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Sample records for cdna library screening

  1. High-Throughput Plasmid cDNA Library Screening

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    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.

  2. 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.

  3. 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.

  4. A human cDNA library for high-throughput protein expression screening.

    Science.gov (United States)

    Büssow, K; Nordhoff, E; Lübbert, C; Lehrach, H; Walter, G

    2000-04-01

    We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses. PMID:10777659

  5. Rapid and Efficient cDNA Library Screening by Self-Ligation ofInverse PCR Products (SLIP)

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    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.

  6. 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.

  7. Screening of a peanut (Arachis hypogaea L.) cDNA library to isolate a Bowman-Birk trypsin inhibitor clone.

    Science.gov (United States)

    Boateng, Judith A; Viquez, Olga M; Konan, Koffi N; Dodo, Hortense W

    2005-03-23

    Peanut crop losses due to insect and pest infestation cost peanut farmers nearly 20% of their annual yields. The conventional use of chemicals to combat this problem is costly and toxic to humans and livestock and leads to the development of resistance by target insects. Transgenic plants expressing a trypsin inhibitor gene in tobacco and cowpea have proven to be efficient for resistance against insects. Therefore, a transgenic peanut overexpressing a trypsin inhibitor gene could be an alternative solution to the use of toxic chemicals. Five Bowman-Birk trypsin inhibitor (BBTI) proteins were previously isolated from peanut. However, to date, neither cDNA nor genomic DNA sequences are available. The objective of this research was to screen a peanut cDNA library to isolate and sequence at least one full-length peanut BBTI cDNA clone. Two heterologous oligonucleotides were constructed on the basis of a garden pea (Pisum sativa) trypsin inhibitor nucleotide sequence and used as probes to screen a peanut lambda gt-11 cDNA library. Two positive and identical cDNA clones were isolated, subcloned into a pBluescript vector, and sequenced. Sequence analysis revealed a full-length BBTI cDNA of about 243 bp, with a start codon ATG at position +1 and a stop codon TGA at position +243. In the 3' end, two poly adenylation signals (AATAAA) were identified at positions +261 and +269. The isolated cDNA clone encodes a protein of 80 amino acid residues including a leader sequence of 11 amino acids. The deduced amino acid sequence is 100% identical to published sequences of peanut BBTI AI, AII, BI, and BIII and 81% identical to BII. PMID:15769131

  8. 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.

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

    OpenAIRE

    Gutjahr, C.; Murphy, D.; Lueking, A.; Koenig, A.; Janitz, M; O'Brien, J.; Korn, B. (Bernhard); S. Horn; Lehrach, H; Cahill, D.

    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...

  10. 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.

  11. 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. PMID:26658002

  12. 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.

  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)

    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.

  14. 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.

  15. 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

    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...... 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...

  16. 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.

  17. 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.

  18. Next-generation cDNA screening for oncogene and resistance phenotypes.

    Directory of Open Access Journals (Sweden)

    Nobuaki Shindoh

    Full Text Available There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo.

  19. Construction and identification of subtracted cDNA library in bone marrow cells of radon-exposed mice

    International Nuclear Information System (INIS)

    Objective: To construct and identify subtracted cDNA library in bone marrow cells of mice exposed to radon inhalation. Methods: Adult male BALB/c mice, weighing 18-22 g, were placed in a multi- functional radon chamber. One group of mice was exposed to radon up to the accumulative dose of 105 work level month (WLM). The control group of mice was housed in a room with an accumulative dose of 1 WLM. To construct a subtracted cDNA library enriched with differentially expressed genes, the SMART technique and the suppression subtractive hybridization were performed. The obtained forward and reverse cDNA fragments were directly inserted into pMD18-T vector and transformed into E. coli JM109. The inserting cDNA fragments were screened by the blue-and-white blot screening and nested PCR of bacterium liquid. Results: The 244 of 285 white bacteria clones obtained randomly were positive clones contained 100-1100 bp inserted cDNA fragments. Conclusions: The forward and reverse subtracted cDNA library in bone marrow cells of mice exposed to radon inhalation is successfully constructed. (authors)

  20. 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.

  1. CDNA library from the Latex of Hevea brasiliensis

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    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%.

  2. Identification of Novel Protein–Ligand Interactions by Exon Microarray Analysis of Yeast Surface Displayed cDNA Library Selection Outputs

    OpenAIRE

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    Yeast surface display is widely utilized to screen large libraries for proteins or protein fragments with specific binding properties. We have previously constructed and utilized yeast surface displayed human cDNA libraries to identify protein fragments that bind to various target ligands. Conventional approaches employ monoclonal screening and sequencing of polyclonal outputs that have been enriched for binding to a target molecule by several rounds of affinity-based selection. Frequently, a...

  3. 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

  4. The function analysis of full-length cDNA sequence from IRM-2 mouse cDNA library

    International Nuclear Information System (INIS)

    Objective: To identify the function of full-length cDNA sequence from IRM-2 mouse cDNA library. Methods: Full-length cDNA products were amplified by PCR from IRM-2 mouse cDNA library according to twenty-one pieces of expressed sequence tag. The expression of full-length cDNAs were detected after mouse embryonic fibroblasts were exposed to 6.5 Gy γ-ray radiation. And the effect on the growth of radiosensitivity cells AT5B1VA transfected with full-length cDNAs was investigated. Results: The expression of No.4, 5 and 2 full-length cDNAs from IRM-2 mouse were higher than that of parental ICR and 615 mouse after mouse embryonic fibroblasts irradiated with γ-ray radiation. And the survival rate of AT5B1VA cells transfected with No.4, 5 and 2 full-length cDNAs was high. Conclusion: No.4, 5 and 2 full-length cDNAs of IRM-2 mouse are of high radioresistance. (authors)

  5. Identification and molecular characterization of numerous Histomonas meleagridis proteins using a cDNA library.

    Science.gov (United States)

    Bilic, I; Leberl, M; Hess, M

    2009-04-01

    SUMMARYHistomonas meleagridis is a protozoan parasite of various galliform birds causing a type of enterohepatitis termed histomonosis or 'blackhead disease'. Due to the ban of chemotherapeutic substances and an increase in free-range poultry production, histomonosis is currently a re-emerging disease. So far limited molecular knowledge is available. In the present work, mRNAs coding for antigenic proteins of H. meleagridis were identified. For this purpose, a cDNA expression library was constructed from a mono-eukaryotic culture of H. meleagridis. The library was screened with polyclonal rabbit serum raised against purified H. meleagridis trophozoites. Polyclonal rabbit serum specifically recognized the same major H. meleagridis antigens as chicken and turkey sera originating from animal trials, but displayed a significantly lower bacteria-dependent background signal. After 2 rounds of screening, a total of 95 positive clones were sequenced. Bioinformatics analyses were performed on nucleotide and deduced amino acid sequences, identifying 37 unique clones. Based on the homology to other protozoan parasites, mostly Trichomonas vaginalis, the clones were grouped according to functional aspects: structural proteins, possible surface proteins, oxygen reducing proteins, ribosomal proteins, protein kinases and various other intracellular proteins. PMID:19154645

  6. 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.

  7. 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.

  8. Cloning vectors for expression of cDNA libraries in mammalian cells.

    OpenAIRE

    Murphy, A.J.; Efstratiadis, A

    1987-01-01

    We have constructed a series of compound cloning vectors (lambda ZD vectors), each consisting of phage lambda arms carrying a modified version of the retroviral expression vector pZIP-neoSV (x)1. cDNA, inserted into a cloning site present in the retroviral vector component, is cloned with high efficiency using the lambda system. A cDNA library in plasmids is then released by homologous recombination between the retroviral long terminal repeats. Retroviral transduction is achieved by transient...

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

    OpenAIRE

    Stefanie Hartman; Greg Touchton; Jessica Wynn; Tuoyu Geng; Chong, Nelson W.; Ed Smith

    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 d...

  10. 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. PMID:26345909

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

    Science.gov (United States)

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

    2009-01-01

    Sequencing of cDNA libraries is an efficient and inexpensive approach to analyze the protein-coding portion of a genome. It is frequently used for surveying the genomes of poorly studied eukaryotes, and is particularly useful for species that are not easily amenable to genome sequencing, because they are nonaxenic and/or difficult to cultivate. In this chapter, we describe protocols that have been applied successfully to construct and normalize a variety of cDNA libraries from many different species of free-living protists and fungi, and that require only small quantities of cell material. PMID:19277563

  12. cDNA library Table: NRPG [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available NRPG NA NRPG p50 pheromone gland adult stage female pBluescript SK- EcoR1 for 5' Xh...o1for 3' sequenced from T3 primer (5' -> 3') BP182009-BP183529 NRPG[number] p50, Normalized Library ...

  13. 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 ...

  14. 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.

  15. 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.

  16. cDNA microarray screening in food safety

    International Nuclear Information System (INIS)

    The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests

  17. 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.

  18. The isolation of transcription factors from lambda gt11 cDNA expression libraries: human steroid 5 alpha-reductase 1 has sequence-specific DNA binding activity.

    OpenAIRE

    Gaston, K; Fried, M

    1992-01-01

    The Surf-1/Surf-2 bi-directional promoter contains binding sites for at least three transcription factors (Su1, Su2, and Su3). By screening a lambda gt11 HeLa cell cDNA expression library with a concatenated Su2 factor binding site, we isolated a cDNA which encodes a protein with sequence-specific DNA binding activity. Gel retardation assays showed that the cloned factor binds specifically to the Su2 factor binding site present in the human Surf-1/Surf-2 promoter but not to an Su2 site contai...

  19. Large-Scale Screens of Metagenomic Libraries

    OpenAIRE

    Pham, Vinh D.; Palden, Tsultrim; DeLong, Edward F.

    2007-01-01

    Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that...

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

    DEFF Research Database (Denmark)

    Rong, Mingqiang; Yang, Shilong; Wen, Bo;

    2015-01-01

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

  1. Identification and molecular characterization of numerous Histomonas meleagridis proteins using a cDNA library

    OpenAIRE

    Bilic, I.; LEBERL, M.; Hess, M.

    2009-01-01

    Histomonas meleagridis is a protozoan parasite of various galliform birds causing a type of enterohepatitis termed histomonosis or ‘blackhead disease’. Due to the ban of chemotherapeutic substances and an increase in free-range poultry production, histomonosis is currently a re-emerging disease. So far limited molecular knowledge is available. In the present work, mRNAs coding for antigenic proteins of H. meleagridis were identified. For this purpose, a cDNA expression library was constructed...

  2. Construction of Full-length cDNA Library of Non-diapause Pupae of the Onion Maggot, Delia antiqua

    Directory of Open Access Journals (Sweden)

    CHEN Bin

    2010-01-01

    Full Text Available The onion maggot, Delia antiqua has the characteristic of summer- and winter-diapause, and is close to Drosophila Melanogaster in phelogenetics. It is an ideal model species for the studies of the molecular mechanism of insect diapause and the comparison of winter- and summer-diapause-specific genes. The study aims to construct full-length cDNA library of summer-diapause pupae of the onion maggot, Delia antiqua, in order to play a base for further screening, cloning and expression analysis of diapause-specific genes. In this study, total RNA was extracted from non-diapause pupae of onion maggot, D. antiqua using RNAiso. Double-stranded cDNAs were synthesized with SMART technique and digested by SfiⅠ, and then the cDNAs were ligated into the vector pDNR-LIB. The ligation mixture was transformed into E. coli DH10B by eletroporation. According to the evaluation on quality, the titer of primary library was 2.3×107 cfu/mL. The results from random picking 15 clones showed that the inserted fragments ranged from 0.4 to 1.2 kb by PCR amplification, with an average size of 0.9 kb, and the recombination rate was 100 %. These results showed that a full-length cDNA library with high quality on Delia antiqua non-diapause pupae was well constructed. This indicates that the library is of high quality for cloning target genes and expressing target proteins.

  3. Rapid Discovery of Functional Small Molecule Ligands against Proteomic Targets through Library-Against-Library Screening.

    Science.gov (United States)

    Wu, Chun-Yi; Wang, Don-Hong; Wang, Xiaobing; Dixon, Seth M; Meng, Liping; Ahadi, Sara; Enter, Daniel H; Chen, Chao-Yu; Kato, Jason; Leon, Leonardo J; Ramirez, Laura M; Maeda, Yoshiko; Reis, Carolina F; Ribeiro, Brianna; Weems, Brittany; Kung, Hsing-Jien; Lam, Kit S

    2016-06-13

    Identifying "druggable" targets and their corresponding therapeutic agents are two fundamental challenges in drug discovery research. The one-bead-one-compound (OBOC) combinatorial library method has been developed to discover peptides or small molecules that bind to a specific target protein or elicit a specific cellular response. The phage display cDNA expression proteome library method has been employed to identify target proteins that interact with specific compounds. Here, we combined these two high-throughput approaches, efficiently interrogated approximately 10(13) possible molecular interactions, and identified 91 small molecule compound beads that interacted strongly with the phage library. Of 19 compounds resynthesized, 4 were cytotoxic against cancer cells; one of these compounds was found to interact with EIF5B and inhibit protein translation. As more binding pairs are confirmed and evaluated, the "library-against-library" screening approach and the resulting small molecule-protein domain interaction database may serve as a valuable tool for basic research and drug development. PMID:27053324

  4. Construction and screening of marine metagenomic libraries

    DEFF Research Database (Denmark)

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

    2010-01-01

    microbial consortia on marine tissues of multicellular organisms are rich sources for isolating novel bioactive compounds and genes. Here we describe the sampling, construction of large-insert metagenomic libraries from marine habitats and exemplarily one function based screen of metagenomic clones....

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Dixon Richard A

    2007-11-01

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

  7. Study on Wusan Granule Anti-tumor Related Target Gene Screened by Cdna Microarray

    Institute of Scientific and Technical Information of China (English)

    YOU Zi-li; SHI Jin-ping; CHEN Hai-hong

    2006-01-01

    To screen Wusan Granule anti-tumor related target gene using cDNA microarray technique, both mRNA from Lewis lung carcinoma tissues treated by Wusan Granule and untreated control are reversibly transcribed to prepare cDNA probes which are labeled by Cy5 and Cy3. Then, the probes are hybridized to the mice cDNA microarray type MGEC-20S. After hybridization, the cDNA microarray is scanned by ScanArray 3 000 scanner and the data is analyzed by ImaGene 3 software to screen the differentially expressed genes. There are 45 differentially expressed genes including 18 known genes and 27 unknown genes between the two groups, and among them, 20 elevated genes and 25 reduced genes are identified. Additionally, the genes related to invasion and metastasis of malignant carcinomas are down-regulated and the genes related to apoptosis are up-regulated. The cDNA microarray technique is a high-throughput approach to screen the Wusan Granule anti-tumor related target genes, which allow us to explore the molecular biological mechanism on a genomic scale.

  8. Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing

    OpenAIRE

    Hafner, Markus; Renwick, Neil; Farazi, Thalia A.; Mihailovi, Aleksandra; Pena, John T.G.; Tuschl, Thomas

    2012-01-01

    The characterization of post-transcriptional gene regulation by small regulatory (20–30 nt) RNAs, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for characterizing small RNAs is their identification and quantification across different developmental stages, and in normal and disease tissues, as well as model cell lines. Here we present a step-by-step protocol for generating barcoded small RNA cDNA libraries compatible with Illumina HiSeq se...

  9. Construction and Characterization of cDNA Library from Water-Stressed Plantlets Regenerated in vitro of Populus hopeiensis

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    In order to isolate and clone water-stress-responsive genes, total RNA was extracted from water-stressed plantlets regenerated in vitro of Populus hopeiensis using a QIAGEN RNeasy Plant Mini Kit. CDNA, synthesized by LD-PCR with the SMART cDNA Library Construction Kit, was in vitro packaged into a phage λTriplEx2 vector. The resulting primary library and amplified library have a titer of 1.68×106 and 1.69×109 pfu·mL-1 respectively. The combination ratio reached 98.8% and the average size of inserts was about 800 bp. In addition, the percentage of inserted fragments (> 400 bp) was approximately 90%. The results indicate that a cDNA library has been successfully constructed.

  10. 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.

  11. Preparation of cDNA libraries for high-throughput RNA sequencing analysis of RNA 5′ ends

    Science.gov (United States)

    Vvedenskaya, Irina O.; Goldman, Seth R.; Nickels, Bryce E.

    2015-01-01

    Summary We provide a detailed protocol for preparing cDNA libraries suitable for high throughput sequencing that are derived specifically from the 5′ ends of RNA (5′ specific RNA-seq). The protocol describes how cDNA libraries for 5′ specific RNA-seq can be tailored to analyze specific classes of RNAs based upon the phosphorylation status of the 5′ end. Thus, the analysis of cDNA libraries generated by these methods provides information regarding both the sequence and phosphorylation status of the 5′ ends of RNAs. 5′ specific RNA-seq can be used to analyze transcription initiation and post-transcriptional processing of RNAs with single base pair resolution on a genome-wide level. PMID:25665566

  12. Screening of TACE Peptide Inhibitors from Phage Display Peptide Library

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To obtain the recombinant tumor necrosis factor-α converting enzyme (TACE) ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RTPCR, and the expression plasmids were constructed by inserting T800 and T1300 into plasmid pET28a and pET-28c respectively. The recombinant T800 and T1300 were induced by IPTG, and SDSPAGE and Western blotting analysis results revealed that T800 and T1300 were highly expressed in the form of inclusion body. After Ni2+-NTA resin affinity chromatography, the recombinant proteins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence (TRWLVYFSRPYLVAT) was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3 %. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE.

  13. cDNA array screening differentially expressed gene in liver regeneration

    Institute of Scientific and Technical Information of China (English)

    强晖; 谢渭芬; 张忠兵; 张新; 张兴荣; 陈岳祥; 杨秀疆

    2003-01-01

    Objective: To screen the differentially expressed gene in liver regeneration with cDNA array and gain insight of the pathogenesis of the acute hepatic failure. Methods: Acute liver failure model in Sprague-Dawley (SD) rat was induced by 95% hepatectomy. The differential expression profiles in regenerating rat liver were studied by a cDNA array representing 1176 cDNA clusters. Some genes were randomly selected from a pool of differentially expressed genes and subjected to RT-PCR to further confirm the result from microarray hybridization. Results: The alterations of transcription levels were noted in the expressions of 188 genes. There were 138 genes with their expression up-regulated such as growth factors, PCNA, ribosomal proteins, IL6 and CDKs which were associated with cell cycle regulation, stress, metabolism and proliferation. Conclusion: cDNA array is a powerful tool to explore the gene expression of acute liver failure and is potential for the diagnosis and treatment of liver regeneration.

  14. In vitro recombination cloning of entire cDNA libraries in Arabidopsis thaliana and its application to the yeast two-hybrid system

    OpenAIRE

    Bürkle, L.; Meyer, S.; Dortay, H.; Lehrach, H; Heyl, A.

    2005-01-01

    In the postgenomic era many experiments rely on the availability of transcript sequence for cloning. As these clones usually originate from cDNA libraries, the quality of these libraries is crucial. If a good library is generated it is desirable to use a versatile cloning system suitable for many different kinds of applications. The cloning systems based on in vitro recombination proves fitting for this task. However, the use of this method for shuttling entire cDNA libraries between differen...

  15. Cheminformatics approaches to analyze diversity in compound screening libraries.

    Science.gov (United States)

    Akella, Lakshmi B; DeCaprio, David

    2010-06-01

    As high-throughput screening matures as a discipline, cheminformatics is playing an increasingly important role in selecting new compounds for diverse screening libraries. New visualization techniques such as multi-fusion similarity maps, scaffold trees, and principal moments of inertia plots provide complementary information on compound libraries and enable identification of unexplored regions of chemical space with potential biological relevance. Quantitative metrics have been developed to analyze libraries for properties such as natural product-likeness and shape complexity. Analysis of high-throughput screening results and drug discovery programs identify compounds problematic for screening. Taken together these approaches allow us to increase the diversity of biological outcomes available in compound screening libraries and improve the success rates of high-throughput screening against new targets without making significant increases in the size of compound libraries. PMID:20457001

  16. ESTS from skin and PBMC cDNA subtractive library of alpaca

    International Nuclear Information System (INIS)

    Full text: As an effort to map and identify genes and genetic markers that influence the fibre quality in alpacas, cDNA subtractive libraries of Alpaca (Vicugna pacos) were constructed in order to find differentially expressed genes in skin. Skin and blood samples were removed from six adult Alpaca (1.5 year old). Total RNA was extracted using Trizol (Invitrogen) and mRNA was purified using the Gene Elute mRNA purification kit (Sigma). Suppression PCR was used to construct the library using mRNA from skin as a tester and the mRNA from PBMC as a driver. The subtracted PCR products were inserted into the TA cloning vector and the ligation reaction was transformed into TOP10 E. coli cells. Randomly selected clones were sequenced and a total of 2280 high quality 5' end sequences were generated. Clustering analysis using StackPACK version 2.2.0 resulted in 1075 unique transcripts, consisting of 347 consensi and 728 singletons. BLAST analysis of the generated sequences revealed skin associated transcripts such as hair keratin 6A, keratin 10, keratin KA27, keratin 34, wool keratin microfibril type I, and collagen. A total of 27 microsatellite loci were also uncovered. Further work is in progress to generate more sequences in order to build an EST database of differentially expressed genes from Alpaca skin and PBMC, and for the generation of genetic molecular markers such as microsatellites and SNP for Alpaca. (author)

  17. 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.

  18. Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici☆

    Science.gov (United States)

    Ma, W.; Kilaru, S.; Collins, C.; Courbot, M.; Steinberg, G.

    2015-01-01

    Pathogenic fungi are constantly emerging resistance to anti-fungal treatments. Therefore, identification of new fungicide targets is important. Good candidates are essential fungal proteins and their regulators. An efficient way to reveal the molecular environment of an essential protein is the search for interacting factors. Here, we establish three yeast two-hybrid libraries, covering yeast and hyphal stages of the wheat pathogen Zymoseptoria tritici. No detectable genomic DNA was present in any of the 3 libraries. Random amplification revealed that the libraries include cDNA fragments of up to 2000 bp, suggesting that small-to-medium sized proteins are represented therein. Indeed, full-length cDNAs of five proteins were found in all libraries. The full-length cDNA of large chitin synthase gene mcs1 (5742 bp with introns; 5568 bp without introns) could not be amplified, but its 5′ and 3′ regions were represented, suggesting that even larger genes are covered in all libraries. Finally, we tested for the expected interaction of the autophagy proteins ZtAtg4 and ZtAtg8 in Z. tritici, and then used ZtAtg4 to screen one of the two-hybrid libraries. Indeed, we found ZtAtg8 as a positive interaction partner, confirming that interacting proteins can be identified. Thus, these molecular tools promise to be useful in identifying novel fungicide target proteins. PMID:26092795

  19. Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici.

    Science.gov (United States)

    Ma, W; Kilaru, S; Collins, C; Courbot, M; Steinberg, G

    2015-06-01

    Pathogenic fungi are constantly emerging resistance to anti-fungal treatments. Therefore, identification of new fungicide targets is important. Good candidates are essential fungal proteins and their regulators. An efficient way to reveal the molecular environment of an essential protein is the search for interacting factors. Here, we establish three yeast two-hybrid libraries, covering yeast and hyphal stages of the wheat pathogen Zymoseptoria tritici. No detectable genomic DNA was present in any of the 3 libraries. Random amplification revealed that the libraries include cDNA fragments of up to 2000bp, suggesting that small-to-medium sized proteins are represented therein. Indeed, full-length cDNAs of five proteins were found in all libraries. The full-length cDNA of large chitin synthase gene mcs1 (5742bp with introns; 5568bp without introns) could not be amplified, but its 5' and 3' regions were represented, suggesting that even larger genes are covered in all libraries. Finally, we tested for the expected interaction of the autophagy proteins ZtAtg4 and ZtAtg8 in Z. tritici, and then used ZtAtg4 to screen one of the two-hybrid libraries. Indeed, we found ZtAtg8 as a positive interaction partner, confirming that interacting proteins can be identified. Thus, these molecular tools promise to be useful in identifying novel fungicide target proteins. PMID:26092795

  20. The construction of a recombinant cDNA library representative of the poly(A)+ mRNA population from normal human lymphocytes.

    OpenAIRE

    Woods, D.; Crampton, J.; Clarke, B.; Williamson, R

    1980-01-01

    A recombinant library has been constructed using the plasmid pAT153 and double stranded cDNA prepared from normal human lymphocyte poly(A)+ RNA. Transformation conditions were optimized to yield approximately 200,000 recombinants per microgram of double stranded cDNA. Statistical analysis as well as sequence complexity analysis of the inserted sequences indicates that the cDNA library is representative of > 99% of the poly(A)+ RNA present in the normal human lymphocyte.

  1. Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library

    OpenAIRE

    Bhore, Subhash J.; Kassim, Amelia; Loh, Chye Ying; Shah, Farida H

    2010-01-01

    It is well known that the nutritional quality of the American oil-palm (Elaeis oleifera) mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we constructed a cDNA library and generated expres...

  2. cDNA library construction and isolation of genes for candidate vaccine antigens from Chrysomya bezziana (the Old World Screwworm fly

    Directory of Open Access Journals (Sweden)

    Tony Voucolo

    2000-10-01

    Full Text Available The construction and use of cDNA libraries for the isolation of genes encoding candidate antigens for use in a recombinant vaccine against Chrysomya bezziana is described. RNA was isolated and mRNA purified from first and third instar larvae of Chrysomya bezziana and used in the synthesis of two cDNA libraries in the bacteriophage vector λ ZAP express®. These libraries were screened using Digoxigenin-labeled DNA probes obtained from two independent approaches. First, a homolog approach used probes designed from previously characterized peritrophic membrane genes identified from the related myiasis fly, Lucilia cuprina. Secondly, a de novo approach used amino-terminal and internal peptide sequence information derived from purified Chrysomya bezziana peritrophic membrane proteins to generate DNA probes. Three peritrophic membrane genes were identified and characterized. Chrysomya bezziana peritrophin-48 was identified using the homolog approach and, Chrysomya bezziana peritrophin-15 and Chrysomya bezziana peritrophin-42 were identified using the de novo approach. The identification of these genes as encoding candidate antigens against Chrysomya bezziana has allowed the production of recombinant proteins for use in vaccination trials

  3. Cloning of Drosophila choline acetyltransferase cDNA.

    OpenAIRE

    Itoh, N; Slemmon, J.R.; Hawke, D.H.; Williamson, R.; Morita, E.; Itakura, K; Roberts, E; Shively, J. E.; Crawford, G D; Salvaterra, P M

    1986-01-01

    Choline acetyltransferase (EC 2.3.1.6) is the biosynthetic enzyme for the neurotransmitter acetylcholine. To isolate choline acetyltransferase cDNA clones, a cDNA library was constructed from poly(A)+ RNA of Drosophila melanogaster heads, these being one of the richest known sources of the enzyme. By screening the cDNA library with a mixture of three different monoclonal antibodies to Drosophila choline acetyltransferase, we isolated 14 positive clones. Only 1 of these clones was identified t...

  4. Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project

    Science.gov (United States)

    Fernández, Paula; Paniego, Norma; Lew, Sergio; Hopp, H Esteban; Heinz, Ruth A

    2003-01-01

    Background Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. Results Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4). The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60%) did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences putatively related to responses

  5. Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project

    Directory of Open Access Journals (Sweden)

    Heinz Ruth A

    2003-09-01

    Full Text Available Abstract Background Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. Results Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4. The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60% did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences

  6. Analysis of expressed sequence tags from a fetal human heart cDNA library

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, D.M.; Fung, Y.W.; Wang, R.X.; Laurenssen, C.M. [Univ. of Toronto, Ontario (Canada)] [and others

    1995-11-20

    Single-pass sequencing of randomly selected cDNA clones to generate expressed sequence tags (ESTs) has been widely used to identify novel genes and to study gene expression in a variety of tissues. We have generated 2244 ESTs from a human fetal heart library (Gen-Bank Accession Nos. R30692-30774 and R56965-58824), which we present in this report. Of these, 51.7% showed no homology to known genes or were similar only to other ESTs, while 48.4% demonstrated homology to known transcripts. A total of 764 ESTs corresponding to known genes were used to study gene expression patterns in the fetal heart and to analyze differences in these patterns from those observed in the adult heart. These analyses demonstrate the utility of ESTs and sequence-tagged clones in comparative studies of gene expression in the cardiovascular system, and they reveal that differential gene expression underlies the structural and functional characteristics of the developing heart. 48 refs., 1 fig., 3 tabs.

  7. Cloning of low dose radiation induced gene RIG1 by RACE based on non-cloned cDNA library

    International Nuclear Information System (INIS)

    Objective: To obtain full-length cDNA of radiation induced new gene RIG1 based on its EST fragment. Methods: Based on non-cloned cDNA library, enhanced nested RACE PCR and biotin-avidin labelled probe for magnetic bead purification was used to obtain full-length cDNA of RIG1. Results: About 1 kb of 3' end of RIG1 gene was successfully cloned by this set of methods and cloning of RIG1 5' end is proceeding well. Conclusion: The result is consistent with the design of experiment. This set of protocol is useful for cloning of full-length gene based on EST fragment

  8. cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family

    International Nuclear Information System (INIS)

    A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase. One positive clone was isolated and the nucleotide sequence was determined. It contained 1,305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3' untranslated end, although a poly(A)+ tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues. This was supported by the synthesis of a Mr 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low Mr PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes

  9. Random sequencing of an induced Taxus cell cDNA library for identification of clones involved in Taxol biosynthesis

    OpenAIRE

    Jennewein, Stefan; Wildung, Mark R.; Chau, MyDoanh; Walker, Kevin; Croteau, Rodney

    2004-01-01

    Biosynthesis of the anticancer drug Taxol involves 19 enzymatic steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methylerythritol phosphate pathway for isoprenoid precursor supply. To gain further insight about Taxol biosynthesis relevant to the improved production of this drug and to draw inferences about the organization, regulation, and origins of this complex natural product pathway, random sequencing of a cDNA library derived from Taxus...

  10. 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

    metabolites. Conclusion SMART technology could be effectively applied to constructing the full-length cDNA library of S. Scandens. Our results indicate that the full-length cDNA library could be further applied to regulation of expression on identification of functional genome, screening of novel genes, and biosynthesis of metabolism genes in S. Scandens.

  11. Rasmuson Library DVD Browser: Fun with Screen Scraping and Drupal

    Directory of Open Access Journals (Sweden)

    Mark Morlino

    2008-12-01

    Full Text Available The DVD Browser is a simple application that lets library patrons browse movie covers, titles, and reviews. It works by screen scraping the the Rasmuson Library catalog for DVD movies and dumps the data into a Drupal MySQL database. This paper describes the process of setting up the DVD Browser.

  12. Rasmuson Library DVD Browser: Fun with Screen Scraping and Drupal

    OpenAIRE

    Mark Morlino; Ilana Kingsley

    2008-01-01

    The DVD Browser is a simple application that lets library patrons browse movie covers, titles, and reviews. It works by screen scraping the the Rasmuson Library catalog for DVD movies and dumps the data into a Drupal MySQL database. This paper describes the process of setting up the DVD Browser.

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

    Science.gov (United States)

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

    2012-06-01

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

  15. Oligonucleotide probes for the screening of recombinant DNA libraries

    International Nuclear Information System (INIS)

    Oligonucleotides of unique sequence are also useful for screening recombinant DNA libraries. The hybridization specificity of oligonucleotide probes allows one to use unique sequences probes to screen for genomic clones or cDNAs encoding a specific number of a multigene family, to screen for a new allele when the sequence of one allele is known, to screen for a specific region of a gene, to screen for specific mutants created by site-directed mutagenesis, or to screen libraries with probes whose sequence represents a consensus coding sequence. This chapter deals with procedures for the use of oligonucleotides as hybridization probes including probe design, labeling, and hybridization to colonies, phage plaques, DNA, and RNA

  16. Construction and analysis of full-lengh and normalized cDNA libraries from citrus

    OpenAIRE

    Marqués, M.Carmen; Pérez-Amador, Miguel A.

    2012-01-01

    We have developed an integrated method to generate a normalized cDNA collection enriched in full-length and rare transcripts from citrus, using different species and multiple tissues and developmental stages. Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. In this regard, the availability of full-length cDNA clones facilitates functional an...

  17. An anti-tumor protein produced by Trichinella spiralis and identified by screening a T7 phage display library, induces apoptosis in human hepatoma H7402 cells

    Science.gov (United States)

    Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic m...

  18. 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.

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

    International Nuclear Information System (INIS)

    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). 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. 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). 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

  20. 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.

  1. Generation of a large scale repertoire of Expressed Sequence Tags (ESTs from normalised rainbow trout cDNA libraries

    Directory of Open Access Journals (Sweden)

    Guiguen Yann

    2006-08-01

    Full Text Available Abstract Background Within the framework of a genomics project on livestock species (AGENAE, we initiated a high-throughput DNA sequencing program of Expressed Sequence Tags (ESTs in rainbow trout, Oncorhynchus mykiss. Results We constructed three cDNA libraries including one highly complex pooled-tissue library. These libraries were normalized and subtracted to reduce clone redundancy. ESTs sequences were produced, and 96 472 ESTs corresponding to high quality sequence reads were released on the international database, currently representing 42.5% of the overall sequence knowledge in this species. All these EST sequences and other publicly available ESTs in rainbow trout have been included on a publicly available Website (SIGENAE and have been clustered into a total of 52 930 clusters of putative transcripts groups, including 24 616 singletons. 57.1% of these 52 930 clusters are represented by at least one Agenae EST and 14 343 clusters (27.1% are only composed by Agenae ESTs. Sequence analysis also reveals that normalization and especially subtraction were effective in decreasing redundancy, and that the pooled-tissue library was representative of the initial tissue complexity. Conclusion Due to present work on the construction of rainbow trout normalized cDNA libraries and their extensive sequencing, along with other large scale sequencing programs, rainbow trout is now one of the major fish models in term of EST sequences available in a public database, just after Zebrafish, Danio rerio. This information is now used for the selection of a non redundant set of clones for producing DNA micro-arrays in order to examine global gene expression.

  2. 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

  3. Identification of hypoxia-responsive genes in a dopaminergic cell line by subtractive cDNA libraries and microarray analysis.

    Science.gov (United States)

    Beitner-Johnson, D; Seta, K; Yuan, Y; Kim, H -W.; Rust, R T.; Conrad, P W.; Kobayashi, S; Millhorn, D E.

    2001-07-01

    Transplantation of dopamine-secreting cells harvested from fetal mesencephalon directly into the striatum has had limited success as a therapy for Parkinson's disease. A major problem is that the majority of the cells die during the first 3 weeks following transplantation. Hypoxia in the tissue surrounding the graft is a potential cause of the cell death. We have used subtractive cDNA libraries and microarray analysis to identify the gene expression profile that regulates tolerance to hypoxia. An improved understanding of the molecular basis of hypoxia-tolerance may allow investigators to engineer cells that can survive in the hypoxic environment of the brain parenchyma following transplantation. PMID:11331199

  4. Leishmania chagasi T-cell antigens identified through a double library screen.

    Science.gov (United States)

    Martins, Daniella R A; Jeronimo, Selma M B; Donelson, John E; Wilson, Mary E

    2006-12-01

    Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1gamma, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines. PMID:17000724

  5. Structure-Based Virtual Screening of Commercially Available Compound Libraries.

    Science.gov (United States)

    Kireev, Dmitri

    2016-01-01

    Virtual screening (VS) is an efficient hit-finding tool. Its distinctive strength is that it allows one to screen compound libraries that are not available in the lab. Moreover, structure-based (SB) VS also enables an understanding of how the hit compounds bind the protein target, thus laying ground work for the rational hit-to-lead progression. SBVS requires a very limited experimental effort and is particularly well suited for academic labs and small biotech companies that, unlike pharmaceutical companies, do not have physical access to quality small-molecule libraries. Here, we describe SBVS of commercial compound libraries for Mer kinase inhibitors. The screening protocol relies on the docking algorithm Glide complemented by a post-docking filter based on structural protein-ligand interaction fingerprints (SPLIF). PMID:27316988

  6. Preparing unbiased T cell receptor and antibody cDNA libraries for the deep next generation sequencing profiling

    Directory of Open Access Journals (Sweden)

    Ilgar Z Mamedov

    2013-12-01

    Full Text Available High-throughput sequencing has the power to reveal the nature of adaptive immunity as represented by the full complexity of T cell receptor (TCR and antibody (IG repertoires, but is at present severely compromised by the quantitative bias, bottlenecks, and accumulated errors that inevitably occur in the course of library preparation and sequencing. Here we report an optimized protocol for the unbiased preparation of TCR and IG cDNA libraries for high-throughput sequencing, starting from thousands or millions of live cells in an investigated sample. Critical points to control are revealed, along with tips that allow researchers to minimize quantitative bias, accumulated errors, and cross-sample contamination at each stage, and to enhance the subsequent bioinformatic analysis. The protocol is simple, reliable, and can be performed in 1–2 days.

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Bendahmane Abdelhafid

    2011-05-01

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

  9. Construction and 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.

  10. 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。

  11. Construction of cDNA libraries by blunt-end ligation: high-frequency cloning of long cDNAs from filamentous fungi.

    Science.gov (United States)

    Teeri, T T; Kumar, V; Lehtovaara, P; Knowles, J

    1987-07-01

    A simplified cDNA synthesis and cloning method, suitable for efficient generation of cDNA libraries at frequencies up to 10(6) clones/micrograms mRNA, is described. Routine synthesis of transcripts of well over 4 kb is facilitated by the use of high-quality RNA template isolated from materials rich in RNases. Laborious cloning steps, like tailing or addition of linkers, can be omitted by the use of efficient blunt-end ligation to plasmid vectors, and rapid verification as well as characterization of the clones is possible by double-stranded plasmid sequencing. Using this method we have constructed several cDNA libraries of different filamentous fungi and show here the synthesis and cloning of cDNA copies larger than 1.8 kb corresponding to three Trichoderma reesei cellulases. PMID:2823635

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

    Science.gov (United States)

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

    2014-02-01

    Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al₂(SO₄)₃ were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al₂(SO₄)₃ for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses. PMID:24078111

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

    2016-01-01

    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.

  20. 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.

  1. 非小细胞肺癌T7噬菌体展示文库的构建%Construction and quality identification of T7 recombination expression cDNA library form human lung cancer

    Institute of Scientific and Technical Information of China (English)

    Wentao Yue; Zitong Wang; Yue Wang; Lina Zhang

    2009-01-01

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

  2. Optimization and comparison of different methods for RNA isolation for cDNA library construction from the reindeer lichen Cladonia rangiferina

    Directory of Open Access Journals (Sweden)

    Lim Kean-Jin

    2009-10-01

    Full Text Available Abstract Background The reindeer lichen is the product of a mutualistic relationship between a fungus and an algae. Lichen demonstrate a remarkable capacity to tolerate dehydration. This tolerance is driven by a variety of biochemical processes and the accumulation of specific secondary metabolites that may be of relevance to the pharmaceutical, biotechnology and agriculture industries. These protective metabolites hinder in vitro enzymatic reactions required in cDNA synthesis. Along with the low concentrations of RNA present within lichen tissues, the process of creating a cDNA library is technically challenging. Findings An evaluation of existing commercial and published protocols for RNA extraction from plant or fungal tissues has been performed and experimental conditions have been optimised to balance the need for the highest quality total ribonucleotides and the constraints of budget, time and human resources. Conclusion We present a protocol that balances inexpensive RNA extraction methods with commercial RNA clean-up kits to yield sufficient RNA for cDNA library construction. Evaluation of the protocol and the construction of, and sampling from, a cDNA library is used to demonstrate the suitability of the RNA extraction method for expressed sequence tag production.

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

    Directory of Open Access Journals (Sweden)

    Huang Lili

    2009-12-01

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

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

    Science.gov (United States)

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

    2010-05-01

    Greek sage (Salvia fruticosa Mill., Syn. Salvia triloba L.) is appreciated for its essential oil which is used as an aromatic spice and active against a wide range of microorganisms and viruses. The essential oil is dominated by terpenoids and flavonoids which are produced and stored in glandular trichomes on the plant surface. The present study aims to give insights into the metabolic activities of S. fruticosa trichomes on a transcriptome level. A total of 2,304 clones were sequenced from a cDNA library from leaves' trichomes of S. fruticosa. Exclusion of sequences shorter than 100 bp resulted in 1,615 high-quality ESTs with a mean length of 592 bp. Cluster analysis indicated the presence of 197 contigs (908 clones) and 707 singletons, generating a total of 904 unique sequences. Of the 904 unique ESTs, 628 (69.5%) had significant hits in the non-redundant protein database and were annotated. A total of 517 (82.3%) sequences were functionally classified using the gene ontologies (GO) and established pathway associations to 220 (24.3%) sequences in Kyoto encyclopedia of genes and genomes (KEGG). In addition, 52 (5.8%) of the unique ESTs revealed a GO biological term with relation to terpenoid (78 ESTs), phenylpropanoid (43 ESTs), flavonoid (18 ESTs) or alkaloid (10 ESTs) biosynthesis or to P450s (26 ESTs). Expression analysis of a selected set of genes known to be involved in the pathways of secondary metabolite synthesis showed higher expression levels in trichomes, validating the tissue specificity of the analyzed glandular trichome library. PMID:20333525

  5. 感染高粱花叶病毒甘蔗叶片cDNA文库构建及评价%Construction and Evaluation of Yeast Two Hybrid cDNA Library of Suagarcane Leaf Infected SrMV Virus

    Institute of Scientific and Technical Information of China (English)

    张雨良; 张树珍; 王健华; 熊国如; 王俊刚; 余乃通; 刘志昕

    2012-01-01

    为了研究甘蔗花叶病与甘蔗寄主致病的互作分子机制,利用SMART技术成功构建了感染高梁花叶病毒甘蔗叶片的cDNA文库,用于后续酵母双杂交互作蛋白筛选试验.采用Omega公司Plant RNA Kit提取感染高粱花叶病毒甘蔗叶片总RNA,经过Oligotex纯化获得mRNA,将其反转录成cDNA第一链,再在DNA聚合酶作用下,通过长距离PCR扩增双链cDNA.经SfiI酶切并去除短片段后,连接到pGADT7-SfiI载体上,成功获得初级cDNA文库,最后以初级文库100万克隆为基数扩增,得到扩增文库并提取质粒.经检测构建的文库容量为1.6×106 cfu,文库滴度2.2× 106 cfu/mL,文库cDNA插入片段长度主要分布在700~2 000 bp,文库重组率约为96%.结果表明,该文库质量较好,为筛选分离抗病功能基因及开展寄主与病毒互作的研究奠定了基础.%Sugarcane (Saccharum officinarum L.) is one of important specie producing sugar and energy cash crop. It plays an important role in sugar jar of raw materials and increases farmers' income. Total RNA was extracted from infected SrMV sugarcane leaf using Omega company RNA extraction reagent. Total RNA was used to purify mRNA with Oligotex kit. The first strand cDNA was synthesized by reverse transcription of mRNA with SMART technique and LI>-PCR was performed to synthesize double strand cDNA. Then double strand cDNA were digested by Sfil enzyme to remove shorter cDNA by running through a CHROMA SPIN TE-400 column. Purified ds cDNA and linear vector pGADT7-SfiI co-transformed into E.coli XL10-GOLD strain to generate sugarcane leaf infected SrMV virus's Yeast-Two-Hybrid primary cDNA library. Then we obtained the amplified library and extracted the plasmids. The results of detection showed that the library contained 1.6xlO6 independent clones, and the titer of library were 2.2X106 cfu/mL. The sizes of most inserts were from 700 to 2 000 bp in the library. The recombination rate of library was 96%. These results

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

    Objective: The aim of this study was to reveal the exact changes during the occurrence of breast cancer to explore significant new and promising genes or factors related to this disease. Methods: We compared the gene expression profiles of breast cancer tissues with its uninvolved normal breast tissues as controls using the cDNA microarray analysis in seven breast cancer patients. Further, one representative gene, named IFI30, was quanti-tatively analyzed by real-time PCR to confirm the result of the cDNA microarray analysis. Results: A total of 427 genes were identified with significantly differential expression, 221 genes were up-regulated and 206 genes were down-regulated. And the result of cDNA microarray analysis was validated by detection of IFI30 mRNA level changes by real-time PCR. Genes for cell proliferation, cell cycle, cell division, mitosis, apoptosis, and immune response were enriched in the up-regulated genes, while genes for cell adhesion, proteolysis, and transport were significantly enriched in the down-regulated genes in breast cancer tissues compared with normal breast tissues by a gene ontology analysis. Conclusion: Our present study revealed a range of differentially expressed genes between breast cancer tissues and normal breast tissues, and provide candidate genes for further study focusing on the pathogenesis and new biomarkers for breast cancer. Copyright © 2015, Chinese Medical Association Production. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

  7. Sequencing and comparative genomics analysis in Senecio scandens Buch.-Ham. Ex D. Don, based on full-length cDNA library

    Science.gov (United States)

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

    2014-01-01

    Senecio scandens Buch.-Ham. ex D. Don, an important antibacterial source of Chinese traditional medicine, has a widespread distribution in a few ecological habitats of China. We generated a full-length complementary DNA (cDNA) library from a sample of elite individuals with superior antibacterial properties, with satisfactory parameters such as library storage (4.30 × 106 CFU), efficiency of titre (1.30 × 106 CFU/mL), transformation efficiency (96.35%), full-length ratio (64.00%) and redundancy ratio (3.28%). The BLASTN search revealed the facile formation of counterparts between the experimental sample and Arabidopsis thaliana in view of high-homology cDNA sequence (90.79%) with e-values <1e – 50. Sequence similarities to known proteins indicate that the entire sequences of the full-length cDNA clones consist of the major of functional genes identified by a large set of microarray data from the present experimental material. For other Compositae species, a large set of full-length cDNA clones reported in the present article will serve as a useful resource to facilitate further research on the transferability of expressed sequence tag-derived simple sequence repeats (EST-SSR) development, comparative genomics and novel transcript profiles. PMID:26740776

  8. Identification of selective enzyme inhibitors by fragment library screening.

    Science.gov (United States)

    Gao, Geng; Liu, Xuying; Pavlovsky, Alexander; Viola, Ronald E

    2010-10-01

    The microbial threat to human health is growing due to the dramatic increase in the number of multidrug-resistant organisms. The decline in effective antibiotics available to treat these growing threats has provided greater urgency to the search for new antibiotics. Clearly, new approaches must be developed against novel targets to control these resistant infectious organisms. The screening of low molecular weight compounds against new protein targets provides an opportunity to identify novel inhibitors as starting points for the development of new antibiotics. Custom fragment libraries have been assembled and screened against 3 representative forms of a key enzyme in an essential microbial biosynthetic pathway. Although each of these aspartate semialdehyde dehydrogenases (ASADHs) catalyzes the same reaction and each shares identical active site functional groups, subtle differences in enzyme structures have led to different binding selectivity among the initial hits from these fragment libraries. Amino acid analogues have been identified that show selectivity for either the gram-negative or gram-positive bacterial enzyme forms. A series of benzophenone analogues selectively inhibit the gram-negative ASADH, whereas some haloacids and substituted aromatic acids have been found to inhibit only the fungal form of ASADH. Each of these low molecular weight compounds possesses high ligand binding efficiency for their target enzyme forms. These results support the goal of designing lead compounds that will selectively target ASADHs from different microbial species. PMID:20855558

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

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jakob;

    2007-01-01

    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...... public databases. The Sino-Danish ESTs were generated from one normalized and 97 non-normalized cDNA libraries representing 35 different tissues and three developmental stages. RESULTS: Using the Distiller package, the ESTs were assembled to roughly 48,000 contigs and 73,000 singletons, of which...... with the greatest number of different expressed genes, whereas tissues with more specialized function, such as developing liver, have fewer expressed genes. There are at least 65 high confidence housekeeping gene candidates and 876 cDNA library-specific gene candidates. We identified differential...

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2013-05-01

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

  12. Analysis of proteins encoded by full-length cDNA sequence from IRM-2 mouse

    International Nuclear Information System (INIS)

    Objective: To screen and isolate radioresistance-related genes from IRM-2 mouse. Methods: Full-length cDNA products were amplified by PCR from IRM-2 mouse cDNA library according to twenty-one pieces of expressed sequence tags. The property of proteins encoded by full-length cDNA were analyzed by comparing with GenBank database. Results: Five pieces of full-length cDNA which were not the same source as the known mice genes were found out from IRM-2 mouse cDNA library.Amino acid sequence and property of proteins encoded by these five pieces of full-length cDNA were obtained. Conclusion: Proteins encoded by full-length cDNA imply that unknown radioresistance-related genes may exist in IR M-2 mouse. (authors)

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

    OpenAIRE

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

    2012-01-01

    Purpose Mutations in the homeobox transcription factor paired-like homeodomain transcription factor 2 (PITX2) cause Axenfeld–Reiger syndrome (ARS), which is associated with anterior segment dysgenesis (ASD) and glaucoma. To understand ARS pathogenesis, it is essential to know the normal functions of PITX2 and the proteins with which PITX2 interacts in the eye. Therefore, we used a unique cDNA library that we created from human trabecular meshwork (TM) primary cells to discover PITX2-interacti...

  14. Constructing and random sequencing analysis of normalized cDNA library of testis tissue from oriental river prawn (Macrobrachium nipponense).

    Science.gov (United States)

    Qiao, Hui; Fu, Hongtuo; Jin, Shubo; Wu, Yan; Jiang, Sufei; Gong, Yongsheng; Xiong, Yiwei

    2012-09-01

    The oriental river prawn, Macrobrachium nipponense, is an important aquaculture species in China. Sexual precocity is a serious problem because of genetic retrogression, which has negative effects on product quality and dramatically affects price. Culture of all-male populations of this species would be economically advantageous, as the males grow faster and reach a much larger size than females. Developing such a culture scheme will require discovery of sex- or reproduction-related genes that affect sexual maturity and sex determination. In this study, a high-quality normalized testis cDNA library was constructed to identify novel transcripts. Of the 5280 successful sequencing reaction yields, 5202 expressed tagged sequences (ESTs) with an average length of 954 bp. Ultimately, 3677 unique sequences, including 891 contigs and 2786 singletons, were identified based on cluster and assembly analyses. Sixteen hundred (43.5%) genes were novel based on the NCBI protein database, thus these unidentified genes may improve basic molecular knowledge about M. nipponense. Of the novel unigenes, 34.4% (715/2077) were homologous to insects, such as Tribolium castaneum, Drosophila spp. and Apis mellifera. Fifty-two genes were identified as sex- or reproduction-related based on Gene Ontology classification and sequence comparison with data from other publications. These genes can be classified into groups based on different functions, including 10 sex-determination related genes, 8 male-reproductive genes, 5 cathepsin-related genes, 20 ubiquitin-related genes, 5 ferritin-related genes, and 4 LRR genes. The results of this study provide new sequence information about M. nipponense, which will be the basis for further genetic studies of this species and other decapods crustaceans. PMID:22632994

  15. cDNA: 40711 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.41523 Mus musculus adult male thymus cDNA, RIKEN full-length enriched library, ... lone:5830492N08 product:hypothetical Mitochondrial energy ... transfer proteins (carrier protein) containing pro ...

  16. cDNA: 36927 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.240850 Mus musculus adult male stomach cDNA, RIKEN full-length enriched library ... MA AMPLIFIED SEQUENCE 1 (NOVEL AMPLIFIED IN BREAST CANCER ... 1) (AMPLIFIED AND OVEREXPRESSED IN BREAST CANCER ) ...

  17. cDNA: 40220 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.207654 Mus musculus adult male olfactory brain ... cDNA, RIKEN full-length enriched ... library, clone:6430704M03 product:similar to BRAIN ... PROTEIN (FRAGMENT) [Homo sapiens], full insert seq ...

  18. cDNA: 52278 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.275648 Mus musculus adult male tongue cDNA, RIKEN full-length enriched library, ... clone:2310073F10 product:SIMILAR TO ENIGMA ... (LIM DOMAIN PROTEIN) homolog [Homo sapiens], full ...

  19. cDNA: 52276 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.275648 Mus musculus adult male brain cDNA, RIKEN full-length enriched library, ... clone:0710007K04 product:SIMILAR TO ENIGMA ... (LIM DOMAIN PROTEIN) homolog [Homo sapiens], full ...

  20. Screening a cDNA library for protein-protein interactions directly in planta

    Czech Academy of Sciences Publication Activity Database

    Lee, L.-Y.; Wu, F.-H.; Hsu, Ch.-T.; Shen, S.-Ch.; Yeh, H.-Y.; Liao, D.-Ch.; Fang, M.-J.; Liu, N.-T.; Yen, Y.-Ch.; Dokládal, Ladislav; Sýkorová, Eva; Gelvin, S.B.; Lin, Ch.-S.

    2012-01-01

    Roč. 24, č. 5 (2012), s. 1746-1759. ISSN 1040-4651 R&D Projects: GA AV ČR(CZ) IAA500040801 Institutional research plan: CEZ:AV0Z50040702 Keywords : bimolecular fluorescence complementation * telomerase-binding-protein * transformation Subject RIV: BO - Biophysics Impact factor: 9.251, year: 2012

  1. 大乳头水螅RACE cDNA文库的构建%Construction of RACE cDNA Library of Hydra Magnipapillata

    Institute of Scientific and Technical Information of China (English)

    赵凤霞; 杨好强; 钱小成; 潘红春

    2013-01-01

    目的:为筛选和克隆大乳头水螅发育调控相关基因的全长cDNA,构建大乳头水螅RACE cDNA文库.方法:提取大乳头水螅总RNA后从其中分离mRNA,运用SMART技术构建RACE cDNA文库.为鉴定所构建文库的质量,根据GenBank中大乳头水螅actin基因cDNA序列设计5'RACE和3'RACE的引物及用于扩增actin基因编码区全长序列的引物.结果:琼脂糖凝胶电泳结果表明,RACE cDNA文库中全长cDNA的长度集中在500-2 000bp之间.5'RACE、3'RACE PCR及扩增actin基因编码区全长序列时均以本文构建的大乳头水螅RACE cDNA文库为模板,这3个PCR反应均能扩增出产物,产物大小与目标片段预计大小相似.PCR产物分别经T/A克隆及测序后证明为大乳头水螅actin基因cDNA的相应序列.结论:RACE cDNA文库的成功构建为通过RACE方法获得大乳头水螅功能基因cDNA全长序列奠定了基础.%Objective: To construct RACE cDNA library of Hydra magnipapillata. Methods: Total RNA was isolated from Hydra magnipapillata, and purified mRNA from total RNA was used to construct RACE cDNA library with the SMART cDNA library construction kit. In order to identify the cDNA library, the polymerase chain reaction (PCR) primers for 5' RACE, 3' RACE and the full-length cDNAs of actin gene were designed based on the pupative cDNA sequence of actin from GenBank. Results: Agarose gel elec-trophoresis showed that the lengths of full-length cDNAs in this library were pooled mainly between 500 and 2 000 base pairs. By RACE PCR, amplified products were obtained with all the gene-specific primers and adaptor primers. Conclusion: The quality of the RACE cD-NA library was high and appropriate for cloning the full-length cDNAs of functional genes in Hydra magnipapillata.

  2. Complementation of radiation-sensitive Ataxia telangiectasia cells after transfection of cDNA expression libraries and cosmid clones from wildtype cells

    International Nuclear Information System (INIS)

    In this Ph.D.-thesis, phenotypic complementation of AT-cells (AT5BIVA) by transfection of cDNA-expression-libraries was adressed: After stable transfection of cDNA-expression-libraries G418 resistant clones were selected for enhanced radioresistance by a fractionated X-ray selection. One surviving transfectant clone (clone 514) exhibited enhanced radiation resistance in dose-response experiments and further X-ray selections. Cell cycle analysis revealed complementation of untreated and irradiated 514-cells in cell cycle progression. The rate of DNA synthesis, however, is not diminished after irradiation but shows the reverse effect. A transfected cDNA-fragment (AT500-cDNA) was isolated from the genomic DNA of 514-cells and proved to be an unknown DNA sequence. A homologous sequence could be detected in genomic DNA from human cell lines, but not in DNA from other species. The cDNA-sequence could be localized to human chromosome 11. In human cells the cDNA sequence is part of two large mRNAs. 4 different cosmid clones containing high molecular genomic DNA from normal human cells could be isolated from a library, each hybridizing to the AT500-cDNA. After stable transfection into AT-cells, one cosmid-clone was able to confer enhanced radiation resistance both in X-ray selections and dose-response experiments. The results indicate that the cloned cDNA-fragment is based on an unknown gene from human chromosome 11 which partially complements the radiosensitivity and the defective cell cycle progression in AT5BIVA cells. (orig.)

  3. Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Keith D.; Pefaur, Noah B.; Baird, Cheryl L.

    2008-07-01

    These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks resulting in a panel of scFvs specific for the target antigen.

  4. Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Keith D.; Pefaur, Noah B.; Baird, Cheryl L.

    2009-08-02

    These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magneticactivated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen.

  5. 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

  6. Cloning and characterization of a novel human zinc finger gene, hKid3, from a C2H2-ZNF enriched human embryonic cDNA library

    International Nuclear Information System (INIS)

    To investigate the zinc finger genes involved in human embryonic development, we constructed a C2H2-ZNF enriched human embryonic cDNA library, from which a novel human gene named hKid3 was identified. The hKid3 cDNA encodes a 554 amino acid protein with an amino-terminal KRAB domain and 11 carboxyl-terminal C2H2 zinc finger motifs. Northern blot analysis indicates that two hKid3 transcripts of 6 and 8.5 kb express in human fetal brain and kidney. The 6 kb transcript can also be detected in human adult brain, heart, and skeletal muscle while the 8.5 kb transcript appears to be embryo-specific. GFP-fused hKid3 protein is localized to nuclei and the ZF domain is necessary and sufficient for nuclear localization. To explore the DNA-binding specificity of hKid3, an oligonucleotide library was selected by GST fusion protein of hKid3 ZF domain, and the consensus core sequence 5'-CCAC-3' was evaluated by competitive electrophoretic mobility shift assay. Moreover, The KRAB domain of hKid3 exhibits transcription repressor activity when tested in GAL4 fusion protein assay. These results indicate that hKid3 may function as a transcription repressor with regulated expression pattern during human development of brain and kidney

  7. Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project

    OpenAIRE

    Heinz Ruth A; Lew Sergio; Hopp H; Paniego Norma; Fernández Paula

    2003-01-01

    Abstract Background Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative str...

  8. High-content screening of yeast mutant libraries by shotgun lipidomics

    DEFF Research Database (Denmark)

    Tarasov, Kirill; Stefanko, Adam; Casanovas, Albert;

    2014-01-01

    To identify proteins with a functional role in lipid metabolism and homeostasis we designed a high-throughput platform for high-content lipidomic screening of yeast mutant libraries. To this end, we combined culturing and lipid extraction in 96-well format, automated direct infusion...... factor KAR4 precipitated distinct lipid metabolic phenotypes. These results demonstrate that the high-throughput shotgun lipidomics platform is a valid and complementary proxy for high-content screening of yeast mutant libraries....

  9. Construction of a yeast two-hybrid cDNA library for gene interaction during heading stage of rice%水稻抽穗期基因酵母双杂交cDNA文库的构建及分析

    Institute of Scientific and Technical Information of China (English)

    彭强; 吴昌银

    2012-01-01

    为研究水稻开花调控的分子网络,构建了水稻品种中花11幼穗分化前倒二叶叶片的酵母双杂交cD-NA文库.制备了高质量的总RNA,用Oligo(dT)引物反转录合成cDNA第一链和Long Distance PCR合成双链cDNA,通过SMART同源重组交换技术在酵母菌株AH109中建立酵母双杂交所需的水稻倒二叶叶片cDNA文库.文库质量检测结果表明:cDNA文库的转化率为1.178×106/3μg pGADT7-Rec,文库滴度为2.65×108cfu/mL,重组率为94.7%,插入片段长度为350~2000bp.该文库可用来筛选水稻抽穗期基因的互作蛋白.%A yeast two-hybrid cDNA library of penultimate leaf harvested from the rice cultivar Zhonghuall before its flowering transition was constructed to study the molecular network of flowering regulation. The high-quality total RNA was isolated and the first-strand cDNA was obtained by reverse transcription using primer Oligo(dT). The double-strand cDNA was amplified by using Long Distance PCR. The cDNA library of rice penultimate leaf was generated by using homologous recombination-mediated SMART technology in yeast strain AH109. The testing results of library's quality showed that the transformation efficiency of cDNA library was 1. 185 × 106 transformants/3 μg pGADT7-Rec with the titers of 2. 65 × 108 cfu/mL and the recombinant rate of 94. 7%. The sizes of inserted fragments were ranged from 350 bp to 2 000 bp. These data indicated that the cDNA library could be used to screen interacting proteins of the flowering time gene in rice.

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

    Directory of Open Access Journals (Sweden)

    Sugantham Priyanka Annabel

    2010-10-01

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

  11. cDNA expression cloning in mammalian cells.

    Science.gov (United States)

    Hoffman, B J

    2001-05-01

    This unit contains protocols for expression cloning in mammalian cells. Either calcium phosphate- or liposome-mediated transfection of mammalian cells, or virus infection and liposome-mediated transfection are used to screen pools derived from a cDNA library. cDNA pools are prepared for cloning from library-transformed E. coli grown in liquid culture medium or on antibiotic-containing selection plates. Results of screening assays for expression can be detected using autoradiography of dishes of cultured cells to identify clones, direct visualization of radiolabeled cells on emulsion-coated and developed chamber slides, detection and quantification of gene activity by a functional (transport) assay with scintillation counting, or detection using a filter-based assay for binding of radioligand to membranes or whole cells. The most critical step of any cDNA cloning project is the establishment of the screening protocol. Therefore, the bioassay for the gene product must be established prior to executing any of these protocols, including construction of the cDNA library. PMID:18428491

  12. Wide screening of phage-displayed libraries identifies immune targets in planta.

    Directory of Open Access Journals (Sweden)

    Cristina Rioja

    Full Text Available Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 2 × 10(7 different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes

  13. 刺参肌肉组织cDNA文库的构建%Construction of cDNA library from the musculature of Apostichopus japonicus

    Institute of Scientific and Technical Information of China (English)

    陈璐; 姜国良; 刘云; 仇磊; 项鹏

    2009-01-01

    用RNA提取试剂--TRIZOL Reagent提取刺参(Apostichopus japonicus)肌肉组织总RNA,用SMART cDNA Library Construction Kit构建cDNA文库.经测定原始文库滴度达到 3.2×10~6,扩增后文库滴度达到 5.1×10~9,重组率达到96.7%,从扩增文库随机挑取12 个克隆进行PCR 扩增鉴定,结果显示,插入片段大小为0.5~2.5 kb.通过各项指标验证成功构建了刺参肌肉组织cDNA 文库.

  14. Isolating Viral and Host RNA Sequences from Archival Material and Production of cDNA Libraries for High-Throughput DNA Sequencing

    Science.gov (United States)

    Xiao, Yongli; Sheng, Zong-Mei; Taubenberger, Jeffery K.

    2015-01-01

    The vast majority of surgical biopsy and post-mortem tissue samples are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Using the protocols described here, the full genome of the 1918 virus at high coverage was determined in one high-throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. This basic methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of infectious diseases. PMID:26344216

  15. Generation and analysis of a large-scale expressed sequence tags from a full-length enriched cDNA library of Siberian tiger (Panthera tigris altaica).

    Science.gov (United States)

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

    2014-05-15

    In this study, a full-length enriched cDNA library was successfully constructed from Siberian tiger, the world's most endangered species. The titers of primary and amplified libraries were 1.28×10(6)pfu/mL and 1.59×10(10)pfu/mL respectively. The proportion of recombinants from unamplified library was 91.3% and the average length of exogenous inserts was 1.06kb. A total of 279 individual ESTs with sizes ranging from 316 to 1258bps were then analyzed. Furthermore, 204 unigenes were successfully annotated and involved in 49 functions of the GO classification, cell (175, 85.5%), cellular process (165, 80.9%), and binding (152, 74.5%) are the dominant terms. 198 unigenes were assigned to 156 KEGG pathways, and the pathways with the most representation are metabolic pathways (18, 9.1%). The proportion pattern of each COG subcategory was similar among Panthera tigris altaica, P. tigris tigris and Homo sapiens, and general function prediction only cluster (44, 15.8%) represents the largest group, followed by translation, ribosomal structure and biogenesis (33, 11.8%), replication, recombination and repair (24, 8.6%), and only 7.2% ESTs classified as novel genes. Moreover, the recombinant plasmid pET32a-TAT-COL6A2 was constructed, coded for the Trx-TAT-COL6A2 fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-COL6A2 recombinant protein was 2.64±0.18mg/mL. This library will provide a useful platform for the functional genome and transcriptome research of for the P. tigris and other felid animals in the future. PMID:24630959

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

    Directory of Open Access Journals (Sweden)

    Lee Hae-Young

    2006-02-01

    Full Text Available Abstract Background Genome research in farm animals will expand our basic knowledge of the genetic control of complex traits, and the results will be applied in the livestock industry to improve meat quality and productivity, as well as to reduce the incidence of disease. A combination of quantitative trait locus mapping and microarray analysis is a useful approach to reduce the overall effort needed to identify genes associated with quantitative traits of interest. Results We constructed a full-length enriched cDNA library from porcine backfat tissue. The estimated average size of the cDNA inserts was 1.7 kb, and the cDNA fullness ratio was 70%. In total, we deposited 16,110 high-quality sequences in the dbEST division of GenBank (accession numbers: DT319652-DT335761. For all the expressed sequence tags (ESTs, approximately 10.9 Mb of porcine sequence were generated with an average length of 674 bp per EST (range: 200–952 bp. Clustering and assembly of these ESTs resulted in a total of 5,008 unique sequences with 1,776 contigs (35.46% and 3,232 singleton (65.54% ESTs. From a total of 5,008 unique sequences, 3,154 (62.98% were similar to other sequences, and 1,854 (37.02% were identified as having no hit or low identity (Sus scrofa. Gene ontology (GO annotation of unique sequences showed that approximately 31.7, 32.3, and 30.8% were assigned molecular function, biological process, and cellular component GO terms, respectively. A total of 1,854 putative novel transcripts resulted after comparison and filtering with the TIGR SsGI; these included a large percentage of singletons (80.64% and a small proportion of contigs (13.36%. Conclusion The sequence data generated in this study will provide valuable information for studying expression profiles using EST-based microarrays and assist in the condensation of current pig TCs into clusters representing longer stretches of cDNA sequences. The isolation of genes expressed in backfat tissue is the

  17. Creation of a CT Image Library for the Lung Screening Study of the National Lung Screening Trial.

    Science.gov (United States)

    Clark, K W; Gierada, D S; Moore, S M; Maffitt, D R; Koppel, P; Phillips, S R; Prior, F W

    2007-03-01

    The CT Image Library (CTIL) of the Lung Screening Study (LSS) network of the National Lung Screening Trial (NLST) consists of up to three annual screens using CT imaging from each of 17,308 participants with a significant history of smoking but no evidence of cancer at trial enrollment (Fall 2002-Spring 2004). Screens performed at numerous medical centers associated with 10 LSS-NLST screening centers are deidentified of protected health information and delivered to the CTIL via DVD, external hard disk, or Internet/Virtual Private Network transmission. The collection will be completed in late 2006. The CTIL is of potential interest to clinical researchers and software developers of nodule detection algorithms. Its attractiveness lies in its very specific, well-defined patient population, scanned via a common CT protocol, and in its collection of evenly spaced serial screens. In this work, we describe the technical details of the CTIL collection process from screening center retrieval through library storage. PMID:16783598

  18. cDNA: 53887 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.196480 Mus musculus adult male testis cDNA, RIKEN full-length enriched library, ... uct:DNA Segment, Chr 15 Massachusetts Institute of Technology ... 260, full insert sequence gnl|UG|Mm#S10837764 AK07 ...

  19. cDNA: 53885 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.196480 Mus musculus adult male testis cDNA, RIKEN full-length enriched library, ... uct:DNA Segment, Chr 15 Massachusetts Institute of Technology ... 260, full insert sequence gnl|UG|Mm#S10837547 AK07 ...

  20. cDNA: 56670 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083407 AK006184 17/9809_56670.png ...

  1. cDNA: 56898 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083407 AK006184 17/9810_56898.png ...

  2. cDNA: 56671 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083172 AK006562 17/9809_56671.png ...

  3. cDNA: 56677 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083407 AK006184 17/9809_56677.png ...

  4. cDNA: 56899 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083172 AK006562 17/9810_56899.png ...

  5. cDNA: 56891 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083407 AK006184 17/9810_56891.png ...

  6. cDNA: 56678 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083172 AK006562 17/9809_56678.png ...

  7. cDNA: 41699 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.246636 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... e/G-protein beta WD-40 repeats containing protein, fu ... ... gnl|UG|Mm#S9075317 AK016965 5/6970_41699.png ...

  8. cDNA: 56892 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.10747 Mus musculus adult male testis cDNA, RIKEN fu ll-length enriched library, ... PONSE FACTOR) (MRF-1) homolog [Rattus norvegicus], fu ... ... gnl|UG|Mm#S9083172 AK006562 17/9810_56892.png ...

  9. cDNA: 49729 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.286963 Mus musculus 10 days lactation, adult female mammary gland cDNA, RIKEN f ... d library, clone:D730027I09 product:similar to LAK-4P ... [Homo sapiens], full insert sequence gnl|UG|Mm#S10 ...

  10. cDNA: 45098 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.334199 Mus musculus adult male aorta and vein cDNA, RIKEN full-length enriched ... library, clone:A530074J19 product:SA rat hypertension -associated homolog, full insert sequence gnl|UG|Mm ...

  11. Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries

    Science.gov (United States)

    Gaida, Stefan M.; Sandoval, Nicholas R.; Nicolaou, Sergios A.; Chen, Yili; Venkataramanan, Keerthi P.; Papoutsakis, Eleftherios T.

    2015-01-01

    A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli. To overcome this limitation, here we generate E. coli strains capable of recognizing heterologous promoters by expressing heterologous sigma factors. Among seven sigma factors tested, RpoD from Lactobacillus plantarum (Lpl) appears to be able of initiating transcription from all sources of DNA. Using the promoter GFP-trap concept, we successfully screen several heterologous and metagenomic DNA libraries, thus enlarging the genomic space that can be functionally sampled in E. coli. For an application, we show that screening fosmid-based Lpl genomic libraries in an E. coli strain with a chromosomally integrated Lpl rpoD enables the identification of Lpl genetic determinants imparting strong ethanol tolerance in E. coli. Transcriptome analysis confirms increased expression of heterologous genes in the engineered strain. PMID:25944046

  12. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

    International Nuclear Information System (INIS)

    The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [32P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

  13. Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries

    Science.gov (United States)

    Kalber, Tammy; Badar, Adam; Lythgoe, Mark; Pule, Martin

    2015-01-01

    The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins. PMID:26536118

  14. Peptide substrate identification for yeast Hsp40 Ydj1 by screening the phage display library

    Directory of Open Access Journals (Sweden)

    Li Jingzhi

    2004-01-01

    Full Text Available We have identified a peptide substrate for molecular chaperone Hsp40 Ydj1 by utilizing the combination of phage display library screening and isothemol titration calirimetry (ITC. The initial peptide substrate screening for Hsp40 Ydj1 has been carried out by utilizing a 7-mer phage display library. The peptide sequences from the bio-panning were synthesized and object to the direct affinity measurement for Hsp40 Ydj1 by isothemol titration calirimetry studies. The peptide which has the measurable affinity with Ydj1 shows enriched hydrophobic residues in the middle of the substrate fragment. The peptide substrate specificity for molecular chaperone Hsp40 has been analyzed.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  16. Automated screening for small organic ligands using DNA-encoded chemical libraries.

    Science.gov (United States)

    Decurtins, Willy; Wichert, Moreno; Franzini, Raphael M; Buller, Fabian; Stravs, Michael A; Zhang, Yixin; Neri, Dario; Scheuermann, Jörg

    2016-04-01

    DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d. PMID:26985574

  17. Next-generation libraries for robust RNA interference-based genome-wide screens

    Science.gov (United States)

    Kampmann, Martin; Horlbeck, Max A.; Chen, Yuwen; Tsai, Jordan C.; Bassik, Michael C.; Gilbert, Luke A.; Villalta, Jacqueline E.; Kwon, S. Chul; Chang, Hyeshik; Kim, V. Narry; Weissman, Jonathan S.

    2015-01-01

    Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity. PMID:26080438

  18. Large-Scale Single-Guide RNA Library Construction and Use for Genetic Screens

    Science.gov (United States)

    Wang, Tim; Lander, Eric S.; Sabatini, David M.

    2016-01-01

    The ability to systematically disrupt genes serves as a powerful tool for understanding their function. The programmable Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system enables efficient targeting of large numbers of genes through the use of single-guide RNA (sgRNA) libraries. In cultured mammalian cells, collections of knockout mutants can be readily generated via transduction of Cas9/sgRNA lentiviral pools, screened for a phenotype of interest, and tracked using high-throughput DNA sequencing. This technique represents the first general method for undertaking systematic loss-of-function genetic screens in mammalian cells. In this chapter, we outline the steps for conducting CRISPR-based screens from the initial library design to final data analysis and provide guidelines for developing an appropriate screening strategy. PMID:26933254

  19. High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing

    OpenAIRE

    Sims, David; Mendes-Pereira, Ana M.; Frankum, Jessica; Burgess, Darren; Cerone, Maria-Antonietta; Lombardelli, Cristina; Mitsopoulos, Costas; Hakas, Jarle; Murugaesu, Nirupa; Isacke, Clare M.; Fenwick, Kerry; Assiotis, Ioannis; Kozarewa, Iwanka; Zvelebil, Marketa; Ashworth, Alan

    2011-01-01

    RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidl...

  20. Identification of Novel Hexapeptides Bioactive against Phytopathogenic Fungi through Screening of a Synthetic Peptide Combinatorial Library

    OpenAIRE

    López-García, Belén; Pérez-Payá, Enrique; Marcos, Jose F.

    2002-01-01

    The purpose of the present study was to improve the antifungal activity against selected phytopathogenic fungi of the previously identified hexapeptide PAF19. We describe some properties of a set of novel synthetic hexapeptides whose d-amino acid sequences were obtained through screening of a synthetic peptide combinatorial library in a positional scanning format. As a result of the screening, 12 putative bioactive peptides were identified, synthesized, and assayed. The peptides PAF26 (Ac-rkk...

  1. 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

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

    Science.gov (United States)

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

    2011-11-01

    Ma bamboo (Dendrocalamus latiflorus Munro) belongs to Dendrocalamus genus, Bambusease tribe, Bambusoideae subfamily, Poaceae family. It is a representative species of clumping bamboo, and a principal commercial species for various construction purposes using mature culms and for human consumption using young shoots. A normalized cDNA library was constructed from young leaves of Ma bamboo and 9,574 high-quality ESTs were generated, from which 5,317 unigenes including 1,502 contigs and 3,815 singletons were assembled. The unigenes were assigned into different gene ontology (GO) categories and summarized into 13 broad biologically functional groups according to similar functional characteristics or cellular roles by BLAST search against public databases. Eight hundred and ninety-one unigenes were assigned by KO identifiers and mapped to six KEGG biochemical pathways. The transcripts involved in biosynthesis of secondary metabolites such as cytochrome 450, flavonol synthase/flavanone 3-hydroxylase, and dihydroflavonol-4-reductase were well represented by 14 unigenes in the unigene set. The candidate genes involved in phytohormone metabolism, signal transduction and encoding cell wall-associated receptor kinases were also identified. Sixty-seven unigenes related to plant resistance (R) genes, including RPP genes, RGAs and RDL/RF genes, were discovered. These results will provide genome-wide knowledge about the molecular physiology of Ma bamboo young leaves and tools for advanced studies of molecular mechanism underlying leaf growth and development. PMID:21713530

  3. Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library

    International Nuclear Information System (INIS)

    In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion

  4. Construction of full-length cDNA library and development of EST-derived simple sequence repeat (EST-SSR) markers in Senecio scandens.

    Science.gov (United States)

    Qian, Gang; Ping, Junjiao; Lu, Jian; Zhang, Zhen; Wang, Lei; Xu, Delin

    2014-12-01

    Senecio scandens Buch.-Ham. ex D. Don (Compositae) is a crucial source of Chinese traditional medicine with antibacterial properties. We constructed a cDNA library and obtained expressed sequence tags (ESTs) to show the distribution of gene ontology annotations for mRNAs, using an individual plant with superior antibacterial characteristics. Analysis of comparative genomics indicates that the putative uncharacterized proteins (21.07%) might be derived from "molecular function unknown" clones or rare transcripts. Furthermore, the Compositae had high cross-species transferability of EST-derived simple sequence repeats (EST-SSR), based on valid amplifications of 206 primer pairs developed from the newly assembled expressed sequence tag sequences in Artemisia annua L. Among those EST-SSR markers, 52 primers showed polymorphic amplifications between individuals with contrasting diverse antibacterial traits. Our sequence data and molecular markers will be cost-effective tools for further studies such as genome annotation, molecular breeding, and novel transcript profiles within Compositae species. PMID:25007751

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

    Science.gov (United States)

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

    2015-01-01

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

  6. Cdc25B Dual-Specificity Phosphatase Inhibitors Identified in a High-Throughput Screen of the NIH Compound Library

    OpenAIRE

    Johnston, Paul A.; Foster, Caleb A.; Tierno, Marni Brisson; Shun, Tong Ying; Shinde, Sunita N.; Paquette, William D.; Brummond, Kay M.; Wipf, Peter; Lazo, John S.

    2009-01-01

    The University of Pittsburgh Molecular Library Screening Center (Pittsburgh, PA) conducted a screen with the National Institutes of Health compound library for inhibitors of in vitro cell division cycle 25 protein (Cdc25) B activity during the pilot phase of the Molecular Library Screening Center Network. Seventy-nine (0.12%) of the 65,239 compounds screened at 10 μM met the active criterion of ≥50% inhibition of Cdc25B activity, and 25 (31.6%) of these were confirmed as Cdc25B inhibitors wit...

  7. Die Rolle des neuentwickelten pYD-Vectors bei der Entwicklung eines Selektionssystems zum Screening von cDNA Bibliotheken auf sezernierte Proteine und Membranproteine

    OpenAIRE

    Duran, Mansur

    2010-01-01

    Introduction: The purpose of this work is to be represented the role of the pYD vector in the yeast invertase screening system for isolating secreted proteins and membrane proteins. The examination of the sensitivity and specificity of the system should be accomplished with a human microvascular endothelial cell library. At the same time new molecules, which play a function in the angiogenesis, should be found. Receptors, membrane proteins, and secreted proteins, are of considerable intere...

  8. Cloning and expression of cDNA for anti-müllerian hormone.

    OpenAIRE

    Picard, J Y; Benarous, R; Guerrier, D.; Josso, N; Kahn, A.

    1986-01-01

    Messenger RNA, prepared from fetal bovine testicular tissue, was used to construct a cDNA library in lambda gt11 phage. The library was screened with an antibody probe directed against bovine anti-Müllerian hormone and three positive clones were isolated. Cross-hybridizing cDNA inserts carried by clones 4 and 5 (1.2 and 0.08 kilobases long, respectively) code for a fragment of authentic anti-Müllerian hormone, as shown by the ability of the anti-epitope antibodies eluted from fusion protein 4...

  9. Molecular cloning of a cDNA for the chicken progesterone receptor B antigen.

    OpenAIRE

    Zarucki-Schulz, T; Kulomaa, M S; Headon, D R; N.L. Weigel; Baez, M; Edwards, D.P.; McGuire, W L; Schrader, W T; O'Malley, B W

    1984-01-01

    A cDNA for the chicken progesterone receptor B subunit antigen (Mr, 108,000) has been isolated from a cDNA library prepared from size-selected chicken oviduct poly(A)+RNA. A specific monoclonal antibody raised against hen progesterone receptor B subunit (alpha PR-B) was used to screen the library. Recombinant clones reacting with the antibody by virtue of antigen expression were used in hybrid-selected translation. A single clone, pPRB-1, hybridized specifically to a mRNA that yielded a Mr 10...

  10. Fully automatized high-throughput enzyme library screening using a robotic platform.

    Science.gov (United States)

    Dörr, Mark; Fibinger, Michael P C; Last, Daniel; Schmidt, Sandy; Santos-Aberturas, Javier; Böttcher, Dominique; Hummel, Anke; Vickers, Clare; Voss, Moritz; Bornscheuer, Uwe T

    2016-07-01

    A fully automatized robotic platform has been established to facilitate high-throughput screening for protein engineering purposes. This platform enables proper monitoring and control of growth conditions in the microtiter plate format to ensure precise enzyme production for the interrogation of enzyme mutant libraries, protein stability tests and multiple assay screenings. The performance of this system has been exemplified for four enzyme classes important for biocatalysis such as Baeyer-Villiger monooxygenase, transaminase, dehalogenase and acylase in the high-throughput screening of various mutant libraries. This allowed the identification of novel enzyme variants in a sophisticated and highly reliable manner. Furthermore, the detailed optimization protocols should enable other researchers to adapt and improve their methods. Biotechnol. Bioeng. 2016;113: 1421-1432. © 2016 Wiley Periodicals, Inc. PMID:26724475

  11. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

    International Nuclear Information System (INIS)

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-Km, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus

  12. Identification of Multiple Stress Responsive Genes by Sequencing a Normalized cDNA Library from Sea-Land Cotton (Gossypium barbadense L..

    Directory of Open Access Journals (Sweden)

    Bin Zhou

    Full Text Available Plants often face multiple stresses including drought, extreme temperature, salinity, nutrition deficiency and biotic stresses during growth and development. All the stresses result in a series of physiological and metabolic reactions and then generate reversible inhibition of metabolism and growth and can cause seriously irreversible damage, even death. At each stage of cotton growth, environmental stress conditions pose devastating threats to plant growth and development, especially yield and quality. Due to the complex stress conditions and unclear molecular mechanisms of stress response, there is an urgent need to explore the mechanisms of cotton response against abiotic stresses.A normalized cDNA library was constructed using Gossypium barbadense Hai-7124 treated with different stress conditions (heat, cold, salt, drought, potassium and phosphorus deficit and Verticillium dahliae infection. Random sequencing of this library generated 6,047 high-quality expressed sequence tags (ESTs. The ESTs were clustered and assembled into 3,135 uniESTs, composed of 2,497 contigs and 638 singletons. The blastx results demonstrated 2,746 unigenes showing significant similarity to known genes, 74 uniESTs displaying significant similarity to genes of predicted proteins, and 315 uniESTs remain uncharacterized. Functional classification unveiled the abundance of uniESTs in binding, catalytic activity, and structural molecule activity. Annotations of the uniESTs by the plant transcription factor database (PlantTFDB and Plant Stress Protein Database (PSPDB disclosed that transcription factors and stress-related genes were enriched in the current library. The expression of some transcription factors and specific stress-related genes were verified by RT-PCR under various stress conditions.Annotation results showed that a huge number of genes respond to stress in our study, such as MYB-related, C2H2, FAR1, bHLH, bZIP, MADS, and mTERF. These results will improve our

  13. Identification of Multiple Stress Responsive Genes by Sequencing a Normalized cDNA Library from Sea-Land Cotton (Gossypium barbadense L.)

    Science.gov (United States)

    Zhou, Bin; Zhang, Lin; Ullah, Abid; Jin, Xin; Yang, Xiyan; Zhang, Xianlong

    2016-01-01

    Background Plants often face multiple stresses including drought, extreme temperature, salinity, nutrition deficiency and biotic stresses during growth and development. All the stresses result in a series of physiological and metabolic reactions and then generate reversible inhibition of metabolism and growth and can cause seriously irreversible damage, even death. At each stage of cotton growth, environmental stress conditions pose devastating threats to plant growth and development, especially yield and quality. Due to the complex stress conditions and unclear molecular mechanisms of stress response, there is an urgent need to explore the mechanisms of cotton response against abiotic stresses. Methodology and Principal Findings A normalized cDNA library was constructed using Gossypium barbadense Hai-7124 treated with different stress conditions (heat, cold, salt, drought, potassium and phosphorus deficit and Verticillium dahliae infection). Random sequencing of this library generated 6,047 high-quality expressed sequence tags (ESTs). The ESTs were clustered and assembled into 3,135 uniESTs, composed of 2,497 contigs and 638 singletons. The blastx results demonstrated 2,746 unigenes showing significant similarity to known genes, 74 uniESTs displaying significant similarity to genes of predicted proteins, and 315 uniESTs remain uncharacterized. Functional classification unveiled the abundance of uniESTs in binding, catalytic activity, and structural molecule activity. Annotations of the uniESTs by the plant transcription factor database (PlantTFDB) and Plant Stress Protein Database (PSPDB) disclosed that transcription factors and stress-related genes were enriched in the current library. The expression of some transcription factors and specific stress-related genes were verified by RT-PCR under various stress conditions. Conclusions/Significance Annotation results showed that a huge number of genes respond to stress in our study, such as MYB-related, C2H2, FAR1, b

  14. Using Yeast Transposon-Insertion Libraries for Phenotypic Screening and Protein Localization.

    Science.gov (United States)

    Kumar, Anuj

    2016-01-01

    This protocol details how to use a transposon-insertion library for phenotypic screening and protein localization. The insertion library was generated by mutagenesis of a plasmid-based yeast genomic DNA library by using a multipurpose transposon; the transposon produces gene disruptions, and, by Cre-mediated recombination at lox sites incorporated within the transposon, alleles with an in-frame insertion can be truncated to a residual transposon encoding multiple copies of the hemagglutinin epitope. Insertions are generated in yeast by shuttle mutagenesis. Yeast genomic DNA containing a transposon insertion is released from the library, and the mutagenized DNA sequences are introduced into a desired strain of yeast, where the insertion alleles replace native loci by homologous recombination. The insertion mutants can be screened for phenotypes, and the site of transposon insertion can subsequently be identified in selected mutants by inverse polymerase chain reaction (PCR). In-frame insertions within genes of interest can be truncated to an epitope-tagged allele by Cre-lox recombination, and the subcellular localization of the encoded protein product can be identified by standard methods of indirect immunofluorescence. In summary, the transposon-insertion libraries represent an informative resource for large-scale mutagenesis, presenting a straightforward alternative to labor-intensive targeted approaches for the construction of deletion alleles and fluorescent protein fusions. PMID:27250939

  15. Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: Application to diagnosis and blood screening for posttransfusion hepatitis

    Energy Technology Data Exchange (ETDEWEB)

    Miyamura, Tatsuo; Saito, Izumu (National Institute of Health, Tokyo (Japan)); Katayama, Tohru (Tokyo National Chest Hospital (Japan)); Kikuchi, Shu; Tateda, Akira (Sendai National Hospital (Japan)); Houghton, M.; Choo, Quilim; Kuo, G. (Chiron Corporation, Emeryville, CA (USA))

    1990-02-01

    A cDNA clone has been derived from the plasma of a chimpanzee with chronic non-A, non-B viral hepatits (NANBH). The authors have assayed for antibodies reacting with the encoded antigen in sera from posttransfusion hepatitis patients (643 samples from 23 patients) and their corresponding donors collected during the past 10 years in Japan. The antibody was detected in 15 out of 17 (88.2%) posttransfusion NANBH (PT-NANBH) patients whose sera over time displayed multiple alanine aminotransferase (ALT) peaks. In general, the antibody was detected after several peaks of serum ALT elevations and, once detected, it persisted for years. Of the 15 well-defined cases of PT-NANBH that showed multiple ALT peaks and hepatitis C virus seroconversions, 11 (73.3%) were shown to be transfused with at least one unit of blood positive for the antibody. The retrospective analysis showed that all tested donor blood found to be positive for the antibody had been transfused to recipients who afterwards developed NANBH. These data strongly suggest that the cloned cDNA originated from an etiological agent of NANBH termed the hepatitis C virus. Furthermore, the present study demonstrates that had the screening been done with the anti-hepatitis C virus assay, 11 out of 17 (64.7%) cases of chronic PT-NANBH and 1 out of 6 (16.6%) acute PT-NANBH would have been prevented.

  16. Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: Application to diagnosis and blood screening for posttransfusion hepatitis

    International Nuclear Information System (INIS)

    A cDNA clone has been derived from the plasma of a chimpanzee with chronic non-A, non-B viral hepatits (NANBH). The authors have assayed for antibodies reacting with the encoded antigen in sera from posttransfusion hepatitis patients (643 samples from 23 patients) and their corresponding donors collected during the past 10 years in Japan. The antibody was detected in 15 out of 17 (88.2%) posttransfusion NANBH (PT-NANBH) patients whose sera over time displayed multiple alanine aminotransferase (ALT) peaks. In general, the antibody was detected after several peaks of serum ALT elevations and, once detected, it persisted for years. Of the 15 well-defined cases of PT-NANBH that showed multiple ALT peaks and hepatitis C virus seroconversions, 11 (73.3%) were shown to be transfused with at least one unit of blood positive for the antibody. The retrospective analysis showed that all tested donor blood found to be positive for the antibody had been transfused to recipients who afterwards developed NANBH. These data strongly suggest that the cloned cDNA originated from an etiological agent of NANBH termed the hepatitis C virus. Furthermore, the present study demonstrates that had the screening been done with the anti-hepatitis C virus assay, 11 out of 17 (64.7%) cases of chronic PT-NANBH and 1 out of 6 (16.6%) acute PT-NANBH would have been prevented

  17. 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.

  18. Design of a general-purpose European compound screening library for EU-OPENSCREEN.

    Science.gov (United States)

    Horvath, Dragos; Lisurek, Michael; Rupp, Bernd; Kühne, Ronald; Specker, Edgar; von Kries, Jens; Rognan, Didier; Andersson, C David; Almqvist, Fredrik; Elofsson, Mikael; Enqvist, Per-Anders; Gustavsson, Anna-Lena; Remez, Nikita; Mestres, Jordi; Marcou, Gilles; Varnek, Alexander; Hibert, Marcel; Quintana, Jordi; Frank, Ronald

    2014-10-01

    This work describes a collaborative effort to define and apply a protocol for the rational selection of a general-purpose screening library, to be used by the screening platforms affiliated with the EU-OPENSCREEN initiative. It is designed as a standard source of compounds for primary screening against novel biological targets, at the request of research partners. Given the general nature of the potential applications of this compound collection, the focus of the selection strategy lies on ensuring chemical stability, absence of reactive compounds, screening-compliant physicochemical properties, loose compliance to drug-likeness criteria (as drug design is a major, but not exclusive application), and maximal diversity/coverage of chemical space, aimed at providing hits for a wide spectrum of drugable targets. Finally, practical availability/cost issues cannot be avoided. The main goal of this publication is to inform potential future users of this library about its conception, sources, and characteristics. The outline of the selection procedure, notably of the filtering rules designed by a large committee of European medicinal chemists and chemoinformaticians, may be of general methodological interest for the screening/medicinal chemistry community. The selection task of 200K molecules out of a pre-filtered set of 1.4M candidates was shared by five independent European research groups, each picking a subset of 40K compounds according to their own in-house methodology and expertise. An in-depth analysis of chemical space coverage of the library serves not only to characterize the collection, but also to compare the various chemoinformatics-driven selection procedures of maximal diversity sets. Compound selections contributed by various participating groups were mapped onto general-purpose self-organizing maps (SOMs) built on the basis of marketed drugs and bioactive reference molecules. In this way, the occupancy of chemical space by the EU-OPENSCREEN library could

  19. Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression.

    OpenAIRE

    Ohkawa, J; Okada, N; Shinmyo, A; Takano, M.

    1989-01-01

    cDNA clones for ascorbate oxidase were isolated from a cDNA library made from cucumber (Cucumis sativus) fruit mRNA. The library was screened with synthetic oligonucleotides that encode the NH2-terminal sequence of this enzyme. Nucleotide sequence analysis of the cloned cDNA inserts revealed a 1761-base-pair open reading frame that encoded an NH2-terminal signal peptide of 33 amino acids and a mature enzyme of 554 amino acids (Mr, 62,258). The amino acid sequence deduced from nucleotide seque...

  20. Proteomic Screening Method for Phosphopeptide Motif Binding Proteins Using Peptide Libraries

    OpenAIRE

    Christofk, Heather R.; Wu, Ning; Cantley, Lewis C.; John M. Asara

    2011-01-01

    Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially-degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC/MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pTh...

  1. Understanding Dermatan Sulfate−Heparin Cofactor II Interaction through Virtual Library Screening

    OpenAIRE

    Raghuraman, Arjun; Mosier, Philip D.; Desai, Umesh R.

    2010-01-01

    Dermatan sulfate, an important member of the glycosaminoglycan family, interacts with heparin cofactor II, a member of the serpin family of proteins, to modulate antithrombotic response. Yet, the nature of this interaction remains poorly understood at a molecular level. We report the genetic algorithm-based combinatorial virtual library screening study of a natural, high-affinity dermatan sulfate hexasaccharide with heparin cofactor II. Of the 192 topologies possible for the hexasaccharide, o...

  2. Epitope Prediction Based on Random Peptide Library Screening: Benchmark Dataset and Prediction Tools Evaluation

    OpenAIRE

    Yanxin Huang; Hongyan Wang; Zhiqiang Ma; Yinghua Lv; Pingping Sun; Wenhan Chen

    2011-01-01

    Epitope prediction based on random peptide library screening has become a focus as a promising method in immunoinformatics research. Some novel software and web-based servers have been proposed in recent years and have succeeded in given test cases. However, since the number of available mimotopes with the relevant structure of template-target complex is limited, a systematic evaluation of these methods is still absent. In this study, a new benchmark dataset was defined. Using this benchmark ...

  3. Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening.

    Science.gov (United States)

    Viña-Gonzalez, Javier; Gonzalez-Perez, David; Alcalde, Miguel

    2016-01-01

    Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps. PMID:27077451

  4. From the ORFeome concept to highly comprehensive, full-genome screening libraries.

    Science.gov (United States)

    Rid, Raphaela; Abdel-Hadi, Omar; Maier, Richard; Wagner, Martin; Hundsberger, Harald; Hintner, Helmut; Bauer, Johann; Onder, Kamil

    2013-02-01

    Recombination-based cloning techniques have in recent times facilitated the establishment of genome-scale single-gene ORFeome repositories. Their further handling and downstream application in systematic fashion is, however, practically impeded because of logistical plus economic challenges. At this juncture, simultaneously transferring entire gene collections in compiled pool format could represent an advanced compromise between systematic ORFeome (an organism's entire set of protein-encoding open reading frames) projects and traditional random library approaches, but has not yet been considered in great detail. In our endeavor to merge the comprehensiveness of ORFeomes with a basically simple, streamlined, and easily executable single-tube design, we have here produced five different pooled screening-ready libraries for both Staphylococcus aureus and Homo sapiens. By evaluating the parallel transfer efficiencies of differentially sized genes from initial polymerase chain reaction (PCR) product amplification to entry and final destination library construction via quantitative real-time PCR, we found that the complexity of the gene population is fairly stably maintained once an entry resource has been successfully established, and that no apparent size-selection bias loss of large inserts takes place. Recombinational transfer processes are hence robust enough for straightforwardly achieving such pooled screening libraries. PMID:22621725

  5. Screening Oligosaccharide Libraries against Lectins Using the Proxy Protein Electrospray Ionization Mass Spectrometry Assay.

    Science.gov (United States)

    Han, Ling; Shams-Ud-Doha, Km; Kitova, Elena N; Klassen, John S

    2016-08-16

    An electrospray ionization mass spectrometry (ESI-MS) assay for screening carbohydrate libraries against lectins is described. The assay is based on the proxy protein ESI-MS method, which combines direct ESI-MS protein-ligand binding measurements and competitive protein binding, to simultaneously detect and quantify protein-carbohydrate interactions. Specific interactions between components of the library and the target protein (PT) are identified from changes in the relative abundances (as measured by ESI-MS) of the carbohydrate complexes of a proxy protein (Pproxy), which binds to all components of the library with known affinity, upon addition of PT to the solution. The magnitude of the change in relative abundance of a given Pproxy-ligand complex provides a quantitative measure of the affinity of the corresponding PT-ligand interaction. A mathematical framework for the implementation of the method in the case of monovalent (single binding site) Pproxy and monovalent and multivalent (multiple equivalent and independent binding sites) PT is described. The application of the method to screen small libraries of oligosaccharides, on the basis of human histo-blood group antigens and milk oligosaccharides, against an N-terminal fragment of the family 51 carbohydrate-binding module, a fucose-binding lectin from Ralstonia solanacearum, and human norovirus VA387 P particle (24-mer of the protruding domain of the capsid protein), serves to demonstrate the reliability and versatility of the assay. PMID:27366913

  6. Screening of an Escherichia coli promoter library for a phenylalanine biosensor.

    Science.gov (United States)

    Mahr, Regina; von Boeselager, Raphael Freiherr; Wiechert, Johanna; Frunzke, Julia

    2016-08-01

    In recent years, the application of transcription factor-based biosensors for the engineering of microbial production strains opened up new opportunities for industrial biotechnology. However, the design of synthetic regulatory circuits depends on the selection of suitable transcription factor-promoter pairs to convert the concentration of effector molecules into a measureable output. Here, we present an efficient strategy to screen promoter libraries for appropriate parts for biosensor design. To this end, we pooled the strains of the Alon library containing about 2000 different Escherichia coli promoter-gfpmut2 fusions, and enriched galactose- and L-phenylalanine-responsive promoters by toggled rounds of positive and negative selection using fluorescence-activated cell sorting (FACS). For both effectors, responsive promoters were isolated and verified by cultivation in microtiter plates. The promoter of mtr, encoding an L-tryptophan-specific transporter, was identified as suitable part for the construction of an L-phenylalanine biosensor. In the following, we performed a comparative analysis of different biosensor constructs based on the mtr promoter. The obtained data revealed a strong influence of the biosensor architecture on the performance characteristics. For proof-of-principle, the mtr sensor was applied in a FACS high-throughput screening of an E. coli MG1655 mutant library for the isolation of L-phenylalanine producers. These results emphasize the developed screening approach as a convenient strategy for the identification of effector-responsive promoters for the design of novel biosensors. PMID:27170323

  7. A Colony Multiplex Quantitative PCR-Based 3S3DBC Method and Variations of It for Screening DNA Libraries

    Science.gov (United States)

    An, Yang; Toyoda, Atsushi; Zhao, Chen; Fujiyama, Asao; Agata, Kiyokazu

    2015-01-01

    A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC) method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements. PMID:25646755

  8. A colony multiplex quantitative PCR-Based 3S3DBC method and variations of it for screening DNA libraries.

    Directory of Open Access Journals (Sweden)

    Yang An

    Full Text Available A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements.

  9. Cloning and chromosomal localization of a human kidney cDNA involved in cystine, dibasic, and neutral amino acid transport.

    OpenAIRE

    Lee, W S; Wells, R G; Sabbag, R V; Mohandas, T K; Hediger, M A

    1993-01-01

    We have recently cloned, sequenced, and characterized a rat kidney cDNA (D2) that stimulates cystine as well as dibasic and neutral amino acid transport. In order to evaluate the role of this protein in human inherited diseases such as cystinuria, we have isolated a human D2 clone (D2H) by low stringency screening of a human kidney cDNA library using the radiolabeled D2 insert as a probe. The D2H cDNA is 2284 nucleotides long and encodes a 663 amino acid protein that is 80% identical to the r...

  10. Syrinx 2A: an improved lambda phage vector designed for screening DNA libraries by recombination in vivo.

    OpenAIRE

    Lutz, C T; Hollifield, W C; Seed, B.; Davie, J M; Huang, H V

    1987-01-01

    The Syrinx 2A phage and pi AN13 plasmid were designed for screening of DNA libraries by homologous recombination in vivo. Syrinx 2A carries multiple cloning sites and a recently identified lambda gene, rap (recombination adept with plasmid), required for efficient phage-plasmid recombination. We describe a rapid, reliable, and technically easy method to screen Syrinx 2A libraries, expand the resulting phage-plasmid cointegrates, and subclone plasmid in as little as 2 days. Recombination scree...

  11. Expression screening using a Medaka cDNA library identifies evolutionarily conserved regulators of the p53/Mdm2 pathway

    OpenAIRE

    P. Zhang; Kratz, A.S.; M. Salama; Elabd, S.; Heinrich, T; Wittbrodt, J.; Blattner, C.; G Davidson

    2015-01-01

    Background The p53 tumor suppressor protein is mainly regulated by alterations in the half-life of the protein, resulting in significant differences in p53 protein levels in cells. The major regulator of this process is Mdm2, which ubiquitinates p53 and targets it for proteasomal degradation. This process can be enhanced or reduced by proteins that associate with p53 or Mdm2 and several proteins have been identified with such an activity. Furthermore, additional ubiquitin ligases for p53 have...

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

    Czech Academy of Sciences Publication Activity Database

    Dokládal, Ladislav; Honys, David; Rana, Rajiv; Lee, L.-Y.; Gelvin, S.B.; Sýkorová, Eva

    2015-01-01

    Roč. 6, NOV2015 (2015). ISSN 1664-462X R&D Projects: GA ČR GA13-06943S; GA MŠk(CZ) ED1.1.00/02.0068 Grant ostatní: GA MŠk(CZ) LH10352 Institutional support: RVO:68081707 ; RVO:61389030 Keywords : telomerase * nuclear poly(A)-binding protein * telobox Subject RIV: BO - Biophysics; EF - Botanics (UEB-Q) Impact factor: 3.948, year: 2014

  13. A Microfluidic Platform for High-Throughput Screening of Small Mutant Libraries.

    Science.gov (United States)

    Lim, Ji Won; Shin, Kwang Soo; Moon, Jaemin; Lee, Sung Kuk; Kim, Taesung

    2016-05-17

    The screening and isolation of target microorganisms from mutated recombinant libraries are crucial for the advancement of synthetic biology and metabolic engineering. However, conventional screening tools present several limitations in throughput, cost, and labor. Herein, we describe a novel microfluidic high-throughput screening (HTS) platform with several advantages. The platform utilizes a fluid array to compartmentalize bacterial cells in well-ordered separated microwells and allows long-term cell culture with high throughput. The platform enables the extraction of selected target cells from the fluid array for additional culture and postanalysis by using a capillary-driven sample relocation method. To confirm the feasibility of the platform, we demonstrated two different types of HTS methods based on the levels of reporter gene expression and cellular growth rate difference. For the reporter gene-based HTS, a spike recovery approach was taken to demonstrate that target cells are successfully screened out from a mixture containing nontarget cells by repeating the culture and extraction processes. Additionally, the same platform allowed us to screen and sort target cells according to their cellular growth rate difference, which seems hard in conventional screening methods. Hence, the platform could be used for various microbiological assays, including the detection of cell-excreted metabolites, microbial biosensors, and other HTS systems. PMID:27104360

  14. 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.

  15. Isolation of a cDNA clone for the type I regulatory subunit of bovine cAMP-dependent protein kinase.

    OpenAIRE

    Lee, D C; Carmichael, D F; Krebs, E G; McKnight, G S

    1983-01-01

    A cDNA clone for the type I regulatory subunit (RI) of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was isolated from bovine testis by a differential screening method. mRNA coding for RI was enriched 50- to 100-fold by polysome immunoadsorption chromatography with affinity-purified rabbit anti-RI and protein A-Sepharose. Poly(A)+ RNA from these polysomes was utilized to construct a cDNA library in pBR322, and this library was screened for hybridization to 32P-la...

  16. Construction and preliminary analysis for a full length cDNA library of the dominant strain of Penicillium marneffei isolated from AIDS patient in yeast phase%艾滋病患者马尔尼菲青霉菌优势株酵母相全长cDNA文库的构建和初步分析

    Institute of Scientific and Technical Information of China (English)

    李凌华; 胡凤玉; 陈万山; 宋伟南; 邝燕玲; 蔡卫平; 唐小平

    2010-01-01

    Objective To construct a full length cDNA library of the dominant strain of Penicillium marneffei (PM) in yeast phase isolated from AIDS patients in Guangdong province and screen UniGenes as well as full-length genes, so as to establish the foundation for the study of PM's functional genes and pathogenic mechanisms. Methods CloneMiner cDNA construction kit was utilized to extract mRNA of the dominant PM strain isolated from AIDS patients in Guangdong province. The mRNA was reversed into cDNA, then cloned into a pDONR222 vector by BP recombination to obtain an Uncut cDNA library, which was homogenized later to construct a normalized cDNA library with the principal of saturation hybridization for DNA genome. 2000 clones were chosen randomly to make a bi-directional sequencing and analyzed with bioinformatics for screening UniGenes and full-length genes. Results The total clone number of the Uncut cDNA library was 1.16 × 107 cfu/mL, with a recombination rate of 95% and an average insertion element being over 1 kb. The total clone number of the normalized cDNA library was 1.18 × 106 cfu/mL, with a recombination rate of 95% and an average insertion element being over 1 kb as well. 1945 genes which DNA length were longer than 1 kb were obtained by sequencing and merged into 1360 UniG enes, of which 632 genes were full-length ones. Conclusions The full-length cDNA library of the dominant strain of PM from AIDS patients in Guangdong province possesses good quality.Meanwhile, the technical routine presents high efficiency in obtaining full-length genes and establishing a gene expression spectrum, which can contentedly meet the needs of future experiments.%目的 构建广东地区艾滋病患者马尔尼菲青霉菌(PM)优势株酵母相全长cDNA文库,筛选UniGene和全长基因,为PM的功能基因组学研究和致病机制的探讨奠定基础.方法 应用CloneMiner cDNA construction kit提取广东地区艾滋病患者PM优势株酵母相mRNA,反转录成cDNA后

  17. Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries.

    Directory of Open Access Journals (Sweden)

    Brian D Hondowicz

    Full Text Available The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.

  18. Newborn Screening: National Library of Medicine Literature Search, January 1980 through March 1987. No. 87-2.

    Science.gov (United States)

    Patrias, Karen

    This bibliography, prepared by the National Library of Medicine through a literature search of its online databases, covers all aspects of newborn screening. It includes references to screening for: inborn errors of metabolism, such as phenylketonuria and galactosemia; hemoglobinopathies, particularly sickle cell disease; congenital hypothyroidism…

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

    Institute of Scientific and Technical Information of China (English)

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

    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 106 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

  1. Characterization of antigen-expressing Plasmodium falciparum cDNA clones that are reactive with parasite inhibitory antibodies.

    Science.gov (United States)

    Horii, T; Bzik, D J; Inselburg, J

    1988-07-01

    A Plasmodium falciparum (FCR3 strain) lambda gt11 cDNA expression library was constructed from trophozoite and schizont poly(A) RNA and was screened immunologically with a pooled human immune serum from Nigeria to form a gene bank of 288 positive clones. The gene bank was subsequently screened with parasite inhibitory mouse monoclonal antibodies (mMAb) and with individual human Liberian sera. Two mMAb, 43E5 and 5H10, strongly reacted with 8 and 3 cDNA clones, respectively. Several of those clones also weakly cross-reacted with the other mMAb. Two of those weakly cross-reactive clones, cDNA#366 and cDNA#22, were shown to be located in different chromosomal regions of the parasite by Southern hybridization and so appeared to represent two different parasite genes. The genomic organization of both cDNA#366 and cDNA#22 sequences were identical in the FCR3 and the Honduras-1 strain. The nucleotide sequence of cDNA#366 and the amino acid sequence it coded for were homologous to a partial DNA and amino acid sequence previously reported for a P. falciparum (Camp strain) exoantigen designated p126. The mRNA for cDNA#366 appeared to represent an abundant message in blood stage trophozoites and schizonts. PMID:2456465

  2. Histidine-containing peptide catalysts developed by a facile library screening method.

    Science.gov (United States)

    Akagawa, Kengo; Sakai, Nobutaka; Kudo, Kazuaki

    2015-02-01

    Although peptide catalysts have a high potential for the use as organocatalysts, the optimization of peptide sequences is laborious and time-consuming. To address this issue, a facile screening method for finding efficient aminocatalysts from a peptide library has been developed. In the screening for the Michael addition of a malonate to an enal, a dye-labeled product is immobilized on resin-bound peptides through reductive amination to visualize active catalysts. This procedure allows for the monitoring of the reactivity of entire peptides without modifying the resin beads beforehand. Peptides containing histidine at an appropriate position were identified by this method. A novel function of the histidyl residue, which enhances the binding of a substrate to the catalyst by capturing an iminium intermediate, was indicated. PMID:25521645

  3. A high throughput screen for biomining cellulase activity from metagenomic libraries.

    Science.gov (United States)

    Mewis, Keith; Taupp, Marcus; Hallam, Steven J

    2011-01-01

    Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system. PMID:21307835

  4. cDNA: 57843 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.1441 Mus musculus adult male testis cDNA, RIKEN full-length enriched library, c ... lone:4930519F16 product:hypothetical Serine proteases , trypsin family/Chymotrypsin serine protease famil ...

  5. cDNA: 44740 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.29756 Mus musculus adult male testis cDNA, RIKEN full-length enriched library, ... 2 product:hypothetical Eukaryotic thiol (cysteine) proteases ... active site containing protein, full insert sequen ...

  6. cDNA: 54640 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.212157 Mus musculus adult male kidney cDNA, RIKEN full-length enriched library, ... 1 product:hypothetical Eukaryotic thiol (cysteine) proteases ... active site containing protein, full insert sequen ...

  7. cDNA: 35981 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.122430 Mus musculus adult retina cDNA, RIKEN full-length enriched library, clon ... 0014I21 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  8. cDNA: 35985 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.122430 Mus musculus adult male testis cDNA, RIKEN full-length enriched library, ... 0440H19 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  9. THE FULL-LENGTH cDNA LIBRARY OF HEMOCYTE INDUCED BY AEROMONAS HYDROPHILA AND MOLECULAR CHARACTERISTICS OF CYCLOPHILINA FROM HYRIOPSIS SCHLEGELH%嗜水气单胞菌诱导的池蝶蚌血细胞cDNA文库的构建和亲环蛋白基因序列的初步分析

    Institute of Scientific and Technical Information of China (English)

    谢凯; 徐灵; 盛军庆; 曾柳根; 王军花; 洪一江

    2011-01-01

    fractionation by CHROMA SPIN-400 columns, ligated with the sfi I digested pDNR-LIB vector and transformed into E. Coli DH10B by thermal shock, the full-length cDNA library was constructed. It was the first time to construct a full-length cDNA library from blood hemocyte of the freshwater mussel. The titer of the primary constructed cDNA library was 4×106 cfu/cm3with a high recombination rate of 90% and the amplified one was 3.55xl09cfu/cm\\ 672 clones were sequenced randomly selected from the library and 436 have the homologous gene information after analyzed by BLASTx in the website of NCBI. Among the sequences, the shortest one was 270 bp and the longest one was 2153 bp, and average length was 608 bp. Results indicated it was a high quality cDNA library. The full length of the Cyp A (HsCyp A), which may provide immune stress function in some species, was isolated from the cDNA library by screening of H. Schlegelii. It has 1229 base pair (bp), containing a 52 bp 5' untranslated region (UTR), a 495 bp open reading frame, a 682 bp 3'-untranslated sequence, and a 29 nucleotide long poly (A) tail. The initiator codon (ATG) and the stop codon (TAA) can be found at the position of 53 and 545, but can not find the putative polyadenylation signal (AATAAA). The predicted protein sequence consisted of 146 amino acids with a calculated molecular mass of 17.26 kD and an isoelectric point of 8.9. The secondary structure, 3D homology-modeled structure and phylogenetic analysis indicated the HsCyp A was extremely conservative during the evolutionary history. All the results showed that the Cyp A was not only a consistent protein, but also may play important roles in the immune system the lower aquatic animal. This work may provide important information about specific genes for mussel immune, and also provide some background for future research about the immune mechanisms of Cyp A in freshwater mussel.

  10. cDNA for R-cognin: homology with a multifunctional protein.

    OpenAIRE

    Krishna Rao, A S; Hausman, R E

    1993-01-01

    Retina cognin (R-cognin) is a developmentally regulated 50-kDa protein that was isolated from chicken embryo retina cell membranes. It mediates the adhesion and reaggregation in vitro of retina cells from chicken and mouse embryos, but not of cells from other tissues, and may be involved in neuronal differentiation. We report here the cloning of a cDNA for R-cognin. A chicken embryo retina cDNA library was constructed in lambda gt11 vector and was screened with polyclonal R-cognin antiserum, ...

  11. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila.

    Directory of Open Access Journals (Sweden)

    Stephen R Spindler

    Full Text Available Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR, platelet-derived growth factor (PDGF/vascular endothelial growth factor (VEGF receptors, G-protein coupled receptor (GPCR, Janus kinase (JAK/signal transducer and activator of transcription (STAT, the insulin and insulin-like growth factor (IGFI receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38, c-Jun N-terminal kinase (JNK and protein kinase C (PKC. If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

  12. Library+

    Science.gov (United States)

    Merrill, Alex

    2011-01-01

    This article discusses possible future directions for academic libraries in the post Web/Library 2.0 world. These possible directions include areas such as data literacy, linked data sets, and opportunities for libraries in support of digital humanities. The author provides a brief sketch of the background information regarding the topics and…

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

    Institute of Scientific and Technical Information of China (English)

    Ignacio Ferrés; Vanesa Amarelle; Francisco Noya; Elena Fabiano

    2015-01-01

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

  14. Functional Screening of Antibiotic Resistance Genes from a Representative Metagenomic Library of Food Fermenting Microbiota

    Directory of Open Access Journals (Sweden)

    Chiara Devirgiliis

    2014-01-01

    Full Text Available Lactic acid bacteria (LAB represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.

  15. Molecular cloning of cDNA for human prothymosin alpha.

    OpenAIRE

    Goodall, G J; Dominguez, F.; Horecker, B L

    1986-01-01

    A cDNA library was constructed from human spleen mRNA and screened for clones containing cDNAs coding for prothymosin alpha. A clone containing a 503-base-pair insert including the entire coding sequence for the translated portion of the mRNA was isolated. The deduced amino acid sequence confirms and completes the partial sequence of human prothymosin alpha determined by protein sequencing methods. The presence of an initiator codon immediately preceding the codon for the NH2-terminal serine ...

  16. New lipolytic enzymes identified by screening two metagenomic libraries derived from the soil of a winter wheat field

    Directory of Open Access Journals (Sweden)

    Stroobants, A.

    2015-01-01

    Full Text Available Description of the subject. Lipolytic enzymes are widely distributed and fulfil important physiological functions in the microorganisms inhabiting diverse environments. Soils are rich, diversified environments containing microbial communities that remain largely unknown. Objectives. This work aimed to discover new lipolytic enzymes. Method. New enzymes were found by functional screening of two seasonal metagenomic libraries (a winter and a spring library constructed from an agricultural soil. Screens were performed on 2xYT medium supplemented with 3% lipase reagent. Results. Nineteen positive clones were isolated. Analysis of the corresponding inserts led to identifying 23 putative lipolytic enzymes (13 for the winter library and 10 for the spring library displaying between 31% and 62% identity to known enzymes and belonging to seven different families. Conclusions. As enzymes show low identity to known enzymes, the encoded enzymes may display novel biochemical features.

  17. High-throughput screen of natural product libraries for hsp90 inhibitors.

    Science.gov (United States)

    Davenport, Jason; Balch, Maurie; Galam, Lakshmi; Girgis, Antwan; Hall, Jessica; Blagg, Brian S J; Matts, Robert L

    2014-01-01

    Hsp90 has become the target of intensive investigation, as inhibition of its function has the ability to simultaneously incapacitate proteins that function in pathways that represent the six hallmarks of cancer. While a number of Hsp90 inhibitors have made it into clinical trials, a number of short-comings have been noted, such that the search continues for novel Hsp90 inhibitors with superior pharmacological properties. To identify new potential Hsp90 inhibitors, we have utilized a high-throughput assay based on measuring Hsp90-dependent refolding of thermally denatured luciferase to screen natural compound libraries. Over 4,000 compounds were screen with over 100 hits. Data mining of the literature indicated that 51 compounds had physiological effects that Hsp90 inhibitors also exhibit, and/or the ability to downregulate the expression levels of Hsp90-dependent proteins. Of these 51 compounds, seven were previously characterized as Hsp90 inhibitors. Four compounds, anthothecol, garcinol, piplartine, and rottlerin, were further characterized, and the ability of these compounds to inhibit the refolding of luciferase, and reduce the rate of growth of MCF7 breast cancer cells, correlated with their ability to suppress the Hsp90-dependent maturation of the heme-regulated eIF2α kinase, and deplete cultured cells of Hsp90-dependent client proteins. Thus, this screen has identified an additional 44 compounds with known beneficial pharmacological properties, but with unknown mechanisms of action as possible new inhibitors of the Hsp90 chaperone machine. PMID:24833337

  18. Users’ Expectation from the User Interface Screen of an Academic Digital Library

    Directory of Open Access Journals (Sweden)

    Akbar Majidi

    2010-09-01

    Full Text Available The present paper investigates the E-learner’s expectations concerning the features incorporated within the user interface screen of an academic digital library. A researcher-made questionnaire was used for the survey. The sample was taken from the E-learners using this technology in Iranian universities. 200 questionnaires were distributed. The data analysis showed a general consensus about the priority of comprehensibility of the terms used in the User Interface Screen (uis as well as the display features and clarity of the navigational functions as the usability criteria for UIS. ANOVA analysis indicated that, with the exception of navigation and guidance functions, there was no significance with respect to three categories of students. In other words, all students had similar expectations and their ICT skill is not a factor influencing the prioritization of these criteria. The results further indicated that except for the browsing page, there is no significant difference between novice, intermediate and advanced students with respect to search screen features.

  19. High-Throughput Screen of Natural Product Libraries for Hsp90 Inhibitors

    Directory of Open Access Journals (Sweden)

    Jason Davenport

    2014-02-01

    Full Text Available Hsp90 has become the target of intensive investigation, as inhibition of its function has the ability to simultaneously incapacitate proteins that function in pathways that represent the six hallmarks of cancer. While a number of Hsp90 inhibitors have made it into clinical trials, a number of short-comings have been noted, such that the search continues for novel Hsp90 inhibitors with superior pharmacological properties. To identify new potential Hsp90 inhibitors, we have utilized a high-throughput assay based on measuring Hsp90-dependent refolding of thermally denatured luciferase to screen natural compound libraries. Over 4,000 compounds were screen with over 100 hits. Data mining of the literature indicated that 51 compounds had physiological effects that Hsp90 inhibitors also exhibit, and/or the ability to downregulate the expression levels of Hsp90-dependent proteins. Of these 51 compounds, seven were previously characterized as Hsp90 inhibitors. Four compounds, anthothecol, garcinol, piplartine, and rottlerin, were further characterized, and the ability of these compounds to inhibit the refolding of luciferase, and reduce the rate of growth of MCF7 breast cancer cells, correlated with their ability to suppress the Hsp90-dependent maturation of the heme-regulated eIF2α kinase, and deplete cultured cells of Hsp90-dependent client proteins. Thus, this screen has identified an additional 44 compounds with known beneficial pharmacological properties, but with unknown mechanisms of action as possible new inhibitors of the Hsp90 chaperone machine.

  20. Human liver mitochondrial carnitine palmitoyltransferase I: characterization of its cDNA and chromosomal localization and partial analysis of the gene.

    OpenAIRE

    Britton, C H; Schultz, R.A.; Zhang, B; Esser, V; Foster, D W; McGarry, J D

    1995-01-01

    Using the cDNA for rat liver mitochondrial carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) as a probe, we isolated its counterpart as three overlapping clones from a human liver cDNA library. Both the nucleotide sequence of the human cDNA and the predicted primary structure of the protein (773 aa) proved to be very similar to those of the rat enzyme (82% and 88% identity, respectively). The CPT I mRNA size was also found to be the same (approximately 4.7 kb) in both species. Screening o...

  1. 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...

  2. Library design and screening protocol for artificial metalloenzymes based on the biotin-streptavidin technology.

    Science.gov (United States)

    Mallin, Hendrik; Hestericová, Martina; Reuter, Raphael; Ward, Thomas R

    2016-05-01

    Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within streptavidin (Sav) combine attractive features of both homogeneous and enzymatic catalysts. To speed up their optimization, we present a streamlined protocol for the design, expression, partial purification and screening of Sav libraries. Twenty-eight positions have been subjected to mutagenesis to yield 335 Sav isoforms, which can be expressed in 24-deep-well plates using autoinduction medium. The resulting cell-free extracts (CFEs) typically contain >1 mg of soluble Sav. Two straightforward alternatives are presented, which allow the screening of ArMs using CFEs containing Sav. To produce an artificial transfer hydrogenase, Sav is coupled to a biotinylated three-legged iridium pianostool complex Cp*Ir(Biot-p-L)Cl (the cofactor). To screen Sav variants for this application, you would determine the number of free binding sites, treat them with diamide, incubate them with the cofactor and then perform the reaction with your test compound (the example used in this protocol is 1-phenyl-3,4-dihydroisoquinoline). This process takes 20 d. If you want to perform metathesis reactions, Sav is coupled to a biotinylated second-generation Grubbs-Hoveyda catalyst. In this application, it is best to first immobilize Sav on Sepharose-iminobiotin beads and then perform washing steps. Elution from the beads is achieved in an acidic reaction buffer before incubation with the cofactor. Catalysis using your test compound (in this protocol, 2-(4-(N,N-diallylsulfamoyl)phenyl)-N,N,N-trimethylethan-1-aminium iodide) is performed using the formed metalloenzyme. Screening using this approach takes 19 d. PMID:27031496

  3. Methods for transforming and expression screening of filamentous fungal cells with a DNA library

    Energy Technology Data Exchange (ETDEWEB)

    Teter, Sarah; Lamsa, Michael; Cherry, Joel; Ward, Connie

    2015-06-02

    The present invention relates to methods for expression screening of filamentous fungal transformants, comprising: (a) isolating single colony transformants of a DNA library introduced into E. coli; (b) preparing DNA from each of the single colony E. coli transformants; (c) introducing a sample of each of the DNA preparations of step (b) into separate suspensions of protoplasts of a filamentous fungus to obtain transformants thereof, wherein each transformant contains one or more copies of an individual polynucleotide from the DNA library; (d) growing the individual filamentous fungal transformants of step (c) on selective growth medium, thereby permitting growth of the filamentous fungal transformants, while suppressing growth of untransformed filamentous fungi; and (e) measuring activity or a property of each polypeptide encoded by the individual polynucleotides. The present invention also relates to isolated polynucleotides encoding polypeptides of interest obtained by such methods, to nucleic acid constructs, expression vectors, and recombinant host cells comprising the isolated polynucleotides, and to methods of producing the polypeptides encoded by the isolated polynucleotides.

  4. Study on the Mitochondrial Genome of Sea Island Cotton (Gossypium barbadense) by BAC Library Screening

    Institute of Scientific and Technical Information of China (English)

    SU Ai-guo; LI Shuang-shuang; LIU Guo-zheng; LEI Bin-bin; KANG Ding-ming; LI Zhao-hu; MA Zhi-ying; HUA Jin-ping

    2014-01-01

    The plant mitochondrial genome displays complex features, particularly in terms of cytoplasmic male sterility (CMS). Therefore, research on the cotton mitochondrial genome may provide important information for analyzing genome evolution and exploring the molecular mechanism of CMS. In this paper, we present a preliminary study on the mitochondrial genome of sea island cotton (Gossypium barbadense) based on positive clones from the bacterial artiifcial chromosome (BAC) library. Thirty-ifve primers designed with the conserved sequences of functional genes and exons of mitochondria were used to screen positive clones in the genome library of the sea island cotton variety called Pima 90-53. Ten BAC clones were obtained and veriifed for further study. A contig was obtained based on six overlapping clones and subsequently laid out primarily on the mitochondrial genome. One BAC clone, clone 6 harbored with the inserter of approximate 115 kb mtDNA sequence, in which more than 10 primers fragments could be ampliifed, was sequenced and assembled using the Solexa strategy. Fifteen mitochondrial functional genes were revealed in clone 6 by gene annotation. The characteristics of the syntenic gene/exon of the sequences and RNA editing were preliminarily predicted.

  5. Screening of efficient siRNA carriers in a library of surface-engineered dendrimers

    Science.gov (United States)

    Liu, Hongmei; Chang, Hong; Lv, Jia; Jiang, Cong; Li, Zhenxi; Wang, Fei; Wang, Hui; Wang, Mingming; Liu, Chongyi; Wang, Xinyu; Shao, Naimin; He, Bingwei; Shen, Wanwan; Zhang, Qiang; Cheng, Yiyun

    2016-01-01

    Polymers are widely used as non-viral carriers for siRNA delivery, but concern has also arisen in their limited efficacy and inherent toxicity. Whilst many of previous efforts have been documented towards improving the performance of polymers via chemical modifications, the structure-activity relationships (SAR) of these ligand-modified polymers are not well understood. To address this issue, we systemically prepared a library of surface-engineered dendrimers (>300) as the screening pool to discover efficient siRNA carriers. The modified ligands include alkyls and fluoroalkyls, amino acids, benzene derivatives and heterocyclic compounds. Gene silencing results showed that the lead material shows excellent efficacy even in hard-to-transfect cells such as mesenchymal stem cells. The SAR studies revealed that ligands containing appropriate hydrophobicity, or ligands with both hydrophobic and functional atoms/groups are essential for polymers to achive efficient knockdown efficacy. A second-generation library designed based on the above principles further confirms the proposed design criteria. The results enable the future rational design of potent siRNA carriers. PMID:27121799

  6. Screening Peptide Inhibitors Using Phage Peptide Library with Isocitrate Lyase in Mycobacterium tuberculosis as Target

    Institute of Scientific and Technical Information of China (English)

    YIN Yu-he; NIU Xue; SUN Bo; TENG Guo-sheng; ZHAO Yun-hui; WU Cong-mei

    2011-01-01

    When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to stop persistent infections by interdicting the glyoxylate bypass in which the isocitrate lyase(ICL) is the key rate-limiting enzyme and a persistence factor.ICL is the target of anti-TB(TB:tubercular) drugs,which could screen ICL out and effectively inhibit the activity of ICL in Mycobacterium tuberculosis,and because of this,anti-TB drugs can be used to kill persistent Mycobacterium tuberculosis.In this study,the ICL gene of the Mycobacterium tuberculosis H37Rv was cloned successfully and recombinant protein with bioactivity was obtained through the enzyme characteristic appraisal.The specific activity of the recombined ICL is 24 μmol·mg-1 -min-1.The recombined ICL protein was used as the target,and phages which can specifically combine to ICL were screened in the phage 7 peptide library.According to the results of the ELISA and DNA sequence detection,eventually three 7-peptide chains were synthesized.Then the peptide chains were reacted with ICL,respectively,to detect their inhibitory effects on ICL.The results show that all the three 7-peptide chains possessed varying inhibitory effects on the activity of ICL.This study provided lead compounds for the research and development of new peptide anti-TB drugs.

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

    Directory of Open Access Journals (Sweden)

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

    2009-01-01

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

  8. Rapid screening of anti-infective drug products for counterfeits using Raman spectral library-based correlation methods.

    Science.gov (United States)

    Loethen, Yvette L; Kauffman, John F; Buhse, Lucinda F; Rodriguez, Jason D

    2015-11-01

    A new spectral library-based approach that is capable of screening a diverse set of finished drug products using only an active pharmaceutical ingredient spectral library is described in this paper. This approach obviates the need for a comprehensive drug product library, thereby streamlining the use of spectral library-based tests for anti-counterfeiting efforts, specifically to target finished drug products containing the wrong active ingredient or no active ingredient at all. Both laboratory-based and portable spectrometers are used in the study to demonstrate the usefulness and transferability of the spectral correlation method for field screening. The spectral correlation between the active pharmaceutical ingredient and finished drug product spectra is calculated using both full spectral analysis and targeted spectral regions analysis of six types of antimalarial, antibiotic and antiviral products. The spectral regions were determined using a moving window spectral correlation algorithm, and the use of specific spectral regions is shown to be crucial in screening finished drug products using only the active pharmaceutical ingredient spectrum. This comprehensive screening spectral correlation method is tested on seven different validation samples from different manufacturers as those used to develop the method, as well as simulated counterfeits which were prepared to mimic falsified drugs containing no active ingredient. The spectral correlation method is successful in correctly identifying 100% of the authentic products and simulated counterfeit samples tested. PMID:26401527

  9. 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.

  10. 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.

  11. Chemical Library Screens Targeting an HIV-1 Accessory Factor/Host Cell Kinase Complex Identify Novel Anti-retroviral Compounds

    OpenAIRE

    Emert-Sedlak, Lori; Kodama, Toshiaki; Lerner, Edwina C.; Dai, Weixiang; Foster, Caleb; Day, Billy W.; Lazo, John S.; Smithgall, Thomas E

    2009-01-01

    Nef is an HIV-1 accessory protein essential for AIDS progression and an attractive target for drug discovery. Lack of a catalytic function makes Nef difficult to assay in chemical library screens. We developed a high-throughput screening assay for inhibitors of Nef function by coupling it to one of its host cell binding partners, the Src-family kinase Hck. Hck activation is dependent upon Nef in this assay, providing a direct readout of Nef activity in vitro. Using this screen, a unique diphe...

  12. Screening of Proteins Interacting with Nonstructural 1 Protein of H5N1 Avian Influenza Virus from T7-phage Display Library

    Institute of Scientific and Technical Information of China (English)

    ZHU Chun-yu; WU Jian-guo; LIU Hong-sheng; SUN Ting-ting; ZHAO Jian; WANG Ning; ZHENG Fang-liang; AI Hai-xin; ZHU Jun-feng; WANG Xiao-ying; ZHU Ying

    2012-01-01

    Avian influenza virus(AIV) nonstructural 1(NS1) gene was amplified by real-time polymerse chain reaction(RT-PCR) and inserted into pET28a,then transformed into E.coli BL21(DE3) competent cell.With the induction of isopropyl-β-D-thiogalactoside(IPTG) and the purification of Ni-NTA column,we finally obtained purified NS1 protein.T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA library.The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in GeneBank.Two proteins were obtained as NS 1 binding proteins,Homo sapiens nucleolar and coiled-body phosphoprotein 1(NOLC1) and Homo sapiens similar to colon cancer-associated antigen.By co-immunoprecipitation and other methods,Homo sapiens NOLC1 was found to interact with the NS1 protein,the results would provide the basis for further studying biological function of NS1 protein.

  13. Development of peritoneal tumor-targeting vector by in vivo screening with a random peptide-displaying adenovirus library.

    Directory of Open Access Journals (Sweden)

    Takeshi Nishimoto

    Full Text Available The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.

  14. A Cell-Based Approach for the Biosynthesis/Screening of Cyclic Peptide Libraries against Bacterial Toxins

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kimura, R; Woo, Y; Cantor, J; Steenblock, E

    2007-10-24

    Available methods for developing and screening small drug-like molecules able to knockout toxins or pathogenic microorganisms have some limitations. In order to be useful, these new methods must provide high-throughput analysis and identify specific binders in a short period of time. To meet this need, we are developing an approach that uses living cells to generate libraries of small biomolecules, which are then screened inside the cell for activity. Our group is using this new, combined approach to find highly specific ligands capable of disabling anthrax Lethal Factor (LF) as proof of principle. Key to our approach is the development of a method for the biosynthesis of libraries of cyclic peptides, and an efficient screening process that can be carried out inside the cell.

  15. CONSTRUCTION AND ANALYSIS OF THE SUBTRACTED cDNA LIBRARY OF APOSTICHOPUS JAPONICUS (SELENKA) UNDER HIGH TEMPERATURE STRESS%刺参(Apostichopus japonicus)高温胁迫消减cDNA文库的构建与分析

    Institute of Scientific and Technical Information of China (English)

    吉成龙; 孙国华; 杨建敏; 宋志乐; 刘相全; 王卫军

    2011-01-01

    采用抑制性消减杂交技术(SSH)研究刺参高温胁迫下差异表达的基因.分别以高温实验组为检测组(tester)、常温对照组为驱动组(driver),进行正向抑制性消减杂交;以常温组为tester、高温组为driver,进行反向消减杂交,成功构建了刺参正反双向差异消减文库.从两个文库随机挑选384个白斑克隆进行斑点杂交进一步筛选,对其中差异显著的50个克隆进行测序分析.测序结果经BLAST比对分析得出:44个克隆与已知基因序列高度同源,主要包括3个上调表达基因和2个下调表达基因;另外5个克隆为未知新基因序列.该消减文库的成功构建对刺参耐高温相关基因的深入研究以及阐述耐高温的分子机理有非常重要的意义.%Suppression subtractive hybridization (SSH) was used to study the differentially expressed genes of Apostichopus japonicus under high temperature stress. The cDNA of A. japonicus underwent heat stress was used as tester and the same cDNA treated under room temperature was used as driver to construct a forward subtractive library. A reverse subtractive library was generated by using the same cNDA but underwent heat stress was the driver and untreated was tester. 384 positive clones were randomly picked from each library and used for dot blotting. 50 significant differentially expression clones were sequenced and analyzed using BLAST. The results show there are 44 sequences have high identities with the known genes. There are 10 major differentially expressed genes: three up-regulated known genes, two down-regulated known genes, and 5 are unknown genes. The constructed libraries in this study play an important role in studying the heat-resistant genes in A. japonicus and the molecular genetic mechanism of its ability in heat tolerance.

  16. Application of a cocktail approach to screen cytochrome P450 BM3 libraries for metabolic activity and diversity.

    Science.gov (United States)

    Reinen, Jelle; Postma, Geert; Tump, Cornelis; Bloemberg, Tom; Engel, Jasper; Vermeulen, Nico P E; Commandeur, Jan N M; Honing, Maarten

    2016-02-01

    In the present study, the validity of using a cocktail screening method in combination with a chemometrical data mining approach to evaluate metabolic activity and diversity of drug-metabolizing bacterial Cytochrome P450 (CYP) BM3 mutants was investigated. In addition, the concept of utilizing an in-house-developed library of CYP BM3 mutants as a unique biocatalytic synthetic tool to support medicinal chemistry was evaluated. Metabolic efficiency of the mutant library towards a selection of CYP model substrates, being amitriptyline (AMI), buspirone (BUS), coumarine (COU), dextromethorphan (DEX), diclofenac (DIC) and norethisterone (NET), was investigated. First, metabolic activity of a selection of CYP BM3 mutants was screened against AMI and BUS. Subsequently, for a single CYP BM3 mutant, the effect of co-administration of multiple drugs on the metabolic activity and diversity towards AMI and BUS was investigated. Finally, a cocktail of AMI, BUS, COU, DEX, DIC and NET was screened against the whole in-house CYP BM3 library. Different validated quantitative and qualitative (U)HPLC-MS/MS-based analytical methods were applied to screen for substrate depletion and targeted product formation, followed by a more in-depth screen for metabolic diversity. A chemometrical approach was used to mine all data to search for unique metabolic properties of the mutants and allow classification of the mutants. The latter would open the possibility of obtaining a more in-depth mechanistic understanding of the metabolites. The presented method is the first MS-based method to screen CYP BM3 mutant libraries for diversity in combination with a chemometrical approach to interpret results and visualize differences between the tested mutants. PMID:26753974

  17. Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.

    OpenAIRE

    Shi, J.; Dixon, R A; Gonzales, R A; Kjellbom, P; Bhattacharyya, M K

    1995-01-01

    An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive ...

  18. Characterization of cDNA encoding a human sperm membrane protein related to A4 amyloid protein.

    OpenAIRE

    Yan, Y C; Bai, Y.(Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China); Wang, L.F.; Miao, S Y; Koide, S S

    1990-01-01

    A rat testis lambda gt11 cDNA library was screened with a monoclonal antibody raised against a human sperm membrane protein designated YWK-II. A clone was found with a cDNA insert composed of 1837 base pairs that contained an open reading frame coding for 191 amino acid residues. The deduced polypeptide contained a segment with high homology to the transmembrane-cytoplasmic domains of the A4 amyloid protein found in brain plaques of Alzheimer disease patients. A sequence of basic amino acid r...

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

    Directory of Open Access Journals (Sweden)

    R.PRABAVATHI

    2012-07-01

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

  20. Identification of siRNA delivery enhancers by a chemical library screen.

    Science.gov (United States)

    Gilleron, Jerome; Paramasivam, Prasath; Zeigerer, Anja; Querbes, William; Marsico, Giovanni; Andree, Cordula; Seifert, Sarah; Amaya, Pablo; Stöter, Martin; Koteliansky, Victor; Waldmann, Herbert; Fitzgerald, Kevin; Kalaidzidis, Yannis; Akinc, Akin; Maier, Martin A; Manoharan, Muthiah; Bickle, Marc; Zerial, Marino

    2015-09-18

    Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2-5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types. PMID:26220182

  1. Identification of siRNA delivery enhancers by a chemical library screen

    Science.gov (United States)

    Gilleron, Jerome; Paramasivam, Prasath; Zeigerer, Anja; Querbes, William; Marsico, Giovanni; Andree, Cordula; Seifert, Sarah; Amaya, Pablo; Stöter, Martin; Koteliansky, Victor; Waldmann, Herbert; Fitzgerald, Kevin; Kalaidzidis, Yannis; Akinc, Akin; Maier, Martin A.; Manoharan, Muthiah; Bickle, Marc; Zerial, Marino

    2015-01-01

    Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2–5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types. PMID:26220182

  2. Protein stability regulators screening assay (Pro-SRSA): protein degradation meets the CRISPR-Cas9 library.

    Science.gov (United States)

    Wu, Yuanzhong; Kang, Tiebang

    2016-01-01

    The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A," recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability. PMID:27357860

  3. Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

    OpenAIRE

    Saxena, A.; Padmanabha, R; Glover, C V

    1987-01-01

    Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic...

  4. A covalent peptide inhibitor of RGS4 identified in a focused one-bead, one compound library screen

    OpenAIRE

    2009-01-01

    Background Regulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs). The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We recently described a focused one-bead, one-compound (OBOC) library screen to identify peptide inhibitors of RGS4. Here we extend our observations to include another peptide with a different m...

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

    Institute of Scientific and Technical Information of China (English)

    LIZONGDONG; MINGYEH

    1997-01-01

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

  6. Screening of antitubercular compound library identifies novel shikimate kinase inhibitors of Mycobacterium tuberculosis.

    Science.gov (United States)

    Rajput, Vikrant S; Mehra, Rukmankesh; Kumar, Sanjay; Nargotra, Amit; Singh, Parvinder Pal; Khan, Inshad Ali

    2016-06-01

    Shikimate kinase of Mycobacterium tuberculosis is involved in the biosynthesis of aromatic amino acids through shikimate pathway. The enzyme is essential for the survival of M. tuberculosis and is absent from mammals, thus providing an excellent opportunity for identifying new chemical entities to combat tuberculosis with a novel mechanism of action. In this study, an antitubercular library of 1000 compounds was screened against M. tuberculosis shikimate kinase (MtSK). This effort led to the identification of 20 inhibitors, among which five promising leads exhibited half maximal inhibitory concentration (IC50) values below 10 μM. The most potent inhibitor ("5631296") showed an IC50 value of 5.10 μM ± 0.6. The leads were further evaluated for the activity against multidrug-resistant (MDR)-TB, Gram-positive and Gram-negative bacterial strains, mode of action, docking simulations, and combinatorial study with three frontline anti-TB drugs. Compound "5491210" displayed a nearly synergistic activity with rifampicin, isoniazid, and ethambutol while compound "5631296" was synergistic with rifampicin. In vitro cytotoxicity against HepG2 cell line was evaluated and barring one compound; all were found to be non-toxic (SI > 10). In order to rule out mitochondrial toxicity, the promising inhibitors were also evaluated for cell cytotoxicity using galactose medium where compounds "5631296" and "5122752" appeared non-toxic. Upon comprehensive analysis, compound "5631296" was found to be the most promising MtSK inhibitor that was safe, synergistic with rifampicin, and bactericidal against M. tuberculosis. PMID:26887318

  7. A targeted library screen reveals a new inhibitor scaffold for protein kinase D.

    Directory of Open Access Journals (Sweden)

    Manuj Tandon

    Full Text Available Protein kinase D (PKD has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD.

  8. Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

    Science.gov (United States)

    van Tegelen, Léon J.P.; Moreno, Paolo R.H.; Croes, Anton F.; Verpoorte, Robert; Wullems, George J.

    1999-01-01

    Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus. PMID:9952467

  9. Genetic factors affecting radiosensitivity and cancer predisposition: application of a continuous low dose-rate irradiation colony formation assay to select radiosensitive retinoblastoma family members for correction with a cDNA library

    International Nuclear Information System (INIS)

    Full text: The aim of this study is to identify new or undescribed functions of radiosensitivity and genomic instability genes using a continuous low dose-rate colony formation assay. This assay expands on the standard colony formation assay, whereby colony formation ability (retention of proliferative capacity) is measured during continuous low dose-rate irradiation rather than 10-14 days following the completion of such exposures. This approach has previously employed by the Bedford laboratory to identify a Prkdc (DNA-PKcs) mutant of CHO cells, irs-20. In this study we examine the growth response of fibroblasts derived from recently identified radiosensitive retinoblastoma family members, both affected probands and their unaffected parents, and various apparently normal fibroblast lines obtained from the NIGMS Human Genetic Cell Repository (Coriell Medical Institute, Camden, NJ). Colony formation was assayed by plating single cells, exposing them at 37 deg C to continuous Cs-137 gamma irradiation at dose rates of 0.5-8.5 cGy/h, and scoring survivors as colonies with >100 viable cells. The retinoblastoma family members display severely limited growth (survival less than 10E-3) at dose rates greater than 2-2.5 cGy/h, while the apparently normal cell lines do not display such inhibited growth until 6-7 cGy/h. Two of the retinoblastoma family cell lines, MF-6F and MF-15F (both unaffected but radiosensitive parents), were selected as targets of transfection with a viral cDNA library (ViraPort human cDNA library, Stratagene Cloning Systems, La Jolla, CA) and subjected to a ∼3 cGy/h selection dose rate, where uncorrected survival relative to normal cells is lower by a factor of 50-150. Colonies recovered will provide valuable information regarding the genetic nature of their radiosensitivity (possibly involving chromosome stability, DNA repair, and/or cell cycle regulatory pathways), that may influence risks for cancer and heritable effects for a previously

  10. Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: application to diagnosis and blood screening for posttransfusion hepatitis.

    OpenAIRE

    Miyamura, T; Saito, I; Katayama, T; Kikuchi, S; Tateda, A; Houghton, M; Choo, Q L; Kuo, G

    1990-01-01

    A cDNA clone has been derived from the plasma of a chimpanzee with chronic non-A, non-B viral hepatitis (NANBH). We have assayed for antibodies reacting with the encoded antigen in sera from posttransfusion hepatitis patients (643 samples from 23 patients) and their corresponding donors collected during the past 10 years in Japan. The antibody was detected in 15 out of 17 (88.2%) posttransfusion NANBH (PT-NANBH) patients whose sera over time displayed multiple alanine aminotransferase (ALT) p...

  11. A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format

    Science.gov (United States)

    Mugabe, Sheila; Gao, Changshou; Tkaczyk, Christine; Mazor, Yariv; Pavlik, Peter; Wu, Herren; Dall’Acqua, William; Chowdhury, Partha Sarathi

    2015-01-01

    High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for

  12. A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

    Science.gov (United States)

    Xiao, Xiaodong; Chen, Yan; Mugabe, Sheila; Gao, Changshou; Tkaczyk, Christine; Mazor, Yariv; Pavlik, Peter; Wu, Herren; Dall'Acqua, William; Chowdhury, Partha Sarathi

    2015-01-01

    High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for

  13. 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.

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

    International Nuclear Information System (INIS)

    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 NH2-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 NH2-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

  15. Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries

    Directory of Open Access Journals (Sweden)

    Mari eNyyssönen

    2013-09-01

    Full Text Available Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26 % cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase families were detected. Using tetranucleotide frequency binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.

  16. 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.

  17. Screening of a virtual mirror-image library of natural products.

    Science.gov (United States)

    Noguchi, Taro; Oishi, Shinya; Honda, Kaori; Kondoh, Yasumitsu; Saito, Tamio; Ohno, Hiroaki; Osada, Hiroyuki; Fujii, Nobutaka

    2016-06-01

    We established a facile access to an unexplored mirror-image library of chiral natural product derivatives using d-protein technology. In this process, two chemical syntheses of mirror-image substances including a target protein and hit compound(s) allow the lead discovery from a virtual mirror-image library without the synthesis of numerous mirror-image compounds. PMID:27198617

  18. cDNA: 37672 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.315626 Mus musculus adult male thymus cDNA, RIKEN full-length enriched library, ... 10 product:MODULATOR OF ANTIGEN RECEPTOR SIGNALING MARS , full insert sequence gnl|UG|Mm#S10847064 AK030877 ...

  19. cDNA: 37677 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.315626 Mus musculus adult retina cDNA, RIKEN full-length enriched library, clon ... 21 product:MODULATOR OF ANTIGEN RECEPTOR SIGNALING MARS , full insert sequence gnl|UG|Mm#S9072626 AK020837 ...

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

    Directory of Open Access Journals (Sweden)

    Rempei Yanagawa

    2001-01-01

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

  1. Screening specific allergens of allergic asthma from DNA expression library of humulus pollen%从葎草花粉cDNA表达文库中筛选过敏性哮喘特异性变应原

    Institute of Scientific and Technical Information of China (English)

    徐晶; 孙秀珍; 刘昀; 尹变玲; 张永红; 李维

    2009-01-01

    目的 从葎草花粉cDNA表达文库中筛选过敏性哮喘特异性变应原.方法 应用葎草花粉过敏性哮喘患者血清对自行构建的葎草花粉cDNA λTripIEx2噬菌体表达文库进行免疫学筛选,提取阳性噬菌体DNA,EeoR I和HindⅢ双酶切、琼脂糖凝胶电泳鉴定后进行DNA序列测定、重复序列分析和同源性分析.结果 经过3轮筛选,从cDNA表达文库中筛选获得3个葎草花粉特异性变应原cDNA克隆,其序列大小分别为868、550和592 bp.测序结果表明这3个克隆代表3个不同的cDNA序列,重复序列分析未发现重复序列,同源性分析表明与现有的基因均无高度同源性.结论 所获得的与过敏性哮喘相关的3个葎草花粉特异性变应原cDNA克隆可能为新基因,此发现为进一步制备重组变应原疫苗或核酸疫苗打下了良好基础.%Objective To screen the allergic asthma-specific allergen from complementary DNA (cDNA)expression library of humulus pollen.Methods Screened the humulus pollen λTripIEx2 cDNA expression library which had been constructed using the method of immunological screening with patients' blood serum of humulus pollen allergic asthma,extracted the positive phage DNA and the positive phage DNA.and identitied by double restriction enzyme with EcoR Ⅰ,Hind Ⅲ and agarose gel electrophoresis.Then the DNA fragments were sequenced and the repeats and sequence homology were analyzed.Results After 3 rounds of screening,three specific allergic cDNA clone of humulus pollen were obtained and the sequences were 868,550 and 592 base pairs respectively.The results of sequencing showed that these three clones represented different cDNA sequences.The repeats analysis found no repeats and these clones had no high homology with any known gene.Conclusions Three specific allergic cDNA clones of humulus pollen obtained may be new genes.It provides a rational basis for constructing a recombinant allergen or nuclear acid vaccine.

  2. Investigation of the incidence of "undesirable" molecular moieties for high-throughput screening compound libraries in marketed drug compounds.

    Science.gov (United States)

    Axerio-Cilies, Peter; Castañeda, Ivan P; Mirza, Amin; Reynisson, Jóhannes

    2009-03-01

    A database of 1070 marketed drug compounds was compiled and analyzed in order to assess the occurrence of moieties described in the literature as "undesirable" for high-throughput screening compound libraries due to their ability to perturb assay formats. The study revealed a total of 277 compounds, 26% of the database, contained at least one of the moieties. As some of the drug compounds contained more than one "undesirable" moiety, the total number was 352. Electrophilic reactive groups, particularly aliphatic esters, were the most abundant type with 55% of the total. Half of the drug compounds incorporating the "undesirable" moieties were synthetic organic molecules. These findings suggest that "undesirable" moieties do not pose a major hindrance during clinical trials, the most expensive phase of drug development. In addition, their early elimination in the preclinical stage excludes large regions of known drug space due to the reliance on biochemical and cell-based assays. In general, it can be concluded that compounds with "undesirable" moieties should not simply be eliminated from compound screening libraries but rather be flagged as potentially problematic. A possible solution is to segregate the compounds containing suspect moieties and screen them when deemed appropriate. PMID:18692938

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

    International Nuclear Information System (INIS)

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors 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 cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA

  4. Development of microsatellite markers for common bean (Phaseolus vulgaris L.) based on screening of non-enriched, small-insert genomic libraries.

    Science.gov (United States)

    Blair, Matthew W; Torres, Monica Muñoz; Pedraza, Fabio; Giraldo, Martha C; Buendía, Hector F; Hurtado, Natalia

    2009-09-01

    Microsatellite markers are useful genetic tools for a wide array of genomic analyses although their development is time-consuming and requires the identification of simple sequence repeats (SSRs) from genomic sequences. Screening of non-enriched, small-insert libraries is an effective method of SSR isolation that can give an unbiased picture of motif frequency. Here we adapt high-throughput protocols for the screening of plasmid-based libraries using robotic colony picking and filter preparation. Seven non-enriched genomic libraries from common bean genomic DNA were made by digestion with four frequently cutting restriction enzymes, double digestion with a frequently cutting restriction enzyme and a less frequently cutting restriction enzyme, or sonication. Library quality was compared and three of the small-insert libraries were selected for further analysis. Each library was plated and picked into 384-well plates that were used to create high-density filter arrays of over 18 000 clones each, which were screened with oligonucleotide probes for various SSR motifs. Positive clones were found to have low redundancy. One hundred SSR markers were developed and 80 were tested for polymorphism in a standard parental survey. These microsatellite markers derived from non-SSR-enriched libraries should be useful additions to previous markers developed from enriched libraries. PMID:19935925

  5. Target based screening of small molecule library identifies pregnelonene, a Nrf2 agonist, as a potential radioprotector in zebrafish

    International Nuclear Information System (INIS)

    Reactive oxygen species, cellular oxidative stress, tissue inflammation and cell death are the downstream consequences of radiation exposures which ultimately could lead to organism death. Present study aims at identifying potential targets and screening of small molecule compound library for identifying novel and effective radioprotectors. In-silco analysis of known radioprotectors revealed three main function, antioxidant, anti-inflammation and antiapoptosis. In this study, a collection of small molecules (John Hopkins Clinical Compound Library, JHCCL) were screened for these different functions using the biological activity database of NCBI with the help of in-house developed python script. Further, filtering of the JHCCL was done by searching for molecules which are known to be active against target of radiobiological significance, Nrf-2. Close observation of potential hits identified, pregnenolone, as an Nrf-2 agonist which was further evaluated for radioprotection in zebrafish model. Pregnenolone rendered significant protection (at 40 μM; added 1 hour prior to 20 Gy gamma radiation) in terms of damage manifestations (pericardial edema, microcephaly, micropthalmia, yolk sac resorption, curvature of spine, blood flow, body length, heart-beat, blood clot, roughness of skin) and survival advantage (60%) when compared to irradiated control. Further, the ability of pregnenolone to act as a neuroprotectant was also carried out using in-house developed software for assessing neuromotor functions. In comparison to radiation alone group, pregnenolone was found to possess significant neuroactive functions and diminished radiation induced neuronal impairment. Over all these results suggests that pregnenolone is an effective radioprotector which warrants further investigation for validation of its radioprotective action in higher vertebrates. Apart from that the utility of approach to screen out bioactivity data base of various chemical compound libraries for possible

  6. Characterization of cDNA for human tripeptidyl peptidase II: The N-terminal part of the enzyme is similar to subtilisin

    International Nuclear Information System (INIS)

    Tripeptidyl peptidase II is a high molecular weight serine exopeptidase, which has been purified from rat liver and human erythrocytes. Four clones, representing 4453 bp, or 90% of the mRNA of the human enzyme, have been isolated from two different cDNA libraries. One clone, designated A2, was obtained after screening a human B-lymphocyte cDNA library with a degenerated oligonucleotide mixture. The B-lymphocyte cDNA library, obtained from human fibroblasts, were rescreened with a 147 bp fragment from the 5' part of the A2 clone, whereby three different overlapping cDNA clones could be isolated. The deduced amino acid sequence, 1196 amino acid residues, corresponding to the longest open rading frame of the assembled nucleotide sequence, was compared to sequences of current databases. This revealed a 56% similarity between the bacterial enzyme subtilisin and the N-terminal part of tripeptidyl peptidase II. The enzyme was found to be represented by two different mRNAs of 4.2 and 5.0 kilobases, respectively, which probably result from the utilziation of two different polyadenylation sites. Futhermore, cDNA corresponding to both the N-terminal and C-terminal part of tripeptidyl peptidase II hybridized with genomic DNA from mouse, horse, calf, and hen, even under fairly high stringency conditions, indicating that tripeptidyl peptidase II is highly conserved

  7. A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo

    OpenAIRE

    Laitinen, Olli H.; Airenne, Kari J; Hytönen, Vesa P; Peltomaa, Erik; Mähönen, Anssi J.; Wirth, Thomas; Lind, Miia M.; Mäkelä, Kari A.; Toivanen, Pyry I.; Schenkwein, Diana; Heikura, Tommi; Nordlund, Henri R.; Kulomaa, Markku S.; Ylä-Herttuala, Seppo

    2005-01-01

    We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the de...

  8. A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format

    OpenAIRE

    Xiao, Xiaodong; Chen, Yan; Mugabe, Sheila; Gao, Changshou; Tkaczyk, Christine; Mazor, Yariv; Pavlik, Peter; Wu, Herren; Dall’Acqua, William; Chowdhury, Partha Sarathi

    2015-01-01

    High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express ...

  9. Acri-2,7-Py, a bright red-emitting DNA probe identified through screening of a distyryl dye library.

    Science.gov (United States)

    Naud-Martin, Delphine; Martin-Benlloch, Xavier; Poyer, Florent; Mahuteau-Betzer, Florence; Teulade-Fichou, Marie-Paule

    2014-02-01

    The identification of DNA sensors is still a challenge since no DNA probe possesses all the photophysical properties required for live-cell imaging: high fluorescence yield, red emission, permeability, no photobleaching and no cytotoxicity. We describe the preparation of a distyryl dye library and its evaluation on a panel of nucleic acids with various structures (duplex DNA, quadruplex DNA and RNA). The screening involved measuring the modification of the fluorescence properties of the dyes with or without nucleic acids on a microplate reader, and allowed the identification of selective quadruplex DNA ligands with good affinities. Using this screening method we discovered a new bright red-emitting DNA stain, Acri-2,7-Py, for fixed cells. In living cells, the staining was not nuclear and photodamage generated through illumination induced cellular death. These processes require further studies to determine the relevance of Acri-2,7-Py in photodynamic therapy. PMID:24323895

  10. 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.

  11. Integration of high-content screening and untargeted metabolomics for comprehensive functional annotation of natural product libraries

    Science.gov (United States)

    Kurita, Kenji L.; Glassey, Emerson; Linington, Roger G.

    2015-01-01

    Traditional natural products discovery using a combination of live/dead screening followed by iterative bioassay-guided fractionation affords no information about compound structure or mode of action until late in the discovery process. This leads to high rates of rediscovery and low probabilities of finding compounds with unique biological and/or chemical properties. By integrating image-based phenotypic screening in HeLa cells with high-resolution untargeted metabolomics analysis, we have developed a new platform, termed Compound Activity Mapping, that is capable of directly predicting the identities and modes of action of bioactive constituents for any complex natural product extract library. This new tool can be used to rapidly identify novel bioactive constituents and provide predictions of compound modes of action directly from primary screening data. This approach inverts the natural products discovery process from the existing ‟grind and find” model to a targeted, hypothesis-driven discovery model where the chemical features and biological function of bioactive metabolites are known early in the screening workflow, and lead compounds can be rationally selected based on biological and/or chemical novelty. We demonstrate the utility of the Compound Activity Mapping platform by combining 10,977 mass spectral features and 58,032 biological measurements from a library of 234 natural products extracts and integrating these two datasets to identify 13 clusters of fractions containing 11 known compound families and four new compounds. Using Compound Activity Mapping we discovered the quinocinnolinomycins, a new family of natural products with a unique carbon skeleton that cause endoplasmic reticulum stress. PMID:26371303

  12. Cloning and Expression of Glucoamylase Genes from Aspergilus niger cDNA Library in Pichia pastrois%黑曲霉糖化酶基因在毕赤酵母中的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    汤斌; 钱鹏

    2012-01-01

    从高产糖化酶的黑曲霉的cDNA文库中筛选出糖化酶基因,并研究在毕赤酵母中的表达情况。运用RT-PCR从黑曲霉cDNA文库中克隆糖化酶基因的cDNA片段与载体pPIC9K相连,构建重组载体,电转化毕赤酵母GS115,筛选阳性克隆并进行研究。阳性克隆在MM培养基中发酵72 h和1%的甲醇的诱导的情况下,重组毕赤酵母产生的糖化酶酶活最大为15.6 U/mL。测定结果显示,其糖化酶大小为1 908 bp,编码636个氨基酸残基组成的蛋白质。经柱分离纯化其发酵上清液后,用SDS-PAGE电泳方法,测得分子质量大约为80 ku。黑曲霉糖化酶基因在毕赤酵母GS115中成功得到了表达。%An expression cDNA library was constructed from high-yielding glucoamylase strains of A.niger and the glucoamylase gene was isolated,then the expression of the gene in Pichia pastoris was studied.The cDNA sequence of glucoamylase from A.niger was obtained by RT-PCR.The cDNA fragment was cloned into the expression vector pPIC9K and the linearized recombinant vector was transformed to Pichia pastrois GS115 by electroporation.The positive clones were analyzed subsequently.The recombined Pichia pastrois were cultured in the MM medium,using 1% methanol to induce the expression of recombinant gene.The results showed that the maximum activity of glucoamylase was 15.6 U/mL after it was fermented for 72 h.Sequence analysis revealed that glucoamylase had 1908 bp,which encodes a putative polypeptide of 636 amino acids.The expressed protein was purified from the fermented supernatant using DEAE column and determined by SDS-PAGE.The result of SDS-PAGE also showed that the molecular weights of the enzyme was 80 kDa.The expression vector of the glucoamylase gene was constructed successfully,and it could express glucoamylase in Pichia pastrois.

  13. 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. PMID:22502908

  14. Identification and characterization of two novel types of non-clip domain serine proteases (PtSP and PtSPH1) from cDNA haemocytes library of swimming crab Portunus trituberculatus.

    Science.gov (United States)

    Li, Qianqian; Cui, Zhaoxia; Liu, Yuan; Wang, Shuangyan; Song, Chengwen

    2012-05-01

    In our previous studies, five serine proteases containing clip domain were characterized from the swimming crab Portunus trituberculatus. To further investigate the characterization and function of serine proteases, one serine protease (PtSP) and one serine protease homolog (PtSPH1) without clip domain were identified from haemocytes cDNA library in this paper. They both possessed an SP or SP-like domain at the C-terminal. In contrast to PtSP, absence of Ser catalytic residue resulted in the loss of serine protease activity of PtSPH1. Phylogenetic analysis suggested either SPs or SPHs might not have a single origin in gene evolution. Six introns presented in PtSP genomic DNA with one uncommon splice site (GG) was discovered at exon 1/intron 1 boundary region. Four introns with common splice sites were found in PtSPH1 genomic DNA. RT-PCR results showed that PtSP mRNA was mainly distributed in haemocytes, gill and eyestalk, whereas PtSPH1 transcript was mainly expressed in stomach. PtSP showed slight increase during the first 48 h compared to control groups except 8 h point after Micrococcus luteus challenge. However, significant up-regulation was observed in the expression level of PtSPH1 challenged by Gram-negative bacteria Vibrio alginolyticus, Gram-positive bacteria M. luteus and fungi Pichia pastoris during the first 48 h. It indicates that PtSPH1 might be more sensitive to microorganism challenges compared with PtSP. PMID:22289714

  15. Microscale Adaptation of In Vitro Transcription/Translation for High-Throughput Screening of Natural Product Extract Libraries

    Science.gov (United States)

    Lowell, Andrew N.; Santoro, Nicholas; Swaney, Steven M.; McQuade, Thomas J.; Schultz, Pamela J.; Larsen, Martha J.; Sherman, David H.

    2016-01-01

    Novel antimicrobials that effectively inhibit bacterial growth are essential to fight the growing threat of antibiotic resistance. A promising target is the bacterial ribosome, a 2.5 MDa organelle susceptible to several biorthogonal modes of action used by different classes of antibiotics. To promote the discovery of unique inhibitors, we have miniaturized a coupled transcription/translation assay using E. coli and applied it to screen a natural product library of ∼30 000 extracts. We significantly reduced the scale of the assay to 2 μL in a 1536-well plate format and decreased the effective concentration of costly reagents. The improved assay returned 1327 hits (4.6% hit rate) with %CV and Z′ values of 8.5% and 0.74, respectively. This assay represents a significant advance in molecular screening, both in miniaturization and its application to a natural product extract library, and we intend to apply it to a broad array of pathogenic microbes in the search for novel anti-infective agents. PMID:26147927

  16. Human kidney amiloride-binding protein: cDNA structure and functional expression

    International Nuclear Information System (INIS)

    Phenamil, an analog of amiloride, is a potent blocker of the epithelial Naplus channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic oligonucleotides derived from partial sequences have been used to screen a human kidney cDNA library and to isolate the cDNA encoding the human amiloride-binding protein. The primary structure was deduced from the DNA sequence analysis. The protein is 713 residues long, with a 19-amino acid signal peptide. The mRNA was expressed in 293-S and NIH 3T3 cells, yielding a glycoprotein (i) that binds amiloride and amiloride analogs with affinities similar to the amiloride receptor associated with the apical Naplus channel in pig kidney membranes and (ii) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein

  17. Comparative analysis of two cDNA libraries from Populus simonii × P. nigra tension wood.%小黑杨应拉木上下侧cDNA文库的比较分析

    Institute of Scientific and Technical Information of China (English)

    张凯旋; 赵桂媛; 刘关君; 刘桂丰; 杨传平; 魏志刚

    2011-01-01

    为了研究小黑杨应拉木的形成机制和相关基因的表达情况,通过模拟重力对小黑杨茎生长产生影响后,进行了以下研究:以小黑杨茎应拉木未成熟木质部组织为材料,分别构建了弯曲茎上侧(TW)与下侧(OW)cDNA文库,共获得了6048条高质量的ESTs序列,代表了5007条单一基因,鉴定出437条可能与应拉木形成有关的ESTs.通过比较TW与OW中的ESTs发现,纤维素合成相关基因、FLA等细胞壁相关蛋白基因以及MYB等转录因子均在TW中高表达,而木质素合成相关基因在OW中表达量较高.此外,一些参与信号转导和多糖代谢的基因在TW和OW中出现不同的表达模式.%To increase our understanding of the tension wood formation and relative gene expression in Populus simonii × P. nigra, we isolated the immature xylem from both tension wood (TW) and opposite wood (OW) of bent poplars after the species growth were affected by simulated gravity. Two cDNA libraries were then constructed and used to generate 6 048 expressed sequence tags ( ESTs), which represented 5 007 unique transcripts and involved 437 ESTs in tension wood formation. The EST distributions were compared between two libraries. The results showed that genes involved in cellulose biosynthesis, cell wall proteins and transcriptional factors were found and expressed at high levels in TW, while genes involved lignin biosynthesis were expressed at high levels in OW. Moreover, genes involved in signal transduction and carbohydrate metabolism were also identified and had different expression models in TW and OW.

  18. Establish an automated flow injection ESI-MS method for the screening of fragment based libraries: Application to Hsp90.

    Science.gov (United States)

    Riccardi Sirtori, Federico; Caronni, Dannica; Colombo, Maristella; Dalvit, Claudio; Paolucci, Mauro; Regazzoni, Luca; Visco, Carlo; Fogliatto, Gianpaolo

    2015-08-30

    ESI-MS is a well established technique for the study of biopolymers (nucleic acids, proteins) and their non covalent adducts, due to its capacity to detect ligand-target complexes in the gas phase and allows inference of ligand-target binding in solution. In this article we used this approach to investigate the interaction of ligands to the Heat Shock Protein 90 (Hsp90). This enzyme is a molecular chaperone involved in the folding and maturation of several proteins which has been subjected in the last years to intensive drug discovery efforts due to its key role in cancer. In particular, reference compounds, with a broad range of dissociation constants from 40pM to 100μM, were tested to assess the reliability of ESI-MS for the study of protein-ligand complexes. A good agreement was found between the values measured with a fluorescence polarization displacement assay and those determined by mass spectrometry. After this validation step we describe the setup of a medium throughput screening method, based on ESI-MS, suitable to explore interactions of therapeutic relevance biopolymers with chemical libraries. Our approach is based on an automated flow injection ESI-MS method (AFI-MS) and has been applied to screen the Nerviano Medical Sciences proprietary fragment library of about 2000 fragments against Hsp90. In order to discard false positive hits and to discriminate those of them interacting with the N-terminal ATP binding site, competition experiments were performed using a reference inhibitor. Gratifyingly, this group of hits matches with the ligands previously identified by NMR FAXS techniques and confirmed by X-ray co-crystallization experiments. These results support the use of AFI-MS for the screening of medium size libraries, including libraries of small molecules with low affinity typically used in fragment based drug discovery. AFI-MS is a valid alternative to other techniques with the additional opportunities to identify compounds interacting with

  19. A 3.5 genome equivalent multi access YAC library: construction, characterisation, screening and storage.

    OpenAIRE

    Anand, R; Riley, J H; Butler, R; Smith, J C; Markham, A F

    1990-01-01

    The construction of a yeast artificial chromosome (YAC) primary gridded library of 35,000 clones from human lymphoblastoid (48,XXXX) cell line DNA is described. The average YAC size is approximately 350kb representing a greater than 3.5 times coverage of the genome. The library is stored at -70 degrees C as gridded clones on nylon filters impregnated with 20% glycerol and as glycerol suspensions of individual clones in microtitre plates providing a prolonged multi-user potential. To date we h...

  20. 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.

  1. Development, screening, and analysis of DNA aptamer libraries potentially useful for diagnosis and passive immunity of arboviruses

    Directory of Open Access Journals (Sweden)

    Bruno John G

    2012-11-01

    Full Text Available Abstract Background Nucleic acid aptamers have long demonstrated the capacity to bind viral envelope proteins and to inhibit the progression of pathogenic virus infections. Here we report on initial efforts to develop and screen DNA aptamers against recombinant envelope proteins or synthetic peptides and whole inactivated viruses from several virulent arboviruses including Chikungunya, Crimean-Congo hemorrhagic fever (CCHF, dengue, tickborne encephalitis and West Nile viruses. We also analyzed sequence data and secondary structures for commonalities that might reveal consensus binding sites among the various aptamers. Some of the highest affinity and most specific aptamers in the down-selected libraries were demonstrated to have diagnostic utility in lateral flow chromatographic assays and in a fluorescent aptamer-magnetic bead sandwich assay. Some of the reported aptamers may also be able to bind viral envelope proteins in vivo and therefore may have antiviral potential in passive immunity or prophylactic applications. Results Several arbovirus DNA aptamer sequences emerged multiple times in the various down selected aptamer libraries thereby suggesting some consensus sequences for binding arbovirus envelope proteins. Screening of aptamers by enzyme-linked aptamer sorbent assay (ELASA was useful for ranking relative aptamer affinities against their cognate viral targets. Additional study of the aptamer sequences and secondary structures of top-ranked anti-arboviral aptamers suggest potential virus binding motifs exist within some of the key aptamers and are highlighted in the supplemental figures for this article. One sequence segment (ACGGGTCCGGACA emerged 60 times in the anti-CCHF aptamer library, but nowhere else in the anti-arbovirus library and only a few other times in a larger library of aptamers known to bind bacteria and rickettsia or other targets. Diagnostic utility of some of the aptamers for arbovirus detection in lateral flow

  2. Studying Screen Interactions Long-term: The Library as a Case

    NARCIS (Netherlands)

    Kanis, Marije; Groen, Maarten; Meijs, Wouter; Veenstra, Mettina

    2012-01-01

    This paper presents the design and long-term study of BiebBeep, a large interactive touchscreen that has been developed with the aim to augment the information and social function of a library. BiebBeep displays user-generated and context-relevant content, such as information about local events and

  3. 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.

  4. 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.

  5. Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene

    International Nuclear Information System (INIS)

    A human umbilical vein endothelial cell cDNA library in λgt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, λHTm10 and λHTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A) + RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of λHTm10. One isolate, λHTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The organization of thrombomodulin is similar to that of the low-density lipoprotein receptor, and the protein is homologous to a large number of other proteins that also contain EGF-like domains, including factor VII, factor IX, factor X, factor XII, protein C, tissue plasminogen activator, and urokinase. The gene for thrombomodulin has been localized to chromosome 20 by hybridization of cDNA probes to purified human chromosomes

  6. CONSTRUCTION AND SCREENING OF PARTIAL CDNA LIBRARIES OF AGARICUS BISPORUS%双孢蘑菇部分cDNA文库的构建及筛选

    Institute of Scientific and Technical Information of China (English)

    王泽生; 陈美元; 廖剑华; 卢政辉; 郭仲杰; 李洪荣; M.P.Wach

    2004-01-01

    构建了双孢蘑菇Agaricus bisporus菌株AA和A41的部分cDNA文库,其滴度分别为1.0×105 pfu/ml和6.0×104pfu/ml.用地高辛标记的丛生差异片段探针对文库进行筛选,分别获得5个和3个阳性克隆.

  7. cDNA: 34099 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.15282 Homo sapiens cDNA FLJ44214 fis, clone THYMU3003309, moderately similar to ... Homo sapiens sarcoma antigen (SAGE ) gnl|UG|Hs#S16886502 AK126202 23/5622_34099.png ...

  8. cDNA: 43397 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.30035 Mus musculus adult male corpora quadrigemina cDNA, RIKEN full-length enri ... FOLATE DEHYDROGENASE (EC 1.5.1.6) (10-FTHFDH) (FBP-CI ) homolog [Rattus norvegicus], full insert sequence ...

  9. cDNA: 33377 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.181243 Homo sapiens full open reading frame cDNA clone RZPDo834H102D for gene AT ... F4, activating transcription factor 4 (tax -responsive enhancer element B67); complete cds; wi ...

  10. cDNA: 17527 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.134229 Homo sapiens cDNA FLJ44146 fis, clone THYMU2027734, weakly similar to Hom ... o sapiens SA hypertension -associated homolog (rat) (SAH) gnl|UG|Hs#S16886570 ...

  11. cDNA: 47992 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.228067 Mus musculus 15 days embryo male testis cDNA, RIKEN full-length enriched ... lone:8030476B22 product:hypothetical Mitochondrial energy ... transfer proteins (carrier protein) containing pro ...

  12. cDNA: 47994 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.228067 Mus musculus 0 day neonate eyeball cDNA, RIKEN full-length enriched libr ... lone:E130118D21 product:hypothetical Mitochondrial energy ... transfer proteins (carrier protein) containing pro ...

  13. cDNA: 47991 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.228067 Mus musculus adult male diencephalon cDNA, RIKEN full-length enriched li ... lone:9330189G22 product:hypothetical Mitochondrial energy ... transfer proteins (carrier protein) containing pro ...

  14. cDNA: 36928 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.240850 Mus musculus adult male medulla oblongata cDNA, RIKEN full-length enrich ... MA AMPLIFIED SEQUENCE 1 (NOVEL AMPLIFIED IN BREAST CANCER ... 1) (AMPLIFIED AND OVEREXPRESSED IN BREAST CANCER ) ...

  15. cDNA: 52275 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.275648 Mus musculus 11 days embryo head cDNA, RIKEN full-length enriched librar ... y, clone:6230400I01 product:SIMILAR TO ENIGMA ... (LIM DOMAIN PROTEIN) homolog [Homo sapiens], full ...

  16. cDNA: 52277 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.275648 Mus musculus 18-day embryo whole body cDNA, RIKEN full-length enriched l ... ibrary, clone:1110003B01 product:SIMILAR TO ENIGMA ... (LIM DOMAIN PROTEIN) homolog [Homo sapiens], full ...

  17. An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast

    OpenAIRE

    Jung-Hoon Bae; Bong Hyun Sung; Hyun-Jin Kim; Soon-Ho Park; Kwang-Mook Lim; Mi-Jin Kim; Cho-Ryong Lee; Jung-Hoon Sohn

    2015-01-01

    To produce rarely secreted recombinant proteins in the yeast Saccharomyces cerevisiae, we developed a novel genome-wide optimal translational fusion partner (TFP) screening system that involves recruitment of an optimal secretion signal and fusion partner. A TFP library was constructed from a genomic and truncated cDNA library by using the invertase-based signal sequence trap technique. The efficiency of the system was demonstrated using two rarely secreted proteins, human interleukin (hIL)-2...

  18. A high-throughput strategy for screening of bacterial artificial chromosome libraries and anchoring of clones on a genetic map constructed with single nucleotide polymorphisms

    OpenAIRE

    Deal Karin R; Ma Yaqin; Xu Kenong; Luo Ming-Cheng; Nicolet Charles M; Dvorak Jan

    2009-01-01

    Abstract Background Current techniques of screening bacterial artificial chromosome (BAC) libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly para...

  19. A high-throughput strategy for screening of bacterial artificial chromosome libraries and anchoring of clones on a genetic map constructed with single nucleotide polymorphisms

    OpenAIRE

    Luo, Ming-Cheng; Xu, Kenong; Ma, Yaqin; Karin R Deal; Nicolet, Charles M.; Dvorak, Jan

    2009-01-01

    Background Current techniques of screening bacterial artificial chromosome (BAC) libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly parallel Illu...

  20. Erkitinib, a novel EGFR tyrosine kinase inhibitor screened using a ProteoChip system from a phytochemical library

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eung-Yoon; Choi, Young-Jin [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of); Park, Chan-Won [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Kang, In-Cheol, E-mail: ickang@hoseo.edu [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of)

    2009-11-20

    Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer. Therefore PTK inhibitors are currently under intensive investigation as potential drug candidates. Herein, we report on a ProteoChip-based screening of an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, Erkitinibs, from phytochemical libraries. PLC-{gamma}-1 was used as a substrate immobilized on a ProteoChip and incubated with an EGFR kinase to phosphorylate tyrosine residues of the substrate, followed by a fluorescence detection of the substrate recognized by a phospho-specific monoclonal antibody. Erkitinibs inhibited HeLa cell proliferation in a dose-dependent manner. In conclusion, these data suggest that Erkitinibs can be a specific inhibitor of an EGFR kinase and can be further developed as a potent anti-tumor agent.

  1. In silico Screening of Chemical Libraries to Develop Inhibitors That Hamper the Interaction of PCSK9 with the LDL Receptor

    Science.gov (United States)

    Min, Dong-Kook; Lee, Hyun-Sook; Lee, Narae; Lee, Chan Joo; Song, Hyun Joo; Yang, Ga Eul; Yoon, Dojun

    2015-01-01

    Purpose Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) and promotes degradation of the LDLR. Inhibition of PCSK9 either by reducing its expression or by blocking its activity results in the upregulation of the LDLR and subsequently lowers the plasma concentration of LDL-cholesterol. As a modality to inhibit PCSK9 action, we searched the chemical library for small molecules that block the binding of PCSK9 to the LDLR. Materials and Methods We selected 100 chemicals that bind to PCSK9 where the EGF-AB fragment of the LDLR binds via in silico screening of the ChemBridge chemical library, using the computational GOLD algorithm analysis. Effects of chemicals were evaluated using the PCSK9-LDLR binding assay, immunoblot analysis, and the LDL-cholesterol uptake assay in vitro, as well as the fast performance liquid chromatography assay for plasma lipoproteins in vivo. Results A set of chemicals were found that decreased the binding of PCSK9 to the EGF-AB fragment of the LDLR in a dose-dependent manner. They also increased the amount of the LDLR significantly and subsequently increased the uptake of fluorescence-labeled LDL in HepG2 cells. Additionally, one particular molecule lowered the plasma concentration of total cholesterol and LDL-cholesterol significantly in wild-type mice, while such an effect was not observed in Pcsk9 knockout mice. Conclusion Our findings strongly suggest that in silico screening of small molecules that inhibit the protein-protein interaction between PCSK9 and the LDLR is a potential modality for developing hypercholesterolemia therapeutics. PMID:26256967

  2. Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C.

    Science.gov (United States)

    Ulferts, Rachel; de Boer, S Matthijn; van der Linden, Lonneke; Bauer, Lisa; Lyoo, Hey Rhyoung; Maté, Maria J; Lichière, Julie; Canard, Bruno; Lelieveld, Daphne; Omta, Wienand; Egan, David; Coutard, Bruno; van Kuppeveld, Frank J M

    2016-05-01

    Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired. PMID:26856848

  3. High-throughput transformation method for Yarrowia lipolytica mutant library screening.

    Science.gov (United States)

    Leplat, Christophe; Nicaud, Jean-Marc; Rossignol, Tristan

    2015-09-01

    As a microorganism of major biotechnological importance, the oleaginous yeast Yarrowia lipolytica is subjected to intensive genetic engineering and functional genomic analysis. Future advancements in this area, however, require a system that will generate a large collection of mutants for high-throughput screening. Here, we report a rapid and efficient method for high-throughput transformation of Y. lipolytica in 96-well plates. We developed plasmids and strains for the large-scale screening of overexpression mutant strains, using Gateway® vectors that were adapted for specific locus integration in Y. lipolytica. As an example, a collection of mutants that overexpressed the alkaline extracellular protease (AEP) was obtained in a single transformation experiment. The platform strain that we developed to receive the overexpression cassette was designed to constitutively express a fluorescent protein as a convenient growth reporter for screening in non-translucid media. An example of growth comparison in skim milk-based medium between AEP overexpression and deletion mutants is provided. PMID:26100263

  4. Screening of early antigen genes of adult-stage Trichinella spiralis using pig serum from different stages of early infection

    Science.gov (United States)

    The goal of this work was to identify novel, early antigens present in Trichinella spiralis. To this end, a cDNA library generated from 3-day old adult worms (Ad3) was immunologically screened using serum from a pig infected with 20,000 muscle larvae. The serum was obtained from multiple, time cours...

  5. MRM screening/biomarker discovery with linear ion trap MS: a library of human cancer-specific peptides

    International Nuclear Information System (INIS)

    The discovery of novel protein biomarkers is essential in the clinical setting to enable early disease diagnosis and increase survivability rates. To facilitate differential expression analysis and biomarker discovery, a variety of tandem mass spectrometry (MS/MS)-based protein profiling techniques have been developed. For achieving sensitive detection and accurate quantitation, targeted MS screening approaches, such as multiple reaction monitoring (MRM), have been implemented. MCF-7 breast cancer protein cellular extracts were analyzed by 2D-strong cation exchange (SCX)/reversed phase liquid chromatography (RPLC) separations interfaced to linear ion trap MS detection. MS data were interpreted with the Sequest-based Bioworks software (Thermo Electron). In-house developed Perl-scripts were used to calculate the spectral counts and the representative fragment ions for each peptide. In this work, we report on the generation of a library of 9,677 peptides (p < 0.001), representing ~1,572 proteins from human breast cancer cells, that can be used for MRM/MS-based biomarker screening studies. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum (p-value, DeltaM, Xcorr, DeltaCn, Sp, no. of matching a, b, y ions in the spectrum), the retention time, and the top 10 most intense product ions that correspond to a given peptide. Only proteins identified by at least two spectral counts are listed. The experimental distribution of protein frequencies, as a function of molecular weight, closely matched the theoretical distribution of proteins in the human proteome, as provided in the SwissProt database. The amino acid sequence coverage of the identified proteins ranged from 0.04% to 98.3%. The highest-abundance proteins in the cellular extract had a molecular weight (MW)<50,000. Preliminary experiments have

  6. Screening of a synthetic peptide combinatorial library to identify inhibitors of the appressorium formation in Magnaporthe oryzae.

    Science.gov (United States)

    Rebollar, Aarón; Marcos, Jose F; López-García, Belén

    2014-11-01

    The rice blast disease caused by Magnaporthe oryzae is one of the most devastating diseases of cultivated rice. One of the most important stages in the infective cycle of M. oryzae is the formation of the dome-shaped structure called appressorium. The purpose of the present study was to identify novel peptides to control the rice blast disease by blocking the appressorium formation through screening of a synthetic peptide combinatorial library. As result of the screening, a set of 29 putative bioactive peptides were identified, synthesized and assayed in comparison with the previously identified peptide PAF104. The peptides MgAPI24, MgAPI40 and MgAPI47 showed improved inhibitory activity on the M. oryzae appressorium formation. Our data show that these peptides have a differential effect on two developmental structures: appressoria and appressorium-like structures. Antimicrobial assays against M. oryzae and other non-target microorganisms showed a weak or no toxicity of these peptides, demonstrating their specific activity blocking the appressorium formation. Therefore, the outcome of this research would be useful in the development of novel target-oriented peptides to use in plant protection. PMID:25450357

  7. The Identification of Genes Important in Pseudomonas syringae pv. phaseolicola Plant Colonisation Using In Vitro Screening of Transposon Libraries.

    Directory of Open Access Journals (Sweden)

    Bharani Manoharan

    Full Text Available The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms around plant cells. If the pathogen can suppress the plant's natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.

  8. A library-screening approach for developing a fluorescence sensing array for the detection of metal ions.

    Science.gov (United States)

    Smith, David G; Sajid, Naveed; Rehn, Simone; Chandramohan, Ramya; Carney, Isaac J; Khan, Misbahul A; New, Elizabeth J

    2016-08-01

    Detection of individual metal ions is of importance across a range of fields of chemistry including environmental monitoring, and health and disease. Fluorescence is a highly sensitive technique and small fluorescent molecules are widely used for the detection and quantification of metal ions in various applications. Achieving specificity for a single metal from a single sensor is always a challenge. An alternative to selective sensing is the use of a number of non-specific sensors, in an array, which together respond in a unique pattern to each analyte. Here we show that screening a library of compounds can give a small sensor set that can be used to identify a range of metal ions following PCA and LDA. We explore a method for screening the initial compounds to identify the best performing sensors. We then present our method for reducing the size of the sensor array, resulting in a four-membered system, which is capable of identifying nine distinct metal ion species in lake water. PMID:27291513

  9. The Identification of Genes Important in Pseudomonas syringae pv. phaseolicola Plant Colonisation Using In Vitro Screening of Transposon Libraries

    Science.gov (United States)

    Hancock, John T.; Jackson, Robert W.; Arnold, Dawn L.

    2015-01-01

    The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant’s natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction. PMID:26325299

  10. Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library

    Science.gov (United States)

    Kumar, Anand; Drozd, Mary; Pina-Mimbela, Ruby; Xu, Xiulan; Helmy, Yosra A.; Antwi, Janet; Fuchs, James R.; Nislow, Corey; Templeton, Jillian; Blackall, Patrick J.; Rajashekara, Gireesh

    2016-01-01

    Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a “bioactive” library of 4182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 μM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 μM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide, and pyridazinone. Exploitation of analogs of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine. PMID:27092106

  11. Screening for genes involved in Klebsiella pneumoniae biofilm formation using a fosmid library

    DEFF Research Database (Denmark)

    Stahlhut, Steen G; Schroll, Casper; Harmsen, Morten;

    2010-01-01

    compared with the E. coli parent strain using a biofilm microtiter plate assay. Nine clones with significantly enhanced biofilm formation were identified, subjected to random Tn5 transposon mutagenesis, screened for biofilm deficiency and the biofilm-promoting genes identified. Five of the clones contained...... the type 3 fimbriae gene cluster, a well-known K. pneumoniae virulence factor and biofilm promoter. Thus, the effectiveness of our approach was confirmed. Furthermore, genes encoding cell surface proteins and proteins involved in metabolism, none of them previously associated with biofilm formation in...

  12. Tissue-targeting lead generation and optimization from random and directed screening of technetium-99m labeled tripeptide complex libraries in vivo

    Institute of Scientific and Technical Information of China (English)

    ZENG Jun; LIU Ci-yi; XIE Wen-hui; HU Si-long; JIN Mu-xiu

    2006-01-01

    Background Screening libraries against a molecular target in vitro are idealized models that cannot reflect the real state in vivo where biomolecules coexist and interact. C-terminal amide tripeptides labelled with Technetium-99m can provide a unique noninvasive approach to trace a large number of compounds in vivo.Methods The C-terminal amide tripeptide libraries were synthesized on Rink Amide-MBHA resin using iterative and pooling protocol. Technetium (Ⅴ) oxo core [TcO3+] was bound to each tripeptide via 4 deprotonated nitrogen atoms to form a library of 8000 99mTc tripeptoid complexes. The radiocombinatorial screening (RCS) in vivo was carried out on SD rats and A549 tumour bearing mice.Results Signals of tissue distribution and metabolism of libraries were recorded by counting or imaging and tissue targeting leads identified by both random and directed RCS. Among them, 99mTc RPA, 99mTc VIG and 99mTc RES had specific tissue targeting in kidney, liver and tumour respectively. The percent injected dose per gram tissue of 99mTc labelled leads in their target tissue was highly structure dependent. Because the nontarget tissue binding and the metabolism of 99mTc tripeptoid sublibraries were simultaneously monitored successfully by RCS, the interference of background activity was limited to the lowest level. Optimization of renal function agent from the labelled libraries was carried out by directed screening. 99mTc DSG was finally identified the most promising agent for renal function studies.Conclusions RCS in vivo is a powerful tool for the discovery of tissue targeting drugs. The potential screening bias is probably the major limitation of labelled libraries.

  13. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

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

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  14. Differential screening of phage-ab libraries by oligonucleotide microarray technology.

    Directory of Open Access Journals (Sweden)

    Paolo Monaci

    Full Text Available A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag in the phagemid coding for each phage-displayed antibody fragment (phage-Ab present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.

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

    Science.gov (United States)

    Finocchiaro, G; Taroni, F; Rocchi, M; Martin, A L; Colombo, I; Tarelli, G T; DiDonato, S

    1991-01-01

    We have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase (CPTase; palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21), 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 NH2-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 NH2-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-hamster somatic cell hybrids. Images PMID:1988962

  16. cDNA: 12295 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.267288 Homo sapiens full open reading ... frame cDNA clone RZPDo834G0212D for gene C ... 6orf55, chromosome 6 open reading ... frame 55; complete cds, incl. stopcodon gnl|UG|Hs# ...

  17. cDNA: 16610 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.62595 Homo sapiens full open reading ... frame cDNA clone RZPDo834H088D for gene C9o ... rf9, chromosome 9 open reading ... frame 9; complete cds, incl. stopcodon gnl|UG|Hs#S ...

  18. cDNA: 3940 [

    Lifescience Database Archive (English)

    Full Text Available H. sapiens - Hs.7188 Homo sapiens cDNA PSEC0078 fis, clone NT2RP2004036, moderately similar to M ... -Sema F=a factor in neural network ... development. gnl|UG|Hs#S4806431 AK075388 2/887_394 ...

  19. cDNA: 41587 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.30133 Mus musculus 18-day embryo whole body cDNA, RIKEN full-length enriched li ... brary, clone:1110004A14 product:ethanol ... induced 6, full insert sequence gnl|UG|Mm#S9085518 ...

  20. cDNA: 39377 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.7091 Mus musculus adult pancreas islet cells cDNA, RIKEN fu ll-length enriched l ... R BETA SUBUNIT) (SSR-BETA) homolog [Homo sapiens], fu ... ... gnl|UG|Mm#S10839939 AK050505 3/6436_39377.png ...

  1. cDNA: 46817 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.30092 Mus musculus adult male cerebellum cDNA, RIKEN fu ll-length enriched libra ... 1 PRECURSOR (EC 3.4.21.-) homolog [Homo sapiens], fu ... ... gnl|UG|Mm#S10838326 AK075719 8/7837_46817.png ...

  2. cDNA: 46327 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.292517 Mus musculus 12 days embryo spinal ganglion cDNA, RIKEN fu ll-length enri ... PHA CHAIN) (PHERS) (CML33) homolog [Homo sapiens], fu ... ... gnl|UG|Mm#S10835133 AK084031 8/7824_46327.png ...

  3. cDNA: 36690 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.345070 Mus musculus 16 days embryo head cDNA, RIKEN fu ll-length enriched librar ... SPLICEOSOME ASSOCIATED PROTEIN 49) [Homo sapiens], fu ... ... gnl|UG|Mm#S10835972 AK081584 2/6005_36690.png ...

  4. cDNA: 40377 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.154312 Mus musculus 0 day neonate kidney cDNA, RIKEN full-length enriched libra ... ry, clone:D630023P19 product:HYPERTENSION ... RELATED PROTEIN 1, full insert sequence gnl|UG|Mm# ...

  5. cDNA: 40378 [

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.154312 Mus musculus 7 days embryo whole body cDNA, RIKEN full-length enriched l ... ibrary, clone:C430046A10 product:HYPERTENSION ... RELATED PROTEIN 1, full insert sequence gnl|UG|Mm# ...

  6. Screening and characterization of a novel cellulase gene from the gut microflora of Hermetia illucens using metagenomic library.

    Science.gov (United States)

    Lee, Chang-Muk; Lee, Young-Seok; Seo, So-Hyeon; Yoon, Sang-Hong; Kim, Soo-Jin; Hahn, Bum-Soo; Sim, Joon-Soo; Koo, Bon-Sung

    2014-09-01

    A metagenomic fosmid library was constructed using genomic DNA isolated from the gut microflora of Hermetia illucens, a black soldier fly. A cellulase-positive clone, with the CS10 gene, was identified by extensive Congo-red overlay screenings for cellulase activity from the fosmid library of 92,000 clones. The CS10 gene was composed of a 996 bp DNA sequence encoding the mature protein of 331 amino acids. The deduced amino acids of CS10 showed 72% sequence identity with the glycosyl hydrolase family 5 gene of Dysgonomonas mossii, displaying no significant sequence homology to already known cellulases. The purified CS10 protein presented a single band of cellulase activity with a molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at 50°C and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of 20~50°C and pH 4.0~10.0. CS10 exhibited little loss of cellulase activity against various chemical reagents such as 10% polar organic solvents, 1% non-ionic detergents, and 0.5 M denaturing agents. Moreover, the substrate specificity and the product patterns by thinlayer chromatography suggested that CS10 is an endo-β-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes. PMID:25022521

  7. MRM screening/biomarker discovery with linear ion trap MS: a library of human cancer-specific peptides

    Directory of Open Access Journals (Sweden)

    Lazar Iulia M

    2009-03-01

    Full Text Available Abstract Background The discovery of novel protein biomarkers is essential in the clinical setting to enable early disease diagnosis and increase survivability rates. To facilitate differential expression analysis and biomarker discovery, a variety of tandem mass spectrometry (MS/MS-based protein profiling techniques have been developed. For achieving sensitive detection and accurate quantitation, targeted MS screening approaches, such as multiple reaction monitoring (MRM, have been implemented. Methods MCF-7 breast cancer protein cellular extracts were analyzed by 2D-strong cation exchange (SCX/reversed phase liquid chromatography (RPLC separations interfaced to linear ion trap MS detection. MS data were interpreted with the Sequest-based Bioworks software (Thermo Electron. In-house developed Perl-scripts were used to calculate the spectral counts and the representative fragment ions for each peptide. Results In this work, we report on the generation of a library of 9,677 peptides (p a, b, y ions in the spectrum, the retention time, and the top 10 most intense product ions that correspond to a given peptide. Only proteins identified by at least two spectral counts are listed. The experimental distribution of protein frequencies, as a function of molecular weight, closely matched the theoretical distribution of proteins in the human proteome, as provided in the SwissProt database. The amino acid sequence coverage of the identified proteins ranged from 0.04% to 98.3%. The highest-abundance proteins in the cellular extract had a molecular weight (MW Conclusion Preliminary experiments have demonstrated that putative biomarkers, that are not detectable by conventional data dependent MS acquisition methods in complex un-fractionated samples, can be reliable identified with the information provided in this library. Based on the spectral count, the quality of a tandem mass spectrum and the m/z values for a parent peptide and its most abundant daughter

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

    International Nuclear Information System (INIS)

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma λgt11 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 λgt11 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

  9. Development of a comprehensive spectral library of sildenafil and related active analogues using LC-QTOF-MS and its application for screening counterfeit pharmaceuticals.

    Science.gov (United States)

    Lee, Sooyeun; Ji, Dajeong; Park, Meejung; Chung, Kyu Hyuck

    2015-12-01

    The abuse or misuse of forged erectile-dysfunction drugs, containing phosphodiesterase type 5 inhibitors (e.g. sildenafil), is a serious issue globally. Therefore, the detection of sildenafil and related active analogues in counterfeit pharmaceuticals or the differentiation between counterfeit and authentic drugs has been performed with a variety of analytical techniques. Recently, a liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS)-based in-house library, consisting of accurate mass ion fragmentation information and retention times, was effectively applied to screen a large number of compounds in field of forensic toxicology. However, a comprehensive LC-QTOF-MS spectral library of sildenafil and related active analogues has not yet been reported. In the present study, a spectral library of 40 compounds of sildenafil and related analogues was developed with accurate mass spectra and retention times using LC-QTOF-MS, and applied to screen nine marketed counterfeit products. The in-house library successfully identified sildenafil, dimethylsildenafil, hydroxyhomosildenafil, demethylhongdenafil, pseudovardenafil and vardenafil in the samples. Our LC-QTOF-MS-based spectral library search is considered a powerful approach for identifying sildenafil and related active analogues in counterfeit pharmaceuticals. PMID:26363440

  10. Genomic library screening for viruses from the human dental plaque revealed pathogen-specific lytic phage sequences.

    Science.gov (United States)

    Al-Jarbou, Ahmed Nasser

    2012-01-01

    Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence. PMID:21969025

  11. Isolation and characterization of a cDNA clone for the Cat2 gene in maize and its homology with other catalases.

    OpenAIRE

    Bethards, L. A.; Skadsen, R W; Scandalios, J G

    1987-01-01

    A 230-base-pair cDNA representing the 3' end of the Cat2 gene in maize was isolated and used to screen a lambda gt11 cDNA library made from scutellar poly(A)+ RNA. Several clones were subcloned into a pUC12 vector; one of the subclones, pCat2.1c, appears to represent a near-complete sequence of the Cat2 coding region. Immunological screening, hybridization-selection translation, and RNA blot analysis confirmed that pCat2.1c was derived from the Cat2 transcript. The DNA sequence of pCat2.1c wa...

  12. Construction of a mutant library of horseradish peroxidase gene by directed evolution and development of an in situ screening method

    Directory of Open Access Journals (Sweden)

    F.M. Mendive

    2003-03-01

    Full Text Available A process of directed evolution applied to obtain a library of mutants of horseradish peroxidase (HRP enzyme is described. We have introduced slight variations into the original DNA shuffling protocol. A DNA template was prepared by PCR amplification and digested with Dnase I during 1 hour. Dnase I products were concentrated by precipitation with isopropanol. Gel electrophoresis showed fragments of the desired size range (20-600 pb without a full-length template remaining in the reaction mixture. A high concentration of fragments was crucial for performing PCR without primers. In this case, a template concentration of 32.5 ng/mu l was appropriate. Amplification of recombinant genes in a standard PCR reaction (template dilution 1:100 produced a smear with a low yield for the full-length sequence. A single product of the correct size was obtained by PCR with nested primers separated from the previously used primers by 40 pb. In our laboratory, native HRP has been functionally expressed in a baculovirus expression vector system. The purpose is to develop the screening of the first generation of random mutants in this system. To facilitate detection of those clones that have high peroxidase activity, we developed a rapid method: after five days postinfection agarose plates with six wells were covered with DAB (3,3´-diaminobenzidine and H2O2. The appearance of brown-black stain allows visualization of up to 100 active clones/well in only 1 min.

  13. Characterization of three new carboxylic ester hydrolases isolated by functional screening of a forest soil metagenomic library.

    Science.gov (United States)

    Biver, Sophie; Vandenbol, Micheline

    2013-02-01

    Three new lipolytic genes were isolated from a forest soil metagenomic library by functional screening on tributyrin agar plates. The genes SBLip1, SBLip2 and SBLip5.1 respectively encode polypeptides of 445, 346 and 316 amino acids. Phylogenetic analyses revealed that SBLip2 and SBLip5.1 belong to bacterial esterase/lipase family IV, whereas SBLip1 shows similarity to class C β-lactamases and is thus related to esterase family VIII. The corresponding genes were overexpressed and their products purified by affinity chromatography for characterization. Analyses of substrate specificity with different p-nitrophenyl esters showed that all three enzymes have a preference for short-acyl-chain p-nitrophenyl esters, a feature of carboxylesterases as opposed to lipases. The β-lactamase activity of SBLip1, measured with the chromogenic substrate nitrocefin, was very low. The three esterases have the same optimal pH (pH 10) and remain active across a relatively broad pH range, displaying more than 60 % activity between pH 6 and 10. The temperature optima determined were 35 °C for SBLip1, 45 °C for SBLip2 and 50 °C for SBLip5.1. The three esterases displayed different levels of tolerance to salts, solvents and detergents, SBLip2 being overall more tolerant to high concentrations of solvent and SBLip5.1 less affected by detergents. PMID:23160923

  14. An Antibody Screen of a Plasmodium vivax Antigen Library Identifies Novel Merozoite Proteins Associated with Clinical Protection.

    Directory of Open Access Journals (Sweden)

    Camila T França

    2016-05-01

    Full Text Available Elimination of Plasmodium vivax malaria would be greatly facilitated by the development of an effective vaccine. A comprehensive and systematic characterization of antibodies to P. vivax antigens in exposed populations is useful in guiding rational vaccine design.In this study, we investigated antibodies to a large library of P. vivax entire ectodomain merozoite proteins in 2 Asia-Pacific populations, analysing the relationship of antibody levels with markers of current and cumulative malaria exposure, and socioeconomic and clinical indicators. 29 antigenic targets of natural immunity were identified. Of these, 12 highly-immunogenic proteins were strongly associated with age and thus cumulative lifetime exposure in Solomon Islanders (P<0.001-0.027. A subset of 6 proteins, selected on the basis of immunogenicity and expression levels, were used to examine antibody levels in plasma samples from a population of young Papua New Guinean children with well-characterized individual differences in exposure. This analysis identified a strong association between reduced risk of clinical disease and antibody levels to P12, P41, and a novel hypothetical protein that has not previously been studied, PVX_081550 (IRR 0.46-0.74; P<0.001-0.041.These data emphasize the benefits of an unbiased screening approach in identifying novel vaccine candidate antigens. Functional studies are now required to establish whether PVX_081550 is a key component of the naturally-acquired protective immune response, a biomarker of immune status, or both.

  15. 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.

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

    DEFF Research Database (Denmark)

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

    1985-01-01

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

  17. A feasibility study of non-targeted adulterant screening based on NIRM spectral library of soybean meal to guarantee quality: The example of non-protein nitrogen.

    Science.gov (United States)

    Shen, Guanghui; Fan, Xia; Yang, Zengling; Han, Lujia

    2016-11-01

    The quality and safety of soybean meal is a key matter for the livestock breeding and food industries, since it is one of the most important and widely used protein feed raw materials. As driven by commercial interests, new illegal adulterants which are unknown to consumers and regulators emerge constantly. In order to make up for the inadequacy of traditional detection methods, a novel non-targeted adulterant screening method based on a near-infrared microscopy spectral library of soybean meal is proposed. This study focused on the feasibility of non-targeted screening methods for the detection of adulteration in soybean meal. Six types of non-protein nitrogen were taken as examples and partial least squares discriminant analysis was employed to verify the feasibility of this novel method. The results showed that the non-targeted screening method could screen out adulterations in soybean meal with satisfactory results. PMID:27211617

  18. Human tissue factor: cDNA sequence and chromosome localization of the gene

    International Nuclear Information System (INIS)

    A human placenta cDNA library in λgt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, λHTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of λHTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of ∼ 3.2 kilobases in poly(A) + RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased several fold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of λHTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(a) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser. The 3'-noncoding region contains an inverted Alu family repetitive sequence. The tissue factor gene was localized to chromosome 1 by hybridization of the cDNA insert of λHTF8 to flow-sorted human chromosomes

  19. A Wide-Field Fluorescence Microscope Extension for Ultrafast Screening of One-Bead One-Compound Libraries Using a Spectral Image Subtraction Approach.

    Science.gov (United States)

    Heusermann, Wolf; Ludin, Beat; Pham, Nhan T; Auer, Manfred; Weidemann, Thomas; Hintersteiner, Martin

    2016-05-01

    The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community. PMID:27057765

  20. One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

    Science.gov (United States)

    Sasso, Emanuele; Paciello, Rolando; D'Auria, Francesco; Riccio, Gennaro; Froechlich, Guendalina; Cortese, Riccardo; Nicosia, Alfredo; De Lorenzo, Claudia; Zambrano, Nicola

    2015-01-01

    Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications. PMID:26649313

  1. One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

    Directory of Open Access Journals (Sweden)

    Emanuele Sasso

    2015-01-01

    Full Text Available Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications.

  2. One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

    OpenAIRE

    Emanuele Sasso; Rolando Paciello; Francesco D’Auria; Gennaro Riccio; Guendalina Froechlich; Riccardo Cortese; Alfredo Nicosia; Claudia De Lorenzo; Nicola Zambrano

    2015-01-01

    Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C ...

  3. Using 99TcM-tripeptoid library as an example of radio-combinatorial screening in vivo for the discovery of tissue targeting drug

    International Nuclear Information System (INIS)

    Libraries against a molecular target in vitro is an artificially idealized system that are not the real picture in vivo where biomolecules coexist to maintain life activities. Furthermore, the diagnostic and therapeutic efficiency of drug depends highly on the drug distribution in target tissues (tumor for example) both specifically and accumulatively. Radio-labeling technology can make a unique opportunity of tracing large number of compounds in a combinatorial way in vivo simultaneously, sensitively, quantitatively, dynamically, and noninvasively. Methods: The C-terminal amide tripeptide libraries were synthesized on Rink Amide-MBHA resin in the OXX aO1OXaO1O2O positional scanning format and iterative protoco. A technetium (V) oxo core[(TcO)3+] was bound to the N4-triligands of tripeptide libraries via four deprotonated amide nitrogen atoms to form a structure of 99Tcm-tripeptoid libraries. The radio-combinatorial screening (RCS) in vivo was then carded out after SD rats and A549 tumor bearing mice received i.v. with 99Tcm-tripeptoid libraries. Results: Using 99Tcm-tripeptoid libraries as an example, a new method of radio-combinatorial screening (RCS) in vivo was demonstrated successful in generation, optimization, and design of tissue targeting leads. Signals of tissue distribution and metabolism of libraries were recorded by g counting or imaging. From library of 8,000 99Tcm-tripeptoid members, the tissue targeting leads had been identified by RCS. Those included 99Tcm-DSG, 99Tcm-VAA, 99Tcm-VIG, and 99Tcm-RES that had specific tissue targeting in kidney, stomach, liver, and tumor respectively. The percent injected dose per gram tissue (%ID/g) of 99Tcm labeled leads in their target tissues was highly structure-dependent Because the nontarget tissue binding and the metabolism of 99Tcm-tripeptoid libraries was simultaneously monitored so successfully by RCS that the background activity was limited to the lowest level when a 99Tcm labeled lead finally generated

  4. An Antibody Screen of a Plasmodium vivax Antigen Library Identifies Novel Merozoite Proteins Associated with Clinical Protection

    Science.gov (United States)

    França, Camila T.; Hostetler, Jessica B.; Sharma, Sumana; White, Michael T.; Lin, Enmoore; Kiniboro, Benson; Waltmann, Andreea; Darcy, Andrew W.; Li Wai Suen, Connie S. N.; Siba, Peter; King, Christopher L.; Rayner, Julian C.; Fairhurst, Rick M.; Mueller, Ivo

    2016-01-01

    Background Elimination of Plasmodium vivax malaria would be greatly facilitated by the development of an effective vaccine. A comprehensive and systematic characterization of antibodies to P. vivax antigens in exposed populations is useful in guiding rational vaccine design. Methodology/Principal Findings In this study, we investigated antibodies to a large library of P. vivax entire ectodomain merozoite proteins in 2 Asia-Pacific populations, analysing the relationship of antibody levels with markers of current and cumulative malaria exposure, and socioeconomic and clinical indicators. 29 antigenic targets of natural immunity were identified. Of these, 12 highly-immunogenic proteins were strongly associated with age and thus cumulative lifetime exposure in Solomon Islanders (P<0.001–0.027). A subset of 6 proteins, selected on the basis of immunogenicity and expression levels, were used to examine antibody levels in plasma samples from a population of young Papua New Guinean children with well-characterized individual differences in exposure. This analysis identified a strong association between reduced risk of clinical disease and antibody levels to P12, P41, and a novel hypothetical protein that has not previously been studied, PVX_081550 (IRR 0.46–0.74; P<0.001–0.041). Conclusion/Significance These data emphasize the benefits of an unbiased screening approach in identifying novel vaccine candidate antigens. Functional studies are now required to establish whether PVX_081550 is a key component of the naturally-acquired protective immune response, a biomarker of immune status, or both. PMID:27182597

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

    International Nuclear Information System (INIS)

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

  6. Rapid Screening of Bovine Milk Oligosaccharides in a Whey Permeate Product and Domestic Animal Milks by Accurate Mass Database and Tandem Mass Spectral Library.

    Science.gov (United States)

    Lee, Hyeyoung; Cuthbertson, Daniel J; Otter, Don E; Barile, Daniela

    2016-08-17

    A bovine milk oligosaccharide (BMO) library, prepared from cow colostrum, with 34 structures was generated and used to rapidly screen oligosaccharides in domestic animal milks and a whey permeate powder. The novel library was entered into a custom Personal Compound Database and Library (PCDL) and included accurate mass, retention time, and tandem mass spectra. Oligosaccharides in minute-sized samples were separated using nanoliquid chromatography (nanoLC) coupled to a high resolution and sensitive quadrupole-Time of Flight (Q-ToF) MS system. Using the PCDL, 18 oligosaccharides were found in a BMO-enriched product obtained from whey permeate processing. The usefulness of the analytical system and BMO library was further validated using milks from domestic sheep and buffaloes. Through BMO PCDL searching, 15 and 13 oligosaccharides in the BMO library were assigned in sheep and buffalo milks, respectively, thus demonstrating significant overlap between oligosaccharides in bovine (cow and buffalo) and ovine (sheep) milks. This method was shown to be an efficient, reliable, and rapid tool to identify oligosaccharide structures using automated spectral matching. PMID:27428379

  7. Direct Phenotypic Screening in Mice: Identification of Individual, Novel Antinociceptive Compounds from a Library of 734,821 Pyrrolidine Bis-piperazines.

    Science.gov (United States)

    Houghten, Richard A; Ganno, Michelle L; McLaughlin, Jay P; Dooley, Colette T; Eans, Shainnel O; Santos, Radleigh G; LaVoi, Travis; Nefzi, Adel; Welmaker, Greg; Giulianotti, Marc A; Toll, Lawrence

    2016-01-11

    The hypothesis in the current study is that the simultaneous direct in vivo testing of thousands to millions of systematically arranged mixture-based libraries will facilitate the identification of enhanced individual compounds. Individual compounds identified from such libraries may have increased specificity and decreased side effects early in the discovery phase. Testing began by screening ten diverse scaffolds as single mixtures (ranging from 17,340 to 4,879,681 compounds) for analgesia directly in the mouse tail withdrawal model. The "all X" mixture representing the library TPI-1954 was found to produce significant antinociception and lacked respiratory depression and hyperlocomotor effects using the Comprehensive Laboratory Animal Monitoring System (CLAMS). The TPI-1954 library is a pyrrolidine bis-piperazine and totals 738,192 compounds. This library has 26 functionalities at the first three positions of diversity made up of 28,392 compounds each (26 × 26 × 42) and 42 functionalities at the fourth made up of 19,915 compounds each (26 × 26 × 26). The 120 resulting mixtures representing each of the variable four positions were screened directly in vivo in the mouse 55 °C warm-water tail-withdrawal assay (ip administration). The 120 samples were then ranked in terms of their antinociceptive activity. The synthesis of 54 individual compounds was then carried out. Nine of the individual compounds produced dose-dependent antinociception equivalent to morphine. In practical terms what this means is that one would not expect multiexponential increases in activity as we move from the all-X mixture, to the positional scanning libraries, to the individual compounds. Actually because of the systematic formatting one would typically anticipate steady increases in activity as the complexity of the mixtures is reduced. This is in fact what we see in the current study. One of the final individual compounds identified, TPI 2213-17, lacked significant respiratory

  8. Human type VII collagen: cDNA cloning and chromosomal mapping of the gene

    Energy Technology Data Exchange (ETDEWEB)

    Parente, M.G.; Chung, L.C.; Ryynaenen, J.; Monli Chu; Uitto, J. (Thomas Jefferson Univ., Philadelphia, PA (United States)); Woodley, D.T.; Wynn, K.C.; Bauer, E.A. (Stanford Univ., CA (United States)); Mattei, M.G. (Institute National de la Sante et de la Recherche Medicale, Marseille (France))

    1991-08-15

    A human keratinocyte cDNA expression library in bacteriophage {lambda}gt11 was screened with the purified IgG fraction of serum from a patient with epidermolysis bullosa acquisita, which had a high titer of anti-type VII collagen antibodies. Screening of {approx}3 {times} 10{sup 5} plaques identified 8 positive clones, the largest one (K-131) being {approx}1.9 kilobases in size. Dideoxynucleotide sequencing of K-131 indicated that it consisted of 1875 base pairs and contained an open reading frame coding for a putative N-terminal noncollagenous domain of 439 amino acids and a collagenous domain was characterized by repeating Gly-Xaa-Yaa sequences that were interrupted in several positions by insertions or deletions of 1-3 amino acids. The deduced amino acid sequence also revealed a peptide segment that had a high degree of identity with a published type VII collagen protein sequence. The results mapped the COL7A1 to the locus 3p21. The cDNA clones characterized in this study will be valuable for understanding the protein structure and gene expression of type VII collagen present in anchoring fibrils and its aberrations in the dystrophic forms of heritable epidermolysis bullosa.

  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. 低密度cDNA Macroarray对干扰素α抗病毒蛋白的筛选%Based on the low-density cDNA Macroarray for screening of antiviral proteins of IFNa tissues

    Institute of Scientific and Technical Information of China (English)

    管世鹤; 杨凯; 王琴; 程中乐; 潘颖; 吴园园; 杨东亮

    2011-01-01

    目的 基于低密度cDNA Macoarray技术筛选出差异表达的干扰素(IFN)α抗病毒基因,以探讨IFN α抗病毒蛋白的表达与HBV复制的关系. 方法 以一定浓度的IFN α处理肝胚瘤细胞株HepG2和HepG2.2.15细胞6h,用cDNA Macroarray分析比较两细胞株IFN α抗病毒基因表达谱,并筛选出差异表达的IFNα抗病毒基因.将表达HBV核心蛋白(HBc)的质粒pHBc-EGFP转染HepG2细胞,RT-PCR法分析HBc对IFN α抗病毒基因表达的影响.将表达抗黏病毒A蛋白(MxA)的表达质粒pcDNA3.1-Flag-MxA转染HepG2.2.15,以酶联免疫吸附试验、Dot blot、Southern blot等方法分别检测HepG2.2.15细胞表达释放的HBsAg与HBeAg、细胞外HBV DNA和细胞内HBV DNA复制中间体(松弛环状DNA、双股线性DNA),以判断HBV复制情况.两组间数据比较采用t检验,组间不同时间点数据比较采用单因素方差分析.结果 cDNA Macroarray分析显示HepG2和HepG2.2.15细胞的抗病毒基因表达谱具有差异性:IFNa抗病毒基因中干扰素诱导跨膜蛋白(IFITM)1、IFITM2、IFITM3、RING4等在HepG2.2.15细胞的表达被部分抑制,而重要的抗病毒蛋白MxA表达被完全抑制.HBc转染组细胞中MxA mRNA表达的相对水平为0.31±0.05,低于空白对照组的0.74±0.04,差异有统计学意义,P<0.05.MxA蛋白转染HepG2.2.15细胞48、72 h后,MxA转染组细胞上清液中HBsAg的S/CO值分别为1.42+0.21和1.58±0.18,HBeAg的S/CO值为1.44±0.14和2.28±0.24,而空白对照组细胞上清液中HBsAg的S/CO值为1.92±0.19和2.79±0.25,HBeAg的S/CO值为2.31±0.46和3.37±0.29,两组细胞上清液中HBV抗原的S/CO值差异均有统计学意义,P值均<0.05.细胞外HBV DNA、胞内HBV复制中间体DNA均无明显变化.结论 HBV及其抗原成分的复制和表达影响着IFNα抗病毒蛋白的表达;HBV通过抑制IFN α抗病毒蛋白的表达而发挥拮抗IFNα的抗病毒活性.%Objective To screen the gene expression profiles of IFN-α antiviral proteins

  11. Isolation and characterisation of the Xenopus laevis albumin genes: loss of 74K albumin gene sequences by library amplification.

    OpenAIRE

    May, F E; Weber, R.; Westley, B. R.

    1982-01-01

    The blood of the frog X.laevis contains 2 albumins of 68,000 and 74,000 daltons which are encoded in the liver by two related mRNAs. When an amplified X.laevis DNA library was screened with cloned albumin cDNA only 68,000 dalton albumin gene sequences were isolated. Hybridisation of the albumin cDNA to Southern-blots of Eco R1 digested X.laevis DNA showed that the sequences present in the recombinants did not account for all the fragments which hybridised on the Southern-blots. This indicated...

  12. Construction of a BAC library and identification of Dmrt1 gene of the rice field eel, Monopterus albus

    International Nuclear Information System (INIS)

    A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from the rice field eel (Monopterus albus). The BAC library consists of a total of 33,000 clones with an average insert size of 115 kb. Based on the rice field eel haploid genome size of 600 Mb, the BAC library is estimated to contain approximately 6.3 genome equivalents and represents 99.8% of the genome of the rice field eel. This is first BAC library constructed from this species. To estimate the possibility of isolating a specific clone, high-density colony hybridization-based library screening was performed using Dmrt1 cDNA of the rice field eel as a probe. Both library screening and PCR identification results revealed three positive BAC clones which were overlapped, and formed a contig covering the Dmrt1 gene of 195 kb. By sequence comparisons with the Dmrt1 cDNA and sequencing of first four intron-exon junctions, Dmrt1 gene of the rice field eel was predicted to contain four introns and five exons. The sizes of first and second intron are 1.5 and 2.6 kb, respectively, and the sizes of last two introns were predicted to be about 20 kb. The Dmrt1 gene structure was conserved in evolution. These results also indicate that the BAC library is a useful resource for BAC contig construction and molecular isolation of functional genes

  13. Screening and analyzing genes associated with Amur tiger placental development.

    Science.gov (United States)

    Li, Q; Lu, T F; Liu, D; Hu, P F; Sun, B; Ma, J Z; Wang, W J; Wang, K F; Zhang, W X; Chen, J; Guan, W J; Ma, Y H; Zhang, M H

    2014-01-01

    The Amur tiger is a unique endangered species in the world, and thus, protection of its genetic resources is extremely important. In this study, an Amur tiger placenta cDNA library was constructed using the SMART cDNA Library Construction kit. A total of 508 colonies were sequenced, in which 205 (76%) genes were annotated and mapped to 74 KEGG pathways, including 29 metabolism, 29 genetic information processing, 4 environmental information processing, 7 cell motility, and 5 organismal system pathways. Additionally, PLAC8, PEG10 and IGF-II were identified after screening genes from the expressed sequence tags, and they were associated with placental development. These findings could lay the foundation for future functional genomic studies of the Amur tiger. PMID:25299101

  14. Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement.

    OpenAIRE

    Weis, J J; Fearon, D T; Klickstein, L B; Wong, W. W.; Richards, S A; de Bruyn Kops, A; Smith, J. A.; Weis, J H

    1986-01-01

    Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low-stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequen...

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-08-01

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

  17. Screening and identification of a novel target specific for hepatoma cell line HepG2 from the FliTrx bacterial peptide library

    Institute of Scientific and Technical Information of China (English)

    Wenhan Li; Ping Lei; Bing Yu; Sha Wu; Jilin Peng; Xiaoping Zhao; Huffen Zhu; Michael Kirschfink; Guanxin Shen

    2008-01-01

    To explore new targets for hepatoma research, we used a surface display library to screen novel tumor cell-specific peptides. The bacterial FliTrx system was screened with living normal liver cell line L02 and hepatoma cell line HepG2 successively to search for hepatoma-specific peptides. Three clones (Hep1, Hep2, and Hep3) were identified to be specific to HepG2 compared with L02 and other cancer cell lines.Three-dimensional structural prediction proved that peptides inserted into the active site of Escherichia coli thioredoxin (TrxA) formed certain loop structures protruding out of the surface. Western blot analysis showed that FliC/TrxA-pepfide fusion proteins could be directly used to detect HepG2 cells.Three different FliC/TrxA-peptide fusion proteins targeted the same molecule, at approximately 140 kDa, on HepG2 cells.This work presented for the first time the application of the FliTrx library in screening living cells. Three peptides were obtained that could be potential candidates for targeted liver cancer therapy.

  18. Pulmonary Targeting of Adeno-associated Viral Vectors by Next-generation Sequencing-guided Screening of Random Capsid Displayed Peptide Libraries.

    Science.gov (United States)

    Körbelin, Jakob; Sieber, Timo; Michelfelder, Stefan; Lunding, Lars; Spies, Elmar; Hunger, Agnes; Alawi, Malik; Rapti, Kleopatra; Indenbirken, Daniela; Müller, Oliver J; Pasqualini, Renata; Arap, Wadih; Kleinschmidt, Jürgen A; Trepel, Martin

    2016-06-01

    Vectors mediating strong, durable, and tissue-specific transgene expression are mandatory for safe and effective gene therapy. In settings requiring systemic vector administration, the availability of suited vectors is extremely limited. Here, we present a strategy to select vectors with true specificity for a target tissue from random peptide libraries displayed on adeno-associated virus (AAV) by screening the library under circulation conditions in a murine model. Guiding the in vivo screening by next-generation sequencing, we were able to monitor the selection kinetics and to determine the right time point to discontinue the screening process. The establishment of different rating scores enabled us to identify the most specifically enriched AAV capsid candidates. As proof of concept, a capsid variant was selected that specifically and very efficiently delivers genes to the endothelium of the pulmonary vasculature after intravenous administration. This technical approach of selecting target-specific vectors in vivo is applicable to any given tissue of interest and therefore has broad implications in translational research and medicine. PMID:27018516

  19. Effects of an amyloid-beta 1-42 oligomers antibody screened from a phage display library in APP/PS1 transgenic mice

    Science.gov (United States)

    Wang, Jianping; Li, Nan; Ma, Jun; Gu, Zhiqiang; Yu, Lie; Fu, Xiaojie; Liu, Xi; Wang, Jian

    2016-01-01

    We screened anti-Aβ1-42 antibodies from a human Alzheimer’s disease (AD) specific single chain variable fragment (scFv) phage display library and assessed their effects in APP/PS1 transgenic mice. Reverse transcription-PCR was used to construct the scFv phage display library, and screening identified 11A5 as an anti-Aβ1-42 antibody. We mixed 11A5 and the monoclonal antibody 6E10 with Aβ1-42 and administered the mixture to Sprague-Dawley rats via intracerebroventricular injection. After 30 days, rats injected with the antibody/ Aβ1-42 mixture and those injected with Aβ1-42 alone were tested on the Morris water maze. We also injected 11A5 and 6E10 into APP/PS1 transgenic mice and assessed the concentrations of Aβ in brain and peripheral blood by ELISA at 1-month intervals for 3 months. Finally we evaluated behavior changes in the Morris water maze. Rats injected with Aβ1-42 and mixed antibodies showed better performance in the Morris water maze than did rats injected with Aβ1-42 alone. In APP/PS1 transgenic mice, Aβ concentration was lower in the brains of the antibody-treated group than in the control group, but higher in the peripheral blood. The antibody-treated mice also exhibited improved behavioral performance in the Morris water maze. In conclusion, anti-Aβ1-42 antibodies (11A5) screened from the human scFv antibody phage display library promoted the efflux or clearance of Aβ1-42 and effectively decreased the cerebral Aβ burden in an AD mouse model. PMID:26820640

  20. Guanine nucleotide-binding proteins that enhance choleragen ADP-ribosyltransferase activity: nucleotide and deduced amino acid sequence of an ADP-ribosylation factor cDNA.

    OpenAIRE

    Price, S R; Nightingale, M.; Tsai, S C; Williamson, K. C.; Adamik, R; H. C. Chen; Moss, J; M. Vaughan

    1988-01-01

    Three (two soluble and one membrane) guanine nucleotide-binding proteins (G proteins) that enhance ADP-ribosylation of the Gs alpha stimulatory subunit of the adenylyl cyclase (EC 4.6.1.1) complex by choleragen have recently been purified from bovine brain. To further define the structure and function of these ADP-ribosylation factors (ARFs), we isolated a cDNA clone (lambda ARF2B) from a bovine retinal library by screening with a mixed heptadecanucleotide probe whose sequence was based on th...

  1. Cloning and expression of cDNA for salmon growth hormone in Escherichia coli

    OpenAIRE

    Sekine, Susumu; Mizukami, Tamio; Nishi, Tatsunari; Kuwana, Yoshihisa; Saito, Akiko; Sato, Moriyuki; Itoh, Seiga; Kawauchi, Hiroshi

    1985-01-01

    cDNA clones encoding chum salmon (Oncorhynchus keta) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)+ RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putativ...

  2. Mapping the Protein-Protein Interactome Networks Using Yeast Two-Hybrid Screens.

    Science.gov (United States)

    Rajagopala, Seesandra Venkatappa

    2015-01-01

    The yeast two-hybrid system (Y2H) is a powerful method to identify binary protein-protein interactions in vivo. Here we describe Y2H screening strategies that use defined libraries of open reading frames (ORFs) and cDNA libraries. The array-based Y2H system is well suited for interactome studies of small genomes with an existing ORFeome clones preferentially in a recombination based cloning system. For large genomes, pooled library screening followed by Y2H pairwise retests may be more efficient in terms of time and resources, but multiple sampling is necessary to ensure comprehensive screening. While the Y2H false positives can be efficiently reduced by using built-in controls, retesting, and evaluation of background activation; implementing the multiple variants of the Y2H vector systems is essential to reduce the false negatives and ensure comprehensive coverage of an interactome. PMID:26621469

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

    Energy Technology Data Exchange (ETDEWEB)

    Zarlenga, D.; Gamble, H.R.

    1987-05-01

    A cDNA expression library was built in lambda gt11 phage using poly A mRNA isolated from Trichinella spiralis muscle stage larvae. This library was screened with rabbit antibodies to parasite excretory-secretory (ES) products and greater than 180 clones were isolated. Thirteen clones producing highly immunogenic protein antigens were plaque purified and rescreened with pig antisera to T.spiralis, Trichuris suis or Ascaris suum to identify clones producing epitopes specific to T.spiralis ES products, only. Two clones, TsAc-2 and TsAc-8, which displayed strong interactions with pig antisera to T. spiralis were lysogenized in E. coli Y1089 and the protein extracted. Western blots of the crude fusion proteins revealed molecular weights of 133 kD and 129 kD, respectively. Northern blot analysis of total RNA with TSP labelled cDNA:lambda gt11 probes indicated single RNA transcripts for each clone with molecular sizes corresponding to 800-850 nucleotides. dscDNA inserts were estimated by southern blot analysis to be 500 bp and 340 bp, respectively, with no cross-hybridization observed between the cloned sequences. Dot blots using pig sera to screen crude fusion protein preparations, total bacterial protein (negative controls) and crude worm extract or ES products from T.spiralis, T.suis and A.suum (positive controls) corroborated the specificity and sensitivity of these clones as potential diagnostic antigens for swine trichinellosis.

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

    International Nuclear Information System (INIS)

    A cDNA expression library was built in lambda gt11 phage using poly A mRNA isolated from Trichinella spiralis muscle stage larvae. This library was screened with rabbit antibodies to parasite excretory-secretory (ES) products and greater than 180 clones were isolated. Thirteen clones producing highly immunogenic protein antigens were plaque purified and rescreened with pig antisera to T.spiralis, Trichuris suis or Ascaris suum to identify clones producing epitopes specific to T.spiralis ES products, only. Two clones, TsAc-2 and TsAc-8, which displayed strong interactions with pig antisera to T. spiralis were lysogenized in E. coli Y1089 and the protein extracted. Western blots of the crude fusion proteins revealed molecular weights of 133 kD and 129 kD, respectively. Northern blot analysis of total RNA with 32P labelled cDNA:lambda gt11 probes indicated single RNA transcripts for each clone with molecular sizes corresponding to 800-850 nucleotides. dscDNA inserts were estimated by southern blot analysis to be 500 bp and 340 bp, respectively, with no cross-hybridization observed between the cloned sequences. Dot blots using pig sera to screen crude fusion protein preparations, total bacterial protein (negative controls) and crude worm extract or ES products from T.spiralis, T.suis and A.suum (positive controls) corroborated the specificity and sensitivity of these clones as potential diagnostic antigens for swine trichinellosis

  5. Human liver mitochondrial carnitine palmitoyltransferase I: characterization of its cDNA and chromosomal localization and partial analysis of the gene.

    Science.gov (United States)

    Britton, C H; Schultz, R A; Zhang, B; Esser, V; Foster, D W; McGarry, J D

    1995-01-01

    Using the cDNA for rat liver mitochondrial carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) as a probe, we isolated its counterpart as three overlapping clones from a human liver cDNA library. Both the nucleotide sequence of the human cDNA and the predicted primary structure of the protein (773 aa) proved to be very similar to those of the rat enzyme (82% and 88% identity, respectively). The CPT I mRNA size was also found to be the same (approximately 4.7 kb) in both species. Screening of a human genomic library with the newly obtained cDNA yielded a positive clone of approximately 6.5 kb which, upon partial analysis, was found to contain at least two complete exons linked by a 2.3-kb intron. Oligonucleotide primers specific to upstream and downstream regions of one of the exon/intron junctions were tested in PCRs with DNA from a panel of somatic cell hybrids, each containing a single human chromosome. The results allowed unambiguous assignment of the human liver CPT I gene to the q (long) arm of chromosome 11. Additional experiments established that liver and fibroblasts express the same isoform of mitochondrial CPT I, legitimizing the use of fibroblast assays in the differential diagnosis of the "muscle" and "hepatic" forms of CPT deficiency. The data provide insights into the structure of a human CPT I isoform and its corresponding gene and establish unequivocally that CPT I and CPT II are distinct gene products. Availability of the human CPT I cDNA should open the way to an understanding of the genetic basis of inherited CPT I deficiency syndromes, how the liver CPT I gene is regulated, and which tissues other than liver express this particular variant of the enzyme. Images Fig. 4 Fig. 5 PMID:7892212

  6. ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSEDIN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

    Institute of Scientific and Technical Information of China (English)

    金虎林; 胡松年; 李光涛; 涂纯; 袁建刚; 强伯勤

    2000-01-01

    Objective. To investigate the differentiation process of the human glioblastoma cells. Methods. Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325. Routine method of cDNA library screening was performed to clone full-length cDNA. Results. Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered, amplified and cloned. Of them,46 ESTs were sequenced and delivered into the GenBank. The homology comparison us-ing BLAST algorithm revealed that 22ESFs are highly homologous with the known genes and many of them play impor-tant roles in the cell differentiation progress. A dot-blot hybridization was conducted to certify the differemiation expres-sion. The result showed that 27 EST clones are expressed at different level in control and all-traus retinoic acid treated BT-325 cells. A full-length cDNA was cloned using the EST-HGBB098.Conclusion. DDRT-PCR was a simple and effective method to segally analyze the differentially expressed genes.

  7. ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSED IN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

    Institute of Scientific and Technical Information of China (English)

    金虎林; 胡松年; 李光涛; 涂纯; 袁建刚; 强伯勤

    2000-01-01

    Objective. To investigate the differentiation process of the human glioblastoma cells. Methods. Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325. Routine method of cDNA library screening was performed to clone full-length cDNA. Results. Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered, amplified and cloned. Of them,46 ESTs were sequenced and delivered into the GenBank. The homology comparison us ing BLAST algorithm revealed that 22ESTs are highly homologous with the known genes and many of them play impor tant roles in the cell differentiation progress. A dot-blot hybridization was conducted to certify the differentiation expres sion. The result showed that 27 EST clones are expressed at different level in control and all-trans retinoi c acid treated BT-325 cells. A full-length cDNA was cloned using the ES T-HGBB098. Conclusion. DDRT-PCR was a simple and effective method to serially analyze the differentially expressed genes.

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

    International Nuclear Information System (INIS)

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 106 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria

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

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J. (Mount Sinai School of Medicine, New York, NY (USA))

    1988-10-01

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

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

    International Nuclear Information System (INIS)

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

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

    International Nuclear Information System (INIS)

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

  12. Acoustic Sample Deposition MALDI-MS (ASD-MALDI-MS): A Novel Process Flow for Quality Control Screening of Compound Libraries.

    Science.gov (United States)

    Chin, Jefferson; Wood, Elizabeth; Peters, Grace S; Drexler, Dieter M

    2016-02-01

    In the early stages of drug discovery, high-throughput screening (HTS) of compound libraries against pharmaceutical targets is a common method to identify potential lead molecules. For these HTS campaigns to be efficient and successful, continuous quality control of the compound collection is necessary and crucial. However, the large number of compound samples and the limited sample amount pose unique challenges. Presented here is a proof-of-concept study for a novel process flow for the quality control screening of small-molecule compound libraries that consumes only minimal amounts of samples and affords compound-specific molecular data. This process employs an acoustic sample deposition (ASD) technique for the offline sample preparation by depositing nanoliter volumes in an array format onto microscope glass slides followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. An initial study of a 384-compound array employing the ASD-MALDI-MS workflow resulted in a 75% first-pass positive identification rate with an analysis time of <1 s per sample. PMID:26203056

  13. Rice bicoid-related cDNA sequence and its expression during early embryogenesis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation.To clone and characterize the rice bicoid-related genes,one cDNA clone,Rb24 (EMBL accession number: AJ2771380),was isolated by screening of rice unmature seed cDNA library.Sequence analysis indicates that Rb24 contains a putative amino acid sequence,which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity,75% similarity) and involves a lys-9 in putative helix 3.Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner.The transcript was detected strongly in young panicles,but less in young leaves and roots.This results are further confirmed with paraffin section in situ hybridization.The signal is intensive in rice globular embryo and located at the apical tip of the embryo,then,along with the development of embryo,the signal is getting reduced and transfers into both sides of embryo.The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

  14. Molecular cloning of a cDNA encoding the human Sm-D autoantigen

    International Nuclear Information System (INIS)

    Antibodies to the Sm-D polypeptide antigen are closely associated with the rheumatic disease systemic lupus erythematosus. Sm-D exists in the cell as one of the core proteins of the small nuclear ribonucleoprotein complexes implicated in RNA processing. The authors have isolated a cDNA clone, D45-2, coding for the Sm-D human nuclear antigen by screening a human B-lymphocyte cDNA library with synthetic oligonucleotide probes. The 1633-base-pair clone contains an open reading frame (ORF) 357 nucleotides long, capable of encoding a 13,282-dalton polypeptide. The Sm-D coding region is initiated at an AUG codon downstream from a sequence with excellent match to the consensus for the eukaryotic ribosome-binding site. The Sm-D ORF is preceded by a 150-nucleotide-long untranslated leader and followed by a 1126-nucleotide-long untranslated region containing four putative poly(A) signals. The predicted amino acid sequence reveals a (Gly-Arg)9 repeated motif at the C terminus, which may constitute one of the Sm-D immunoreactive determinants. Moreover, this C terminus shows interesting features: (i) a good homology to protamines as expected for a nucleic acid binding protein and (ii) a striking similarity to a region in the Epstein-Barr nuclear antigen

  15. A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

    Science.gov (United States)

    Wang, W.; Takezawa, D.; Poovaiah, B. W.

    1996-01-01

    A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

  16. Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes

    International Nuclear Information System (INIS)

    A method is presented for screening fragment libraries using acoustic droplet ejection to co-crystallize proteins and chemicals directly on micromeshes with as little as 2.5 nl of each component. This method was used to identify previously unreported fragments that bind to lysozyme, thermolysin, and trypsin. Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment-screening strategy is described that uses ADE to co-crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co-crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid-handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co-crystallization experiments to be discretely positioned on a single data-collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co-crystallized with a small 33-compound mini-library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure-based drug discovery using as little as 2.5 nl of each major component

  17. Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Xingyu [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Stony Brook University, NY 11794-5215 (United States); Nanjing University, Nanjing, Jiangsu (China); Scalia, Alexander [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Binghamton University, 4400 Vestal Parkway East, NY 13902 (United States); Leroy, Ludmila [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Ministry of Education of Brazil, 70040-020 Brasilia-DF (Brazil); Universidade Federal de Minas Gerais, 6627 Av. Antonio Carlos, 31270-901 Belo Horizonte-MG (Brazil); Cuttitta, Christina M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); The City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314 (United States); Polizzo, Gina M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); St Joseph’s College, 155 West Roe Boulevard, East Patchogue, NY 11772 (United States); Ericson, Daniel L. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); University at Buffalo, SUNY, 12 Capen Hall, Buffalo, NY 14260 (United States); Roessler, Christian G. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Campos, Olven [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33414 (United States); Ma, Millie Y. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Comsewogue High School, 565 Bicycle Path, Port Jefferson Station, NY 11776 (United States); Agarwal, Rakhi; Jackimowicz, Rick; Allaire, Marc [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Orville, Allen M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Sweet, Robert M.; Soares, Alexei S., E-mail: soares@bnl.gov [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States)

    2014-05-01

    A method is presented for screening fragment libraries using acoustic droplet ejection to co-crystallize proteins and chemicals directly on micromeshes with as little as 2.5 nl of each component. This method was used to identify previously unreported fragments that bind to lysozyme, thermolysin, and trypsin. Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment-screening strategy is described that uses ADE to co-crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co-crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid-handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co-crystallization experiments to be discretely positioned on a single data-collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co-crystallized with a small 33-compound mini-library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure-based drug discovery using as little as 2.5 nl of each major component.

  18. Screening study on new tumor marker periplakin for lung cancer

    Institute of Scientific and Technical Information of China (English)

    Shuqin Dai; Wei Li; Mian Kong; Yuzhen Zheng; Shuying Chen; Junye Wang; Linquan Zang

    2013-01-01

    Objective: The aim of this study was to use lung cancer targeting binding polypeptide ZS-9 to screen cDNA library of human lung cancer and obtain ZS-9 specific ligand to confirm tumor marker of non small-cell lung cancer. Methods: Artificially synthesize biotin labeled peptide ZS-9, anchored ZS-9 in the enzyme label plate coupled by avidin, used ZS-9 as probe to screen cDNA library of human lung cancer, after screening, obtained bacteriophage clone specifically binding with anchored polypeptide ZS-9. Extracted plasmid of bacteriophage and performed sequencing after amplified by PCR. Results: It was demonstrated by bioinformatic analysis on the sequence of ligand binded by lung cancer specific peptide ZS-9 that the ligand was the cytoskeletal protein periplakin on the surface of lung cancer cells, suggesting that periplakin might be a new marker for non-small-cell lung cancer in lung cancer. Conclusion: Use specific lung cancer binding peptide to screen new tumor marker periplakin in lung cancer and further studies on its biologic functions in genesis and development of lung cancer are still needed.

  19. Construction of a mutant library of horseradish peroxidase gene by directed evolution and development of an in situ screening method

    OpenAIRE

    F.M. Mendive; M.M. Segura; Targovnik, H M; O. Cascone; M.V. Miranda

    2003-01-01

    A process of directed evolution applied to obtain a library of mutants of horseradish peroxidase (HRP) enzyme is described. We have introduced slight variations into the original DNA shuffling protocol. A DNA template was prepared by PCR amplification and digested with Dnase I during 1 hour. Dnase I products were concentrated by precipitation with isopropanol. Gel electrophoresis showed fragments of the desired size range (20-600 pb) without a full-length template remaining in the reaction mi...

  20. Cloning, tissue expression pattern characterization and chromosome localization of human peptide methionine sulfoxide reductase cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Oxidation and reduction of some amino acids are one of the molecular mechanisms for regulating the function of proteins. The oxidation of methionine (Met) to methionine sulfoxide (Met(O)) results in decreasing or loss of the biological activity of related proteins. It was found that peptide methionine sulfoxide reductase (msrA) can reduce Met(O) to Met and therefore restored the biological function of the oxidized proteins. To reveal the methionine oxidation-reduction mechanism in human body, in this study, the cDNA sequence of bovine msrA was used as an information-probe to screen the human EST database. Based on a contig assembled from homologous ESTs, a 1 256-bp human MSRA cDNA was cloned from several human cDNA libraries. The cDNA contains an open reading frame (ORF) of 705 bp in length, which encodes 235 amino acid residues. Homology comparison revealed that human MSRA shares 88% and 61% identities with bovine and Escherichia coli msrA protein respectively. Expression pattern analysis revealed a single 1.6-kb transcript of human MSRA in most human tissues and with highest expression in kidney. By radiation hybrid panel mapping, the gene was localized to human chromosome 8p22-23 between markers D8S518 and D8S550. There are 2 human inherited diseases Keratolytic Winter Erythema and Microcephaly related genes in this region, it is inferred that human MSRA might be the candidate of the two diseases.

  1. High-throughput screening of thin-film semiconductor material libraries I: system development and case study for Ti-W-O.

    Science.gov (United States)

    Sliozberg, Kirill; Schäfer, Dominik; Erichsen, Thomas; Meyer, Robert; Khare, Chinmay; Ludwig, Alfred; Schuhmann, Wolfgang

    2015-04-13

    An automated optical scanning droplet cell (OSDC) enables high-throughput quantitative characterization of thin-film semiconductor material libraries. Photoelectrochemical data on small selected measurement areas are recorded including intensity-dependent photopotentials and -currents, potentiodynamic and potentiostatic photocurrents, as well as photocurrent (action) spectra. The OSDC contains integrated counter and double-junction reference electrodes and is fixed on a precise positioning system. A Xe lamp with a monochromator is coupled to the cell through a thin poly(methyl methacrylate) (PMMA) optical fiber. A specifically designed polytetrafluoroethylene (PTFE) capillary tip is pressed on the sample surface and defines through its diameter the homogeneously illuminated measurement area. The overall and wavelength-resolved irradiation intensities and the cell surface area are precisely determined and calibrated. System development and its performance are demonstrated by means of screening of a TiWO thin film. PMID:25727402

  2. Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)

    Institute of Scientific and Technical Information of China (English)

    LI Bi-feng; MAO Hua-ming; YANG Shu-Li; ZHU Ya-xin; GU Zhao-bing; CHEN Yuan; LENG Jing; GOU Xiao; FENG Li; LI Qing; XI Dong-mei

    2016-01-01

    Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as wel as reeds and other plant species, and grow to higher mature live weights than Yunnan Yelow cattle maintained in similar harsh environments. The aim of this study was to identify speciifc celulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38400 clones with an average insert size of 35.5 kb. TheUmcel-1 gene was isolated from this library. Investigation of the celulase activity of 24 random clones led to the identiifcation of theUmcel-1 gene, which exhibited the most potent celulase activity. Sequencing theUmcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putativegene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the celulase (GenBank accession no. YP_004310852.1) fromClostridium lentocelum DSM 5427, with 44% identity and 62% similarity. TheUmcel-1 gene was heterologously expressed inEscherichia coli BL21, and recombinant Umcel-1 was puriifed. The activity of puriifed recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl celulose with optimal activity at pH 5.5 and 45°C. To our knowledge, this study provides the ifrst evidence for a celulase produced by bacteria in gayal rumen.

  3. Screening a wide host-range, waste-water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes.

    Science.gov (United States)

    Li, Youguo; Wexler, Margaret; Richardson, David J; Bond, Philip L; Johnston, Andrew W B

    2005-12-01

    A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors. PMID:16309391

  4. 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

  5. High-Throughput Screening Platform for the Discovery of New Immunomodulator Molecules from Natural Product Extract Libraries.

    Science.gov (United States)

    Pérez Del Palacio, José; Díaz, Caridad; de la Cruz, Mercedes; Annang, Frederick; Martín, Jesús; Pérez-Victoria, Ignacio; González-Menéndez, Víctor; de Pedro, Nuria; Tormo, José R; Algieri, Francesca; Rodriguez-Nogales, Alba; Rodríguez-Cabezas, M Elena; Reyes, Fernando; Genilloud, Olga; Vicente, Francisca; Gálvez, Julio

    2016-07-01

    It is widely accepted that central nervous system inflammation and systemic inflammation play a significant role in the progression of chronic neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, neurotropic viral infections, stroke, paraneoplastic disorders, traumatic brain injury, and multiple sclerosis. Therefore, it seems reasonable to propose that the use of anti-inflammatory drugs might diminish the cumulative effects of inflammation. Indeed, some epidemiological studies suggest that sustained use of anti-inflammatory drugs may prevent or slow down the progression of neurodegenerative diseases. However, the anti-inflammatory drugs and biologics used clinically have the disadvantage of causing side effects and a high cost of treatment. Alternatively, natural products offer great potential for the identification and development of bioactive lead compounds into drugs for treating inflammatory diseases with an improved safety profile. In this work, we present a validated high-throughput screening approach in 96-well plate format for the discovery of new molecules with anti-inflammatory/immunomodulatory activity. The in vitro models are based on the quantitation of nitrite levels in RAW264.7 murine macrophages and interleukin-8 in Caco-2 cells. We have used this platform in a pilot project to screen a subset of 5976 noncytotoxic crude microbial extracts from the MEDINA microbial natural product collection. To our knowledge, this is the first report on an high-throughput screening of microbial natural product extracts for the discovery of immunomodulators. PMID:26962874

  6. 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.

  7. EST Express: PHP/MySQL based automated annotation of ESTs from expression libraries

    Directory of Open Access Journals (Sweden)

    Pardinas Jose R

    2008-04-01

    Full Text Available Abstract Background Several biological techniques result in the acquisition of functional sets of cDNAs that must be sequenced and analyzed. The emergence of redundant databases such as UniGene and centralized annotation engines such as Entrez Gene has allowed the development of software that can analyze a great number of sequences in a matter of seconds. Results We have developed "EST Express", a suite of analytical tools that identify and annotate ESTs originating from specific mRNA populations. The software consists of a user-friendly GUI powered by PHP and MySQL that allows for online collaboration between researchers and continuity with UniGene, Entrez Gene and RefSeq. Two key features of the software include a novel, simplified Entrez Gene parser and tools to manage cDNA library sequencing projects. We have tested the software on a large data set (2,016 samples produced by subtractive hybridization. Conclusion EST Express is an open-source, cross-platform web server application that imports sequences from cDNA libraries, such as those generated through subtractive hybridization or yeast two-hybrid screens. It then provides several layers of annotation based on Entrez Gene and RefSeq to allow the user to highlight useful genes and manage cDNA library projects.

  8. cDNA cloning and disruption of the major vault protein alpha gene (mvpA) in Dictyostelium discoideum.

    Science.gov (United States)

    Vasu, S K; Kedersha, N L; Rome, L H

    1993-07-25

    Vaults are large cytoplasmic ribonucleoprotein particles found in nearly all eukaryotic cells. Dictyostelium vaults contain two major proteins, MVP alpha (94.2 kDa) and MVP beta (approximately 92 kDa). Using an anti-rat vault antibody, we screened a Dictyostelium cDNA expression library and isolated a 2.8-kilobase pair clone that contained a single full-length reading frame. The identity of the clone was established by the presence of a predicted 20-amino acid sequence identical to that found in a peptide sequenced from purified MVP alpha. We have disrupted the single copy gene using homologous recombination and have demonstrated a loss of MVP alpha. Although the cells still produce MVP beta, they do not contain characteristic vault particles, suggesting that MVP alpha is required for normal vault structure. These cells should be a valuable tool for elucidating the function of vaults. PMID:8340365

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

    Science.gov (United States)

    Johnson, Alex; Elya, Carolyn; Anderson, Johanna; Clark, Julie; Connelly, Michele; Yang, Lei; Min, Jaeki; Sato, Yuko; Guy, R. Kiplin; Landfear, Scott M.

    2015-01-01

    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. PMID:25894322

  10. A Prospective Virtual Screening Study: Enriching Hit Rates and Designing Focus Libraries To Find Inhibitors of PI3Kδ and PI3Kγ.

    Science.gov (United States)

    Damm-Ganamet, Kelly L; Bembenek, Scott D; Venable, Jennifer W; Castro, Glenda G; Mangelschots, Lieve; Peeters, Daniëlle C G; Mcallister, Heather M; Edwards, James P; Disepio, Daniel; Mirzadegan, Taraneh

    2016-05-12

    Here, we report a high-throughput virtual screening (HTVS) study using phosphoinositide 3-kinase (both PI3Kγ and PI3Kδ). Our initial HTVS results of the Janssen corporate database identified small focused libraries with hit rates at 50% inhibition showing a 50-fold increase over those from a HTS (high-throughput screen). Further, applying constraints based on "chemically intuitive" hydrogen bonds and/or positional requirements resulted in a substantial improvement in the hit rates (versus no constraints) and reduced docking time. While we find that docking scoring functions are not capable of providing a reliable relative ranking of a set of compounds, a prioritization of groups of compounds (e.g., low, medium, and high) does emerge, which allows for the chemistry efforts to be quickly focused on the most viable candidates. Thus, this illustrates that it is not always necessary to have a high correlation between a computational score and the experimental data to impact the drug discovery process. PMID:27043133

  11. An Applied Study on Touch Screen in Library%触摸屏在图书馆中的应用研究

    Institute of Scientific and Technical Information of China (English)

    张肖; 杨广锋

    2013-01-01

    近年来,随着网络技术的发展、多媒体技术的进步,人们的阅读习惯也随之发生变化,从纸质阅读逐渐向数字阅读倾斜,触摸屏就为读者开启了在图书馆内进行数字阅读的新篇章。本文先详细介绍触摸屏种类和在图书馆服务应用方式,而后简单介绍了在应用过程中需要注意的问题,并展望未来应用的发展。%In recent years,with the development of network technology and the advance of multi_media technique, people’s reading habits also have been changed,moved from paper reading to digital reading,the touch screen open a new era of digital reading for the reader in library. This paper details the kinds of touch screen and the application in li-brary,and then introduces some issues that might attention during the application,and hopes for development in the fu-ture.

  12. Characterization of the sporulation control protein SsgA by use of an efficient method to create and screen random mutant libraries in streptomycetes.

    Science.gov (United States)

    Traag, Bjørn A; Seghezzi, Nicolas; Vijgenboom, Erik; van Wezel, Gilles P

    2007-04-01

    Filamentous actinomycetes are commercially widely used as producers of natural products. However, the mycelial lifestyle of actinomycetes has been a major bottleneck in their commercialization, and screening is difficult due to their poor growth on microtiter plates. We previously demonstrated that the enhanced expression of the cell division activator protein SsgA results in the fragmented growth of streptomycetes, with enhanced growth rates and improved product formation. We here describe a novel and efficient method to create, maintain, and screen mutant libraries in streptomycetes and the application of this method for the functional analysis of Streptomyces coelicolor ssgA. The variants were amplified directly from deep-frozen biomass suspensions. Around 800 ssgA variants, including single-amino-acid-substitution mutants corresponding to more than half of all SsgA residues, were analyzed for their abilities to restore sporulation to an ssgA mutant. The essential residues were clustered in three main sections, and hardly any were in the carboxy-terminal third of the protein. The majority of the crucial residues were conserved among all SsgA-like proteins (SALPs). However, the essential residues L29, D58, and S89 were conserved only in SsgA orthologues and not in other SALPs, suggesting an SsgA-specific function. PMID:17293502

  13. Cloning and expression of human neuron-specific enolase cDNA in Escherichia coli.

    Science.gov (United States)

    Pavlov, K A; Gurina, O I; Antonova, O M; Semenova, A V; Chekhonin, V P

    2011-12-01

    cDNA fragment encoding neuron-specific enolase was amplified from the cDNA library of human brain. Then the fragment was cloned for expression in E. coli using the vector pET28-a. High level of neuron-specific enolase expression was confirmed by SDS-PAAG electrophoresis and immunochemical identity by immunoblot analysis. The constructed producer strain is the cheapest source of neuron-specific enolase suitable for the use in diagnostic applications. PMID:22808461

  14. In-bead screening

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to screening of one-bead-one-compound (OBOC) combinatorial libraries which is useful for the discovery of compounds displaying molecular interactions with a biological or a physicochemical system, such as substrates and inhibitors of enzymes and the like. The invention...... provides a method for screening a library of compounds for their interaction with a physico- chemical or biological system and a corresponding kit for performing the method of screening a one-bead-one-compound library of compounds....

  15. Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis.

    Science.gov (United States)

    Matsuoka, Kazuhiro; Komori, Hiroaki; Nose, Masato; Endo, Yaeta; Sawasaki, Tatsuya

    2010-08-01

    Autoimmune diseases are a heterogeneous group of diseases characterized by immune reactions against either a major or a limited number of the bodies own autoantigens, causing inflammation and damage to tissues and organs. Thus, identification of autoantigens is an important first step to understanding autoimmune diseases. Here we demonstrate a simple screening method for identification of autoantigens reacting with patient serum antibodies by combination of an N-terminal biotinylated protein library (BPL), produced using a wheat cell-free protein production system, and a commercially available luminescence system. Optimization studies using well-characterized autoantigens showed specific interactions between N-terminal biotinylated proteins and antibody that were sensitively detected under homogeneous reaction conditions. In this optimized assay, 1 microL of the translation mixture expressing the biotinylated proteins produced significant luminescence signal by addition of diluted serum between 1:500 and 1:10 000 in 25 microL of reaction volume. For the BPL construction, 214 mouse genes, consisting of 103 well-known autoantigens and 111 genes in the mouse autoimmune susceptibility loci, and the sera of MRL/lpr mouse were used as an autoimmune model. By this screening method, 25 well-known autoantigens and 71 proteins in the loci were identified as autoantigen proteins specifically reacting with sera antibodies. Cross-referencing with the Gene Ontology Database, 26 and 38 of autoantigen proteins were predicted to have nuclear localization and identified as membrane and/or extracellular proteins. The immune reaction of six randomly selected proteins was confirmed by immunoprecipitation and/or immunoblot analyses. Interestingly, three autoantigen proteins were recognized by immunoprecipitation but not by immunoblot analysis. These results suggest that the BPL-based method could provide a simple system for screening of autoantigen proteins and would help with

  16. Expression cloning of a cDNA encoding the bovine histamine H1 receptor.

    OpenAIRE

    Yamashita, M; Fukui, H; Sugama, K; Horio, Y; Ito, S.; Mizuguchi, H.; Wada, H

    1991-01-01

    A functional cDNA clone for the histamine H1 receptor was isolated from a cDNA library of bovine adrenal medulla by a combination of molecular cloning in an expression vector and electrophysiological assay in Xenopus oocytes. The H1 receptor cDNA encodes a protein of 491 amino acids (Mr 55,954) with seven putative transmembrane domains, illustrating the similarity to other receptors that couple with guanine nucleotide-binding regulatory proteins (G protein-coupled receptors). The sequence hom...

  17. Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.

    OpenAIRE

    Devos, René; Plaetinck, Geert; Cheroutre, Hilde; Simons, Guus; Degrave, Wim,; Tavernier, Jan; Remaut, Erik; Fiers, Walter

    1983-01-01

    A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal seq...

  18. Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata

    Directory of Open Access Journals (Sweden)

    Jin-Xin Gao

    2015-06-01

    Full Text Available Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5′ end of the RNA Transcript technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 6.39 ×10⁵ transformants/3 μg pGADT7-Rec. The titer of the primary cDNA library was 2.5×10⁸ cfu/mL. The numbers for the cDNA library was 2.46×10⁵. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase was used as a “bait” to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

  19. Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family.

    OpenAIRE

    Grove, G; Zarlengo, R P; Timmerman, K P; Li, N Q; Tam, M F; Tu, C P

    1988-01-01

    We have isolated from a constructed lambda gt11 expression library two classes of cDNA clones encoding the entire sequence of the maize GSH S-transferases GST I and GST III. Expression of a full-length GST I cDNA in E. coli resulted in the synthesis of enzymatically active maize GST I that is immunologically indistinguishable from the native GST I. Another GST I cDNA with a truncated N-terminal sequence is also active in heterospecific expression. Our GST III cDNA sequence differs from the ve...

  20. MOLECULAR-CLONING AND CHARACTERIZATION OF A CDNA FOR THE BETA-SUBUNIT OF HUMAN ALCOHOL-DEHYDROGENASE

    OpenAIRE

    Duester, G; Hatfield, G.; Buhler, R; Hempel, J; Jornvall, H; Smith, M.

    1984-01-01

    Human alcohol dehydrogenase (ADH) is encoded by at least five genes that fall into three classes. The class I ADH genes encode the three closely related alpha, beta, and gamma polypeptides. Molecular genetic analysis of class I ADH genes has been initiated by isolating a cDNA clone from a human adult liver cDNA library. A synthetic oligonucleotide mixture encoding a portion of the beta subunit of ADH was used as an in situ hybridization probe for the cDNA library. One positively hybridizing c...

  1. Gene expression in the pulp of ripening bananas. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products and cDNA cloning of 25 different ripening-related mRNAs.

    Science.gov (United States)

    Medina-Suárez, R; Manning, K; Fletcher, J; Aked, J; Bird, C R; Seymour, G B

    1997-10-01

    mRNA was extracted from the pulp and peel of preclimacteric (d 0) bananas (Musa AAA group, cv Grand Nain) and those exposed to ethylene gas for 24 h and stored in air alone for a further 1 (d 2) and 4 d (d 5). Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products from the pulp and peel of these fruits revealed significant up-regulation of numerous transcripts during ripening. The majority of the changes were initiated by d 2, with the level of these messages increasing during the remainder of the ripening period. Pulp tissue from d 2 was used for the construction of a cDNA library. This library was differentially screened for ripening-related clones using cDNA from d-0 and d-2 pulp by a novel microtiter plate method. In the primary screen 250 up- and down-regulated clones were isolated. Of these, 59 differentially expressed clones were obtained from the secondary screen. All of these cDNAs were partially sequenced and grouped into families after database searches. Twenty-five nonredundant groups of pulp clones were identified. These encoded enzymes were involved in ethylene biosynthesis, respiration, starch metabolism, cell wall degradation, and several other key metabolic events. We describe the analysis of these clones and their possible involvement in ripening. PMID:9342865

  2. An FDA-Drug Library Screen for Compounds with Bioactivities against Meticillin-Resistant Staphylococcus aureus (MRSA

    Directory of Open Access Journals (Sweden)

    Qiu Ying Lau

    2015-10-01

    Full Text Available The lack of new antibacterial drugs entering the market and their misuse have resulted in the emergence of drug-resistant bacteria, posing a major health crisis worldwide. In particular, meticillin-resistant Staphylococcus aureus (MRSA, a pathogen responsible for numerous human infections, has become endemic in hospitals worldwide. Drug repurposing, the finding of new therapeutic indications for approved drugs, is deemed a plausible solution to accelerate drug discovery and development in this area. Towards this end, we screened 1163 drugs approved by the Food and Drug Administration (FDA for bioactivities against MRSA in a 10 μM single-point assay. After excluding known antibiotics and antiseptics, six compounds were identified and their MICs were determined against a panel of clinical MRSA strains. A toxicity assay using human keratinocytes was also conducted to gauge their potential for repurposing as topical agents for treating MRSA skin infections.

  3. Comparative molecular surface analysis (CoMSA) for virtual combinatorial library screening of styrylquinoline HIV-1 blocking agents.

    Science.gov (United States)

    Niedbala, Halina; Polanski, Jaroslaw; Gieleciak, Rafal; Musiol, Robert; Tabak, Dominik; Podeszwa, Barbara; Bak, Andrzej; Palka, Anna; Mouscadet, Jean-Francois; Gasteiger, Johann; Le Bret, Marc

    2006-12-01

    We used comparative molecular surface analysis to design molecules for the synthesis as part of the search for new HIV-1 integrase inhibitors. We analyzed the virtual combinatorial library (VCL) constituted from various moieties of styrylquinoline and styrylquinazoline inhibitors. Since imines can be applied in a strategy of dynamic combinatorial chemistry (DCC), we also tested similar compounds in which the -C=N- or -N=C- linker connected the heteroaromatic and aromatic moieties. We then used principal component analysis (PCA) or self-organizing maps (SOM), namely, the Kohonen neural networks to obtain a clustering plot analyzing the diversity of the VCL formed. Previously synthesized compounds of known activity, used as molecular probes, were projected onto this plot, which provided a set of promising virtual drugs. Moreover, we further modified the above mentioned VCL to include the single bond linker -C-N- or -N-C-. This allowed increasing compound stability but expanded also the diversity between the available molecular probes and virtual targets. The application of the CoMSA with SOM indicated important differences between such compounds and active molecular probes. We synthesized such compounds to verify the computational predictions. PMID:17168681

  4. cDNA sequence for human erythrocyte ankyrin

    International Nuclear Information System (INIS)

    The cDNA for human erythrocyte ankyrin has been isolated from a series of overlapping clones obtained from a reticulocyte cDNA library. The composite cDNA sequence has a large open reading frame of 5636 base pairs (bp) with the complete coding sequence for a polypeptide of 1879 amino acids with a predicted molecular mass of 206 kDa. The derived amino acid sequence contained 194 residues that were identical to those obtained by direct amino acid sequencing of 11 ankyrin proteolytic peptides. The primary sequence contained 23 highly homologous repeat units of 33 amino acids within the 90-kDa band 3 binding domain. Two cDNA clones showed evidence of apparent mRNA processing, resulting in the deletions of 486 bp and 135 bp, respectively. The 486-bp deletion resulted in the removal of a 16-kDa highly acidic peptide, and the smaller deletion had the effect of altering the COOH terminus of the molecule. Radiolabeled ankyrin cDNAs recognized two erythroid message sizes by RNA blot analysis, one of which was predominantly associated with early erythroid cell types. An ankyrin message was also observed in RNA from the human cerebellum by the same method. The ankyrin gene is assigned to chromosome 8 using genomic DNA from a panel of sorted human chromosomes

  5. Molecular cloning of cDNA for the B beta subunit of Xenopus fibrinogen, the product of a coordinately-regulated gene family.

    Science.gov (United States)

    Bhattacharya, A; Shepard, A R; Moser, D R; Roberts, L R; Holland, L J

    1991-02-01

    Fibrinogen, the principal blood-clotting protein, is made up of three different subunits synthesized in the liver. In vitro administration of glucocorticoids to liver cells from the frog Xenopus laevis causes a dramatic increase in fibrinogen synthesis. Investigations of molecular mechanisms underlying this hormonal stimulation at the mRNA level require cDNA clones complementary to the mRNAs coding for the three fibrinogen subunits, called A alpha, B beta, and gamma. We describe here the isolation and characterization of cDNA clones for the B beta subunit of Xenopus fibrinogen. cDNA libraries in both plasmid (pBR322) and phage (lambda gt10) cloning vectors were constructed from frog liver mRNA and screened with a rat B beta cDNA. Clones thus isolated hybridized to two Xenopus liver mRNAs 2500 and 1800 bases long, the previously-determined sizes for B beta mRNAs. The identity of the plasmid clone B beta-27 was confirmed by hybridization-selection of complementary mRNA which translated in vitro into the B beta polypeptide, as determined by size and susceptibility to thrombin cleavage. lambda/B beta 10, a clone representing nearly all of the 2500-base B beta mRNA, was isolated from the phage cDNA library. The 3'-end of this clone includes a polyadenylation signal about 20 residues upstream of a stretch of 34 adenosine residues, which probably represents the 3'-poly(A) tail of the messenger RNA. lambda/B beta 10 lacks only 20 nucleotides of full-length B beta mRNA at the 5'-end and there is one major start site of transcription. The 2500-base B beta mRNA has a 700-base extension at the 3'-end that is not present in the 1800-base mRNA. The Xenopus laevis genome contains two or three genes for the B beta fibrinogen subunit. Using the cDNA clone as a probe, B beta mRNA was shown to be induced at least 20-fold by glucocorticoid treatment of purified parenchymal cells of Xenopus liver maintained in primary culture. PMID:2050271

  6. Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.).

    Science.gov (United States)

    Yamaguchi, S; Saito, T; Abe, H; Yamane, H; Murofushi, N; Kamiya, Y

    1996-08-01

    The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene. ent-Kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB). A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full-length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize [3H]CDP to [3H]ent-kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region. PMID:8771778

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

    International Nuclear Information System (INIS)

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

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

    International Nuclear Information System (INIS)

    Screening of a λgt11 human melanocyte cDNA library with antibodies against hamster tyrosinase resulted in the isolation of 16 clones. The cDNA inserts from 13 of the 16 clones cross-hybridized with each other, indicating that they were form related mRNA species. One of the cDNA clones, Pmel34, detected one mRNA species with an approximate length of 2.4 kilobases that was expressed preferentially in normal and malignant melanocytes but not in other cell types. The amino acid sequence deduced from the nucleotide sequence showed that the putative human tyrosinase is composed of 548 amino acids with a molecular weight of 62,610. The deduced protein contains glycosylation sites and histidine-rich sites that could be used for copper binding. Southern blot analysis of DNA derived from newborn mice carrying lethal albino deletion mutations revealed that Pmel34 maps near or at the c-albino locus, the position of the structural gene for tyrosinase

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

    Indian Academy of Sciences (India)

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

    2001-09-01

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

  10. 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.

  11. 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

  12. A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil.

    Science.gov (United States)

    Cretoiu, Mariana Silvia; Berini, Francesca; Kielak, Anna Maria; Marinelli, Flavia; van Elsas, Jan Dirk

    2015-10-01

    Here, we report on the construction of a metagenomic library from a chitin-amended disease-suppressive agricultural soil and its screening for genes that encode novel chitinolytic enzymes. The library, constructed in fosmids in an Escherichia coli host, comprised 145,000 clones containing inserts of sizes of 21 to 40 kb, yielding a total of approximately 5.8 GB of cloned soil DNA. Using genetic screenings by repeated PCR cycles aimed to detect gene sequences of the bacterial chitinase A-class (hereby named chi A genes), we identified and characterized five fosmids carrying candidate genes for chitinolytic enzymes. The analysis thus allowed access to the genomic (fosmid-borne) context of these genes. Using the chiA-targeted PCR, which is based on degenerate primers, the five fosmids all produced amplicons, of which the sequences were related to predicted chitinolytic enzyme-encoding genes of four different host organisms, including Stenotrophomonas maltophilia. Sequencing and de novo annotation of the fosmid inserts confirmed that each one of these carried one or more open reading frames that were predicted to encode enzymes active on chitin, including one for a chitin deacetylase. Moreover, the genetic contexts in which the putative chitinolytic enzyme-encoding genes were located were unique per fosmid. Specifically, inserts from organisms related to Burkholderia sp., Acidobacterium sp., Aeromonas veronii, and the chloroflexi Nitrolancetus hollandicus and/or Ktedonobacter racemifer were obtained. Remarkably, the S. maltophilia chiA-like gene was found to occur in two different genetic contexts (related to N. hollandicus/K. racemifer), indicating the historical occurrence of genetic reshufflings in this part of the soil microbiota. One fosmid containing the insert composed of DNA from the N. hollandicus-like organism (denoted 53D1) was selected for further work. Using subcloning procedures, its putative gene for a chitinolytic enzyme was successfully brought to

  13. A Drug Combination Screen Identifies Drugs Active against Amoxicillin-induced Round Bodies of Borrelia burgdorferi Persisters from an FDA Drug Library

    Directory of Open Access Journals (Sweden)

    Jie eFeng

    2016-05-01

    Full Text Available Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10-20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that are not killed by current Lyme antibiotics. To identify more effective drugs that are active against the round bodies of B. burgdorferi, we established a round body persister model induced by amoxicillin and screened the Food and Drug Administration (FDA drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide (PI viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven of these scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. While some drug candidates such as daptomycin and clofazimine overlapped with a previous screen against stationary phase B. burgdorferi persisters, additional drug candidates active against round bodies we identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi persisters in vitro, even if pre-treated with amoxicillin. These findings may have implications for improved treatment of Lyme disease.

  14. A Drug Combination Screen Identifies Drugs Active against Amoxicillin-Induced Round Bodies of In Vitro Borrelia burgdorferi Persisters from an FDA Drug Library

    Science.gov (United States)

    Feng, Jie; Shi, Wanliang; Zhang, Shuo; Sullivan, David; Auwaerter, Paul G.; Zhang, Ying

    2016-01-01

    Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10–20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under experimental stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that appear resistant in vitro to customary first-line antibiotics for Lyme disease. To identify more effective drugs with activity against the round body form of B. burgdorferi, we established a round body persister model induced by exposure to amoxicillin (50 μg/ml) and then screened the Food and Drug Administration drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven individual drugs scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. In this amoxicillin-induced round body model, some drug candidates such as daptomycin and clofazimine also displayed enhanced activity which was similar to a previous screen against stationary phase B. burgdorferi persisters not exposure to amoxicillin. Additional candidate drugs active against round bodies identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against amoxicillin-induced round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi

  15. Abiotic stresses induce a thaumatin-like protein in maize; cDNA isolation and sequence analysis

    International Nuclear Information System (INIS)

    The cDNA clone (CHEM4) coding for a stress-induced protein has been isolated from a mercuric chloride-treated maize (Zea mays L.) cDNA library by differential screening. Nucleotide sequence analysis of the 694-bp-cDNA insert predicts an open reading frame of 522 nucleotides encoding a mature protein of 149 amino acids and a signal peptide of 25 amino acid residues. The sequence of CHEM4-encoded protein shows 50 to 60% sequence identity with thaumatin, bifunctional α-amylase/trypsin inhibitor from maize and thaumatin-like (TL) proteins from dicotyledonous plants. Higher sequence identities are found to wheat PWIR2 and barley Hv-1b TL-proteins. CHEM4-mRNA is not only induced by mercuric chloride treatment but also by other stresses such as high salt concentration, high temperatures, UV light, wounding and polluted rainwater. CHEM4 genes appear to be highly inducible by UV light and mercuric chloride, moderately by wounding, salt stress, polluted rainwater and heat shock and very weakly by cooling. (author)

  16. Application of parallel liquid chromatography/mass spectrometry for high throughput microsomal stability screening of compound libraries.

    Science.gov (United States)

    Xu, Rongda; Nemes, Csaba; Jenkins, Kelly M; Rourick, Robyn A; Kassel, Daniel B; Liu, Charles Z C

    2002-02-01

    Solution-phase and solid-phase parallel synthesis and high throughput screening have enabled biologically active and selective compounds to be identified at an unprecedented rate. The challenge has been to convert these hits into viable development candidates. To accelerate the conversion of these hits into lead development candidates, early assessment of the physicochemical and pharmacological properties of these compounds is being made. In particular, in vitro absorption, distribution, metabolism, and elimination (ADME) assays are being conducted at earlier and earlier stages of discovery with the goal of reducing the attrition rate of these potential drug candidates as they progress through development. In this report, we present an eight-channel parallel liquid chromatography/mass spectrometry (LC/MS) system in combination with custom Visual Basic and Applescript automated data processing applications for high throughput early ADME. The parallel LC/MS system was configured with one set of gradient LC pumps and an eight-channel multiple probe autosampler. The flow was split equivalently into eight streams before the multiple probe autosampler and recombined after the eight columns and just prior to the mass spectrometer ion source. The system was tested for column-to-column variation and for reproducibility over a 17 h period (approximately 500 injections per column). The variations in retention time and peak area were determined to be less than 2 and 10%, respectively, in both tests. The parallel LC/MS system described permits time-course microsomal incubations (t(o), t5, t15, t30) to be measured in triplicate and enables estimations of t 1/2 microsomal stability. The parallel LC/MS system is capable of analyzing up to 240 samples per hour and permits the complete profiling up to two microtiter plates of compounds per day (i.e., 176 test substrate compounds + sixteen controls). PMID:11841071

  17. Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library.

    Science.gov (United States)

    Choi, Kyoung-Jae; Yu, Yeon Gyu; Hahn, Hoh Gyu; Choi, Jung-Do; Yoon, Moon-Young

    2005-08-29

    Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The genes of AHAS from Mycobacterium tuberculosis were cloned, and overexpressed in E. coli and purified to homogeneity. The purified AHAS from M. tuberculosis is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC(50) (inhibitory concentration 50%) of 0.87 microM. The kinetic parameters of M. tuberculosis AHAS were determined, and an enzyme activity assay system using 96-well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC(50) in the range of 1.8-2.6 microM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of M. tuberculosis. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti-tuberculosis therapeutics. PMID:16111681

  18. Identification of Eusynstyelamide B as a Potent Cell Cycle Inhibitor Following the Generation and Screening of an Ascidian-Derived Extract Library Using a Real Time Cell Analyzer

    Directory of Open Access Journals (Sweden)

    Michelle S. Liberio

    2014-10-01

    Full Text Available Ascidians are marine invertebrates that have been a source of numerous cytotoxic compounds. Of the first six marine-derived drugs that made anticancer clinical trials, three originated from ascidian specimens. In order to identify new anti-neoplastic compounds, an ascidian extract library (143 samples was generated and screened in MDA-MB-231 breast cancer cells using a real-time cell analyzer (RTCA. This resulted in 143 time-dependent cell response profiles (TCRP, which are read-outs of changes to the growth rate, morphology, and adhesive characteristics of the cell culture. Twenty-one extracts affected the TCRP of MDA-MB-231 cells and were further investigated regarding toxicity and specificity, as well as their effects on cell morphology and cell cycle. The results of these studies were used to prioritize extracts for bioassay-guided fractionation, which led to the isolation of the previously identified marine natural product, eusynstyelamide B (1. This bis-indole alkaloid was shown to display an IC50 of 5 µM in MDA-MB-231 cells. Moreover, 1 caused a strong cell cycle arrest in G2/M and induced apoptosis after 72 h treatment, making this molecule an attractive candidate for further mechanism of action studies.

  19. Chemical Library Screening and Structure-Function Relationship Studies Identify Bisacodyl as a Potent and Selective Cytotoxic Agent Towards Quiescent Human Glioblastoma Tumor Stem-Like Cells.

    Directory of Open Access Journals (Sweden)

    Maria Zeniou

    Full Text Available Cancer stem-like cells reside in hypoxic and slightly acidic tumor niches. Such microenvironments favor more aggressive undifferentiated phenotypes and a slow growing "quiescent state" which preserves them from chemotherapeutic agents that essentially target proliferating cells. Our objective was to identify compounds active on glioblastoma stem-like cells, including under conditions that mimick those found in vivo within this most severe and incurable form of brain malignancy. We screened the Prestwick Library to identify cytotoxic compounds towards glioblastoma stem-like cells, either in a proliferating state or in more slow-growing "quiescent" phenotype resulting from non-renewal of the culture medium in vitro. Compound effects were assessed by ATP-level determination using a cell-based assay. Twenty active molecules belonging to different pharmacological classes have thus been identified. Among those, the stimulant laxative drug bisacodyl was the sole to inhibit in a potent and specific manner the survival of quiescent glioblastoma stem-like cells. Subsequent structure-function relationship studies led to identification of 4,4'-dihydroxydiphenyl-2-pyridyl-methane (DDPM, the deacetylated form of bisacodyl, as the pharmacophore. To our knowledge, bisacodyl is currently the only known compound targeting glioblastoma cancer stem-like cells in their quiescent, more resistant state. Due to its known non-toxicity in humans, bisacodyl appears as a new potential anti-tumor agent that may, in association with classical chemotherapeutic compounds, participate in tumor eradication.

  20. Novel HIV-1 knockdown targets identified by an enriched kinases/phosphatases shRNA library using a long-term iterative screen in Jurkat T-cells.

    Directory of Open Access Journals (Sweden)

    Sylvie Rato

    Full Text Available HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies.

  1. 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 ...

  2. 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] ...

  3. 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 ...

  4. cDNA library Table: wd-- [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available wd-- NA wd-- p50T wing disc mixture of fifth instar larval stage to spinning stage ...mixed pCMVFL3, Oligo-cap, full-length Unknown sequenced from 5' DC552315-DC563654,DC563655-DC574923 E_FL_wd--_[number]_F_0E_FL_wd--_[number]_R_0 ...

  5. Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones

    OpenAIRE

    Halgren, Robert G.; Fielden, Mark R.; Fong, Cora J.; Zacharewski, Timothy R

    2001-01-01

    This report documents the error rate in a commercially distributed subset of the IMAGE Consortium mouse cDNA clone collection. After isolation of plasmid DNA from 1189 bacterial stock cultures, only 62.2% were uncontaminated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones. An agarose gel electrophoresis pre-screening strategy identified 361 stock cultures that appeared to contain two or more plasmid species. Is...

  6. Construction and screening of genomic library of Haemophilus paragallinarum%副鸡嗜血杆菌基因文库的构建与筛选

    Institute of Scientific and Technical Information of China (English)

    王雪敏; 梁玉荣; 吕学泽; 张培君; 龚玉梅; 王宏俊

    2012-01-01

    为了筛选副鸡嗜血杆菌的体内表达基因,提取了副鸡嗜血杆菌的全基因组,构建了副鸡嗜血杆菌基因组的pET系统表达文库。运用PCR及核酸内切酶(SalⅠ+NdeⅠ)鉴定基因文库,并以病原菌吸附后的康复血清作为探针,采用菌落原位杂交的方法对基因文库进行筛选。结果显示,重组质粒中有0.5~2kb的片段插入,99%的基因包含在基因文库中;重复筛选后得到的阳性克隆再经过PCR与SalⅠ+NdeⅠ酶切鉴定后定向测序,并对测序结果在NCBI上进行分析后发现筛选获得的基因中,有1个表达为转运谷氨酰还原酶、1个表达为转录终止因子,1个表达为荚膜合成域2,还有2个表达为保守假想蛋白。结果表明,本研究应用体内诱导抗原技术(IVIAT)筛选到了一些副鸡嗜血杆菌体内诱导表达基因,并对基因的功能做了初步探讨,在找寻副鸡嗜血杆菌在体内生存以及致病关键基因的道路上前进了一步,为传染性鼻炎的预防和治疗积累了有价值的资料。%In order to select in vivo expression genes of Haemophilus paragallinarum,the total DNA of the bacterium was extracted and the genomic library was constructed with pET system.The positive clones in the library were identified by PCR and SalⅠ-NdeⅠ-digestion.The expressed library was screened by colony hybridization using rehabilitation serum,which was absorbed with H.paragallinarum,as the probe.In result,the recombinant plasmids contained from 0.5 to 2 kb of target fragments,and 99% of H.paragallinarum genes were involved in the library.The 17 positive clones obtained by colony hybridization were confirmed by PCR and SalⅠ-NdeⅠ digestion followed by direct-sequencing.Sequences were then blasted in the NCBI GenBank.The sequence analysis revealed 5 ORFs encoded glutamyl tRNA reductase,transcription termination factor,capsule biosynthesis region 2 gene cluster,and two hypothetical proteins

  7. Characterization of a Pinus pinaster cDNA encoding an auxin up-regulated putative peroxidase in roots.

    Science.gov (United States)

    Charvet-Candela, V; Hitchin, S; Reddy, M S; Cournoyer, B; Marmeisse, R; Gay, G

    2002-03-01

    As part of a study to identify host plant genes regulated by fungal auxin during ectomycorrhiza formation, we differentially screened a cDNA library constructed from roots of auxin-treated Pinus pinaster (Ait.) Sol. seedlings. We identified three cDNAs up-regulated by auxin. Sequence analysis of one of these cDNAs, PpPrx75, revealed the presence of an open reading frame of 216 amino acids with the characteristic consensus sequences of plant peroxidases. The deduced amino acid sequence showed homology with Arabidopsis thaliana (L.) Heynh., Arachis hypogaea L. and Stylosanthes humilis HBK cationic peroxidases. Amino acid sequence identities in the conserved domains of plant peroxidases ranged from 60 to 100%. In PpPrx75, there are five cysteine residues and one histidine residue that are found at conserved positions among other peroxidases. A potential glycosylation site (NTS) is present in the deduced sequence. Phylogenetic analysis showed that PpPrx75 is closely related to two A. thaliana peroxidases. The PpPrx75 cDNA was induced by active auxins, ethylene, abscisic acid and quercetin, a flavonoid possibly involved in plant-microorganism interactions. Transcript accumulation was detected within 3 h following root induction by auxin, and the amount of mRNA increased over the following 24 h. The protein synthesis inhibitor cycloheximide did not inhibit indole-3-acetic acid-induced transcript accumulation, suggesting that PpPrx75 induction is a primary (direct) response to auxin. This cDNA can be used to study expression of an auxin-regulated peroxidase during ectomycorrhiza formation. PMID:11874719

  8. 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

  9. Transcriptome screen for fast evolving genes by Inter-Specific Selective Hybridization (ISSH

    Directory of Open Access Journals (Sweden)

    Bahechar Ilham

    2010-02-01

    Full Text Available Abstract Background Fast evolving genes are targets of an increasing panel of biological studies, from cancer research to population genetics and species specific adaptations. Yet, their identification and isolation are still laborious, particularly for non-model organisms. We developed a method, named the Inter-Specific Selective Hybridization (ISSH method, for generating cDNA libraries enriched in fast evolving genes. It utilizes transcripts of homologous tissues of distinct yet related species. Experimental hybridization conditions are monitored in order to discard transcripts that do not find their homologous counterparts in the two species sets as well as transcripts that display a strong complementarity between the two species. Only heteroduplexes that disanneal at low stringency are used for constructing the resulting cDNA library. Results We demonstrate the efficiency of the ISSH method by generating a brain cDNA library enriched in fast evolving transcripts of a non-model catfish species as well as a control, non-enriched library. Our results indicate that the enriched library contains effectively more fast evolving sequences than the control library. Gene annotation analyses also indicate enrichment in genes with low expression levels and non-ubiquitously expressed genes, both categories encompassing the majority of fast evolving genes. Furthermore, most of the identified transcripts show higher sequence divergence between two closely related catfish species as compared to recognized fast evolving DNA markers. Conclusions The ISSH method offers a simple, inexpensive and efficient way to screen the transcriptome for isolating fast evolving genes. This method opens new opportunities in the investigation of biological mechanisms that include fast evolving genes, such as the evolution of lineage specific processes and traits responsible for species adaptation to their environment.

  10. Construction and Screening of a cDNA library of Taenia solium on cospheres%猪带绦虫六钩蚴cDNA文库的构建与筛选

    Institute of Scientific and Technical Information of China (English)

    唐雨德; 陆承平

    2003-01-01

    在体外将猪带绦虫虫卵孵化为有活力的六钩蚴.采用商品化试剂盒抽提六钩蚴总RNA、mRNA,反转录为双链cDNA,再加EcoR I Adapter.在去除小片段后,dscDNA与λgt11噬菌体DNA连接,经蛋白包装,构建了猪带绦虫六钩蚴λgt11 cDNA文库.该文库效价为3.5×10 9pfu/mL,重组DNA片段大小为0.5~3.2kb,平均为1.6kb,蓝白斑比1:9.用抗体探针对文库进行免疫学筛选,在消除非特异性反应的基础上筛选约106重组子,共得到118株强阳性克隆;应用PCR鉴定上述部分阳性克隆,均扩增到0.5kb以上的片段.结果显示,构建的文库合格,含六钩蚴所有抗原基因,可用于六钩蚴cDNA克隆的筛选;用免疫学与PCR联合筛选cDNA文库,可消除假阳性.

  11. Construction and screening of the cDNA expression library for muscle larvae of Trichinella pseudospiralis%伪旋毛虫肌幼虫cDNA表达文库的构建及筛选

    Institute of Scientific and Technical Information of China (English)

    吴秀萍; 王峰; 刘明远; 赫荣华; 高丽芳; 付宝权; 王学林; R. Blaga; 邓洪宽; 于申业; P.Boireau

    2008-01-01

    目的 获取伪旋毛虫(Trichinella pseudospiralis)肌幼虫时期抗原性基因.方法 利用λZAP载体构建伪旋毛虫肌幼虫cDNA表达文库;以感染伪旋毛虫60天后的猪血清为抗体探针,对伪旋毛虫肌幼虫cDNA文库进行免疫学筛选,对筛选出的强反应原性克隆进行测序,利用相关分子生物学软件进行序列分析.结果 构建的伪旋毛虫cDNA表达文库的库容量为0.55×106pfu,重组率为95.6%,扩增后文库滴度为1.2×1010 pfu/mL.从2.0×105个重组噬菌体筛选获得阳性克隆27个.序列分析结果表明这27个阳性克隆共编码7种cDNA分子,其编码的类似蛋白分别为伪旋毛虫Tp21kDa蛋白、伪旋毛虫Tp28kDa蛋白、蛋白酶体激活因子亚单位(Proteasome activator subunit)类蛋白、Nudix水解酶(Nudix hydrolyase)类蛋白、凝集因子(Condensin)类蛋白、尿嘧啶糖基化酶(uracil glycosylase)类蛋白和一个未知蛋白.结论 获得两个反应原性较强且高拷贝的抗原基因,其分别编码Tp21kDa蛋白及蛋白酶体激活因子亚单位.

  12. Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Thrombopoietin(TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl.The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl.First,the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2,and the resulting plasmid is designated as pASMM.Then a human placenta cDNA library was screened using the pASMM as a target plasmid.Seven positive clones were isolated from 150 000 independent transformants.Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3'end and flanking non-translation region was obtained.Therefore,there is an interaction between vimentin and TPO receptor.The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.

  13. Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    赵新燕[1; 冯丽冰[2; 周伟国[3; 戴卫列[4; 李昌本[5; 赵寿元[6

    2000-01-01

    Thrombopoietin (TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl. The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl. First, the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2, and the resulting plasmid is designated as pASMM. Then a human placenta cDNA library was screened using the pASMM as a target plasmid. Seven positive clones were isolated from 150 000 independent transformants. Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3’ end and flanking non-translation region was obtained. Therefore, there is an interaction between vimentin and TPO receptor. The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.

  14. Screening a phage display library for a novel FGF8b-binding peptide with anti-tumor effect on prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wenhui; Chen, Xilei; Li, Tao; Li, Yanmei; Wang, Ruixue; He, Dan; Luo, Wu [Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632 (China); Li, Xiaokun [Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632 (China); School of Pharmaceutical Science, Wenzhou Medical College, Wenzhou 325035 (China); Wu, Xiaoping, E-mail: twxp@jnu.edu.cn [Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632 (China); School of Pharmaceutical Science, Wenzhou Medical College, Wenzhou 325035 (China)

    2013-05-01

    Fibroblast growth factor 8b (FGF8b) is the major isoform of FGF8 expressed in prostate cancer and it correlates with the stage and grade of the disease. FGF8b has been considered as a potential target for prostate cancer therapy. Here we isolated 12 specific FGF8b-binding phage clones by screening a phage display heptapeptide library with FGF8b. The peptide (HSQAAVP, named as P12) corresponding to one of these clones showed high homology to the immunoglobulin-like (Ig-like) domain II(D2) of high-affinity FGF8b receptor (FGFR3c), contained 3 identical amino acids (AVP) to the authentic FGFR3 D2 sequence aa 163–169 (LLAVPAA) directly participating in ligand binding, carried the same charges as its corresponding motif (aa163–169) in FGFR3c, suggesting that P12 may have a greater potential to interrupt FGF8b binding to its receptors than other identified heptapeptides do. Functional analysis indicated that synthetic P12 peptides mediate significant inhibition of FGF8b-induced cell proliferation, arrest cell cycle at the G0/G1 phase via suppression of Cyclin D1 and PCNA, and blockade of the activations of Erk1/2 and Akt cascades in both prostate cancer cells and vascular endothelial cells. The results demonstrated that the P12 peptide acting as an FGF8b antagonist may have therapeutic potential in prostate cancer. - Highlights: ► A novel FGF8b-binding peptide P12 was isolated from a phage display library. ► The mechanisms for P12 peptide inhibiting cell proliferation were proposed. ► P12 caused cell cycle arrest at G0/G1 phase via suppression of Cyclin D1 and PCNA. ► P12 suppressed FGF8b-induced activations of Akt and MAP kinases. ► P12 acting as an FGF8b antagonist may have therapeutic potential in prostate cancer.

  15. Screening a phage display library for a novel FGF8b-binding peptide with anti-tumor effect on prostate cancer

    International Nuclear Information System (INIS)

    Fibroblast growth factor 8b (FGF8b) is the major isoform of FGF8 expressed in prostate cancer and it correlates with the stage and grade of the disease. FGF8b has been considered as a potential target for prostate cancer therapy. Here we isolated 12 specific FGF8b-binding phage clones by screening a phage display heptapeptide library with FGF8b. The peptide (HSQAAVP, named as P12) corresponding to one of these clones showed high homology to the immunoglobulin-like (Ig-like) domain II(D2) of high-affinity FGF8b receptor (FGFR3c), contained 3 identical amino acids (AVP) to the authentic FGFR3 D2 sequence aa 163–169 (LLAVPAA) directly participating in ligand binding, carried the same charges as its corresponding motif (aa163–169) in FGFR3c, suggesting that P12 may have a greater potential to interrupt FGF8b binding to its receptors than other identified heptapeptides do. Functional analysis indicated that synthetic P12 peptides mediate significant inhibition of FGF8b-induced cell proliferation, arrest cell cycle at the G0/G1 phase via suppression of Cyclin D1 and PCNA, and blockade of the activations of Erk1/2 and Akt cascades in both prostate cancer cells and vascular endothelial cells. The results demonstrated that the P12 peptide acting as an FGF8b antagonist may have therapeutic potential in prostate cancer. - Highlights: ► A novel FGF8b-binding peptide P12 was isolated from a phage display library. ► The mechanisms for P12 peptide inhibiting cell proliferation were proposed. ► P12 caused cell cycle arrest at G0/G1 phase via suppression of Cyclin D1 and PCNA. ► P12 suppressed FGF8b-induced activations of Akt and MAP kinases. ► P12 acting as an FGF8b antagonist may have therapeutic potential in prostate cancer

  16. cDNA cloning and gene expression of ascorbate oxidase in tobacco.

    Science.gov (United States)

    Kato, N; Esaka, M

    1996-02-01

    A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Nothern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant. PMID:8624413

  17. Broad screening and identification of β-agonists in feed and animal body fluid and tissues using ultra-high performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry combined with spectra library search.

    Science.gov (United States)

    Li, Tingting; Cao, Jingjing; Li, Zhen; Wang, Xian; He, Pingli

    2016-02-01

    Broad screening and identification of β-agonists in feed, serum, urine, muscle and liver samples was achieved in a quick and highly sensitive manner using ultra high performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) combined with a spectra library search. Solid-phase extraction technology was employed for sample purification and enrichment. After extraction and purification, the samples were analyzed using a Q-Orbitrap high-resolution mass spectrometer under full-scan and data-dependent MS/MS mode. The acquired mass spectra were compared with an in-house library (compound library and MS/MS mass spectral library) built with TraceFinder Software which contained the M/Z of the precursor ion, chemical formula, retention time, character fragment ions and the entire MS/MS spectra of 32 β-agonist standards. Screening was achieved by comparing 5 key mass spectral results and positive matches were marked. Using the developed method, the identification results from 10 spiked samples and 238 actual samples indicated that only 2% of acquired mass spectra produced false identities. The method validation results showed that the limit of detection ranged from 0.021-3.854 μg kg(-1)and 0.015-1.198 ng mL(-1) for solid and liquid samples, respectively. PMID:26304337

  18. A two-channel detection method for autofluorescence correction and efficient on-bead screening of one-bead one-compound combinatorial libraries using the COPAS fluorescence activated bead sorting system

    International Nuclear Information System (INIS)

    One-bead one-compound combinatorial library beads exhibit varying levels of autofluorescence after solid phase combinatorial synthesis. Very often this causes significant problems for automated on-bead screening using TentaGel beads and fluorescently labeled target proteins. Herein, we present a method to overcome this limitation when fluorescence activated bead sorting is used as the screening method. We have equipped the COPAS bead sorting instrument with a high-speed profiling unit and developed a spectral autofluorescence correction method. The correction method is based on a simple algebraic operation using the fluorescence data from two detection channels and is applied on-the-fly in order to reliably identify hit beads by COPAS bead sorting. Our method provides a practical tool for the fast and efficient isolation of hit beads from one-bead one-compound library screens using either fluorescently labeled target proteins or biotinylated target proteins. This method makes hit bead identification easier and more reliable. It reduces false positives and eliminates the need for time-consuming pre-sorting of library beads in order to remove autofluorescent beads. (technical note)

  19. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  1. Uroporphyrinogen-III synthase: Molecular cloning, nucleotide sequence, expression of a mouse full-length cDNA, and its localization on mouse chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Xu, W.; Desnick, R.J. [Mount Sinai School of Medicine, New York, NY (United States); Kozak, C.A. [National Institute of Health, Bethesda, MD (United States)

    1995-04-10

    Uroporphyrinogen-III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for the conversion of hydroxymethylbilane to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-S is the enzymatic defect in congenital erythropoietic porphyria (CEP), an autosomal recessive disorder. For the generation of a mouse model of CEP, the human URO-S cDNA was used to screen 2 X 10{sup 6} recombinants from a mouse adult liver cDNA library. Ten positive clones were isolated, and dideoxy sequencing of the entire 1.6-kb insert of clone pmUROS-1 revealed 5{prime} and 3{prime} untranslated sequences of 144 and 623 bp, respectively, and an open reading frame of 798 bp encoding a 265-amino-acid polypeptide with a predicted molecular mass of 28,501 Da. The mouse and human coding sequences had 80.5 and 77.8% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active monomeric enzyme in Escherichia coli. In addition, the analysis of two multilocus genetic crosses localized the mouse gene on chromosome 7, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 10. The isolation, expression, and chromosomal mapping of this full-length cDNA should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of CEP for characterization of the disease pathogenesis and evaluation of gene therapy. 38 refs., 1 tab.

  2. Brain tubulin and actin cDNA sequences: isolation of recombinant plasmids.

    OpenAIRE

    Ginzburg, I.(Sobolev Institute of Mathematics and Novosibirsk State University, 630090, Novosibirsk, Russia); de Baetselier, A; Walker, M D; Behar, L; Lehrach, H; Frischauf, A M; Littauer, U Z

    1980-01-01

    Rat brain mRNA enriched for tubulin and actin sequences was used to prepare double stranded cDNA. A library of recombinant clones was constructed by inserting the dsDNA into the Pst1 site of pBR322 plasmid and transformation of E. coli chi 1776 host. Clones bearing sequences coding for tubulin and actin were identified and characterized.

  3. 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...

  4. Cypriot libraries

    OpenAIRE

    John F. Harvey

    1982-01-01

    Describes the current state of librarianship and bibliography in Cyprus, with separate sections for the Greek and Turkish sectors. Although there is no national library in the Greek sector there are 5 types of public library: Nicosia public library; Limassol, Larnaca and Paphos public libraries; community libraries; mobile libraries; and foreign cultural centre libraries. Schools and colleges in the Greek centre are well provided with libraries and most government departments sponsor special ...

  5. Cloning of cDNA and expression analysis of a DnaJ-like gene under heavy metal stress in bean

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A clone of PvSR6 encoding a new member of the DnaJ-like protein family was isolated from a mer curic-chloride-treated bean ( Phaseolus vulgaris L. )cDNA library by differential screening using cDNAs derived from treated and untreated plants. The predicted protein contains the highly conserved J domain only, which is present in all DnaJ-like proteins and is considered to play a critical role in DnaJ protein-protein interactions. PvSR6 gene is constitu tively expressed in roots but weakly expressed in stems and leaf tissue. Northern blot analysis revealed the transcripts of PvSR6 were at low levels in unstressed bean leaves, but the genes expression was strongly stimulated by heavy metals. These suggest that the PvSR6 might play an important role in resistance to the damage caused by heavy metals.

  6. Expression cloning of cDNA encoding a seven-helix receptor from human placenta with affinity for opioid ligands

    OpenAIRE

    1992-01-01

    Here we report the expression cloning of cDNA encoding a putative opioid receptor from a human placenta cDNA library. Placental opioid receptors are of the kappa type. As the dynorphin opioid peptides are kappa-selective, a dynorphin ligand was used in an affinity-enrichment (panning) procedure to select transiently transfected COS-7 cells expressing kappa receptor binding sites. The cloned cDNA encodes a 440-residue protein of the seven-helix guanine nucleotide-binding protein (G-protein)-co...

  7. 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.

  8. Identification of two linear B-cell epitopes from West Nile virus NS1 by screening a phage-displayed random peptide library

    Directory of Open Access Journals (Sweden)

    Qin Yong-Li

    2011-07-01

    Full Text Available Abstract Background The West Nile virus (WNV nonstructural protein 1 (NS1 is an important antigenic protein that elicits protective antibody responses in animals and can be used for the serological diagnosis of WNV infection. Although previous work has demonstrated the vital role of WNV NS1-specific antibody responses, the specific epitopes in the NS1 have not been identified. Results The present study describes the identification of two linear B-cell epitopes in WNV NS1 through screening a phage-displayed random 12-mer peptide library with two monoclonal antibodies (mAbs 3C7 and 4D1 that directed against the NS1. The mAbs 3C7 and 4D1 recognized phages displaying peptides with the consensus motifs LTATTEK and VVDGPETKEC, respectively. Exact sequences of both motifs were found in the NS1 (895LTATTEK901 and 925VVDGPETKEC934. Further identification of the displayed B cell epitopes were conducted using a set of truncated peptides expressed as MBP fusion proteins. The data indicated that 896TATTEK901 and925VVDGPETKEC934 are minimal determinants of the linear B cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. Antibodies present in the serum of WNV-positive horses recognized the minimal linear epitopes in Western blot analysis, indicating that the two peptides are antigenic in horses during infection. Furthermore, we found that the epitope recognized by 3C7 is conserved only among WNV strains, whereas the epitope recognized by 4D1 is a common motif shared among WNV and other members of Japanese encephalitis virus (JEV serocomplex. Conclusions We identified TATTEK and VVDGPETKEC as NS1-specific linear B-cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against WNV and other viruses of JEV serocomplex.

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

    Directory of Open Access Journals (Sweden)

    Regenbogen Johannes

    2002-03-01

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

  10. Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization.

    OpenAIRE

    Foster, D C; Sprecher, C A; Grant, F J; Kramer, J M; Kuijper, J L; Holly, R D; Whitmore, T E; Heipel, M D; Bell, L A; Ching, A F

    1994-01-01

    Thrombopoietin (TPO), a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells, is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe, we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure si...

  11. Analysis of Expressed Sequence Tags from Skeletal Muscle specific cDNA Library of Chinese Native Xiang Pig%中国地方品种香猪的肌肉特异组织表达序列标签(ESTs)的

    Institute of Scientific and Technical Information of China (English)

    王秀利; 吴克亮; 李宁; 李长绿; 仇雪梅; 王爱华; 吴常信

    2006-01-01

    通过构建香猪肌肉组织cDNA文库,并在文库中随机挑选克隆进行测序的方法,获得了131个香猪肌肉EST序列.在这131个EST序列所代表的109个单一克隆中,有99个为人类及其他物种的同源序列,3个为已知的猪的ESTs,7个为未知ESTs.对这10个已知、未知ESTs进行开放阅读框预测并进行B1ast分析,没有找到高度同源的氨基酸序列.对上述EST所对应的基因功能分析结果表明,除去27.27%的EST未能分类外,克隆到的EST大多来自与基因/蛋白的表达调控相关的基因(占45.46%).来自具有其他功能的基因的EST依次是细胞代谢占10.10%、细胞结构/迁移占10.10%、细胞/机体防御占5.05%和细胞信号/传导占2.02%.没有发现和细胞分裂相关的已知功能基因.本研究结果为中国地方品种香猪提供了第一个骨骼肌的基因表达谱,为今后寻找猪肌肉生长和肉用品质的候选基因奠定了基础.%A Longissimus Dorsi muscle cDNA library of Xiang Pig was constructed, and 131 randomly isolated clones were sequenced in this study. The results of bioinformatics analysis showed that 131 ESTs represented 109 unique clones sequences, of which 99 showed homology to previously identified genes in humans or other mammals, 3 matched other uncharacterized expressed sequence tags (ESTs), and 7 showed no significant matches to sequences already present in DNA databases. No protein matches were found for 10 ESTs. Functional analysis of the ESTs showed that a considerable proportion of them encoded proteins involved in gene/protein expression (45.46%). Other classes included genes involved in metabolism (10.10%), cell structure/motility (10.10%), cell/organism defense (5.05%), cell signaling/communication (2.02%), and cell division (0.0%).Unclassified genes constituted the remaining 27.27%. This study reported the results of the first gene expression profile analysis of Chinese native Xiang Pig skeletal muscle cells, thereby greatly

  12. 陆地棉开花后20d纤维抑制性消减文库的构建及分析%Construction and Analysis of SSH cDNA Library of Fiber in 20 Days Post Anthesis of Upland Cotton

    Institute of Scientific and Technical Information of China (English)

    王少干; 王涛; 袁有禄; 商海红; 计志斌; 闫恒超; 李俊文; 刘爱英; 石玉真; 龚举武; 巩万奎

    2012-01-01

    本研究以高比强度纤维材料0-153和转基因抗虫棉sGK9708为亲本构建的高代重组自交系群体(F6∶9)中选育出的高比强度纤维品系(69307)作为材料,利用抑制性消减杂交技术,以15 DPA纤维为driver、20 DPA纤维为tester,成功构建出陆地棉开花后20 d纤维的cDNA消减文库.通过蓝白斑筛选、菌落PCR及反向Northern技术最终筛选出差异表达的阳性克隆340个.通过对阳性克隆测序及序列分析,共得到1 15个单一序列,其中35个重叠群,80个单拷贝.利用Blast2GO等对差异表达基因进行生物信息学分析,结果表明这些差异表达基因广泛参与糖类、脂类、氨基酸等物质的代谢,以及纤维素生物合成、细胞壁合成与修饰、氧化还原、细胞信号转导等生物学过程.%In this study, one of excellent recombinant inbred line (69307) with high fiber strength was chosen as material from an F6:9 generation of upland cotton (Gossypium Hirsutum L.) derived from the combination between sGK9708 and 0-153. sGk9708 is a commercial transgenic cultivar with resistant to budworm, and 0-153 is a line with high fiber strength. An SSH cDNA library of fiber in 20 days post-anthesis of upland cotton had been successfully constructed via the approach of Suppressive Subtractive Hybridization (SSH), in which the mRNAs isolated from the fibers in 15 days post anthesis (dpa) were used as driver, and the mRNAs from the fibers in 20 days post anthesis (dpa) as tester. Finally, the 340 positive clones expressed differentially had been chosen by Blue-white bolting, colony PCR and Reverse Northern Dot-Blot. 115 unigenes were obtained based on sequencing of the positive clones and analysis of the sequences, which included 35 contigs and 80 singlets. Bioinformatic analysis with Blast2GO software revealed that these differentially expressed genes extensively involved in metabolism of carbohydrate, lipid, amino acid and other substances, and in biological process

  13. Screening and identification of proteins interacting with nucleostemin

    Institute of Scientific and Technical Information of China (English)

    Hai-Xia Yang; Geng-Lin Jin; Ling Meng; Jian-Zhi Zhang; Wen-Bin Liu; Cheng-Chao Shou

    2005-01-01

    AIM: To identify the proteins interacting with nucleostemin (NS), thereby gaining an insight into the function of NS.METHODS: Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and β-galactosidase filter assay were subsequently conducted to check the positive clones and the gene was identified by DNA sequencing.To further confirm the interaction of two proteins, the DNA fragment coding NS and the DNA fragment isolated from the positive clone were inserted into the mammalian expression vector pcDNA3 and pcDNA3-myc, respectively.Then, two plasmids were cotransfected into the COS-7 cells by DEAE-dextron. The total protein from the cotransfected cells was extracted and coimmunoprecipitation and Western blot were performed with suitable antibodies sequentially.RESULTS: Two positive clones that interacted with NS were obtained from human placenta cDNA library. One was an alpha isoform of human protein phosphatase 2 regulatory subunit B (B56) (PPP2R5A) and the other was a novel gene being highly homologous to the gene associated with spondylo paralysis. The co-immunoprecipitation also showed that NS specifically interacted with PPP2R5A.CONCLUSION: NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.

  14. Developing New Tools for the in vivo Generation/Screening of Cyclic Peptide Libraries. A New Combinatorial Approach for the Detection of Bacterial Toxin Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2006-11-28

    A new combinatorial approach for the biosynthesis and screening of small drug-like toxin inhibitors inside living cells is presented. This approach has been initially used as proof of principle for finding inhibitors against the LF factor from Bacillus anthracis. Key to our ''living combinatorial'' approach is the use of a living cell as a micro-chemical factory for both synthesis and screening of potential inhibitors for a given molecular recognition event (see Scheme 1). This powerful technique posses the advantage that both processes synthesis and screening happen inside the cell thus accelerating the whole screening/selection process.

  15. Molecular characterization of a cDNA encoding Cu/Zn superoxide dismutase from Deschampsia antarctica and its expression regulated by cold and UV stresses

    Directory of Open Access Journals (Sweden)

    Gidekel Manuel

    2009-09-01

    Full Text Available Abstract Background The Copper/Zinc superoxide dismutase (Cu/ZnSOD gene, SOD gene, was isolated from a Deschampsia antarctica Desv. by cDNA library screening. The expression of SOD gene in the leaves of D. antarctica was determined by RT-PCR and its differential expression of gene transcripts in conditions of cold and UV radiation stresses was revealed by northern blot. Findings The molecular characterization shows that SOD cDNA is 709 bp in length, which translates an ORF of 152 amino acids that correspond to a protein of predicted molecular mass of 15 kDa. The assay shows that the expression of SOD gene increases when D. antarctica is acclimatised to 4°C and exposed to UV radiation. These results indicate that the SOD gene of D. antarctica is involved in the antioxidative process triggered by oxidative stress induced by the conditions of environmental change in which they live. Conclusion The present results allow us to know the characteristics of Cu/ZnSOD gene from D. antarctica and understand that its expression is regulated by cold and UV radiation.

  16. Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.

    Directory of Open Access Journals (Sweden)

    Hideki Hayashi

    Full Text Available Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026;We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy.Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.

  17. Bioactivity Screening of Fermentation Crude Extracts from Deep Sea Metagenomic Library Clones%深海宏基因组文库克隆子发酵产物的生物活性筛选

    Institute of Scientific and Technical Information of China (English)

    汤熙翔; 易志伟; 李宁; 马群; 李慧; 秦丹; 肖湘

    2011-01-01

    目的:对前期构建的深海宏基因组文库克隆子发酵产物进行生物活性筛选.方法:利用CCK8法及琼脂扩散法和双层琼脂法进行了细胞毒活性及抗菌活性筛选,利用荧光定量PCR法进行了抗乙肝病毒活性筛选.结果:筛选发现3个具有细胞毒活性的混合克隆子.HPLC指纹图谱分析表明这3个混合克隆子具有与大肠杆菌宿主对照不同的指纹图谱,其活性与出现的指纹峰成分可能相关.结论:研究结果提示对深海宏基因组文库克隆子发酵产物进行化学筛选为常规的生物活性功能筛选之外的有意义途径.%Objective: Detect bioactivities of fermentation crude extracts originated from previously constructed deep sea metagenomic libraries. Methods : Functional screening of antitumor( cytotoxic ) ,antibacterial and anti HBV activity from fermentation crude extracts was performed by CCK-8 method, agar diffusion method,double agar method and real time PCR method. Results: No antibacterial and anti HBV clones were identified and three mixed clones showed good cytotoxic activity. HPLC analysis of these three mixed clones fingerprints indicated they possess obviously different fingerprints with E. coli host which could be useful to next isolation of cytotoxic compounds from deep sea metagenomic library. Conclusion: These results indicated that chemical screening could be potential way for screening bioactivity substance from deep sea metagenomic libraries besides functional screening and homology screening.

  18. cDNA: 27908 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available H. sapiens - Hs.406397 Homo sapiens cDNA FLJ45472 fis, clone BRSTN2016918, highly similar to Gli ... al fibrillary acidic protein, astrocyte ... gnl|UG|Hs#S16883914 AK128790 17/4578_27908.png ...

  19. cDNA: 55929 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.244143 Mus musculus adult male small intestine cDNA, RIKEN full-length enriched ... rary, clone:2010001P08 product:hypothetical Serine proteases , trypsin family containing protein, full insert se ...

  20. cDNA: 43675 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.333219 Mus musculus 8 days embryo whole body cDNA, RIKEN full-length enriched l ... 0 product:hypothetical Eukaryotic thiol (cysteine) proteases ... active site containing protein, full insert sequen ...

  1. cDNA: 53523 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.277582 Mus musculus 16 days neonate cerebellum cDNA, RIKEN full-length enriched ... rary, clone:9630053E09 product:hypothetical Serine proteases , subtilase family/Leucine-rich repeat containing p ...

  2. cDNA: 42035 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.274255 Mus musculus adult male urinary bladder cDNA, RIKEN full-length enriched ... rary, clone:9530081K03 product:hypothetical Serine proteases , trypsin family containing protein, full insert se ...

  3. cDNA: 43679 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.333219 Mus musculus 10 days neonate cerebellum cDNA, RIKEN full-length enriched ... 0 product:hypothetical Eukaryotic thiol (cysteine) proteases ... active site containing protein, full insert sequen ...

  4. cDNA: 44744 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.29756 Mus musculus 0 day neonate thymus cDNA, RIKEN full-length enriched librar ... 1 product:hypothetical Eukaryotic thiol (cysteine) proteases ... active site containing protein, full insert sequen ...

  5. cDNA: 35986 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.122430 Mus musculus adult male corpora quadrigemina cDNA, RIKEN full-length enr ... 0341B09 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  6. 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 ...

  7. Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement.

    Science.gov (United States)

    Weis, J J; Fearon, D T; Klickstein, L B; Wong, W W; Richards, S A; de Bruyn Kops, A; Smith, J A; Weis, J H

    1986-08-01

    Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low-stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequences, two clones were identified that hybridized at low, but not at high, stringency. Redundant oligonucleotides specific for CR2 sequences were synthesized and used to establish that the two cDNA clones weakly hybridizing with the CR1 cDNA contained CR2 sequences. One of these CR2 cDNA clones hybridized to oligonucleotides derived from two distinct CR2 tryptic peptides, whereas the other, smaller cDNA clone hybridized to oligonucleotides derived from only one of the CR2 peptides. Nucleotide sequence analysis of the CR2 cDNA confirmed that the site of oligonucleotide hybridization was identical to that predicted from the peptide sequence, including flanking sequences not included within the oligonucleotide probes. The CR2-specific cDNA sequences identified a poly(A)+ RNA species of 5 kilobases in RNA extracted from human B cells but did not hybridize to any RNA obtained from the CR2-negative T-cell line HSB-2, thus confirming the appropriate size and tissue-specific distribution for the CR2 mRNA. The striking peptide sequence homology between CR2 and CR1 and the cross-hybridization of the CR2 cDNA with the CR1-specific sequences allow the placement of CR2 in a recently defined gene family of C3- and C4-binding proteins consisting of CR1, C4-binding protein, factor H, and now, CR2. PMID:3016712

  8. cDNA and deduced amino acid sequence of human pulmonary surfactant-associated proteolipid SPL(Phe)

    International Nuclear Information System (INIS)

    Hydrophobic surfactant-associated protein of M/sub r/ 6000-14,000 was isolated from either/ethanol or chloroform/methanol extracts of mammalian pulmonary surfactant. Automated Edman degradation in a gas-phase sequencer showed the major N-terminus of the human low molecular weight protein to be Phe-Pro-Ile-Pro-Leu-Pro-Try-Cys-Trp-Leu-Cys-Arg-Ala-Leu-. Because of the N-terminal phenylalanine, the surfactant protein was designated SPL(Phe). Antiserum generated against hydrophobic surfactant protein(s) from bovine pulmonary surfactant recognized protein of M/sub r/ 6000-14,000 in immunoblot analysis and was used to screen a λgt11 expression library constructed from adult human lung poly(A)+ RNA. This resulted in identification of a 1.4-kilobase cDNA clone that was shown to encode the N-terminus of the surfactant polypeptide SPL(Phe) (Phe-Pro-Ile-Pro-Leu-Pro-) within an open reading frame for a larger protein. Expression of a fused β-galactosidase-SPL (Phe) gene in Escherichia coli yielded an immunoreactive M/sub r/ 34,000 fusion peptide. Hybrid-arrested translation with the cDNA and immunoprecipitation of [35S]methionine-labeled in vitro translation products of human poly(A)+ RNA with a surfactant polyclonal antibody resulted in identification of a M/sub r/ 40,000 precursor protein. Blot hybridization analysis of electrophoretically fractionated RNA from human lung detected a 2.0-kilobase RNA that was more abundant in adult lung than in fetal lung. These proteins, and specifically SPL(Phe), may therefore be useful for synthesis of replacement surfactants for treatment of hyaline membrane disease in newborn infants or of other surfactant-deficient states

  9. cDNA and deduced amino acid sequence of human pulmonary surfactant-associated proteolipid SPL(Phe)

    Energy Technology Data Exchange (ETDEWEB)

    Glasser, S.W.; Korfhagen, T.R.; Weaver, T.; Pilot-Matias, T.; Fox, J.L.; Whitsett, J.A.

    1987-06-01

    Hydrophobic surfactant-associated protein of M/sub r/ 6000-14,000 was isolated from either/ethanol or chloroform/methanol extracts of mammalian pulmonary surfactant. Automated Edman degradation in a gas-phase sequencer showed the major N-terminus of the human low molecular weight protein to be Phe-Pro-Ile-Pro-Leu-Pro-Try-Cys-Trp-Leu-Cys-Arg-Ala-Leu-. Because of the N-terminal phenylalanine, the surfactant protein was designated SPL(Phe). Antiserum generated against hydrophobic surfactant protein(s) from bovine pulmonary surfactant recognized protein of M/sub r/ 6000-14,000 in immunoblot analysis and was used to screen a lambdagt11 expression library constructed from adult human lung poly(A)/sup +/ RNA. This resulted in identification of a 1.4-kilobase cDNA clone that was shown to encode the N-terminus of the surfactant polypeptide SPL(Phe) (Phe-Pro-Ile-Pro-Leu-Pro-) within an open reading frame for a larger protein. Expression of a fused ..beta..-galactosidase-SPL (Phe) gene in Escherichia coli yielded an immunoreactive M/sub r/ 34,000 fusion peptide. Hybrid-arrested translation with the cDNA and immunoprecipitation of (/sup 35/S)methionine-labeled in vitro translation products of human poly(A)/sup +/ RNA with a surfactant polyclonal antibody resulted in identification of a M/sub r/ 40,000 precursor protein. Blot hybridization analysis of electrophoretically fractionated RNA from human lung detected a 2.0-kilobase RNA that was more abundant in adult lung than in fetal lung. These proteins, and specifically SPL(Phe), may therefore be useful for synthesis of replacement surfactants for treatment of hyaline membrane disease in newborn infants or of other surfactant-deficient states.

  10. Structural of the class II enzyme of human liver alcohol dehydrogenase: combined cDNA and protein sequence determination of the π subunit

    International Nuclear Information System (INIS)

    The class II enzyme of human liver alcohol dehydrogenase was isolated, carboxymethylated, and cleaved with CNBr and proteolytic enzymes. Sequence analysis of peptides established structures corresponding to the π subunit. Two segments from the C-terminal region unique to π were selected for synthesis of oligodeoxyribonucleotide probes to screen a human liver cDNA library constructed in plasmid pT4. Sequence analysis of two identical hybridization-positive clones with cDNA inserts of about 2000 nucleotides gave the entire coding region of the π subunit, a 61-nucleotide 5' noncoding region and a 741-nucleotide 3' noncoding region containing four possible polyadenylation sites. Translation of the coding region yields a 391-residue polypeptide, which in all regions except the C-terminal segment corresponds to the protein structure as determined directly by peptide analysis. With the class I numbering system, the exception concerns a residue exchange at position 368, the actual C-terminus which is Phe-374 by peptide data but a 12 residue extension by cDNA data, and possibly two further residue exchanges at positions 303 and 312. The size difference might indicate the existence of posttranslational modifications of the mature protein or, in combination with the residue exchanges, the existence of polymorphism at the locus for class II subunits. The π subunit analyzed directly results in a 379-residue polypeptide and is the only class II size thus far known to occur in the mature protein. Comparison of the π structure with those of the class I subunits (α, β, and γ) reveals a homology with extensive differences. Large variations in segments affecting relationships at the active site and the area of subunit interactions account for the significant alterations of enzymatic specificities and other properties that differentiate class II from class I enzymes

  11. Automation of cDNA Synthesis and Labelling Improves Reproducibility

    Directory of Open Access Journals (Sweden)

    Daniel Klevebring

    2009-01-01

    Full Text Available Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

  12. 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.

  13. Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

    DEFF Research Database (Denmark)

    Becker, F.; Schnorr, K.; Wilting, R.; Tolstrup, N.; Bendtsen, Jannick Dyrløv; Olsen, P.B.

    2004-01-01

    To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage MU...

  14. 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.

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

    International Nuclear Information System (INIS)

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

  16. Induction of pigmentation in mouse fibroblasts by expression of human tyrosinase cDNA

    OpenAIRE

    1989-01-01

    A distinguishing characteristic of cells of the melanocyte lineage is the expression of the melanosomal enzyme tyrosinase that catalyzes the synthesis of the pigment melanin. A tyrosinase cDNA clone, designated BBTY-1, was isolated from a library constructed from the pigmented TA99+/CF21+ melanoma cell line SK-MEL-19. Expression of BBTY-1 in mouse L929 fibroblasts led to synthesis and expression of active tyrosinase, and, unexpectedly, to stable production of melanin. Melanin was synthesized ...

  17. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system

    International Nuclear Information System (INIS)

    CD28 (Tp44) is a human T-cell-specific homodimer surface protein that may participate in T-cell activation. The authors have isolated a cDNA clone encoding CD28 by a simple and highly efficient cloning strategy based on transient expression in COS cells. Central to this strategy is the use of an efficient method to prepare large plasmid cDNA libraries. The libraries are introduced into COS cells, where transient expression of surface antigen allows the isolation of cDNAs by way of monoclonal antibody binding. The CD28 cDNA encodes a highly glycosylated membrane protein with homology to the immunoglobulin superfamily and directs the production of a homodimer in transfected COS cells

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

    Science.gov (United States)

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

    2013-03-01

    The Urodacidae scorpions are the most widely distributed of the four families in Australia and represent half of the species in the continent, yet their venoms remain largely unstudied. This communication reports the first results of a proteome analysis of the venom of the scorpion Urodacus yaschenkoi performed by mass fingerprinting, after high performance liquid chromatography (HPLC) separation. A total of 74 fractions were obtained by HPLC separation allowing the identification of approximately 274 different molecular masses with molecular weights varying from 287 to 43,437 Da. The most abundant peptides were those from 1 K Da and 4-5 K Da representing antimicrobial peptides and putative potassium channel toxins, respectively. Three such peptides were chemically synthesized and tested against Gram-positive and Gram-negative bacteria showing minimum inhibitory concentration in the low micromolar range, but with moderate hemolytic activity. It also reports a transcriptome analysis of the venom glands of the same scorpion species, undertaken by constructing a cDNA library and conducting random sequencing screening of the transcripts. From the resultant cDNA library 172 expressed sequence tags (ESTs) were analyzed. These transcripts were further clustered into 120 unique sequences (23 contigs and 97 singlets). The identified putative proteins can be assorted in several groups, such as those implicated in common cellular processes, putative neurotoxins and antimicrobial peptides. The scorpion U. yaschenkoi is not known to be dangerous to humans and its venom contains peptides similar to those of Opisthacanthus cayaporum (antibacterial), Scorpio maurus palmatus (maurocalcin), Opistophthalmus carinatus (opistoporines) and Hadrurus gerstchi (scorpine-like molecules), amongst others. PMID:23182832

  19. Binding proteins for the regulatory subunit (RII-B) of brain cAMP-dependent protein kinase II: isolation and initial characterization of cDNA clones

    International Nuclear Information System (INIS)

    In mammalian brain several proteins bind RII-B with high affinity. An example is P75, which co-purifies with RII-B and also complexes Ca2+-calmodulin. Thus, RII-B binding proteins (RBPs) might play a role in integrating the Ca2+ and cAMP signalling pathways in the CNS. In order to study the structure and function of these polypeptides they have isolated cloned cDNAs for RBPs by screening brain λgt11 expression libraries using a functional assay: the binding of 32P-labeled RII to fusion proteins produced by recombinants expressing RII binding domains. Inserts from rat brain recombinant clones λ7B and λ10B both hybridize to a brain mRNA of 7000 nucleotides. Northern gel analyses indicate that the putative RBP mRNA is also expressed in lung, but not in several other tissues. The λ7B insert was subcloned into the expression plasmid pINIA. A 50 kDa high affinity RII-B binding polypeptide accumulated in E. coli transformed with pINIA-7B. Two RBP cDNAs (λ77, λ100A) have been retrieved from a bovine λgt 11 library using a monoclonal antibody directed against P75 and the binding assay respectively. On Southern blots the insert from λ100A hybridizes to the cDNA insert from clones λ77, suggesting that λ 77 cDNA might contain sequences coding for both an RII binding domain and a P75 epitope. The bovine λ100A insert also hybridizes with the rat λ7B clone indicating that an RII binding domain is conserved in the two species

  20. The characterization of cDNA clones coding for wheat storage proteins.

    OpenAIRE

    Bartels, D.; Thompson, R D

    1983-01-01

    Poly(A)+ RNA isolated from the developing wheat endosperm var. Chinese Spring, has been used as template for the construction of a cDNA library. Within the library, clones have been identified by in vitro translation of hybrid-selected mRNA which encode alpha/beta gliadin related sequences and gamma-gliadin related sequences. The DNA sequence of one such clone has been determined and it shows homology with that of a clone encoding a barley storage protein, B-hordein. The sequence includes a t...

  1. PIPERIDINE OLIGOMERS AND COMBINATORIAL LIBRARIES THEREOF

    DEFF Research Database (Denmark)

    1999-01-01

    The present invention relates to piperidine oligomers, methods for the preparation of piperidine oligomers and compound libraries thereof, and the use of piperidine oligomers as drug substances. The present invention also relates to the use of combinatorial libraries of piperidine oligomers for...... libraries (arrays) of compounds especially suitable for screening purposes....

  2. Paramyosin from the parasitic mite Sarcoptes scabiei: cDNA cloning and heterologous expression.

    Science.gov (United States)

    Mattsson, J G; Ljunggren, E L; Bergström, K

    2001-05-01

    The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies. PMID:11393829

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

    International Nuclear Information System (INIS)

    Human mammary epithelial cells secrete and express on their cell surfaces complex mucin glycoproteins that are developmentally regulated, tumor-associated, and highly immunogenic. Studies using monoclonal antibodies directed to these glycoproteins suggest that their molecular structures can vary with differentiation stages in the normal gland and in malignancy. To analyze the molecular nature of these glycoproteins, milk mucin was affinity-purifed and deglycosylated with hydrogen fluoride, yielding bands at 68 and 72 kDa on silver-stained gels. Polyclonal and monoclonal antibodies to the stripped core protein were developed and used to screen a λgt11 expression library of cDNA made from mRNA of the mammary tumor cell line MCF-7. Seven crossreacting clones were isolated, with inserts 0.1-1.8 kilobases long. RNA blot analysis, using as a probe the 1.8-kilobase insert subcloned in plasmid pUC8 (pMUC10), revealed transcripts of 4.7 and 6.4 kilobases in MCF-7 and T47D mammary tumor cells, whereas normal mammary epithelial cells from pooled milks have additional transcripts. The expression of mRNA correlates with antigen expression as determined by binding of two previously characterized anti-mucin monoclonal antibodies (HMFG-1 and HMFG-2) to seven cell lines. Restriction enzyme analysis detected a restriction fragment length polymorphism when human genomic DNA was digested with EcoRI or HinfI

  4. Generation of EST and cDNA Microarray Resources for the Study of Bovine Immunobiology*

    Directory of Open Access Journals (Sweden)

    Coussens PM

    2003-03-01

    Full Text Available Recent developments in expressed sequence tag (EST and cDNA microarray technology have had a dramatic impact on the ability of scientists to study the responses of thousands of genes to external stimuli, such as infection, nutrient flux, and stress. To date however, these studies have largely been limited to human and rodent systems. Despite the tremendous potential benefit of EST and cDNA microarray technology to studies of complex problems in domestic animal species, a lack of integrated resources has precluded application of these technologies to domestic species. To address this problem, the Center for Animal Functional Genomics (CAFG at Michigan State University has developed a normalized bovine total leukocyte (BOTL cDNA library, generated EST clones from this library, and printed cDNA microarrays suitable for studying bovine immunobiology. Our data revealed that the normalization procedure successfully reduced highly abundant cDNA species while enhancing the relative percentage of clones representing rare transcripts. To date, a total of 932 EST sequences have been generated from this library (BOTL and the sequence information plus BLAST results made available through a web-accessible database http://gowhite.ans.msu.edu. Cluster analysis of the data indicates that a total of 842 unique cDNAs are present in this collection, reflecting a low redundancy rate of 9.7%. For creation of first generation cDNA microarrays, inserts from 720 unique clones in this library were amplified and microarrays were produced by spotting each insert or amplicon 3 times on glass slides in a 48-patch arrangement with 64 total spots (including blanks and positive controls per patch. To test our BOTL microarray, we compared gene expression patterns of concanavalin A stimulated and unstimulated peripheral blood mononuclear cells (PBMCs. In total, hybridization signals on over 90 amplicons showed upregulation (>3× in response to Con A stimulation, relative to

  5. 肠道微生物宏基因组文库构建及酯解酶基因筛选%Screening of lipolytic genes from the metagenomic library of human intestine microbes

    Institute of Scientific and Technical Information of China (English)

    段嘉; 程功; 胡永飞; 李晶; 律娜; 朱宝利

    2013-01-01

    利用宏基因组学方法,以人肠道微生物样品为原材料,构建了1个约30 000个克隆的fosmid文库.以三丁酸甘油酯为底物,通过功能筛选,获得1个酯解酶阳性克隆.对该阳性克隆构建亚克隆,挑选具有酯解酶活性的阳性亚克隆进行测序分析,最终获得1个肠道微生物来源的酯解酶基因(GenBank登录号:JQ972699).结果表明,文库克隆的平均插入片段约为40 kb,没有重复插入片段克隆.获得的酯解酶基因推演蛋白与Pyramidobacter piscolens W5455的patatin样磷脂酶同源性最高,氨基酸一致性为95%.生物信息学分析结果表明该基因可能通过Vd型分泌方式进行分泌并发挥功能.本研究是通过构建人肠道微生物宏基因组大片段文库并结合重组子功能筛选获得酯解酶的首次报道,可为食品工业提供新的酯解酶来源和筛选方法.%Metagenomic DNA was extracted from a human fecal sample and DNA fragments around 40 kb was isolated and purified. A fosmid library containing approximately 30 000 clones was created. Restriction analysis by EcoR I revealed a high diversity of the cloned DNA fragments and the estimated average insert size of the library was 40 kb. Tributyrin was used as substrate for activity-based screening of the library and one lipolytic enzyme positive fosmid clone was obtained. The lipolytic gene sequence was obtained after subcloning and sequencing, which showed high homology to the patatin-like phospholipase from Pyramidobacter piscolens W5455 with an amino acid identity of 95% . This is the first report of function-based metagenomics on screening of lipolytic genes from human gut microbiota. It also provides food industry with a new source of lipolytic genes and screening method.

  6. 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.

  7. Identification of novel pro-migratory, cancer-associated genes using quantitative, microscopy-based screening.

    Directory of Open Access Journals (Sweden)

    Suha Naffar-Abu-Amara

    Full Text Available BACKGROUND: Cell migration is a highly complex process, regulated by multiple genes, signaling pathways and external stimuli. To discover genes or pharmacological agents that can modulate the migratory activity of cells, screening strategies that enable the monitoring of diverse migratory parameters in a large number of samples are necessary. METHODOLOGY: In the present study, we describe the development of a quantitative, high-throughput cell migration assay, based on a modified phagokinetic tracks (PKT procedure, and apply it for identifying novel pro-migratory genes in a cancer-related gene library. In brief, cells are seeded on fibronectin-coated 96-well plates, covered with a monolayer of carboxylated latex beads. Motile cells clear the beads, located along their migratory paths, forming tracks that are visualized using an automated, transmitted-light screening microscope. The tracks are then segmented and characterized by multi-parametric, morphometric analysis, resolving a variety of morphological and kinetic features. CONCLUSIONS: In this screen we identified 4 novel genes derived from breast carcinoma related cDNA library, whose over-expression induces major alteration in the migration of the stationary MCF7 cells. This approach can serve for high throughput screening for novel ways to modulate cellular migration in pathological states such as tumor metastasis and invasion.

  8. cDNA: 13750 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available H. sapiens + Hs.183373 Homo sapiens full open reading frame cDNA clone RZPDo834C1012D for gene HIP ... IP-55, src homology 3 domain-containing protein HIP -55; complete cds, incl. stopcodon gnl|UG|Hs#S20347 ...

  9. cDNA: 46042 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.290868 Mus musculus 12 days embryo spinal ganglion cDNA, RIKEN full-length enri ... , clone:D130020P04 product:target of myb1 homolog (chicken ), full insert sequence gnl|UG|Mm#S10839663 AK05124 ...

  10. cDNA: 37673 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.315626 Mus musculus 2 days neonate thymus thymic cells cDNA, RIKEN full-length ... 24 product:MODULATOR OF ANTIGEN RECEPTOR SIGNALING MARS , full insert sequence gnl|UG|Mm#S10833741 AK088672 ...

  11. cDNA: 53525 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.277582 Mus musculus 10 days neonate cerebellum cDNA, RIKEN full-length enriched ... rary, clone:B930059G06 product:hypothetical Serine proteases , subtilase family/Leucine-rich repeat containing p ...

  12. cDNA: 53519 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.277582 Mus musculus 0 day neonate eyeball cDNA, RIKEN full-length enriched libr ... ary, clone:E130306I01 product:hypothetical Serine proteases , subtilase family/Leucine-rich repeat containing p ...

  13. cDNA: 57842 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.1441 Mus musculus adult male spinal cord cDNA, RIKEN full-length enriched libra ... ry, clone:A330093C04 product:hypothetical Serine proteases , trypsin family/Chymotrypsin serine protease famil ...

  14. cDNA: 35980 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.122430 Mus musculus 0 day neonate cerebellum cDNA, RIKEN full-length enriched l ... 0027A22 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  15. cDNA: 35899 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus + Mm.337238 Mus musculus 13 days embryo liver cDNA, RIKEN full-length enriched libra ... 0010F15 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  16. cDNA: 35982 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.122430 Mus musculus 14 days embryo thymus cDNA, RIKEN full-length enriched libr ... 0401J04 product:hypothetical Ubiquitin-conjugating enzymes ... containing protein, full insert sequence gnl|UG|Mm ...

  17. Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicans enolase, an abundant, immunodominant glycolytic enzyme.

    OpenAIRE

    Sundstrom, P; Aliaga, G R

    1992-01-01

    We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the ...

  18. Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA.

    OpenAIRE

    Ruggli, N; Tratschin, J D; Mittelholzer, C.; Hofmann, M A

    1996-01-01

    The complete nucleotide sequence of the genome of classical swine fever virus (CSFV) strain Alfort/187 was determined from three cDNA libraries constructed by cloning of DNA fragments obtained from independent sets of reverse transcription and PCR. The cDNA fragments were then assembled and inserted downstream of a T7 promoter in a P15A-derived plasmid vector to obtain the full-length cDNA clone pA187-1. The first nucleotide of the CSFV genome was positioned at the transcription start site of...

  19. Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

    International Nuclear Information System (INIS)

    Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A)+ RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A)+ RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A)+ RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding

  20. Simple Screening Method for Autoantigen Proteins Using the N-Terminal Biotinylated Protein Library Produced by Wheat Cell-Free Synthesis

    OpenAIRE

    Matsuoka, Kazuhiro; Komori, Hiroaki; Nose, Masato; Endo, Yaeta; Sawasaki, Tatsuya

    2010-01-01

    Autoimmune diseases are a heterogeneous group of diseases characterized by immune reactions against either a major or a limited number of the bodies own autoantigens, causing inflammation and damage to tissues and organs. Thus, identification of autoantigens is an important first step to understanding autoimmune diseases. Here we demonstrate a simple screening method for identification of autoantigens reacting with patient serum antibodies by combination of an N-terminal biotinylated protein ...