and salt from Leymus multicaulis,the two TAC genomic libraries I and II were constructed in vector pYLTAC17 and pYLTAC747H/sacB,which contain about 165000 and 236000 recombinant clones sepa-rately.The genome coverage of the two libraries was totally estimated to be about 3―5 haploid genome equivalents,as size selection of genomic DNA fragments was approximately from 9 to 300 kb.Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so,stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern using a GeneTAC?G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates.Nineteen positive clones were detected by using the probe glutahione reductase gene of L.multicaulis.TAC libraries constructed here can be used to isolate genomic clones containing target genes,and to carry out genome walking for positional cloning.Once the target TAC clones were isolated,they could be immediately transferred into plant genomes with the Agrobacterium system.

  9. On the construction of a new stellar classification template library for the LAMOST spectral analysis pipeline

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Peng; Luo, Ali; Li, Yinbi; Tu, Liangping; Wang, Fengfei; Zhang, Jiannan; Chen, Xiaoyan; Hou, Wen; Kong, Xiao; Wu, Yue; Zuo, Fang; Yi, Zhenping; Zhao, Yongheng; Chen, Jianjun; Du, Bing; Guo, Yanxin; Ren, Juanjuan [Key Laboratory of Optical Astronomy, National Astronomical Observatories, Chinese Academy of Sciences, Beijing 100012 (China); Pan, Jingchang; Jiang, Bin; Liu, Jie, E-mail: lal@nao.cas.cn, E-mail: weipeng@nao.cas.cn [School of Mechanical, Electrical, and Information Engineering, Shandong University, Weihai 264209 (China); and others

    2014-05-01

    The LAMOST spectral analysis pipeline, called the 1D pipeline, aims to classify and measure the spectra observed in the LAMOST survey. Through this pipeline, the observed stellar spectra are classified into different subclasses by matching with template spectra. Consequently, the performance of the stellar classification greatly depends on the quality of the template spectra. In this paper, we construct a new LAMOST stellar spectral classification template library, which is supposed to improve the precision and credibility of the present LAMOST stellar classification. About one million spectra are selected from LAMOST Data Release One to construct the new stellar templates, and they are gathered in 233 groups by two criteria: (1) pseudo g – r colors obtained by convolving the LAMOST spectra with the Sloan Digital Sky Survey ugriz filter response curve, and (2) the stellar subclass given by the LAMOST pipeline. In each group, the template spectra are constructed using three steps. (1) Outliers are excluded using the Local Outlier Probabilities algorithm, and then the principal component analysis method is applied to the remaining spectra of each group. About 5% of the one million spectra are ruled out as outliers. (2) All remaining spectra are reconstructed using the first principal components of each group. (3) The weighted average spectrum is used as the template spectrum in each group. Using the previous 3 steps, we initially obtain 216 stellar template spectra. We visually inspect all template spectra, and 29 spectra are abandoned due to low spectral quality. Furthermore, the MK classification for the remaining 187 template spectra is manually determined by comparing with 3 template libraries. Meanwhile, 10 template spectra whose subclass is difficult to determine are abandoned. Finally, we obtain a new template library containing 183 LAMOST template spectra with 61 different MK classes by combining it with the current library.

  10. BAC libraries construction from the ancestral diploid genomes of the allotetraploid cultivated peanut

    Directory of Open Access Journals (Sweden)

    Chaine Christian

    2008-01-01

    Full Text Available Abstract Background Cultivated peanut, Arachis hypogaea is an allotetraploid of recent origin, with an AABB genome. In common with many other polyploids, it seems that a severe genetic bottle-neck was imposed at the species origin, via hybridisation of two wild species and spontaneous chromosome duplication. Therefore, the study of the genome of peanut is hampered both by the crop's low genetic diversity and its polyploidy. In contrast to cultivated peanut, most wild Arachis species are diploid with high genetic diversity. The study of diploid Arachis genomes is therefore attractive, both to simplify the construction of genetic and physical maps, and for the isolation and characterization of wild alleles. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaënsis with genome types AA and BB respectively. Results We constructed and characterized two large-insert libraries in Bacterial Artificial Chromosome (BAC vector, one for each of the diploid ancestral species. The libraries (AA and BB are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb. Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes, and resistance gene analogues. Conclusion These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map.

  11. 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 fra...

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

    Science.gov (United States)

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

    2016-02-01

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

  13. The hemiptera (insecta of Canada: constructing a reference library of DNA barcodes.

    Directory of Open Access Journals (Sweden)

    Rodger A Gwiazdowski

    Full Text Available DNA barcode reference libraries linked to voucher specimens create new opportunities for high-throughput identification and taxonomic re-evaluations. This study provides a DNA barcode library for about 45% of the recognized species of Canadian Hemiptera, and the publically available R workflow used for its generation. The current library is based on the analysis of 20,851 specimens including 1849 species belonging to 628 genera and 64 families. These individuals were assigned to 1867 Barcode Index Numbers (BINs, sequence clusters that often coincide with species recognized through prior taxonomy. Museum collections were a key source for identified specimens, but we also employed high-throughput collection methods that generated large numbers of unidentified specimens. Many of these specimens represented novel BINs that were subsequently identified by taxonomists, adding barcode coverage for additional species. Our analyses based on both approaches includes 94 species not listed in the most recent Canadian checklist, representing a potential 3% increase in the fauna. We discuss the development of our workflow in the context of prior DNA barcode library construction projects, emphasizing the importance of delineating a set of reference specimens to aid investigations in cases of nomenclatural and DNA barcode discordance. The identification for each specimen in the reference set can be annotated on the Barcode of Life Data System (BOLD, allowing experts to highlight questionable identifications; annotations can be added by any registered user of BOLD, and instructions for this are provided.

  14. Recursive construction and error correction of DNA molecules and libraries from synthetic and natural DNA.

    Science.gov (United States)

    Yehezkel, Tuval Ben; Linshiz, Gregory; Kaplan, Shai; Gronau, Ilan; Ravid, Sivan; Adar, Rivka; Shapiro, Ehud

    2011-01-01

    Making error-free, custom DNA assemblies from potentially faulty building blocks is a fundamental challenge in synthetic biology. Here, we show how recursion can be used to address this challenge using a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone synthetic oligonucleotides and naturally existing DNA. Specifically, we describe how divide and conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide target DNA sequences into overlapping, albeit error prone, oligonucleotides, and how recursive construction is applied in vitro to combine them to form error-prone DNA molecules. To correct DNA sequence errors, error-free fragments of these molecules are then identified, extracted, and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. The method allows combining synthetic and natural DNA fragments into error-free designer DNA libraries, thus providing a foundation for the design and construction of complex synthetic DNA assemblies.

  15. Construction of an extended library of adult male 3D models: rationale and results

    Science.gov (United States)

    Broggio, D.; Beurrier, J.; Bremaud, M.; Desbrée, A.; Farah, J.; Huet, C.; Franck, D.

    2011-12-01

    In order to best cover the possible extent of heights and weights of male adults the construction of 25 whole body 3D models has been undertaken. Such a library is thought to be useful to specify the uncertainties and relevance of dosimetry calculations carried out with models representing individuals of average body heights and weights. Representative 3D models of Caucasian body types are selected in a commercial database according to their height and weight, and 3D models of the skeleton and internal organs are designed using another commercial dataset. A review of the literature enabled one to fix volume or mass target values for the skeleton, soft organs, skin and fat content of the selected individuals. The composition of the remainder tissue is fixed so that the weight of the voxel models equals the weight of the selected individuals. After mesh and NURBS modelling, volume adjustment of the selected body shapes and additional voxel-based work, 25 voxel models with 109 identified organs or tissue are obtained. Radiation transport calculations are carried out with some of the developed models to illustrate potential uses. The following points are discussed throughout this paper: justification of the fixed or obtained models' features regarding available and relevant literature data; workflow and strategy for major modelling steps; advantages and drawbacks of the obtained library as compared with other works. The construction hypotheses are explained and justified in detail since future calculation results obtained with this library will depend on them.

  16. Construction of a linker library with widely controllable flexibility for fusion protein design.

    Science.gov (United States)

    Li, Gang; Huang, Ziliang; Zhang, Chong; Dong, Bo-Jun; Guo, Ruo-Hai; Yue, Hong-Wei; Yan, Li-Tang; Xing, Xin-Hui

    2016-01-01

    Flexibility or rigidity of the linker between two fused proteins is an important parameter that affects the function of fusion proteins. In this study, we constructed a linker library with five elementary units based on the combination of the flexible (GGGGS) and the rigid (EAAAK) units. Molecular dynamics (MD) simulation showed that more rigid units in the linkers lead to more helical conformation and hydrogen bonds, and less distance fluctuation between the N- and C-termini of the linker. The diversity of linker flexibility of the linker library was then studied by fluorescence resonance energy transfer (FRET) of cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) fusion proteins, which showed that there is a wide range of distribution of the FRET efficiency. Dissipative particle dynamics (DPD) simulation of CFP-YFP with different linkers also gave identical results with that of FRET efficiency analysis, and we further found that the combination manner of the linker peptide had a remarkable effect on the orientation of CFP and YFP domains. Our studies demonstrated that the construction of the linker library with the widely controllable flexibility could provide appropriate linkers with the desirable characteristics to engineer the fusion proteins with the expected functions.

  17. Fosmid library construction and screening for the maize mutant gene Vestigial glume 1

    Institute of Scientific and Technical Information of China (English)

    Chaoxian Liu; Xiaoli Liu; Lei Lei; Haiying Guan; Yilin Cai

    2016-01-01

    The maize mutant gene Vestigial glume 1 (Vg1) has been fine-mapped to a narrow region by map-based cloning and the candidate gene for Vg1 spanned 19.5 kb. Here we report Vg1 genomic fosmid library construction and screening. The fosmid library of Vg1 consisted of 574,000 clones with an average insert size of 36.4 kb, representing 7.9-fold coverage of the maize genome. Fosmid stability assays indicated that clones were stable during propagation in the fosmid system. Using Vg1 candidate gene-specific primers, a positive clone was successfully identified. This discovery will pave the way for identifying the function of Vg1 in maize development.

  18. Fosmid library construction and screening for the maize mutant gene Vestigial glume 1

    Institute of Scientific and Technical Information of China (English)

    Chaoxian Liu; Xiaoli Liu; Lei Lei; Haiying Guan; Yilin Cai

    2016-01-01

    The maize mutant gene Vestigial glume 1(Vg1) has been fine-mapped to a narrow region by map-based cloning and the candidate gene for Vg1 spanned 19.5 kb. Here we report Vg1 genomic fosmid library construction and screening. The fosmid library of Vg1 consisted of574,000 clones with an average insert size of 36.4 kb, representing 7.9-fold coverage of the maize genome. Fosmid stability assays indicated that clones were stable during propagation in the fosmid system. Using Vg1 candidate gene-specific primers, a positive clone was successfully identified. This discovery will pave the way for identifying the function of Vg1 in maize development.

  19. The construction of a library of synthetic promoters revealed some specific features of strong Streptomyces promoters

    DEFF Research Database (Denmark)

    Seghezzi, Nicolas; Amar, Patrick; Købmann, Brian;

    2011-01-01

    cloned into the promoter-probe plasmid pIJ487 just upstream of the promoter-less aphII gene that confers resistance to neomycin. This synthetic promoter library was transformed into Streptomyces lividans, and the resulting transformants were screened for their ability to grow in the presence of different......Streptomyces are bacteria of industrial interest whose genome contains more than 73% of bases GC. In order to define, in these GC-rich bacteria, specific sequence features of strong promoters, a library of synthetic promoters of various sequence composition was constructed in Streptomyces. To do so......, the sequences located upstream, between and downstream of the −35 and −10 consensus promoter sequences were completely randomized and some variability was introduced in the −35 (position 6) and −10 (positions 3, 4 and 5) hexamers recognized by the major vegetative sigma factor HrdB. The synthetic promoters were...

  20. Fosmid library construction and screening for the maize mutant gene Vestigial glume 1

    Directory of Open Access Journals (Sweden)

    Chaoxian Liu

    2016-02-01

    Full Text Available The maize mutant gene Vestigial glume 1 (Vg1 has been fine-mapped to a narrow region by map-based cloning and the candidate gene for Vg1 spanned 19.5 kb. Here we report Vg1 genomic fosmid library construction and screening. The fosmid library of Vg1 consisted of 574,000 clones with an average insert size of 36.4 kb, representing 7.9-fold coverage of the maize genome. Fosmid stability assays indicated that clones were stable during propagation in the fosmid system. Using Vg1 candidate gene-specific primers, a positive clone was successfully identified. This discovery will pave the way for identifying the function of Vg1 in maize development.

  1. [Construction of Frankia genomic libraries and isolation of clones homologous to nodulation genes from Rhizobium leguminosarum].

    Science.gov (United States)

    Cui, Y H; Qin, M; Wang, Y L; Ding, J; Ma, Q S

    1990-01-01

    High molecular genomic DNAs were isolated by using the lysozyme plus achromopeptidase system from Frankia strains At4, Ccol and Hr16, the root nodule endophytes of Alnus, Casuarina and Hippophae respectively, and used to construct genomic libraries in pLAFR1, a broad host range cosmid vector within many gram-negative hosts. The genomic libraries were screened by in situ colony hybridization to identify clones homologous to common nodulation genes of Rhizobium leguminosarum, based on the sequence homology of EcoRI-digested Frankia total DNA to nodABC from Rhizobium meliloti. Several clones showing relatively strong hybridization were found, the recombinant plasmid was isolated, and their homology with Rhizobium nodulation genes was confirmed by spot hybridization. Further work on these positive clones is now underway.

  2. Concept and construction process of the ceramic curtain of Vila-real Library

    Directory of Open Access Journals (Sweden)

    A. Peñín Llobell

    2016-12-01

    Full Text Available The construction of the Library of Vila-real, selected in 2012 in the VIII Latin American Biennial of Architecture and Urbanism, highlights the importance of collaboration with industry for the development and application of its outer membrane. The analysis of the construction process of the ceramic cylindrical curtain that defines it, performed by white glazed ceramic, 5 cm diameter and 7.5 m height, reveals this fact. The system builds an interstitial space, essential for its use and environmental integration. At the same time it links the building to both local industrial fabric, which aims to establish itself as one of its exponents, and to the Mediterranean culture of filters. The procedure followed is ascribed to the postartesanal and pragmatic perspective that beyond the modern heroes, introduced characters like Jean Prouvé and that today, we state, should find natural and legal channels for its development, on behalf of the progress of the construction sector.

  3. Research on the Service Mode of Primary Level Libraries Through the Co-Construction of University Libraries and Public Libraries%中小城市校地共建基层图书馆服务模式探讨

    Institute of Scientific and Technical Information of China (English)

    杨洁

    2012-01-01

    Presently the construction of primary level libraries is still in the infant stage, primary level libraries have many problems such as the imbalanced regional development, lack of resources, the single fund source, inadequate layout, the unstable team, and so on. The mode of primary level libraries of the co-construction of university libraries and public libraries is an innovative mode. In the construction of primary level libraries, university libraries and public libraries should promote the application and the development of network information resources, build the training base for librarians of primary level libraries, carry out volunteer activities, expand fund source channels, and construct the uniform acquisition center, in order to promote the development of primary level libraries continuously and stably.%目前,基层图书馆的建设仍处于起步阶段,存在区域发展不均衡、资源不足、资金来源单一、布局规划不合理、从业队伍不稳定等问题。高校图书馆和公共图书馆共建基层图书馆是图书馆服务模式的一种创新。在基层图书馆建设过程中,高校图书馆和公共图书馆应推进网络信息资源的开发和应用,创建基层图书馆管理员培训基地,开展大学生志愿者服务基层图书馆活动,拓展资金来源渠道,构建统一采购中心,以推进基层图书馆持续、稳定的发展。

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

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2014-03-01

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

  5. Construction and analysis of Siberian tiger bacterial artificial chromosome library with approximately 6.5-fold genome equivalent coverage.

    Science.gov (United States)

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

    2014-03-07

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

  6. 医院电子图书馆的建设%Construction of Electronic Library in Hospital

    Institute of Scientific and Technical Information of China (English)

    陈祖林; 石兵; 邹宏

    2012-01-01

    阐述电子图书馆的内涵和医院建立电子图书馆的优势,以武警江西省总队医院为例介绍电子图书馆建设情况,阐述规划医院电子图书馆建设方案应重点考虑的问题。%The paper elaborates the connotation of electronic library and the advantages of hospital constructing electronic library. Taking Jiangxi Province Hospital of Chinese Armed Police Force as an example, it analyzes the construction status of electronic library, e- laborates the problems which should be well-considered in planning hospital electronic library construction.

  7. Construction of cosmid genomic libraries for the normal and myopathic Syrian hamsters.

    Science.gov (United States)

    McCully, J D; Jandreski, M A; Liew, J; Sole, M J; Liew, C C

    1987-11-01

    Cosmid genomic libraries from both normal and myopathic Syrian hamsters have been constructed. MboI was used to generate 35- to 50-kilobase DNA fragments which were isolated from a 5-25% NaCl gradient. The 35- to 50-kilobase DNA fragments were ligated to the cosmid vector pCV108 and packaged into Escherichia coli DK1. Approximately 3 X 10(5) - 4 X 10(5) clones were obtained per microgram of ligated DNA. Thirteen clones have been isolated from 2 X 10(5) colonies using a cardiac myosin heavy chain clone as a probe. Restriction maps of two of these clones are presented here.

  8. 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...

  9. 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...

  10. Construction and screening of a functional metagenomic library to identify novel enzymes produced by Antarctic bacteria

    Institute of Scientific and Technical Information of China (English)

    Ignacio Ferrés; Vanesa Amarelle; Francisco Noya; Elena Fabiano

    2015-01-01

    A metagenomic fosmid library of approximately 52 000 clones was constructed to identify functional genes encoding cold-adapted enzymes. Metagenomic DNA was extracted from a sample of glacial meltwater, collected on the Antarctic Peninsula during the ANTARKOS XXIX Expedition during the austral summer of 2012–2013. Each clone contained an insert of about 35–40 kb, so the library represented almost 2 Gb of genetic information from metagenomic DNA. Activity-driven screening was used to detect the cold-adapted functions expressed by the library. Fifty lipase/esterase and two cellulase-producing clones were isolated, and two clones able to grow on Avicel® as the sole carbon source. Interestingly, three clones formed a brown precipitate in the presence of manganese (II). Accumulation of manganese oxides was determined with a leucoberbelin blue assay, indicating that these three clones had manganese-oxidizing activity. To the best of our knowledge, this is the first report of a manganese oxidase activity detected with a functional metagenomic strategy.

  11. Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.

    Science.gov (United States)

    Koo, Byoung-Mo; Kritikos, George; Farelli, Jeremiah D; Todor, Horia; Tong, Kenneth; Kimsey, Harvey; Wapinski, Ilan; Galardini, Marco; Cabal, Angelo; Peters, Jason M; Hachmann, Anna-Barbara; Rudner, David Z; Allen, Karen N; Typas, Athanasios; Gross, Carol A

    2017-03-22

    A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.

  12. Construction of a full bacterial artificial chromosome (BAC) library of Oryza sativa genome

    Institute of Scientific and Technical Information of China (English)

    TAOQUANZHOU; HAIYINGZHAO; 等

    1994-01-01

    We have constructed a full BAC library for the superior early indica variety of Oryza sativa,Guang Lu Ai 4.The MAX Efficiency DH10B with increased stability of inserts was used as BAC host cells.The potent pBelo BACII with double selection markers was used as cloning vector.The cloning efficiency we have reached was as high as 98%,and the transformation efficiency was raised up to 106 transformants/μg of large fragment DNA.The BAC recombinant transformants were picked at random and analyzed for the size of inserts,which turned out to be of 120 kb in length on average.We have obtained more than 20,000 such BAC clones.According to conventional probability equation,they covered the entire rice genome of 420,000 kb in length.The entire length of inserts of the library obtained has the 5-to 6-fold coverage of the genome.To our knowledge,this is the first reported full BAC library for a complex genome.

  13. Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Bao-Ping Wu; Bing Xiao; Tian-Mo Wan; Ya-Li Zhang; Zhen-Shu Zhang; Dian-Yuan Zhou; Zhuo-Sheng Lai; Chun-Fang Gao

    2001-01-01

    AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and light chain к and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and к gained by RT-PCR were about 650bp. Fd and к PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 106. The libraries were enriched about 120-fold by 3 cycles of adsorption-elution- multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

  14. Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes.

    Directory of Open Access Journals (Sweden)

    Man Cheng

    Full Text Available Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3. The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx. Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N protein of SARS-associated coronavirus (SCoV were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.

  15. A polymerase chain reaction-based method for isolating clones from a complimentary DNA library in sheep.

    Science.gov (United States)

    Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon; Hutmacher, Dietmar W

    2014-10-01

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

  16. Research on Cultural Construction in University Libraries%高校图书馆文化建设研究

    Institute of Scientific and Technical Information of China (English)

    李红旗

    2012-01-01

    Library culture is a modern management mode of the library in a new stage, is fi'om the traditional system of management, target management to culture management as the center of the strategic transfer. The library culture is the soul of an excellent library, library culture, can create a positive, harmonious, progressive work atmosphere, is generated ever fount power source, to the library cause development plays a huge role in promoting and facilitating. Pay attention to the library culture construction, is helpful for us from new highly promoting the library scientific mangement, promote the reform of management system in library.%图书馆文化是现代图书馆管理模式的一个新阶段,是由传统的制度管理、目标管理向文化管理为中心的战略转移。优良的图书馆文化,能够创造出积极、和谐、上进的工作氛围,是产生源源不断的动力源泉,对图书馆事业的发展起着巨大的推动和促进作用。重视图书馆文化建设,有利于我们从新的高度推进图书馆管理科学化,促进图书馆内部管理体制改革。

  17. The libraries that made SUCEST

    Directory of Open Access Journals (Sweden)

    André L. Vettore

    2001-12-01

    Full Text Available A large-scale sequencing of sugarcane expressed sequence tags (ESTs was carried out as a first step in depicting the genome of this important tropical crop. Twenty-six unidirectional cDNA libraries were constructed from a variety of tissues sampled from thirteen different sugarcane cultivars. A total of 291,689 cDNA clones were sequenced in their 5’ and 3’end regions. After trimming low-quality sequences and removing vector and ribosomal RNA sequences, 237,954 ESTs potentially derived from protein-encoding messenger RNA (mRNA remained. The average insert size in all libraries was estimated to be 1,250bp with the insert length varying from 500 to 5,000 bp. Clustering the 237,954 sugarcane ESTs resulted in 43,141clusters, from which 38% had no matches with existing sequences in the public databases. Around 53% of the clusters were formed by ESTs expressed in at least two libraries while 47% of the clusters are formed by ESTs expressed in only one library. A global analysis of the ESTs indicated that around 33% contain cDNA clones with full-length insert.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  19. Construction of a large synthetic human Fab antibody library on yeast cell surface by optimized yeast mating.

    Science.gov (United States)

    Baek, Du-San; Kim, Yong-Sung

    2014-03-28

    Yeast surface-displayed antibody libraries provide an efficient and quantitative screening resource for given antigens, but suffer from typically modest library sizes owing to low yeast transformation efficiency. Yeast mating is an attractive method for overcoming the limit of yeast transformation to construct a large, combinatorial antibody library, but the optimal conditions have not been reported. Here, we report a large synthetic human Fab (antigen binding fragment) yeast surface-displayed library generated by stepwise optimization of yeast mating conditions. We first constructed HC (heavy chain) and LC (light chain) libraries, where all of the six CDRs (complementarity-determining regions) of the variable domains were diversified mimicking the human germline antibody repertoires by degenerate codons, onto single frameworks of VH3-23 and Vkappa1-16 germline sequences, in two haploid cells of opposite mating types. Yeast mating conditions were optimized in the order of cell density, media pH, and cell growth phase, yielding a mating efficiency of ~58% between the two haploid cells carrying HC and LC libraries. We constructed two combinatorial Fab libraries with CDR-H3 of 9 or 11 residues in length with colony diversities of more than 10(9) by one round of yeast mating between the two haploid HC and LC libraries, with modest diversity sizes of ~10(7). The synthetic human Fab yeast-displayed libraries exhibited relative amino acid compositions in each position of the six CDRs that were very similar to those of the designed repertoires, suggesting that they are a promising source for human Fab antibody screening.

  20. A Case Study of Construction of Special Database on Urban Agriculture in Library of Beijing University of Agriculture

    Institute of Scientific and Technical Information of China (English)

    Qianning; LIU

    2013-01-01

    With the development of urban agriculture and digital library, the theoretical research and exploitation of special database on urban agriculture has become an inevitable trend. On the basis of analyzing the advantages of the special database on urban agriculture constructed by the library of Beijing University of Agriculture, the author has analyzed the status and the problems of the special database on urban agriculture developed by Beijing University of Agriculture and proposed the development path of special database on urban agriculture.

  1. A Case Study of Construction of Special Database on Urban Agriculture in Library of Beijing University of Agriculture

    OpenAIRE

    2013-01-01

    With the development of urban agriculture and digital library, the theoretical research and exploitation of special database on urban agriculture has become an inevitable trend. On the basis of analyzing the advantages of the special database on urban agriculture constructed by the library of Beijing University of Agriculture, the author has analyzed the status and the problems of the special database on urban agriculture developed by Beijing University of Agriculture and proposed the develop...

  2. Website Construction of University Libraries%浅论高校图书馆网站建设

    Institute of Scientific and Technical Information of China (English)

    蔡明

    2011-01-01

    从图书馆工作人员的角度,文章就目前高校图书馆网站建设的定位、存在的问题及对策等方面问题,结合本馆网站改版工作情况谈了谈看法。%From the perspective of library workers, combined with the library website rcvision in his own school, the author talks about personal views on the current site location, existing problems and countermeasures and other issues of university library website construction.

  3. New polymorphic microsatellite markers derived from hemocyte cDNA library of Manila clam Ruditapes philippinarum challenged by the protozoan parasite Perkinsus olseni

    Science.gov (United States)

    Kang, Hyun-Sil; Hong, Hyun-Ki; Park, Kyung-Il; Cho, Moonjae; Youn, Seok-Hyun; Choi, Kwang-Sik

    2017-02-01

    Manila clam Ruditapes philippinarum is one of the most important benthic animals in the coastal north Pacific region, where clam populations have been mixed genetically through trade and aquaculture activities. Accordingly, identification of the genetically different clam populations has become one of the most important issues to manage interbreeding of the local and introduced clam populations. To identify genetically different populations of clam populations, we developed 11 expressed sequence tag (EST)-microsatellite loci (i.e., simple sequence repeat, SSR) from 1,128 clam hemocyte cDNA clones challenged by the protozoan parasite Perkinsus olseni. Genotype analysis using the markers developed in this study demonstrated that clams from a tidal flat on the west coast contained 6 to 19 alleles per locus, and a population from Jeju Island had 4 to 20 alleles per locus. The expected heterozygosity of the 2 clam populations ranged from 0.472 to 0.919 for clams from the west coast, and 0.494 to 0.919 for clams from Jeju Island, respectively. Among the 11 loci discovered in this study, 7 loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction. The 5 loci developed in this study also successfully amplified the SSRs of R. variegatus, a clam species taxonomically very close to R. philippinarum, from Hong Kong and Jeju Island. We believe that the 11 novel polymorphic SSR developed in this study can be utilized successfully in Manila clam genetic diversity analysis, as well as in genetic discrimination of different clam populations.

  4. New polymorphic microsatellite markers derived from hemocyte cDNA library of Manila clam Ruditapes philippinarum challenged by the protozoan parasite Perkinsus olseni

    Science.gov (United States)

    Kang, Hyun-Sil; Hong, Hyun-Ki; Park, Kyung-Il; Cho, Moonjae; Youn, Seok-Hyun; Choi, Kwang-Sik

    2017-03-01

    Manila clam Ruditapes philippinarum is one of the most important benthic animals in the coastal north Pacific region, where clam populations have been mixed genetically through trade and aquaculture activities. Accordingly, identification of the genetically different clam populations has become one of the most important issues to manage interbreeding of the local and introduced clam populations. To identify genetically different populations of clam populations, we developed 11 expressed sequence tag (EST)-microsatellite loci (i.e., simple sequence repeat, SSR) from 1,128 clam hemocyte cDNA clones challenged by the protozoan parasite Perkinsus olseni. Genotype analysis using the markers developed in this study demonstrated that clams from a tidal flat on the west coast contained 6 to 19 alleles per locus, and a population from Jeju Island had 4 to 20 alleles per locus. The expected heterozygosity of the 2 clam populations ranged from 0.472 to 0.919 for clams from the west coast, and 0.494 to 0.919 for clams from Jeju Island, respectively. Among the 11 loci discovered in this study, 7 loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction. The 5 loci developed in this study also successfully amplified the SSRs of R. variegatus, a clam species taxonomically very close to R. philippinarum, from Hong Kong and Jeju Island. We believe that the 11 novel polymorphic SSR developed in this study can be utilized successfully in Manila clam genetic diversity analysis, as well as in genetic discrimination of different clam populations.

  5. Screening of genes for proteins interacting with the PS1TP5 protein of hepatitis B virus: probing a human leukocyte cDNA library using the yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-kang; ZHAO Long-feng; CHENG Jun; GUO Jiang; LUN Yong-zhi; HONG Yuan

    2006-01-01

    Background The hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS 1TP5 protein.Methods The reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS 1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization.After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH 109 with Y 187 containing a leukocyte cDNA library plasmid.The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis.Results Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes

  6. Construction and characterization of a 10-genome equivalent yeast artificial chromosome library for the laboratory rat, Rattus norvegicus

    Energy Technology Data Exchange (ETDEWEB)

    Cai, L.; Zee, R.Y.L. [Harvard Medical School, Boston, MA (United States); Schalkwyk, L.C. [Max Planck Institute for Molecular Genetics, Berlin (Germany)] [and others

    1997-02-01

    Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescence in situ hybridization were found to be chimeric, indicating a proportion of about 20-30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species. 35 refs., 4 figs.

  7. Construction and Preliminary Characterization Analysis of Wuzhishan Miniature Pig Bacterial Artificial Chromosome Library with Approximately 8-Fold Genome Equivalent Coverage

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2013-01-01

    Full Text Available Bacterial artificial chromosome (BAC libraries have been invaluable tools for the genome-wide genetic dissection of complex organisms. Here, we report the construction and characterization of a high-redundancy BAC library from a very valuable pig breed in China, Wuzhishan miniature pig (Sus scrofa, using its blood cells and fibroblasts, respectively. The library contains approximately 153,600 clones ordered in 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 152.3 kb, representing approximately 7.68 genome equivalents of the porcine haploid genome and a 99.93% statistical probability of obtaining at least one clone containing a unique DNA sequence in the library. 19 pairs of microsatellite marker primers covering porcine chromosomes were used for screening the BAC library, which showed that each of these markers was positive in the library; the positive clone number was 2 to 9, and the average number was 7.89, which was consistent with 7.68-fold coverage of the porcine genome. And there were no significant differences of genomic BAC library from blood cells and fibroblast cells. Therefore, we identified 19 microsatellite markers that could potentially be used as genetic markers. As a result, this BAC library will serve as a valuable resource for gene identification, physical mapping, and comparative genomics and large-scale genome sequencing in the porcine.

  8. Probe into the Construction of Community Digital Library%社区数字图书馆建设探讨

    Institute of Scientific and Technical Information of China (English)

    陈彤芳

    2015-01-01

    The reading pattern in digital era speeds up the construction of digital library, and the construction of community digital library is imperative. Based on introducing the advantages of the digital works, this paper expounds the significance of and countermeasures for the construction of community digital library in the era of digital reading.%数字化时代的阅读方式加快了数字图书馆建设,建设社区数字图书馆势在必行。在介绍数字读物优势的基础上,阐述了数字阅读时代社区数字图书馆建设的意义及对策。

  9. Identification of novel glycosyl hydrolases with cellulolytic activity against crystalline cellulose from metagenomic libraries constructed from bacterial enrichment cultures.

    Science.gov (United States)

    Mori, Toshio; Kamei, Ichiro; Hirai, Hirofumi; Kondo, Ryuichiro

    2014-01-01

    To obtain cellulases that are capable of degrading crystalline cellulose and cedar wood, metagenomic libraries were constructed from raw soil sample which was covered to pile of cedar wood sawdust or from its enrichment cultures. The efficiency of screening of metagenomic library was improved more than 3 times by repeating enrichment cultivation using crystalline cellulose as a carbon source, compared with the library constructed from raw soil. Four cellulase genes were obtained from the metagenomic libraries that were constructed from the total genome extracted from an enrichment culture that used crystalline cellulose as a carbon source. A cellulase gene and a xylanase gene were obtained from the enrichment culture that used unbleached kraft pulp as a carbon source. The culture supernatants of Escherichia coli expressing three clones that were derived from the enrichment culture that used crystalline cellulose showed activity against crystalline cellulose. In addition, these three enzyme solutions generated a reducing sugar from cedar wood powder. From these results, the construction of a metagenomic library from cultures that were repetition enriched using crystalline cellulose demonstrated that this technique is a powerful tool for obtaining cellulases that have activity toward crystalline cellulose.

  10. The construction and preliminary analysis of a Tn5 transposon based random mutant library of baculovirus

    Institute of Scientific and Technical Information of China (English)

    Li Hui; Zhao Minglei; Yin Juan; Zhong Jiang

    2006-01-01

    A transposon-based random mutation library of AcMNPV,the type species of baculovirus,was constructed using a Tn5 transposon.The green fluorescence protein gene under the control of the Drosophila hsp70 promoter was inserted into the transposon for easy tracking in insect cells.In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon into the virus genome.The transposed genome was then used to transfect Sf21 insect cells,and a library of mutant viruses capable of expressing green fluorescence protein was obtained.Two mutant viruses,B9F and Li6A were isolated,and the sites of transposon insertion were determined to be within the coding regions of the 94k and p10 genes,respectively.Both genes were determined to be nonessential in viral replication and infection.This technique will be very useful in the functional study of baculovirus genes.

  11. BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.

    Directory of Open Access Journals (Sweden)

    Wu Cheng-Cang

    2011-05-01

    Full Text Available Background Although second generation sequencing (2GS technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. Result Five new BAC libraries were constructed for barley (Hordeum vulgare L. cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast, empty wells and off-scale clones (clones with 250 fragments. Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. Conclusion BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.

  12. Directed construction and analysis of a Sinorhizobium meliloti pSymA deletion mutant library.

    Science.gov (United States)

    Yurgel, Svetlana N; Mortimer, Michael W; Rice, Jennifer T; Humann, Jodi L; Kahn, Michael L

    2013-03-01

    Resources from the Sinorhizobium meliloti Rm1021 open reading frame (ORF) plasmid libraries were used in a medium-throughput method to construct a set of 50 overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except the replicon region. Each resulting pSymA derivative carried a defined deletion of approximately 25 ORFs. Various phenotypes, including cytochrome c respiration activity, the ability of the mutants to grow on various carbon and nitrogen sources, and the symbiotic effectiveness of the mutants with alfalfa, were analyzed. This approach allowed us to systematically evaluate the potential impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.

  13. An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries.

    Science.gov (United States)

    Verma, Digvijay; Satyanarayana, T

    2011-09-01

    An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification of DNA while minimizing the loss of DNA with respect to other available protocols for metagenomic DNA extraction. Moreover, the purity levels are significant, which are difficult to attain with any of the methods reported in the literature for DNA extraction from soils. The DNA thus extracted was free from humic substances and, therefore, could be processed for restriction digestion, PCR amplification as well as for the construction of metagenomic libraries.

  14. Construction of a DNA library from chromosome 4 of rice (Oryza sativa) by microdissection

    Institute of Scientific and Technical Information of China (English)

    MAOYINGWEI; SIYUANLIANG; 等

    1998-01-01

    A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.

  15. 区域图书馆数据中心IaaS建设研究%The Research on the Construction of Library Regional Library Datacenter IaaS

    Institute of Scientific and Technical Information of China (English)

    郑晓军

    2014-01-01

    文章从图书馆IT系统的应用现状切入,讨论了云计算IaaS的整合优化基础资源,提高资源利用率,降低IT系统的建设、管理和维护总成本等方面的优势,探索了图书馆区域性云计算IaaS的构建和应用。%This paper reviews current status of IT system applications in library sector and thus discusses some advantages of cloud computing IaaS in terms of Infrastructure integration and utilization, reduction of IT system construction, management and maintenance costs, etc. It further explores the construction and application of library regional cloud computing IaaS.

  16. 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...

  17. 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

  18. Higher vocational college library culture construction%高职院校图书馆文化建设探讨

    Institute of Scientific and Technical Information of China (English)

    王萍

    2015-01-01

    The main cause of the current higher vocational colleges are not good development is the serious lag in the development process of library culture. Therefore, higher vocational colleges must be based on their actual situation to construct reasonable library culture ecosystem, to trigger a new ideas and thoughts. Today, many higher vocational colleges to the importance of the construction of library culture, have the right consciousness, also have already started to strengthen the pace of the construction of the library culture. Based on the present situation in the construction of higher vocational college library culture as the starting point, analyzes the current problems existing in the construction of library culture, and proposes the corresponding advice.%当前高职院校没有得到良好发展的主要原因就是图书馆文化在发展过程中严重滞后。因此,高职院校必须要根据自身的实际情况来构建出合理的图书馆文化生态系统,使之能够触发出新的观念与思想。如今,很多高职院校对图书馆文化建设的重要性已经有了正确的意识,也已经开始着手对图书馆文化加强了建设的步伐。文章以高职院校图书馆文化建设现状为着手点,分析了当前图书馆文化建设中存在的问题,并提出了相应的建设意见。

  19. Brand service construction of Ningxia Library%宁夏图书馆品牌服务建设分析

    Institute of Scientific and Technical Information of China (English)

    杨文琴

    2014-01-01

    针对图书馆品牌建设问题,采用归纳分析的方法,以宁夏图书馆为例,分析了图书馆品牌建设作用、品牌建设内涵,提出打造图书馆服务品牌、提供图书馆创新服务、提供图书馆特色服务、提供图书馆满意服务4个方面的图书馆品牌服务建设实现途径,为图书馆品牌服务建设提供借鉴。%In view of problems in constructing library brand, an inductive analysis was made of the role, content of library brand construction based on the situation of Ningxia Library. Corresponding approaches are offered as follows: building the brand of library ser-vices and providing innovative, feature and satisfactory library services, which provides ref-erences for building brand services of libraries.

  20. Construction of a BAC library from cucumber (Cucumis sativus L.) and identification of linkage group specific clones

    Institute of Scientific and Technical Information of China (English)

    Yuan Guan; Qi Chen; Junsong Pan; Zheng Li; Huanle He; Aizhong Wu; Rentao Song; Run Cai

    2008-01-01

    A bacterial artificial chromosome (BAC) library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber (Cucumis sativus L.) inbred line S94, derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions (SCAR) and seven simple sequence repeats (SSR) markers from each link-age group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction (PCR). Fifteen mark-ers gave at least two positive clones. As a result, 22 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.

  1. 公共图书馆专题图书馆的建设与服务%The Construction and Service of Subject Library in Public Lilbrary

    Institute of Scientific and Technical Information of China (English)

    李彩萍

    2012-01-01

    专题图书馆是收藏专题文献资源,面向特定读者开展专题服务的图书馆。专题图书馆的建设,是公共图书馆服务创新的新举措。结合我国图书馆专题图书馆的建设实践,探讨了公共图书馆专题图书馆的建设与服务。%Subject library is the kind of libraries that carries out subject services on the basis of subject literatures. The construction of subject library is a new measure of service innovation in public library.This paper discusses the construction and service of subject library in public library combining with the practice of the construction of subject libraries in China.

  2. Construction of bacterial artificial chromosome libraries for the Lake Malawi cichlid (Metriaclima zebra), and the blind cavefish (Astyanax mexicanus).

    Science.gov (United States)

    Di Palma, Federica; Kidd, Celeste; Borowsky, Richard; Kocher, Thomas D

    2007-01-01

    Teleost fishes have become important models for studying the evolution of the genetic mechanisms of development. A key resource for comparative genomics and positional cloning are large-insert libraries constructed in bacterial artificial chromosomes. We have constructed bacterial artificial chromosome libraries for two species of teleost fish that are important models for the study of developmental evolution. Metriaclima zebra is one of several hundred closely related, morphologically diverse, haplochromine cichlids which have evolved over the last one million years in Lake Malawi, East Africa. The Mexican tetra, Astyanax mexicanus, is well known for adaptations related to the recent evolution of blind cave-dwelling forms. Clones and high-density filters for each library are available to the scientific community through the Hubbard Center for Genome Studies.

  3. A dedicated vector for efficient library construction and high throughput screening in the hyphal fungus Chrysosporium lucknowense

    NARCIS (Netherlands)

    Verdoes, J.C.; Punt, P.J.; Burlingame, R.; Bartels, J.; Dijk, R. van; Slump, E.; Meens, M.; Joosten, R.; Emalfarb, M.

    2007-01-01

    A self-replicating vector was designed that enables the construction of complex libraries in the fungus Chiysosporium lucknowense. The circular vector is linearized in vivo and results in a transformation frequency up to 13,000 transformants/ug ofplasmid DNA. Upon prolonged cultivation of the transf

  4. Construction and Creation of Culture of Library in College%高校图书馆文化建设与创新的研究

    Institute of Scientific and Technical Information of China (English)

    孙红艳

    2011-01-01

    高校图书馆文化建设与创新在高校图书馆建设中具有非常重要的意义.一般认为,高校图书馆文化建设涉及文化、图书馆文化、高校图书馆文化以及高校图书馆文化的建设与创新等问题.%Construction and creation of culture of library in college has an important significance in the construction of library in college.Construction of culture of library in college is related to the culture, culture of library, culture of library in college and construction and creation of culture of library in college etc.

  5. Microwave-Assisted Solid Phase Organic Synthesis.Application to Indole Library Construction

    Institute of Scientific and Technical Information of China (English)

    DAI Wei-Min; SUN Li-Ping; GUO Dian-Shun; HUANG Xiang-Hong

    2004-01-01

    Microwave-assisted organic synthesis (MAOS) has attained increasing popularity due to recent advancement in the instrumentation of microwave technology. Now, MAOS can be performed under controlled temperature and pressure to yield reproducible results. For combinatorial chemistry,the dramatically increased reaction rate under microwave irradiation at high temperature provides an ideal solution to those sluggish reactions, in particular the combinatorial reactions carried out on solid supports. In this presentation, we describe our results on microwave-assisted solid-phase organic synthesis (MASPOS) applied to the construction of indole libraries such as 5. Compounds 4 were synthesized on the Rink amide resins using IRORI MicroKanTM reactors encoded with a radio-frequency (Rf) tag. The resin-bound terminal alkynes 2, prepared via the amide bond, were cross-coupled with the nitroaryl triflate under the conditions adopted from the solution reactions developed by us1,2. The nitro group of 3 was then reduced and sulfonylated to give 4. Ring closure reactions within 4 with Cu(OAc)2 were examined initially in refluxing DCE for 24 h, but no indole product was detected after cleavage from the resin. Therefore, the same reactions were carried out under microwave irradiation at 200 ℃ for 10 min on a Personal Chemistry Emrys Creator, the desired indoles 5 were obtained in 60-95% overall yields calculated from 1 and in >90% purities in most cases3. It is necessary to mention that the IRORI microreactors cannot tolerate the high temperature and the resin-bound 4 must be transferred to the reaction vials for the microwave-assisted ring closure reactions. A traceless synthesis of an indole library via MASPOS will be discussed as well.4

  6. The Rural Libraries' Construction Needs to Be Upgraded%乡镇图书馆建设亟待升级

    Institute of Scientific and Technical Information of China (English)

    陈景朝

    2016-01-01

    Along with the acceleration of urbanization in our country, various functions of infrastructure are continuously upgraded and perfected, and the performance-cost ratio of rural libraries' accessible services is increased constantly. Connecting with the service status of rural libraries and the working experiences in grassroots libraries, this paper discusses the necessity and urgency of the upgrade of the construction of rural libraries, and puts forward some suggestions on the construction of rural libraries.%随着我国城镇化建设步伐的加快,基础设施的各种功能不断升级完善,乡镇图书馆可及性服务的功能价值比不断增高.结合乡镇图书馆服务现状及基层图书馆工作经验,论述了乡镇图书馆建设升级的必要性和紧迫性,提出了关于乡镇图书馆建设的若干建议.

  7. Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genic male-sterile rice 5460S

    Institute of Scientific and Technical Information of China (English)

    邱芳; 金德敏; 伏健民; 张超良; 谢纬武; 王斌; 杨仁崔; 张洪斌

    1999-01-01

    In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondrial and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tmsl gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.

  8. Design and construction of a new human naïve single-chain fragment variable antibody library, IORISS1.

    Science.gov (United States)

    Pasello, Michela; Zamboni, Silvia; Mallano, Alessandra; Flego, Michela; Picci, Piero; Cianfriglia, Maurizio; Scotlandi, Katia

    2016-04-20

    Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated.

  9. 新疆高校图书馆建设中亚文库的思考%Thoughts on Constructing Central Asia Library by University Libraries in Xinjiang

    Institute of Scientific and Technical Information of China (English)

    顾燕玲; 刘晖; 张沛黎

    2015-01-01

    中亚是中国的重要邻邦,也是中国周边合作的重点地区,更是中国提升安全与能源保障的重要地缘战略依托.文章阐述了新疆高校图书馆建设中亚文库的必要性及意义.%Central Asia is an important neighbor of China, as well as the key area for China's cooperation and the more important geopolitical strategy support on enhancing China's security and energy security. The article discussed the significance of constructing Central Asia Library by university libraries in Xinjiang.

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

    Directory of Open Access Journals (Sweden)

    Pan Hui-Juan

    2007-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-09-01

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

  12. Construction and sequence sampling of deep-coverage, large-insert BAC libraries for three model lepidopteran species

    Directory of Open Access Journals (Sweden)

    Zhao Shaying

    2009-06-01

    Full Text Available Abstract Background Manduca sexta, Heliothis virescens, and Heliconius erato represent three widely-used insect model species for genomic and fundamental studies in Lepidoptera. Large-insert BAC libraries of these insects are critical resources for many molecular studies, including physical mapping and genome sequencing, but not available to date. Results We report the construction and characterization of six large-insert BAC libraries for the three species and sampling sequence analysis of the genomes. The six BAC libraries were constructed with two restriction enzymes, two libraries for each species, and each has an average clone insert size ranging from 152–175 kb. We estimated that the genome coverage of each library ranged from 6–9 ×, with the two combined libraries of each species being equivalent to 13.0–16.3 × haploid genomes. The genome coverage, quality and utility of the libraries were further confirmed by library screening using 6~8 putative single-copy probes. To provide a first glimpse into these genomes, we sequenced and analyzed the BAC ends of ~200 clones randomly selected from the libraries of each species. The data revealed that the genomes are AT-rich, contain relatively small fractions of repeat elements with a majority belonging to the category of low complexity repeats, and are more abundant in retro-elements than DNA transposons. Among the species, the H. erato genome is somewhat more abundant in repeat elements and simple repeats than those of M. sexta and H. virescens. The BLAST analysis of the BAC end sequences suggested that the evolution of the three genomes is widely varied, with the genome of H. virescens being the most conserved as a typical lepidopteran, whereas both genomes of H. erato and M. sexta appear to have evolved significantly, resulting in a higher level of species- or evolutionary lineage-specific sequences. Conclusion The high-quality and large-insert BAC libraries of the insects, together

  13. 与人巨细胞病毒UL128两种不同结构蛋白相互作用蛋白的筛选与分析%Screening of protein interacting with the transcript of UL128 gene showed two protein patterns by yeast two-hybrid from human fetus brain cDNA library

    Institute of Scientific and Technical Information of China (English)

    任高伟; 崔鑫; 马艳萍; 齐莹; 阮强; 孙峥嵘

    2010-01-01

    Objective Using yeast two-hybrid system to screen the proteins which can interact with the human cytomegalovirus (HCMV) UL128 which have two difference transcription structure from human fetus brain cDNA library, and compare the difference with structure and function of interacting proteins. Methods Two fragments of UL128 were amplified by 3'RACE and 5'RACE technology, the length are 519 bp and 642 bp, respectively. The "bait plasmid" (named as pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp) was constructed successfully. Using pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp as a bait, a human fetus brain cDNA was screened and the proteins interacting with UL128-519 bp and UL128-642 bp encoded protein were searched, and the positive clones were sequenced and analyzed by bioinformatic methods. Results EFEMP2 interacting with HCMV UL128-519 bp were identified, THY-1 interacting with HCMV UL128-642 bp were identified. Conclusion EFEMP2 and THY-1 proteins interacting with HCMV UL128-519 bp and UL128-642 bp in human fetus brain cDNA library were successfully screened, but same proteins weren't found from the proteins interacting with UL128-519 bp and UL128-642 bp protein, UL128-519 bp and UL128-642 bp protein may be play an different effect in the process of infect by HCMV.%目的 利用酵母双杂交系统从人胎脑cDNA文库中筛选与两种不同转录结构的人巨细胞病毒(HCMV)UL128编码蛋白相互作用的蛋白,比较两者相互作用蛋白之间的异同点.方法 通过3'RACE和5'RACE技术扩增出两种HCMV UL128片段,其大小分别为519 bp和642 bp,并将其成功构建到酵母诱饵表达载体pGBKT7中.将以上两种酵母表达载体分别转化到酵母菌AH109中,再将文库DNA转化到已含有酵母表达载体的AH109中,筛选与两种片段大小不同的UL128编码蛋白相互作用的人胎脑蛋白,并对筛选得到的阳性克隆进行测序和生物信息学分析.结果 筛出EFEMP2与UL128-519 bp编码蛋白相互作用,THY-1

  14. A Study on Service System Construction of Dalian Children Main Library and Branch Libraries%大连市少儿图书馆总分馆服务体系建设研究

    Institute of Scientific and Technical Information of China (English)

    曲岩红

    2014-01-01

    大连市少儿图书馆总分馆服务体系建设是以大连市少儿图书馆图书馆为中心馆,以本地区中小学特别是偏远涉农区市县中小学图书馆为分馆,构建的集群化图书馆服务网络。目前,总分馆服务体系已初见成效。本文通过回顾与总结,探索大连市少儿图书馆服务体系建设与“少儿阅读资源全域共享”的经验与不足,以期为少儿图书馆总分馆服务体系建设提供参考。%Service system construction of Dalian children main library and branch libraries is Children Library of Dalian City as the center library and libraries of middle schools, primary schools especially in remote rural area as branch libraries that build cluster library service network .The current service system of children main library and branch libraries has achieved initial success .By reviewing the system construction this paper discusses the lack of children reading resources in order to provide reference for service system of children main library and branch libraries.

  15. Ouachita Parish Public Library, Final Performance Report for Library Services and Construction Act (LSCA) Title VI, Library Literacy Program, 1992-1993.

    Science.gov (United States)

    Camp, Gloria S.

    The Ouachita Parish Public Library (Louisiana) conducted a project that involved recruitment, coalition building, public awareness, training, basic literacy, collection development, tutoring, technology, and English as a Second Language (ESL) programs. The project served a community of over 200,000 people, and targeted the learning disabled,…

  16. SCREENING OF PROTEASE ENZYME BY CONSTRUCTION OF METAGENOMIC LIBRARY FROM MARINE SOIL SEDIMENTS

    Directory of Open Access Journals (Sweden)

    R.PRABAVATHI

    2012-07-01

    Full Text Available Nonetheless, the cultivable microorganisms constituting these resources correspond to only a small fraction of the microbial diversity less than 1% of the microorganisms in various environments are readily cultivable (Amann et al., 1995. This limits the range of a search for new biocatalysts for the bioprocess industry, so the use of complex communities and the effort to overcome the problem of noncultivability attract not only scientific attention but also biotechnological innovation. Methods have been developed and used toovercome the non-cultivability of environmental microorganisms for biotechnology, namely cloning and the expression of metagenomes in suitable expression hosts. Proteases are present in all living forms as they are involved in various metabolic processes. They are mainly involved in hydrolysis of the peptide bonds (Gupta et al., 2002. Proteases find a wide range of applications in food, pharmaceutical, leather and textile, detergent, diagnostics industries and also in waste management. In order to discover new proteases from metagenomiclibraries, we screened for proteolytic activity from a constructed metagenomic library by direct cloning of environmental DNA of large DNA inserts. A novel gene encoding proteolytic enzyme was picked up,sequenced, expressed in E. coli and characterized. Several microbial proteases from the culturable organisms have been characterized. However, very few proteases have been identified through culture independent metagenomic approach.

  17. 我国登革2型病毒43株基因组全长cDNA的构建%Construction of the full-length cDNA of dengue type 2 virus isolated in China

    Institute of Scientific and Technical Information of China (English)

    欧武; 秦鄂德; 杨翠红; 杨佩英; 于曼

    2001-01-01

    目的构建我国登革2型病毒43株基因组全长cDNA,为进一步研究其体外RNA转录产物的感染性,阐明致病机理及探索新型疫苗奠定基础。方法根据登革2型病毒参考株NGC株的核苷酸序列,利用DNASTAR软件设计覆盖登革2型病毒43株基因组的6对重叠引物。从感染登革2型病毒43株的乳鼠脑中提取病毒基因组RNA,采用RT-PCR分别扩增6条基因片段,并将其分别与pGEM-T载体进行连接。重组质粒用PCR进行快速鉴定,并在377A型自动测序仪进行序列分析。然后利用单一酶切位点,分别自阳性重组子上切下各基因片段,在体外分别进行5′半分子和3′半分子的连接,最后将5′和3′半分子连接成基因组全长的cDNA。扩增各接头两侧长约457~691bp的基因片段,连接至T载体后测序,从而对全长cDNA进行鉴定。结果共扩增出6条约1.5~2.5kb的基因片段,并在体外进行连接,获得了全长cDNA。结论通过测序证实成功地构建了我国登革2型病毒43株基因组全长cDNA分子。本研究结果将为阐明我国登革病毒株的毒力及致病机理奠定基础。%Objective To construct the full-length cDNA of Chinese strain 43 of dengue 2 virus, and thus to set up basis for investigating the infectivity of its in vitro RNA transcript, elucidating the mechanism of pathogenesis of dengue virus infection, and developing novel vaccine against dengue. Methods Using the software DNASTAR, we devised six pairs of over-lapping primers which cover the whole genome of strain 43, according to the nucleotide sequence of international standard strain NGC. After extracting the RNA of virus from the infected brain tissue of the new-born mice, we amplified six cDNA fragments of D2-43 strain by reverse transcription PCR. The cDNA fragments were cloned into vector pGEM-T and then transformed into competent E.coli DH5α cells. Positive clones were screened by PCR and restriction

  18. A method to simultaneously construct up to 12 differently sized Illumina Nextera long mate pair libraries with reduced DNA input, time, and cost.

    Science.gov (United States)

    Heavens, Darren; Accinelli, Gonzalo Garcia; Clavijo, Bernardo; Clark, Matthew Derek

    2015-07-01

    Long mate pair (LMP) or "jump" libraries are invaluable for producing contiguous genome assemblies and assessing structural variation. However the consistent production of high quality (low duplication rate, accurately sized) LMP libraries has proven problematic in many genome projects. Input DNA length and quantity are key issues that can affect success. Here we demonstrate how 12 libraries covering a wide range of jump sizes can be constructed from DNA, thus ensuring production of the best LMP libraries from a given DNA sample. Finally, we demonstrate the accuracy of the insert sizes by mapping reads from each library back to an existing assembly.

  19. 地方高校图书馆文化建设的现状分析%Current Situation of Cultural Construction of Local University Library

    Institute of Scientific and Technical Information of China (English)

    杨月; 费晶

    2015-01-01

    地方高校图书馆文化建设是高校校园文化建设的的重要组成部分。因此,应加强图书馆文化建设。%The cultural construction of local university library is an important part of the construction of university campus culture. So, the cultural construction of library should be strengthened.

  20. A New Combination of Mutated loxPs in a Vector for Construction of Phage Antibody Libraries

    Institute of Scientific and Technical Information of China (English)

    Yu GAN; Xin-Tai ZHAO

    2005-01-01

    In the construction of large antibody libraries by in vivo recombination, two non-homogeneous loxP sites are required for the exchange of Vgenes between phagemids to create many new VH-VL combinations.The mutated loxP511 was designed not to recombine with the wild-type loxP (loxPwt) in early studies and a combination of the two has been used to construct antibody libraries. But recent reports have shown that recombination occurs between loxPwt and loxP511. This suggests that the combinational use of loxP511 and loxPwt might lead to the loss of the V gene diversity of antibody libraries. Therefore, it is necessary to find a new combination of loxPs to avoid the excision recombination in the antibody library. In this study,we found that the excision recombination between loxP511 and loxP2272, another mutated loxP sequence,was undetectable within one phagemid, while the excision recombination between loxP511 and loxPwt occurred at a frequency of 40%, higher than that reported previously. Furthermore, the in vivo recombination of different phagemids with loxP511 and loxP2272 showed that the V gene exchange was efficiently mediated to produce new VH-VL combinations. It was concluded that the loxP511 and loxP2272 combination was more favorable for reducing the excision recombination and constructing large phage antibody libraries with high diversity.

  1. Construction of a quinoa (Chenopodium quinoa Willd.) BAC library and its use in identifying genes encoding seed storage proteins.

    Science.gov (United States)

    Stevens, M R; Coleman, C E; Parkinson, S E; Maughan, P J; Zhang, H-B; Balzotti, M R; Kooyman, D L; Arumuganathan, K; Bonifacio, A; Fairbanks, D J; Jellen, E N; Stevens, J J

    2006-05-01

    Quinoa (Chenopodium quinoa Willd.) is adapted to the harsh environments of the Andean Altiplano region. Its seeds have a well-balanced amino acid composition and exceptionally high protein content with respect to human nutrition. Quinoa grain is a staple in the diet of some of the most impoverished people in the world. The plant is an allotetraploid displaying disomic inheritance (2n=4x=36) with a di-haploid genome of 967 Mbp (megabase pair), or 2C=2.01 pg. We constructed two quinoa BAC libraries using BamHI (26,880 clones) and EcoRI (48,000 clones) restriction endonucleases. Cloned inserts in the BamHI library average 113 kb (kilobase) with approximately 2% of the clones lacking inserts, whereas cloned inserts in the EcoRI library average 130 kb and approximately 1% lack inserts. Three plastid genes used as probes of high-density arrayed blots of 73,728 BACs identified approximately 2.8% of the clones as containing plastid DNA inserts. We estimate that the combined quinoa libraries represent at least 9.0 di-haploid nuclear genome equivalents. An average of 12.2 positive clones per probe were identified with 13 quinoa single-copy ESTs as probes of the high-density arrayed blots, suggesting that the estimate of 9.0x coverage of the genome is conservative. Utility of the BAC libraries for gene identification was demonstrated by probing the library with a partial sequence of the 11S globulin seed storage protein gene and identifying multiple positive clones. The presence of the 11S globulin gene in four of the clones was verified by direct comparison with quinoa genomic DNA on a Southern blot. Besides serving as a useful tool for gene identification, the quinoa BAC libraries will be an important resource for physical mapping of the quinoa genome.

  2. Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing.

    Science.gov (United States)

    Yang, Tae-Jin; Yu, Yeisoo; Nah, Gyoungju; Atkins, Michael; Lee, Seunghee; Frisch, David A; Wing, Rod A

    2003-08-01

    Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.

  3. Construction and characterization of two Citrus BAC libraries and identification of clones containing the phytoene synthase gene.

    Science.gov (United States)

    Baig, M N R; Yu, An; Guo, Wenwu; Deng, Xiuxin

    2009-05-01

    Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck 'Cara Cara' navel orange and Citrus reticulata (L.) Blanco 'Egan No. 1' Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 x 109 bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 x 109 bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in genomics research in the two important species C. sinensis and C. reticulata. Resources, high-density filters, individual clones, and whole libraries are available for public distribution and are accessible at the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  5. 高校图书馆文化建设研究%On Construction of University Library Culture

    Institute of Scientific and Technical Information of China (English)

    邓胤龙; 赵维平

    2012-01-01

    校园文化建设是高校发展、繁荣的一项重要工作。图书馆要以校园文化建设为契机,加强图书馆精神文化、制度文化、行为文化、馆藏等方面的建设,充分发挥图书馆文化在图书馆建设、发展、服务中的引领作用,不断提高办馆质量,提升馆员素质,完善服务水平。%The construction of campus culture is an important work for the development and prosperity of the university. Li- brary should take the opportunity of the construction of campus culture, strengthen the building of the library spirit culture, system culture, behavior culture, collection and other aspects. The library also should make full use of the leading role in building library culture, development and service, and constantly improve the quality of library, raise librarians quality and im- prove the service level.

  6. Cloning and identification of full-length DCC cDNA and construction of its eukaryotic expression vector%人类DCC基因克隆及真核表达载体构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    翟保平; 李文涛; 张斌; 于洋; 李育红

    2011-01-01

    目的 克隆人类DCC基因并构建其真核表达载体pIRES2-AcGFPI/DCC.方法 从正常皮肤组织中提取总RNA,采用逆转录-聚合酶链反应(RT-PCR)方法扩增DCC基因全长cDNA(4351bp),克隆人pMD18-T载体并转化大肠杆菌JM109,经PCR、酶切鉴定均为阳性的克隆,进行核苷酸测序分析,再将DCC基因定向克隆人pIRES2-AcGFP1载体中构建表达载体pIRES2-AcGFP1/DCC.结果 RT-PCR扩增后的产物在约4351 bp处出现明显的特异性条带,DCC基因的cDNA片段被成功插入真核表达载体pIRES2-AcGFP1质粒的多克隆位点,经鉴定与GenBank收录的DCC cDNA序列一致.结论 DCC基因的cDNA片段被成功克隆.%Objective To clone the full-length cDNA of human tumor suppressor DCC gene and construct its eukaryotic expression vector. Methods Total RNA was isolated from human foreskin tissue.Full-length DCC cDNA fragment (4351 bp) was amplified by reverse-transcription polymerase chain reaction (RT-PCR) and inserted into pMD18-T vector. The recombinant pMD18-T/DCC cDNA was transformed into E. coli JM109 host bacteria. The positive clones were confirmed by RT-PCR and doule-enzyme digestion assay. Orientation-based sub-cloning into pIRES2-AcGFP1 was performed as above followed by sequencing. Results Product of RT-PCR showed a clear specific band at 4341bp. pIRES2-AcGFP1/DCC was successfully constructed and transformed into E. coli JM109 host bacteria. Conclusion DCC gene cDNA has been inserted into eukaryotic expression vector pIRES2-AcGFP1 and successfully expressed.

  7. Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics

    Directory of Open Access Journals (Sweden)

    Hui eYuan

    2015-09-01

    Full Text Available Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of Pyrococcus furiosus proteins at whole genome level, we constructed expression plasmids of each Pyrococcus furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using phosphorothioate-modified primers and digesting PCR products by λ exonuclease. Our cloning method had a positive clone percentage of ≥ 80% in 96-well plate cloning format. Small-scale expression experiment showed that 55 out of 80 genes were efficiently expressed in Escherichia coli Strain Rosetta 2(DE3pLysS. In summary, this recombinant expression library of Pyrococcus furiosus provides a platform for functional and structural studies, as well as developing novel industrial enzymes. Our cloning scheme is adaptable to constructing recombinant expression library of other sequenced organisms.

  8. Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library.

    Science.gov (United States)

    Le Paslier, M C; Pierce, R J; Merlin, F; Hirai, H; Wu, W; Williams, D L; Johnston, D; LoVerde, P T; Le Paslier, D

    2000-04-15

    A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen.

  9. Constructing virtual combinatorial fragment libraries based upon MDL Drug Data Report database

    Institute of Scientific and Technical Information of China (English)

    ZHANG Min; SHENG ChunQuan; XU Hui; SONG YunLong; ZHANG WanNian

    2007-01-01

    Structural analysis of known drugs or drug-like compounds provides important information for drug design. The 142553 drug molecules in the MDL Drug Data Report database were analyzed, and then the common structural features were extracted. According to the common structural features, drug molecules were segmented into 32017 fragments, including 13642 ring fragments, 10076 linker fragments,and 8299 side chain fragments. These fragments were further used to establish three types of virtual combinatorial fragment libraries: a basic framework library containing 13574 rings; a linker library of 8051 linkers and a pharmacophore library of 34244 fragments combined by rings and side chains. After energy minimization, all fragments in the above three libraries maintain reasonable geometrical features and spatial conformations, and would be useful for building a virtual combinatorial database and de novo drug design.

  10. Constructing virtual combinatorial fragment libraries based upon MDL Drug Data Report database

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Structural analysis of known drugs or drug-like compounds provides important information for drug design. The 142553 drug molecules in the MDL Drug Data Report database were analyzed, and then the common structural features were extracted. According to the common structural features, drug molecules were segmented into 32017 fragments, including 13642 ring fragments, 10076 linker fragments, and 8299 side chain fragments. These fragments were further used to establish three types of virtual combinatorial fragment libraries: a basic framework library containing 13574 rings; a linker library of 8051 linkers and a pharmacophore library of 34244 fragments combined by rings and side chains. After energy minimization, all fragments in the above three libraries maintain reasonable geometrical features and spatial conformations, and would be useful for building a virtual combinatorial database and de novo drug design.

  11. 面向MOOC的数字图书馆建设%The Construction of the Digital Library for MOOC

    Institute of Scientific and Technical Information of China (English)

    吴丁; 刘丽

    2016-01-01

    The support function of university libraries for the MOOC curriculum is gradually reflected under the background of MOOC's vigorous development. This paper points out the problem of platform construction, digital resource management and network social environment support of MOOC localization. According to the objective law of the construction of digital resources, it considers that MOOC and the construction of the digital library have a unified direction. On the basis of de⁃tailed analysis of the large data features, Web3.0 features, and fragmentation characteristics of the MOOC curriculum,it dis⁃cusses how to strengthen the promotion effect of the digital library on MOOC from the construction of network learning com⁃munity and one stop resource discovery system from the perspective of the digital library.%在MOOC蓬勃发展的背景下,高校图书馆对于MOOC课程的支持作用开始逐渐体现出来。首先指出MOOC本土化发展面临的平台建设、数字资源管理和网络社交环境支持方面的问题。根据数字化资源建设的客观规律,认为MOOC与数字图书馆的建设方向具有统一性。在详细分析MOOC课程的大数据特征、Web3.0特征、碎片化特征的基础上,以数字图书馆的视角探讨如何从网络学习社区建设、一站式资源发现系统的建设入手,强化高校数字图书馆对MOOC的促进作用。

  12. Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

    Science.gov (United States)

    Ufarté, Lisa; Bozonnet, Sophie; Laville, Elisabeth; Cecchini, Davide A; Pizzut-Serin, Sandra; Jacquiod, Samuel; Demanèche, Sandrine; Simonet, Pascal; Franqueville, Laure; Veronese, Gabrielle Potocki

    2016-01-01

    Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.

  13. Construction of Yeast One-Hybrid Library for Screening of G-box Binding Proteins%筛选G-box结合蛋白的酵母单杂交文库的构建

    Institute of Scientific and Technical Information of China (English)

    杨鹏程; 周波; 李玉花

    2012-01-01

    目的:筛选花青素合成中的关键基因查尔酮合成酶基因CHS启动子中G-box的结合蛋白,从而找到调节CHS表达的转录因子.方法:采用Matchmaker Gold Yeast One-Hybrid Library Screening System,将CHS启动子G-box序列串联后整合入酵母染色体,构建诱饵菌株;采用SMART技术合成芜菁幼苗下胚轴cDNA,将该cDNA与pGA DT7-Rec表达载体共同转化诱饵菌株,通过同源重组在酵母细胞内同步进行cDNA文库的构建和筛选;用酵母菌落PCR法获得阳性克隆中的cDNA插入片段,测序后在NCBI网站进行Blast分析.结果:共筛选了2.52×106个酵母克隆,得到94个阳性克隆,菌落PCR获得了长度为0.4~2.0 kb的cDNA插入片段,并通过Blast推测了其编码蛋白.结论:实验结果证明酵母单杂交文库构建成功,初步筛选获得了G-box结合蛋白的候选蛋白,为研究CHS的表达调控奠定了基础.%Objective: In order to screen binding proteins of G-box, an important element in chalcone synthase (CHS) promoter region, and find transcriptional regulators of CHS gene. Methods: Matchmaker Gold Yeast One-Hybrid Library Screening System was employed in this study. Bait yeast strain was constructed by synthesizing oligonucleotides containing three tandem copies of G-box core sequences and integrating it into the genome of yeast. The cDNA for hypocotyls of turnip(Brassica rapa L. subsp. rapa Tsuda) was synthesized via SMART technology and co-transformed into bait yeast strain with pGADT7-Rec vector, one-hybrid cDNA library was simultaneously constructed and screened directly in yeast as a result of in vivo plasmid recombination. cDNA inserts in positive clones was amplified by yeast colony PCR and analyzed through NCBI Blast after sequencing. Results: Based on the experiments, we screened 2.52×106 yeast clones and got 94 positive clones. Colony PCR amplification products were 0.4~2.0 kb in length and proteins encoded by them were inferred by NCBI Blast analysis

  14. Some Considerations about the Construction of University 's Digital Library%对于高校数字图书馆建设的几点思考

    Institute of Scientific and Technical Information of China (English)

    罗绘秀

    2015-01-01

    This paper introduces the features and construction principles of the digital library, analyzes some problems existing in the construction of university's digital library, and probes into the strategies for the construction of university's digital library.%介绍了数字图书馆的特征与建设原则,分析了高校数字图书馆建设中存在的问题,探讨了高校数字图书馆的建设策略.

  15. 对高校移动图书馆 APP 建设的探讨%Discussion on the Construction of University Mobile Library APP

    Institute of Scientific and Technical Information of China (English)

    任海鹏

    2016-01-01

    本文从我国移动图书馆 APP 建设现状着手,重点分析了移动图书馆 APP 建设中存在的不足,进而探讨了怎样才能真正做好我国高校移动图书馆 APP 建设。%This article,from the current situation of the mobile library APP construction,focuses on the lack of construction in the presence of APP mobile library,and then discusses how to really do a good job moving APP university library construction in China.

  16. 音乐院校图书馆联盟建设的思考%Reflections on the Construction of Music College Library Consortia

    Institute of Scientific and Technical Information of China (English)

    刘纪刚

    2012-01-01

    在阐述音乐院校图书馆联盟建设必要性的基础之上,应用SWOT进行分析,提出建设音乐院校图书馆联盟的措施,并提出建设音乐院校图书馆联盟应注意的问题。%This paper expounds the necessity of the construction of music college library consortia,and analyzes the construction of music college library consortia by SWOT.Furthermore,it brings forward the measures to construct music college library consortia and some matters to be pay attention to.

  17. A good social work: women's clubs, libraries, and the construction of a secular society in Utah, 1890-1920.

    Science.gov (United States)

    Stauffer, Suzanne M

    2011-01-01

    After the renunciation of polygamy, Mormon women formed secular women's clubs as a means of collaborating with non-Mormon women in the construction of a shared secular society. Their common goal was the establishment and maintenance of the mainstream American social order. Activity in these clubs extended women's sphere into the public realm through socially acceptable public activities such as the temperance cause, civic improvements, political reform movements, and child welfare. The women campaigned for public support of libraries as institutions that would construct, preserve, and transmit American culture, educate the young, strengthen the home and family, and reform society.

  18. Construction of Chinese adult male phantom library and its application in the virtual calibration of in vivo measurement

    Science.gov (United States)

    Chen, Yizheng; Qiu, Rui; Li, Chunyan; Wu, Zhen; Li, Junli

    2016-03-01

    In vivo measurement is a main method of internal contamination evaluation, particularly for large numbers of people after a nuclear accident. Before the practical application, it is necessary to obtain the counting efficiency of the detector by calibration. The virtual calibration based on Monte Carlo simulation usually uses the reference human computational phantom, and the morphological difference between the monitored personnel with the calibrated phantom may lead to the deviation of the counting efficiency. Therefore, a phantom library containing a wide range of heights and total body masses is needed. In this study, a Chinese reference adult male polygon surface (CRAM_S) phantom was constructed based on the CRAM voxel phantom, with the organ models adjusted to match the Chinese reference data. CRAMS phantom was then transformed to sitting posture for convenience in practical monitoring. Referring to the mass and height distribution of the Chinese adult male, a phantom library containing 84 phantoms was constructed by deforming the reference surface phantom. Phantoms in the library have 7 different heights ranging from 155 cm to 185 cm, and there are 12 phantoms with different total body masses in each height. As an example of application, organ specific and total counting efficiencies of Ba-133 were calculated using the MCNPX code, with two series of phantoms selected from the library. The influence of morphological variation on the counting efficiency was analyzed. The results show only using the reference phantom in virtual calibration may lead to an error of 68.9% for total counting efficiency. Thus the influence of morphological difference on virtual calibration can be greatly reduced using the phantom library with a wide range of masses and heights instead of a single reference phantom.

  19. Construction of an Ultrahigh Pressure Liquid Chromatography-Tandem Mass Spectral Library of Plant Natural Products and Comparative Spectral Analyses.

    Science.gov (United States)

    Lei, Zhentian; Jing, Li; Qiu, Feng; Zhang, Hua; Huhman, David; Zhou, Zhiqin; Sumner, Lloyd W

    2015-07-21

    A plant natural product tandem mass spectral library has been constructed using authentic standards and purified compounds. Currently, the library contains 1734 tandem mass spectra for 289 compounds, with the majority (76%) of the compounds being plant phenolics such as flavonoids, isoflavonoids, and phenylpropanoids. Tandem mass spectra and chromatographic retention data were acquired on a triple quadrupole mass spectrometer coupled to an ultrahigh pressure liquid chromatograph using six different collision energies (CEs) (10-60 eV). Comparative analyses of the tandem mass spectral data revealed that the loss of ring substituents preceded the C-ring opening during the fragmentation of flavonoids and isoflavonoids. At lower CE (i.e., 10 and 20 eV), the flavonoids and isoflavonoid central ring structures typically remained intact, and fragmentation was characterized by the loss of the substituents (i.e., methyl and glycosyl groups). At higher CE, the flavonoid and isoflavonoid core ring systems underwent C-ring cleavage and/or rearrangement depending on the structure, particularly hydroxylation patterns. In-source electrochemical oxidation was observed for phenolics that had ortho-diphenol moieties (i.e., vicinal hydroxyl groups on the aromatic rings). The ortho-diphenols were oxidized to ortho-quinones, yielding an intensive and, in most cases, a base ion peak corresponding to a [(M - 2H) - H](-) ion in their mass spectra. The library also contains reverse-phase retention times, allowing for the construction, validation, and testing of an artificial neural network retention prediction of other flavonoids and isoflavonoids not contained within the library. The library is freely available for nonprofit, academic use and it can be downloaded at http://www.noble.org/apps/Scientific/WebDownloadManager/DownloadArea.aspx.

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

    Directory of Open Access Journals (Sweden)

    Inoko Hidetoshi

    2006-05-01

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

  1. Construction and characterization of a full-length infectious cDNA clone of foot-and-mouth disease virus strain O/JPN/2010 isolated in Japan in 2010.

    Science.gov (United States)

    Nishi, Tatsuya; Onozato, Hiroyuki; Ohashi, Seiichi; Fukai, Katsuhiko; Yamada, Manabu; Morioka, Kazuki; Kanno, Toru

    2016-06-01

    A full-length infectious cDNA clone of the genome of a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan was constructed and designated pSVL-f02. Transfection of Cos-7 or IBRS-2 cells with this clone allowed the recovery of infectious virus. The recovered virus had the same in vitro characterization as the parental virus with regard to antigenicity in neutralization and indirect immunofluorescence tests, plaque size and one-step growth. Pigs were experimentally infected with the parental virus or the recombinant virus recovered from pSVL-f02 transfected cells. There were no significant differences in clinical signs or antibody responses between the two groups, and virus isolation and viral RNA detection from clinical samples were similar. Virus recovered from transfected cells therefore retained the in vitro characteristics and the in vivo pathogenicity of their parental strain. This cDNA clone should be a valuable tool to analyze determinants of pathogenicity and mechanisms of virus replication, and to develop genetically engineered vaccines against foot-and-mouth disease virus.

  2. Accelerated construction of a regional DNA-barcode reference library: Caddisflies (Trichoptera) in the Great Smoky Mountains National Park

    Science.gov (United States)

    Zhou, X.; Robinson, J.L.; Geraci, C.J.; Parker, C.R.; Flint, O.S.; Etnier, D.A.; Ruiter, D.; DeWalt, R.E.; Jacobus, L.M.; Hebert, P.D.N.

    2011-01-01

    Deoxyribonucleic acid (DNA) barcoding is an effective tool for species identification and lifestage association in a wide range of animal taxa. We developed a strategy for rapid construction of a regional DNA-barcode reference library and used the caddisflies (Trichoptera) of the Great Smoky Mountains National Park (GSMNP) as a model. Nearly 1000 cytochrome c oxidase subunit I (COI) sequences, representing 209 caddisfly species previously recorded from GSMNP, were obtained from the global Trichoptera Barcode of Life campaign. Most of these sequences were collected from outside the GSMNP area. Another 645 COI sequences, representing 80 species, were obtained from specimens collected in a 3-d bioblitz (short-term, intense sampling program) in GSMNP. The joint collections provided barcode coverage for 212 species, 91% of the GSMNP fauna. Inclusion of samples from other localities greatly expedited construction of the regional DNA-barcode reference library. This strategy increased intraspecific divergence and decreased average distances to nearest neighboring species, but the DNA-barcode library was able to differentiate 93% of the GSMNP Trichoptera species examined. Global barcoding projects will aid construction of regional DNA-barcode libraries, but local surveys make crucial contributions to progress by contributing rare or endemic species and full-length barcodes generated from high-quality DNA. DNA taxonomy is not a goal of our present work, but the investigation of COI divergence patterns in caddisflies is providing new insights into broader biodiversity patterns in this group and has directed attention to various issues, ranging from the need to re-evaluate species taxonomy with integrated morphological and molecular evidence to the necessity of an appropriate interpretation of barcode analyses and its implications in understanding species diversity (in contrast to a simple claim for barcoding failure).

  3. Construction of a microbial natural product library for chemical biology studies.

    Science.gov (United States)

    Kato, Naoki; Takahashi, Shunji; Nogawa, Toshihiko; Saito, Tamio; Osada, Hiroyuki

    2012-04-01

    The RIKEN Natural Products Depository (NPDepo) is a public depository of small molecules. Currently, the NPDepo chemical library contains 39,200 pure compounds, half of which are natural products and their derivatives. In order to reinforce the uniqueness of our chemical library, we have improved our strategies for the collection of microbial natural products. Firstly, a microbial metabolite fraction library coupled with an MP (microbial products) plot database provides a powerful resource for the efficient isolation of microbial metabolites. Secondly, biosynthetic studies of microbial metabolites have enabled us to not only access ingenious biosynthetic machineries, but also obtain a variety of biosynthetic intermediates. Our chemical library contributes to the discovery of molecular probes for increasing our understanding of complex biological processes and for eventually developing new drug leads.

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

    Directory of Open Access Journals (Sweden)

    Christensen Knud

    2011-07-01

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

  5. Construction and Characterization of a Bacterial Artificial Chromosome Library for the A-Genome of Cotton (G. arboreum L.

    Directory of Open Access Journals (Sweden)

    Yan Hu

    2011-01-01

    Full Text Available A bacterial artificial chromosome (BAC library for the A-genome of cotton has been constructed from the leaves of G. arboreum L cv. Jianglinzhongmian. It is used as elite A-genome germplasm resources in the present cotton breeding program and has been used to build a genetic reference map of cotton. The BAC library consists of 123,648 clones stored in 322 384-well plates. Statistical analysis of a set of 103 randomly selected BAC clones indicated that each clone has an average insert length of 100.2 kb per plasmid, with a range of 30 to 190 kb. Theoretically, this represents 7.2 haploid genome equivalents based on an A-genome size of 1697 Mb. The BAC library has been arranged in column pools and superpools allowing screening with various PCR-based markers. In the future, the A-genome cotton BAC library will serve as both a giant gene resource and a valuable tool for map-based gene isolation, physical mapping and comparative genome analysis.

  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. Construction and analysis of gonad suppression subtractive hybridization library of rice field eel(Monopterus albus)%黄鳝性腺抑制差减文库的构建和分析

    Institute of Scientific and Technical Information of China (English)

    曲宪成; 尚晓莉; 程翠; 曲学伟; 张勇; 张开岳; 蒋骄云

    2011-01-01

    By using suppression subtractive hybridization (SSH), subtracted cDNA libraries were constructed from rice field eel (Monopterus albus) gonad at Ⅳ ovary and ovotestis stage. Two-directional (forward and backward) SSH was performed under cDNAs of rice field eel gonad at Ⅳ ovary and ovotestis stage. The cDNA fragments were inserted into plasmid vectors, and then transformed into Escherichia coli DHSα. Finally, forward and backward subtracted cDNA libraries (461 and 674 clones, respectively) were obtained. By PCR detection, length of the subtractive cDNA fragments cloned into the vector ranged from 200 bp to 2 000 bp. One hundred positive clones were selected and sequenced randomly from the forward and the backward libraries, and ninety gene fragment sequences were obtained. EST analysis of these sequences was performed with Blastx and Blaxtn by comparing sequences with GenBank database. Two sequenced genes were further analyzed by semi-quantitative RT-PCR method. The results showed that the library can enrich differentially expressed genes in the two phases gonad.%应用抑制性差减杂交技术构建了黄鳝(Monopterus albus)Ⅳ期卵巢和间期性腺的差减cDNA文库.正向差减杂交以间期性腺为试验方,Ⅳ期卵巢为驱动方;反向差减杂交以Ⅳ期卵巢为试验方,间期性腺为驱动方.将获得的差减cDNA片段连接质粒载体,然后转化大肠杆菌DH5α,最后获得的正、反向差减文库分别含461、678个重组子.经PCR扩增鉴定插入片段范嗣为200~2000 bp.随机选取正、反文库共100个阳性克隆测序分析,得到90个有效基因片段,与GenBank进行BLASTx和BLASTn同源比对.进一步从文库中选取2个基因用于半定量RT-PCR,对文库质量进行验证.结果表明,所建文库能够达到富集这两期性腺差异表达基因的目的.黄鳝性腺差减文库的构建,为进一步分离、鉴定黄鳝性腺发育和性逆转的相关基因奠定了基础.

  8. 运用抑制消减杂交构建辐射诱导EST库%Suppression subtractive hybridization in construction of radiation-induced EST library

    Institute of Scientific and Technical Information of China (English)

    铁轶; 罗瑛; 隋建丽; 周平坤

    2001-01-01

    目的 克隆并鉴定低剂量辐射诱导基因,为探讨低剂量辐射生物学效应分子机理提供有益信息。方法 0.5 Gy γ射线照射人胚肺细胞后,用抑制消减杂交技术(Suppression Subtractive Hybridization,SSH)构建低剂量辐射诱导EST库。用反向Northern杂交法对库中的EST片段进行筛选,挑取阳性克隆测序,并在GenBank序列数据库中进行同源性比较。结果 得到受照后表达升高90个EST序列,代表21个基因的转录产物,部分基因的功能已明确。从功能上分析,这些基因产物的生物学作用涵盖了细胞周期、信号转导、细胞骨架、代谢、蛋白合成等几个重要的细胞生物学代谢反应过程,显示出细胞对低剂量辐射反应过程的多样性。另外,实验还获得4个新cDNA序列。结论 利用抑制消减杂交技术,从辐射前后HEL细胞中,得到低剂量辐射诱导EST片段,它们与细胞周期、增殖、代谢、信号转导、应激反应等相关,这些基因可能在细胞的低剂量辐射效应中起到重要作用。%Objective To clone and identify radiation-induced genes from 0.5 Gy γ-ray irradiated human embryo lung cells(HEL).The identification and functional studies of radiation-induced genes will prompt the elucidation of the molecular mechanisms of low dose radiation-induced biological effects.It will also profit the understanding of the basic processes of cellular metabolisms. Methods  A low dose radiation-induced differentially displaying EST library has been constructed by suppression subtractive hybridization from HEL cells irradiated with 0.5 Gy γ-rays.The EST library was screened by reverse Northern hybridization analysis.Positive clones were sequenced and the similarity was searched against the DNA database in GenBank. Results  Altogether 90 positive cDNA clones with increased expression after 0.5 Gy irradiation were identified which corresponded to 21 individual genes

  9. Study on the Construction of Community Family Library%社区家庭图书馆建设探讨

    Institute of Scientific and Technical Information of China (English)

    王慧

    2016-01-01

    In view of the present situation of the public library inadequate, this paper puts forward a creative idea that using the study of families to construct community family libraries, which are open for outside, then analyzes the feasibility and so⁃cial significance of the idea such as promotion potential and conditions, etc. Finally, this paper puts forward concrete ways and methods.%针对公共图书馆覆盖不足的现状,本文提出利用家庭书房建设对外开放的社区家庭图书馆的创意,并分析了推广潜力、条件等可行性及社会意义,最后提出具体的途径、方法。

  10. A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes.

    Science.gov (United States)

    Osiewacz, H D

    1994-07-01

    A shuttle cosmid vector, pANsCos1, has been constructed for Escherichia coli and filamentous fungi. This vector contains two cos sequences separated by a single XbaI restriction site. pANsCos1 allows the efficient construction of representative genomic libraries from as little as 15-20 micrograms of genomic DNA. Due to the presence of a functional hygromycin B phosphotransferase gene (hph) transformation of fungal protoplasts with pAN-sCos1, or derivatives of it, results in the formation of hygromycin B-resistant transformants. The T7 and T3 RNA polymerase promoter sequences flanking the cloning site, in combination with two adjacent NotI sites facilitate genomic walking and the rapid construction of restriction maps of cloned inserts.

  11. Book Review: American libraries and the Internet: The social construction of Web appropriation and use

    Directory of Open Access Journals (Sweden)

    Hamid R. Jamali

    2008-06-01

    Full Text Available One can hardly find any aspect of human life that has not been affected one way or another by the Internet. Most of the Internet’s impact is because of the changes it has brought about in the areas of communication and availability of information. Facilitating information availability and information communication are among core activities of libraries. Since the advent of the Internet, libraries have tried to adopt this technology and make the best of it for their services. The Internet has been considered by librarians both as a source of concern and as a gate to fascinating opportunities. It is of much interest to see how librarians have reacted to this technology in its early days and how they have perceived it and made effort to apply it in their libraries. The book ‘American Libraries and the Internet’ by Bin Li, a research-based book, aims to answer questions such as: how do librarians define their roles in the changing environment? How do they understand and appropriate the Web in their profession and workplace? How have they perceived the World Wide Web from its early period of its implementation? And finally how the Web is appropriated and used in libraries?

  12. 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.

  13. 小议构建手机图书馆%On the Construction of Cellphone Library

    Institute of Scientific and Technical Information of China (English)

    段慧娇

    2011-01-01

    With the constant perfecting of cellphone functions, cellphone library correspondingly emerges.This paper elaborates on the advantages of the development of cellphone library and its merits and shortcomings,hoping to provide theoretical references for the development of China's cellphone library.%随着手机性能的不断提升,手机图书馆随即诞生。本文论述了手机图书馆发展的有利条件以及手机图书馆的优缺点,以期为我国手机图书馆的发展提供理论参考。

  14. Construction of a full-length cDNA library from Acanthamoeba%棘阿米巴原虫全长cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    王月华; 冯宪敏; 李瑶; 鞠晓红; 孙艳美

    2014-01-01

    目的 快速构建棘阿米巴滋养体全长cDNA文库,为棘阿米巴滋养体抗原的筛选和基因结构研究奠定基础.方法 以棘阿米巴滋养体分离株总RNA为模板,以经修饰的SMART寡聚核苷酸引物逆转录合成单链cDNA,采用长距PCR(long-Distance PCR,LD PCR)方法扩增得到双链cDNA,经蛋白酶K消化和Sf Ⅰ酶切后通过色谱柱CHROMA SPON-400按分子质量进行分离,进行1%琼脂糖凝胶电泳鉴定,回收纯化0.4~2.5 kb分离产物.取1μl PCR产物与λ噬菌体连接,以XL1-BLUE为受体菌扩增得到cDNA文库,分别用梯度法和随机测序法检测文库滴度与重组率. 结果 成功构建棘阿米巴cDNA文库,文库滴度为3.85×107 pfu/ml,插入片段大小在0.4~2.5 kb之间,重组率为100%(20/20). 结论 棘阿米巴滋养cDNA文库构建成功,为棘阿米巴滋养体抗原的筛选和基因结构研究奠定了基础.

  15. Construction and Identification of cDNA Library of Trichomonas vaginalis%阴道毛滴虫cDNA文库的构建和鉴定

    Institute of Scientific and Technical Information of China (English)

    章家新; 傅玉才; 郑晓虹; 刘红

    2003-01-01

    目的:构建阴道毛滴虫(Trichomonas vaginalis,Tv)全长cDNA表达文库,筛选全长目的基因,研究与其细胞周期相关基因的结构和功能.方法:从Tv中提取总RNA,用ClonTech公司的SMARTTMcDNA文库试剂盒构建其全长cDNA表达文库. 结果:所建文库的初始滴度为6.78×106pfu/mL,经一次扩增后的滴度为1.68×109pfu/mL,重组率>98%.结论:成功构建Tv全长cDNA文库,并从该文库中筛选出了与细胞周期相关的基因.

  16. Construction and Analysis of 'Tsuda' Turnip Subtracted cDNA Library%'津田'芜菁消减cDNA文库的构建及分析

    Institute of Scientific and Technical Information of China (English)

    许志茹; 李玉花

    2006-01-01

    以'津田'芜菁红色块根和白色块根为材料,利用SSH技术成功构建了消减cDNA文库并富集相关基因片段.从消减文库中筛选到960个阳性克隆,PCR鉴定插入片段长度主要分布于300~800 bp之间.克隆测序后与GenBank中的同源序列进行比较,特异基因片段为302个,其中99个与数据库中的序列具有相似区域且功能已知,与数据库中序列没有显著同源性及功能未知的序列为203条.

  17. 骨质疏松症骨组织cDNA差减文库的构建%Construction on subtractive cDNA library of osteporosis mice

    Institute of Scientific and Technical Information of China (English)

    魏小勇

    2009-01-01

    目的 构建人抑制差减文库,为筛选骨质疏松骨相关基因奠定基础.方法 分别从正常骨和骨质疏松症骨组织分离mRNA并反转录成cDNA,酶切后骨质疏松cDNA分成两组,分别与不同的接头连接.经过两轮差减杂交和两次抑制性PCR后得到两者之间差异表达的cDNA挑取克隆进行PCR扩增鉴定.结果 成功地构建了骨质疏松相关的的差减cDNA文库,获得的正、反向差减文库分别含652、345个重组子;插入片段的平均大小为526 bp.结论 该差减cDNA文库的建立为进一步在分子水平上阐明骨质疏松症的分子机制奠定了基础.

  18. Construction of cDNA Library of Newborn Larvae of Trichinella spiralis%旋毛虫新生幼虫cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    刘明远; 付宝权; 卢强; 吴秀萍; 姚春雨; 宇丽; 窦兰清; P.Boireau

    2001-01-01

    为了克隆和研究旋毛虫新生幼虫功能性抗原基因,采用酸性异硫氰酸胍-酚-氯仿一步法提取新生幼虫总RNA,Oligo(dT)纤维素柱纯化mRNA,反转录合成第一链cDNA及第二链cDNA,用CHROMA SPIN-400柱离心层析纯化后,与载体λZAP Express连接.体外包装后得到中国猪旋毛虫(Trichinella spiralis)分离株新生幼虫cDNA文库.文库容量为2.0×106,重组率为98.6%,插入片段长度在0.4×103-2.0×103bp.

  19. 伊氏锥虫cDNA表达文库的构建%Construction of Trypanosoma evansi cDNA expression library

    Institute of Scientific and Technical Information of China (English)

    刘全; 刘文森; 王祥生; 陈丽凤

    2006-01-01

    目的 构建伊氏锥虫cDNA表达文库.方法 利用DEAE-纤维素柱从小鼠血液中纯化伊氏锥虫,Trizol法提取总RNA,采用poly (A) Quik mRNA Isolation kit 分离纯化mRNA.运用cDNA Synthesis Kit 合成cDNA,以XR-λZAP为载体,构建伊氏锥虫cDNA表达文库.结果伊氏锥虫cDNA表达文库重组率为98%,滴度为0.9×106 pfu/ml,扩增后滴度为1×109 pfu/ml结论成功构建伊氏锥虫cDNA表达文库,为伊氏锥虫免疫相关因子的基因筛选奠定了基础.

  20. CONSTRUCTION AND CHARACTERIZATION OF GOLDEN HAMSTER NEURAL TUBE cDNA LIBRARY%金仓鼠神经管cDNA文库的建立

    Institute of Scientific and Technical Information of China (English)

    于丽; 管英俊; 高英茂; 李少玲

    2005-01-01

    目的构建金仓鼠神经管cDNA文库,以筛选与神经管发育及神经管畸形发生相关的基因.方法用TRIZOL Reagent提取金仓鼠神经管总RNA;用LD-PCR法反转录合成双链cDNA;经蛋白酶K消化、Sfi Ⅰ酶切、CHROMA SPIN-400柱分离去除小于500bp的片段;将cDNA与载体λ Tripl EX2按一定比例连接;经体外包装,建立cDNA的噬菌体表达文库.结果cDNA文库容量为1.5×106pfu/ml,重组率为99%,平均插入片段大于1kb.结论我们初步构建的金仓鼠神经管cDNA文库为高效的cDNA文库.

  1. Use of a recombination-deficient phage lambda system to construct wheat genomic libraries.

    Science.gov (United States)

    Murray, M G; Kennard, W C; Drong, R F; Slightom, J L

    1984-10-01

    The poor cloning efficiency of wheat (Triticum aestivum cv. Yamhill) DNA in conventional cloning vectors has previously prevented the preparation of complete genomic libraries. We show here that while wheat DNA does not clone efficiently using the vector Ch4A, it can be cloned efficiently using Ch32. Ch32 clones are red- gam+ and therefore can be propagated on recombination-deficient hosts. These results suggest that instability of wheat sequences in conventional lambda vector systems has frustrated previous attempts to prepare libraries.

  2. 艺术院校教学资源库建设策略研究%Strategy Research on Teaching Resource Library Construction in Art Colleges

    Institute of Scientific and Technical Information of China (English)

    夏军

    2015-01-01

    The article expounds the significance of constructing teaching resource library combining the feature of art college. In this article, the writer proposes some tactics about the construction of teaching resource library, including constructing content, constructing ways, organization and manage, etc. The article would perhaps provide some references for the construction of teaching resource library in art colleges.%结合艺术院校特点,阐述建设教学资源库的意义;对艺术院校教学资源库的建设提出几点策略,就建设内容、建设方式、组织与管理等问题进行研究探讨,以期为艺术院校教学资源库的建设提供参考.

  3. Probe into the New Scheme for Constructing Taiyuan Library 's Website%构建太原市图书馆网站新方案的探索

    Institute of Scientific and Technical Information of China (English)

    闫冰冰

    2016-01-01

    The rapid development of information technology industry and the popularity of computer hardware technology bring about new challenges and opportunities to the construction of library website. And how to construct well library website keeping in step with the situation is a problem faced by many libraries. Connecting with the practice, this paper Probe into the new scheme for constructing Taiyuan Library's Website.%信息技术产业的迅猛发展与计算机硬件技术的普及,为图书馆网站建设带来了新的挑战及机遇,如何紧跟形势做好图书馆网站建设是不少图书馆面临的问题. 结合实际,探索了太原市图书馆网站建设新方案.

  4. Design, construction, and validation of a modular library of sequence diversity standards for polymerase chain reaction.

    Science.gov (United States)

    Baum, Paul D; Young, Jennifer J; Zhang, Qianjun; Kasakow, Zeljka; McCune, Joseph M

    2011-04-01

    Methods to measure the sequence diversity of polymerase chain reaction (PCR)-amplified DNA lack standards for use as assay calibrators and controls. Here we present a general and economical method for developing customizable DNA standards of known sequence diversity. Standards ranging from 1 to 25,000 sequences were generated by directional ligation of oligonucleotide "words" of standard length and GC content and then amplified by PCR. The sequence accuracy and diversity of the library were validated using AmpliCot analysis (DNA hybridization kinetics) and Illumina sequencing. The library has the following features: (i) pools containing tens of thousands of sequences can be generated from the ligation of relatively few commercially synthesized short oligonucleotides; (ii) each sequence differs from all others in the library at a minimum of three nucleotide positions, permitting discrimination between different sequences by either sequencing or hybridization; (iii) all sequences have identical length, GC content, and melting temperature; (iv) the identity of each standard can be verified by restriction digestion; and (v) once made, the ends of the library may be cleaved and replaced with sequences to match any PCR primer pair. These standards should greatly improve the accuracy and reproducibility of sequence diversity measurements.

  5. The Contingent Constructed Nature of Social Life: Suggested Implications for Library and Information Science.

    Science.gov (United States)

    Hall, Peter M.

    1990-01-01

    Discusses a variety of topics and their implications for library and information science professionals and their education. Highlights include the nature of science; contingency theories; the assumptions of sociology; the approach of symbolic interaction; recent scholarship on organizations; and the advantages of qualitative research methods.…

  6. 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.

  7. Construction, Characterization, and Chromosomal Mapping of a Fosmid Library of the White-Cheeked Gibbon (Nomascus leucogenys)

    Institute of Scientific and Technical Information of China (English)

    Liping; Chen; Jianping; Ye; Yan; Liu; Jinghuan; Wang; Weiting; Su; Fengtang; Yang; Wenhui; Nie

    2007-01-01

    Gibbons have experienced extensive karyotype rearrangements during evolution and represent an ideal model for studying the underlying molecular mechanism of evolutionary chromosomal rearrangements. It is anticipated that the cloning and sequence characterization of evolutionary chromosomal breakpoints will provide vital insights into the molecular force that has driven such a radical karyotype reshuffle in gibbons. We constructed and characterized a high-quality fosmid li- brary of the white-cheeked gibbon (Nomascus leucogenys) containing 192,000 non- redundant clones with an average insert size of 38 kb and 2.5-fold genome coverage. By end sequencing of 100 randomly selected fosmid clones, we generated 196 se- quence tags for the library. These end-sequenced fosmid clones were then mapped onto the chromosomes of the white-cheeked gibbon by fluorescence in situ hy- bridization, and no spurious chimeric clone was detected. BLAST search against the human genome showed a good correlation between the number of hit clones and the number of chromosomes, an indication of unbiased chromosomal distribu- tion of the fosmid library. The chromosomal distribution of the mapped clones is also consistent with the BLAST search result against human and white-cheeked gibbon genomes. The fosmid library and the mapped clones will serve as a valu- able resource for further studying gibbons' chromosomal rearrangements and the underlying molecular mechanism as well as for comparative genomic study in the lesser apes.

  8. Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

    OpenAIRE

    Decai Tuo; Wentao Shen; Pu Yan; Xiaoying Li; Peng Zhou

    2015-01-01

    Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length ...

  9. Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations.

    Science.gov (United States)

    Fu, Glenn K; Xu, Weihong; Wilhelmy, Julie; Mindrinos, Michael N; Davis, Ronald W; Xiao, Wenzhong; Fodor, Stephen P A

    2014-02-01

    We present a simple molecular indexing method for quantitative targeted RNA sequencing, in which mRNAs of interest are selectively captured from complex cDNA libraries and sequenced to determine their absolute concentrations. cDNA fragments are individually labeled so that each molecule can be tracked from the original sample through the library preparation and sequencing process. Multiple copies of cDNA fragments of identical sequence become distinct through labeling, and replicate clones created during PCR amplification steps can be identified and assigned to their distinct parent molecules. Selective capture enables efficient use of sequencing for deep sampling and for the absolute quantitation of rare or transient transcripts that would otherwise escape detection by standard sequencing methods. We have also constructed a set of synthetic barcoded RNA molecules, which can be introduced as controls into the sample preparation mix and used to monitor the efficiency of library construction. The quantitative targeted sequencing revealed extremely low efficiency in standard library preparations, which were further confirmed by using synthetic barcoded RNA molecules. This finding shows that standard library preparation methods result in the loss of rare transcripts and highlights the need for monitoring library efficiency and for developing more efficient sample preparation methods.

  10. 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)

  11. Construction and characterization of a non-immune Llama variable heavy chain phage display antibody library%羊驼非免疫重链单域抗体库的构建和鉴定

    Institute of Scientific and Technical Information of China (English)

    吴标; 王树军; 夏立亮; 季萍; 葛海良; 赵国屏; 王颖

    2011-01-01

    本研究旨在通过构建羊驼非免疫重链单域抗体库,完成抗体库多样性的鉴定,为进一步筛选抗原特异性重链抗体奠定基础.我们从未经免疫的羊驼外周血中分离外周血单个核细胞(PBMC),抽提RNA后,用RT-PCR方法特异性扩增羊驼重链抗体可变区(VHH)片段;并采用两步连接方法将重链抗体可变区片段与噬菌粒载体pCANTAB5E连接获得重组子,多次电转感受态大肠杆菌TG1后获得VHH抗体基因库;并采用稀释计数法测定抗体库库容量,随机挑取克隆测序验证抗体库多样性.结果显示,我们所构建的羊驼非免疫重链单域抗体库的库容量为1.5×109,随机克隆测序验证多样性良好,独立克隆所占比例为80%,并显示出和人源抗体较高的同源性.上述结果表明,我们已经成功构建获得大容量的羊驼非免疫重链单域抗体库,为进一步筛选抗原特异性重链抗体奠定基础.%To construct a non-immune Llama variable domain of heavy chain antibody(VHH) phage display antibody library (VHH antibody library). Llama peripheral blood mononuclear lymphocytes were isolated from whole blood by Ficoll-hypaque density gradient centrifugation. Total RNA was extracted from PBMCs and reverse-transcripted into cDNA by using specific primers. VHH were amplified by nested PCR. PCR products of VHH fragments were then purified and ligated with phagemid vector pCANTAB5E by a modified two-step ligation method. Recombinant pCANTAB5E-VHH vectors were electroporated into competent TGI E.coli cells to obtain the primary VHH antibody library. The library capacity was titrated through limited dilution. Recombination efficacy and diversity of VHH antibody library was determined by sequencing analysis. Alignment a-nalysis was performed to compare the homology between Llama VHH domain and human/mouse variable regions of heavy chains. By using a modified strategy, we have constructed a non-immune Llama VHH antibody library with 1. 5

  12. Constructing gene-enriched plant genomic libraries using methylation filtration technology.

    Science.gov (United States)

    Rabinowicz, Pablo D

    2003-01-01

    Full genome sequencing in higher plants is a very difficult task, because their genomes are often very large and repetitive. For this reason, gene targeted partial genomic sequencing becomes a realistic option. The method reported here is a simple approach to generate gene-enriched plant genomic libraries called methylation filtration. This technique takes advantage of the fact that repetitive DNA is heavily methylated and genes are hypomethylated. Then, by simply using an Escherichia coli host strain harboring a wild-type modified cytosine restriction (McrBC) system, which cuts DNA containing methylcytosine, repetitive DNA is eliminated from these genomic libraries, while low copy DNA (i.e., genes) is recovered. To prevent cloning significant proportions of organelle DNA, a crude nuclear preparation must be performed prior to purifying genomic DNA. Adaptor-mediated cloning and DNA size fractionation are necessary for optimal results.

  13. Genes expressed in cotton (Gossypium hirsutum) buds isolated with a subtractive library.

    Science.gov (United States)

    Pinheiro, M P N; Batista, V G L; Martins, N F; Santos, R C; Melo Filho, P A; Silva, C R C; Lima, L M

    2013-01-16

    A subtractive cDNA library from cotton buds was constructed to prospect for differentially expressed genes related to early bud development. A library was constructed and 768 cDNA sequences were obtained, comprising 168 clusters, with 126 contigs and 42 singlets. Both the Gossypium as well as Arabidopsis databases were utilized for the in silico analysis, since some genes identified in cotton have not yet been studied for functionality, although they have homology with genes from other species. The transcriptome revealed a large number of transcripts, some of them with unknown function, and others related to pollen development, pollen tubes, ovules, and fibers at different stages. The most populated contig was identified as fiber from 0-10 days after anthesis, with 12 reads. The success and novelty rates generated from the library were 67 and 51%, respectively. The information obtained here will provide a framework for research on functional cotton genomics.

  14. Construction of white spot syndrome virus (WSSV) whole genome phage display library

    Institute of Scientific and Technical Information of China (English)

    ZHU Yanbing; YANG Feng

    2007-01-01

    A rebuilt vector pCANTAB 5 EE was obtained by inserting a 34 bp double-stranded oligonucleotide which contained a EcoRV recognition sequence into pCANTAB 5 E. White spot syndrome virus (WSSV) genome DNA was fragmented by sonication to isolate fragments mainly in the range of 0.8 ~2.0 kb, then the fragments were blunt-ended with T4 DNA polymerase and cloned into the EcoRV site of pCANTAB 5 EE. The primary recombinant clone of the library was 3.0 × 105.Colony PCR of random selected recombinants showed that the size of the inserts was 0.12 ~ 1.77 kb. After the whole library recombinant phages infected Escherichia coli HB2151 cells, the extracellular and periplasmic extracts were dropped on PVDF membranes to perform dot blot, using polyclonal mouse anti-VP24 serum,anti-WSV026 serum,anti-WSV063 serum,anti-WSV069 serum,anti-WSV112 serum, anti WSV238 serum,anti-WSV303 serum and anti-VP26 serum as the primary antibody, respectively. The results showed that the display library could express the viral proteins.

  15. Bias in ligation-based small RNA sequencing library construction is determined by adaptor and RNA structure.

    Directory of Open Access Journals (Sweden)

    Ryan T Fuchs

    Full Text Available High-throughput sequencing (HTS has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA and other small RNAs (sRNA. Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps, specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material.

  16. The hunt for original microbial enzymes: an initiatory review on the construction and functional screening of (metagenomic libraries

    Directory of Open Access Journals (Sweden)

    Martin, M.

    2016-01-01

    Full Text Available Introduction. Discovering novel enzymes is of interest in both applied and basic science. Microbial enzymes, which are incredibly diverse and easy to produce, are increasingly sought by diverse approaches. Literature. This review first distinguishes culture-based from culture-independent methods, detailing within each group the advantages and drawbacks of sequence- and function-based methods. It then discusses the main factors affecting the success of endeavors to identify novel enzymes through construction and functional screening of genomic or metagenomic libraries: the sampled environment, how DNA is extracted and processed, the vector used (plasmid, cosmid, fosmid, BAC, or shuttle vector, the host cell chosen from the available prokaryotic and eukaryotic ones and the main screening steps. Conclusions. Library construction and screening can be tricky and requires expertise. Combining different strategies, such as working with cultivable and non-cultivable organisms, using sequence- and function-based approaches, or performing multihost screenings, is probably the best way to identify novel and diverse enzymes from an environmental sample.

  17. Discussion on occupation morality construction of library management%浅谈图书管理职业道德建设

    Institute of Scientific and Technical Information of China (English)

    杨艳飞

    2013-01-01

      This article from the basic connotation, importance of occupation moral of library management, and the way improving the quality to elaborate modern library occupation moral construction. On the construction of library management ethics is more and more important, the moral strength to make books management personnel shoulder the responsibility, to feel important social meanings of the library, so as to master the attitude to keep pace with the times continuous learning progress, do a good job.%  本文从图书管理职业道德基本内涵、重要性及提高素质的途径阐述现代图书馆的职业道德建设。

  18. Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae.

    Science.gov (United States)

    Clark-Curtiss, J E; Jacobs, W R; Docherty, M A; Ritchie, L R; Curtiss, R

    1985-03-01

    Molecular analysis of DNA from Mycobacterium leprae, "Mycobacterium lufu," and Mycobacterium vaccae has demonstrated that the G + C (guanine plus cytosine) contents of the DNAs are 56, 61, and 65%, respectively, and that the genome sizes are 2.2 X 10(9), 3.1 X 10(9), and 3.1 X 10(9) daltons, respectively. Because of the significant differences in both G + C content and genome size among M. leprae, "M. lufu," and M. vaccae DNAs, these species are not related, although hybridization experiments under nonstringent conditions, with two separate cloned M. leprae DNA inserts as probes, indicate that there are some conserved sequences among the DNAs. The G + C content of Dasypus novemcinctus (armadillo, the animal of choice for cultivating M. leprae) DNA was determined to be 36%. Genomic libraries potentially representing more than 99.99% of each genome were prepared by cloning into the cosmid vector, pHC79, in Escherichia coli K-12. A genomic library representing approximately 95% of the genome of M. vaccae was prepared in pBR322. M. leprae DNA was subcloned from the pHC79::M. leprae library into an expression vector, pYA626. This vector is a 3.8-kilobase derivative of pBR322 in which the promoter region of the asd (aspartate semialdehyde dehydrogenase) gene from Streptococcus mutans has been inserted in place of the EcoRI-to-PstI fragment of pBR322. Several (44% of those tested) pYA626::M. leprae recombinants and one pBR322::M. vaccae recombinant synthesized new polypeptides in minicells of E. coli, indicating that mycobacterial DNA can be expressed in E. coli K-12, although expression is probably dependent upon use of nonmycobacterial promoters recognized by the E. coli transcription-translation apparatus.

  19. 罗汉果果实不同发育时期SSH文库的构建%Construction of SSH-cDNA library from different developmental stages of Siraitia grosvenorii

    Institute of Scientific and Technical Information of China (English)

    唐其; 邱德有; 马小军; 莫长明; 付伟

    2011-01-01

    In order to reveal the differentially-expressed genes involved in triterpenoid saponin biosynthesis of Siraitia grosvenorii, 50 DAF(days after flowering)and 70 DAF fruits from different developmental stages were employed to construct subtractive cDNA library by suppression subtractive hybridization(SSH)in this study. Total 641 positive clones were selected randomly and sequenced from the forward-subtracted cDNA library(70 DAF as the tester and 50 DAF as the driver), and 622 high quality sequences were obtained. The rate of the recombination fraction was above 96%. The distribution of inserted fragments ranged from 101 bp and 934 bp,and the average fragment size was 500 bp. Followed by BLASTN and BLASTX for sequences dates,201 ESTs showed no significant matches to any other sequences in NCBI nucleotide sequence database,they were probably novel genes. The other 421 ESTs carried with remarkable identity to proteins with known function in the database, which fell into several functional categories including energy and secondary metabolism, transcription factors,ripening, senescence and pathogen-resistance. Further results indicated that the cDNA library quality were conformed to SSH library standard and could be used in further studies, also would provide reference for studying the differentially expressed genes, exploring molecular mechanism of triterpenoid saponin biosynthesis,and increasing productivity and quality of mogrosides in S. grosvenorii.%以罗汉果授粉后50 d和70 d果实为材料,利用抑制消减杂交技术构建了罗汉果果实在不同生育时期皂苷生物合成相关基因的差减cDNA文库.在正向差减文库(70 d为tester,50 d果实为driver)中随机挑选了641个cDNA阳性克隆测序,得到622条有效序列,重组率在96%以上.外源片段的长度分布在101~934 bp之间,平均长度约500 bp.BLASTN结果显示:201个ESTs序列没有同源序列,可能是新基因;另外421个ESTs序列在核酸数据库中存在同源

  20. Construction of a Sequencing Library from Circulating Cell-Free DNA.

    Science.gov (United States)

    Fang, Nan; Löffert, Dirk; Akinci-Tolun, Rumeysa; Heitz, Katja; Wolf, Alexander

    2016-04-01

    Circulating DNA is cell-free DNA (cfDNA) in serum or plasma that can be used for non-invasive prenatal testing, as well as cancer diagnosis, prognosis, and stratification. High-throughput sequence analysis of the cfDNA with next-generation sequencing technologies has proven to be a highly sensitive and specific method in detecting and characterizing mutations in cancer and other diseases, as well as aneuploidy during pregnancy. This unit describes detailed procedures to extract circulating cfDNA from human serum and plasma and generate sequencing libraries from a wide concentration range of circulating DNA.

  1. 乌金猪基因组文库的构建%Construction of Genomic Library of Wujin Pig

    Institute of Scientific and Technical Information of China (English)

    张永云; 李卫真; 赵素梅; 黄英; 高士争

    2011-01-01

    为探究与我国优良地方猪种乌金猪特性相关的基因机制,拟构建其基因组文库.取乌金猪肝脏组织,提取大小50 kb以上的基因组DNA,利用限制性内切酶Bcl I对基因组DNA进行随机酶切和低熔点琼脂糖电泳方法回收10~23 kb的DNA片段.以EMBL3作为载体,经BamH I酶切和去磷酸化处理后,与上述纯化的目的DNA片段连接,在体外包装成重组噬菌体,重组噬菌体转染宿主菌KW251,构建成乌金猪基因组文库.文库的效价为2.4 x 109 pfu/mL.乌金猪基因组文库的成功构建,为进行其相关功能基因和基因组区域的识别,基因组DNA和调控元件的克隆与功能分析等后续研究奠定了良好的基础.%To identify the genomic mechanisms for specific traits of Wujin pig, a domestic Chinese swine breed, we constructed its genomic library. The genomic DNA larger than 50 kb was extracted from Wujin pig liver tissue and was digested randomly by restriction enzyme Bel I. DNA fragments ranging from 10 to 23 kb were recovered by agarose gel electrophoresis. EMBL3 vector was digested by BamH I, treated with calf intestine alkaline phosphatase, and then liga-ted with purified DNA fragments forementioned. The genomic library of Wujin pig was constructed by packing recombinant DNA in vitro with phage package protein and transecting KW251 host cells. The result showed that the titer of the library was 2. 4 × 109 pfu/mL. The genomic library of Wujin pig could be used for identification of genes and genomic regions of interest, and provide valuable data for further study such as cloning of genomic DNA and functional analysis of regulatory element.

  2. Schistosoma japonicum:construction of phage display antibody library and its application in the immunodiagnosis of infection

    Institute of Scientific and Technical Information of China (English)

    陈代雄; 何蔼; 詹希美; 俞慕华; 雷智刚; 孟锦绣; 李卓雅; 梁瑜; 张瑞琳

    2004-01-01

    Background A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj).Methods A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients.Results Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07×106 individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%.Conc