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Sample records for cdna library selected

  1. 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.

  2. 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.

  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 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.

  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. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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...

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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...

  17. 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%.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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 ...

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

    Institute of Scientific and Technical Information of China (English)

    Jun Shu; Xiaojuan He; Guancheng Li

    2006-01-01

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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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...

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  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 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.

  18. 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.

  19. 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.

  20. Library Materials: Selection and Processing.

    Science.gov (United States)

    Freeman, Michael; And Others

    This script of a slide-tape presentation, which describes the selection and processing of materials for a university library, includes commentary with indicators for specific slide placement. Distinction is made between books and serial publications and the materials are followed from the ordering decision through processing. The role of the…

  1. 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.

  2. 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.

  3. [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.

  4. 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.

  5. 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.

  6. Methods for Selecting Phage Display Antibody Libraries.

    Science.gov (United States)

    Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2016-01-01

    The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

  7. 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.

  8. 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.

  9. [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.

  10. 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.

  11. 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.

  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. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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...

  19. 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.

  20. 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.

  1. 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...

  2. 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...

  3. Isolation of novel non-HLA gene fragments from the hemochromatosis region (6p21. 3) by cDNA hybridization selection

    Energy Technology Data Exchange (ETDEWEB)

    Goei, V.L.; Capossela, A.; Gruen, J.R.; Parimoo, S.; Chu, T.W. (Yale Univ. School of Medicine, New Haven, CT (United States))

    1994-02-01

    It has previously been shown that cDNA hybridization selection can identify and recover novel genes from large cloned genomic DNA such as cosmids or YACs. In an effort to identify candidate genes for hemochromatosis, this technique was applied to a 320-kb YAC containing the HLA-A gene. A short fragment cDNA library derived from human duodenum was selected with the YAC DNA. Ten novel gene fragments were isolated, characterized, and localized on the physical map of the YAC. 39 refs., 4 figs., 3 tabs.

  4. 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.

  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. 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.

  7. 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.

  8. 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.

  9. 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...

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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...

  19. 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.

  20. 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

  1. 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).

  2. 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.

  3. 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.

  4. Selection of antibodies from synthetic antibody libraries.

    Science.gov (United States)

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

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

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    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.

  11. 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.

  12. 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.

  13. 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。

  14. 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.

  15. 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.

  16. [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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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...

  5. [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.

  6. 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.

  7. 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.

  8. Rational principles of compound selection for combinatorial library design.

    Science.gov (United States)

    Tropsha, Alexander; Zheng, Weifan

    2002-03-01

    It is practically impossible in a short period of time to synthesize and test all compounds in any large exhaustive chemical library. We discuss rational approaches to selecting representative subsets of virtual libraries that help direct experimental synthetic efforts for both targeted and diverse library design. For targeted library design, we consider principles based on the similarity to lead molecules. In the case of diverse library design, we discuss algorithms aimed at the selection of both diverse and representative subsets of the entire chemical library space. We illustrate methodologies with several practical examples.

  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. 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

  11. Colony color assay coupled with 5FOA negative selection greatly improves yeast threehybrid library screening efficiency

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The recently developed yeast three-hybrid system is a powerful tool for analyzing RNA-protein interactions in vivo. However, large numbers of false positives are frequently met due to bait RNA-independent activation of the reporter gene in the library screening using this system. In this report, we coupled the colony color assay with the 5FOA (5-fluoroorotic acid) negative selection in the library screening, and found that this coupled method effectively eliminated bait RNA-independent false positives and hence greatly improved library screening efficiency. We used this method successfully in isolation of cDNA of an RNA-binding protein that might play important roles in certain cellular process. This improvement will facilitate the use of the yeast three-hybrid system in analyzing RNA-protein interaction.

  12. 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.

  13. 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.

  14. 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%.

  15. 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.

  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. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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].

  2. 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.

  3. 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.

  4. 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

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

    Science.gov (United States)

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

    2015-12-01

    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.

  6. 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....

  7. Software Migration in Selected University and Special Libraries in Nigeria

    Directory of Open Access Journals (Sweden)

    Benson Oghenevwogaga Adogbeji

    2013-04-01

    Full Text Available The work has surveyed migration in selected University and Special Libraries in Nigeria, which was based on the experience of seven automated libraries in which four have changed from one library software to another. The objective of the study is to ascertain the major consideration in the choice of software by the selected library, to examine the problems encountered and reasons for the change of software. Questionnaire was used in data collection; a total of (70% of the questionnaire were used for analysis. Simple percentage was used to compute statistics of the findings. The study revealed that among the seven libraries, four have changed from one library application software to another while some are still in search of the library software to adopt. This was attributed to factors such as lack of systems analysis before and after embarking on the purchase of the software, lack of maintenance or technical support, limitation of software, absolute nature of the operating systems on which the software run. Recommendations made in the study include systems analysis study of library operations before embarking on automation, providing electricity, setting aside some fund for maintenance among others while it is recommended for the library software developers to endeavor to always develop upgrade that will not wipe not away old data, software that will be compatible with other library software sponsor users group conference among as this will help in automation process in Nigeria libraries.

  8. Presentation of the selected French libraries and the French library system

    Directory of Open Access Journals (Sweden)

    Moja Kotar

    2007-01-01

    Full Text Available The Selected French libraries (Bibliotheque nationale de France, Bibliotheque publique d’information, multimedia library of the Cité des sciences et de l’industrie as well as Paris public libraries are presented in the article. France does not have a union catalogue at the national level, therefore libraries use different platforms for shared cataloguing and compile more union catalogues. According to their needs, French libraries join into consortia for the acquisition of electronic resources, which can be either geographically or thematically delimited or formed by the institutions of the same status. The author believes that the Slovenian library network works well considering much smaller budget for culture and higher education in comparison with France. To improve its performance, more funds would have to be allocated and higher reputation of the library profession achieved, comparable to the situation in France. Digitization of resources is the area where the Slovenian librarianship lags most behind the French one.

  9. 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

  10. [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.

  11. 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

  12. 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

  13. 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。

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. Artificial promoter libraries for selected organisms and promoters derived from such libraries

    DEFF Research Database (Denmark)

    1998-01-01

    An artificial promoter library for a selected organism or group of organisms is constructed as a mixture of double stranded DNA fragments, the sense strands of which comprise at least two consensus sequences of efficient promoters from said organism or group of organisms, or parts thereof...... imparting a specific regulatory feature, such as activation by a change in the growth conditions, to the promoters of the library. Further, they may have a sequence comprising one or more recognition sites for restriction endonucleases added to one of or both their ends. The selected organism or group...... in eukaryotes said consensus sequences should comprise a TATA box and at least one upstream activation sequence (UAS). Such artificial promoter libraries can be used i.a. for optimizing the expression of specific genes in various selected organisms....

  19. ISOLATION OF ENDOTOXIN-SPECIFIC ANTIBODIES BY SELECTION OF AN SINGLE CHAIN PHAGE ANTIBODY LIBRARY

    Institute of Scientific and Technical Information of China (English)

    陈鸣; 俞丽丽; 张雪; 府伟灵

    2002-01-01

    Objective: To isolate murine anti endotoxin single chain phage antibody from a constructed library. Methods: Total RNA was firstly extracted from murine splenic cells and mRNA was reverse-transcribed into cDNA. Then the designed primers were used to amplify the variable region genes of the heavy and light chain (VH, VL) with polymerase chain reaction. The linker was used to assemble the VH and VL into ScFv, and the NotI and SfiI restriction enzymes were used to digest the ScFv in order to ligate into the pCANTAB5E phagemid vector that was already digested with the same restriction enzymes. The ligated vector was then introduced into competent E.coli TG1 cells to construct a single-chain phage antibody library. After rescued with M13KO7 helper phage, recombinant phages displaying ScFv fragments were harvested from the supernatant and selected with endotoxin. The enriched positive clones were reinfected into TG1 cells. Finally, 190 clones were randomly selected to detect the anti endotoxin antibody with indirect ELISA. Results: The titer of anti endotoxin in murine sera was 1:12,800. The concentration of total RNA was 12.38 μg/ml. 1.9×107 clones were obtained after transformed into TG1. 3×104 colonies were gotten after one round panning. Two positive colonies were confirmed with indirect ELISA among 190 randomly selected colonies. Conclusion: A 1.9×107 murine anti endotoxin single chain phage antibody library was successfully constructed. Two anti endotoxin antibodies were obtained from the library.

  20. CLA Selection Guidelines: How to Choose Materials for Parish and School Libraries.

    Science.gov (United States)

    Weathers, Barbara

    1991-01-01

    Reviews selection guidelines suggested by the Catholic Library Association (CLA) for parish and school libraries. Highlights include who develops the selection policy; American Library Association selection guidelines for school libraries; criteria for both print and nonprint materials; how to handle censorship challenges; and issues concerning…

  1. 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

  2. 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

  3. Novel selection methods for DNA-encoded chemical libraries.

    Science.gov (United States)

    Chan, Alix I; McGregor, Lynn M; Liu, David R

    2015-06-01

    Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

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

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    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.

  11. 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

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

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    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.

  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. Halk Kütüphanelerinde Koleksiyon Seçimi / Selection Process for the Library Collections of Public Libraries

    Directory of Open Access Journals (Sweden)

    Dilek Köprülü

    2006-04-01

    Full Text Available The task of collection development of public libraries requires the fulfilment of a selection process. In this context, main characteristics of the collection of public libraries, assessment of the user’s information needs, the selection process and its policy of implementation, and the related responsibilities of the professional librarians were examined. Finally, the selection process undertaken by public libraries in Turkey was examined, and certain suggestions were forwarded.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. Standardization in library and information science in selected European countries

    Science.gov (United States)

    Matysek, Anna

    2015-02-01

    Standardization plays an important role in library and information science (LIS), because it gives rules to identify, classify, access, select, exploit, communicate, exchange and preserve information. Standards are developed by national, European and international organizations. The objective of the study is to present the situation of standardization in library and information science in the countries that joined the European Union in 2004. The research covered Technical Committees that take the problems of LIS, their cooperation with European Committee for Standardization (CEN) and International Organization for Standardization (ISO). The second part of the study is an analysis of LIS standards published in the last 10 years. Data on published documents were gathered from online standards directories. The documents were searched using International Classification for Standards. Retrieved standards were analyzed for their origin and status. The research illustrates the changes in the national standardization, most popular topics and the growing importance of international cooperation in standardization.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Hyperopt: a Python library for model selection and hyperparameter optimization

    Science.gov (United States)

    Bergstra, James; Komer, Brent; Eliasmith, Chris; Yamins, Dan; Cox, David D.

    2015-01-01

    Sequential model-based optimization (also known as Bayesian optimization) is one of the most efficient methods (per function evaluation) of function minimization. This efficiency makes it appropriate for optimizing the hyperparameters of machine learning algorithms that are slow to train. The Hyperopt library provides algorithms and parallelization infrastructure for performing hyperparameter optimization (model selection) in Python. This paper presents an introductory tutorial on the usage of the Hyperopt library, including the description of search spaces, minimization (in serial and parallel), and the analysis of the results collected in the course of minimization. This paper also gives an overview of Hyperopt-Sklearn, a software project that provides automatic algorithm configuration of the Scikit-learn machine learning library. Following Auto-Weka, we take the view that the choice of classifier and even the choice of preprocessing module can be taken together to represent a single large hyperparameter optimization problem. We use Hyperopt to define a search space that encompasses many standard components (e.g. SVM, RF, KNN, PCA, TFIDF) and common patterns of composing them together. We demonstrate, using search algorithms in Hyperopt and standard benchmarking data sets (MNIST, 20-newsgroups, convex shapes), that searching this space is practical and effective. In particular, we improve on best-known scores for the model space for both MNIST and convex shapes. The paper closes with some discussion of ongoing and future work.

  14. 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.

  15. 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

  16. An expressed sequence tag (EST library for Drosophila serrata, a model system for sexual selection and climatic adaptation studies

    Directory of Open Access Journals (Sweden)

    McGraw Elizabeth A

    2009-01-01

    Full Text Available Abstract Background The native Australian fly Drosophila serrata belongs to the highly speciose montium subgroup of the melanogaster species group. It has recently emerged as an excellent model system with which to address a number of important questions, including the evolution of traits under sexual selection and traits involved in climatic adaptation along latitudinal gradients. Understanding the molecular genetic basis of such traits has been limited by a lack of genomic resources for this species. Here, we present the first expressed sequence tag (EST collection for D. serrata that will enable the identification of genes underlying sexually-selected phenotypes and physiological responses to environmental change and may help resolve controversial phylogenetic relationships within the montium subgroup. Results A normalized cDNA library was constructed from whole fly bodies at several developmental stages, including larvae and adults. Assembly of 11,616 clones sequenced from the 3' end allowed us to identify 6,607 unique contigs, of which at least 90% encoded peptides. Partial transcripts were discovered from a variety of genes of evolutionary interest by BLASTing contigs against the 12 Drosophila genomes currently sequenced. By incorporating into the cDNA library multiple individuals from populations spanning a large portion of the geographical range of D. serrata, we were able to identify 11,057 putative single nucleotide polymorphisms (SNPs, with 278 different contigs having at least one "double hit" SNP that is highly likely to be a real polymorphism. At least 394 EST-associated microsatellite markers, representing 355 different contigs, were also found, providing an additional set of genetic markers. The assembled EST library is available online at http://www.chenowethlab.org/serrata/index.cgi. Conclusion We have provided the first gene collection and largest set of polymorphic genetic markers, to date, for the fly D. serrata. The EST

  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. 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.

  19. 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.

  20. SELECTION OF NEW EPITOPES FROM MONOVALENT DISPLAYED PHAGE OCTAPEPTIDE LIBRARY

    Institute of Scientific and Technical Information of China (English)

    李全喜; 王琰; 李竞; 王雅明; 徐建军; 王力民; 董志伟

    1998-01-01

    A library of 2×l07 random oetspaptides was constructed by use of phegemid-based monovaient phage display system. The randomly synthesized degenerated oilgodeoxyribonucleotides (oligos) were fused to the truncated gⅢ (p210-p408). Sequeraze analysis of 11 randomly chosen clones suggested that the degenerated inserts and its deduced amino acid (an) sequences are randomly distributed. The library was used to select binding paptides to the morroeloncl antlhody (mAb) 9E10, which recognizes a continuous decapaptide epitope of denatured human c-myc protein. After four to five rounds of panning, most of the eluted clones could bind to 9E10. Sequerlce analysis of the selected positive clones indlcated that the binding sequences could fall into two chsses, one class (clone 1) shares a consensus motif, ISE x x L, with c-mire decapeprider and the sequences of the other class are entirely different. The binding of both classes to 9E10 could be specifically lnhlhited by froe c-myc deeapeptide. The immunogenlcitF cff the phage peptide was further investigsted h5, construction of multivalent displayed phage peptides and immunization of animals with or without adjuvant. ELISA and competitive ELISA showed that anti-serum from both mice and rabbit immunized with either done could bind to the original antigen, c-myc decapeptide. These results denote that in spite of the dissimilarity of the selected psptides with c-myc decapeptide, they are capable of inducing similar immune respones in vivo, thus actually mimicking the antigen epitope.

  1. 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.

  2. Generation and selection of immunized Fab phage display library against human B cell lymphoma

    Institute of Scientific and Technical Information of China (English)

    Yongmei Shen; Xiaochun Yang; Ningzheng Dong; Xiaofang Xie; Xia Bai; Yizhen Shi

    2007-01-01

    The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production of monoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the K light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94×107. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (VH) and variable light domains (VL) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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

    ( blue/white) was determined , and 24 clones were picked randomly from the library for screening size of cDNA inserts through PCR reaction. By identification, the primary constructed cDNA library contained 1.021 × 106 independent clones. The titer of the cDNA library was estimated as 1. 078 × 106/mL. the recombinant rate was above 94.71 %. The inserts varied from 0.7 to 3.0 kb with average size was about 1500 bp. 24 clones were sequenced randomly-selected from each lihrary and 18 contigs were acquired and analyzed by BLASTx. The resulta indicated that 15 contigs had the homologous gene information which 10 were full-length cDNA with 66. 7 % of full-ratio. These results shoWed that the full-length cDNA library meet the requirement of a standard full-length cDNA library. and it could lay a strong basis for subsequent work of clone and selection of some immune functional genes.

  9. 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

  10. 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。

  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. 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

  13. 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.

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

    Institute of Scientific and Technical Information of China (English)

    王燕红; 李兵; 王东; 朱莎; 赵华强; 卫正国; 沈卫德

    2008-01-01

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

  15. Selecting a Relational Database Management System for Library Automation Systems.

    Science.gov (United States)

    Shekhel, Alex; O'Brien, Mike

    1989-01-01

    Describes the evaluation of four relational database management systems (RDBMSs) (Informix Turbo, Oracle 6.0 TPS, Unify 2000 and Relational Technology's Ingres 5.0) to determine which is best suited for library automation. The evaluation criteria used to develop a benchmark specifically designed to test RDBMSs for libraries are discussed. (CLB)

  16. 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

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

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Yong Li; You-Yong Lu

    2002-01-01

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

  19. 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.

  20. 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.

  1. 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。

  2. 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

  3. 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

  4. Selection of DNA-encoded small molecule libraries against unmodified and non-immobilized protein targets.

    Science.gov (United States)

    Zhao, Peng; Chen, Zitian; Li, Yizhou; Sun, Dawei; Gao, Yuan; Huang, Yanyi; Li, Xiaoyu

    2014-09-15

    The selection of DNA-encoded libraries against biological targets has become an important discovery method in chemical biology and drug discovery, but the requirement of modified and immobilized targets remains a significant disadvantage. With a terminal protection strategy and ligand-induced photo-crosslinking, we show that iterated selections of DNA-encoded libraries can be realized with unmodified and non-immobilized protein targets.

  5. Online Information. Selected Databases at the New York State Library.

    Science.gov (United States)

    New York State Library, Albany. Database Services.

    This brochure describes the online information services at the New York State Library, which has online access to over 250 databases covering a broad range of subject areas, including current events, law, science, medicine, public affairs, grants, business, computer technology, education, social welfare, and humanities. Many of these databases are…

  6. 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.

  7. Design, synthesis and selection of DNA-encoded small-molecule libraries.

    Science.gov (United States)

    Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

    2009-09-01

    Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

  8. 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...

  9. 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

  10. 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.

  11. 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

  12. Sequencing and identification of expressed Schistosoma mansoni genes by random selection of cDNA clones from a directional library

    Directory of Open Access Journals (Sweden)

    Glória R. Franco

    1995-04-01

    Full Text Available We have initiated a gene discovery program in Schistosoma mansoni based on the technique of Expressed Sequence Tags (ESTs, i.e. partial sequences of cDNAs obtained from single passes in automatic DNA sequencers. ESTs can be used to identify genese onf the basis of their homology whith sequences from other species deposited in DNA or protein databases. Trasncripts with sequences without matches in teh databases may represent novel parasite-specific genes. This approach has shown to be very efficient and in less than two years a broad range of novel genes has already been ascertained, more than doubling the number of known S. mansoni genes.

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

    Institute of Scientific and Technical Information of China (English)

    钟云; 姜波; 易干军; 曾继吾; 王辉; 蒋侬辉; 周碧容

    2012-01-01

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

  14. 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。

  15. 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.

  16. 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

  17. 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.

  18. Selecting a Web Content Management System for an Academic Library Website

    OpenAIRE

    Elizabeth L. Black

    2011-01-01

    This article describes the selection of a web content management system (CMS) at the Ohio State University Libraries. The author outlines the need for a CMS, describes the system requirements to support a large distributed content model and shares the CMS trial method used, which directly included content provider feedback side-by-side with the technical experts. The selected CMS is briefly described.

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

    DEFF Research Database (Denmark)

    Kaufman, J; Salomonsen, J; Skjødt, K

    1989-01-01

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

  20. Computer selection of oligonucleotide probes from amino acid sequences for use in gene library screening.

    Science.gov (United States)

    Yang, J H; Ye, J H; Wallace, D C

    1984-01-11

    We present a computer program, FINPROBE, which utilizes known amino acid sequence data to deduce minimum redundancy oligonucleotide probes for use in screening cDNA or genomic libraries or in primer extension. The user enters the amino acid sequence of interest, the desired probe length, the number of probes sought, and the constraints on oligonucleotide synthesis. The computer generates a table of possible probes listed in increasing order of redundancy and provides the location of each probe in the protein and mRNA coding sequence. Activation of a next function provides the amino acid and mRNA sequences of each probe of interest as well as the complementary sequence and the minimum dissociation temperature of the probe. A final routine prints out the amino acid sequence of the protein in parallel with the mRNA sequence listing all possible codons for each amino acid.

  1. 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...

  2. 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...

  3. 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.

  4. 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

  5. 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.

  6. Selection of library services by post-registration nursing, midwifery and health visiting students.

    Science.gov (United States)

    Yeoh, J; Morrissey, C

    1996-06-01

    This paper describes a small-scale survey undertaken with 300 nurses, midwives and health visitors on post-registration courses and registered for the ENB Higher Award. The aim was threefold: first to examine reasons for electing to use particular libraries; second, the relationship between workplace, home, teaching site and choice of library services; and third, the use of library enquiry services. There was a 56% response rate to the postal questionnaire and results indicated that the key factor in selecting a library was availability of resources rather than convenience to home or workplace. The third largest group of respondents worked in community organizations with little or no on-site access to resources and were dependent on access to other libraries. Most who used the library for literature searching needed to seek help in spite of preliminary user education sessions. Similar levels of assistance were needed to locate journals and use the computerized book catalogue. The paper recommends explicit information on strategic entry points, necessity of a broad central collection, integration of information retrieval into post-registration courses, use of self-study packs, better trust/university liaison and recognition of the need for resource transfer from teaching to learning.

  7. A Survey of User Attitudes Toward Selected Services Offered by the OISE Library.

    Science.gov (United States)

    Ontario Inst. for Studies in Education, Toronto.

    A survey of faculty and students to assess user needs and satisfaction with collections and selected library services is described and the resulting data are reported. Questionnaire construction, sampling and distribution, and response rate and factors are discussed. Data analysis is broken into major categories, which include responses to general…

  8. The International Federation of Library Associations. A Selected List of References.

    Science.gov (United States)

    Cambio, Edward P., Comp.

    Prepared in anticipation of the 40th session of the General Council of the International Federation of Library Associations (IFLA), this bibliography cites materials issued by or under the auspices of the federation and its council, sections, and committees, as well as selected works about IFLA and its various subdivisions. The citations are…

  9. 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.

  10. 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.

  11. Spectral Band Selection for Urban Material Classification Using Hyperspectral Libraries

    Science.gov (United States)

    Le Bris, A.; Chehata, N.; Briottet, X.; Paparoditis, N.

    2016-06-01

    In urban areas, information concerning very high resolution land cover and especially material maps are necessary for several city modelling or monitoring applications. That is to say, knowledge concerning the roofing materials or the different kinds of ground areas is required. Airborne remote sensing techniques appear to be convenient for providing such information at a large scale. However, results obtained using most traditional processing methods based on usual red-green-blue-near infrared multispectral images remain limited for such applications. A possible way to improve classification results is to enhance the imagery spectral resolution using superspectral or hyperspectral sensors. In this study, it is intended to design a superspectral sensor dedicated to urban materials classification and this work particularly focused on the selection of the optimal spectral band subsets for such sensor. First, reflectance spectral signatures of urban materials were collected from 7 spectral libraires. Then, spectral optimization was performed using this data set. The band selection workflow included two steps, optimising first the number of spectral bands using an incremental method and then examining several possible optimised band subsets using a stochastic algorithm. The same wrapper relevance criterion relying on a confidence measure of Random Forests classifier was used at both steps. To cope with the limited number of available spectra for several classes, additional synthetic spectra were generated from the collection of reference spectra: intra-class variability was simulated by multiplying reference spectra by a random coefficient. At the end, selected band subsets were evaluated considering the classification quality reached using a rbf svm classifier. It was confirmed that a limited band subset was sufficient to classify common urban materials. The important contribution of bands from the Short Wave Infra-Red (SWIR) spectral domain (1000-2400 nm) to material

  12. Selecting, Acquiring, and Using Small Molecule Libraries for High-Throughput Screening.

    Science.gov (United States)

    Dandapani, Sivaraman; Rosse, Gerard; Southall, Noel; Salvino, Joseph M; Thomas, Craig J

    The selection, acquisition and use of high quality small molecule libraries for screening is an essential aspect of drug discovery and chemical biology programs. Screening libraries continue to evolve as researchers gain a greater appreciation of the suitability of small molecules for specific biological targets, processes and environments. The decisions surrounding the make-up of any given small molecule library is informed by a multitude of variables and opinions vary on best-practices. The fitness of any collection relies upon upfront filtering to avoiding problematic compounds, assess appropriate physicochemical properties, install the ideal level of structural uniqueness and determine the desired extent of molecular complexity. These criteria are under constant evaluation and revision as academic and industrial organizations seek out collections that yield ever improving results from their screening portfolios. Practical questions including cost, compound management, screening sophistication and assay objective also play a significant role in the choice of library composition. This overview attempts to offer advice to all organizations engaged in small molecule screening based upon current best practices and theoretical considerations in library selection and acquisition.

  13. Selecting a Web Content Management System for an Academic Library Website

    Directory of Open Access Journals (Sweden)

    Elizabeth L. Black

    2011-12-01

    Full Text Available This article describes the selection of a web content management system (CMS at the Ohio State University Libraries. The author outlines the need for a CMS, describes the system requirements to support a large distributed content model and shares the CMS trial method used, which directly included content provider feedback side-by-side with the technical experts. The selected CMS is briefly described.

  14. Theoretical principles of in vitro selection using combinatorial nucleic acid libraries.

    Science.gov (United States)

    Vant-Hull, B; Gold, L; Zichi, D A

    2000-02-01

    A new paradigm for drug discovery and biological research has developed from technologies that integrate combinatorial chemistry with rounds of selection and amplification, a technique called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). This overview unit discusses nucleic acid libraries that can be used, affinity probability distributions, an equilibrium model for SELEX, and optimal conditions including concentrations and signal-to-noise ratios.

  15. 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.

  16. 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.

  17. 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

  18. An Ontology-based Context Aware System for Selective Dissemination of Information in a Digital Library

    CERN Document Server

    De Giusti, Marisa R; Vosou, Agustín; Martínez, Juan P

    2010-01-01

    Users of Institutional Repositories and Digital Libraries are known by their needs for very specific information about one or more subjects. To characterize users profiles and offer them new documents and resources is one of the main challenges of today's libraries. In this paper, a Selective Dissemination of Information service is described, which proposes an Ontology-based Context Aware system for identifying user's context (research subjects, work team, areas of interest). This system enables librarians to broaden users profiles beyond the information that users have introduced by hand (such as institution, age and language). The system requires a context retrieval layer to capture user information and behavior, and an inference engine to support context inference from many information sources (selected documents and users' queries).

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

    Directory of Open Access Journals (Sweden)

    Osamu Kitahara

    2002-01-01

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

  20. Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): application to sequencing 6.5 kb genome segment of hantavirus strain B-1.

    Science.gov (United States)

    Isegawa, Y; Sheng, J; Sokawa, Y; Yamanishi, K; Nakagomi, O; Ueda, S

    1992-12-01

    A method, referred to as cassette-ligation mediated polymerase chain reaction (PCR), has been developed to permit selective and specific amplification of cDNA sequence from total cellular RNA. This technique comprises (i) digestion of cDNA with multiple restriction enzymes, (ii) ligation of cleavage products to double-stranded DNA cassettes possessing a corresponding restriction site and (iii) amplification of cassette-ligated restriction fragments containing a short, known sequence (but not all the other ligation products) by PCR using the specific and cassette primers; the specific primer is designed to prime synthesis from the known sequence of the cDNA whereas the cassette primer anneals to one strand of the cassette. Sequencing from the cassette primer provides information to design a new primer for the next walking step. The amplified cDNA fragments are often larger than the maximum DNA fragments (500-600 bp) that can be sequenced without the need of synthesizing internal sequencing primer. Each of such large cDNA fragments is dissected into smaller DNA fragments by repeating cassette-ligation mediated PCR exploiting different restriction sites and different sets of cassette primers. This dissection process reduces the number of specific primers to a minimum, thereby increasing the speed of sequencing and minimizing the overall cost. We have successfully applied this cDNA walking and sequencing by the cassette-ligation mediated PCR to the sequencing of an entire 6.5 kb genome segment of hantavirus strain B-1.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Selective dissemination of health information: Bulletins of the National Medical Library of Cuba

    Directory of Open Access Journals (Sweden)

    Sonia Santana Arroyo

    2016-10-01

    Full Text Available The National Medical Library of Cuba (BMN has the mission to ensure the provision of scientific and technology information to the community of professionals that make up the healthcare sector in the country, primarily the Ministry of Public Health, identified as prioritized user of BMN, in order to elevate the quality of care, research, management and teaching, as well as to encourage the finding of new solutions that enable to face health problems in Cuba and in the world. This article aims to describe the making process of the bulletins: Bibliomed, Bibliomed Supplemento, BiblioDir and Factográfico de Salud, which are offered by specialists from Selective Dissemination of Information Service that resides in the BMN area of Customer Service, in order to support the decision making of health managers of Cuba Ministry of Public Health. For this purpose, the paper details scope, content, sections, team responsibility, procedures for preparing and processing the bulletins. Most used sources of information and skills of staff are also presented. Such Informative scientific work strengthen BMN as a paradigm of the cuban medical libraries network, transforming the pattern of a traditional library into a modern library, characterized by an effective knowledge management and development processes in virtual environments.

  2. Access or Censorhip? Libraries and Pornography. An Annotated Selective Bibliography on the Theme of the LACUNY 1987 Institute.

    Science.gov (United States)

    Apterbach, Evelyn, Ed.; And Others

    This bibliography lists selected publications both from areas that relate pornography as a concept to libraries and, by extension, those that relate it to censorship. Introductory material includes listings of the Library Association of the City University of New York (LACUNY) 1987 Institute committee and emcees and LACUNY officers, and the 1987…

  3. Decision-Making in the Selection of Science Library Materials for Higher Education: Empirical Findings and Guidelines.

    Science.gov (United States)

    Farina, Alfred J.; Snyder, Monroe B.

    The project which resulted in this report was a study of decision making in the selection of science library materials in college and university libraries. Questionnaires, interviews and objective collection descriptions were used to collect information in the fields of chemistry, physics and biology. Part I is an analytic description of the…

  4. An efficient strategy for cell-based antibody library selection using an integrated vector system

    Directory of Open Access Journals (Sweden)

    Yoon Hyerim

    2012-09-01

    Full Text Available Abstract Background Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9. Results A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9. Conclusions This study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets.

  5. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    Science.gov (United States)

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants.

  6. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.

    Science.gov (United States)

    James, P; Halladay, J; Craig, E A

    1996-12-01

    The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.

  7. 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.

  8. 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.

  9. Partial protection induced by phage library-selected peptides mimicking epitopes of Schistosoma japonicum

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective To obtain peptide mimicking epitopes of Schistosoma japonicum (S.japonicum) through screening of a phage peptide library and to test their potential for induction of protection. Methods S.japonicum infected sera from Microtus fortis (IMFS) and normal sera from Microtus fortis (NMFS) were used respectively to screen a 12-mers random peptide library by testing the reactivity of anti-S.japonicum serum with the phagotopes. After three rounds of biopanning, the pooled phages were used to immunize mice, after which challenge infection was performed. Results Of 12 randomly picked clones, 10 clones selected using IMFS and 7 clones selected using NMFS were shown to be antigenic. Significant reduction in adult worms (22.6%) and a high reduction (68.9%) in liver eggs were achieved following immunization with phages screened with IMFS. However, no protection was elicited by those selected with NMFS. Conclusion The results show that the phagotopes are both antigenic and immunogenic, suggesting a potential use of phage displayed peptide as novel vaccines against S. japonicum.

  10. Methods to determine the transcriptomes of trypanosomes in mixtures with mammalian cells: the effects of parasite purification and selective cDNA amplification.

    Directory of Open Access Journals (Sweden)

    Julius Mulindwa

    2014-04-01

    Full Text Available Patterns of gene expression in cultured Trypanosoma brucei bloodstream and procyclic forms have been extensively characterized, and some comparisons have been made with trypanosomes grown to high parasitaemias in laboratory rodents. We do not know, however, to what extent these transcriptomes resemble those in infected Tsetse flies - or in humans or cattle, where parasitaemias are substantially lower. For clinical and field samples it is difficult to characterize parasite gene expression because of the large excess of host cell RNA. We have here examined two potential solutions to this problem for bloodstream form trypanosomes, assaying transcriptomes by high throughput cDNA sequencing (RNASeq. We first purified the parasites from blood of infected rats. We found that a red blood cell lysis procedure affected the transcriptome substantially more than purification using a DEAE cellulose column, but that too introduced significant distortions and variability. As an alternative, we specifically amplified parasite sequences from a mixture containing a 1000-fold excess of human RNA. We first purified polyadenylated RNA, then made trypanosome-specific cDNA by priming with a spliced leader primer. Finally, the cDNA was amplified using nested primers. The amplification procedure was able to produce samples in which 20% of sequence reads mapped to the trypanosome genome. Synthesis of the second cDNA strand with a spliced leader primer, followed by amplification, is sufficiently reproducible to allow comparison of different samples so long as they are all treated in the same way. However, SL priming distorted the abundances of the cDNA products and definitely cannot be used, by itself, to measure absolute mRNA levels. The amplification method might be suitable for clinical samples with low parasitaemias, and could also be adapted for other Kinetoplastids and to samples from infected vectors.

  11. 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.

  12. Selection of trkB-binding peptides from a phage-displayed random peptide library

    Institute of Scientific and Technical Information of China (English)

    马仲才; 吴晓兰; 曹明媚; 潘卫; 朱分禄; 陈景山; 戚中田

    2003-01-01

    Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurodegenerative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to permeate blood-brain barrier. To develop low-molecular-weight BDNF-like peptides, we selected a phage-displayed random peptide library using trkB expressed on NIH 3T3 cells as target in the study. With the strategy of peptide library incubation with NIH 3T3 cells and competitive elution with 1 υg/mL of BDNF in the last round of selection, the specific phages able to bind to the natural conformation of trkB and antagonize BDNF binding to trkB were enriched effectively. Five trkB-binding peptides were obtained, in which a core sequence of CRA/TXφXXφXXC (X represents the random amino acids, φ represents T, L or I) was identified. The BDNF-like activity of these five peptides displayed on phages was not observed, though all of them antagonized the activity of BDNF in a dose-dependent manner. Similar results were obtained with the synthetic peptide of C1 clone, indicating that the 5 phage-derived peptides were trkB antagonists. These low-molecular-weight antagonists of trkB may be of potential application in the treatment of neuroblastoma and chronic pain. Meanwhile, the obtained core sequence also could be used as the base to construct the secondary phage-displayed peptide library for further development of small peptides mimicking BDNF activity.

  13. Targeting Phosphatidylserine on Apoptotic Cells with Phages and Peptides Selected from a Bacteriophage Display Library

    Directory of Open Access Journals (Sweden)

    Ruping Shao

    2007-11-01

    Full Text Available Phosphatidylserine (PS is a well-characterized biomarker for apoptosis. Ligands that bind to PS can be used for noninvasive imaging of therapy-induced cell death, particularly apoptosis. In this study, we screened a random 12-mer peptide phage library on liposomes prepared from PS. One clone displaying the peptide SVSVGMKPSPRP (designated as PS3-10 bound to PS approximately 4-fold better than its binding to phosphatidylcholine and 18-fold better than to bovine serum albumin in a solid-phase binding assay. In addition, the binding of the corresponding PS3-10 peptide to PS was significantly higher than that of a scrambled peptide. PS3-10 phages, but not a control 4-2-2 phage, bound to aged red blood cells that had PS exposed on their surface. Binding of PS3-10 phages and PS3-10 peptide to TRAIL-induced apoptotic DLD1 cells was 3.2 and 5.4 times higher than their binding to untreated viable cells, respectively. Significantly, immunohistochemical staining confirmed selective binding of PS3-10 phages to apoptotic cells. Our data suggest that panning of phage display libraries may allow the selection of suitable peptide ligands for apoptotic cells and that PS3-10 peptide may serve as a template for further development of molecular probes for in vitro and in vivo imaging of apoptosis.

  14. A novel phage-library-selected peptide inhibits human TNF-α binding to its receptors.

    Science.gov (United States)

    Brunetti, Jlenia; Lelli, Barbara; Scali, Silvia; Falciani, Chiara; Bracci, Luisa; Pini, Alessandro

    2014-06-03

    We report the identification of a new human tumor necrosis factor-alpha (TNF-α) specific peptide selected by competitive panning of a phage library. Competitive elution of phages was obtained using the monoclonal antibody adalimumab, which neutralizes pro-inflammatory processes caused by over-production of TNF-α in vivo, and is used to treat severe symptoms of rheumatoid arthritis. The selected peptide was synthesized in monomeric and branched form and analyzed for binding to TNF-α and competition with adalimumab and TNF-α receptors. Results of competition with TNF-α receptors in surface plasmon resonance and melanoma cells expressing both TNF receptors make the peptide a candidate compound for the development of a novel anti-TNF-α drug.

  15. Censorship or Selection? Academic Library Holdings of the Top Ten Most Challenged Books of 2007

    Science.gov (United States)

    Best, Rickey

    2010-01-01

    Academic libraries often serve as the protectors of challenged books. Pressure on public libraries to remove controversial works can and often does result in the restriction of access or removal of the work from those libraries shelves. Academic libraries, however, operate with a stronger sense of academic and intellectual freedom. To analyze how…

  16. Collection-based analysis of selected medical libraries in the Philippines using Doody’s Core Titles

    Science.gov (United States)

    Torres, Efren

    2017-01-01

    Objectives This study assessed the book collection of five selected medical libraries in the Philippines, based on Doodys’ Essential Purchase List for basic sciences and clinical medicine, to compare the match and non-match titles among libraries, to determine the strong and weak disciplines of each library, and to explore the factors that contributed to the percentage of match and non-match titles. Method List checking was employed as the method of research. Results Among the medical libraries, De La Salle Health Sciences Institute and University of Santo Tomas had the highest percentage of match titles, whereas Ateneo School of Medicine and Public Health had the lowest percentage of match titles. University of the Philippines Manila had the highest percentage of near-match titles. Conclusion De La Salle Health Sciences Institute and University of Santo Tomas had sound medical collections based on Doody’s Core Titles. Collectively, the medical libraries shared common collection development priorities, as evidenced by similarities in strong areas. Library budget and the role of the library director in book selection were among the factors that could contribute to a high percentage of match titles. PMID:28096742

  17. 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

  18. Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

    Science.gov (United States)

    Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

    1997-02-01

    Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

  19. Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples.

    Science.gov (United States)

    Uyaguari-Diaz, Miguel I; Slobodan, Jared R; Nesbitt, Matthew J; Croxen, Matthew A; Isaac-Renton, Judith; Prystajecky, Natalie A; Tang, Patrick

    2015-04-17

    Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

  20. 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

  1. 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.

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

    Science.gov (United States)

    Rinaldy, A R; Steiner, M S

    1999-11-01

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

  3. Detection and isolation of selected genes of interest from metagenomic libraries by a DNA microarray approach.

    Science.gov (United States)

    Pathak, Gopal P; Gärtner, Wolfgang

    2010-01-01

    A DNA microarray-based approach is described for screening metagenomic libraries for the presence of selected genes. The protocol is exemplified for the identification of flavin-binding, blue-light-sensitive biological photoreceptors (BL), based on a homology search in already sequenced, annotated genomes. The microarray carried 149 different 54-mer oligonucleotides, derived from consensus sequences of BL photoreceptors. The array could readily identify targets carrying 4% sequence mismatch, and allowed unambiguous identification of a positive cosmid clone of as little as 10 ng against a background of 25 μg of cosmid DNA. The protocol allows screening up to 1,200 library clones in concentrations as low as ca. 20 ng, each with a ca. 40 kb insert size readily in a single batch. Calibration and control conditions are outlined. This protocol, when applied to the thermophilic fraction of a soil sample, yielded the identification and functional characterization of a novel, BL-encoding gene that showed a 58% similarity to a known, BL-encoding gene from Kineococcus radiotolerans SRS30216 (similarity values refer to the respective LOV domains).

  4. 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...

  5. 人胰腺细胞cDNA 文库中丙型肝炎病毒NS4B 结合蛋白基因的筛选%Screening of proteins binding to hepatitis C virus NS4B protein from human pancreas cDNA library

    Institute of Scientific and Technical Information of China (English)

    温少芳; 张锦前; 孙荣华; 李卓; 高萍; 王琦; 刘顺爱; 成军

    2011-01-01

    Objective To screen proteins from human pancreas cDNA library , which interact with the protein coded hy hepatitis C virus ( HCV) NS4B. Methods The reconstructed plasmid pGBKT7-NS4B was transformed into yeast strain AH109 and identified. The transformed AH109 ( pGBKT7-NS4B) mated with Y187 ( plasmids of pancreatic cDNA library ) . The diploid yeast cells were plated on nutrient deficiency medium containing X-α-gal for selecting. The plasmids in diploid yeast cells were extracted and electrotransformed into E. c.oli DH5a. The plasmids in DH5α were extracted, sequenced and blasted.Results Seven proteins interacting with HCV NS4B were found . such as CDK5RAP3 , colipase , pancreatic stone protein ,lactoprotein . elastase2A , chymotrypsin and bile salt-stimulated esterase . Conclusions Some of the eight pancreatic proteins may be related with metaholisms of glucose and lipid .%目的 从人胰腺细胞cDNA 文库中,采用酵母双杂交方法 筛选HCV NS4B 包膜蛋白的相互作用蛋白.方法 人胰腺细胞cDNA 文库先进行扩增,随后纯化及鉴定,然后将鉴定好的人胰腺细胞cDNA 文库质粒转化入酵母菌株Y187.构建pGBKT7-NS4B 作为诱饵质粒,随之转化酵母菌株AH109,用色氨酸缺陷型培养基(SD/-Trp)筛选阳性菌落.配合两种重组酵母菌株AH109 与Y187,用四缺培养基及X-α-gal 筛选蓝色酵母菌落,提取相应质粒,电转化感受态菌DH5α后再提取质粒,测序仪测序,结果 使用PUBMED 进行序列比对.结果 成功构建pGBKT7-HCV NS4B 重组质粒,并从人胰腺cDNA 文库中筛选出7 种与HCV NS4B 蛋白相结合的蛋白基因,包括CDK5RAP3、辅脂肪酶、胰石蛋白、凝乳蛋白、弹性蛋白酶2A、糜蛋白酶和胆盐刺激酯酶.结论 HCV NS4B 蛋白可能通过与所筛选出前述蛋白中参与代谢过程的相关蛋白结合,影响糖、脂类代谢过程.

  6. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality

    DEFF Research Database (Denmark)

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J

    2016-01-01

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform...... reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples...

  7. Identification of peptides that selectively bind to myoglobin by biopanning of phage displayed-peptide library.

    Science.gov (United States)

    Padmanaban, Guruprasath; Park, Hyekyung; Choi, Ji Suk; Cho, Yong-Woo; Kang, Woong Chol; Moon, Chan-Il; Kim, In-San; Lee, Byung-Heon

    2014-10-10

    Biopanning of phage displayed-peptide library was performed against myoglobin, a marker for the early assessment of acute myocardial infarction (AMI), to identify peptides that selectively bind to myoglobin. Using myoglobin-conjugated magnetic beads, phages that bound to myoglobin were collected and amplified for the next round of screening. A 148-fold enrichment of phage titer was observed after five rounds of screening relative to the first round. After phage binding ELISA, three phage clones were selected (3R1, 3R7 and 3R10) and the inserted peptides were chemically synthesized. The analysis of binding affinity showed that the 3R7 (CPSTLGASC) peptide had higher binding affinity (Kd=57 nM) than did the 3R1 (CNLSSSWIC) and 3R10 (CVPRLSAPC) peptide (Kd=125 nM and 293 nM, respectively). Cross binding activity to other proteins, such as bovine serum albumin, troponin I, and creatine kinase-MB, was minimal. In a peptide-antibody sandwich ELISA, the selected peptides efficiently captured myoglobin. Moreover, the concentrations of myoglobin in serum samples measured by a peptide-peptide sandwich assay were comparable to those measured by a commercial antibody-based kit. These results indicate that the identified peptides can be used for the detection of myoglobin and may be a cost effective alternative to antibodies.

  8. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

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

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  9. Perceptions of Personnel at Selected Texas Community Colleges Regarding the Impact of Technology on Their Libraries

    Science.gov (United States)

    Kenan, Sharon K.

    2012-01-01

    Technological innovations have transformed all areas of community college libraries. Automated library systems, office software, and Internet access have altered work processes for library personnel and have changed research methodologies for students and faculty. The purpose of this bounded multiple case study was to explore how the adoption of…

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

    Institute of Scientific and Technical Information of China (English)

    遇珑; 胡海; 冉宇靓; 彭良平; 李江伟; 杨治华

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    张帅; 崔金杰; 王春义; 雒珺瑜; 吕丽敏

    2012-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  13. 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.

  14. Pacific Visions: Finding, Selecting, and Using Resources for Your Libraries, Archives, and Museums. Selected Papers from PIALA 2009, Pacific Islands Association of Libraries, Archives, and Museums Annual Conference (19th, Pohnpei, Federated States of Micronesia, November 16-21, 2009)

    Science.gov (United States)

    Drake, Paul B., Ed.

    2011-01-01

    This publication follows the tradition of publishing selected papers from Pacific Islands Association of Libraries, Archives and Museums (PIALA) annual conferences. This 19th annual conference was held in Pohnpei, Federated States of Micronesia, November 16-21, 2009. The volume begins with a listing of the members of the PIALA 2009 Planning…

  15. 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

  16. Blocking peptides against HBV: PreS1 protein selected from a phage display library

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wei; Liu, Yang; Zu, Xiangyang; Jin, Rui [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China)

    2011-09-09

    Highlights: {yields} Successfully selected specific PreS1-interacting peptides by using phage displayed library. {yields} Alignment of the positive phage clones revealed a consensus PreS1 binding motif. {yields} A highly enriched peptide named P7 had a strong binding ability for PreS1. {yields} P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX{sub n}HX{sub m}HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.

  17. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones.

    Science.gov (United States)

    Ginolhac, Aurélien; Jarrin, Cyrille; Gillet, Benjamin; Robe, Patrick; Pujic, Petar; Tuphile, Karine; Bertrand, Hélène; Vogel, Timothy M; Perrière, Guy; Simonet, Pascal; Nalin, Renaud

    2004-09-01

    The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.

  18. The Availability and Utilization of School Library Resources in Some Selected Secondary Schools (High School) in Rivers State

    Science.gov (United States)

    Owate, C. N.; Iroha, Okpa

    2013-01-01

    This study investigates the availability and utilization of school library resources by Secondary School (High School) Students. Eight Selected Secondary Schools in Rivers State, Nigeria were chosen based on their performance in external examinations and geographic locations. In carrying out the research, questionnaires were administered to both…

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-04-01

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

  20. 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

  1. Selective agonists and antagonists of formylpeptide receptors: duplex flow cytometry and mixture-based positional scanning libraries.

    Science.gov (United States)

    Pinilla, Clemencia; Edwards, Bruce S; Appel, Jon R; Yates-Gibbins, Tina; Giulianotti, Marc A; Medina-Franco, Jose L; Young, Susan M; Santos, Radleigh G; Sklar, Larry A; Houghten, Richard A

    2013-09-01

    The formylpeptide receptor (FPR1) and formylpeptide-like 1 receptor (FPR2) are G protein-coupled receptors that are linked to acute inflammatory responses, malignant glioma stem cell metastasis, and chronic inflammation. Although several N-formyl peptides are known to bind to these receptors, more selective small-molecule, high-affinity ligands are needed for a better understanding of the physiologic roles played by these receptors. High-throughput assays using mixture-based combinatorial libraries represent a unique, highly efficient approach for rapid data acquisition and ligand identification. We report the superiority of this approach in the context of the simultaneous screening of a diverse set of mixture-based small-molecule libraries. We used a single cross-reactive peptide ligand for a duplex flow cytometric screen of FPR1 and FPR2 in color-coded cell lines. Screening 37 different mixture-based combinatorial libraries totaling more than five million small molecules (contained in 5,261 mixture samples) resulted in seven libraries that significantly inhibited activity at the receptors. Using positional scanning deconvolution, selective high-affinity (low nM K(i)) individual compounds were identified from two separate libraries, namely, pyrrolidine bis-diketopiperazine and polyphenyl urea. The most active individual compounds were characterized for their functional activities as agonists or antagonists with the most potent FPR1 agonist and FPR2 antagonist identified to date with an EC₅₀ of 131 nM (4 nM K(i)) and an IC₅₀ of 81 nM (1 nM K(i)), respectively, in intracellular Ca²⁺ response determinations. Comparative analyses of other previous screening approaches clearly illustrate the efficiency of identifying receptor selective, individual compounds from mixture-based combinatorial libraries.

  2. 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%

  3. Analytical approach for selecting normalizing genes from a cDNA microarray platform to be used in q-RT-PCR assays: a cnidarian case study.

    Science.gov (United States)

    Rodriguez-Lanetty, Mauricio; Phillips, Wendy S; Dove, Sophie; Hoegh-Guldberg, Ove; Weis, Virginia M

    2008-04-24

    Research in gene function using Quantitative Reverse Transcription PCR (q-RT-PCR) and microarray approaches are emerging and just about to explode in the field of coral and cnidarian biology. These approaches are showing the great potential to significantly advance our understanding of how corals respond to abiotic and biotic stresses, and how host cnidarians/dinoflagellates symbioses are maintained and regulated. With these genomic advances, however, new analytical challenges are also emerging, such as the normalization of gene expression data derived from q-RT-PCR. In this study, an effective analytical method is introduced to identify candidate housekeeping genes (HKG) from a sea anemone (Anthopleura elegantissima) cDNA microarray platform that can be used as internal control genes to normalize q-RT-PCR gene expression data. It is shown that the identified HKGs were stable among the experimental conditions tested in this study. The three most stables genes identified, in term of gene expression, were beta-actin, ribosomal protein L12, and a Poly(a) binding protein. The applications of these HKGs in other cnidarian systems are further discussed.

  4. Selection of functional T cell receptor mutants from a yeast surface-display library.

    Science.gov (United States)

    Kieke, M C; Shusta, E V; Boder, E T; Teyton, L; Wittrup, K D; Kranz, D M

    1999-05-11

    The heterodimeric alphabeta T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues. These residues are located in two regions of the TCR, at the interface of the alpha- and beta-chains and in the beta-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeast-displayed mutant TCRs bind specifically to the peptide/MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.

  5. Automating the School Library Media Center. A Select ERIC Bibliography. Mini-Bib.

    Science.gov (United States)

    Preston, Nancy R., Comp.

    This 13-item annotated bibliography was compiled through a search of the Educational Resources Information Center (ERIC) database using the following descriptors: Library Automation, Bibliographic Databases, Bibliographic Utilities, Online Catalogs, Machine Readable Cataloging, and Integrated Library Systems. Among the issues addressed are the…

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

    OpenAIRE

    Jiang, H P; Serrero, G

    1992-01-01

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

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

    OpenAIRE

    Jiang, Hui-Ping; Serrero, Ginette

    1992-01-01

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

  8. 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.

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

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen;

    1991-01-01

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

  10. Biochemical evaluation of a 108-member deglycobleomycin library: viability of a selection strategy for identifying bleomycin analogues with altered properties.

    Science.gov (United States)

    Ma, Qian; Xu, Zhidong; Schroeder, Benjamin R; Sun, Wenyue; Wei, Fang; Hashimoto, Shigeki; Konishi, Kazuhide; Leitheiser, Christopher J; Hecht, Sidney M

    2007-10-17

    The bleomycins (BLMs) are clinically used glycopeptide antitumor antibiotics that have been shown to mediate the sequence-selective oxidative damage of both DNA and RNA. Previously, we described the solid-phase synthesis of a library of 108 unique analogues of deglycoBLM A6, a congener that cleaves DNA analogously to BLM itself. Each member of the library was assayed for its ability to effect single- and double-strand nicking of duplex DNA, sequence-selective DNA cleavage, and RNA cleavage in the presence and absence of a metal ion cofactor. All of the analogues tested were found to mediate concentration-dependent plasmid DNA relaxation to some extent, and a number exhibited double-strand cleavage with an efficiency comparable to or greater than deglycoBLM A6. Further, some analogues having altered linker and metal-binding domains mediated altered sequence-selective cleavage, and a few were found to cleave a tRNA3Lys transcript both in the presence and in the absence of a metal cofactor. The results provide insights into structural elements within BLM that control DNA and RNA cleavage. The present study also permits inferences to be drawn regarding the practicality of a selection strategy for the solid-phase construction and evaluation of large libraries of BLM analogues having altered properties.

  11. Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection.

    Directory of Open Access Journals (Sweden)

    Yongping Wang

    Full Text Available RNA interference (RNAi is a mechanism for interfering with gene expression through the action of small, non-coding RNAs. We previously constructed a short-hairpin-loop RNA (shRNA encoding library that is random at the nucleotide level [1]. In this library, the stems of the hairpin are completely complementary. To improve the potency of initial hits, and therefore signal-to-noise ratios in library screening, as well as to simplify hit-sequence retrieval by PCR, we constructed a second-generation library in which we introduced random mismatches between the two halves of the stem of each hairpin, on a random template background. In a screen for shRNAs that protect an interleukin-3 (IL3 dependent cell line from IL3 withdrawal, our second-generation library yielded hit sequences with significantly higher potencies than those from the first-generation library in the same screen. Our method of random mutagenesis was effective for a random template and is likely suitable, therefore, for any DNA template of interest. The improved potency of our second-generation library expands the range of possible unbiased screens for small-RNA therapeutics and biologic tools.

  12. Development of a porcine skeletal muscle cDNA microarray: analysis of differential transcript expression in phenotypically distinct muscles

    Directory of Open Access Journals (Sweden)

    Stear Michael

    2003-03-01

    Full Text Available Abstract Background Microarray profiling has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production. Results We report on the de novo development of a composite skeletal muscle cDNA microarray, comprising 5500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig. Clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony hybridisation. We profiled the differential expression of genes between the psoas (red muscle and the longissimus dorsi (white muscle, by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Results from seven microarray slides (replicates correctly identified genes that were expected to be differentially expressed, as well as a number of novel candidate regulatory genes. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray. Conclusion We have developed a porcine skeletal muscle cDNA microarray and have identified a number of candidate genes that could be involved in muscle phenotype determination, including several members of the casein kinase 2 signalling pathway.

  13. Cloning and expression of cDNA for human poly(ADP-ribose)polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Alkhatib, H.M.; Chen, D.; Cherney, B.; Bhatia, K.; Notario, V.; Giri, C.; Stein, G.; Slattery, E.; Roeder, R.G.; Smulson, M.E.

    1987-03-01

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambdagt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly(ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambdagt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.

  14. 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

  15. Rapid isolation and characterization of hybridization selected recombinants from lambda genomic libraries.

    Science.gov (United States)

    Porteous, D J

    1986-11-15

    This paper describes an efficient protocol for the screening of lambda genomic libraries, plaque and DNA purification, and probe characterization by a combination of new and recently described techniques. The protocol has allowed large numbers of human subchromosome-specific probes to be rapidly generated from an EMBL3 library of human-mouse somatic cell hybrid DNA. The protocol affords considerable savings in time and effort over previous procedures.

  16. Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo.

    OpenAIRE

    Seed, B

    1983-01-01

    Cloned genes have been purified from recombinant DNA bacteriophage libraries by a method exploiting homologous reciprocal recombination in vivo. In this method 'probe' sequences are inserted in a very small plasmid vector and introduced into recombination-proficient bacterial cells. Genomic bacteriophage libraries are propagated on the cells, and phage bearing sequences homologous to the probe acquire an integrated copy of the plasmid by reciprocal recombination. Phage bearing integrated plas...

  17. The Use of Personal Value Estimations to Select Images for Preservation in Public Library Digital Community Collections

    Directory of Open Access Journals (Sweden)

    Andrea Copeland

    2014-05-01

    Full Text Available A considerable amount of information, particularly in image form, is shared on the web through social networking sites. If any of this content is worthy of preservation, who decides what is to be preserved and based on what criteria. This paper explores the potential for public libraries to assume this role of community digital repositories through the creation of digital collections. Thirty public library users and thirty librarians were solicited from the Indianapolis metropolitan area to evaluate five images selected from Flickr in terms of their value to public library digital collections and their worthiness of long-term preservation. Using a seven-point Likert scale, participants assigned a value to each image in terms of its importance to self, family and society. Participants were then asked to explain the reasoning behind their valuations. Public library users and librarians had similar value estimations of the images in the study. This is perhaps the most significant finding of the study, given the importance of collaboration and forming partnerships for building and sustaining community collections and archives.

  18. Indoor Air Quality and Sick Building Syndrome Study at Two Selected Libraries in Johor Bahru, Malaysia

    Directory of Open Access Journals (Sweden)

    Z. Sulaiman

    2011-01-01

    Full Text Available This study was carried out to investigate the association between sick building syndrome (SBS and indoor air pollutants in two libraries. 101 workers in both libraries responded to the questionnaire, which was based on Malaysian Industry Code of Practice on Indoor Air Quality 2010 (MCPIAQ for the measurement of SBS occurrences. Measurements of indoor air quality were also performed according to the MCPIAQ methods. Higher prevalence of SBS recorded in Perpustakaan Sultanah Zanariah (PSZ, Universiti Teknologi Malaysia, compared to Perpustakaan Sultan Ismail (PSI (X2 = 38.81, p = 0.000, Johor Bahru City. Significantly higher levels of indoor air pollutants were detected in PSZ compare to PSI for CO, CO2, temperature, bacteria, fungi and Total Volatile Organic Compounds (TVOC, while PSI indicated higher level of relative humidity (RH. The levels of CO2, temperature, humidity, TVOC and bacteria counts were the possible major factors contributing to SBS complaints among the workers of both libraries.

  19. 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.

  20. Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase.

    Science.gov (United States)

    Giallongo, A; Feo, S; Moore, R; Croce, C M; Showe, L C

    1986-01-01

    We previously purified a 48-kDa protein (p48) that specifically reacts with an antiserum directed against the 12 carboxyl-terminal amino acids of the c-myc gene product. Using an antiserum directed against the purified p48, we have cloned a cDNA from a human expression library. This cDNA hybrid-selects an mRNA that translates to a 48-kDa protein that specifically reacts with anti-p48 serum. We have isolated a full-length cDNA that encodes p48 and spans 1755 bases. The coding region is 1299 bases long; 94 bases are 5' noncoding and 359 bases are 3' noncoding. The cDNA encodes a 433 amino acid protein that is 67% homologous to yeast enolase and 94% homologous to the rat non-neuronal enolase. The purified protein has been shown to have enolase activity and has been identified to be of the alpha type by isoenzyme analysis. The transcriptional regulation of enolase expression in response to mitogenic stimulation of peripheral blood lymphocytes and in response to heat shock is also discussed. Images PMID:3529090

  1. 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

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

    Science.gov (United States)

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

    2011-04-01

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

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

    Science.gov (United States)

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

    2006-01-01

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

  4. Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library.

    Science.gov (United States)

    Liu, Jinny L; Anderson, George P; Delehanty, James B; Baumann, Richard; Hayhurst, Andrew; Goldman, Ellen R

    2007-03-01

    Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

  5. Isolation and characterization of goat ovarian aromatase cDNA: assessment of the activity using an intact cell system and placental expression.

    Science.gov (United States)

    Bobes, Raúl José; Miranda, Carolina; Pérez-Martinez, Mario; Luu-The, Van; Romano, Marta C

    2004-08-01

    Goat ovarian follicles produce estrone and estradiol from androgens. The synthesis of C18 estrogens from C19 androgens requires cytochrome P450 aromatase, but little information about this key enzyme is available in the goat. We report here for the first time the cDNA sequence of the goat ovarian aromatase, the activity of the enzyme in a cell system, and its expression in the term goat placenta. A cDNA library from goat ovarian poly(A)+ RNA was constructed. Human aromatase cDNA was selected as probe to screen the library; several clones were isolated, but none was complete. The longest clone was 3.1 kb long, but it lacked the sequence coding for a few amino acids in the NH(2)-terminal. To obtain the missing sequence, we performed reverse amplification of the cDNA end (RACE). Sequence analysis indicated that goat aromatase possessed a very long 3'-untranslated region ( approximately 1790 bp), and a polyadenylation signal (AATAAA) located at position 3320 downstream from the ATG start codon. The coding region of goat cDNA was inserted in an expression vector and transfected into HEK-293 cells that were cultured in presence of [14C]-androstenedione, steroids extracted and further separated by TLC. The transfected cells efficiently transformed [14C]-androstenedione into estrone. This activity was inhibited by 4-hydroxyandrostenedione. We also investigated the presence of mRNA for P450 aromatase in the goat placenta, using reverse transcription-polymerase chain reaction (RT-PCR) and primers derived from the cDNA ovarian sequence and confirmed the expression of the mRNA in term placenta.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. Single domain antibodies with VH hallmarks are positively selected during panning of llama (Lama glama) naïve libraries.

    Science.gov (United States)

    Monegal, Ana; Olichon, Aurelien; Bery, Nicolas; Filleron, Thomas; Favre, Gilles; de Marco, Ario

    2012-01-01

    Independent variable domains with VH hallmarks have been repeatedly identified in immune and pre-immune VHH libraries. In some cases, stable independent VH domains have been also isolated in mouse and human recombinant antibody repertoires. However, we have come to realize that VHs were selected with a higher efficiency than VHHs during biopanning of a pre-immune (VHH) library. The biochemical and biophysical comparison did not indicate a presence of any feature that would favor the VH binders during the selection process. In contrast, selected VHHs seemed to be more stable than the VHs, ruling out the existence of a thermodynamically - favored VH sub-class. Therefore, we reasoned that a certain degree of thermodynamic instability may be beneficial for both displaying and expression of VH(H)s when the Sec-pathway is used for their secretion to avoid the cytoplasmic trapping of fast-folding polypeptides. Indeed, VHHs, but not VHs, were accumulated at higher concentrations when expressed fused to the dsbA leader peptide, a sequence that drives the linked polypeptides to the co-translational SRP secretion machinery. These data suggest that the thermodynamically favored VHHs can be lost during biopanning, as previously observed for DARPins and in contrast to the recombinant antibodies in scFv format.

  11. A novel approach for immunization, screening and characterization of selected scFv libraries using membrane fractions of tumor cells.

    Science.gov (United States)

    Tur, Mehmet Kemal; Rothe, Achim; Huhn, Michael; Goerres, Ute; Klimka, Alexander; Stöcker, Michael; Engert, Andreas; Fischer, Rainerr; Finner, Ricarda; Barth, Stefan

    2003-04-01

    Isolation of cell-surface specific antibodies prerequisites the functional expressing of antigens on intact cells, which are maintained routinely by cell culturing. However, long-term culturing of tumor cells could alter their antigen expression patterns and stable fixation of whole cells is not guaranteed on plastic surfaces during stringent screening procedures. We prepared functional breast cancer cell-membrane fractions that express surface molecules in their native conformation. Specific binding phages were isolated from phage antibody libraries constructed from the spleen messenger RNA of mice immunized with breast cancer cell-membrane fractions. After negative selection on non-mammary carcinoma cells and four rounds of positive selection on breast carcinoma cell lines, phage antibodies were enriched that bound specifically to breast cancer cell lines as confirmed by phage enzyme linked immunosorbent assay (ELISA) using 96-well plates coated with breast cancer cell membranes. The isolated phage antibodies were highly specific for the breast cancer cell line 8701-BC but not on other carcinoma such as the Hodgkin-derived cell line L540Cy as demonstrated by ELISA and flow cytometry. This report describes a rapid and more versatile method for isolating antibody fragments compared to whole cell screening procedures. One single membrane preparation can be stored for at least 15 months at -80 degrees C and used to immunize mice or for screening of antibody libraries. The selection and screening strategy used should be generally applicable to identify novel cell-surface antigens and their corresponding antibodies.

  12. 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.

  13. Cloning and expression analysis of MBLL cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  14. The "Integrated Library System."

    Science.gov (United States)

    Dowlin, Kenneth E.

    1985-01-01

    Reviews internal and external dimensions of library environment that must be taken into account by library managers when choosing an integrated library system. The selection, acquisition, and implementation stages of Maggie III--a computerized library system sensitive to the internal and external organizational environment--are described. (MBR)

  15. 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

  16. 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.

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-08-01

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

  19. 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...

  20. Application of high-throughput affinity-selection mass spectrometry for screening of chemical compound libraries in lead discovery.

    Science.gov (United States)

    Zehender, Hartmut; Mayr, Lorenz M

    2007-02-01

    High-throughput screening of chemical libraries for compounds that interfere with a particular molecular target is among the most powerful methodologies applied in lead discovery at present. In this review, the authors describe a label-free, homogeneous, affinity-selection-based technology developed at Novartis, termed SpeedScreen, which is compared with similar technologies used for high-throughput screening in the pharmaceutical and biotechnology industries. The focus at present of SpeedScreen is twofold: first, this technology is applied to orphan genomic targets and to those targets that are non-tractable by a functional assay; second, this technology is applied complementary to the well-established traditional methodologies for the screening of molecular targets. In summary, the authors discuss the value of affinity-selection-based high-throughput screening as a complementary technology to the common functional screening platforms and the benefits as well as the limitations of this new technology are outlined.

  1. Synthesis and Evaluation of a Rationally Designed Click-Based Library for G-Quadruplex Selective DNA Photocleavage

    Directory of Open Access Journals (Sweden)

    Dominic McBrayer

    2015-09-01

    Full Text Available DNA containing repeating G-rich sequences can adopt higher-order structures known as G-quadruplexes (G4. These structures are believed to form within telomeres and the promoter regions of some genes, particularly in a number of proto-oncogenes, where they may play a role in regulating transcription. Alternatively, G4 DNA may act as a barrier to replication. To investigate these potential biological roles, probes that combine highly selective G4 DNA targeting with photocleavage activity can allow temporal detection of G4 DNA, providing opportunities to obtain novel insights about the biological roles of G4 DNA. We have designed, synthesized, and screened a small library of potential selective G-quadruplex DNA photocleavage agents incorporating the G-quadruplex targeting moiety of 360A with known photocleavage groups linked via “click” chemistry.

  2. 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.

  3. 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

  4. Library and Information Services for Literacy. A Select ERIC Bibliography Prepared on a Theme of the 1991 White House Conference on Library and Information Services. ERIC/IR Mini-Bib.

    Science.gov (United States)

    Preston, Nancy R., Comp.

    Documents and journal articles featured in this nine-item annotated bibliography concern the role of library and information services in developing and expanding literacy at all levels in an increasingly information-oriented society. Citations were selected through a computer search of the Educational Resources Information Center (ERIC) database.…

  5. Library and Information Services for Productivity. A Select ERIC Bibliography Prepared on a Theme of the 1991 White House Conference on Library and Information Services. ERIC/IR Mini-Bib.

    Science.gov (United States)

    Preston, Nancy R., Comp.

    Documents and journal articles featured in this nine-item annotated bibliography concern the role of the library and information services, in both public and private settings, in promoting the competitiveness and economic well-being of businesses, regions, and the nation. Citations were selected through a computer search of the Educational…

  6. Library and Information Services for Democracy. A Select ERIC Bibliography Prepared on a Theme of the 1991 White House Conference on Library and Information Services. ERIC/IR Mini-Bib.

    Science.gov (United States)

    Preston, Nancy R., Comp.

    Documents and journal articles featured in this nine-item annotated bibliography concern information as a critical resource in a democratic society and the role of the library and information services in ensuring access to information, especially that which is government produced. Citations were selected through a computer search of the…

  7. New Beginnings: The Library as an Information and Resource Basket. Selected Papers from PIALA 2008: Pacific Islands Association of Libraries, Archives and Museums Annual Conference (18th, Colonia, Yap, Federated States of Micronesia, November 17-21, 2001)

    Science.gov (United States)

    Horie, Ruth H., Ed.

    2009-01-01

    This publication follows the tradition of publishing selected papers from Pacific Islands Association of Libraries, Archives and Museums (PIALA) annual conferences. The volume begins with a listing of the members of the PIALA 2008 Planning Committee, the Officers and Executive Board, and Acknowledgements, followed by the Schedule of Events and a…

  8. Discover the Hidden Jewels in Your Library and Sharing the Wealth through Collaboration. Selected Papers from PIALA 2011, Pacific Islands Association of Libraries, Archives, and Museums Annual Conference (21st, Kosrae, Federated States of Micronesia, November 14-17, 2011)

    Science.gov (United States)

    Drake, Paul B., Ed.

    2012-01-01

    This publication follows the tradition of publishing selected papers from Pacific Islands Association of Libraries, Archives and Museums (PIALA) annual conferences. This 21st annual conference was held in Kosrae, Federated States of Micronesia, November 14-17, 2011. The volume begins with a listing of the members of the PIALA 2011 Planning…

  9. Collecting Poetry for the Academic Library: An Evaluation of Poetry Prizes as Selection Tools

    Science.gov (United States)

    Golomb, Liorah

    2011-01-01

    This paper examines the usefulness of poetry book prizes as a selection tool by evaluating their fairness, meaningfulness, and reliability as an indication of quality. The results of two surveys, one collecting data on poetry book prizes and the other asking librarians about their collecting practices, suggest that selecting on the basis of prizes…

  10. 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

  11. Screening of tissues pooled cDNA library using probes by restricted fragments of BAC positive clones of ovine MHC%以绵羊MHC 区段BAC 克隆酶切片段为探针杂交筛选绵羊混合组织cDNA 文库

    Institute of Scientific and Technical Information of China (English)

    杨小亮; 白大章; 邱巍; 董慧芹; 李大全; 陈芳; 马润林; Hugh T Blair; 高剑峰

    2012-01-01

    Under the premise what we have known bacterial artificial chromosome(BAC) clone sequence information and gene annotation predicted in the Chinese Merino sheep major histocompatibility complex (MHC) region, the digested fragments from 6 BAC clones that were located in the MHC region of the Chinese Merino sheep genome BAC library, which were used to screen the cDNA library using plaque in situ hybridization as probes. The full length of positive cDNA clones (sequences) isolated were completely sequenced, and the sequences obtained were aligned with the corresponding known sequence information and the BAC clones with gene annotation. Meanwhile, the sequence similarity was searched in NCBI Blastn database. This work aimed at verification of accuracy of the gene annotation results and initial analysis of gene (sequence) function. At last, 27 positive cDNA clones (sequences) in total were screened through two runs of hybridization. It was also found that these sequences could be positioned in the corresponding BAC clones, and 25 sequences were located in exon area of the annotated gene. It was verified that 23 sequences had the highest sequence similarity with those in the Bos taurus by searching against the NCBI Blastn database; moreover, the function of these sequences were closely relate to immunology.%在已知中国美利奴羊MHC(Major histocompatibility complex)区段BAC(Bacterial artificial chromosome)克隆序列信息和预测的基因注释前提下,用位于中国美利奴羊基因组BAC 文库MHC 区段的6 个BAC 克隆酶切片段为探针,以噬菌斑原位杂交筛选法筛选中国美利奴羊混合组织cDNA 文库(库库杂交),对分离到的cDNA阳性克隆进行全序列测定,并与相应的已知序列信息和基因注释的BAC 克隆比对以及在NCBI Blastn 数据库中序列相似性检索,旨在验证基因注释结果的准确性和对基因(序列)功能的初步分析.实验中,经过两轮杂交共筛选出27 个cDNA 阳性克隆(序

  12. Structural characterization and optimization of antibody-selected phage library mimotopes of an antigen associated with autoimmune recurrent thrombosis.

    Science.gov (United States)

    Sem, D S; Baker, B L; Victoria, E J; Jones, D S; Marquis, D; Yu, L; Parks, J; Coutts, S M

    1998-11-17

    The presence of high titers of anti-cardiolipin antibodies (ACA's) of autoimmune origin, which are known to bind to plasma beta2-glycoprotein I (aka apolipoprotein H), correlates clinically with autoimmune recurrent thrombosis. Soluble beta2-glycoprotein I binds to solid-phase ACA (immobilized on a surface plasmon resonance chip) with a Kd of 1.4 microM, but if the reactants are reversed and beta2-glycoprotein I is on the solid-phase support, then the Kd is 52 nM. This 27-fold difference in affinity reflects the avidity/entropic advantage obtained for an antibody binding to an antigen that is made multivalent because it is attached to a solid phase. A mimotope of this antigen, selected from a phage display peptide library screen with an ACA, has been shown to bind to solid-phase ACA as a phage, using surface plasmon resonance. This peptide is representative of the motif from 37 peptides obtained in a previously reported phage library screen with this ACA (1). A synthetic version of this peptide, referred to as P4, has the sequence: A1G2P3C4I5L6L7A8R9D10R11C12P13G14, and binds to its selecting antibody with a Kd of 42 nM. NMR data indicate that proline-13 is present in both cis and trans configurations, and that these two geometries dramatically affect the overall tertiary structure of the molecule. The peptide lacking this proline binds severalfold better to the ACA, consistent with at least one of these structures having low affinity for binding ACA. Replacement of the arginine-9 position with a proline decreases binding affinity to ACA 10-fold. Another phage library-selected peptide has a proline in position 9, but also has a leucine in position 5, instead of isoleucine. Since its affinity for ACA is nearly as good as that for peptide P4, the phage library screening must have selected for a non-beta-branched amino acid in this position to compensate for the adverse effects of the arginine-9 to proline-9 substitution. The solution structure of a modified version

  13. Large-insert BAC/YAC libraries for selective re-isolation of genomic regions by homologous recombination in yeast.

    Science.gov (United States)

    Zeng, C; Kouprina, N; Zhu, B; Cairo, A; Hoek, M; Cross, G; Osoegawa, K; Larionov, V; de Jong, P

    2001-09-01

    We constructed representative large-insert bacterial artificial chromosome (BAC) libraries of two human pathogens (Trypanosoma brucei and Giardia lamblia) using a new hybrid vector, pTARBAC1, containing a yeast artificial chromosome (YAC) cassette (a yeast selectable marker and a centromere). The cassette allows transferring of BACs into yeast for their further modification. Furthermore, the new hybrid vector provides the opportunity to re-isolate each DNA insert without construction of a new library of random clones. Digestion of a BAC DNA by an endonuclease that has no recognition site in the vector, but which deletes most of the internal insert sequence and leaves the unique flanking sequences, converts a BAC into a TAR vector, thus allowing direct gene isolation. Cotransformation of a TAR vector and genomic DNA into yeast spheroplasts, and subsequent recombination between the TAR vector's flanking ends and a specific genomic fragment, allows rescue of the fragment as a circular YAC/BAC molecule. Here we prove a new cloning strategy by re-isolation of randomly chosen genomic fragments of different size from T. brucei cloned in BACs. We conclude that genomic regions of unicellular eukaryotes can be easily re-isolated using this technique, which provides an opportunity to study evolution of these genomes and the role of genome instability in pathogenicity.

  14. A theoretical model to predict customer satisfaction in relation to service quality in selected university libraries in Sri Lanka

    Directory of Open Access Journals (Sweden)

    Chaminda Jayasundara

    2009-01-01

    Full Text Available University library administrators in Sri Lanka have begun to search for alternative ways to satisfy their clientele on the basis of service quality. This article aims at providing a theoretical model to facilitate the identification of service quality attributes and domains that may be used to predict customer satisfaction from a service quality perspective. The effectiveness of existing service quality models such as LibQUAL, SERVQUAL and SERVPREF have been questioned. In that regard, this study developed a theoretical model for academic libraries in Sri Lanka based on the disconfirmation and performance-only paradigms. These perspectives were considered by researchers to be the core mechanism to develop service quality/customer satisfaction models. The attributes and domain identification of service quality was carried out with a stratified sample of 263 participants selected from postgraduate and undergraduate students and academic staff members from the faculties of Arts in four universities in Sri Lanka. The study established that responsiveness, supportiveness, building environment, collection and access, furniture and facilities, technology, Web services and service delivery were quality domains which can be used to predict customer satisfaction. The theoretical model is unique in its domain structure compared to the existing models. The model needs to be statistically tested to make it valid and parsimonious.

  15. Size-selected genomic libraries: the distribution and size-fractionation of restricted genomic DNA fragments by gel electrophoresis.

    Science.gov (United States)

    Gondo, Y

    1995-02-01

    By using one-dimensional genome scanning, it is possible to directly identify the restricted genomic DNA fragment that reflects the site of genetic change. The subsequent strategies to obtain the molecular clones of the corresponding restriction fragment are usually as follows: (i) the restriction of a mass quantity of an appropriate genomic DNA, (ii) the size-fractionation of the restricted DNA on a preparative electrophoresis gel in order to enrich the corresponding restriction fragment, (iii) the construction of the size-selected libraries from the fractionated genomic DNA, and (iv) the screening of the library to obtain an objective clone which is identified on the analytical genome scanning gel. A knowledge of the size distribution pattern of restriction fragments of the genomic DNA makes it possible to calculate the heterogeneity or complexity of the restriction fragment in each size-fraction. This manuscript first describes the distribution of the restriction fragments with respect to their length. Some examples of the practical application of this theory to genome scanning is then discussed using presumptive genome scanning gels. The way to calculate such DNA complexities in the prepared size-fractionated samples is also demonstrated. Such information should greatly facilitate the design of experimental strategies for the cloning of a certain size of genomic DNA after digestion with restriction enzyme(s) as is the case with genome scanning.

  16. 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

  17. Hits identified in library screening demonstrate selective CYP17A1 lyase inhibition.

    Science.gov (United States)

    Krug, Sebastian J; Hu, Qingzhong; Hartmann, Rolf W

    2013-03-01

    A screening of structurally different steroid hormone synthesis inhibitors was performed in order to find a starting point for the development of a new inhibitor of the bifunctional steroidogenic enzyme CYP17A1. Emphasis was placed on determination of selectivity between the two catalytic steps, namely 17α-hydroxylase and C(17,20)-lyase. For that purpose a new inhibition assay has been developed. Hits identified within this novel assay demonstrated selective inhibition of CYP17A1 lyase activity, and thus mark the basis for the development of selective C(17,20)-lyase inhibitors for the treatment of prostate cancer.

  18. 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.

  19. 大學圖書館選書人員之探討 The study of Selectors in Book Selection in University Libraries

    Directory of Open Access Journals (Sweden)

    Pi-ching Chu

    1994-09-01

    Full Text Available 無It should be the goal of a university library to maintain a high quality and frequently used book collection. It depends on the intellectual judgement of selectors for the quality control of the book collection to support education and research. Whether a faculty or a librarian should be the selector is always an arguing issue. This article describes the characteristics of book selection in a university library, and discusses the advantages and disadvantages of book collection selected by faculties and librarians. Also try to propose some solutions.

  20. A novel peptide, selected from phage display library of random peptides, can efficiently target into human breast cancer cell

    Institute of Scientific and Technical Information of China (English)

    DONG Jian; LIU WeiQing; JIANG AiMei; ZHANG KeJian; CHEN MingQing

    2008-01-01

    To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 (9 aa) phage display library of random peptides. In multiple inde-pendent peptide-presenting phage screening trials, subtilisin was used as a protease to inactivate ex-tra-cellular phages. The internalized phages were collected by cell lysising and amplified in E. coli XLI-Blue. Through five rounds of selection, the peptide-presenting phages which could be internalized in MDA-MB-231 cells were isolated. A comparison was made between internalization capacities of pep-tide-presenting phages isolated from MDA-MB-231 cells and RGD-integrin binding phage by cocultur-ing them with other human tumor cell lines and normal cells. The nucleotide sequences of isolated peptide-presenting phages were then determined by DNA sequencing. To uncover whether phage coat protein or amino acid order was required for the character of the peptide to MDA-MB-231 cells, three peptides were synthesized. They are CASPSGALRSC, ASPSGALRS and CGVIFDHSVPC (the shifted sequence of CASPSGALRSC), and after coculturing them with different cell lines, their targeting ca-pacities to MDA-MB-231 cells were detected. These data suggested that the internalization process was highly selective, and capable of capturing a specific peptide from parent peptide variants. Moreover, the targeting internalization event of peptides was an amino acid sequence dependent manner. The results demonstrated the feasibility of using phage display library of random peptides to develop new targeting system for intracellular delivery of macromolecules, and the peptide we obtained might be modified as a targeting vector for breast cancer gene therapy.

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

  2. Human recombinant beta-secretase immobilized enzyme reactor for fast hits' selection and characterization from a virtual screening library.

    Science.gov (United States)

    De Simone, Angela; Mancini, Francesca; Cosconati, Sandro; Marinelli, Luciana; La Pietra, Valeria; Novellino, Ettore; Andrisano, Vincenza

    2013-01-25

    In the present work, a human recombinant BACE1 immobilized enzyme reactor (hrBACE1-IMER) has been applied for the sensitive fast screening of 38 compounds selected through a virtual screening approach. HrBACE1-IMER was inserted into a liquid chromatograph coupled with a fluorescent detector. A fluorogenic peptide substrate (M-2420), containing the β-secretase site of the Swedish mutation of APP, was injected and cleaved in the on-line HPLC-hrBACE1-IMER system, giving rise to the fluorescent product. The compounds of the library were tested for their ability to inhibit BACE1 in the immobilized format and to reduce the area related to the chromatographic peak of the fluorescent enzymatic product. The results were validated in solution by using two different FRET methods. Due to the efficient virtual screening methodology, more than fifty percent of the selected compounds showed a measurable inhibitory activity. One of the most active compound (a bis-indanone derivative) was characterized in terms of IC(50) and K(i) determination on the hrBACE1-IMER. Thus, the hrBACE1-IMER has been confirmed as a valid tool for the throughput screening of different chemical entities with potency lower than 30μM for the fast hits' selection and for mode of action determination.

  3. REAL-Select: full-length antibody display and library screening by surface capture on yeast cells.

    Science.gov (United States)

    Rhiel, Laura; Krah, Simon; Günther, Ralf; Becker, Stefan; Kolmar, Harald; Hock, Björn

    2014-01-01

    We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1:1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.

  4. REAL-Select: full-length antibody display and library screening by surface capture on yeast cells.

    Directory of Open Access Journals (Sweden)

    Laura Rhiel

    Full Text Available We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1:1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.

  5. Immunodiagnosis of Canine Visceral Leishmaniasis Using Mimotope Peptides Selected from Phage Displayed Combinatorial Libraries

    Directory of Open Access Journals (Sweden)

    Christina Monerat Toledo-Machado

    2015-01-01

    Full Text Available ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL. The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL.

  6. 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.

  7. Chemical library subset selection algorithms: a unified derivation using spatial statistics.

    Science.gov (United States)

    Hamprecht, Fred A; Thiel, Walter; van Gunsteren, Wilfred F

    2002-01-01

    If similar compounds have similar activity, rational subset selection becomes superior to random selection in screening for pharmacological lead discovery programs. Traditional approaches to this experimental design problem fall into two classes: (i) a linear or quadratic response function is assumed (ii) some space filling criterion is optimized. The assumptions underlying the first approach are clear but not always defendable; the second approach yields more intuitive designs but lacks a clear theoretical foundation. We model activity in a bioassay as realization of a stochastic process and use the best linear unbiased estimator to construct spatial sampling designs that optimize the integrated mean square prediction error, the maximum mean square prediction error, or the entropy. We argue that our approach constitutes a unifying framework encompassing most proposed techniques as limiting cases and sheds light on their underlying assumptions. In particular, vector quantization is obtained, in dimensions up to eight, in the limiting case of very smooth response surfaces for the integrated mean square error criterion. Closest packing is obtained for very rough surfaces under the integrated mean square error and entropy criteria. We suggest to use either the integrated mean square prediction error or the entropy as optimization criteria rather than approximations thereof and propose a scheme for direct iterative minimization of the integrated mean square prediction error. Finally, we discuss how the quality of chemical descriptors manifests itself and clarify the assumptions underlying the selection of diverse or representative subsets.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-05-01

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

  9. Comparison of Approval Plan Profiles and Supplementary Collection Development Activities in Selected ARL Libraries. A Report to the Council on Library Resources.

    Science.gov (United States)

    Snoke, Helen Lloyd; Loup, Jean L.

    This study was conducted to learn the extent to which member institutions of the Association of Research Libraries (ARL) with Blackwell North America (B/NA) approval plans covering philosophy and political science are duplicating collections, and to determine what supplementary collection development practices are being used. Initial data were…

  10. Rapid Characterization of S. mansoni Expression Library Clones of Potential Interest

    Directory of Open Access Journals (Sweden)

    KANAMURA Herminia Yohko

    1997-01-01

    Full Text Available A S. mansoni adult worm cDNA expression library was screened with sera from baboons in a early phase after infection. The clones that were positive with the early infection sera were examined for reactivity with pre-infection sera and heterologous infection sera. In order to discriminate a positive antibody reaction from the reactivity due to residual anti-E. coli antibodies, an unrelated cDNA clone was plated with the positive clone. The unrelated clone provided the negative background and the contrast necessary to discern a positive antibody reaction. In this way, we were able to eliminate selected clones that were positive with the pre-infection sera or heterologous infection sera. This characterization of the expression library clones enabled us to quickly target only clones with the desired pattern of antibody reactivity for sequencing, subcloning, and expressing

  11. Selection of Immunogenic Peptide Mimics of Male Worm Origin of Schistosoma Japonicum using Phage Display Peptide Library

    Institute of Scientific and Technical Information of China (English)

    陈欲晓; 易新元; 曾宪芳; 王林纤; 唐连飞; 章洁; McreynoldsLarry

    2004-01-01

    To select the immunogenic short peptide mimics of male worm origin of Schistosoma japonicum (Sj) and to explore their protection effect against schistosomiasis in mice, the random phage display peptide hbrary of 12 - mer was screened with IgG to soluble male worm antigen of Sj, and the specific positive clones selected through three rounds of screenings were detected by Dot-ELISA, and then injected subcutaneously into mice for vaccination and protection assessment against Sj. It was found that 18 randomly picked phage displayed clones all showed definite antigenicity with various intensities. The pooled phages displayed clones could induce production of specific antibodies and cause 31.72% of worm reduction rate and 51.54 % of egg reduction rate in mice, revealing a significant difference ( P < 0. 001 ) in comparison with those of the controls. It concludes that the short peptide mimics of male worm origin of Sj obtained by affinity screening phage display ptide library can elicit partial protection against this pathogen.

  12. 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.

  13. 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.

  14. Five eighteenth and nineteenth century books significant in vision science selected from the collection of the Lilly Library at Indiana University.

    Science.gov (United States)

    Goss, David A

    2007-07-01

    This paper discusses five eighteenth and nineteenth century books selected from the collection of the Lilly Library at Indiana University in Bloomington, Indiana. The five were selected because they all contained at least some material of significance in clinical vision science (some contained a broad range of scientific topics) and they were all important publications in their time. The authors of the books, in alphabetical order, were Frans Cornelis Donders, Joseph Priestley, William Charles Wells, Charles Wheatstone, and Thomas Young.

  15. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones and quality ...scores Data detail Data name 5'-end sequences of budding yeast full-length cDNA clones and quality scores De...from the budding yeast full-length cDNA library by the vector-capping method, the sequence quality score gen...s accession only. Sequence 5'-end sequence data of budding yeast full-length cDNA clones. FASTA format. Quality Phred's quality... Update History of This Database Site Policy | Contact Us 5'-end sequences of budding yeast full-length cDNA clones and quality

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

    OpenAIRE

    1988-01-01

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

  17. A Study on the use of Facebook, RSS, Blogs and Twitter (Web2.0) among selected academic libraries from 6 Gulf countries namely: Kingdom of Saudi Arabia, United Arab Emirates, Qatar, Bahrain, Oman and Kuwait

    KAUST Repository

    Ramli, Rindra M.

    2014-04-01

    This paper aims to explore and study the current usage trends of Web2.0 namely Facebook, RSS, Blogs and Twitter among selected higher education institutions’ libraries in 6 gulf countries namely: Kingdom of Saudi Arabia, United Arab Emirates, Qatar, Bahrain, Oman and Kuwait. Websites of the selected libraries would be compared on the extent of the usage of these tools, the participation level and their purpose. The author would also share his opinion and suggestions on improving the current trends pertaining to the area of Web2.0 and libraries. The impact and importance of Web2.0 on libraries cannot be disputed. Since gaining popularity in mid-2000, libraries around the globe have jumped onto the Web2.0 bandwagon. Among the common examples of Web2.0 used by libraries today are namely: social networking sites, blogs, wikis, folksonomies and video sharing sites. Libraries are using Web2.0 to (among others): • market their services / resources to their community, • announce latest library news, • provide their online guides / notes for their resources among others. Though such tools have been implemented by most libraries around the world, some of the challenges faced by libraries are: •participation level – casting the net to a wider audience •selection of web2.0 tools •effectiveness of present web2.0 tools used by the libraries

  18. The generation and selection of single-domain, v region libraries from nurse sharks.

    Science.gov (United States)

    Flajnik, Martin F; Dooley, Helen

    2009-01-01

    The cartilaginous fish (sharks, skates, and rays) are the oldest phylogenetic group in which a human-type adaptive immune system and immunoglobulins (Igs) have been found. In addition to their conventional (heavy-light chain heterodimeric) isotypes, IgM and IgW, sharks produce the novel isotype, IgNAR, a heavy chain homodimer that does not associate with light chains. Instead, its variable (V) regions act as independent, soluble units in order to bind antigen. In this chapter, we detail our immunization protocol in order to raise a humoral IgNAR response in the nurse shark (Ginglymostoma cirratum) and the subsequent cloning of the single-domain V regions from this isotype in order to select antigen-specific binders by phage display.

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

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

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

  20. Information Needs and Use of Library Resources by Special Needs Students in Selected Government Schools in Kaduna State and Federal Capital Territory, Abuja, Nigeria

    Directory of Open Access Journals (Sweden)

    Christiana Alami Atabor

    2015-12-01

    Full Text Available This study is on the information needs and use of library resources by special needs students in selected government schools in Kaduna State and Federal Capital Territory, Abuja, Nigeria. The survey technique was employed in the study. Five schools (i.e., Kaduna State Special Education School; Government Technical College, Malali, Kaduna; Alhudahuda College Zaria; Government Secondary School, Kwali and Government Secondary School, Kuje were purposefully selected out of a total of seven. Data were collected using a structured questionnaire for two respondent groups (i.e., special needs students and teachers/librarians. A total of 5 teachers/librarians and 345 special needs students were selected for the study. The data collected were analyzed using descriptive statistics. The findings of the study show that the special needs students have high needs for information on education, employment opportunities, health matter, and human rights. The major information resources in the libraries are books, magazines, newspapers, and few Braille materials. In general, special needs students have a moderate level of satisfaction with the resources in the libraries. The major issue identified by special needs students is insufficient information resources, especially in Braille. On the part of the teachers/librarians, inadequate funds for the acquisition of information resources and the employment of qualified librarians/teachers are found to be the major challenges faced by the libraries.

  1. Selection and Application of ssDNA Aptamers against Clenbuterol Hydrochloride Based on ssDNA Library Immobilized SELEX.

    Science.gov (United States)

    Duan, Nuo; Gong, Wenhui; Wu, Shijia; Wang, Zhouping

    2017-03-01

    Clenbuterol hydrochloride (CLB) is often abused as additive feed for livestock to decrease adipose tissue deposition and to increase growth rate. It raises a potential risk to human health through the consumption of animal product. In this study, aptamers with higher affinity and specificity were screened through 16 selection rounds based on the ssDNA library immobilized systematic evolution of ligands by exponential enrichment (SELEX) technique. After cloning and sequencing, five aptamer candidates were picked out for affinity and specificity assays based on a graphene oxide (GO) adsorption method. The results showed that the aptamer CLB-2 binds specifically against CLB with a dissociation constant, Kd, value of 76.61 ± 12.70 nM. In addition, an aptamer-based fluorescence bioassay was established for CLB analysis. The correlation between the CLB concentration and fluorescent signal was found to be linear within the range of 0.10 to 50 ng/mL with a limit of detection of 0.07 ng/mL. It has been further applied for the determination of CLB in pork samples, showing its great potential for sensitive analysis in food safety control.

  2. The GPO and the Depository Library Program as Structured Are Needed: Views of a Selective Depository Librarian.

    Science.gov (United States)

    Walshak, Lynn G.

    1998-01-01

    Addresses perceived problems of the Federal Depository Library Program (FDLP) and the Government Printing Office (GPO) relative to a foundering program, electronic capabilities, costs to participating libraries, inefficient service, and accountability requirements. Unfavorable images have resulted and have been used by advocacy groups to bring the…

  3. cDNA library Table: fufe [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available fufe NA fufe p50T embryo unfertilized egg stage mixed pGCAP10, V-capping, full-leng...th Unknown Sequenced from 5' with PGCAP-F-21 primer; Sequenced from 3' with degenerate oligo dT primer FS904111-FS921205 E_FL_fufe_[number]_F_0 ...

  4. cDNA library Table: fbpv [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available for 5' Xho1for 3' sequenced from T3 primer (5' -> 3') BP124845-BP125532,BY915277-BY915334 fbpv[number],fbpv[number]_1 2hr after infection of BmNPV ... ...fbpv NA fbpv Shuko x Ryuhaku fat body spinning stage S2 mixed pBluescript SK- EcoR1

  5. cDNA library Table: mg [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available mg NA mg-- p50 midgut fifth instar larval stage D3 mixed pBluescript SK- EcoR1 for ...5' Xho1for 3' sequenced from T3 primer (5' -> 3') AU001868-AU002476,BY916287-BY916543 mg[number],mg[number]_1 ...

  6. cDNA library Table: phe- [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available phe- NA phe- p50T pheromone gland adult stage D0 female pGCAPI, G-capping, full-len...gth Unknown Sequenced from 5' with T7 primer; Sequenced from 3' with modified pT primer DC544856-DC552314 E_FL_phe-_[number]_F_0 ...

  7. cDNA library Table: ffbm [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available ffbm NA ffbm p50T fat body fifth instar larval stage male pGCAP10, V-capping, full-...length Unknown Sequenced from 5' with PGCAP-F-21 primer; Sequenced from 3' with degenerate oligo dT primer FS789515-FS799357 E_FL_ffbm_[number]_F_0 ...

  8. cDNA library Table: dpe- [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available dpe- NA dpe- NA diapause-destined embryo fertilized egg stage H12-40 mixed pGCAPI, ...G-capping, full-length Unknown Sequenced from 5' with T7 primer DC539445-DC544855 E_FL_dpe-_[number]_F_0 ...

  9. cDNA library Table: ce-- [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available ce-- NA ce-- C202 x J201 compound eyes mixture of fifth instar larval stage to pupa...l stage mixed pBluescript SK- EcoR1 for 5' Xho1for 3' sequenced from T3 primer (5' -> 3') BP117205-BP118782 ce--[number] ...

  10. cDNA library Table: JFsb [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available JFsb NA JFsb J150-F1 midgut fifth instar larval stage D3 mixed pBluescript SK- EcoR...1 for 5' Xho1for 3' sequenced from T3 primer (5' -> 3') BY921003-BY921254,BY929473-BY929787 E_ET_JFsb_[number]_F_0,E_ET_JFsb_[number]_R_0 ...

  11. cDNA library Table: cesb [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available cesb NA cesb C202 x J201 compound eyes mixture of fifth instar larval stage to pupa...l stage mixed pBluescript SK- EcoR1 for 5' Xho1for 3' sequenced from T3 primer (5' -> 3') FS939461-FS939542 cesb

  12. cDNA library Table: FJsb [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available FJsb NA FJsb F1-J150 midgut fourth instar larval stage mixed pBluescript SK- EcoR1 ...for 5' Xho1for 3' sequenced from T3 primer (5' -> 3') BY920389-BY921002,BY928826-BY929472 E_ET_FJsb_[number]_F_0,E_ET_FJsb_[number]_R_0 ...

  13. 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.

  14. Integrated Library Information Systems in ARL Libraries. SPEC Kit 90.

    Science.gov (United States)

    Hirshon, Arnold

    Based on an October 1982 survey of 31 selected members of the Association of Research Libraries (ARL), this report presents library planning documents, general system descriptions and reviews, and examples of library specifications--all dealing with integrated library information systems (ILIS). An ILIS is defined as a fully interactive integrated…

  15. Canadian Literature in American Libraries

    Science.gov (United States)

    Rogers, A. Robert

    1973-01-01

    Acquisition of Canadian literature by American libraries was investigated in three ways: questionnaires were sent to selected large libraries, titles were checked against the National Union Catalog'' and published literature describing major collections was examined. With the exception of the Library of Congress, American libraries purchase…

  16. Sports Education Library Information Services

    Institute of Scientific and Technical Information of China (English)

    许晓峰

    2014-01-01

    Library website, is the first window for readers to understand library information services. Sports Education academy library fully take advantage of homepage, combine open access resource searched with the library collections, after targeted collection, selection, sorting, processing, clustering or reorganization, establish a navigation system of open access resource of physical Sports Education.

  17. Application of Fuzzy Set Theory and SPOT Satellite Images in Site Selection of Public Libraries (Case Study: Saqqez City, Iran

    Directory of Open Access Journals (Sweden)

    Heydar Dashti Nasserabadi

    2013-06-01

    Full Text Available Easy availability to public libraries is one of the main keys in successful presentation of public library services with high quality will be valueless for those have not access to them. Availability to services should be configured in such a way that maximizes easiness and facility for potential and de facto users. In present research the required number of public libraries and establishment situation of public libraries in Saqqez city in Kurdistan province in North West of Iran were investigated and evaluated by using fuzzy set theory and SPOT 5 satellite images in 2009. In addition, optimum regions for generation of new public libraries were distinguished in city area in form of recommended map. Results showed that weighting variables classes were determined between 0-1 (profusion proportion. Fuzzification each one of effective factors using IDRISI software terminated and after was assigned type and shape membership function. Final map of suitable regions for construction of new public libraries in saqqez city area indicates that none of the libraries were situated in suitable regions.

  18. Molecular Cloning and Sequencing of Channel Catfish, Ictalurus punctatus, Cathepsin H and L cDNA

    Science.gov (United States)

    Cathepsin H and L, a lysosomal cysteine endopeptidase of the papain family, are ubiquitously expressed and involve in antigen processing. In this communication, the channel catfish cathepsin H and L transcripts were sequenced and analyzed. Total RNA from tissues was extracted and cDNA libraries we...

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed......, was determined to be on distal mouse Chromosome (Chr) 9 by analysis of two sets of multilocus crosses....

  20. Cloning and expression analysis of human reticulon 4c cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Zhang Qifa

    2008-11-01

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

  2. Effectively designed molecularly imprinted polymers for selective extraction of glabridin from Glycyrrhiza glabra L. residues by screening the library of non-imprinted polymers.

    Science.gov (United States)

    Chen, Lingxiao; Ji, Wenhua; Duan, Wenjuan; Wang, Xiao; Gao, Qianshan; Geng, Yanling; Huang, Luqi

    2014-08-15

    Molecularly imprinted polymers (MIPs) with high selectivity and affinity to glabridin were designed based on the screening results of the library of non-imprinted polymers (NIPs). The NIP library contained 48 polymers that were polymerized with the combinations of different functional monomers, cross-linkers, and porogenic solvents. The distribution coefficient (k) values were used to estimate the affinity of NIPs to glabridin. The corresponding MIPs of the best three NIPs were prepared. After evaluating the imprinting effects and selectivity of the three MIPs, the performance of the best MIP as solid phase extraction sorbent was investigated. Glabridin with percent recovery of 83 was obtained from the extract of Glycyrrhiza glabra L. (G. glabra L.) residues by molecularly imprinted solid phase extraction (MISPE). Thus, this material can be successfully used for the extraction and purification of glabridin from G. glabra L. residues.

  3. Phage display selection of tight specific binding variants from a hyperthermostable Sso7d scaffold protein library.

    Science.gov (United States)

    Zhao, Ning; Schmitt, Margaret A; Fisk, John D

    2016-04-01

    Antibodies, the quintessential biological recognition molecules, are not ideal for many applications because of their large size, complex modifications, and thermal and chemical instability. Identifying alternative scaffolds that may be evolved into tight, specific binding molecules with improved physical properties is of increasing interest, particularly for biomedical applications in resource-limited environments. Hyperthermophilic organisms, such as Sulfolobus solfataricus, are an attractive source of highly stable proteins that may serve as starting points for alternative molecular recognition scaffolds. We describe the first application of phage display to identify binding proteins based on the S. solfataricus protein Sso7d scaffold. Sso7d is a small cysteine-free DNA-binding protein (approximately 7 kDa, 63 amino acids), with a melting temperature of nearly 100 °C. Tight-binding Sso7d variants were selected for a diverse set of protein targets from a 10(10) member library, demonstrating the versatility of the scaffold. These Sso7d variants are able to discriminate among closely related human, bovine and rabbit serum albumins. Equilibrium dissociation constants in the nanomolar to low micromolar range were measured via competitive ELISA. Importantly, the Sso7d variants continue to bind their targets in the absence of the phage context. Furthermore, phage-displayed Sso7d variants retain their binding affinity after exposure to temperatures up to 70 °C. Taken together, our results suggest that the Sso7d scaffold will be a complementary addition to the range of non-antibody scaffold proteins that may be utilized in phage display. Variants of hyperthermostable binding proteins have potential applications in diagnostics and therapeutics for environments with extreme conditions of storage and deployment.

  4. Selection of binding targets in parasites using phage-display and aptamer libraries in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Renata Rosito Tonelli

    2013-01-01

    Full Text Available Parasite infections are largely dependent on interactions between pathogen and different host cell populations to guarantee a successful infectious process. This is particularly true for obligatory intracellular parasites as Plasmodium, Toxoplasma, Leishmania, to name a few. Adhesion to and entry into the cell are essential steps requiring specific parasite and host cell molecules. The large amount of possible involved molecules poses additional difficulties for their identification by the classical biochemical approaches. In this respect, the search for alternative techniques should be pursued. Among them two powerful methodologies can be employed, both relying upon the construction of highly diverse combinatorial libraries of peptides or oligonucleotides that randomly bind with high affinity to targets on the cell surface and are selectively displaced by putative ligands. These are, respectively, the peptide-based phage display and the oligonucleotide-based aptamer techniques.The phage display technique has been extensively employed for the identification of novel ligands in vitro and in vivo in different areas such as cancer, vaccine development and epitope mapping. Particularly, phage display has been employed in the investigation of pathogen-host interactions. Although this methodology has been used for some parasites with encouraging results, in trypanosomatids its use is, as yet, scanty. RNA and DNA aptamers, developed by the SELEX process (Systematic Evolution of Ligands by Exponential Enrichment, were described over two decades ago and since then contributed to a large number of structured nucleic acids for diagnostic or therapeutic purposes or for the understanding of the cell biology. Similarly to the phage display technique scarce use of the SELEX process has been used in the probing of parasite-host interaction.In this review, an overall survey on the use of both phage display and aptamer technologies in different pathogenic

  5. Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two β-domains of opposite topologies.

    Directory of Open Access Journals (Sweden)

    Valencio Salema

    Full Text Available Screening of antibody (Ab libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM. In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs from camelids (nanobodies or VHH on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS. We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirM(EHEC. We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirM(EHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirM(EHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.

  6. FOX-superroots of Lotus corniculatus, overexpressing Arabidopsis full-length cDNA, show stable variations in morphological traits.

    Science.gov (United States)

    Himuro, Yasuyo; Tanaka, Hidenori; Hashiguchi, Masatsugu; Ichikawa, Takanari; Nakazawa, Miki; Seki, Motoaki; Fujita, Miki; Shinozaki, Kazuo; Matsui, Minami; Akashi, Ryo; Hoffmann, Franz

    2011-01-15

    Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in bird's-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant.

  7. The Primary Sequence of Acetylcholinesterase and Selective Antibodies for the Detection of Organophosphate Toxicity

    Science.gov (United States)

    1987-01-01

    cDNA library screening . b. To employ the sequence to verify inferred amino acid sequence resulting from cDNA sequencing and correlate the cDNA qequenr-s...the same laboratory. Our library screening strategems rely on hybridization with tandem but not overlapping probes, since we initially found that

  8. Discovery of highly potent and selective small molecule ADAMTS-5 inhibitors that inhibit human cartilage degradation via encoded library technology (ELT).

    Science.gov (United States)

    Deng, Hongfeng; O'Keefe, Heather; Davie, Christopher P; Lind, Kenneth E; Acharya, Raksha A; Franklin, G Joseph; Larkin, Jonathan; Matico, Rosalie; Neeb, Michael; Thompson, Monique M; Lohr, Thomas; Gross, Jeffrey W; Centrella, Paolo A; O'Donovan, Gary K; Bedard, Katie L Sargent; van Vloten, Kurt; Mataruse, Sibongile; Skinner, Steven R; Belyanskaya, Svetlana L; Carpenter, Tiffany Y; Shearer, Todd W; Clark, Matthew A; Cuozzo, John W; Arico-Muendel, Christopher C; Morgan, Barry A

    2012-08-23

    The metalloprotease ADAMTS-5 is considered a potential target for the treatment of osteoarthritis. To identify selective inhibitors of ADAMTS-5, we employed encoded library technology (ELT), which enables affinity selection of small molecule binders from complex mixtures by DNA tagging. Selection of ADAMTS-5 against a four-billion member ELT library led to a novel inhibitor scaffold not containing a classical zinc-binding functionality. One exemplar, (R)-N-((1-(4-(but-3-en-1-ylamino)-6-(((2-(thiophen-2-yl)thiazol-4-yl)methyl)amino)-1,3,5-triazin-2-yl)pyrrolidin-2-yl)methyl)-4-propylbenzenesulfonamide (8), inhibited ADAMTS-5 with IC(50) = 30 nM, showing >50-fold selectivity against ADAMTS-4 and >1000-fold selectivity against ADAMTS-1, ADAMTS-13, MMP-13, and TACE. Extensive SAR studies showed that potency and physicochemical properties of the scaffold could be further improved. Furthermore, in a human osteoarthritis cartilage explant study, compounds 8 and 15f inhibited aggrecanase-mediated (374)ARGS neoepitope release from aggrecan and glycosaminoglycan in response to IL-1β/OSM stimulation. This study provides the first small molecule evidence for the critical role of ADAMTS-5 in human cartilage degradation.

  9. Variable domain antibodies specific for viral hemorrhagic septicemia virus (VHSV) selected from a randomized IgNAR phage display library.

    Science.gov (United States)

    Ohtani, Maki; Hikima, Jun-ichi; Jung, Tae-Sung; Kondo, Hidehiro; Hirono, Ikuo; Takeyama, Haruko; Aoki, Takashi

    2013-02-01

    Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 × His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.

  10. Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis.

    Science.gov (United States)

    Hairul Bahara, Nur Hidayah; Chin, Siang Tean; Choong, Yee Siew; Lim, Theam Soon

    2016-01-01

    The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline. In short, the semisynthetic library generated was able to select monoclonal VH domain antibodies against full MTb α-crystalline protein with complete semisynthetic CDRs displayed on a single scaffold. The library has the potential to be applied for the isolation of antibodies against other pathogenic proteins.

  11. The participatory public library

    DEFF Research Database (Denmark)

    Rasmussen, Casper Hvenegaard

    2016-01-01

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

  12. The participatory public library

    DEFF Research Database (Denmark)

    Rasmussen, Casper Hvenegaard

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  14. Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

    Energy Technology Data Exchange (ETDEWEB)

    Peabody, David S.; Chackerian, Bryce; Ashley, Carlee; Carnes, Eric; Negrete, Oscar

    2017-01-24

    The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referred to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.

  15. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

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

  17. Mutational library analysis of selected amino acids in the receptor binding domain of envelope of Akv murine leukemia virus by conditionally replication competent bicistronic vectors

    DEFF Research Database (Denmark)

    Bahrami, Shervin; Pedersen, Finn Skou; Duch, Mogens R.

    2003-01-01

    The envelope protein of retroviruses is responsible for viral entry into host cells. Here, we describe a mutational library approach to dissect functional domains of the envelope protein involving a retroviral vector, which expresses both the envelope protein of Akv murine leukemia virus (MLV...... mutational library of Arg 85 and Asp 86 in the first variable region of Akv envelope protein. Homologous amino acids to Asp 86 in Moloney and Friend murine leukemia viruses are thought to be directly involved in receptor binding. Subsequent selection of mutants capable of infecting murine NIH 3T3 cells...... indicated that the wild type aspartic acid or another hydrophilic residue at position 86 is an important determinant for envelope function....

  18. Library news

    CERN Multimedia

    CERN Library

    2010-01-01

    The CERN Library has been providing electronic access to the "Techniques de l'Ingénieur" database for the past 8 months. As a reminder, this is a multidisciplinary database of over 4000 technical and scientific articles in French, covering a broad range of topics such as mechanical engineering, safety, electronics and the environment. In a few simple steps, you can create your own account, select the types of documents you are interested in and configure your settings so as to receive alerts when articles in your field of activity are published. You can now access this resource from outside CERN using the "remote access to electronic resources" service. Further information is available here. Direct access to the database. Remote access to electronic resources. If you have any questions or comments, don't hesitate to contact us at: library.desk@cern.ch.

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

    Science.gov (United States)

    Shen

    1999-01-01

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

  20. Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Glutathione peroxidase (GPX) plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening. Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immunosorbent assay(ELISA) analysis with 4 rounds of selection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester(GSH-s-DNP-Bu) and S-2,4-dinitriphenyl t-hexyl ester(GSH-s-DNP-He). Nevertheless, several studies need to be conducted to determine whether scFv-B8 and scFv-H6 possess GPX activity. To enhance the speed of the selection, selenocysteine(Sec, the catalytic group of GPX) was incorporated directly into the phages, scFv-B8 and scFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-scFv with GPX activity.

  1. cDNA cloning of the beta subunit of teleost thyrotropin.

    OpenAIRE

    Ito, M.; Koide, Y.; Takamatsu, N; Kawauchi, H.; Shiba, T.

    1993-01-01

    cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human fo...

  2. 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...

  3. Selecting and Using Information Sources: Source Preferences and Information Pathways of Israeli Library and Information Science Students

    Science.gov (United States)

    Bronstein, Jenny

    2010-01-01

    Introduction: The study investigated the source preference criteria of library and information science students for their academic and personal information needs. Method: The empirical study was based on two methods of data collection. Eighteen participants wrote a personal diary for four months in which they recorded search episodes and answered…

  4. Popular Music: A Selected Bibliography of Materials in the California State University, Sacramento Library. Bibliographic Series No. 22.

    Science.gov (United States)

    Smith, Donna Ridley, Comp.

    The bibliography lists over 400 works in the California State University Library, Sacramento, on pop, rock, country, folk, blues, and soul music from 1950 to the present. Books, periodicals, and non-book materials noted in the bibliography are appropriate for history, communication studies, and popular culture studies as well as for music. Items…

  5. Public Library Internet Services and the Digital Divide: The Role and Impacts from Selected External Funding Sources.

    Science.gov (United States)

    McClure, Charles R.; Ryan, Joe; Bertot, John Carlo

    This study examines the role of external funding in the development of public library network resources and services, their identification, and their benefit and impact, particularly as they affect the digital divide. The study was conducted between February 2001 and January 2002, employing a range of data collection techniques, including site…

  6. 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.

  7. 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.

  8. Brighton's Toy Library.

    Science.gov (United States)

    Rand, Duncan

    1978-01-01

    Describes a successful toy library in a public library in terms of staffing and patrons, toy selection, toy breakage problem, and sources of toys. In addition to children, users include an adult literacy group, a school for the mentally handicapped, the local social services department, and home for the elderly. (JAB)

  9. Trotsky's Vision of Libraries.

    Science.gov (United States)

    Baker, William; Hurych, Jitka

    1991-01-01

    Discusses Leon Trotsky's views on libraries, librarians, and librarianship in Soviet society in the 1920s as expressed in a speech, "Leninism and Library Work." Topics discussed include role of librarian in promoting literacy, including the use of maps and reference books to explain newspaper stories; the selection of books; and fighting…

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

    Directory of Open Access Journals (Sweden)

    Koopmann Birger

    2005-04-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  12. Using focus groups to investigate service quality determinants for customer satisfaction in selected university libraries in Sri Lanka

    Directory of Open Access Journals (Sweden)

    Chaminda Jayasundara

    2010-01-01

    Full Text Available This study aimed at establishing service quality determinants which may affect customer satisfaction in university libraries in Sri Lanka. Using the literature, 113 service quality determinants were identified. These were then reviewed by eight focus groups in four different universities. Forty of the determinants were perceived to be applicable to their context. The participants also added 14 quality requirements which they thought were not provided for in the list. Finally, the content and face validity of the 54 determinants were evaluated by a panel of experts who ultimately reduced them to 50. This study recommends the use of the identified quality determinants by library administrators and policymakers in the higher education sector in Sri Lanka to gauge the levels of customer satisfaction and assure quality of service.

  13. Selected Aspects Of Modelling Of Non-Linear Behaviour Of Concrete During Tensile Test Using Multiplas Library

    Directory of Open Access Journals (Sweden)

    Hokeš Filip

    2015-12-01

    Full Text Available The subject of this paper is to describe some of the aspects manifesting in the use of the elastoplastic material model library multiPlas, which was developed to support non-linear computations in the ANSYS system. The text focuses on the analysis of numerical simulations of a virtual tension test in several case studies, thereby the text endeavours to describe the problems connected with modelling non-linear behaviour of concrete in a tensile area.

  14. Screening of a chemical library by HT-G4-FID for discovery of selective G-quadruplex binders.

    Science.gov (United States)

    Largy, Eric; Saettel, Nicolas; Hamon, Florian; Dubruille, Sylvie; Teulade-Fichou, Marie-Paule

    2012-01-01

    Due to the lack of structural guidelines about G-quadruplex ligands, rational design cannot be the only approach to discover potent G4-ligands. As a complementary approach, screening of chemical library may provide interesting scaffolds known as hits provided that specific tools are available. In this work, the Institut Curie-CNRS chemical library was firstly screened by chemoinformatics methods. Similarity estimations by comparison with reference compounds (Phen-DC3, 360A, MMQ12) provided a set of molecules, which were then evaluated by high-throughput G4-FID (HT-G4-FID) against various G-quadruplex DNA. A full investigation of the most interesting molecules, using the HT-G4-FID assay and molecular modeling, supplied an interesting structure-activity relationship confirming the efficiency of this general approach. Overall, we demonstrated that HT-G4-FID coupled with screening of chemical libraries is a powerful tool to identify new G4-DNA binding scaffolds.

  15. Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection: biotin biosynthesis in the marine microorganism Chromohalobacter.

    Science.gov (United States)

    Kim, Eun Jin; Angell, Scott; Janes, Jeff; Watanabe, Coran M H

    2008-06-01

    Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.

  16. Characterisation of full-length cDNA sequences provides insights into the Eimeria tenellatranscriptome

    Directory of Open Access Journals (Sweden)

    Amiruddin Nadzirah

    2012-01-01

    Full Text Available Abstract Background Eimeria tenella is an apicomplexan parasite that causes coccidiosis in the domestic fowl. Infection with this parasite is diagnosed frequently in intensively reared poultry and its control is usually accorded a high priority, especially in chickens raised for meat. Prophylactic chemotherapy has been the primary method used for the control of coccidiosis. However, drug efficacy can be compromised by drug-resistant parasites and the lack of new drugs highlights demands for alternative control strategies including vaccination. In the long term, sustainable control of coccidiosis will most likely be achieved through integrated drug and vaccination programmes. Characterisation of the E. tenella transcriptome may provide a better understanding of the biology of the parasite and aid in the development of a more effective control for coccidiosis. Results More than 15,000 partial sequences were generated from the 5' and 3' ends of clones randomly selected from an E. tenella second generation merozoite full-length cDNA library. Clustering of these sequences produced 1,529 unique transcripts (UTs. Based on the transcript assembly and subsequently primer walking, 433 full-length cDNA sequences were successfully generated. These sequences varied in length, ranging from 441 bp to 3,083 bp, with an average size of 1,647 bp. Simple sequence repeat (SSR analysis identified CAG as the most abundant trinucleotide motif, while codon usage analysis revealed that the ten most infrequently used codons in E. tenella are UAU, UGU, GUA, CAU, AUA, CGA, UUA, CUA, CGU and AGU. Subsequent analysis of the E. tenella complete coding sequences identified 25 putative secretory and 60 putative surface proteins, all of which are now rational candidates for development as recombinant vaccines or drug targets in the effort to control avian coccidiosis. Conclusions This paper describes the generation and characterisation of full-length cDNA sequences from E

  17. 5' coding region of the follicular epithelium yolk polypeptide 2 cDNA in the moth, Plodia interpunctella, contains an extended coding region.

    Science.gov (United States)

    Shirk, P D; Perera, O P

    1998-01-01

    The 5' region of YP2 cDNA, a follicular epithelium yolk protein subunit in the moth, Plodia interpunctella, shows that the polypeptide contains an extended internal coding region. Partial cDNA clones for YP2 were isolated from a pharate adult female ovarian cDNA expression library in Lambda Zap II by screening with antigen selected YP2 antiserum. The 5' sequence of the YP2 transcript was determined by 5' RACE PCR of ovarian mRNA using YP2 sequence-specific nested primers. The combined cDNA and 5' RACE sequencing showed the YP2 transcript to be 1971 bp in length up to the poly(A) tail with a single open reading frame for a predicted polypeptide of 616 amino acids. Northern analysis showed a single YP2 transcript to be present in ovarian RNA that was approximately 2 kb in length. The predicted amino acid sequence for YP2 from P. interpunctella is most closely related to egg specific protein (ESP) from Bombyx mori and the partial YP2 sequence from Galleria mellonella. YP2 from P. interpunctella also is similar to vertebrate lipases and contains a conserved lipid binding region. However, the 5' coding region of YP2 from P. interpunctella contains an in-frame insert of approximately 438 bp that had replaced an approximately 270-bp region as compared with ESP from B. mori and YP2 of G. mellonella. This suggests that the insert occurred by a recombinational event internal to the YP2 structural gene of P. interpunctella.

  18. 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.

  19. 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

  20. Characterization of the porcine carboxypeptidase E cDNA

    DEFF Research Database (Denmark)

    Hreidarsdôttir, G.E.; Cirera, Susanna; Fredholm, Merete

    2007-01-01

    Carboxypeptidase E (CPE) is an important enzyme responsible for the proteolytic processing of prohormone intermediates. A naturally occurring point mutation, leading to an accumulation of many neuroendocrine peptides has been characterized within exon 5 of the CPE gene in mice. In the present study...... the sequence of the cDNA for the porcine CPE gene including all the coding region and the 3'-UTR region was generated. Comparisons with bovine, human, mouse, and rat CPE cDNA sequences showed that the coding regions of the gene are highly conserved both at the nucleotide and at the amino acid level. A very low...... nonsynonymousl/synonymous substitution ratios between the proteins was found indicating that purifying selection os acting on the CPE gene. A nonsynonymous SNP identified at position 1272 in the transcript resulting in a codon change from TCA (Serine) to TTA (Leucine) was genotyped in the Danish pig populations...

  1. Managing Quality: TQM in Libraries.

    Science.gov (United States)

    Riggs, Donald E.

    1993-01-01

    Discusses the use of total quality management (TQM) principles in libraries. Topics addressed include commitment from the library director; strategic planning; training needs for library staff; the identification and selection of targeted areas that would benefit from TQM; quality improvement teams; analysis; proposed solutions; and evaluation…

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

    Lifescience Database Archive (English)

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

  3. Library Automation

    OpenAIRE

    Dhakne, B. N.; Giri, V. V.; Waghmode, S. S.

    2010-01-01

    New technologies library provides several new materials, media and mode of storing and communicating the information. Library Automation reduces the drudgery of repeated manual efforts in library routine. By use of library automation collection, Storage, Administration, Processing, Preservation and communication etc.

  4. Characterization of cDNA encoding human placental anticoagulant protein (PP4): Homology with the lipocortin family

    Energy Technology Data Exchange (ETDEWEB)

    Grundmann, U.; Abel, K.J.; Bohn, H.; Loebermann, H.; Lottspeich, F.; Kuepper, H. (Research Institutes, Postfach (West Germany))

    1988-06-01

    A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10{sup 6} independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA the authors identified 9 other recombinants encoding a protein with considerable similarity (74%) to PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I.

  5. Screening of proteins binding to mouse cytomegalovirus M122 protein from mouse brain cDNA library by yeast two-hybrid system%酵母双杂交技术筛选小鼠脑cDNA文库中鼠巨细胞病毒即刻早期蛋白M122相互作用蛋白的研究

    Institute of Scientific and Technical Information of China (English)

    王慧; 周玉峰; 舒赛男; 罗丹; 田佳; 张慧娟; 杜小弋; 方峰

    2010-01-01

    histone acetyltransferase(monocytic leukemia)4,Myst4]、透明质酸和蛋白多糖连接蛋白1(hyaluronan and proteoglycan link protein 1,Hapln1)、自噬相关蛋白3(autophagy-related 3,Atg3)、精氨酸/色氨酸富集的剪切因子5(splicing factor,arginine/serine-rich 5,Sfrs5)、C3HC型锌指蛋白(zinc finger,C3HC-type containing 1,Zc3hc1)、硫氧还蛋白相关的跨膜蛋白1(thioredoxin-related transmembrane protein 1,Txndc1)、接头蛋白复合物AP-1亚单位(adaptor protein complex AP-1,gamma 1 subunit,Aplg1)和Cul1蛋白(cullin 1,Cul1).回返验证实验进一步证实这些蛋白与M122蛋白能够在酵母细胞AH109发生相互作用,但Aplg1和Cul1被证实具有自激活作用.结论 筛选到的其中19种已知基因编码的蛋白可能与巨细胞病毒的致病机制相关,但仍需进一步的验证.%Objective To investigate the biological function of M122 in pathogenesis of MCMV in developmental brain disorders and brain damage, screening for mouse brain cDNA library interacting with M122 was performed by a yeast two-hybrid system. Methods The reconstructed bait plasmid pGBKT7-M122 was transformed into yeast cells AH109 and screened on the nutrient deficiency medium SD/-Trp. After express of the bait protein in AH109 yeast strains was detected by Western blot analysis, yeast-two hybrid screening was performed by mating AH109 with Y187 containing mouse brain cDNA library plasmid. The diploid yeast cells were plated on the nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade. The second screening was performed with SD/-Trp/-Leu/-His/-Ade containing X-α-gal. The plasmids in positive colonies were extracted and transformed into E. coli JM109 cells. After plasmid DNA in JM109 cells were extracted form positive colonies and sequenced, the results were analyzed by bioinformatic methods. The interactions between M122 protein and the protein obtained from positive colonies were further confirmed by repeating yeast-two hybrid. Then, autoactivations of

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

    NARCIS (Netherlands)

    Lücker, J.; El Tamer, M.K.; Schwab, W.; Verstappen, F.W.A.; Plas, van der L.H.W.; Bouwmeester, H.J.; Verhoeven, H.A.

    2002-01-01

    Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the pe

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-01-15

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

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

    Science.gov (United States)

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

    1993-04-15

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2000-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    李权; 闻宏; 敖世洲

    2000-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  13. Integrated Library Systems in Canadian Public, Academic and Special Libraries: Fourth Annual Survey.

    Science.gov (United States)

    Merilees, Bobbie

    1990-01-01

    Reports the results of a survey of integrated library system vendors that examined installations in Canadian academic, public and special libraries during 1989. Findings discussed include large library system versus PC-based system market shares, an analysis of system selection by type of library, and other factors that affect system selection. A…

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

    Directory of Open Access Journals (Sweden)

    Waddington David

    2006-09-01

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

  15. Adaptive Correction from Virtually Complex Dynamic Libraries: The Role of Noncovalent Interactions in Structural Selection and Folding.

    Science.gov (United States)

    Lafuente, Maria; Atcher, Joan; Solà, Jordi; Alfonso, Ignacio

    2015-11-16

    The hierarchical self-assembling of complex molecular systems is dictated by the chemical and structural information stored in their components. This information can be expressed through an adaptive process that determines the structurally fittest assembly under given environmental conditions. We have set up complex disulfide-based dynamic covalent libraries of chemically and topologically diverse pseudopeptidic compounds. We show how the reaction evolves from very complex mixtures at short reaction times to the almost exclusive formation of a major compound, through the establishment of intramolecular noncovalent interactions. Our experiments demonstrate that the systems evolve through error-check and error-correction processes. The nature of these interactions, the importance of the folding and the effects of the environment are also discussed.

  16. Human IgA-binding peptides selected from random peptide libraries: affinity maturation and application in IgA purification.

    Science.gov (United States)

    Hatanaka, Takaaki; Ohzono, Shinji; Park, Mirae; Sakamoto, Kotaro; Tsukamoto, Shogo; Sugita, Ryohei; Ishitobi, Hiroyuki; Mori, Toshiyuki; Ito, Osamu; Sorajo, Koichi; Sugimura, Kazuhisa; Ham, Sihyun; Ito, Yuji

    2012-12-14

    Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random peptide libraries constructed with the T7 phage display system to isolate peptides specific to human IgA. The binding clones (A1-A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic peptide derived from the A2 clone exhibited a low specificity/affinity (K(d) = 1.3 μm). Therefore, we tried to improve the peptide using a partial randomized phage display library and mutational studies on the synthetic peptides. The designed Opt-1 peptide exhibited a 39-fold higher affinity (K(d) = 33 nm) than the A2 peptide. An Opt-1 peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating nonspecific binding. To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water. The simulation results revealed that the Opt-1 peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to nonspecific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination.

  17. Selection and fine mapping of chromosome-specific cDNAs: application to human chromosome 1.

    Science.gov (United States)

    Mancini, M; Sala, C; Rivella, S; Toniolo, D

    1996-12-01

    We have developed a methodology for identification and fine mapping of chromosome-specific transcripts. Combining digestion of DNA with different restriction enzymes, ligation to "bubble" linkers, and PCR amplification from Alu and "bubble" primers, we have synthesized human chromosome 1-specific sequences from DNA of a somatic cell hybrid, A9Neol. After hybridization to human fetal brain cDNA, we could efficiently capture chromosome 1-specific cDNAs. The cDNAs were sequenced and used as probes in hybridizations to high-density filters containing the arrayed CEPH Mega-YAC library and to the arrayed cDNA library from infant brain made by B. Soares, which has been extensively sequenced. By this approach we have been able to select chromosome 1-specific cDNAs, to map them to chromosome 1 YAC contigs, and to identify and map corresponding longer cDNAs and ESTs.

  18. Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

    Science.gov (United States)

    Wu, Zining; Graybill, Todd L; Zeng, Xin; Platchek, Michael; Zhang, Jean; Bodmer, Vera Q; Wisnoski, David D; Deng, Jianghe; Coppo, Frank T; Yao, Gang; Tamburino, Alex; Scavello, Genaro; Franklin, G Joseph; Mataruse, Sibongile; Bedard, Katie L; Ding, Yun; Chai, Jing; Summerfield, Jennifer; Centrella, Paolo A; Messer, Jeffrey A; Pope, Andrew J; Israel, David I

    2015-12-14

    DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

  19. Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen.

    Science.gov (United States)

    Lang, Qiaolin; Wang, Fei; Yin, Long; Liu, Mingjun; Petrenko, Valery A; Liu, Aihua

    2014-03-01

    Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

  20. Induction of immunity in sheep to Fasciola hepatica with mimotopes of cathepsin L selected from a phage display library.

    Science.gov (United States)

    Villa-Mancera, A; Quiroz-Romero, H; Correa, D; Ibarra, F; Reyes-Pérez, M; Reyes-Vivas, H; López-Velázquez, G; Gazarian, K; Gazarian, T; Alonso, R A

    2008-10-01

    An M13 phage random 12-mers peptide library was used to screen cathepsin L mimotopes of Fasciola hepatica and to evaluate their immunogenicity in sheep. Seven clones showed positive reactivity to a rabbit anti-cathepsin L1/L2 antiserum in ELISA, and their amino acid sequences deduced by DNA sequencing were tentatively mapped on the protein. Twenty sheep were randomly allocated into 4 groups of 5 animals each, for immunization with 1x10(14) phage particles of clones 1, 20, a mixture of 7 clones and PBS, without adjuvant at the beginning, and 4 weeks later. All groups were challenged with 300 metacercariae at week 6 and slaughtered 16 weeks later. The mean worm burdens after challenge were reduced by 47.61% and 33.91% in sheep vaccinated with clones 1 and 20, respectively; no effect was observed in animals inoculated with the clone mixture. Also, a significant reduction in worm size and burden was observed for those sheep immunized with clone 1. Animals receiving clone 20, showed a significant reduction in egg output. Immunization induced a reduction of egg viability ranging from 58.92 to 82.11%. Furthermore, vaccinated animals produced clone-specific antibodies which were boosted after challenge with metacercariae of F. hepatica.

  1. Biomedical Libraries

    Science.gov (United States)

    Pizer, Irwin H.

    1978-01-01

    Biomedical libraries are discussed as a distinct and specialized group of special libraries and their unique services and user interactions are described. The move toward professional standards, as evidenced by the Medical Library Association's new certification program, and the current state of development for a new section of IFLA established…

  2. Digital Libraries

    CERN Document Server

    Papy, Fabrice

    2008-01-01

    Of vital interest to all librarians and information specialists, this book presents all aspects of the effects of digitization of today's and tomorrow's libraries. From social to technical issues, Digital Libraries includes chapters on the growth of the role of librarian, the reader experience, cataloging, search engines, OPAC, law, ergonomic studies, and the future of libraries.

  3. Evolutional selection of a combinatorial phage library displaying randomly-rearranged various single domains of immunoglobulin (Ig-binding proteins (IBPs with four kinds of Ig molecules

    Directory of Open Access Journals (Sweden)

    Jia Jian-An

    2008-08-01

    Full Text Available Abstract Background Protein A, protein G and protein L are three well-defined immunoglobulin (Ig-binding proteins (IBPs, which show affinity for specific sites on Ig of mammalian hosts. Although the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. The single domains of protein A, protein G and protein L were all demonstrated to have function to bind to Ig. Whether combinations of Ig-binding domains of various IBPs could exhibit useful novel binding is interesting. Results We used a combinatorial phage library which displayed randomly-rearranged various-peptide-linked molecules of D and A domains of protein A, designated PA(D and PA(A respectively, B2 domain of protein G (PG and B3 domain of protein L (PL for affinity selection with human IgG (hIgG, human IgM (hIgM, human IgA (hIgA and recombinant hIgG1-Fc as bait respectively. Two kinds of novel combinatorial molecules with characteristic structure of PA(A-PG and PA(A-PL were obtained in hIgG (hIgG1-Fc and hIgM (hIgA post-selection populations respectively. In addition, the linking peptides among all PA(A-PG and PA(A-PL structures was strongly selected, and showed interestingly divergent and convergent distribution. The phage binding assays and competitive inhibition experiments demonstrated that PA(A-PG and PA(A-PL combinations possess comparable binding advantages with hIgG/hIgG1-Fc and hIgM/hIgA respectively. Conclusion In this work, a combinatorial phage library displaying Ig-binding domains of protein A, protein G, or protein L joined by various random linking peptides was used to conducted evolutional selection in vitro with four kinds of Ig molecules. Two kinds of novel combinations of Ig-binding domains, PA(A-PG and PA(A-PL, were obtained, and demonstrate the novel Ig binding properties.

  4. 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.

  5. Universal strategies for the DNA-encoding of libraries of small molecules using the chemical ligation of oligonucleotide tags.

    Science.gov (United States)

    Litovchick, Alexander; Clark, Matthew A; Keefe, Anthony D

    2014-01-01

    The affinity-mediated selection of large libraries of DNA-encoded small molecules is increasingly being used to initiate drug discovery programs. We present universal methods for the encoding of such libraries using the chemical ligation of oligonucleotides. These methods may be used to record the chemical history of individual library members during combinatorial synthesis processes. We demonstrate three different chemical ligation methods as examples of information recording processes (writing) for such libraries and two different cDNA-generation methods as examples of information retrieval processes (reading) from such libraries. The example writing methods include uncatalyzed and Cu(I)-catalyzed alkyne-azide cycloadditions and a novel photochemical thymidine-psoralen cycloaddition. The first reading method "relay primer-dependent bypass" utilizes a relay primer that hybridizes across a chemical ligation junction embedded in a fixed-sequence and is extended at its 3'-terminus prior to ligation to adjacent oligonucleotides. The second reading method "repeat-dependent bypass" utilizes chemical ligation junctions that are flanked by repeated sequences. The upstream repeat is copied prior to a rearrangement event during which the 3'-terminus of the cDNA hybridizes to the downstream repeat and polymerization continues. In principle these reading methods may be used with any ligation chemistry and offer universal strategies for the encoding (writing) and interpretation (reading) of DNA-encoded chemical libraries.

  6. Public Library Trends Analysis: Fiscal Years 1992-1996.

    Science.gov (United States)

    Glover, Denise

    2001-01-01

    Identifies and describes trends in public libraries for 24 selected variables, including library collections, services, operating income and expenditures, and staffing. Data are from the National Center for Education Statistics Public Libraries Survey. (Author/SLD)

  7. Nucleotide sequence of cDNA coding for dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus.

    Science.gov (United States)

    Legname, G; Bellosta, P; Gromo, G; Modena, D; Keen, J N; Roberts, L M; Lord, J M

    1991-08-27

    Rabbit antibodies raised against dianthin 30, a ribosome inactivating protein from carnation (Dianthus caryophyllus) leaves, were used to identify a full length dianthin precursor cDNA clone from a lambda gt11 expression library. N-terminal amino acid sequencing of purified dianthin 30 and dianthin 32 confirmed that the clone encoded dianthin 30. The cDNA was 1153 basepairs in length and encoded a precursor protein of 293 amino acid residues. The first 23 N-terminal amino acids of the precursor represented the signal sequence. The protein contained a carboxy-terminal region which, by analogy with barley lectin, may contain a vacuolar targeting signal.

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

    OpenAIRE

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

    1987-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-09-01

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

  10. Efficient and simple protocol employing borohydride systems to design a selective osthol-zirconium (OST-Zr library from potential natural products

    Directory of Open Access Journals (Sweden)

    Radhakrishnan Viswanathan

    2016-04-01

    Full Text Available “Drug likeness” of a molecule is the prime criterion for a molecule to exhibit the desired pharmaceutical activity. A pharmacophore, which describes molecular features that are necessary for molecular recognition of a ligand by a biological macromolecule, is well altered by the Structure Activity Relationship (SAR guidelines through Hydrophobic Lipophilic Balance (HLB demonstrated by the system. The tailoring is best accomplished by organic functional group interconversion on a potent natural product via a variety of synthetic methodologies available to date. Metal borohydrides (MBH4 in particular are promising compounds as they can potentially serve varying HLB systems. The reagent acts on the substrate to cause reduction, hydroboration, or a combination of both outcomes for the purpose of rearrangement and fragmentation. Indeed, Zr(BH44 is expected to be more active and selective as a reducing agent compared to NaBH4. This study aims at evaluating zirconium borohydride (Zr(BH44 in tetrahydrofuran (THF as a reducing system to realize a more selective, meaningful and combinatorial osthol (OST library from potential natural products and attempt to alternate preparation of the same in THF from known metal borohydrides, limiting reduction of the metal center versus metathesis. 

  11. Deep sequencing and Circos analyses of antibody libraries reveal antigen-driven selection of Ig VH genes during HIV-1 infection.

    Science.gov (United States)

    Xiao, Madelyne; Prabakaran, Ponraj; Chen, Weizao; Kessing, Bailey; Dimitrov, Dimiter S

    2013-12-01

    The vast diversity of antibody repertoires is largely attributed to heavy chain (V(H)) recombination of variable (V), diversity (D) and joining (J) gene segments. We used 454 sequencing information of the variable domains of the antibody heavy chain repertoires from neonates, normal adults and an HIV-1-infected individual, to analyze, with Circos software, the VDJ pairing patterns at birth, adulthood and a time-dependent response to HIV-1 infection. Our comparative analyses of the Ig VDJ repertoires from these libraries indicated that, from birth to adulthood, VDJ recombination patterns remain the same with some slight changes, whereas some V(H) families are selected and preferentially expressed after long-term infection with HIV-1. We also demonstrated that the immune system responds to HIV-1 chronic infection by selectively expanding certain HV families in an attempt to combat infection. Our findings may have implications for understanding immune responses in pathology as well as for development of new therapeutics and vaccines.

  12. Identification of Selective Inhibitors of the Plasmodium falciparum Hexose Transporter PfHT by Screening Focused Libraries of Anti-Malarial Compounds.

    Directory of Open Access Journals (Sweden)

    Diana Ortiz

    Full Text Available Development of resistance against current antimalarial drugs necessitates the search for novel drugs that interact with different targets and have distinct mechanisms of action. Malaria parasites depend upon high levels of glucose uptake followed by inefficient metabolic utilization via the glycolytic pathway, and the Plasmodium falciparum hexose transporter PfHT, which mediates uptake of glucose, has thus been recognized as a promising drug target. This transporter is highly divergent from mammalian hexose transporters, and it appears to be a permease that is essential for parasite viability in intra-erythrocytic, mosquito, and liver stages of the parasite life cycle. An assay was developed that is appropriate for high throughput screening against PfHT based upon heterologous expression of PfHT in Leishmania mexicana parasites that are null mutants for their endogenous hexose transporters. Screening of two focused libraries of antimalarial compounds identified two such compounds that are high potency selective inhibitors of PfHT compared to human GLUT1. Additionally, 7 other compounds were identified that are lower potency and lower specificity PfHT inhibitors but might nonetheless serve as starting points for identification of analogs with more selective properties. These results further support the potential of PfHT as a novel drug target.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-10-31

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

  14. Libraries, Archives, and Museums Helping Create Futures: Building on Culture, Knowledge, and Information through Collaboration and Resource Sharing. Selected Papers from PIALA 2010, Pacific Islands Association of Libraries, Archives, and Museums Annual Conference (20th, Weno, Chuuk State, Federated States of Micronesia, November 15-19, 2010)

    Science.gov (United States)

    Drake, Paul B., Ed.

    2012-01-01

    This publication follows the tradition of publishing selected papers from Pacific Islands Association of Libraries, Archives and Museums (PIALA) annual conferences. This 20th annual conference was held in Weno, Chuuk State, Federated States of Micronesia, November 15-19, 2010. The volume begins with a listing of the members of the PIALA 2010…

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-04-01

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

  16. Adoption of Library 2.0 Functionalities by Academic Libraries and Users: A Knowledge Management Perspective

    Science.gov (United States)

    Kim, Yong-Mi; Abbas, June

    2010-01-01

    This study investigates the adoption of Library 2.0 functionalities by academic libraries and users through a knowledge management perspective. Based on randomly selected 230 academic library Web sites and 184 users, the authors found RSS and blogs are widely adopted by academic libraries while users widely utilized the bookmark function.…

  17. Library Architecture: Some Observations

    Directory of Open Access Journals (Sweden)

    Bernhard Fabian

    2002-07-01

    Full Text Available There are plenty of libraries (among them many university and research libraries which do not provide adequate work-places. Chairs may have been selected for their stylish look rather than for their physical comfort. Desk lamps may have been deemed unnecessary (they might have distorted the overall impression which the reading room was expected to make And so on. I keep wondering how many librarians have spent some time in their libraries as readers, and have assessed their reading rooms from the user’s point of view. Have they been in a cubicle? Or have they read a book under glaring neon lights? Do they know how well their air-conditioning works? I know a library in which the only window that can be opened is in the librarian’s office.

  18. America's Star Libraries: Top-Rated Libraries

    Science.gov (United States)

    Lance, Keith Curry; Lyons, Ray

    2009-01-01

    "Library Journal"'s national rating of public libraries, the "LJ" Index of Public Library Service 2009, Round 2, identifies 258 "star" libraries. Created by Keith Curry Lance and Ray Lyons and based on 2007 data from the IMLS, it rates 7,268 public libraries. The top libraries in each group get five, four, or three stars. All included libraries,…

  19. Library Locations

    Data.gov (United States)

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — Carnegie Library of Pittsburgh locations including address, coordinates, phone number, square footage, and standard operating hours.

  20. PvuII RFLP detected by a human. beta. ADH cDNA probe

    Energy Technology Data Exchange (ETDEWEB)

    Parsian, A.; Burgess, A.K.; Khan, M.A.; Devor, E.J. (Washington Univ. School of Medicine, St. Louis, MO (USA))

    1989-12-11

    A 0.97 kb cDNA (ADH12) fragment encoding human exons 7, 8, 9 of the ADH{sub 2} gene was isolated from an adult human liver cDNA library. The insert can be excised by Pst I digestion. Pvu II identifies a two-allele polymorphism with bands at 4.4 kb (A{sub 1}) and 3.0 kb (A{sub 2}) and invariant bands at 5.1, 4.0, 2.8, and 2.3 kb. It was localized on Chromosome 4q21-q25 by in situ hybridization. Co-dominant segregation was observed in 18 informative families.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-05-01

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

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

    Institute of Scientific and Technical Information of China (English)

    种康; 谭克辉; 黄华梁; 梁厚果

    1995-01-01

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

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

    DEFF Research Database (Denmark)

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

    1991-01-01

    A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C......-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed...

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

    Science.gov (United States)

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

    1990-09-25

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

  5. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

    Science.gov (United States)

    Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

    2016-02-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 ‑ 2.0 × 108 cells mL‑1), a low limit of detection (79 cells mL‑1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

  6. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library.

    Science.gov (United States)

    Han, Lei; Liu, Pei; Petrenko, Valery A; Liu, Aihua

    2016-02-24

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 10(2) - 2.0 × 10(8) cells mL(-1)), a low limit of detection (79 cells mL(-1), S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

  7. Cloning and sequencing of murine T3 gamma cDNA from a subtractive cDNA library.

    Science.gov (United States)

    Haser, W G; Saito, H; Koyama, T; Tonegawa, S

    1987-10-01

    The coding sequences of the murine and human T3 gamma chains are of identical length (182 amino acids) and contain a remarkable conservation of residues. The most striking observation is the high degree of homology between the murine and human cytosolic domains (89%), suggesting that the effector function of the T3 complex may be extremely similar or identical within human and murine lymphocytes. Both murine and human T lymphocytes can express two T3 gamma mRNA transcripts, suggesting that a second polyadenylation signal is present downstream. A poly(A) tail is not found in the 3' untranslated region of the murine gamma presented here, indicating that the murine clones analyzed represent mRNA generated by reading through the overlapping poly(A) signals at position 850-860 and possibly terminating at a position that would produce the 1.0 kb transcript.

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

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Knudsen, Steen

    2002-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jesionek-Kupnicka Dorota

    2009-08-01

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

  10. The Library Space Utilization Methodology.

    Science.gov (United States)

    Hall, Richard B.

    1978-01-01

    Describes the Library Space Utilization (LSU) methodology, which demonstrates that significant information about the functional requirements of a library can be measured and displayed in a quantitative and graphic form. It measures "spatial" relationships between selected functional divisions; it also determines how many people--staff and…

  11. Benchmarked Library Websites Comparative Study

    KAUST Repository

    Ramli, Rindra M.

    2015-01-01

    This presentation provides an analysis of services provided by the benchmarked library websites. The exploratory study includes comparison of these websites against a list of criterion and presents a list of services that are most commonly deployed by the selected websites. In addition to that, the investigators proposed a list of services that could be provided via the KAUST library website.

  12. Marketing Your Library: Outreach, Public Relations. An Annotated Selective Bibliography on the Theme of the ARLIS/SLA Conference (New York City, New York, April 28, 1990).

    Science.gov (United States)

    Penchansky, Mimi B., Comp.

    This annotated bibliography on public relations and libraries includes sources published primarily within the last decade, with the exception of a few key works published in earlier years. Arranged in topical sections, most of the sources originate from the library and information science literature; however, a few sources are included from the…

  13. Privatizing Libraries

    Science.gov (United States)

    Jerrard, Jane; Bolt, Nancy; Strege, Karen

    2012-01-01

    This timely special report from ALA Editions provides a succinct but comprehensive overview of the "privatization" of public libraries. It provides a history of the trend of local and state governments privatizing public services and assets, and then examines the history of public library privatization right up to the California legislation…

  14. Library Use

    DEFF Research Database (Denmark)

    Konzack, Lars

    2012-01-01

    A seminar paper about a survey of role-playing games in public libraries combined with three cases and a presentation of a model.......A seminar paper about a survey of role-playing games in public libraries combined with three cases and a presentation of a model....

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  16. PNA-encoded chemical libraries.

    Science.gov (United States)

    Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

    2015-06-01

    Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70.

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

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Aleksandr Zhernakov

    2017-03-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, E.K.; Horton, R.; Bloem, L.; Bach, R.; Williams, K.R.; Guha, A.; Kraus, J.; Lin, T.C.; Nemerson, Y.; Konigsberg, W.H.

    1987-08-01

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

  1. Libraries, Archives and Museums: What's in Them for Us? PIALA '98. Selected Papers from the Pacific Islands Association of Libraries and Archives Conference (8th, Tofol, Kosrae, Federated States of Micronesia, November 17-20, 1998).

    Science.gov (United States)

    Cohen, Arlene, Ed.

    This proceedings contains papers from the 1998 annual conference of the Pacific Islands Association of Libraries and Archives (PIALA). After welcoming remarks from Henry Robert and Isabel Rungrad, the following papers are included: "Sharing Our Successes, Discussing Our Future: A Survey of Pacific Collections Activities--Report from the…

  2. Consulting on Academic Library Buildings.

    Science.gov (United States)

    Mason, Ellsworth

    1980-01-01

    Addresses the following aspects of consulting on library buildings: new trends, the need for a consultant, selecting a consultant, timeliness in hiring, expectations, following through, and the cost of consulting. (FM)

  3. Sex, Kids, and the Public Library.

    Science.gov (United States)

    Mason, Marilyn Gell

    1997-01-01

    Considers issues relating to children's access to information on the Internet in public libraries. Topics include censorship versus selection; First Amendment rights; the American Library Association's challenge to the Communications Decency Act; filtering software; and the role of the public library. (LRW)

  4. The Challenges in Developing Academic Library Collections.

    Science.gov (United States)

    Sankowski, Andrew

    1987-01-01

    Defines collection development and discusses several issues related to this function in the academic library: (1) the importance of a collection development policy; (2) material selection; (3) faculty-library cooperation; (4) cooperation with other library departments; (5) weeding; and (6) effects of automation. Nine references are listed. (MES)

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

    Science.gov (United States)

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

    1988-08-01

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

  6. Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations.

    Science.gov (United States)

    Oikonomopoulos, Spyros; Wang, Yu Chang; Djambazian, Haig; Badescu, Dunarel; Ragoussis, Jiannis

    2016-08-24

    To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.

  7. Cloning a Full-length cDNA Encoding UDP-glucose Pyrophosphorylase from Amorpha fruticosa by PCR-based Methods

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A method based on degenerate Oligo-primed polymerase chain reaction (PCR) and random amplification of cDNA end (RACE) PCR for cloning a full-length cDNA is described. An Amorpha fruticosa cDNA clone encoding UDP-glucose pyrophosphorylase (UGP), a key enzyme producing UDP-glucose in the synthesis of sucrose and cell ulose, is cloned by using this method. We design 5' RACE primers based on UGP A1 fragment, which obtains from degenerate PCR. Inverse PCR and nested PCR enable cloning of the remainder 5' and 3' end fragments of the gene. The deduced amino acid sequence exhibits significant homology with the other UGP genes cloned. This method is more simple and inexpensive than screening cDNA library, and can be easily adapted to clone other genes.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-05-01

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

  9. 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.

  10. Selection of affinity-improved neutralizing human scFv against HBV PreS1 from CDR3 VH/VL mutant library.

    Science.gov (United States)

    Chen, YanMin; Bai, Yin; Guo, XiaoChen; Wang, WenFei; Zheng, Qi; Wang, FuXiang; Sun, Dejun; Li, DeShan; Ren, GuiPing; Yin, JieChao

    2016-07-01

    A CDR3 mutant library was constructed from a previously isolated anti-HBV neutralizing Homo sapiens scFv-31 template by random mutant primers PCR. Then the library was displayed on the inner membrane surface in Escherichia coli periplasmic space. Seven scFv clones were isolated from the mutant library through three rounds of screening by flow cytometry. Competition ELISA assay indicates that isolated scFv fragments show more efficient binding ability to HBV PreS1 compared with parental scFv-31. HBV neutralization assay indicated that two clones (scFv-3 and 59) show higher neutralizing activity by blocking the HBV infection to Chang liver cells. Our method provides a new strategy for rapid screening of mutant antibody library for affinity-enhanced scFv clones and the neutralizing scFvs obtained from this study provide a potential alternative of Hepatitis B immune globulin.

  11. Academic Libraries

    Science.gov (United States)

    Library Journal, 1970

    1970-01-01

    Building data is given for the following academic libraries: (1) Rosary College, River Forest, Illinois; (2) Abilene Christian College, Abilene, Texas; (3) University of California, San Diego, La Jolla, California. (MF)

  12. Screening of genomic libraries.

    Science.gov (United States)

    Novelli, Valdenice M; Cristofani-Yaly, Mariângela; Bastianel, Marinês; Palmieri, Dario A; Machado, Marcos A

    2013-01-01

    Microsatellites, or simple sequence repeats (SSRs), have proven to be an important molecular marker in plant genetics and breeding research. The main strategies to obtain these markers can be through genomic DNA and from expressed sequence tags (ESTs) from mRNA/cDNA libraries. Genetic studies using microsatellite markers have increased rapidly because they can be highly polymorphic, codominant markers and they show heterozygous conserved sequences. Here, we describe a methodology to obtain microsatellite using the enrichment library of DNA genomic sequences. This method is highly efficient to development microsatellite markers especially in plants that do not have available ESTs or genome databases. This methodology has been used to enrich SSR marker libraries in Citrus