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Sample records for cdna library screening

  1. Screening of cDNA libraries on glass slide microarrays.

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    Berger, Dave K; Crampton, Bridget G; Hein, Ingo; Vos, Wiesner

    2007-01-01

    A quantitative screening method was developed to evaluate the quality of cDNA libraries constructed by suppression subtraction hybridization (SSH) or other enrichment techniques. The SSH technique was adapted to facilitate screening of the resultant library on a small number of glass slide microarrays. A simple data analysis pipeline named SSHscreen using "linear models for microarray data" (limma) functions in the R computing environment was developed to identify clones in the cDNA libraries that are significantly differentially expressed, and to determine if they were rare or abundant in the original treated sample. This approach facilitates the choice of clones from the cDNA library for further analysis, such as DNA sequencing, Northern blotting, RT-PCR, or detailed expression profiling using a custom cDNA microarray. Furthermore, this strategy is particularly useful for studies of nonmodel organisms for which there is little genome sequence information.

  2. 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.

  3. [cDNA libraries construction and screening in gene expression profiling of disease resistance in wheat].

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    Luo, Meng; Kong, Xiu-Ying; Liu, Yue; Zhou, Rong-Hua; Jia, Ji-Zeng

    2002-09-01

    A wheat line, Bai Nong 3217/Mardler BC5F4 with resistance to powdery mildew, was used to construct a conventional cDNA library and a suppression subtractive hybridization (SSH) cDNA library from wheat leaves inoculated by Erysiphe graminis DC. Three hundred and eighty-seven non-redundant ESTs from the conventional cDNA library and 760 ESTs from the SSH cDNA library were obtained, and the ESTs similarity analysis using BLASTn and BLASTx were conducted by comparing these ESTs with sequences in GenBank. The results showed that the redundancy of some kinds of genes such as photosynthesis related genes and ribosome related genes was higher in the conventional cDNA library but the varieties and quantities of disease resistance genes were less than those in the SSH cDNA library. The SSH cDNA library was found to have obvious advantages in gene expression profiling of disease resistance such as simple library construction procedure, rich specific DRR (disease-resistance-related) genes and decreased sequencing amount. To acquire genes that were involved in the powdery mildew resistance of wheat, hybridization with high-density dots membranes was used to screen the two libraries. The result showed that the method was relatively simple in operation, and the membranes could be used for many times. But some problems also existed with this screening method. For instance, a large amount of mRNA and radioactive isotope were needed and the hybridization procedure must be repeated several times to obtain stable hybridization results. About 54.1% function-known ESTs in the SSH cDNA library were identified to be DRR genes by screening. There were 247 clones of the SSH cDNA library that had positive signal in the repeated hybridizations with the pathogen uninfected probe. The identified DRR genes distributed in the whole procedure of powdery mildew resistance, but mainly focused on the SAR (systemic of acquired resistance).

  4. [Construction of cDNA expression library of unfed female Haemaphysalis longicornis and immuno-screening].

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    Chai, Hui-ping; Liu, Guang-yuan; Zhang, Lin; Gong, Zhen-li; Xie, Jun-ren; Tian, Zhan-cheng; Wang, Lu; Jia, Ning

    2009-02-28

    To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes. Total RNA was isolated from unfed female ticks, mRNA was purified and a library of oligo (dT) -primed cDNA with added directional EcoR I /Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I /Hind III arms of the lambda SCREEN vector. Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8. Recombinant plasmids that were subcloned to E. coli BM25.8 were isolated and transformed into E. coli JM109. Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing. The recombinant phage DNA was packaged by using phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.8 x 10(6) pfu. Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4 x 10(9) pfu/ml. Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library. Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H. longicornis tropomyosin mRNA, Rhipicephalus annulatus unknown larval protein mRNA, chromosome 2R of Drosophila melanogaster, mitochondrial DNA of H. flava, clones HqL09 unkown mRNA and Hq05 mRNA of H. qinghaiensis, and myosin alkali light chain protein mRNA. The cDNA expression library from unfed female H. longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.

  5. Design and Screening of M13 Phage Display cDNA Libraries

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    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.

  6. Screening a cDNA Library for Protein–Protein Interactions Directly in Planta[W

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    Lee, Lan-Ying; Wu, Fu-Hui; Hsu, Chen-Tran; Shen, Shu-Chen; Yeh, Hsuan-Yu; Liao, De-Chih; Fang, Mei-Jane; Liu, Nien-Tze; Yen, Yu-Chen; Dokládal, Ladislav; Sýkorová, Eva; Gelvin, Stanton B.; Lin, Choun-Sea

    2012-01-01

    Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein– or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ∼2 × 105 cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species. PMID:22623495

  7. Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.

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    Yang, XinChao; Li, MengHui; Liu, JianHua; Ji, YiHong; Li, XiangRui; Xu, LiXin; Yan, RuoFeng; Song, XiaoKai

    2017-02-16

    Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines. With the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed. Adopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 10 6 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 10 7 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite. Our

  8. High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis.

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    van den Berg, Noëlani; Crampton, Bridget G; Hein, Ingo; Birch, Paul R J; Berger, Dave K

    2004-11-01

    Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-color cyanin dye labeling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis.

  9. [Screening of specifically expressed genes in amphioxus neurula by construction of a subtractive cDNA library].

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    Zhang, Lei; Yang, Yong-Jie; Zhang, Yan-Jun

    2010-12-01

    To screen specifically expressed genes in the development of nerve, muscle, and body axis of amphioxus, Branchiostoma belcheri tsingtauenese. A subtractive cDNA library was constructed from the 12-hour amphioxus neurula cDNA after subtractively hybridized with the 6-hour amphioxus gastrula cDNA. The total RNA was extracted from the 12-hour neurula and 6-hour gastrula, then reverse transcribed into cDNA. The 12-hour neurula cDNA was designated as the experimental group (the tester) and the 6-hour gastrula cDNA as the control group (the driver). The differentially expressed sequences were exponentially amplified using suppression PCR. Background was subtracted and differentially expressed sequences were further enriched. The PCR products were ligated to the T Vector. After transformation of the recombinant plasmid carrying inserted amphioxus cDNA into E.coli host cells, the cDNA library was constructed successfully. Two hundred randomly chosen positive clones were sequenced and some of neurula-specifically expressed genes were obtained. SSH is an effective method for searching differentially expressed genes. The subtractive cDNA library we generated provides a tool for further study of regulatory mechanisms of amphioxus early embryonic development.

  10. 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.

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

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

    2006-02-28

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

  12. Normalized cDNA libraries

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    Soares, Marcelo B.; Efstratiadis, Argiris

    1997-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

  13. Novel transcripts of human cytomegalovirus clinical strain found by cDNA library screening.

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    Ma, Y P; Ruan, Q; Ji, Y H; Wang, N; Li, M L; Qi, Y; He, R; Sun, Z R; Ren, G W

    2011-04-05

    Human cytomegalovirus (HCMV) is a double-stranded DNA virus with the largest genome (~235 kb) of the known human herpes viruses. The coding potential and transcript structures of most HCMV predicted genes have not been identified. New or unknown genes could exist in clinical strains. The SMART (switching mechanism at 5' end of RNA template of reverse transcriptase) technique was used to construct a full-length cDNA library of an HCMV clinical strain in the late expression phase. Randomly selected clones were sequenced. The sequenced expressed sequence tags were used to identify the expression and transcript structures of some predicted and unpredicted genes of HCMV. The transcripts of the UL99, TRL5/IRL5, UL73 to UL75, UL4, and UL115 genes, which were previously detected, were obtained with full-length structures from this library. Some novel transcripts, including several transcripts of UL/b' genes and three antisense transcripts of UL83, UL87 and UL31 were found. The novel transcripts that were found, particularly the antisense transcripts of UL83, UL87 and UL31, showed that the transcription of HCMV genes is more complex than previously predicted. Our study highlights the usefulness of the full-length cDNA library for discovering new genes and transcripts of HCMV.

  14. Normalizing cDNA libraries.

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    Bogdanov, Ekaterina A; Shagina, Irina; Barsova, Ekaterina V; Kelmanson, Ilya; Shagin, Dmitry A; Lukyanov, Sergey A

    2010-04-01

    The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses. (c) 2010 by John Wiley & Sons, Inc.

  15. Construction of a full-length cDNA library from castor endosperm for high-throughput functional screening.

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    Lu, Chaofu; Wallis, James G; Browse, John

    2011-01-01

    It is desirable to produce high homogeneity of novel fatty acids in oilseeds through genetic engineering to meet increasing demands by the oleo-chemical industry. However, expression of key enzymes for biosynthesis of industrial fatty acids usually results in low levels of desired fatty acids in transgenic oilseeds. The abundance of unusual fatty acids in their natural species suggests that additional genes are needed for high production in transgenic plants. We used the model oilseed plant Arabidopsis thaliana expressing a castor fatty acid hydroxylase (FAH12) to identify genes that can boost hydroxy fatty acid accumulation in transgenic seeds. We described previously a high-throughput approach that in principle can allow testing of the entire transcriptome of developing castor seed endosperm by shotgun transforming a full-length cDNA library into a FAH12-expressing Arabidopsis line. The resulting transgenic seeds can be screened by high-throughput gas chromatography. The most critical step of the approach is the construction of a full-length cDNA library. In this chapter, we describe in detail the construction of the cloning vectors and a full-length cDNA library from developing castor seed endosperms. The approach we describe has broad applicability in many areas of biology.

  16. cDNA library preparation.

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    Kooiker, Maarten; Xue, Gang-Ping

    2014-01-01

    The construction of full-length cDNA libraries allows researchers to study gene expression and protein interactions and undertake gene discovery. Recent improvements allow the construction of high-quality cDNA libraries, with small amounts of mRNA. In parallel, these improvements allow for the incorporation of adapters into the cDNA, both at the 5' and 3' end of the cDNA. The 3' adapter is attached to the oligo-dT primer that is used by the reverse transcriptase, whereas the 5' adapter is incorporated by the template switching properties of the MMLV reverse transcriptase. This allows directional cloning and eliminates inefficient steps like adapter ligation, phosphorylation, and methylation. Another important step in the construction of high-quality cDNA libraries is the normalization. The difference in the levels of expression between genes might be several orders of magnitude. Therefore, it is essential that the cDNA library is normalized. With a recently discovered enzyme, duplex-specific nuclease, it is possible to normalize the cDNA library, based on the fact that more abundant molecules are more likely to reanneal after denaturation compared to rare molecules.

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

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    Mei Zhang

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

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

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

    2016-01-01

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

  19. [Construction and screening of the subtracted cDNA library of human large cell lung cancer lines with different metastatic potentials].

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    Liao, Li; Zhou, Qinghua; Chen, Jun; Zhu, Daxing; Ma, Li; Yan, Huiqin; Zhu, Wen; Liu, Hongyu

    2007-06-20

    Screening metastatic-related genes of lung cancer is helpful to understand the molecular mechanisms of lung cancer invasion and metastasis. In order to screen the differential expression genes related to metastasis of lung cancer, we constructed and preliminarily screened the subtracted cDNA libraries of human large cell lung cancer cell lines with different metastatic potentials in this study. Subtracted cDNA library was constructed in the different metastastic potential cell lines NL9980 and L9981 by suppression subtractive hybridization (SSH) method. The positive clones were preliminarily screened by blue-white colony based on the α-complementary principal, and precisely identified by PCR. The forward and reverse subtracted libraries were screened and identified by dot blot to obtain the clones corresponding to differential expression segments. The subtracted cDNA libraries were successfully constructed in the different metastastic potential cell lines NL9980 and L9981. Three hundred and seven positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained by the dot blot method. SSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse subtracted cDNA libraries of different metastastic potential cell lines are constructed by this method. The differential expression genes related to tumor metastasis might exist in the human large cell lung cancer cell lines with different metastasis potential.

  20. [Construction and preliminary screening of a forward-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state].

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    Fang, Ding-Zhi; Liu, Bing-Wen; Shen, Tao; Bai, Huai

    2005-11-01

    To construct and preliminarily screen the forward-subtracted cDNA library of differentially expressed genes in rat liver of prothrombotic state (PTS). The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization using cDNAs synthesized from mRNA of PTS rat as Tester and cDNAs from mRNA of control rat as Driver. The products from the last PCR amplification of suppression subtractive hybridization were inserted into a T/A plasmid vectors to transform the Escherichia coli JM109 cells. To produce the library, the transformed cells were incubated at 37 C overnight on a LB agar plate containing ampicillin (50 microg/ml), IPTG and X-gal. Forward-subtracted cDNA probes and reverse-subtracted cDNA probes were prepared by nested PCR amplification, which were labeled with HRP. Positive clones were selected by differential screening in which forward-subtracted and reverse-subtracted cDNA probes were separately hybridized with the membranes slot-blotted by plasmid DNAs amplified and isolated from the library. Inserts in the positive clones were submitted to DNA sequencing. Nucleic acid sequence homology search was performed against the GenBank DNA database (non-redundant, and non-mouse and non-human EST entries) using the Standard nucleotide-nucleotide BLAST [blastn] program via a network connection to the National Center for Biotechnology information. The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed. Two differentially expressed cDNA fragments were found after preliminary screening. The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed in the present study.

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

    Science.gov (United States)

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

    2015-04-01

    Though a large number of salt-tolerant genes were identified from Glycophyte in previous study, genes involved in salt-tolerance of halophyte were scarcely studied. In this report, an important halophyte turfgrass, Zoysia matrella, was used for systematic excavation of salt-tolerant genes using full-length cDNA expression library in yeast. Adopting the Gateway-compatible vector system, a high quality entry library was constructed, containing 3 × 10(6) clones with an average inserted fragments length of 1.64 kb representing a 100% full-length rate. The yeast expression library was screened in a salt-sensitive yeast mutant. The screening yielded dozens of salt-tolerant clones harboring 16 candidate salt-tolerant genes. Under salt-stress condition, these 16 genes exhibited different transcription levels. According to the results, we concluded that the salt-tolerance of Z. matrella might result from known genes involved in ion regulation, osmotic adjustment, as well as unknown pathway associated with protein folding and modification, RNA metabolism, and mitochondrial membrane translocase, etc. In addition, these results shall provide new insight for the future researches with respect to salt-tolerance. Crown Copyright © 2015. Published by Elsevier Masson SAS. All rights reserved.

  2. [Screening of drug resistent gene by cyclical packaging rescue of hepatocellular carcinoma retroviral cDNA libraries].

    Science.gov (United States)

    Dai, Wenyan; Zhu, Ruiyu; Jin, Jian

    2016-02-01

    Multidrug resistant genes are highly expressed in hepatocellular carcinoma that seriousty affects the effect of chemotherapy. Screening of resistant genes from HCC cells and studying its mechanism of drug resistance will be helpful to improve the effecacy of chemotherapy for hepatocellular carcinoma. Here we described an alternative method called cyclical packaging rescue (CPR). First we constructed a retrovirus cDNA library of hepatoma cells and used it to infect fibroblasts. Then we added drugs to screen survival cells. The survival cells, stably integrated helper-free retroviral libraries, were recovered rapidly after transfection with plasmids expressing retroviral gag-pol and env genes. Through this method, retroviral RNAs were directly repackaged into new infectious virions. Recovered retroviral supernatant was then used to reinfect fresh target cells. When performed in concert with selection using functional assays, cDNAs regulating functional responses could be identified by enrichment through multiple rounds of retroviral library recovery and retransmission. Using CPR, we obtained several cDNAs. After a preliminary detection, we found Ribosomal protein S11 (RPS11), Ribosomal protein L6 (RPL6), Ribosomal protein L11 (RPL11), Ribosomal protein L24 (RPL24) possibly had drug resistant function.

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

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    Hou, Q; Chen, K; Shan, Z

    2015-01-01

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

  4. An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences.

    Science.gov (United States)

    Pollier, Jacob; González-Guzmán, Miguel; Ardiles-Diaz, Wilson; Geelen, Danny; Goossens, Alain

    2011-01-01

    cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis. A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts. The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.

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

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Leffers, H

    1993-01-01

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

  6. [Construction of anti-sense cDNA library of human breast cancer cells during apoptosis induced by trichostatin A and preliminary screening of essential genes].

    Science.gov (United States)

    Ma, Xiao-Li; Wang, Bei-Bei; Wu, Peng; Lu, Yun-Ping; Zhou, Jian-Feng; Ma, Ding

    2009-02-24

    To construct an anti-sense cDNA library of human breast cancer cells to screen essential genes with anti-tumor effects on apoptosis of human breast cancer cells induced by trichostatin A. Poly (A)(+)RNA was extracted from human breast cancer cells of the line MCF-7 treated by trichostatin A for 0, 12, 24, 36, 48, 60, or 72 h. cDNA were synthesized and inserted reversely into PCEP 4 vector to construct an anti-sense cDNA library. HeLa cells were transfected with the library DNA or blank PCEP 4 vector as control group. All the transfected cells were screened by 200 nmol/L trichostatin A and 200 microg/ml hygromycin B. Screening was stopped when the control cells died. Then the surviving cell clones were amplified and Hirt DNA was extracted. Several expressed sequence tags were thus obtained. The data were analyzed by bioinformatics and interested EST fragment was chosen for preliminary functional screening. An anti-sense cDNA library was constructed containing 2 x 10(6) independent clones with an insert efficiency of more than 90%; DNA sequencing and bioinformatic analysis suggested that the No.27 survival clone was zinc transporter LIV1 showing a strong resistance against trichostatin A-induced apoptosis during functional screening. An anti-sense cDNA library with high quantity and quality has been successfully constructed; LIV1 gene may be one of the essential genes with anti-tumor effects on apoptosis induced by trichostatin A.

  7. High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis

    CSIR Research Space (South Africa)

    Van den Berg, N

    2004-11-01

    Full Text Available DNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up...

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

    Science.gov (United States)

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

    2007-11-01

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

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

    DEFF Research Database (Denmark)

    Shi, Shuobo; Ji, Haichuan; Siewers, Verena

    2016-01-01

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

  10. [Screening of high taxol producing fungi by mutagenesis and construction of subtracted cDNA library by suppression subtracted hybridization for differentially expressed genes].

    Science.gov (United States)

    Zhao, Kai; Sun, Lixin; Wang, Xuan; Li, Xiuliang; Wang, Xin; Zhou, Dongpo

    2011-07-01

    To screen mutants with high yield of taxol, and construct cDNA subtractive library of obtained mutant and primary strain HD(1-3). The spores of taxol-producing fungus HD(1-3) were treated by diethyl sulphate (DES), ultraviolet radiation and diethyl sulphate (UV + DES). cDNA subtractive library of taxol producing fungi from the mRNA of obtained mutant with high yield of taxol tester and HD(1-3) driver was constructed by using suppression subtracted hybridization (SSH). The optimal conditions for mutagenesis of strain HD(1-3) were as follows: the spore suspension was treated with 8% DES for 15 min, followed by UV irradiation (30 w, 30 cm distance) for 45 sec under magnetic stirring, a mutant UD(14-1) which was able to produce taxol with high yield and could be stably passed on genetics was found. Its ability to produce taxol was improved from 232.73 +/- 4.61 microg/L (strain HD(1-3)) to 312.81 +/- 7.51 microg/L (strain UD(14-1)). The tilter of the constructed cDNA library was 1.2 x 10(7) cfu/mL, the recombinant rate reached to 75.3% and the length of the inserted fragments was mostly 300 bp-1.0 kb. A mutant UD(14-11) with high yield was obtained, and cDNA subtractive library of the mutant UD(14-11) and strain HD(1-3) was constructed. The study laid solid foundation for isolation of taxol biosynthesis related genes and construction of engineering strains with high yield of taxol by genetic techniques.

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

    OpenAIRE

    Soliman, Hatem Mohamed Touhan

    2005-01-01

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

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

    Science.gov (United States)

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

    2011-10-01

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

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

    Science.gov (United States)

    Eißmann, Moritz; Schwamb, Bettina; Melzer, Inga Maria; Moser, Julia; Siele, Dagmar; Köhl, Ulrike; Rieker, Ralf Joachim; Wachter, David Lukas; Agaimy, Abbas; Herpel, Esther; Baumgarten, Peter; Mittelbronn, Michel; Rakel, Stefanie; Kögel, Donat; Böhm, Stefanie; Gutschner, Tony; Diederichs, Sven; Zörnig, Martin

    2013-01-01

    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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Tuomas Rönnberg

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

  16. Construction of yeast surface-displayed cDNA libraries.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2011-01-01

    Using yeast display, heterologous protein fragments can be efficiently displayed at high copy levels on the Saccharomyces cerevisiae cell wall. Yeast display can be used to screen large expressed protein libraries for proteins or protein fragments with specific binding properties. Recently, yeast surface-displayed cDNA libraries have been constructed and used to identify proteins that bind to various target molecules such as peptides, small molecules, and antibodies. Because yeast protein expression pathways are similar to those found in mammalian cells, human protein fragments displayed on the yeast cell wall are likely to be properly folded and functional. Coupled with fluorescence-activated cell sorting, yeast surface-displayed cDNA libraries potentially allow the selection of protein fragments or domains with affinity for any soluble molecule that can be fluorescently detected. In this report, we describe protocols for the construction and validation of yeast surface-displayed cDNA libraries using preexisting yeast two-hybrid cDNA libraries as a starting point.

  17. cDNA libraries for virus-induced gene silencing.

    Science.gov (United States)

    Todd, Andrea T; Liu, Enwu; Page, Jonathan E

    2010-01-01

    Virus-induced gene silencing (VIGS) exploits endogenous plant antiviral defense mechanisms to posttranscriptionally silence the expression of targeted plant genes. VIGS is quick and relatively easy to perform and therefore serves as a powerful tool for high-throughput functional genomics in plants. Combined with the use of subtractive cDNA libraries for generating a collection of VIGS-ready cDNA inserts, VIGS can be utilized to screen a large number of genes to determine phenotypes resulting from the knockdown/knockout of gene function. Taking into account the optimal insert design for VIGS, we describe a methodology for producing VIGS-ready cDNA libraries enriched for inserts relevant to the biological process of interest.

  18. Identification of breast cancer-restricted antigens by antibody screening of SKBR3 cDNA library using a preselected patient's serum.

    Science.gov (United States)

    Forti, Stefania; Scanlan, Matthew J; Invernizzi, Annamaria; Castiglioni, Fabio; Pupa, Sandro; Agresti, Roberto; Fontanelli, Rosanna; Morelli, Daniele; Old, Lloyd J; Pupa, Serenella M; Ménard, Sylvie

    2002-06-01

    Screening of a breast cancer cDNA library from SKBR3 human breast cancer cells by SEREX (serological analysis of cDNA expression library) using a preselected serum from a breast cancer patient revealed 13 genes, two of which, INT-MI-1 and INT-MI-2, encode novel gene products, while the remaining 11 genes and their products are identical with or highly homologous to known GenBank entries. Immunoscreening of the 13 clones using 20 allogeneic sera from breast cancer patients and 20 samples from age- and gender-matched healthy donors showed that lactate dehydrogenase-A (LDH-A), lactate dehydrogenase-B (LDH-B), fibulin-1, and thyroid hormone-binding protein (THBP) were recognized principally by the breast cancer patient sera, indicating the immunogenicity of these molecules in vivo. The other antigens were similarly recognized by normal and patients sera, and thus not tumor-restricted immunologically. RT-PCR analysis revealed strong expression of fibulin-1 in tumor cell lines and surgical specimen whereas in the same experimental conditions, normal tissues scored negative. Also THBP expression was found in various tumors whereas in normal tissues, its expression is restricted to the testis and, at lower levels, in ovary, liver, and spleen. In contrast, LDH-A and LDH-B were ubiquitously expressed in normal and tumor tissues, with LDH-B levels considerably lower and heterogeneous in normal samples compared to those expressed in tumor cell lines. The differential expression of fibulin-1 between the normal tissues and breast carcinoma cell lines (5/6) and surgical specimens (5/6) suggests the possible involvement of the overexpression of this extracellular matrix-associated glycoprotein in the pathogenesis of this neoplasm.

  19. Microarray screening of suppression subtractive hybridization-PCR cDNA libraries identifies novel RNAs regulated by dehydration in the rat supraoptic nucleus.

    Science.gov (United States)

    Ghorbel, Mohamed T; Sharman, Greig; Hindmarch, Charles; Becker, Kevin G; Barrett, Tanya; Murphy, David

    2006-01-12

    The magnocellular neurons (MCNs) of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus are the principal site of biosynthesis of prepropeptide precursor of the antidiuretic hormone vasopressin (VP). This precursor is processed during anterograde axonal transportation to terminals in the posterior pituitary gland, where biologically active VP is stored until release into the general circulation in response to physiological activation of the SON by osmotic cues. By binding to V2-type receptors located in the kidney, VP decreases the amount of water lost in urine. Osmotic activation of the SON is accompanied by a dramatic morphological and functional remodeling. We have sought to understand the mechanistic basis of this plasticity in terms of the differential expression of genes. To identify such genes, we adopted an unbiased global approach based on suppressive subtractive hybridization-polymerase chain reaction (SSH-PCR) Using this method, we generated libraries of clones putatively differentially expressed in control vs. dehydrated SON. To rapidly screen these libraries, 1,152 clones were subjected to microarray analysis, resulting in the identification of 459 differentially expressed transcripts. cDNA clones corresponding to 56 of these RNAs were sequenced, revealing many of them to be novel expressed sequence tags (ESTs). Four transcripts were shown by in situ hybridization (ISH) to be significantly up- or downregulated in the SON after dehydration. These genes may represent novel effectors or mediators of SON physiological remodeling.

  20. Functional cloning using pFB retroviral cDNA expression libraries.

    Science.gov (United States)

    Felts, Katherine A; Chen, Keith; Zaharee, Kim; Sundar, Latha; Limjoco, Jamie; Miller, Anna; Vaillancourt, Peter

    2002-09-01

    Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt's lymphoma (Daudi) cDNA library inserted into the high-titer retroviral vector pFB.

  1. Construction of cDNA libraries in vaccinia virus.

    Science.gov (United States)

    Smith, Ernest S; Shi, Shuying; Zauderer, Maurice

    2004-01-01

    Poxvirus expression vectors have gained widespread use for expression of foreign proteins and as delivery vehicles for vaccine antigens. We have developed a novel method using the poxvirus as a library vector for functional selection of specific cDNA. Poxviruses have several unique and useful properties as a library vector. Most importantly, because poxviruses are packaged into fully infectious particles in the cell cytoplasm, specific recombinants can be readily recovered even from a very small number of selected cells. Moreover, in contrast to libraries constructed in retrovirus or plasmid-based vectors, recombinant vaccinia virus can be efficiently recovered even from cells that have been induced to undergo apoptosis or cessation of cell growth. In the past, the major obstacle in this application to poxviruses has been the low frequency with which recombinants can be generated. The most commonly used method to construct recombinant poxvirus is homologous recombination. The frequency of recombinants derived in this manner is of the order of 0.1%, sufficient to recover a recombinant of a purified DNA clone in a transfer plasmid, but far too low to permit construction of a representative cDNA library. We have developed a method that generates nearly 100% recombinant vaccinia viruses at good titer. We have termed this method trimolecular recombination. cDNA libraries of as many as 107 or more independent viral recombinants can be constructed by trimolecular recombination. For the first time, large, diverse, and representative cDNA libraries can be screened in a vaccinia virus-based expression vector.

  2. [Construction and identification of the expression library of album pollen allergens cDNA].

    Science.gov (United States)

    Zhang, Jie; Sun, Xiu-zhen; Yan, Hong; Zhang, Ni; Feng, Xiang-li

    2011-05-01

    To construct and identify the express library of album pollen allergens cDNA. Total RNA were extracted from the album pollen with TRIzol reagent and the mRNA was isolate for the amplify followed. A double stranded cDNA (ds cDNA) was synthesized using primers containing Xho I and Poly(dT) sequence by ZAP Express®cDNA synthesis kit. The ds cDNA was modified and purified by gel chromatography, and then the cDNA fragment with the length of more than 400 bp containing sticky ends was obtained. The cDNA fragment was ligated with Uni-ZAP XR vector and subsequently treated with in vitro packaging using phage by ZAP-cDNA express GigapackIII Gold cloning kit. The express library of album pollen cDNA was constructed by in vitro packaging. The recombination rate and the lengths of fragments inserted of the cDNA library were detected by polymerase chain reaction. The titer and the recombination rate of cDNA expression library constructed were 9.7×10(5) and 100%, respectively. The capacity of the library was 4.85 Pfu. The average length of cDNA fragments inserted was about 1.0 kb. Based on the capacity of cDNA expression library constructed and the length of cDNA insertion fragments, the cDNA expression library constructed is qualified to screening target cDNA clone, laying the foundation for preparation of gene recombinant allergen pollen vaccine.

  3. PCR-based cDNA library construction: general cDNA libraries at the level of a few cells.

    OpenAIRE

    Belyavsky, A; Vinogradova, T; Rajewsky, K

    1989-01-01

    A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector a controlled trimming of the 3' ends of the cDNA by Klenow enzyme was used. Starting from 10 J558L ...

  4. Construction of full-length cDNA library of white flower Salvia ...

    African Journals Online (AJOL)

    In order to screen and isolate secondary metabolite biosynthesis related gene, we construct a cDNA library of white flower Salvia miltiorrhiza bge. f.alba. High quality of total RNA was successfully isolated from roots of white flower S. miltiorrhiza using modified CTAB method. Double strand cDNA was cloned into pDNR-LIB ...

  5. [Construction and Identification of the cDNA Expression Library for Human Esophageal Cancer Cells].

    Science.gov (United States)

    Zhang, Zhe; Wu, Xiang-Yu; Feng, Lu; Huang, Shang-Ke; Luo, Min-Na; Shao, Shan; Zhao, Xin-Han

    2016-09-01

    To construct a cDNA phage expression library for human esophageal cancer cells. After the total RNA were obtained from esophageal cancer cells, the mRNA were separated with magnetic beads adsorption method, and the single-strand and double-strand cDNA were synthesized through reverse transcription. With the undesirable cDNA fragments removed, the remaining cDNA (linked with Eco R1 aptamer and phosphorylated its 5'end) combined with the carrier of T7 Select10-3b. The recombinant phage were packaged in vitro for preliminary cDNA library. PCR was used to identify the size of inserted cDNA. The constructed original cDNA phage expression library for human esophageal cancer cells was consisted of 2.01×10⁶ pfu/mL bacteriophages with a recombination rate of 100%. The length of the inserted cDNA fragments were range from 300 bp to 1 500 bp. The cDNA phage expression library of human esophageal cell is successfully constructed to meet the currently recognized standards, and can be well used to screen cDNA-cloned genes of human esophageal cancer antigens by serological analysis of recombinantly expressed cDNA clone (SEREX).

  6. [cDNA library constructing and specific antigen expression of Streptomyces thermohydroscopicus].

    Science.gov (United States)

    Xu, Lei; Wang, Ling-ling; Liu, Shuo; Ling, Yuan; Ma, Lie; Wang, Qun; Zhang, Li-jiao; He, Xiao-yu; Zhao, Ming-jing; Wang, Xiao-ge

    2012-03-01

    To construct a cDNA library from Streptomyces thermohydroscopicus and screen genes with virulence, obtain the recombinant fusion virulence proteins by prokaryotic expression system. The Streptomyces thermohydroscopicus cDNA library was constructed by switching mechanism at 5'end of RNA transcript approach. A total of 1020 clones randomly selected from the cDNA library were sequenced and these expressed sequence tags (EST) were further analyzed for the screen of antigen-specific genes. The two candidate genes were subcloned into expression vector pET-28a. The recombinants were transformed into BL2 and proteins were expressed by the induction of isopropyl-β-D-1-thiogalactopyranoside (IPTG). A high-quality cDNA library from Streptomyces thermohydroscopicus was constructed and a set of 978 valid sequences were obtained. Clustering and assembly of these cDNA sequences resulted in 347 unique genes, among which 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein (51%) and transferring-binding protein B (42%) from Actinobacillus pleuropneumoniae serotype (APP). The open reading frame (ORF) of the two candidate genes are 1554 bp and 726 bp, which coded two peptides with 517 and 241 amino acids, respectively. The molecular weights of the recombinant fusion proteins were 63 000 and 30 000. The cDNA library of Streptomyces thermohydroscopicus reached the quality requirement of gene library. EST database in the library would greatly facilitate further screening of virulence genes.

  7. Method for construction of normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1998-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

  8. [Construction of chicken embryo fibroblasts cDNA expression library].

    Science.gov (United States)

    Liu, Wei; Gao, Yu-long; Gao, Hong-lei; Wang, Xiao-mei; Xu, Xiu-hong

    2007-06-01

    Chicken embryo fibroblast (CEF) is a primary cellular material to research the infectious bursal disease virus (IBDV). Constructing the cDNA expression library of CEF is the foundation to research cell tropism and find cell receptors of IBDV from CEF. In order to achieve that purpose, a high-quality cDNA expression library of CEF was constructed by Gateway technology, which could avoid using the restriction enzyme for cloning to solve technical limitation of roution method. The mRNA was extracted from chicken embryonic fibroblast. Moreover, single-strand cDNA and double-strand cDNA were synthesized by using biotin-conjugated Oligo (dT) primer in turn. The double-strand cDNA was ligated Adapter and then purified by the cDNA Size Fractionation Columns. After BP recombination reaction, a cDNA entry library was constructed with a titer of 1 x 10(6) cfu/mL, total clones of 1.2 x 10(7) cfu and an average insertion size of about 2243 bp. After LR recombination reaction, the cDNA entry library was transformed into expression library which took on a titer of 5 x 10(5) cfu/mL, total clones of 5.5 x 10(6) cfu and an average insertion size of about 2411bp. The results indicate that the constructed cDNA expression library performs a remarkable high value in both recombination rate and library coverage. As a result, the cDNA expression library, with its good quality, may facilitate to identify the receptors associated with the resistance against IBDV in chicken embryonic fibroblast and to cast new light on the mechanism of cellular tropism. Moreover, it may also provide data of chicken embryonic fibroblast in transcription level and may be helpful to study its biological functions.

  9. Constructing and detecting a cDNA library for mites.

    Science.gov (United States)

    Hu, Li; Zhao, YaE; Cheng, Juan; Yang, YuanJun; Li, Chen; Lu, ZhaoHui

    2015-10-01

    RNA extraction and construction of complementary DNA (cDNA) library for mites have been quite challenging due to difficulties in acquiring tiny living mites and breaking their hard chitin. The present study is to explore a better method to construct cDNA library for mites that will lay the foundation on transcriptome and molecular pathogenesis research. We selected Psoroptes cuniculi as an experimental subject and took the following steps to construct and verify cDNA library. First, we combined liquid nitrogen grinding with TRIzol for total RNA extraction. Then, switching mechanism at 5' end of the RNA transcript (SMART) technique was used to construct full-length cDNA library. To evaluate the quality of cDNA library, the library titer and recombination rate were calculated. The reliability of cDNA library was detected by sequencing and analyzing positive clones and genes amplified by specific primers. The results showed that the RNA concentration was 836 ng/μl and the absorbance ratio at 260/280 nm was 1.82. The library titer was 5.31 × 10(5) plaque-forming unit (PFU)/ml and the recombination rate was 98.21%, indicating that the library was of good quality. In the 33 expressed sequence tags (ESTs) of P. cuniculi, two clones of 1656 and 1658 bp were almost identical with only three variable sites detected, which had an identity of 99.63% with that of Psoroptes ovis, indicating that the cDNA library was reliable. Further detection by specific primers demonstrated that the 553-bp Pso c II gene sequences of P. cuniculi had an identity of 98.56% with those of P. ovis, confirming that the cDNA library was not only reliable but also feasible.

  10. [Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

    Science.gov (United States)

    Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

    2017-04-12

    Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

  11. [Construction of a cDNA library from liver tissue of rhesus monkey, Macaca mulatta].

    Science.gov (United States)

    Qin, Sheng-fang; Tan, Wei-dong; Chen, You-nan; Ding, Yang; Li, Sheng-fu; Li, Hong-xia; Wang, Li; Yang, Rong; Lu, Yan-rong

    2007-06-01

    To screen the target rhesus genes and give some basic genetic evidences to its value as one of the most important animal model in biomedical study, we constructed a cDNA expression library from liver tissue of a healthy rhesus monkey. With Trizol reagent, the total RNA was extracted from healthy rhesus liver tissue. By mutant Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), the first-strand cDNA was synthesized from purified mRNA, and subsequently the second-strand cDNA was generated via E. coli DNA polymerase I . Then, the EcoR I adapter was added to the synthesized double-strand cDNA, which was subsequently digested by Xho I restriction enzyme and fractionated with CHROMA APIN-400 column. The fractionated cDNA fragments to be longer than 0. 5 kb were ligated into lambda ZAP express vector to form the phagemid cDNA recombinants, which were further packaged into the lambda ZAP cDNA library according to the standard protocol with phage lambda Gold packaging extract. In order to get more stable clones with larger quantity, the primary library was amplified through infecting the host strain XL1-Blue MRF'. Then, the library titre, recombinant rate and length of inserted cDNA were measured, respectively. The capacity of the primary stand or unamplified library was 1. 2X 10(6) pfu. The titers of the unamplified library or the amplified library was 1.1 X 10(6) mixture, pfu/mL or 7. 7 X 10(9) pfu/mL respectively, the percentages of recombinants were 99. 3% and 98. 2%, and the average lengths of the inserts were 2.0 kb and 2. 3 kb, respectively. An excellent cDNA expression library has been constructed successfully, which would lay solid foundation for transplantation study and pre-clinic evaluation of related drugs.

  12. [Construction of phage display cDNA library from adult worms of Schistosoma japonicum].

    Science.gov (United States)

    Sun, Yi; Jia, Ren-chu; Liu, Jin-ming; Yuan, Chun-xiu; Shi, Yao-jun; Lu, Ke; Fu, Zhi-qiang; Sun, Huan; Cai, You-min; Lin, Jiao-jiao

    2007-10-01

    To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum. Total RNA was extracted from adult worms of S. japonicum by Trizol reagent anti mRNA was isolated from the total RNA. The ds cDNA was synthesized by reverse transcription using random primer. Directional EcoR I/ Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoR I anti Hind III, which resulted in ds cDNA with EcoR I and Hind III adhering ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector. After packaging in citro, the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library. Plaque assay and PCR were used to evaluate the library. Seven known objective genes of S. japonicum were screened by PCR to detect the representation of the library. Primary library capacity was 4.98 x 10(6) pfu, and the titer of amplified library was 3.85 x 10(11) pfu/mL. The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%, in which 95.6% inserted cDNA fragments were longer than 300 bp in length. All the seven known objective genes of S. japonicum were amplified from the library. The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

  13. cDNA library construction of two human Demodexspecies.

    Science.gov (United States)

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

    2017-06-01

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

  14. [Construction of cDNA expression library of salivary gland from Boophilus microplus].

    Science.gov (United States)

    Tian, Zhan-Cheng; Liu, Guang-Yuan; Xie, Jun-Ren; Gong, Zhen-Li

    2008-10-30

    Total RNA were isolated from salivary gland dissected from partially engorged Boophilus microplus. The mRNA was purified. A library of oligo (dT)-primed cDNA with added directional EcoR I/Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I/Hind III arms of the lambda SCREEN vector. The recombinant phage DNA was packaged by phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.38x10(6) PFU. Data showed 100% of the library were recombinant and the titer of the amplified library was 2x10(9) PFU/ml. A partial cDNA encoding cytochrome oxidase C subunit II of B. microplus was screened from the expression library with rabbit serum against B. microplus salivary gland proteins. The results is suggested that the cDNA expression library has been constructed.

  15. Construction of cDNA library of cotton mutant Xiangmian-18 library during gland forming stage.

    Science.gov (United States)

    Xie, Yong-Fang; Wang, Bo-Chu; Li, Biao; Cai, Ying-Fan; Xie, Lei; Xia, Yu-Xian; Chang, Ping-An; Jiang, Huai-Zhong

    2007-11-15

    Gossypol, a secondary metabolite stored in the glands of cotton, protecting cottonseed from consumption of human and monogastric animal. This ability is unique to the tribe Gossypieae. Although the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been investigated at molecular level. Here we described a simple and efficient method for constructing a normalized cDNA library from a cotton mutant, Xiangmian-18, during its pigments gland forming stage. It combined switching mechanism at 5'-end of RNA transcript (SMART) technique and duplex-specific nuclease (DSN) normalization methods. In a model experiment, double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into E. coli JM109 by electroporation. Counting the number of colonies, the titer of the original library was 5.86x10(5)cfu/ml in this library. Electrophoresis gel results indicated the fragments ranged from 800bp to 2kb, with the average size of 1400bp. Random picking clones showed that the recombination rate was 94%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of pigments gland cottons.

  16. cDNA library Table - KAIKOcDNA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data List Contact us KAIKOcDNA... cDNA library Table Data detail Data name cDNA library Table DOI 10.18908/lsdba.nbd...c00951-005 Description of data contents List of Bombyx mori cDNA libraries. Data file File name: kaiko_cdna_...iption Registered library name Registered name of the partial cDNA library Library synonym Another name for cDNA... Download License Update History of This Database Site Policy | Contact Us cDNA library Table - KAIKOcDNA | LSDB Archive ...

  17. Procedure for normalization of cDNA libraries

    Science.gov (United States)

    Bonaldo, Maria DeFatima; Soares, Marcelo Bento

    1997-01-01

    This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

  18. Generation of cDNA expression libraries enriched for in-frame sequences.

    Science.gov (United States)

    Davis, C A; Benzer, S

    1997-03-18

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.

  19. Construction of equalized short hairpin RNA library from human brain cDNA.

    Science.gov (United States)

    Xu, Lei; Li, Jingqi; Liu, Li; Lu, Lixia; Gao, Jingxia; Li, Xueli

    2007-02-20

    Short hairpin RNA (shRNA) library is a powerful new tool for high-throughput loss-of-function genetic screens in mammalian cells. An shRNA library can be constructed from synthetic oligonucleotides or enzymatically cleaved natural cDNA. Here, we describe a new method for constructing equalized shRNA libraries from cDNA. First, enzymatically digested cDNA fragments are equalized by a suppression PCR-based method modified from suppression subtractive hybridization. The efficiency of equalization was confirmed by quantitative real-time PCR. The fragments are then converted into an shRNA library by a series of enzymatic treatments. With this new technology, we constructed a library from human brain cDNA. Sequence analysis showed that most of the randomly selected clones had inverted repeat sequences converted from different cDNA. After transfecting HEK 293T cells and detecting gene expression, three out of eight clones were demonstrated to significantly inhibit their target genes.

  20. [Construction and characterization of a cDNA library from human liver tissue of cirrhosis].

    Science.gov (United States)

    Chen, Xiao-hong; Chen, Zhi; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan; Yao, Hang-ping

    2005-03-01

    To construct a cDNA library from human liver tissue of cirrhosis. The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb). A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

  1. [Construction and immunoscreening of cDNA library of Babesia orientalis].

    Science.gov (United States)

    Liu, Qin; Zhou, Dan-Na; Zhou, Yan-Qin; Zhang, Ying; He, Lan; Yao, Bao-An; Zhao, Jun-Long

    2009-06-01

    To construct a cDNA library for Babesia orientalis and screen immunologically positive clones. Total RNA of B. orientalis in red blood cells from an infected calf was isolated. cDNA was synthesized by reverse transcriptase, amplified by PCR and ligated into lambdaTriplEx2 vector. The recombined vectors were packaged and the unamplified cDNA library was constructed. The cDNA library was then amplified and immunologically screened with rabbit anti-B. orientalis serum. The recombinant lambdaTriplEx2 of positive clones were converted to the corresponding recombinant pTriplEx2. The inserted fragments were identified by PCR amplification. The plasmids were sequenced and compared against GenBank database by Blast. The titer of the unamplified library was 2.0 x 10(6) pfu/ml. The inserted fragment length of the library ranged from 500 to 3,000 bp, and the recombination efficiency accounted for 98.8%. The titer of the amplified library was 5.8 x 10(8) pfu/ml. Three positive clones were selected by serum immunological screening and named B04, B05, and B41, respectively. The inserted fragments of the B04, B05 and B41 were about 1,300 bp, 1,000 bp, and 2,400 bp, respectively. Sequence analysis revealed that the 3 clones contained open reading frames. Blast results showed that they were highly homologous to the nuclear movement protein gene, the hypothetical protein gene and the heat shock protein 70 (HSP70) gene, respectively. The deduced amino acid sequences of B04, B05 and B41 contained 310, 192 and 647 amino acid residues, with Mr of 34,000, 21,000, and 70,700, respectively. A qualified cDNA library of B. orientalis has been constructed and three positive clones of B. orientalis discovered.

  2. Study on construction of cDNA library of the treated changliver cell and quality analysis

    OpenAIRE

    Juntang, Lin; Pramanik, Jogenananda; Congrui, Wang; Huiyong, Zhang; Huigen, Feng; Baosheng, Yang; Yuchang, Li; Cunshuan, Xu

    2004-01-01

    The study aims to construct cDNA library of Changliver cell by SMART (switching mechanism at 5′ end of RNA transcript) technique and analyze its quality. cDNA of Changliver cell was made with RT-PCR and LD-PCR (long-distance PCR), the cDNA library was constructed with SMART cDNA library construction kit. Through testing, the high quality cDNA library containing whole long cDNA of Changliver cell had been constructed. The titer of the amplified cDNA library was 4.5 × 1010 pfu/ml and the averag...

  3. [Construction and analysis of suppression subtractive cDNA libraries of continuous monoculture Rehmannia glutinosa].

    Science.gov (United States)

    Zhang, Zhongyi; Fan, Huamin; Yang, Yanhui; Li, Mingjie; Li, Juan; Xu, Haixia; Chen, Junying; Chen, Xinjian

    2011-02-01

    To explore the molecular mechanism of continuous monoculture problem by constructing the cDNA libraries of continuous monoculture Rehmannia glutinosa. To use the suppression subtractive hybridization (SSH) technique to construct the forward and reverse subtractive cDNA libraries of continuous monoculture R. glutinosa to adopt blue-white colony screening and PCR to detect the positive clones which would be sequenced and analyzed by bioinformatics. The subtracted cDNA libraries of continuous monoculture R. glutinosa. were successfully constructed, and the result showed that the forward and reverse subtracted libraries obtained 300 positive clones, respectively. The forward and reverse libraries got different ESTs, and produced 232 (forward library) and 214 (reverse library) unique ESTs by sequencing. Based on homology search of BLASTX and BLASTN in NCBI, 200 and 195 of unique ESTs were homologous to known genes in the forward and reverse libraries, respectively. Categories of orthologous group (COG) showed that the forward and reverse libraries got 60 and 61 ESTs with the corresponding gene annotation, involving 21 metabolic pathways. The information of differential expression genes in continuous monoculture R. glutinosa, and their functional annotation of differentially expressed genes indicate that continuous monoculture has a profound effect on expression of the genes in R. glutinosa. Furthermore, the research analyzed several key genes in response to replant problem, which provided a foundation for revealing the molecular mechanism of continuous monoculture R. glutinosa.

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

    Science.gov (United States)

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

    2003-11-01

    To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database. Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMART PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder, cDNA xProfiler, Digital GENE Expression Displayer (DGED), and Digital Differential Display (DDD) were used. Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed. Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocarcinoma.

  5. Display of a maize cDNA library on baculovirus infected insect cells.

    Science.gov (United States)

    Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

    2008-08-12

    Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

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

    Lifescience Database Archive (English)

    Full Text Available (C) 5) sexually fusion-competent KAX3 cells (Gamete phase) (F) cDNA library construction method How to const...dir) 2) Full-length cDNA libraries (oligocapped method)(fl) 3) Gamete-specific subtraction library (sub) cDNA library construction

  7. cDNA library generation from ribonucleoprotein particles.

    Science.gov (United States)

    Rederstorff, Mathieu; Hüttenhofer, Alexander

    2011-02-01

    Most, if not all, known noncoding RNAs (ncRNAs) are associated with RNA binding proteins, thus forming ribonucleoprotein particles or RNPs. Here we describe a protocol for the generation of a specialized cDNA library from RNPs, thereby increasing the proportion of functional ncRNA species in the library. To that end, cellular extracts are fractionated on 10-30% glycerol gradients. Subsequently, RNP-derived ncRNAs are isolated and 3'-tailed by cytidine triphosphate and poly(A) polymerase; this is followed by 5' adapter ligation by T4 RNA ligase. Reverse transcription of ncRNAs into cDNAs is carried out with an oligo-d(G) anchor primer. The generated cDNA libraries are subsequently submitted to high-throughput sequencing. This RNP selection procedure increases the probability of the presence of biologically relevant ncRNA species in the library compared with libraries generation methods that use size-selected, protein-devoid ncRNAs. The protocol enables the generation of deep-sequencing-compatible cDNA libraries that code for functional ncRNAs within 1 week.

  8. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    Science.gov (United States)

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

  9. [Construction of a yeast two-hybrid cDNA library from the human testis].

    Science.gov (United States)

    Zheng, Ying; Zhang, Lu-Ping; Jia, Xiao-Qin; Wang, Hai-Yan

    2012-04-01

    To construct a human testis cDNA library for yeast two-hybrid screening. Human normal testis mRNA was purified from total RNA, and ds cDNA was synthesized and amplified using primers SMART III and CDS III oligo (dT) as the base of recombination. The purified PCR products and linearized plasmid pGADT7-Rec were co-transformed into the competent yeast Y187 and recombined by yeast homologous recombinase in the yeast cells to form an active cyclic plasmid. All the clones growing on the SD/-Leu plates were harvested to constitute a human testis cDNA library. We constructed a human testis cDNA library with high multiplication and adequate capacity, from which 2.0 x 10(6) recombinants were obtained. The amplified PCR fragments were between 0.3 kb and 4.0 kb in length. The yeast two-hybrid cDNA library of human testis was successfully constructed by the Clontech SMART method, which has prepared a ground for further studies on the molecular mechanism of spermatogenesis.

  10. Construction of cDNA library of Pyrocystis lunula (Pyrophyta)

    Science.gov (United States)

    Sui, Zhenghong; Kowallik, Klaus V.

    2004-10-01

    Complementary DNA library of a dinoflagellate Pyrocystis lunula was constructed for the purpose of expression sequence tags analysis. The RNA isolated from this alga was about 20µgg-1 net cells, and the band intensity ratio of 28S/18 S in electrophoresis pattern was nearly 1 to 1. Different cDNA/vector molar ratios were exploited in the ligating reaction to be optimized. The clones produced by cDNA/vector molar ratio of 3.75 to 1 were desirable, most of whose inserts were longer than 300 bp. The recombinants insert length of the unfractionation cDNA library was largely shorter than 500 bp. However, in the fractionation library made from high molecule weight cDNA parts, over seventy percent of the recombinants contained inserts longer than 1 kb, some of which were even longer than 3 kb. Operating concerns were discussed at the end.

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

    NARCIS (Netherlands)

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

    2001-01-01

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

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

    Science.gov (United States)

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

    1997-01-01

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

  13. Construction and characterization of a cDNA library from head kidney of Japanese sea bass (Lateolabrax japonicus).

    Science.gov (United States)

    Shao, Zhan-tao; Cong, Xiao; Yuan, Jin-duo; Yang, Gui-wen; Chen, Ying; Pan, Jie; An, Li-guo

    2009-09-01

    In this paper, a cDNA expression library from head kidney of Japanese sea bass (Lateolabrax japonicus) was constructed for the first time. The first-strand cDNA was synthesized with Moloney Murine Leukaemia virus reverse transcriptase and the double-stranded cDNA was digested by Xho I enzyme. Size fractionation was performed on CHROMA SPIN-400 columns. cDNA fragments longer than 500 bps were ligated into the lambdaZAPExpress vector. The recombinant DNA was packaged in vitro with Gigapack III gold packaging extract. The titers of the primary and amplified library were 1.0 x 10(5) and 5.0 x 10(9) pfu/ml, respectively. To characterize the constructed cDNA library, 15 phage plaques were selected randomly to test the inserted fragments. The results showed that the inserts were mostly longer than 500 bps. To test the utility, the library was screened with primers designed for three immune-related genes of, Myxovirus resistant (Mx), tumor necrosis factor-alpha (TNF-alpha) and Toll-like receptor (TLR). Results of Blastn and alignment showed that they are members of Mx, TNF-alpha and TLR gene families, respectively, which meets our anticipates for this cDNA library as an immune-related one. These results confirmed that the cDNA library constructed will provide a useful tool for gene cloning and expression analysis in immune system of Japanese sea bass.

  14. Construction and primary characterization of Echinococcus multilocularis protoscolex cDNA expression library.

    Science.gov (United States)

    Li, S; Chen, Y

    2001-02-01

    To construct a lambda gt11 cDNA expression library of Echinococcus multilocularis protoscolex isolated in China. Echinococcus multilocularis protoscolex mRNA was extracted using a Quickprep MicromRNA purification kit based on combining of the disruptive and protective properties of guanidinium thiocyanate (GTC) with the speed and selectivity of oligo (dT)-cellulose chromatography in a spum-column with some modification. Purified mRNA (1.8 micrograms) was submitted to reverse transcription using random hexamers [pd(N6)]. The double-strand blunt-ended cDNAs were ligated with an EcoRI/Notl adaptor to form a cohesive EcoRI end. Subsequently the synthesized cDNA was inserted into vector lambda gt11 EcoRI arms. After being packaged in vitro, lambda gt11 was put to an infectious bacteria Echinococcus coli (E. coli) strain Y1090; the recombinants were screened by color selection. PCR amplification was performed to evaluate the size of insertion DNA fragments. The recombinant ratio was nearly 100% and approximately 1 x 10(6) clones could be derived from this lambda gt11 cDNA library. PCR results indicated that the insertion DNAs were about 1.48 kb. A lambda gt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multicularis protoscolex mRNA. Further studies on this library are deserved.

  15. Construction and characterization of a normalized cDNA library.

    OpenAIRE

    Soares, M B; Bonaldo, M F; Jelene, P; Su, L; Lawton, L; Efstratiadis, A

    1994-01-01

    We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each...

  16. [The construction of rapid amplification of cDNA ends cDNA libraries from human fetal bone and joint].

    Science.gov (United States)

    Liang, X; Gong, Y; Liu, Q; Li, J; Chen, B; Guo, C

    2001-02-01

    To construct rapid amplification cDNA ends(RACE) cDNA libraries from human fetal bone and joint and provide resources for isolation of bone- and joint-specific development-related genes. Total RNA of bone and joint were extracted with the modified single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. The double-stranded end-blunted cDNA were synthesized using TaKaRa's cDNA synthesis kit and ligated to cassette adaptors. All of the cDNA molecules were amplified by a pair of common primers. A protocol for RACE cDNA library construction from bone and joint was established and two RACE cDNA libraries from human fetal bone and joint were successfully constructed. The protocol of RACE cDNA library construction from limited materials proved to be simple and efficient and the library was suitable for RACE to isolate tissue-specific genes.

  17. Solid-phase cDNA library construction, a versatile approach.

    OpenAIRE

    Roeder, T

    1998-01-01

    A rapid and versatile method for cDNA library construction was developed. It is based on conventional cDNA library synthesis including all enzymatic steps usually required, but is performed on a solid support. The cDNA is immobilised via a biotin residue to streptavidin coupled magnetic beads, which allows rapid and easy to perform changes of buffers and enzymes. Therefore, it combines speed (library construction within a single day) with high quality libraries, making it ideally suited for m...

  18. Construction and characterization of yeast two-hybrid cDNA library derived from LFBK cell line.

    Science.gov (United States)

    Mahajan, Sonalika; Sharma, Gaurav Kumar; Matura, Rakesh; Subramaniam, Saravanan; Mohapatra, Jajati Keshari; Pattnaik, Bramhadev

    2015-05-01

    The cDNA libraries are indispensable and critical tools for performing protein-protein interaction studies. In this study, a high quality yeast two-hybrid cDNA library from the LFBK cell line was constructed and characterized. LFBK cell line was originally derived from the swine kidney cells and is highly susceptible to foot-and-mouth disease virus (FMDV) infection. The total RNA was extracted from the LFBK cells and the switching mechanism at the 5' end of RNA template (SMART) technique was employed for the cDNA synthesis. Subsequently, double stranded cDNA was amplified by long-distance PCR, purified and co-transformed with pGADT7-rec vector in yeast strain Y187. The quality parameters of the constructed library were evaluated to qualify the constructed library. Nucleotide sequencing of the randomly selected clones from the library confirmed the swine genotype of LFBK cell line. The LFBK cDNA library was mated with the 2C protein of FMDV in yeast two-hybrid (YTH) system and several putative interaction partners were identified in the preliminary screening. The LFBK library was observed to be of high quality and could potentially be applied to protein interaction studies between FMDV and the host cells using YTH system. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  19. Construction and characterization of a normalized cDNA library.

    Science.gov (United States)

    Soares, M B; Bonaldo, M F; Jelene, P; Su, L; Lawton, L; Efstratiadis, A

    1994-09-27

    We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C0t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.

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

    International Nuclear Information System (INIS)

    Li Jianxiang; Nie Jihua; Tong Jian; Fu Chunling; Zhou Jianwei

    2008-01-01

    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)

  1. Construction of C35 gene bait recombinants and T47D cell cDNA library.

    Science.gov (United States)

    Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

    2017-11-20

    C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

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

    Science.gov (United States)

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

    2014-01-01

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

  3. Construction and analysis of a cDNA library from yellow-fruit ginseng

    African Journals Online (AJOL)

    The total RNA was isolated from yellow-fruit ginseng (Panax ginseng C.A. Meyer) leaf tissue. A cDNA library of panax ginseng leaves was constructed by using pDNR-LIB vector according to the SMART cDNA library construction kit protocol. We obtained 378 high quality sequences (GenBank accession number: ...

  4. [Construction and sequence analysis of a normalized full-length cDNA library of Dendrobium officinale].

    Science.gov (United States)

    Jiang, Min; Wang, Jiang; Wen, Guo-Song; Xu, Shao-Zhong; Zha, Ying-Hong; Rong, Tian-Ju; Qian, Xiong

    2013-02-01

    In order to obtain functional genes, a normalized stems cDNA library was constructed from medicinal plant Dendrobium officinale. SMART (switching mechanism at 5' end of RNA transcript) cDNA synthesis combined with DSN (duplex-specific nuclease) normalization was applied to construct the normalized full-length cDNA library of D. officinale. The titer of cDNA library was about 1.3 x 10(6) cfu x mL(-1) and the average insertion size was about 1.5 kb with high recombination rate (93.9%). Random selected 163 positive clones were sequenced at single side. Bio-information analysis indicated that 147 from 150 high-quality unique sequences matched corresponding homologous proteins, and they participated in various biological processes based on GO (gene ontology). There were 8 clones with complete coding sequence, which presumed to be full-length genes. These results showed preliminarily that we successfully constructed a normalized full-length cDNA library of D. officinale which could be used to screen the functional genes related to metabolic pathways of medicinal ingredients.

  5. Preparation of full-length cDNA libraries: focus on metazoans.

    Science.gov (United States)

    Harada, Masako; Hayashizaki, Yoshihide

    2009-01-01

    Critical steps in a cDNA library preparation include efficient cDNA synthesis, selection of full-length cDNAs, normalizing their abundance, and the subtraction of redundant transcripts. The use of trehalose and sorbiol stabilizes the activity of the reverse transcriptase leading to efficient cDNA synthesis and the cap-trapping method is used for efficient full-length cDNA selection. Through the incorporation of additional normalization and subtraction steps that eliminate the size bias and expressed gene frequency, it is possible to attain cDNA libraries that include larger or rarely expressed genes. This chapter describes an efficient method to construct a full-length cDNA library, with a focus on metazoan samples.

  6. Construction of a T7 Human Lung Cancer cDNA Library

    Directory of Open Access Journals (Sweden)

    Wentao YUE

    2008-10-01

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

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

    OpenAIRE

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

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.

  8. [Construction and preliminary analysis of a full-length cDNA library for Paris polyphylla var. yunnanensis].

    Science.gov (United States)

    Zhao, Shuang; Dong, Xu; Ma, Teng

    2014-01-01

    A full-length cDNA library of Paris polyphylla var. yunnanensis was constructed in order to research the genes relating to growing development and the genes regulation of its secondary metabolite biosynthesis. The total RNA was extracted from Paris polyphylla var. yunnanensis using modified Trizol method. The SMART (switching mechanism at 5' end of RNA transcript )technology was appliedl to construct the full-length cDNA library. The library titer,recombinant rate and length of insert fragments were determined,the sequences of the library were analyzed by Blastx and were compared to GenBank database. The capacity of the library was 2. 5 x 107 cfu/mL, the recombinant rate was 98.5% and the average size of the inserted fragment was 1.5 kb. 9 ESTs (Expressed Sequence Tags) were relating to growing development and 5 ESTs were relating to regulation of secondary metabolite biosynthesis among 149 ESTs obtained from 192 clones sequenced. A full-length cDNA library of Paris polyphylla var. yunnanensis is constructed by SMART technology successfully, and the library has enough capacity, high recombinant rate and long insert fragment for the further research to screen and identify the functional genes of Paris polyphylla var. yunnanensis.

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

    Science.gov (United States)

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

    2009-09-01

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

  10. Generation of full-length cDNA libraries: focus on plants.

    Science.gov (United States)

    Seki, Motoaki; Kamiya, Asako; Carninci, Piero; Hayashizaki, Yoshihide; Shinozaki, Kazuo

    2009-01-01

    Full-length cDNAs are essential for the correct annotation of transcriptional units and gene products from genomic sequence data and for functional analysis of the genes. Full-length cDNA libraries are very important resources for isolation of the full-length cDNAs. The biotinylated cap trapper method using the trehalose-thermostabilized reverse transcriptase has been developed and has become an efficient method for construction of high-content full-length cDNA libraries. We have constructed full-length cDNA libraries from various plants and animals using this method. The protocol of the method is described in this chapter.

  11. Construction and characteristics of 3-end enriched cDNA library from individual embryos of cattle.

    Science.gov (United States)

    Long, Jian-Er; He, Li-Qiang; Cai, Xia; Ren, Zhao-Rui; Huang, Shu-Zhen; Zeng, Yi-Tao

    2006-11-01

    To analyze stage-specific gene expression profiles of pre-implantation embryos and evaluate potential viability, techniques were adapted to generate 3-end enriched cDNA libraries from individual embryos of cattle based on RT-PCR methodology. The reproducibility of constructing a cDNA library was tested by five independent PCR experiments with specific primers for the presence of several rare genes such as DNMT1 (DNA methylation transferase 1), DNMT2, DNMT3A, Oct-4/3 (octmer-binding transcription factor), IFN-iota, IGF-2r (insulin like growth factor 2 receptor), and the housekeeping genes, H2A and beta-actin. Results indicated repeatability and that a proportion of expressed genes in the cDNA library from an individual embryo was not affected by limited PCR amplification. From the cDNA library, 134 clones were randomly selected for sequencing and showed that structure related elements accounted for 33.5% of transcripts and the energy- and metabolism-related genes were also an important component being 11.9% in the cDNA library. Approximately 14% of genes in the library were functionally unknown including greater than 5% of genes that were likely novel because there was no identity in Genbank. The frequency of structure-related genes such as beta-actin and ribosomal proteins in the cDNA library corresponded to other reports and suggested that the cDNA library constructed by RT-PCR might be proportional to the mRNA populations. The cDNA libraries constructed from different stage embryos will provide a powerful tool to explore novel genes relevant to embryogenesis, determine the profiling of stage-specific gene expression, and evaluate the potential viability of embryos.

  12. High-quality RNA preparation from Rhodosporidium toruloides and cDNA library construction therewith.

    Science.gov (United States)

    Yang, Fan; Tan, Haidong; Zhou, Yongjin; Lin, Xinping; Zhang, Sufang

    2011-02-01

    Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the addition of β-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 μg total RNA per gram of cells was obtained with an average purity measured as A₂₆₀/A₂₈₀ of 2.09. A titer of 10⁵ cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for screening the genes related to lipid accumulation.

  13. [Construction of cDNA library of Magnaporthe grisea with magnetic bead].

    Science.gov (United States)

    Feng, Xu; Xiaoli, Wu; Dewen, Qiu

    2008-06-01

    We constructed cDNA library of Magnaporthe grisea. The good quality cDNA library could facilitate finding proteinaceous elicitors of M. grisea, and elucidating the mechanisms of the M. grisea--rice interaction. The Oligo(dT) combined with the magnetic bead was used to extract mRNA from total RNA of Magnaporthe grisea and as primers to synthesize the first-strand cDNA. Terminal transferase introduced PolyA into 3'terminal of the first cDNA strand, then the PolyA was used for amplifying the second-strand cDNA. Restriction enzyme and adapter were avoided in this research, which could solve technical limitation of the traditional method. Because all reactions were done in one centrifuge tube, this process could reduce the risk of cDNA loss and cross-contamination. The primers designed in this research could clone the amplified cDNAs into expression vector in a desirable orientation. The cDNA library constructed had a high titer of 8.9 x 10(6) cfu/mL, and contained a total clones of 8.9 x 10(7) cfu, with an average inserts size of about 1380 bp. Constructing cDNA library with magnetic bead was a highly efficient method using only small amount of experimental materials within a short period.

  14. [Construction of suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus].

    Science.gov (United States)

    Wu, Jia-hong; Zhao, Tong-yan; Dong, Yan-de

    2006-08-01

    To construct the suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus. Total RNA was extracted from the deltamethrin-resistant (R-lab) and -sensitive (S-lab) isolates, mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Two subtractions were performed by suppression subtractive hybridization with S-lab as tester and R-lab as driver or S-lab as driver and R-lab as tester. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. The subtracted cDNA libraries contained 580 and 477 positive clones respectively. The PCR results of 150 clones picked randomly from each library showed that the positive ratio of constructed cDNA libraries was 93%, with a length of cDNA fragments ranged from 150bp to 750bp. The suppression subtracted cDNA library of deltamethrin-resistant Ae. albopictus is constructed.

  15. Generation of cDNA expression libraries enriched for in-frame sequences

    OpenAIRE

    Davis, Claytus A.; Benzer, Seymour

    1997-01-01

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore deve...

  16. [A novel vector for construction of a cDNA library].

    Science.gov (United States)

    Fedchenko, V I; Kaloshin, A A; Medvedev, A E

    2010-01-01

    A new original vector pEM-(dT)40(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The pGEM-(dT)40f(+) is initially transformed into single stranded and then into a linear form and its (dT)40 tail at 3'-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector cyclization and synthesis of the second strand cDNA. This approach significantly simplifies cDNA library construction, it does not require PCR reaction (which can induce artifact mutations in cDNA sequences) and restrictase treatment.

  17. Construction and characterization of a cDNA expression library from the endangered Hu sheep.

    Science.gov (United States)

    Hu, P-F; Li, X-C; Liu, H-K; Guan, W-J; Ma, Y-H

    2014-10-31

    Hu sheep is one of the most important species in China; it is also listed as one of the 78 nationally protected domestic animals by the Chinese government in 2000. The construction of cDNA expression library of Hu sheep is of great significance for protecting individual genomes, generating transgenic sheep, and conducting clinical research using cDNA from Hu sheep. In this study, the total RNA from the ear tissue of Hu sheep was extracted, and a cDNA expression library was constructed using the SMART(TM) technique. The titer of amplified cDNA library was 1.09 x 10(10) PFU/mL, the rate of recombination was above 91.6%, and the average size of fragments was 1.1 kb. This study has an important significance for the preservation of Hu sheep resources at the genome level.

  18. [Construction of the subtracted cDNA libraries related to artemisinin-resistance of Plasmodium berghei].

    Science.gov (United States)

    Bei, Zhu-chun; Wang, Jing-yan

    2004-06-01

    To construct the subtracted cDNA libraries related to artemisinin-resistance of Plasmodium berghei using suppression subtractive hybridization PCR (SSH PCR). Total RNA was extracted from the artemisinin-sensitive (NS) and artemisinin-resistant (AR) strains of Plasmodium berghei K173. The cDNA synthesis followed the protocol of super SMART cDNA synthesis kit. Taking the NS as driver, AR as tester and reverse, two subtractions were performed by SSH PCR. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. The subtracted cDNA libraries of NS-AR and AR-NS contained 395 and 506 positive clones respectively. The PCR results of 108 clones picked randomly from each library showed 100 and 104 positive inserts contained in the plasmids respectively, and distributing in 250-2000 bp. The successful construction of the subtracted cDNA libraries related to artemisinin-resistance of P. berghei enable us to identify the different expressed genes involved in the resistance mechanism.

  19. Construction of primary and subtracted cDNA libraries from early embryos.

    Science.gov (United States)

    Rothstein, J L; Johnson, D; Jessee, J; Skowronski, J; DeLoia, J A; Solter, D; Knowles, B B

    1993-01-01

    By modifying current cDNA cloning and electroporation methods, large and representative murine cDNA libraries were synthesized from 10 to 100 ng mRNA isolated from unfertilized egg and preimplantation mouse embryos. High cloning efficiency is essential for complete representation of genes expressed in egg and preimplantation embryos and for the isolation of stage-specific genes using subtractive hybridization. Because the mouse embryo contains no more than 50 pg of poly(A)+ mRNA at any stage of preimplantation development, approximately 5000-10,000 embryos are required to obtain enough mRNA to synthesize libraries using current methods. To obtain a representative library that also includes rare transcripts, the size of the library should be at least 10(6) clones. The average percent conversion of mRNA to single-stranded cDNA was 20-40%, so that a cloning efficiency of nearly 2 x 10(8) cfu/microgram cDNA is required for such a cDNA library. No previous methods have provided directional cloning of cDNA into plasmids with these high efficiencies. The advent of electroporation methods for the introduction of nucleic acids into bacteria has made possible the use of standard plasmid vectors for high-efficiency cDNA cloning. Plasmid vectors are currently available that can accommodate the directional cloning of cDNA such that T7 and T3 RNA polymerase promoter sequences can be used to generate sense and anti-sense transcripts for subtractive hybridization and riboprobe synthesis. The cDNA libraries we derived using this methodology are a reusable and abundant source of genetic information about the control of preimplantation development. Specialized subtractive cDNA libraries enriched for genes expressed exclusively at a predetermined time in development give access to genes expressed in a stage-specific manner. The ability to construct new cDNA libraries from limited amounts of starting material ensures the provision of new and important resources for the identification

  20. Expressed sequence tags: normalization and subtraction of cDNA libraries expressed sequence tags\\ normalization and subtraction of cDNA libraries.

    Science.gov (United States)

    Soares, Marcelo Bento; de Fatima Bonaldo, Maria; Hackett, Jeremiah D; Bhattacharya, Debashish

    2009-01-01

    Expressed Sequence Tags (ESTs) provide a rapid and efficient approach for gene discovery and analysis of gene expression in eukaryotes. ESTs have also become particularly important with recent expanded efforts in complete genome sequencing of understudied, nonmodel eukaryotes such as protists and algae. For these projects, ESTs provide an invaluable source of data for gene identification and prediction of exon-intron boundaries. The generation of EST data, although straightforward in concept, requires nonetheless great care to ensure the highest efficiency and return for the investment in time and funds. To this end, key steps in the process include generation of a normalized cDNA library to facilitate a high gene discovery rate followed by serial subtraction of normalized libraries to maintain the discovery rate. Here we describe in detail, protocols for normalization and subtraction of cDNA libraries followed by an example using the toxic dinoflagellate Alexandrium tamarense.

  1. Size-selection of cDNA libraries for the cloning of cDNAs after suppression subtractive hybridization.

    Science.gov (United States)

    Lévesque, V; Fayad, T; Ndiaye, K; Nahé Diouf, M; Lussier, J G

    2003-07-01

    Here we describe the establishment of size-selected cDNA libraries for the cloning of full-length cDNAs that were initially identified by suppression subtractive hybridization (SSH) technology as being differentially expressed. First, the SSH-cDNA fragments were used as 32P-probes to verify their level and differential pattern of expression by virtual Northern and to establish their corresponding full-length cDNA size. Second, cDNAs were separated by size on agarose gels and used to construct size-selected cDNA plasmid libraries, which were then screened by colony hybridization with the SSH-cDNA fragments. We conclude that the described approach complements SSH technology by allowing efficient cloning and characterization of the corresponding full-length cDNA from any desired cell type or species. This approach will give researchers the ability to specifically target and study differentially expressed genes in an efficient manner for functional genomic studies.

  2. [Construction of the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene.].

    Science.gov (United States)

    Ye, Sujuan; Feng, Zhihua; Zhu, Wen; Cai, Chunji; Li, Lu; Sun, Liya; Wan, Haisu; Ma, Li; Zhou, Qinghua

    2008-08-20

    It has been proven that nm23-H1 gene is an important metastaticsuppressed gene of lung cancer. In order to screen the differential expression genes related to nm23-H1 , we constructed the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene by suppression subtractive hybridization (SSH) in this study, which lay a solid foundation for further screening and cloning metastatic-related genes of nm23-H1. The forward and reverse suppression subtractive cDNA libraries were constructed in the human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene (L9981 and L9981-nm23-H1) by SSH method. The positive clones were preliminarily screened by bluewhite colony, and precisely identified by PCR. The suppression subtractive cDNA libraries were successfully constructed in the human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981). After the blue-white screening, about three hundred positive clones in the forward subtracted library and four hundred positive clones in the reverse subtracted library were obtained. Ramdom analysis of 96 clones in each library with colony PCR methods showed that 84 clones in the forward subtracted library and 83 clones in the reverse subtracted library contained (300-750) bp inserts. SSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981) are successfully constructed by SSH and T/A cloning technology. The expression of nm23-H1 gene in the human large cell lung cancer cell lines may affect the differential expression of some metastatic-related genes.

  3. Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments

    NARCIS (Netherlands)

    Hakvoort, T. B.; Spijkers, J. A.; Vermeulen, J. L.; Lamers, W. H.

    1996-01-01

    We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes

  4. [Construction and identification of a full-length cDNA library from Spirometra erinaceieuropaei].

    Science.gov (United States)

    Lv, Gang; Lu, Ya-Jun; Fan, Zhi-Gang; Shi, Da-Zhong; Gan, Xiu-Feng; Zhong, Sai-Feng

    2010-10-30

    The full-length pBluescript II SK cDNA library of adult Spirometra erinaceieuropaei was constructed by using the SMART method. Data showed that 95.5% of the library was recombinant and the titer of the library was 1.06 x 10(6). The average insert size of the library was about 1.4 kb. Forty-eight randomly selected clones were sequenced. A set of 36 effective expressed sequence tags (ESTs) with the average size of 674 bp was obtained after excluding clones shorter than 450 bp. The unigenes occupied 58.3% of the 36 ESTs. The rate of full-length cDNAs were 57.7% (15/26). The high-quality of full-length cDNA library could be used for large scale EST sequencing.

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

    Science.gov (United States)

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

    2008-04-07

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

  6. 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.

  7. 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 ...

  8. Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA

    OpenAIRE

    Yadav, Rajiv Kumar; Barbi, Florian; Ziller, Antoine; Luis, Patricia; Marmeisse, Roland; Reddy, M Sudhakara; Fraissinet-Tachet, Laurence

    2014-01-01

    Background: Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. Results: We report here a ...

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

    Science.gov (United States)

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

    2010-10-01

    In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×10(6) pfu/ml and 1.62×10(9) pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142 bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers.

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

    Science.gov (United States)

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

    2010-01-01

    In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×106 pfu/ml and 1.62×109 pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers. PMID:20941376

  11. Construction and characterization of a cDNA library from human ...

    African Journals Online (AJOL)

    The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network, and the challenge to understand the network is to identify p53 downstream genes. In order to isolate and identify new p53 regulated genes, we constructed and characterized a normalized cDNA library from human brain ...

  12. [Construction of a subtracted cDNA library of differentially expressed genes in human normal liver tissue and primary hepatocellular carcinoma tissue].

    Science.gov (United States)

    Li, J; Xu, X; Han, B; Huang, G; Qian, G; Liang, P; Yang, T

    2001-12-01

    To construct a subtracted cDNA library of differentially expressed genes in human normal liver tissue and primary hepatocellular carcinoma (HCC) tissue. Using the suppression subtractive hybridization (SSH), a novel technique has been described recently. cDNA fragments of missing or low expressing tumor suppressor genes in HCC tissue were isolated using paracancerous normal liver tissue and HCC tissue as targets. Then these cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of E.coli by high voltage electroperforation. One hundred positive bacteria clones were randomly picked and identified using enzyme restriction method. The amplified library contained more than 4,000 positive bacteria clones. Random analysis of 100 clones with enzyme restriction method showed that all clones contained 200-600 bp inserts. A subtracted cDNA library of differentially expressed genes in human normal liver tissue and HCC tissue is constructed successfully with SSH and T/A cloning techniques. The library is efficient and lays solid foundation for screening and cloning new and specific missing or low expressing tumor suppressor genes of HCC.

  13. Optimized cDNA libraries for virus-induced gene silencing (VIGS using tobacco rattle virus

    Directory of Open Access Journals (Sweden)

    Page Jonathan E

    2008-01-01

    Full Text Available Abstract Background Virus-induced gene silencing (VIGS has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV. Results NbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A or poly(G homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated N. benthamiana roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401–500 bp in length and 99.5% lacked poly(A tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine N-methyltransferase (PMT, with ten different VIGS-NbPMT constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs. Conclusion Based on the silencing of NbPDS and NbPMT, we suggest the following design guidelines for constructs in TRV vectors: (1 Insert lengths should be in the range of ~200 bp to ~1300 bp, (2 they should be positioned in

  14. Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA.

    Science.gov (United States)

    Yadav, Rajiv Kumar; Barbi, Florian; Ziller, Antoine; Luis, Patricia; Marmeisse, Roland; Reddy, M Sudhakara; Fraissinet-Tachet, Laurence

    2014-09-03

    Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

  15. Trehalose as a good candidate for enriching full-length cDNAs in cDNA library construction.

    Science.gov (United States)

    Chen, Lei; Cao, Lixue; Zhou, Longhai; Jing, Yudong; Chen, Zuozhou; Deng, Cheng; Shen, Yu; Chen, Liangbiao

    2007-01-10

    It has been reported that the disaccharide trehalose is capable of increasing the thermostability and thermoactivity of reverse transcriptase, and therefore improving the length of cDNA synthesis. However, no test has been done on how the disaccharide trehalose performs in the context of the entire cDNA synthesis processes, or whether it can seamlessly integrate into the commercially available cDNA synthesis kit. In this report, we optimized a protocol to incorporate trehalose in the Stratagene's cDNA library construction kit in order to demonstrate great improvement in cDNA's length (average length of 1.8 kb in the trehalose group versus 1.0 kb in the control). Sequence analysis of the cDNA clones showed that the addition of trehalose did not increase the error rate of the RT products but greatly increase the quantity of full-length in cDNA library.

  16. [Construction of subtractive cDNA library of apoptosis-related genes in NB4 cells treated by arsenic trioxide].

    Science.gov (United States)

    Di, Chunhong; Gu, Shaohua; Tan, Xiaohua; Xian, Lingling; Wu, Qihan; Yang, Lei

    2009-02-01

    Construct the gene library of apoptosis related genes in acute promyelocytic leukemia (APL) cell line NB4 cells treated by arsenic trioxide to clarify the apoptotic mechanism of NB4 cells. APL cell line NB4 cells treated with or without arsenic trioxide for 24 hours. Total RNA was extracted and suppress subtractive hybridization (SSH) was conducted according to the manual. With the cDNA of the apoptosis cells as the tester and that of control cells as the driver, forward and reverse hybridization was performed. Differentially expressed genes were linked with pGEM-Teasy cloning vector and transformed into E. coli DH5alpha. The positive clones were screened by blue and white spot. PCR were used to amplify these genes. The subtractive cDNA libraries related with apoptosis of NB4 cells were successfully constructed. The constructed subtractive libraries are suitable for further study on the functional genes associated with apoptosis ofNB4 cells induced by arsenic trioxide.

  17. A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA.

    Science.gov (United States)

    Davis, Claytus; Barvish, Zeev; Gitelman, Inna

    2007-10-09

    The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts. We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed) total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci. We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired.

  18. A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA

    Directory of Open Access Journals (Sweden)

    Gitelman Inna

    2007-10-01

    Full Text Available Abstract Background The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts. Results We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci. Conclusion We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired.

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

    Science.gov (United States)

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

    2007-11-01

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

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

    Science.gov (United States)

    Guan, Yunyan; Huang, Liangmin; He, Maoxian

    2011-05-01

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

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

    DEFF Research Database (Denmark)

    Rong, Mingqiang; Yang, Shilong; Wen, Bo

    2015-01-01

    the 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......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...

  2. [Construction and characterization of normalized cDNA library of maize inbred Mo17 from multiple tissues and developmental stages].

    Science.gov (United States)

    Zhang, Z X; Zhang, F D; Tang, W H; Pi, Y J; Zheng, Y L

    2005-01-01

    Comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10.830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarry was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate gene, and developing microarrays in maize genomics research.

  3. CONSTRUCTION AND APPLICATIONS OF A MYCORRHIZAL ARBUSCULAR SPECIFIC cDNA LIBRARY.

    Science.gov (United States)

    Isayenkov, S; Maathuis, F J M

    2016-01-01

    To exploit the potential benefits of mycorrhizas, we need to investigate the processes that occur in these symbiotic interactions, particularly in the arbuscular compartment where nutrients are exchanged between the plant and the fungus. Progress in this area is restricted due to the intricacy and complexity of this plant-fungus interface and many techniques that have been employed successfully in other plants and animal systems cannot be used. An effective approach to study processes in arbuscules is to examine transcript composition and dynamics. We applied laser capture microdissection (LCM) to isolate approximately 3000 arbuscules from Glomus intraradices colonised Me- dicago truncatula roots. Total RNA was extracted from microdissected arbuscules and subjected to T7 RNA polymerase-based linear amplification. Amplified RNA was then usedfor construction of a cDNA library. The presence and level of enrichment of mycorrhiza-specific transcripts was determined by quantitative Real-time and conventional PCR. To improve enrichment a cDNA library subtraction was performed. Complementation of yeast mutants deficient in the uptake of.potassium, phosphate, sulphate, amino acids, ammonium and of a Mn²⁺sensitive strain, demonstrates the functionality of our cDNA library.

  4. Sequencing of first-strand cDNA library reveals full-length transcriptomes.

    Science.gov (United States)

    Agarwal, Saurabh; Macfarlan, Todd S; Sartor, Maureen A; Iwase, Shigeki

    2015-01-21

    Massively parallel strand-specific sequencing of RNA (ssRNA-seq) has emerged as a powerful tool for profiling complex transcriptomes. However, many current methods for ssRNA-seq suffer from the underrepresentation of both the 5' and 3' ends of RNAs, which can be attributed to second-strand cDNA synthesis. The 5' and 3' ends of RNA harbour crucial information for gene regulation; namely, transcription start sites (TSSs) and polyadenylation sites. Here we report a novel ssRNA-seq method that does not involve second-strand cDNA synthesis, as we Directly Ligate sequencing Adaptors to the First-strand cDNA (DLAF). This novel method with fewer enzymatic reactions results in a higher quality of the libraries than the conventional method. Sequencing of DLAF libraries followed by a novel analysis pipeline enables the profiling of both 5' ends and polyadenylation sites at near-base resolution. Therefore, DLAF offers the first genomics tool to obtain the 'full-length' transcriptome with a single library.

  5. An efficient and sensitive method for preparing cDNA libraries from scarce biological samples.

    Science.gov (United States)

    Sterling, Catherine H; Veksler-Lublinsky, Isana; Ambros, Victor

    2015-01-01

    The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Directory of Open Access Journals (Sweden)

    Zhang Chao-Jun

    2009-07-01

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

  7. cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing.

    Science.gov (United States)

    Cartolano, Maria; Huettel, Bruno; Hartwig, Benjamin; Reinhardt, Richard; Schneeberger, Korbinian

    2016-01-01

    The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.

  8. Construction of the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene

    Directory of Open Access Journals (Sweden)

    Sujuan YE

    2008-08-01

    Full Text Available Background and objective It has been proven that nm23-H1 gene is an important metastatic-suppressed gene of lung cancer. In order to screen the differential expression genes related to nm23-H1, we constructed the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene by suppression subtractive hybridization (SSH in this study, which lay a solid foundation for further screening and cloning metastatic-related genes of nm23-H1. Methods The forward and reverse suppression subtractive cDNA libraries were constructed in the human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene (L9981 and L9981-nm23-H1 by SSH method. The positive clones were preliminarily screened by blue-white colony, and precisely identified by PCR. Results The suppression subtractive cDNA libraries were successfully constructed in the human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981. After the blue-white screening, about three hundred positive clones in the forward subtracted library and four hundred positive clones in the reverse subtracted library were obtained. Ramdom analysis of 96 clones in each library with colony PCR methods showed that 84 clones in the forward subtracted library and 83 clones in the reverse subtracted library contained (300-750 bp inserts. Conclusion SSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981 are successfully constructed by SSH and T/A cloning technology. The expression of nm23-H1 gene in the human large cell lung cancer cell lines may affect the differential expression of some metastatic-related genes.

  9. Construction and characterization of a goat mammary gland cDNA library.

    Science.gov (United States)

    Han, Xue Feng; Luo, Jun; Wu, Ning; Matand, Kanyand; Yang, Bao Jin; Wu, Hui Juan; Zhang, Li Juan; Wang, Hai Bin

    2008-03-01

    A lactating goat mammary gland cDNA library was constructed by using a modified commercially available cDNA library construction kit protocol. The resulting clones were sequenced and functionally analyzed through cross-species genomic comparison to assess (1) the capacity and functional quality of the constructed library for subsequent research and (2) the efficiency of the procedural modifications. The study resulted in the construction of a high-quality mammary gland cDNA library, which was characterized by (1) the total recombinants number of 1.4 x 10(7) colony-forming units (cfus) that was at least 10 times greater than the number expected from the application of the standard kit protocol, (2) the recombinants rate of 96%, and (3) the average insert size of 1,082 bp. BLAST analysis of sequenced clones against GenBank databases determined 55.7% of clone redundancy, 22 known function gene clusters, and 29 novel gene clusters. The analysis of the primary gene expression profile showed that 59% of the tested clones were genes that coded for milk proteins while 16% of the clones coded for ribosomal, metabolism, immune response, and translation proteins. The remaining 25% of the tested clones were described as novel genes. Cross-species comparison showed that 77% of characterized gene clusters were successfully identified by using resources from other ruminants and unrelated species. This outcome is in consonance with the common belief that the genomic resources that have been generated across species are potentially powerful tools that could be used for enhancing the molecular understanding of less genomically studied species, such as goat.

  10. Identification of differential genes by suppression subtractive hybridization: I. Preparation of subtracted cDNA or genomic DNA library.

    Science.gov (United States)

    Rebrikov, Denis V

    2008-07-01

    INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes the preparation of a subtracted cDNA or genomic DNA library, and includes methods for cDNA synthesis, tester and driver DNA digestion, and adapter ligation.

  11. 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.

  12. Construction and analysis of full-length cDNA library of Cryptosporidium parvum.

    Science.gov (United States)

    Yamagishi, Junya; Wakaguri, Hiroyuki; Sugano, Sumio; Kawano, Suguru; Fujisaki, Kozo; Sugimoto, Chihiro; Watanabe, Junichi; Suzuki, Yutaka; Kimata, Isao; Xuan, Xuenan

    2011-06-01

    A full-length cDNA library was constructed from the sporozoite of Cryptosporidium parvum. Normalized clones were subjected to Solexa shotgun sequencing, and then complete sequences for 1066 clones were reconfigured. Detailed analyses of the sequences revealed that 13.5% of the transcripts were spliced; the average and median 5' UTR lengths were 213.5 and 122 nucleotides, respectively. There were 148 inconsistencies out of 562 examined genes between the experimentally described cDNA sequence and the predicted sequence from its genome. In addition, we identified 118 sequences that had little homology against annotated genes of C. parvum as prospective candidates for addable genes. These observations should improve the reliability of C. parvum transcriptome and provide a versatile resource for further studies. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  13. [Construction of forward and reverse subtracted cDNA libraries between muscle tissue of Meishan and Landrace pigs].

    Science.gov (United States)

    Xu, De-Quan; Zhang, Yi-Bing; Xiong, Yuan-Zhu; Gui, Jian-Fang; Jiang, Si-Wen; Su, Yu-Hong

    2003-07-01

    Using suppression subtractive hybridization (SSH) technique, forward and reverse subtracted cDNA libraries were constructed between Longissimus muscles from Meishan and Landrace pigs. A housekeeping gene, G3PDH, was used to estimate the efficiency of subtractive cDNA. In two cDNA libraries, G3PDH was subtracted very efficiently at appropriate 2(10) and 2(5) folds, respectively, indicating that some differentially expressed genes were also enriched at the same folds and the two subtractive cDNA libraries were very successful. A total of 709 and 673 positive clones were isolated from forward and reverse subtracted cDNA libraries, respectively. Analysis of PCR showed that most of all plasmids in the clones contained 150-750 bp inserts. The construction of subtractive cDNA libraries between muscle tissue from different pig breeds laid solid foundations for isolating and identifying the genes determining muscle growth and meat quality, which will be important to understand the mechanism of muscle growth, determination of meat quality and practice of molecular breeding.

  14. Construction and characterization of a normalized cDNA library of Nannochloropsis oculata (Eustigmatophyceae)

    Science.gov (United States)

    Yu, Jianzhong; Ma, Xiaolei; Pan, Kehou; Yang, Guanpin; Yu, Wengong

    2010-07-01

    We constructed and characterized a normalized cDNA library of Nannochloropsis oculata CS-179, and obtained 905 nonredundant sequences (NRSs) ranging from 431-1 756 bp in length. Among them, 496 were very similar to nonredundant ones in the GenBank ( E ≤1.0e-05), and 349 ESTs had significant hits with the clusters of eukaryotic orthologous groups (KOG). Bases G and/or C at the third position of codons of 14 amino acid residues suggested a strong bias in the conserved domain of 362 NRSs (>60%). We also identified the unigenes encoding phosphorus and nitrogen transporters, suggesting that N. oculata could efficiently transport and metabolize phosphorus and nitrogen, and recognized the unigenes that involved in biosynthesis and storage of both fatty acids and polyunsaturated fatty acids (PUFAs), which will facilitate the demonstration of eicosapentaenoic acid (EPA) biosynthesis pathway of N. oculata. In comparison with the original cDNA library, the normalized library significantly increased the efficiencies of random sequencing and rarely expressed genes discovering, and decreased the frequency of abundant gene sequences.

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

    Science.gov (United States)

    Cavazzini, Fabrizio; Magistroni, Riccardo; Furci, Luciana; Lupo, Valentina; Ligabue, Giulia; Granito, Maria; Leonelli, Marco; Albertazzi, Alberto; Cappelli, Gianni

    2012-01-01

    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.

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

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    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.

  17. Construction of small RNA cDNA libraries for deep sequencing.

    Science.gov (United States)

    Lu, Cheng; Meyers, Blake C; Green, Pamela J

    2007-10-01

    Small RNAs (21-24 nucleotides) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) are potent regulators of gene expression in both plants and animals. Several hundred genes encoding miRNAs and thousands of siRNAs have been experimentally identified by cloning approaches. New sequencing technologies facilitate the identification of these molecules and provide global quantitative expression data in a given biological sample. Here, we describe the methods used in our laboratory to construct small RNA cDNA libraries for high-throughput sequencing using technologies such as MPSS, 454 or SBS.

  18. Construction of small RNA cDNA libraries for high-throughput sequencing.

    Science.gov (United States)

    Lu, Cheng; Shedge, Vikas

    2011-01-01

    Small RNAs (smRNAs) play an essential role in virtually every aspect of growth and development, by regulating gene expression at the post-transcriptional and/or transcriptional level. New high-throughput sequencing technology allows for a comprehensive coverage of smRNAs in any given biological sample, and has been widely used for profiling smRNA populations in various developmental stages, tissue and cell types, or normal and disease states. In this article, we describe the method used in our laboratory to construct smRNA cDNA libraries for high-throughput sequencing.

  19. Construction of high-quality Caco-2 three-frame cDNA library and its application to yeast two-hybrid for the human astrovirus protein-protein interaction.

    Science.gov (United States)

    Zhao, Wei; Li, Xin; Liu, Wen-Hui; Zhao, Jian; Jin, Yi-Ming; Sui, Ting-Ting

    2014-09-01

    Human epithelial colorectal adenocarcinoma (Caco-2) cells are widely used as an in vitro model of the human small intestinal mucosa. Caco-2 cells are host cells of the human astrovirus (HAstV) and other enteroviruses. High quality cDNA libraries are pertinent resources and critical tools for protein-protein interaction research, but are currently unavailable for Caco-2 cells. To construct a three-open reading frame, full length-expression cDNA library from the Caco-2 cell line for application to HAstV protein-protein interaction screening, total RNA was extracted from Caco-2 cells. The switching mechanism at the 5' end of the RNA transcript technique was used for cDNA synthesis. Double-stranded cDNA was digested by Sfi I and ligated to reconstruct a pGADT7-Sfi I three-frame vector. The ligation mixture was transformed into Escherichia coli HST08 premium electro cells by electroporation to construct the primary cDNA library. The library capacity was 1.0×10(6)clones. Gel electrophoresis results indicated that the fragments ranged from 0.5kb to 4.2kb. Randomly picked clones show that the recombination rate was 100%. The three-frame primary cDNA library plasmid mixture (5×10(5)cfu) was also transformed into E. coli HST08 premium electro cells, and all clones were harvested to amplify the cDNA library. To detect the sufficiency of the cDNA library, HAstV capsid protein as bait was screened and tested against the Caco-2 cDNA library by a yeast two-hybrid (Y2H) system. A total of 20 proteins were found to interact with the capsid protein. These results showed that a high-quality three-frame cDNA library from Caco-2 cells was successfully constructed. This library was efficient for the application to the Y2H system, and could be used for future research. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Construction of cDNA expression library of watermelon for isolation of ClWRKY1 transcription factors gene involved in resistance to Fusarium wilt.

    Science.gov (United States)

    Yang, Bing-Yan; Huo, Xiu-Ai; Li, Peng-Fei; Wang, Cui-Xia; Duan, Hui-Jun

    2014-08-01

    Full-length cDNAs are very important for genome annotation and functional analysis of genes. The number of full-length cDNAs from watermelon remains limited. Here we report first the construction of a full-length enriched cDNA library from Fusarium wilt stressed watermelon (Citrullus lanatus Thunb.) cultivar PI296341 root tissues using the SMART method. The titer of primary cDNA library and amplified library was 2.21 x 10(6) and 2.0 x 10(10) pfu/ml, respectively and the rate of recombinant was above 85%. The size of insert fragment ranged from 0.3 to 2.0 kb. In this study, we first cloned a gene named ClWRKY1, which was 1981 bp long and encoded a protein consisting of 394 amino acids. It contained two characteristic WRKY domains and two zinc finger motifs. Quantitative real-time PCR showed that ClWRKY1 expression levels reached maximum level at 12 h after inoculation with Fusarium oxysporum f. sp. niveum. The full-length cDNA library of watermelon root tissues is not only essential for the cloning of genes which are known, but also an initial key for the screening and cloning of new genes that might be involved in resistance to Fusarium wilt.

  1. Construction of the subtracted cDNA library of striatal neurons treated with long-term morphine.

    Science.gov (United States)

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

    2011-03-01

    To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization (SSH). Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons (10-5 mol/L for 72 hours). To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein (CREB) mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons (62.85 ± 1.98) compared with normal striatal neurons (28.43 ± 1.46, P library of striatal neurons treated with long-term morphine is constructed. Mtch1 and Akt1 might be the candidate genes for the development of morphine tolerance.

  2. In-frame cDNA library combined with protein complementation assay identifies ARL11-binding partners.

    Directory of Open Access Journals (Sweden)

    Sangkyou Lee

    Full Text Available The cDNA expression libraries that produce correct proteins are essential in facilitating the identification of protein-protein interactions. The 5'-untranslated regions (UTRs that are present in the majority of mammalian and non-mammalian genes are predicted to alter the expression of correct proteins from cDNA libraries. We developed a novel cDNA expression library from which 5'-UTRs were removed using a mixture of polymerase chain reaction primers that complement the Kozak sequences we refer to as an "in-frame cDNA library." We used this library with the protein complementation assay to identify two novel binding partners for ras-related ADP-ribosylation factor-like 11 (ARL11, cellular retinoic acid binding protein 2 (CRABP2, and phosphoglycerate mutase 1 (PGAM1. Thus, the in-frame cDNA library without 5'-UTRs we describe here increases the chance of correctly identifying protein interactions and will have wide applications in both mammalian and non-mammalian detection systems.

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

    Science.gov (United States)

    Soares, Marcelo Bento; Bonaldo, Maria de Fatima

    1998-01-01

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

  4. Discovery of Phytophthora infestans Genes Expressed in Planta through Mining of cDNA Libraries

    Science.gov (United States)

    Chaves, Diego; Pinzón, Andrés; Grajales, Alejandro; Rojas, Alejandro; Mutis, Gabriel; Cárdenas, Martha; Burbano, Daniel; Jiménez, Pedro; Bernal, Adriana; Restrepo, Silvia

    2010-01-01

    Background Phytophthora infestans (Mont.) de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora – Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta. Methodology/Principal Findings We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family. Conclusions/Significance We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant – oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta. PMID:20352100

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

    Directory of Open Access Journals (Sweden)

    Roberto Sierra

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

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

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    Jing Zhang

    Full Text Available BACKGROUND: Pentastomiasis is a rare zoonotic disease caused by pentastomids. Despite their worm-like appearance, they are commonly placed into a separate sub-class of the subphylum Crustacea, phylum Arthropoda. However, until now, the systematic classification of the pentastomids and the diagnosis of pentastomiasis are immature, and genetic information about pentastomid nylum is almost nonexistent. The objective of this study was to obtain information on pentastomid nymph genes and identify the gene homologues related to host-parasite interactions or stage-specific antigens. METHODOLOGY/PRINCIPAL FINDINGS: Total pentastomid nymph RNA was used to construct a cDNA library and 500 colonies were sequenced. Analysis shows one hundred and ninety-seven unigenes were identified. In which, 147 genes were annotated, and 75 unigenes (53.19% were mapped to 82 KEGG pathways, including 29 metabolism pathways, 29 genetic information processing pathways, 4 environmental information processing pathways, 7 cell motility pathways and 5 organismal systems pathways. Additionally, two host-parasite interaction-related gene homologues, a putative Kunitz inhibitor and a putative cysteine protease. CONCLUSION/SIGNIFICANCE: We first successfully constructed a cDNA library and gained a number of expressed sequence tags (EST from pentastomid nymphs, which will lay the foundation for the further study on pentastomids and pentastomiasis.

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

    Science.gov (United States)

    Chen, Xiao-hong; Chen, Zhi; Yao, Hang-ping; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan

    2005-04-01

    To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript (SMART) technique and CDS III/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to lambdaTriplEx2 vector. Then lambda phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.94 x 10(6) pfu/ml and 1.49 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%. A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

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

    Science.gov (United States)

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

    2017-08-01

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

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

    Science.gov (United States)

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

    2005-03-01

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

  10. 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.

  11. Construction and characterization of a cDNA library from liver tissue of Chinese Banna minipig inbred line.

    Science.gov (United States)

    Tan, W; Chen, Y; Zhang, L; Lu, Y; Li, S; Zeng, R; Zeng, Y; Li, Y; Cheng, J

    2006-09-01

    A xenograft that performs efficient functions is an essential premise for successful xenotransplantation. Our early study indicated that Chinese Banna minipig inbred line (BMI) was an ideal xenograft donor. However, the activities of some proteins synthesized by the BMI liver are different from the human, which could lead to functional disorders in coagulation, fibrinolysis, and anticoagulation after liver xenotransplantation. Therefore, it is important to investigate the genetic background of protein incompatibility and to provide new strategies for gene manipulation. In this study we constructed a cDNA expression library using BMI liver tissue to obtain an understanding of nucleic acid and protein differences between the two species. We extracted total RNA and purified mRNA of the liver tissue from one of the sixteenth inbred generation of BMI/JS 151 substrain. After double-strand cDNA synthesis, we fractionated it on a CHROMA APIN-400 column; ligated the longer than 500bp cDNA into a ZAP Express Vector; and performed a lambda: phage packaging reaction, library amplification, and titer. We randomly picked 12 plaques and tested the length of inserts. The titers of the primary and amplified libraries were 1.0 x 10(6) pfu/mL and 5.0 x 10(9) pfu/mL, respectively. The percentages of recombinants were 97.0% in the primary library and 98.0% in the amplified library. The lengths of most inserts were between 750 bp and 2.0 kb. Thus, we successfully constructed a cDNA expression library from BMI liver tissue. Using the library, we hope to get a full-length cDNA of some important genes and conduct further studies on porcine liver function in xenotransplantation.

  12. Construction of a cDNA library from human retinal pigment epithelial cells challenged with rod outer segments.

    Science.gov (United States)

    Cavaney, D M; Rakoczy, P E; Constable, I J

    1995-05-01

    To study genes expressed by retinal pigment epithelial (RPE) cells during phagocytosis and digestion of rod outer segments (ROS), a complementary (c)DNA library was produced using an in-vitro model. The cDNA library can be used to study molecular changes which contribute to the development of diseases due to a failure in outer segment phagocytosis and digestion by RPE cells. Here we demonstrate a way to study genes and their functions using a molecular biological approach and describing the first step involved in this process, the construction of a cDNA library. Human RPE cells obtained from the eyes of a seven-year-old donor were cultured and challenged with bovine ROS. The culture was harvested and total RNA was extracted. Complementary DNA was transcribed from the messenger (m)RNA and was directionally cloned into the LambdaGEM-4 bacteriophage vector successfully. Some clones were picked and the DNA extracted, to determine the size of the inserts as a measure of the quality of the library. Molecular biology and cell culture are important tools to be used in eye research, especially in areas where tissue is limiting and animal models are not available. We now have a ROS challenged RPE cDNA library which will be used to identify genes responsible for degrading phagocytosed debris within the retinal pigment epithelium.

  13. Bioinformatic methods for finding differentially expressed genes in cDNA libraries, applied to the identification of tumour vascular targets.

    Science.gov (United States)

    Herbert, John M J; Stekel, Dov J; Mura, Manuela; Sychev, Michail; Bicknell, Roy

    2011-01-01

    The aim of this method is to guide a bench scientist to maximise cDNA library analyses to predict biologically relevant genes to pursue in the laboratory. Many groups have successfully utilised cDNA libraries to discover novel and/or differentially expressed genes in pathologies of interest. This is despite the high cost of cDNA library production using the Sanger method of sequencing, which produces modest numbers of expressed sequences compared to the total transcriptome. Both public and propriety cDNA libraries can be utilised in this way, and combining biologically relevant data can reveal biologically interesting genes. Pivotal to the quality of target identification are the selection of biologically relevant libraries, the accuracy of Expressed Sequence Tag to gene assignment, and the statistics used. The key steps, methods, and tools used to this end will be described using vascular targeting as an example. With the advent of next-generation sequencing, these or similar methods can be applied to find novel genes with this new source of data.

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

    Science.gov (United States)

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

    2016-01-01

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

  15. [Construction of a subtracted cDNA library of chronic intermittent hypoxia rabbit liver by suppression subtractive hybridization].

    Science.gov (United States)

    Wu, Yue-tao; Liu, Rui-hong; Yang, Yu; Luo, Ying-quan; Rong, Yao

    2007-12-01

    To construct a subtracted cDNA library of chronic intermittent hypoxia (CIH) rabbit liver by suppression subtractive hybridization (SSH). Twenty-four rabbits were divided into 4 groups: ordinary feeding group, full-fat food group, ordinary feeding in chronic intermittent hypoxia group, and full-fat food in chronic intermittent hypoxia group. The mRNAs were extracted from different rabbit livers and converted into double-strand cDNA. After digestion with restriction enzyme, the cDNA of hyperlipidemia-sensitive rabbit group was subdivided into 2 portions and each one was lighted with different adaptors. Two rounds of both hybridization and suppression PCR obtained the differentially expressed cDNA. The PCR products were inserted into T/A vector to set up the subtractive cDNA library. The clones were selected and amplified by PCR and identified. Based on the pathology of the abdominal aorta and liver, and the amplified library contained 500 positive bacteria clones, including 462 clones, which had inserts from 250 to 700 bp by PCR analysis. A novel rabbit gene, Cthrc1, involved in CHI had been cloned. The GenBank Accession Number is XM_418373. The molecular mechanism of CIH promoting atherogenesis formation is made clear.

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

    Directory of Open Access Journals (Sweden)

    Milady Mendoza-Rodríguez

    2004-01-01

    Full Text Available Molecular studies of plant-pathogen interaction are very important for the identification of gene (s related with the pathogenic process, as well as with the plant resistance. These gene (s could be use for the genetic improvement programs in order to obtain resistant cultivars. The aim of this work was to construct complementary DNA (cDNA libraries from infected leaves with Pseudocercospora fijiensis CCIBP-Pf1 isolated of two banana cultivars (a resistant one Calcutta4 and another one susceptible Niyarma Yik. First-strand cDNA synthesis, was made beginning with one microgram of total RNA by using oligo dT primer and cDNA quality was checked by Polimerase chain reaction (PCR with cytochrome b specific primers. Second-strand cDNA synthesis was performed by using the homopolymeric tailing with dC-BamH I + dT-Not I primer combination. Four cDNA libraries of infected plants at different times of infection with the pathogen were obtained. Forty one clones of one of the libraries of Niyarma Yik were sequenced and the obtained sequences correspond with genes related to fungi. Key words: Banana-Mycosphaerella fijiensis interaction,Black Sigatoka, Musa spp.

  17. High-efficiency system for the construction of adenovirus vectors and its application to the generation of representative adenovirus-based cDNA expression libraries.

    Science.gov (United States)

    Hillgenberg, Moritz; Hofmann, Christian; Stadler, Herbert; Löser, Peter

    2006-06-01

    We here describe a convenient system for the production of recombinant adenovirus vectors and its use for the construction of a representative adenovirus-based cDNA expression library. The system is based on direct site-specific insertion of transgene cassettes into a replicating donor virus. The transgene is inserted into a donor plasmid containing the viral 5' inverted terminal repeat, the complete viral packaging signal, and a single loxP site. The plasmid is then transfected into a Cre recombinase-expressing packaging cell line that has been infected with a donor virus containing a partially deleted packaging signal flanked by loxP sites. Cre recombinase, by two steps of action, sequentially catalyzes the generation of a nonpackageable donor virus acceptor substrate and the generation of the desired recombinant adenovirus vector. Due to its growth impairment, residual donor virus can efficiently be counterselected during amplification of the recombinant adenovirus vector. By using this adenovirus construction system, a plasmid-based human liver cDNA library was converted by a single step into an adenovirus-based cDNA expression library with about 10(6) independent adenovirus clones. The high-titer purified library was shown to contain about 44% of full-length cDNAs with an average insert size of 1.3 kb. cDNAs of a gene expressed at a high level (human alpha(1)-antitrypsin) and a gene expressed at a relatively low level (human coagulation factor IX) in human liver were isolated from the adenovirus-based library using an enzyme-linked immunosorbent assay-based screening procedure.

  18. Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors.

    Science.gov (United States)

    Kottke, Timothy; Errington, Fiona; Pulido, Jose; Galivo, Feorillo; Thompson, Jill; Wongthida, Phonphimon; Diaz, Rosa Maria; Chong, Heung; Ilett, Elizabeth; Chester, John; Pandha, Hardev; Harrington, Kevin; Selby, Peter; Melcher, Alan; Vile, Richard

    2011-06-19

    Effective cancer immunotherapy requires the release of a broad spectrum of tumor antigens in the context of potent immune activation. We show here that a cDNA library of normal tissue, expressed from a highly immunogenic viral platform, cures established tumors of the same histological type from which the cDNA library was derived. Immune escape occurred with suboptimal vaccination, but tumor cells that escaped the immune pressure were readily treated by second-line virus-based immunotherapy. This approach has several major advantages. Use of the cDNA library leads to presentation of a broad repertoire of (undefined) tumor-associated antigens, which reduces emergence of treatment-resistant variants and also permits rational, combined-modality approaches in the clinic. Finally, the viral vectors can be delivered systemically, without the need for tumor targeting, and are amenable to clinical-grade production. Therefore, virus-expressed cDNA libraries represent a novel paradigm for cancer treatment addressing many of the key issues that have undermined the efficacy of immuno- and virotherapy to date.

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

    Science.gov (United States)

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

    2004-01-01

    AIM: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential. METHODS: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver. cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5 α competent cells. Individual clones were randomly selected and used for PCR amplification. Vector DNA from positive clones was isolated for sequencing. RESULTS: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13 ,ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes. CONCLUSION: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential. PMID:15285011

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

    Science.gov (United States)

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

    2013-03-01

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

  1. Errors in CGAP xProfiler and cDNA DGED: the importance of library parsing and gene selection algorithms.

    Science.gov (United States)

    Milnthorpe, Andrew T; Soloviev, Mikhail

    2011-04-15

    The Cancer Genome Anatomy Project (CGAP) xProfiler and cDNA Digital Gene Expression Displayer (DGED) have been made available to the scientific community over a decade ago and since then were used widely to find genes which are differentially expressed between cancer and normal tissues. The tissue types are usually chosen according to the ontology hierarchy developed by NCBI. The xProfiler uses an internally available flat file database to determine the presence or absence of genes in the chosen libraries, while cDNA DGED uses the publicly available UniGene Expression and Gene relational databases to count the sequences found for each gene in the presented libraries. We discovered that the CGAP approach often includes libraries from dependent or irrelevant tissues (one third of libraries were incorrect on average, with some tissue searches no correct libraries being selected at all). We also discovered that the CGAP approach reported genes from outside the selected libraries and may omit genes found within the libraries. Other errors include the incorrect estimation of the significance values and inaccurate settings for the library size cut-off values. We advocated a revised approach to finding libraries associated with tissues. In doing so, libraries from dependent or irrelevant tissues do not get included in the final library pool. We also revised the method for determining the presence or absence of a gene by searching the UniGene relational database, revised calculation of statistical significance and sorted the library cut-off filter. Our results justify re-evaluation of all previously reported results where NCBI CGAP expression data and tools were used.

  2. Construction and characterization of a cDNA library from wheat infected with Fusarium graminearum Fg 2.

    Science.gov (United States)

    Al-Taweel, Khaled; Dilantha Fernando, W G; Brûlé-Babel, Anita L

    2011-01-18

    Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART) technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.

  3. Construction and Characterization of a cDNA Library from Wheat Infected with Fusarium graminearum Fg 2

    Directory of Open Access Journals (Sweden)

    Anita L. Brûlé-Babel

    2011-01-01

    Full Text Available Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.

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

    Science.gov (United States)

    Gadkar, Vijay J; Filion, Martin

    2013-06-01

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

  5. Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

    Science.gov (United States)

    2010-01-01

    Background Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression. Results The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system. Conclusions In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts

  6. 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

  7. 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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    International Nuclear Information System (INIS)

    Luo Ying; Sui Jianli; Tie Yi; Zhang Yuanping; Zhou Pingkun; Sun Zhixian

    2001-01-01

    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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kaur Jatinder

    2010-05-01

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

  12. Efficient pooling designs for library screening

    OpenAIRE

    Bruno, William J.; Knill, Emanuel; Balding, David J.; Bruce, D. C.; Doggett, N. A.; Sawhill, W. W.; Stallings, R. L.; Whittaker, Craig C.; Torney, David C.

    1994-01-01

    We describe efficient methods for screening clone libraries, based on pooling schemes which we call ``random $k$-sets designs''. In these designs, the pools in which any clone occurs are equally likely to be any possible selection of $k$ from the $v$ pools. The values of $k$ and $v$ can be chosen to optimize desirable properties. Random $k$-sets designs have substantial advantages over alternative pooling schemes: they are efficient, flexible, easy to specify, require fewer pools, and have er...

  13. Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage.

    Science.gov (United States)

    Archer, Stuart K; Shirokikh, Nikolay E; Preiss, Thomas

    2014-05-26

    A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN). Targeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets. We find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms.

  14. Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda.

    Science.gov (United States)

    Thanh, Tran; Chi, Vu Thi Quynh; Abdullah, Mohd Puad; Omar, Hishamuddin; Noroozi, Mostafa; Ky, Huynh; Napis, Suhaimi

    2011-01-01

    Green microalga Ankistrodesmus convolutus Corda is a fast growing alga which produces appreciable amount of carotenoids and polyunsaturated fatty acids. To our knowledge, this is the first report on the construction of cDNA library and preliminary analysis of ESTs for this species. The titers of the primary and amplified cDNA libraries were 1.1×10(6) and 6.0×10(9) pfu/ml respectively. The percentage of recombinants was 97% in the primary library and a total of 337 out of 415 original cDNA clones selected randomly contained inserts ranging from 600 to 1,500 bps. A total of 201 individual ESTs with sizes ranging from 390 to 1,038 bps were then analyzed and the BLASTX score revealed that 35.8% of the sequences were classified as strong match, 38.3% as nominal and 25.9% as weak match. Among the ESTs with known putative function, 21.4% of them were found to be related to gene expression, 14.4% ESTs to photosynthesis, 10.9% ESTs to metabolism, 5.5% ESTs to miscellaneous, 2.0% to stress response, and the remaining 45.8% were classified as novel genes. Analysis of ESTs described in this paper can be an effective approach to isolate and characterize new genes from A. convolutus and thus the sequences obtained represented a significant contribution to the extensive database of sequences from green microalgae.

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

    Science.gov (United States)

    Kato, Seishi; Oshikawa, Mio; Ohtoko, Kuniyo

    2011-01-01

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

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

    Science.gov (United States)

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

    2011-11-25

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

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

    Directory of Open Access Journals (Sweden)

    Blair Matthew W

    2011-11-01

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

  18. Mouse brain full-length cDNA library construction by negative selection of intact mRNAs.

    Science.gov (United States)

    Wu, Ning; Wu, Huijuan; Li, Yandong; Matand, Kanyand

    2010-06-01

    Synthesis of full-length cDNA libraries is an essential step for the study of gene function. The method for selecting the intact mRNA directly affects the number of full-length transcripts. We have developed a novel method for intact mRNA selection based on the elimination of uncapped mRNAs. A negative-selection strategy that removes both uncapped mRNA and other non-mRNA molecules that present a phosphate at the 5'-end has been applied in the mRNA purification procedures. Briefly, after performing a standard mRNA purification, a biotinylated oligoribonucleotide is ligated to the 5-end phosphate of uncapped mRNAs. Streptavidin extraction is then performed to remove truncated and non-mRNAs from the intact mRNAs. By comparing random sequencing results of mouse brain full-length and standard cDNA libraries, there was a significant increase of full-length clones with the modified procedure. The results showed that the full-length library contained more than 68% full-length clones with the 5'-end positions ranging between -485 to +100 compared to the standard library with 33% of full-length clones and 5'-end positions ranging between -233 to +100. The data were analyzed using the t-test with the significance level set at plibraries in both 5'-end position and mRNA size (p<0.05).

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

    Directory of Open Access Journals (Sweden)

    Wallis James G

    2007-07-01

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

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

    Science.gov (United States)

    Tougan, Takahiro; Okuzaki, Daisuke; Nojima, Hiroshi

    2008-09-01

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

  1. Model for a transcript map of human chromosome 21: isolation of new coding sequences from exon and enriched cDNA libraries.

    Science.gov (United States)

    Yaspo, M L; Gellen, L; Mott, R; Korn, B; Nizetic, D; Poustka, A M; Lehrach, H

    1995-08-01

    The construction of a transcriptional map for human chromosome 21 requires the generation of a specific catalogue of genes, together with corresponding mapping information. Towards this goal, we conducted a pilot study on a pool of random chromosome 21 cosmids representing 2 Mb of non-contiguous DNA. Exon-amplification and cDNA selection methods were used in combination to extract the coding content from these cosmids, and to derive expressed sequences libraries. These libraries and the source cosmid library were arrayed at high density for hybridisation screening. A strategy was used which related data obtained by multiple hybridisations of clones originating from one library, screened against the other libraries. In this way, it was possible to integrate the information with the physical map and to compare the gene recovery rate of each technique. cDNAs and exons were grouped into bins delineated by EcoRI cosmid fragments, and a subset of 91 cDNAs and 29 exons have been sequenced. These sequences defined 79 non-overlapping potential coding segments distributed in 24 transcriptional units, which were mapped along 21q. Northern blot analysis performed for a subset of cDNAs indicated the existence of a cognate transcript. Comparison to databases indicated three segments matching to known chromosome 21 genes: PFKL, COL6A1 and S100B and six segments matching to unmapped anonymous expressed sequence tags (ESTs). At the translated nucleotide level, strong homologies to known proteins were found with ATP-binding transporters of the ABC family and the dihydroorotase domain of pyrimidine synthetases. These data strongly suggest that bona fide partial genes have been isolated. Several of the newly isolated transcriptional units map to clinically important regions, in particular those involved in Down's syndrome, progressive myoclonus epilepsia and auto-immune polyglandular disease. The study presented here illustrates the complementarity of exon-amplification and cDNA

  2. 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.

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

    Science.gov (United States)

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

    2009-06-01

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

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

    Science.gov (United States)

    Zhou, Sun; Ji, Guoli; Liu, Xiaolin; Li, Pei; Moler, James; Karro, John E; Liang, Chun

    2012-05-03

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

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

    Directory of Open Access Journals (Sweden)

    Zhou Sun

    2012-05-01

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

  6. 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.

  7. Construction and analysis of full-length and normalized cDNA libraries from citrus.

    Science.gov (United States)

    Marques, M Carmen; Perez-Amador, Miguel A

    2012-01-01

    We have developed an integrated method to generate a normalized cDNA collection enriched in full-length and rare transcripts from citrus, using different species and multiple tissues and developmental stages. Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. In this regard, the availability of full-length cDNA clones facilitates functional analysis of the corresponding genes enabling manipulation of their expression and the generation of a variety of tagged versions of the native protein. The development of full-length cDNA sequences has the power to improve the quality of genome annotation, as well as provide tools for functional characterization of genes.

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

    Science.gov (United States)

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

    2012-10-01

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

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

    Science.gov (United States)

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

    2014-08-01

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

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

    Science.gov (United States)

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

    2015-08-01

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

  11. Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library

    NARCIS (Netherlands)

    Akkerdaas, Jaap H.; Schocker, Frauke; Vieths, Stefan; Versteeg, Serge; Zuidmeer, Laurian; Hefle, Sue L.; Aalberse, Rob C.; Richter, Klaus; Ferreira, Fatima; van Ree, Ronald

    2006-01-01

    The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA

  12. Construction of a muscle cDNA library of Chinese shrimp Fenneropenaeus chinensis and sequence analysis of the troponin I gene

    Science.gov (United States)

    Li, Jitao; Chen, Ping; Li, Jian; Liu, Ping; He, Yuying; Wang, Qingyin

    2010-03-01

    A muscle cDNA library of Chinese shrimp ( Fenneropenaeus chinensis) was constructed with the SMART™ cDNA Library Construction Kit. The titer of optimal primary library was 7.7×105 pfu mL-1 and that of the amplified library was 3.0×109 pfu mL-1. The percentages of the recombinant clones of primary and amplified libraries were over 98%. The insert sizes were longer than 400 bp with an average of 1000 bp. A positive clone containing a 794 bp insert was sequenced and identified encoding fast skeletal troponin I gene. This library provided a useful resource for the functional genomic research of F. chinensis.

  13. 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

  14. 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.

  15. Development of a simple and powerful method, cDNA AFLP-SSPAG ...

    African Journals Online (AJOL)

    Differential cDNAs were easily obtained from silver stained cDNA-AFLP separated on polyacylamide gels. The cDNA was then reamplified, cloned and fragments were sequenced. Sequenced clones were used as probes in northern dot blot analyses and library screening. Full-length cDNA was cloned from a library ...

  16. 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.

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

    Science.gov (United States)

    He, Tao; Jia, Jing Fen

    2008-12-01

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

  18. Functional characterization of an acidic SK(3) dehydrin isolated from an Opuntia streptacantha cDNA library.

    Science.gov (United States)

    Ochoa-Alfaro, A E; Rodríguez-Kessler, M; Pérez-Morales, M B; Delgado-Sánchez, P; Cuevas-Velazquez, C L; Gómez-Anduro, G; Jiménez-Bremont, J F

    2012-03-01

    Cactus pears are succulent plants of the Cactaceae family adapted to extremely arid, hot and cold environments, making them excellent models for the study of molecular mechanisms underlying abiotic stress tolerance. Herein, we report a directional cDNA library from 12-month-old cladodes of Opuntia streptacantha plants subjected to abiotic stresses. A total of 442 clones were sequenced, representing 329 cactus pear unigenes, classified into eleven functional categories. The most abundant EST (unigen 33) was characterized under abiotic stress. This cDNA of 905 bp encodes a SK(3)-type acidic dehydrin of 248 amino acids. The OpsDHN1 gene contains an intron inserted within the sequence encoding the S-motif. qRT-PCR analysis shows that the OpsDHN1 transcript is specifically accumulated in response to cold stress, and induced by abscisic acid. Over-expression of the OpsDHN1 gene in Arabidopsis thaliana leads to enhanced tolerance to freezing treatment, suggesting that OpsDHN1 participates in freezing stress responsiveness. Generation of the first EST collection for the characterization of cactus pear genes constitutes a useful platform for the understanding of molecular mechanisms of stress tolerance in Opuntia and other CAM plants.

  19. Toxicity evaluation of benzo[a]pyrene on the polychaete Perinereis nuntia using subtractive cDNA libraries.

    Science.gov (United States)

    Zheng, Senlin; Qiu, Xiaoyan; Chen, Bin; Yu, Xingguang; Lin, Kangli; Bian, Mei; Liu, Zhenghua; Huang, Hao; Yu, Weiwei

    2011-10-01

    To gain insight into the toxic effects of the carcinogenic PAH benzo[a]pyrene (BaP) on the typical marine benthic polychaete Perinereis nuntia, we amplified and sequenced genes by creating subtractive cDNA libraries between worms exposed to BaP and solvent control. We assigned functions to the identified sequences and further analyzed the transcriptional profile changes of a set of 50 selected potential marker genes using quantitative real time PCR. A total of 2422 new high quality ESTs (GenBank accession number GT629654-GT632075) were obtained in the P. nuntia subtracted cDNA libraries, and assembled into 1594 unique sequences. Blastx results showed 700 of the unique sequences shared high similarity with existing genes in the GenBank nr database. Functional annotation of these enriched gene segments suggested that P. nuntia shows a wide range of toxicological responses to BaP. Comparison of the transcriptional profiles of the 50 potential marker genes in worms exposed to BaP and the control suggested that BaP significantly changed the expression of genes involved in xenobiotics metabolism, reactive oxygen species (ROS) elimination, DNA repair, apoptosis, cell division cycle, neurodegeneration, neurotransmitter metabolism and carcinogenesis. It also shows that there are significant correlations between these potential marker genes. The results support the prediction that the polychaete P. nuntia also has a set of tumor-related genes, while other responses influenced by BaP involve detoxification, antioxidation, DNA repair and apoptosis. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. 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.

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

    Science.gov (United States)

    Ling, Peng; Wang, Meinan; Chen, Xianming; Campbell, Kimberly Garland

    2007-06-04

    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. 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. The full-length cDNA library is useful in identifying functional genes of P. striiformis.

  2. 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...

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

    Science.gov (United States)

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

    2012-08-01

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

  4. Determination of a Screening Metric for High Diversity DNA Libraries.

    Directory of Open Access Journals (Sweden)

    Nicholas J Guido

    Full Text Available The fields of antibody engineering, enzyme optimization and pathway construction rely increasingly on screening complex variant DNA libraries. These highly diverse libraries allow researchers to sample a maximized sequence space; and therefore, more rapidly identify proteins with significantly improved activity. The current state of the art in synthetic biology allows for libraries with billions of variants, pushing the limits of researchers' ability to qualify libraries for screening by measuring the traditional quality metrics of fidelity and diversity of variants. Instead, when screening variant libraries, researchers typically use a generic, and often insufficient, oversampling rate based on a common rule-of-thumb. We have developed methods to calculate a library-specific oversampling metric, based on fidelity, diversity, and representation of variants, which informs researchers, prior to screening the library, of the amount of oversampling required to ensure that the desired fraction of variant molecules will be sampled. To derive this oversampling metric, we developed a novel alignment tool to efficiently measure frequency counts of individual nucleotide variant positions using next-generation sequencing data. Next, we apply a method based on the "coupon collector" probability theory to construct a curve of upper bound estimates of the sampling size required for any desired variant coverage. The calculated oversampling metric will guide researchers to maximize their efficiency in using highly variant libraries.

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

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jacob

    2007-01-01

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

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

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jacob

    2007-01-01

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

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

    Science.gov (United States)

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

    2012-07-01

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

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

    Science.gov (United States)

    Tripathy, S; Maiti, N K

    2014-03-01

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

  9. 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.

  10. Sequencing over 13 000 expressed sequence tags from six subtractive cDNA libraries of wild and modern wheats following slow drought stress.

    Science.gov (United States)

    Ergen, Neslihan Z; Budak, Hikmet

    2009-03-01

    A deeper understanding of the drought response and genetic improvement of the cultivated crops for better tolerance requires attention because of the complexity of the drought response syndrome and the loss of genetic diversity during domestication. We initially screened about 200 wild emmer wheat genotypes and then focused on 26 of these lines, which led to the selection of two genotypes with contrasting responses to water deficiency. Six subtractive cDNA libraries were constructed, and over 13 000 expressed sequence tags (ESTs) were sequenced using leaf and root tissues of wild emmer wheat genotypes TR39477 (tolerant) and TTD-22 (sensitive), and modern wheat variety Kiziltan drought stressed for 7 d. Clustering and assembly of ESTs resulted in 2376 unique sequences (1159 without hypothetical proteins and no hits), 75% of which were represented only once. At this level of EST sampling, each tissue shared a very low percentage of transcripts (13-26%). The data obtained indicated that the genotypes shared common elements of drought stress as well as distinctly differential expression patterns that might be illustrative of their contrasting ability to tolerate water deficiencies. The new EST data generated here provide a highly diverse and rich source for gene discovery in wheat and other grasses.

  11. 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.

  12. 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

  13. 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.

  14. TcTASV: a novel protein family in trypanosoma cruzi identified from a subtractive trypomastigote cDNA library.

    Science.gov (United States)

    García, Elizabeth A; Ziliani, María; Agüero, Fernán; Bernabó, Guillermo; Sánchez, Daniel O; Tekiel, Valeria

    2010-10-05

    The identification and characterization of antigens expressed in Trypanosoma cruzi stages that parasitize mammals are essential steps for the development of new vaccines and diagnostics. Genes that are preferentially expressed in trypomastigotes may be involved in key processes that define the biology of trypomastigotes, like cell invasion and immune system evasion. With the initial aim of identifying trypomastigote-specific expressed tags, we constructed and sequenced an epimastigote-subtracted trypomastigote cDNA library (library TcT-E). More than 45% of the sequenced clones of the library could not be mapped to previously annotated mRNAs or proteins. We validated the presence of these transcripts by reverse northern blot and northern blot experiments, therefore providing novel information about the mRNA expression of these genes in trypomastigotes. A 280-bp consensus element (TcT-E element, TcT-Eelem) located at the 3' untranslated region (3' UTR) of many different open reading frames (ORFs) was identified after clustering the TcT-E dataset. Using an RT-PCR approach, we were able to amplify different mature mRNAs containing the same TcT-Eelem in the 3' UTR. The proteins encoded by these ORFs are members of a novel surface protein family in T. cruzi, (which we named TcTASV for T. cruzi Trypomastigote, Alanine, Serine and Valine rich proteins). All members of the TcTASV family have conserved coding amino- and carboxy-termini, and a central variable core that allows partitioning of TcTASV proteins into three subfamilies. Analysis of the T. cruzi genome database resulted in the identification of 38 genes/ORFs for the whole TcTASV family in the reference CL-Brener strain (lineage II). Because this protein family was not found in other trypanosomatids, we also looked for the presence of TcTASV genes in other evolutionary lineages of T. cruzi, sequencing 48 and 28 TcTASVs members from the RA (lineage II) and Dm28 (lineage I) T. cruzi strains respectively. Detailed

  15. TcTASV: a novel protein family in trypanosoma cruzi identified from a subtractive trypomastigote cDNA library.

    Directory of Open Access Journals (Sweden)

    Elizabeth A García

    Full Text Available BACKGROUND: The identification and characterization of antigens expressed in Trypanosoma cruzi stages that parasitize mammals are essential steps for the development of new vaccines and diagnostics. Genes that are preferentially expressed in trypomastigotes may be involved in key processes that define the biology of trypomastigotes, like cell invasion and immune system evasion. METHODOLOGY/PRINCIPAL FINDINGS: With the initial aim of identifying trypomastigote-specific expressed tags, we constructed and sequenced an epimastigote-subtracted trypomastigote cDNA library (library TcT-E. More than 45% of the sequenced clones of the library could not be mapped to previously annotated mRNAs or proteins. We validated the presence of these transcripts by reverse northern blot and northern blot experiments, therefore providing novel information about the mRNA expression of these genes in trypomastigotes. A 280-bp consensus element (TcT-E element, TcT-Eelem located at the 3' untranslated region (3' UTR of many different open reading frames (ORFs was identified after clustering the TcT-E dataset. Using an RT-PCR approach, we were able to amplify different mature mRNAs containing the same TcT-Eelem in the 3' UTR. The proteins encoded by these ORFs are members of a novel surface protein family in T. cruzi, (which we named TcTASV for T. cruzi Trypomastigote, Alanine, Serine and Valine rich proteins. All members of the TcTASV family have conserved coding amino- and carboxy-termini, and a central variable core that allows partitioning of TcTASV proteins into three subfamilies. Analysis of the T. cruzi genome database resulted in the identification of 38 genes/ORFs for the whole TcTASV family in the reference CL-Brener strain (lineage II. Because this protein family was not found in other trypanosomatids, we also looked for the presence of TcTASV genes in other evolutionary lineages of T. cruzi, sequencing 48 and 28 TcTASVs members from the RA (lineage II and Dm28

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

    Science.gov (United States)

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

    2014-09-03

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

  17. 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.

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

    Science.gov (United States)

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

    2013-05-24

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

  19. 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

  20. A study of regulatory small RNAs in Vibrio salmonicida: construction of a knock-out mutant and a cDNA library

    OpenAIRE

    Nyrud, May Liss Julianne

    2008-01-01

    The marine fish pathogen Vibrio salmonicida is the causative agent for vibriosis in Atlantic salmon (Salmo salar L.), Rainbow trout (Oncorhynchus mykiss) and Atlantic cod (Gadus morhua L.). V. salmonicidas virulence is regulated by Quorum sensing (QS) systems, which includes important regulatory small RNAs (sRNAs). sRNAs have the last years been identified in large numbers, and mostly in pathogenic bacteria strains. A cDNA library from small RNA species (120-340 nt) was constru...

  1. Construction of a full-length cDNA library and preliminary analysis of expressed sequence tags from lymphocytes of half-pipe snowboarding athletes.

    Science.gov (United States)

    Zhao, Y H; Zhang, Z B; Zhao, C Q; Zhang, Y; Wang, Y F; Guan, W J; Zhu, Z Q

    2015-10-21

    The genes of top athletes are a valuable genetic resource for the human race, and could be exploited to identify novel genes related to sports ability, as well as other functions. We analyzed the expressed sequence tags from top half-pipe snowboarding athletes using the SMART complementary DNA (cDNA) library construction method to elucidate the characteristics of the athlete genome and the differential expression of the genes it contains. Overall, we established a full-length cDNA library from the lymphocytes of half-pipe snowboarding athletes and analyzed the inserted gene fragments. We also classified those genes according to molecular function, biological characteristics, cellular composition, protein types, and signal paths. A total of 201 functional genes were noted, which were distributed in 27 pathways. TXN, MDH1, ARL1, ARPC3, ACTG1, and other genes measured in sequence may be associated with physical ability. This suggests that the SMART cDNA library constructed from the genetic material from top athletes is an effective tool for preserving genetic sports resources and providing genetic markers of physical ability for athlete selection.

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

    Science.gov (United States)

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

    2012-10-01

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

  3. Genomic and cDNA cloning of a novel mouse lipoxygenase gene

    NARCIS (Netherlands)

    Willems van Dijk, K.; Steketee, K.; Havekes, L.; Frants, R.; Hofker, M.

    1995-01-01

    A novel 12- and 15-lipoxygenase related gene was isolated from a mouse strain 129 genomic phage library in a screen with a human 15-lipoxygenase cDNA probe. The complete genomic sequence revealed 14 exons and 13 introns covering 7.3 kb of DNA. The splice junctions were verified from the cDNA

  4. RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries

    Science.gov (United States)

    Hafner, Markus; Renwick, Neil; Brown, Miguel; Mihailović, Aleksandra; Holoch, Daniel; Lin, Carolina; Pena, John T.G.; Nusbaum, Jeffrey D.; Morozov, Pavel; Ludwig, Janos; Ojo, Tolulope; Luo, Shujun; Schroth, Gary; Tuschl, Thomas

    2011-01-01

    Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3′ and 5′ ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1–249) and Rnl2(1–249)K227Q, for 3′-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5′-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3′-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5′-terminal 5-nt barcode extensions for a set of 20 barcoded 3′ adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling. PMID:21775473

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

    Science.gov (United States)

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

    2009-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Yu-Juan Xiang

    2015-06-01

    Full Text Available Objective: The aim of this study was to reveal the exact changes during the occurrence of breast cancer to explore significant new and promising genes or factors related to this disease. Methods: We compared the gene expression profiles of breast cancer tissues with its uninvolved normal breast tissues as controls using the cDNA microarray analysis in seven breast cancer patients. Further, one representative gene, named IFI30, was quantitatively analyzed by real-time PCR to confirm the result of the cDNA microarray analysis. Results: A total of 427 genes were identified with significantly differential expression, 221 genes were up-regulated and 206 genes were down-regulated. And the result of cDNA microarray analysis was validated by detection of IFI30 mRNA level changes by real-time PCR. Genes for cell proliferation, cell cycle, cell division, mitosis, apoptosis, and immune response were enriched in the up-regulated genes, while genes for cell adhesion, proteolysis, and transport were significantly enriched in the down-regulated genes in breast cancer tissues compared with normal breast tissues by a gene ontology analysis. Conclusion: Our present study revealed a range of differentially expressed genes between breast cancer tissues and normal breast tissues, and provide candidate genes for further study focusing on the pathogenesis and new biomarkers for breast cancer. Keywords: Breast neoplasms, Candidate genes, Microarray

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

    Science.gov (United States)

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

    2012-09-10

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

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

    Science.gov (United States)

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

    2009-04-01

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

  9. Construction and Screening of a Lentiviral Secretome Library.

    Science.gov (United States)

    Liu, Tao; Jia, Panpan; Ma, Huailei; Reed, Sean A; Luo, Xiaozhou; Larman, H Benjamin; Schultz, Peter G

    2017-06-22

    Over 2,000 human proteins are predicted to be secreted, but the biological function of the many of these proteins is still unknown. Moreover, a number of these proteins may act as new therapeutic agents or be targets for the development of therapeutic antibodies. To further explore the extracellular proteome, we have developed a secretome-enriched open reading frame (ORF) library that can be readily screened for autocrine activity in cell-based phenotypic or reporter assays. Next-generation sequencing (NGS) and database analysis predict that the library contains approximately 900 ORFs encoding known secreted proteins (accounting for 77.8% of the library), as well as genes encoding potentially unknown secreted proteins. In a proof-of-principle study, human TF-1 cells were screened for proliferative factors, and the known cytokine GMCSF was identified as a dominant hit. This library offers a relatively low-cost and straightforward approach for functional autocrine screens of secreted proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. PAR-CLIP and streamlined small RNA cDNA library preparation protocol for the identification of RNA binding protein target sites.

    Science.gov (United States)

    Benhalevy, Daniel; McFarland, Hannah L; Sarshad, Aishe A; Hafner, Markus

    2017-04-15

    The study of protein-RNA interactions is critical for our understanding of cellular processes and regulatory circuits controlled by RNA binding proteins (RBPs). Recent next generation sequencing-based approaches significantly promoted our understanding of RNA biology and its importance for cell function. We present a streamlined protocol for Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), a technique that allows for the characterization of RBP binding sites on target RNAs at nucleotide resolution and transcriptome-wide scale. PAR-CLIP involves irreversible UV-mediated crosslinking of RNAs labeled with photoreactive nucleosides to interacting proteins, followed by stringent purification steps and the conversion of crosslinked RNA into small RNA cDNA libraries compatible with next-generation sequencing. The defining hallmark of PAR-CLIP is a diagnostic mutation at the crosslinking site that is introduced into cDNA during the library preparation process. This feature allows for efficient computational removal of contaminating sequences derived from non-crosslinked fragments of abundant cellular RNAs. In the following, we present two different step-by-step procedures for PAR-CLIP, which differ in the small RNA cDNA library preparation procedure: (1) Standard library preparation involving gel size selections after each enzymatic manipulation, and (2) A modified PAR-CLIP procedure ("on-beads" PAR-CLIP), where most RNA manipulations including the necessary adapter ligation steps are performed on the immobilized RNP. This streamlined procedure reduces the protocol preparation time by three days compared to the standard workflow. Copyright © 2016. Published by Elsevier Inc.

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

    Directory of Open Access Journals (Sweden)

    Sebastian Boltaña

    2017-02-01

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

  12. Single-step colony assay for screening antibody libraries.

    Science.gov (United States)

    Kato, Mieko; Hanyu, Yoshiro

    2017-08-10

    We describe a method, single-step colony assay, for simple and rapid screening of single-chain Fv fragment (scFv) libraries. Colonies of Escherichia coli expressing the scFv library are formed on a hydrophilic filter that is positioned in contact with a membrane coated with an antigen. scFv expression is triggered upon treatment of colonies with an induction reagent, following which scFvs are secreted from the cells and diffused to the antigen-coated membrane. scFvs that exhibit binding affinity for the antigen are captured by the membrane-immobilized antigen. Lastly, detection of scFv binding of the antigen on the membrane allows identification of the clones on the filter that express antigen-specific scFvs. We tested this methodology by using an anti-rabbit IgG scFv, scFv(A10B), and a rat immune scFv library. Experiments conducted using scFv(A10B) revealed that this method improves scFv expression during the colony assay. By using our method to screen an immune library of 3×10 3 scFv clones, we established several clones exhibiting affinity for the antigen. Moreover, we tested 7 other antigens, including peptides, and successfully identified positive clones. We believe that this simple procedure and controlled scFv expression of the single-step colony assay could make the antibody screening both rapid and reliable and lead to successful isolation of positive clones from antibody libraries. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Efficient Management of High-Throughput Screening Libraries with SAVANAH

    DEFF Research Database (Denmark)

    List, Markus; Elnegaard, Marlene Pedersen; Schmidt, Steffen

    2017-01-01

    High-throughput screening (HTS) has become an indispensable tool for the pharmaceutical industry and for biomedical research. A high degree of automation allows for experiments in the range of a few hundred up to several hundred thousand to be performed in close succession. The basis...... to be serially diluted before they can be used as assay plates. This process, however, leads to an explosion in the number of plates and samples to be tracked. Here, we present SAVANAH, the first tool to effectively manage molecular screening libraries across dilution series. It conveniently links (connects......) sample information from the library to experimental results from the assay plates. All results can be exported to the R statistical environment or piped into HiTSeekR (http://hitseekr.compbio.sdu.dk) for comprehensive follow-up analyses. In summary, SAVANAH supports the HTS community in managing...

  14. Screening library evolution through automation of solution preparation.

    Science.gov (United States)

    Schopfer, U; Höhn, F; Hueber, M; Girod, M; Engeloch, C; Popov, M; Muckenschnabel, I

    2007-08-01

    The quality of the compound library is a critical success factor in every high-throughput screening campaign. Screening solutions have to be prepared with a high level of process control to ensure the correct identity and initial concentration of each compound. However, even under optimized storage conditions, a certain level of degradation in solution cannot be avoided. Therefore, regular quality control and eventual removal of solutions from the screening deck is necessary. Because solution preparation, especially the weighing of compounds, is a tedious and often manual task, a regular resolubilization of compounds is difficult to achieve. By complete automation of the solution preparation, the authors have laid the foundation for a life cycle management of screening solutions. They demonstrate how a combination of quality and process control leads to a continuous improvement of the screening library. In presenting an automation concept, they show how a series of innovative process optimizations led to a high-performance system that achieves full industrialization of solution preparation.

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Alamar Santiago

    2009-09-01

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

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

    Science.gov (United States)

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

    2009-09-11

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

  18. 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

  19. Polymorphism of the thrombostasin gene in the horn fly (Haematobia irritans) revealed in a cDNA library and in genomic DNA.

    Science.gov (United States)

    Zhang, D; Cupp, M S; Cupp, E W

    2001-10-01

    Thrombostasin (TS) is a newly described thrombin-inhibiting protein isolated from the saliva of the horn fly (Haematobia irritans), a blood-sucking ectoparasite of cattle. This report provides a detailed characterization of the TS gene and the first analysis of the allelic complexity of a gene for an anti-hemostatic protein from a blood-feeding insect. Multiple point mutations at fixed positions in the TS gene were identified in a cDNA library prepared from mRNA isolated from horn fly salivary glands. When translated, the variant mRNAs would specify five biochemically active peptides that differ in molecular weight, isoelectric point and predicted secondary structure. Allelic variation with the same mutation pattern was revealed in the genomes of individual flies collected in the field and sampled from a long-standing laboratory colony. Approximately 60% of flies examined carried heterozygous alleles, including five additional alleles not found in the cDNA library. Comparative analysis of the allelic mutations and the predicted effects on secondary structures of the active proteins produced suggest that the TS gene may be undergoing evolutionary selection.

  20. 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)

    Gao Li; Sun Chong; Qiu Hongling; Liu Hui; Shao Huanjie; Wang Jun; Li Wenxin

    2004-01-01

    To investigate the zinc finger genes involved in human embryonic development, we constructed a C 2 H 2 -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 C 2 H 2 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

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

    Science.gov (United States)

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

    2014-04-19

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

  2. Analysis of Current DNA Encoded Library Screening Data Indicates Higher False Negative Rates for Numerically Larger Libraries.

    Science.gov (United States)

    Satz, Alexander L; Hochstrasser, Remo; Petersen, Ann C

    2017-04-10

    To optimize future DNA-encoded library design, we have attempted to quantify the library size at which the signal becomes undetectable. To accomplish this we (i) have calculated that percent yields of individual library members following a screen range from 0.002 to 1%, (ii) extrapolated that ∼1 million copies per library member are required at the outset of a screen, and (iii) from this extrapolation predict that false negative rates will begin to outweigh the benefit of increased diversity at library sizes >10 8 . The above analysis is based upon a large internal data set comprising multiple screens, targets, and libraries; we also augmented our internal data with all currently available literature data. In theory, high false negative rates may be overcome by employing larger amounts of library; however, we argue that using more than currently reported amounts of library (≫10 nmoles) is impractical. The above conclusions may be generally applicable to other DNA encoded library platforms, particularly those platforms that do not allow for library amplification.

  3. Probe-Directed Degradation (PDD) for Flexible Removal of Unwanted cDNA Sequences from RNA-Seq Libraries.

    Science.gov (United States)

    Archer, Stuart K; Shirokikh, Nikolay E; Preiss, Thomas

    2015-04-01

    Most applications for RNA-seq require the depletion of abundant transcripts to gain greater coverage of the underlying transcriptome. The sequences to be targeted for depletion depend on application and species and in many cases may not be supported by commercial depletion kits. This unit describes a method for generating RNA-seq libraries that incorporates probe-directed degradation (PDD), which can deplete any unwanted sequence set, with the low-bias split-adapter method of library generation (although many other library generation methods are in principle compatible). The overall strategy is suitable for applications requiring customized sequence depletion or where faithful representation of fragment ends and lack of sequence bias is paramount. We provide guidelines to rapidly design specific probes against the target sequence, and a detailed protocol for library generation using the split-adapter method including several strategies for streamlining the technique and reducing adapter dimer content. Copyright © 2015 John Wiley & Sons, Inc.

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

    Directory of Open Access Journals (Sweden)

    Sugantham Priyanka Annabel

    2010-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Yoshinobu Hayashi

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

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

    Science.gov (United States)

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

    2013-01-01

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

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Burgess J Grant

    2009-06-01

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

  9. Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

    Science.gov (United States)

    Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly; Hajighasemi, Mahbod; Nocek, Boguslaw; Khusnutdinova, Anna N.; Brown, Greg; Glinos, Julia; Flick, Robert; Skarina, Tatiana; Chernikova, Tatyana N.; Yim, Veronica; Brüls, Thomas; Paslier, Denis Le; Yakimov, Michail M.; Joachimiak, Andrzej; Ferrer, Manuel; Golyshina, Olga V.; Savchenko, Alexei; Golyshin, Peter N.; Yakunin, Alexander F.

    2017-01-01

    Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools. PMID:28272521

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

    Science.gov (United States)

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

    2014-05-15

    In this study, a full-length enriched cDNA library was successfully constructed from Siberian tiger, the world's most endangered species. The titers of primary and amplified libraries were 1.28×10(6)pfu/mL and 1.59×10(10)pfu/mL respectively. The proportion of recombinants from unamplified library was 91.3% and the average length of exogenous inserts was 1.06kb. A total of 279 individual ESTs with sizes ranging from 316 to 1258bps were then analyzed. Furthermore, 204 unigenes were successfully annotated and involved in 49 functions of the GO classification, cell (175, 85.5%), cellular process (165, 80.9%), and binding (152, 74.5%) are the dominant terms. 198 unigenes were assigned to 156 KEGG pathways, and the pathways with the most representation are metabolic pathways (18, 9.1%). The proportion pattern of each COG subcategory was similar among Panthera tigris altaica, P. tigris tigris and Homo sapiens, and general function prediction only cluster (44, 15.8%) represents the largest group, followed by translation, ribosomal structure and biogenesis (33, 11.8%), replication, recombination and repair (24, 8.6%), and only 7.2% ESTs classified as novel genes. Moreover, the recombinant plasmid pET32a-TAT-COL6A2 was constructed, coded for the Trx-TAT-COL6A2 fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-COL6A2 recombinant protein was 2.64±0.18mg/mL. This library will provide a useful platform for the functional genome and transcriptome research of for the P. tigris and other felid animals in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2015-08-01

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

  12. Construction and identification of a cDNA library for use in the yeast two-hybrid system from duck embryonic fibroblast cells post-infected with duck enteritis virus.

    Science.gov (United States)

    Gao, Xinghong; Jia, Renyong; Wang, Mingshu; Zhu, Dekang; Chen, Shun; Lin, Meng; Yin, Zhongqiong; Wang, Yin; Chen, Xiaoyue; Cheng, Anchun

    2014-01-01

    To explore and isolate genes related to duck embryonic fibroblast cells (DEFs) post-infected with duck enteritis virus (DEV), a cDNA library was established using SMART (Switching Mechanism At 5' end of the RNA Transcript) technique coupling with a homologous recombination method. The cells were harvested and total RNA was extracted at 48 h post infection. Then the mRNAs were purified and reverse transcribed to first-strand cDNAs using oligo (dT) primers (CDS III). Subsequently, long distance-PCR was performed, the double-stranded cDNAs were purified, and a transformation assay was carried out in that order. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 2.33 × 10(6) transformants/4.34 μg pGADT7-Rec (>1.0 × 10(6)). The cell density of the library was 1.75 × 10(9) cells/mL (>2 × 10(7) cells/mL). The titer of the primary cDNA library and amplified cDNA library was 6.75 × 10(5) and 2.33 × 10(7) CFU/mL respectively. The numbers for the primary cDNA library and amplified cDNA library were 1.01 × 10(7) and 1.14 × 10(9), respectively, and the recombinant rate was 97.14 %. The sequence results of 27 randomly picked independent clones revealed the insert ranged from 0.323 to 2.017 kb with an average insert size of 0.807 kb. Full-length transcripts of DEV-CHv LORF3, UL26 and UL35 genes were acquired through sequence similarity analysis from the non-redundant nucleic acid or protein database. Five polyA sites were identified in the DEV-CHv genome. Also, a new transcript of 668 bp was found between the IRS gene and US1 gene of the DEV-CHv genome. Thus, we concluded that the constructed cDNA library will be a useful tool in proteomic analysis of interactions between the DEV and host DEFs, and discovery of biomarkers studies on the mechanism of DEV and subsequently exploitation original vaccines and antiviral drugs to prevent or cure diseases.

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

    International Nuclear Information System (INIS)

    Han, J.H.; Stratowa, C.; Rutter, W.J.

    1987-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Quanze He

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

  15. Construction of a normalized full-length cDNA library of cephalopod Amphioctopus fangsiao and development of microsatellite markers

    Science.gov (United States)

    Feng, Yanwei; Liu, Wenfen; Xu, Xin; Yang, Jianmin; Wang, Weijun; Wei, Xiumei; Liu, Xiangquan; Sun, Guohua

    2017-10-01

    Amphioctopus fangsiao is one of the most economically important species and has been considered to be a candidate for aquaculture. In order to facilitate its fine-scale genetic analyses, we constructed a normalized full-length library successfully and developed a set of microsatellite markers in this study. The normalized full-length library had a storage capacity of 6.9×105 independent clones. The recombination efficiency was 95% and the average size of inserted fragments was longer than 1000 bp. A total of 3440 high quality ESTs were obtained, which were assembled into 1803 unigenes. Of these unigenes, 450 (25%) were assigned into 33 Gene Ontology terms, 576 (31.9%) into 153 Kyoto Encyclopedia of Genes and Genomes pathways, and 275 (15.3%) into 22 Clusters of Orthologous Groups. Seventy-six polymorphic microsatellite markers were identified. The number of alleles per locus ranged from 4 to 17, and the observed and expected heterozygosities varied between 0.167 and 0.967 and between 0.326 and 0.944, respectively. Twelve loci were significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no linkage disequilibrium was found between different loci. This study provided not only a useful resource for the isolation of the functional genes, but also a set of informative microsatellites for the assessment of population structure and conservation genetics of A. fangsiao.

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

    Science.gov (United States)

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

    2012-10-12

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

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

    Directory of Open Access Journals (Sweden)

    Jun-Feng Li

    2012-10-01

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

  18. Screening a Natural Product-Based Library against Kinetoplastid Parasites.

    Science.gov (United States)

    Zulfiqar, Bilal; Jones, Amy J; Sykes, Melissa L; Shelper, Todd B; Davis, Rohan A; Avery, Vicky M

    2017-10-12

    Kinetoplastid parasites cause vector-borne parasitic diseases including leishmaniasis, human African trypanosomiasis (HAT) and Chagas disease. These Neglected Tropical Diseases (NTDs) impact on some of the world's lowest socioeconomic communities. Current treatments for these diseases cause severe toxicity and have limited efficacy, highlighting the need to identify new treatments. In this study, the Davis open access natural product-based library was screened against kinetoplastids ( Leishmania donovani DD8, Trypanosoma brucei brucei and Trypanosoma cruzi ) using phenotypic assays. The aim of this study was to identify hit compounds, with a focus on improved efficacy, selectivity and potential to target several kinetoplastid parasites. The IC 50 values of the natural products were obtained for L. donovani DD8, T. b. brucei and T. cruzi in addition to cytotoxicity against the mammalian cell lines, HEK-293, 3T3 and THP-1 cell lines were determined to ascertain parasite selectivity. Thirty-one compounds were identified with IC 50 values of ≤ 10 µM against the kinetoplastid parasites tested. Lissoclinotoxin E ( 1 ) was the only compound identified with activity across all three investigated parasites, exhibiting IC 50 values natural products with the potential to be new chemical starting points for drug discovery efforts for kinetoplastid diseases were identified.

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

    Directory of Open Access Journals (Sweden)

    Czosnek Henryk

    2006-04-01

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

  20. 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

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    2012-11-01

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

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

    International Nuclear Information System (INIS)

    Permutt, M.A.; Koranyi, L.; Keller, K.; Lacy, P.E.; Scharp, D.W.; Mueckler, M.

    1989-01-01

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

  4. Size unbiased representative enzymatically generated RNAi (SURER) library and application for RNAi therapeutic screens.

    Science.gov (United States)

    Li, Tiejun; Zhu, York Yuanyuan; Chen, Li; Sun, Yuncheng; Yuan, Jian; Graham, Michael; French, Peter

    2015-02-01

    RNA interference (RNAi) libraries screens have become widely used for small RNA (sRNA) therapeutic targets development. However, conventional enzymatically libraries, typically prepared using the type 2 restriction enzyme MmeI, produce sRNAs between 18 and 20 bp, much shorter than the usual lengths of 19-23 bp. Here we develop a size unbiased representative enzymatically generated RNAi (SURER) library, which employs type 3 restriction modification enzyme EcoP15I to produce sRNAs ranging from 19 to 23 bp using a group of rationally designed linkers, which can completely mimic the length of sRNAs naturally generated by Dicer enzyme in living cells, and the screening results of SURER libraries showed high recombination rate and knockdown efficiency. SURER library provides a useful tool for RNAi therapeutics screening in a fast and simple way.

  5. A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

    Science.gov (United States)

    Moser, Lindsey A.; Ramirez-Carvajal, Lisbeth; Puri, Vinita; Pauszek, Steven J.; Matthews, Krystal; Dilley, Kari A.; Mullan, Clancy; McGraw, Jennifer; Khayat, Michael; Beeri, Karen; Yee, Anthony; Dugan, Vivien; Heise, Mark T.; Frieman, Matthew B.; Rodriguez, Luis L.; Bernard, Kristen A.; Wentworth, David E.

    2016-01-01

    ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all

  6. 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

  7. cDNA sequence of the long mRNA for human glutamine synthase

    NARCIS (Netherlands)

    van den Hoff, M. J.; Geerts, W. J.; Das, A. T.; Moorman, A. F.; Lamers, W. H.

    1991-01-01

    Screening a human liver cDNA library in lambda ZAP revealed several clones for the mRNA of glutamine synthase. The longest clone was completely sequenced and consists of a 109 bp 5' untranslated region, a 1119 bp protein coding region, a 1498 bp 3' untranslated region and a poly(A) tract of 12 bp

  8. Toxin Inhibition - Deconvolution Strategies and Assay Screening of Combinatorial Peptide Libraries

    National Research Council Canada - National Science Library

    Lee, W. E; Chan, N. W; Marenco, A. J; Moore, G. J; Moore, D; Hayden, Lawrence J; Laing, T. D; Gregory, M; Mah, D. C; Hamilton, M. G

    2007-01-01

    .... We report on development of a capillary electrophoresis laser-induced fluorescence (CE LIF) method for measuring BoNTIA peptidase activity and for screening peptide libraries for inhibitory effects...

  9. Deiodinase 1 Screening of ToxCast Phase 1 Chemical Library

    Data.gov (United States)

    U.S. Environmental Protection Agency — This excel spreadsheet contains the resultant data for over from inhibition assays with human Deiodinase 1 screened against the ToxCast Phase 1 chemical library and...

  10. Cloning of the cDNA for human 12-lipoxygenase

    International Nuclear Information System (INIS)

    Izumi, T.; Hoshiko, S.; Radmark, O.; Samuelsson, B.

    1990-01-01

    A full-length cDNA clone encoding 12-lipoxygenase was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme

  11. 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....

  12. Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens

    Directory of Open Access Journals (Sweden)

    Olivier Loudig

    2017-03-01

    Full Text Available Formalin-fixed paraffin-embedded (FFPE specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases and women who did not develop invasive breast cancer within the same time interval (control. Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

  13. Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens.

    Science.gov (United States)

    Loudig, Olivier; Wang, Tao; Ye, Kenny; Lin, Juan; Wang, Yihong; Ramnauth, Andrew; Liu, Christina; Stark, Azadeh; Chitale, Dhananjay; Greenlee, Robert; Multerer, Deborah; Honda, Stacey; Daida, Yihe; Spencer Feigelson, Heather; Glass, Andrew; Couch, Fergus J; Rohan, Thomas; Ben-Dov, Iddo Z

    2017-03-14

    Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

  14. Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens

    Science.gov (United States)

    Loudig, Olivier; Wang, Tao; Ye, Kenny; Lin, Juan; Wang, Yihong; Ramnauth, Andrew; Liu, Christina; Stark, Azadeh; Chitale, Dhananjay; Greenlee, Robert; Multerer, Deborah; Honda, Stacey; Daida, Yihe; Spencer Feigelson, Heather; Glass, Andrew; Couch, Fergus J.; Rohan, Thomas; Ben-Dov, Iddo Z.

    2017-01-01

    Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens. PMID:28335433

  15. Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries.

    Science.gov (United States)

    Ong, Swee Hoe; Li, Yilong; Koike-Yusa, Hiroko; Yusa, Kosuke

    2017-08-07

    Genome-wide CRISPR-based knockout (CRISPR-KO) screening is an emerging technique which enables systematic genetic analysis of a cellular or molecular phenotype in question. Continuous improvements, such as modifications to the guide RNA (gRNA) scaffold and the development of gRNA on-target prediction algorithms, have since been made to increase their screening performance. We compared the performance of three available second-generation human genome-wide CRISPR-KO libraries that included at least one of the improvements, and examined the effect of gRNA scaffold, number of gRNAs per gene and number of replicates on screen performance. We identified duplicated screens using a library with 6 gRNAs per gene as providing the best trade-off. Despite the improvements, we found that each improved library still has library-specific false negatives and, for the first time, estimated the false negative rates of CRISPR-KO screens, which are between 10% and 20%. Our newly-defined optimal screening parameters would be helpful in designing screens and constructing bespoke gRNA libraries.

  16. Random small interfering RNA library screen identifies siRNAs that induce human erythroleukemia cell differentiation.

    Science.gov (United States)

    Fan, Cuiqing; Xiong, Yuan; Zhu, Ning; Lu, Yabin; Zhang, Jiewen; Wang, Song; Liang, Zicai; Shen, Yan; Chen, Meihong

    2011-03-01

    Cancers are characterized by poor differentiation. Differentiation therapy is a strategy to alleviate malignant phenotypes by inducing cancer cell differentiation. Here we carried out a combinatorial high-throughput screen with a random siRNA library on human erythroleukemia K-562 cell differentiation. Two siRNAs screened from the library were validated to be able to induce erythroid differentiation to varying degrees, determined by CD235 and globin up-regulation, GATA-2 down-regulation, and cell growth inhibition. The screen we performed here is the first trial of screening cancer differentiation-inducing agents from a random siRNA library, demonstrating that a random siRNA library can be considered as a new resource in efforts to seek new therapeutic agents for cancers. As a random siRNA library has a broad coverage for the entire genome, including known/unknown genes and protein coding/non-coding sequences, screening using a random siRNA library can be expected to greatly augment the repertoire of therapeutic siRNAs for cancers.

  17. Cloning of a cDNA for steroid sulfatase: frequent occurrence of gene deletions in patients with recessive X chromosome-linked ichthyosis

    International Nuclear Information System (INIS)

    Bonifas, J.M.; Morley, B.J.; Oakey, R.E.; Kan, Y.W.; Epstein, E.J. Jr.

    1987-01-01

    A human steroid sulfatase cDNA 2.4 kilobases long was isolated from a human placental λ gt11 cDNA expression library. The library was screened with monospecific rabbit antibodies elicited by injection of steroid sulfatase protein purified from human placentas. Hybridization of the cDNA with EcoRI-digested genomic DNA indicated that patients from 14 of 15 apparently unrelated families have gross deletions of the gene for steroid sulfatase. One patient had genomic DNA fragments that were identical to those from normal individuals, indicating the absence of any major deletions as the cause of his lack of steroid sulfatase enzyme activity

  18. Cloning of a cDNA for steroid sulfatase: frequent occurrence of gene deletions in patients with recessive X chromosome-linked ichthyosis

    Energy Technology Data Exchange (ETDEWEB)

    Bonifas, J.M.; Morley, B.J.; Oakey, R.E.; Kan, Y.W.; Epstein, E.J. Jr.

    1987-12-01

    A human steroid sulfatase cDNA 2.4 kilobases long was isolated from a human placental lambda gt11 cDNA expression library. The library was screened with monospecific rabbit antibodies elicited by injection of steroid sulfatase protein purified from human placentas. Hybridization of the cDNA with EcoRI-digested genomic DNA indicated that patients from 14 of 15 apparently unrelated families have gross deletions of the gene for steroid sulfatase. One patient had genomic DNA fragments that were identical to those from normal individuals, indicating the absence of any major deletions as the cause of his lack of steroid sulfatase enzyme activity.

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

    Science.gov (United States)

    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; Lord, Christopher J

    2011-10-21

    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 rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology.

  20. Enthalpy-Based Screening of Focused Combinatorial Libraries for the Identification of Potent and Selective Ligands.

    Science.gov (United States)

    Baggio, Carlo; Udompholkul, Parima; Barile, Elisa; Pellecchia, Maurizio

    2017-12-15

    In modern drug discovery, the ability of biophysical methods, including nuclear magnetic resonance spectroscopy or surface plasmon resonance, to detect and characterize ligand-protein interactions accurately and unambiguously makes these approaches preferred versus conventional biochemical high-throughput screening of large collections of compounds. Nonetheless, ligand screening strategies that address simultaneously potency and selectivity have not yet been fully developed. In this work, we propose a novel method for screening large collections of combinatorial libraries using enthalpy measurements as a primary screening technique. We demonstrate that selecting binders that are driven by enthalpy (ΔH) results in agents that are not only potent but also more selective for a given target. This general and novel approach, we termed ΔH screening of fPOS (enthalpy screening of focused positional scanning library), combines the principles of focused combinatorial chemistry with rapid calorimetry measurements to efficiently identify potent and selective inhibitors.

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

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

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

  2. Screening of a Brassica napus bacterial artificial chromosome library using highly parallel single nucleotide polymorphism assays

    Science.gov (United States)

    2013-01-01

    Background Efficient screening of bacterial artificial chromosome (BAC) libraries with polymerase chain reaction (PCR)-based markers is feasible provided that a multidimensional pooling strategy is implemented. Single nucleotide polymorphisms (SNPs) can be screened in multiplexed format, therefore this marker type lends itself particularly well for medium- to high-throughput applications. Combining the power of multiplex-PCR assays with a multidimensional pooling system may prove to be especially challenging in a polyploid genome. In polyploid genomes two classes of SNPs need to be distinguished, polymorphisms between accessions (intragenomic SNPs) and those differentiating between homoeologous genomes (intergenomic SNPs). We have assessed whether the highly parallel Illumina GoldenGate® Genotyping Assay is suitable for the screening of a BAC library of the polyploid Brassica napus genome. Results A multidimensional screening platform was developed for a Brassica napus BAC library which is composed of almost 83,000 clones. Intragenomic and intergenomic SNPs were included in Illumina’s GoldenGate® Genotyping Assay and both SNP classes were used successfully for screening of the multidimensional BAC pools of the Brassica napus library. An optimized scoring method is proposed which is especially valuable for SNP calling of intergenomic SNPs. Validation of the genotyping results by independent methods revealed a success of approximately 80% for the multiplex PCR-based screening regardless of whether intra- or intergenomic SNPs were evaluated. Conclusions Illumina’s GoldenGate® Genotyping Assay can be efficiently used for screening of multidimensional Brassica napus BAC pools. SNP calling was specifically tailored for the evaluation of BAC pool screening data. The developed scoring method can be implemented independently of plant reference samples. It is demonstrated that intergenomic SNPs represent a powerful tool for BAC library screening of a polyploid genome

  3. 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.

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

    Science.gov (United States)

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

    2011-12-24

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

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

    Directory of Open Access Journals (Sweden)

    Ke Tao

    2011-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Cassandra Stowe

    Full Text Available 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.

  7. Comparative analysis of machine learning methods in ligand-based virtual screening of large compound libraries.

    Science.gov (United States)

    Ma, Xiao H; Jia, Jia; Zhu, Feng; Xue, Ying; Li, Ze R; Chen, Yu Z

    2009-05-01

    Machine learning methods have been explored as ligand-based virtual screening tools for facilitating drug lead discovery. These methods predict compounds of specific pharmacodynamic, pharmacokinetic or toxicological properties based on their structure-derived structural and physicochemical properties. Increasing attention has been directed at these methods because of their capability in predicting compounds of diverse structures and complex structure-activity relationships without requiring the knowledge of target 3D structure. This article reviews current progresses in using machine learning methods for virtual screening of pharmacodynamically active compounds from large compound libraries, and analyzes and compares the reported performances of machine learning tools with those of structure-based and other ligand-based (such as pharmacophore and clustering) virtual screening methods. The feasibility to improve the performance of machine learning methods in screening large libraries is discussed.

  8. High-throughput, image-based screening of pooled genetic-variant libraries.

    Science.gov (United States)

    Emanuel, George; Moffitt, Jeffrey R; Zhuang, Xiaowei

    2017-12-01

    We report a high-throughput screening method that allows diverse genotypes and corresponding phenotypes to be imaged in individual cells. We achieve genotyping by introducing barcoded genetic variants into cells as pooled libraries and reading the barcodes out using massively multiplexed fluorescence in situ hybridization. To demonstrate the power of image-based pooled screening, we identified brighter and more photostable variants of the fluorescent protein YFAST among 60,000 variants.

  9. 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 - others: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: 4.495, year: 2015

  10. Synthesis and screening of materials libraries of buried compound semiconductors by ion beam implantation

    OpenAIRE

    Stritzker, Bernd

    2004-01-01

    Synthesis and screening of materials libraries of buried compound semiconductors by ion beam implantation / I. Großhans, H. Karl and B. Stritzker. - In: Combinatorial and artificial intelligence methods in materials science II : symposium held December 1 - 4, 2003, Boston, Massachusetts, USA / ed.: Radislav A. Potyrailo ... - Warrendale, Pa. : Materials Research Society, 2004. - (Materials Research Society symposium proceedings ; 804)

  11. Systematic hybrid LOH: a new method to reduce false positives and negatives during screening of yeast gene deletion libraries

    DEFF Research Database (Denmark)

    Alvaro, D.; Sunjevaric, I.; Reid, R. J.

    2006-01-01

    We have developed a new method, systematic hybrid loss of heterozygosity, to facilitate genomic screens utilizing the yeast gene deletion library. Screening is performed using hybrid diploid strains produced through mating the library haploids with strains from a different genetic background...... complementation of any spurious recessive mutations in the library strain, facilitating attribution of the observed phenotype to the documented gene deletion and dramatically reducing false positive results commonly obtained in library screens. The systematic hybrid LOH method can be applied to virtually any...

  12. Using ZINC to acquire a virtual screening library.

    Science.gov (United States)

    Irwin, John J

    2008-06-01

    The ZINC database of commercially available compounds contains biologically relevant representations of purchasable compounds in ready-to-screen formats. ZINC uses catalogs from over 50 compound vendors, changing from time to time to reflect newly available compounds and depleted stock. ZINC is available in subsets that reflect current opinion in the field such as "fragment-like" and "lead-like," and has a facility to create additional small ad hoc subsets. ZINC has a search facility, as well as a service to process molecules that are not in ZINC by uploading them to a server. Copyright 2008 by John Wiley & Sons, Inc.

  13. Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH)

    NARCIS (Netherlands)

    Leveau, J.H.J.; Gerards, S.; De Boer, W.; Van Veen, J.A.

    2004-01-01

    We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia

  14. Screening Phage-Display Antibody Libraries Using Protein Arrays.

    Science.gov (United States)

    Jara-Acevedo, Ricardo; Díez, Paula; González-González, María; Dégano, Rosa María; Ibarrola, Nieves; Góngora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2018-01-01

    Phage-display technology constitutes a powerful tool for the generation of specific antibodies against a predefined antigen. The main advantages of phage-display technology in comparison to conventional hybridoma-based techniques are: (1) rapid generation time and (2) antibody selection against an unlimited number of molecules (biological or not). However, the main bottleneck with phage-display technology is the validation strategies employed to confirm the greatest number of antibody fragments. The development of new high-throughput (HT) techniques has helped overcome this great limitation. Here, we describe a new method based on an array technology that allows the deposition of hundreds to thousands of phages by micro-contact on a unique nitrocellulose surface. This setup comes in combination with bioinformatic approaches that enables simultaneous affinity screening in a HT format of antibody-displaying phages.

  15. Immunoassays: biological tools for high throughput screening and characterisation of combinatorial libraries.

    Science.gov (United States)

    Taipa, M Angela

    2008-05-01

    In the demanding field of proteomics, there is an urgent need for affinity-catcher molecules to implement effective and high throughput methods for analysing the human proteome or parts of it. Antibodies have an essential role in this endeavour, and selection, isolation and characterisation of specific antibodies represent a key issue to meet success. Alternatively, it is expected that new, well-characterised affinity reagents generated in rapid and cost-effective manners will also be used to facilitate the deciphering of the function, location and interactions of the high number of encoded protein products. Combinatorial approaches combined with high throughput screening (HTS) technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries and the lead discovery of active/mimic molecules in large chemical libraries. Phage and yeast display provide the means for engineering a multitude of antibody-like molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterisation of ligand-receptor specific interactions, and the search for novel ligands for protein purification. Further improvement of chemical and biological resistance of affinity ligands encouraged the "intelligent" design and synthesis of chemical libraries of low-molecular-weight bio-inspired mimic compounds. No matter what the ligand source, selection and characterisation of leads is a most relevant task. Immunological assays, in microtiter plates, biosensors or microarrays, are a biological tool of inestimable value for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. Particularly, enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies, for both biological and chemical libraries.

  16. Role of normalization in the elimination of abundant myelin sequences in spinal cord cDNA libraries produced by suppression subtractive hybridization.

    Science.gov (United States)

    Lathia, K B; Yan, Z; Clapshaw, P A

    2009-12-01

    Spinal cord libraries subtracted against visual cortex using suppression subtractive hybridization SSH are dominated by abundant gene sequences derived from myelin elements. We compared our subtracted library results of three of these abundant sequences to published expressed sequence tag libraries that are not normalized and not subtracted and presumed representatives of murine spinal cord mRNA abundance. We show that: all three abundant sequences, myelin basic protein (Mbp), proteolipid protein (Plp1) and Ferretin heavy chain (Fth1) are highly expressed in spinal cord when this structure is compared to visual cortex; myelin basic protein is represented in our subtracted libraries but at a low frequency, whereas Plp1 and Fth1 represent nearly one-third of all sequences in these libraries; mirror orientation selection, a procedure designed to reduce background sequences, generates libraries very similar in abundance to SSH; proteolipid protein can be reduced in these libraries by adding Plp1 sequences to the driver in the SSH procedure and also by subtracting Plp1 directly from tester and driver. We conclude that adequate normalization is essential to reduce the presence of abundant sequences in SSH libraries.

  17. Discovering novel enzymes by functional screening of plurigenomic libraries from alga-associated Flavobacteriia and Gammaproteobacteria.

    Science.gov (United States)

    Martin, Marjolaine; Vandermies, Marie; Joyeux, Coline; Martin, Renée; Barbeyron, Tristan; Michel, Gurvan; Vandenbol, Micheline

    2016-01-01

    Alga-associated microorganisms, in the context of their numerous interactions with the host and the complexity of the marine environment, are known to produce diverse hydrolytic enzymes with original biochemistry. We recently isolated several macroalgal-polysaccharide-degrading bacteria from the surface of the brown alga Ascophyllum nodosum. These active isolates belong to two classes: the Flavobacteriia and the Gammaproteobacteria. In the present study, we constructed two "plurigenomic" (with multiple bacterial genomes) libraries with the 5 most interesting isolates (regarding their phylogeny and their enzymatic activities) of each class (Fv and Gm libraries). Both libraries were screened for diverse hydrolytic activities. Five activities, out of the 48 previously identified in the natural polysaccharolytic isolates, were recovered by functional screening: a xylanase (GmXyl7), a beta-glucosidase (GmBg1), an esterase (GmEst7) and two iota-carrageenases (Fvi2.5 and Gmi1.3). We discuss here the potential role of the used host-cell, the average DNA insert-sizes and the used restriction enzymes on the divergent screening yields obtained for both libraries and get deeper inside the "great screen anomaly". Interestingly, the discovered esterase probably stands for a novel family of homoserine o-acetyltransferase-like-esterases, while the two iota-carrageenases represent new members of the poorly known GH82 family (containing only 19 proteins since its description in 2000). These original results demonstrate the efficiency of our uncommon "plurigenomic" library approach and the underexplored potential of alga-associated cultivable microbiota for the identification of novel and algal-specific enzymes. Copyright © 2016 Elsevier GmbH. All rights reserved.

  18. A platform for high-throughput screening of DNA-encoded catalyst libraries in organic solvents.

    Science.gov (United States)

    Hook, K Delaney; Chambers, John T; Hili, Ryan

    2017-10-01

    We have developed a novel high-throughput screening platform for the discovery of small-molecules catalysts for bond-forming reactions. The method employs an in vitro selection for bond-formation using amphiphilic DNA-encoded small molecules charged with reaction substrate, which enables selections to be conducted in a variety of organic or aqueous solvents. Using the amine-catalysed aldol reaction as a catalytic model and high-throughput DNA sequencing as a selection read-out, we demonstrate the 1200-fold enrichment of a known aldol catalyst from a library of 16.7-million uncompetitive library members.

  19. How to Successfully Screen Random Adeno-Associated Virus Display Peptide Libraries In Vivo.

    Science.gov (United States)

    Körbelin, Jakob; Trepel, Martin

    2017-06-01

    Adeno-associated virus (AAV) has emerged as a very promising gene therapy vector. To enable tissue-directed gene expression, many artificially generated AAV variants have been established, often isolated from large pools of mutated capsids. Random peptide libraries displayed on AAV capsids have been used successfully to select vectors targeted to a given target cell or tissue in vitro and in vivo. However, the published methodology for screening of AAV libraries to isolate vectors with selective tissue tropism after intravenous administration in vivo has not been described in sufficient detail to address all critical steps. A step-by-step protocol is provided here.

  20. 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

  1. Arrayed mutant haploid embryonic stem cell libraries facilitate phenotype-driven genetic screens.

    Science.gov (United States)

    Liu, Guang; Wang, Xue; Liu, Yufang; Zhang, Meili; Cai, Tao; Shen, Zhirong; Jia, Yuyan; Huang, Yue

    2017-12-15

    Forward genetic screens using mammalian embryonic stem (ES) cells have identified genes required for numerous cellular processes. However, loss-of-function screens are more difficult to conduct in diploid cells because, in most cases, both alleles of a gene must be mutated to exhibit a phenotype. Recently, mammalian haploid ES cell lines were successfully established and applied to several recessive genetic screens. However, all these screens were performed in mixed pools of mutant cells and were mainly based on positive selection. In general, negative screening is not easy to apply to these mixed pools, although quantitative deep sequencing of mutagen insertions can help to identify some 'missing' mutants. Moreover, the interplay between different mutant cells in the mixed pools would interfere with the readout of the screens. Here, we developed a method for rapidly generating arrayed haploid mutant libraries in which the proportion of homozygous mutant clones can reach 85%. After screening thousands of individual mutant clones, we identified a number of novel factors required for the onset of differentiation in ES cells. A negative screen was also conducted to discover mutations conferring cells with increased sensitivity to DNA double-strand breaks induced by the drug doxorubicin. Both of these screens illustrate the value of this system. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Directory of Open Access Journals (Sweden)

    Elnaiem Dia-Eldin

    2008-01-01

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

  3. Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology.

    Science.gov (United States)

    Duarte, José M; Barbier, Içvara; Schaerli, Yolanda

    2017-11-17

    Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g., being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g., expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.

  4. Engineering of Immunoglobulin Fc Heterodimers Using Yeast Surface-Displayed Combinatorial Fc Library Screening.

    Directory of Open Access Journals (Sweden)

    Hye-Ji Choi

    Full Text Available Immunoglobulin Fc heterodimers, which are useful scaffolds for the generation of bispecific antibodies, have been mostly generated through structure-based rational design methods that introduce asymmetric mutations into the CH3 homodimeric interface to favor heterodimeric Fc formation. Here, we report an approach to generate heterodimeric Fc variants through directed evolution combined with yeast surface display. We developed a combinatorial heterodimeric Fc library display system by mating two haploid yeast cell lines, one haploid cell line displayed an Fc chain library (displayed FcCH3A with mutations in one CH3 domain (CH3A on the yeast cell surface, and the other cell line secreted an Fc chain library (secreted FcCH3B with mutations in the other CH3 domain (CH3B. In the mated cells, secreted FcCH3B is displayed on the cell surface through heterodimerization with the displayed FcCH3A, the detection of which enabled us to screen the library for heterodimeric Fc variants. We constructed combinatorial heterodimeric Fc libraries with simultaneous mutations in the homodimer-favoring electrostatic interaction pairs K370-E357/S364 or D399-K392/K409 at the CH3 domain interface. High-throughput screening of the libraries using flow cytometry yielded heterodimeric Fc variants with heterodimer-favoring CH3 domain interface mutation pairs, some of them showed high heterodimerization yields (~80-90% with previously unidentified CH3 domain interface mutation pairs, such as hydrogen bonds and cation-π interactions. Our study provides a new approach for engineering Fc heterodimers that could be used to engineer other heterodimeric protein-protein interactions through directed evolution combined with yeast surface display.

  5. Development of an HIV-based cDNA expression cloning system.

    Science.gov (United States)

    van Maanen, Marc; Tidwell, Jennie K; Donehower, Lawrence A; Sutton, Richard E

    2003-07-01

    Expression cloning of cDNAs is a powerful tool with which to identify genes based on their specific functional properties. Here we describe the development of a cDNA library transfer system based on the human immunodeficiency virus type-1 (HIV). This system represents an improvement over current oncoretroviral cDNA expression systems in terms of target cell range and the inclusion of a selectable marker. By use of a simple packaging system, we were able to produce high-titer vector stocks from HIV vector-based cDNA libraries and demonstrate highly efficient cDNA expression cloning in three model experiments. First, HOS TK(-) cells, which are null for thymidine kinase (TK) expression, were transduced with an HIV-based cDNA library derived from primary human foreskin fibroblasts (HFFs) and functionally selected for TK expression. In a second experiment, hypoxanthine guanine phosphoribosyltransferase-1-deficient (HPRT(-)) fibroblasts were transduced with a T cell (PM1) line-derived cDNA library and selected for HPRT expression. Both TK (frequency 1 in 5.0 x 10(4)) and HPRT (frequency 1 in 2.0 x 10(4)) cDNAs were readily isolated from these HIV-based cDNA libraries. As a third example, we demonstrated the ability of this vector system to allow functional cDNA library screens to be performed in primary, mitotically inactive cell types. Using senescent HFFs as a target cell population, we were able to isolate SV40 large T antigen cDNA-containing clones (frequency 1 in 2.5 x 10(4)) based on their ability to overcome the senescence-induced block to cell proliferation. Thus, this system can be used to clone relatively low-abundance cDNAs based upon their expression. Because of the ability of HIV-based vectors to transduce primary and nondividing cells efficiently, this vector system will further broaden the range of cell types in which expression cloning studies can be performed.

  6. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Evans, D.M.; Williams, K.P.; McGuinness, B. [PerSeptive Biosystems, Framingham, MA (United States)] [and others

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  7. Synthetic molecular evolution of pore–forming peptides by Iterative combinatorial library screening

    Science.gov (United States)

    Krauson, Aram J.; He, Jing; Wimley, Andrew W.; Hoffmann, Andrew R.; Wimley, William C

    2013-01-01

    We previously reported the de novo design of a combinatorial peptide library that was subjected to high–throughput screening to identify membrane permeabilizing antimicrobial peptides that have β–sheet–like secondary structure. Those peptides do not form discreet pores in membranes but instead partition into membrane interfaces and cause transient permeabilization by membrane disruption, but only when present at high concentration. In this work, we used a consensus sequence from that initial screen as a template to design an iterative, second generation library. In the 24–26–residue, 16,200 member second generation library we varied six residues. Two diad repeat motifs of alternating polar and nonpolar amino acids were preserved to maintain a propensity for non–helical secondary structure. We used a new high–throughput assay to identify members that self–assemble into equilibrium pores in synthetic lipid bilayers. This screen was done at a very stringent peptide to lipid ratio of 1:1000 where most known membrane permeabilizing peptides, including the template peptide, are not active. In a screen of 10,000 library members we identified 16 (~0.2%) that are equilibrium pore–formers at this high stringency. These rare and highly active peptides, which share a common sequence motif, are as potent as the most active pore–forming peptides known. Furthermore, they are not α–helical, which makes them unusual, as most of the highly potent pore–forming peptides are amphipathic α–helices. Here we demonstrate that this synthetic molecular evolution–based approach, taken together with the new high–throughput tools we have developed, enables the identification, refinement and optimization of unique membrane active peptides. PMID:23394375

  8. Synthetic molecular evolution of pore-forming peptides by iterative combinatorial library screening.

    Science.gov (United States)

    Krauson, Aram J; He, Jing; Wimley, Andrew W; Hoffmann, Andrew R; Wimley, William C

    2013-04-19

    We previously reported the de novo design of a combinatorial peptide library that was subjected to high-throughput screening to identify membrane-permeabilizing antimicrobial peptides that have β-sheet-like secondary structure. Those peptides do not form discrete pores in membranes but instead partition into membrane interfaces and cause transient permeabilization by membrane disruption, but only when present at high concentration. In this work, we used a consensus sequence from that initial screen as a template to design an iterative, second generation library. In the 24-26-residue, 16,200-member second generation library we varied six residues. Two diad repeat motifs of alternating polar and nonpolar amino acids were preserved to maintain a propensity for non-helical secondary structure. We used a new high-throughput assay to identify members that self-assemble into equilibrium pores in synthetic lipid bilayers. This screen was done at a very stringent peptide to lipid ratio of 1:1000 where most known membrane-permeabilizing peptides, including the template peptide, are not active. In a screen of 10,000 library members we identified 16 (~0.2%) that are equilibrium pore-formers at this high stringency. These rare and highly active peptides, which share a common sequence motif, are as potent as the most active pore-forming peptides known. Furthermore, they are not α-helical, which makes them unusual, as most of the highly potent pore-forming peptides are amphipathic α-helices. Here we demonstrate that this synthetic molecular evolution-based approach, taken together with the new high-throughput tools we have developed, enables the identification, refinement, and optimization of unique membrane active peptides.

  9. Characterization of the BPI-like gene from a subtracted cDNA library of large yellow croaker (Pseudosciaena crocea) and induced expression by formalin-inactivated Vibrio alginolyticus and Nocardia seriolae vaccine challenges.

    Science.gov (United States)

    Huang, Yanqing; Lou, Huifang; Wu, Xinzhong; Chen, Yanxia

    2008-12-01

    One expressed sequence tag (EST 64LF004 clone), which is from the subtracted cDNA library of the head kidney of large yellow croaker (Pseudosciaena crocea) stimulated with peptidoglycan (PG) by suppression subtractive hybridization (SSH) method, was cloned using RACE-PCR. The full length cDNA, which possesses typical structural features of a signal peptide, a conserved LPS binding domain and two bactericidal permeability-increasing (BPI) motifs as in higher vertebrates, was identified as a novel homologue, namely of the large yellow croaker BPI-like molecule (Pc-BPI-L). Phylogenetic analysis showed this Pc-BPI-L of large yellow croaker as the most ancestral branch in bony fish clade. The recombinant Pc-BPI-L protein expressed in the Tn-5B1-4 insect cells was successfully produced and confirmed to have the predicted size of 52 kDa by Western blot analysis. At the message level, Pc-BPI-L mRNA was ubiquitously expressed in all tissues examined. Following formalin-inactivated Vibrio alginolyticus and Nocardia seriolae treatment, Pc-BPI-L message was differentially up-regulated in primary immune organs. These results indicate that Pc-BPI-L might be involved in the immune response to bacterial infection.

  10. Construction and analysis of antennal cDNA library from rice striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), and expression profiles of putative odorant-binding protein and chemosensory protein genes.

    Science.gov (United States)

    Gong, Zhong-Jun; Liu, Su; Jiang, Yan-Dong; Zhou, Wen-Wu; Liang, Qing-Mei; Cheng, Jiaan; Zhang, Chuan-Xi; Zhu, Zeng-Rong; Gurr, Geoff M

    2015-05-01

    In this study, we constructed a high-quality cDNA library from the antennae of the Chilo suppressalis (Walker) (Lepidoptera: Pyralidae). A total of 1,235 colonies with inserts greater than 0.7 kb were sequenced and analyzed. Homology searching coupled with bioinformatics analysis identified 15 and 7 cDNA sequences, respectively, encoding putative odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). A phylogenetic tree of CsupCSPs showed that each CsupCSP has orthologs in Manduca sexta and Bombyx mori with strong bootstrapping support. One CSP was either very specific or more related to the CSPs of another species than to conspecific CSP. The expression profiles of the OBPs and CSPs in different tissues were measured by real-time quantitative PCR. The results revealed that of the 11 OBP genes, the transcript levels of CsupOBP1, CsupOBP5, and CsupOBP7 were higher in both male and female antennae than those in other tissues. And CsupCSP7 was highly expressed in both male and female antennae. Based on these results, the possible physiological functions of CsupOBPs and CsupCSPs were discussed. © 2015 Wiley Periodicals, Inc.

  11. Construction, arraying, and high-density screening of large insert libraries of human chromosomes X and 21: Their potential use as reference libraries

    Energy Technology Data Exchange (ETDEWEB)

    Nizetic, D.; Zehetner, G.; Monaco, A.P.; Gellen, L.; Lehrach, H. (Imperial Cancer Research Fund, London (England)); Young, B.D. (St. Bartholomew' s Hospital, London (England))

    1991-04-15

    The authors have constructed cosmid libraries from flow-sorted human chromosomes X and 21, each of which contains {gt}30 genome equivalents, and have developed systems allowing permanent storage of primary clones, easy screening of libraries in high-density filter formats, and the simultaneous generation of fingerprinting and mapping data on the same set of cosmid clones. Clones are picked into microtiter plate wells and stored at {minus}70C. A semiautomatic robot system allows the generation of filter replicas containing up to 10,000 clones per membrane. Sets of membranes containing 15-20 chromosome equivalents of both chromosomes will be used for the construction of ordered clone libraries by hybridization fingerprinting protocols. The authors describe the construction of the libraries and demonstrate the use of high-density screening filters in oligonucleotide probe hybridizations and the isolation of cosmids by hybridization with probes from the X chromosome.

  12. A new method for the screening of solid-phase combinatorial libraries for affinity chromatography.

    Science.gov (United States)

    Roque, A Cecília A; Taipa, M Angela; Lowe, Christopher R

    2004-01-01

    A new methodology for the rapid assessment of affinity ligands synthesized by combinatorial solid-phase chemistry is reported. This screening strategy utilizes the target protein conjugated to FITC, and represents an almost universal technique for the preliminary screening of solid-phase combinatorial libraries. The assessment of a triazine-scaffolded solid-phase combinatorial library of ligands, designed to bind to human IgG, was performed with FITC-human IgG, and the results compared with those obtained by conventional affinity chromatographic screening assays. The effect of different molar conjugation ratios of FITC-IgG (F/P) was evaluated. Independently of the F/P ratio, no false negative results were observed, although lower F/P ratios diminished non-specific interactions and the number of false positives. The nature of the substituents on the triazine scaffold was not related to the number of false positive IgG-binding ligands. The reproducibility of the FITC technique, using FITC-human IgG conjugates with low F/P ratio (F/P=2), was also evaluated. The FITC-based technique proved to be efficient and accurate in the identification of strongly binding ligands (binding >50% of loaded protein, by standard affinity chromatographic assays), and is envisaged as a versatile and cost-effective method to screen other systems, and evaluate several binding/elution conditions at small-scale, prior to scale-up to standard affinity chromatography. Copyright 2004 John Wiley & Sons, Ltd.

  13. The programmed cell death GLuc PCA library – a powerful tool for pathway discovery and drug screening

    Science.gov (United States)

    Gilad, Yuval; Kimchi, Adi

    2014-01-01

    A programmed cell death library based on the Gaussia luciferase protein-fragment complementation assay (GLuc PCA) enables detection of protein–protein interactions (PPI) within the cell death network and quantitative assessments of these interactions. Among future applications for the GLuc PCA cell death library is its potential use as a platform for PPI-targeted drug screening. PMID:27308378

  14. BiebBeep : an interactive screen for supporting public library 2.0 information and social services

    NARCIS (Netherlands)

    Kanis, Marije; Meys, Wouter; Groen, Maarten; Slakhorst, Wout; Veenstra, Mettina

    2011-01-01

    This video presents BiebBeep, an interactive touchscreen system that has been developed with the aim to support information and social services for the New Library in Almere, The Netherlands. The constantly updated information displayed on the interactive screen concerns not only the library itself,

  15. Screening of a library of FDA-approved drugs identifies several enterovirus replicaton inhibitors that target viral protein 2C

    NARCIS (Netherlands)

    Ulferts, Rachel; de Boer, 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

    2016-01-01

    Enteroviruses (EV) 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 and identified pirlindole as

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

    NARCIS (Netherlands)

    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.

    2016-01-01

    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

  17. [Screening of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) by cDNA microarray and influence of overexpression of PAG1 on biologic behavior of human metastatic prostatic cancer cell line in vitro].

    Science.gov (United States)

    Yu, Wen-juan; Wang, Yue-wei; Xie, Zhi-gang; You, Jiang-feng; Wang, Jie-liang; Cui, Xiang-lin; Pei, Fei; Zheng, Jie

    2010-02-01

    To screen for novel gene(s) associated with tumor metastasis, and to investigate the effect of overexpression of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behaviors of human prostatic cancer cell line PC-3M-1E8 in vitro. Four cDNA microarrays were constructed using cDNA library of prostatic cancer cells PC-3M-1E8 (high metastatic potential), PC-3M-2B4 (low metastatic potential), lung cancer cells PG-BE1 (high metastatic potential)and PG-LH7 (low metastatic potential)to screen genes which were differentially expressed according to their different metastatic properties. From a battery of differentially expressed genes, PAG1, which was markedly downregulated in both high metastatic sublines of PC-3M and PG was chosen for further investigation. Real-time PCR and Western blot were used to confirm the gene expression of PAG1 at mRNA and protein levels. Full-length coding sequence of human PAG1 was subcloned into plasmid pcDNA3.0 and the recombinant plasmids were stably transfected into PC-3M-1E8. The cell proliferation ability, anchorage-independent growth, cell cycle distribution, apoptosis rates and invasive ability were detected by MTT, and in addition, soft agar colony formation, flow cytometry analysis and matrigel invasion assay using Boyden chamber were also carried out respectively. All experiments contained pcDNA3.0-PAG1-transfected clones, vector transfected clones and non-transfected parental cells. A total of 327 differentially expressed genes were obtained between the high and low metastatic sublines of PC-3M cells, including 123 upregulated and 204 downregulated genes in PC-3M-1E8. A total of 281 genes, including 167 upregulated and 114 downregulated genes were obtained in PG-BE1 cells. Nine genes were simultaneously downregulated and 8 genes were upregulated in both high metastatic cell lines of PC-3M and PG. The expression of PAG1 at mRNA and protein level were decreased in the high metastatic subline PC-3M-1

  18. Automated panning and screening procedure on microplates for antibody generation from phage display libraries.

    Science.gov (United States)

    Turunen, Laura; Takkinen, Kristiina; Söderlund, Hans; Pulli, Timo

    2009-03-01

    Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human gamma-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 gamma-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and beta-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 beta-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency.

  19. [Combining SSH and cDNA microarray for identification of lung cancer related genes].

    Science.gov (United States)

    Fan, Baoxing; Zhang, Kaitai; Da, Jiping; Xie, Ling; Wang, Shengqi; Wu, Dechang

    2003-04-20

    To screen and identify differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and some other kinds of tumor tissues using suppression subtractive hybridization (SSH) and cDNA Microarray. One cDNA chip was made by gathering clones of three differentially expressed cDNA libraries which came from BEP2D cell lines during three different malignant transformed phases. Then the clones were hybridizated with cDNA probes which extracted from 15 cases of lung cancer tissues, 5 cases of paracancerous pulmonary tissues and 24 cases of other 8 kinds of tumor tissues respectively. Twenty-six cDNAs were obtained which expressed higher in lung cancer tissues than that in paracancerous pulmonary tissues. Thirty-one cDNAs expressed remarkably higher in paracancerous tissues than those in cancer tissues. Compared with other 8 kinds of tumors, paracancerous tissues had 63 overexpressed cDNAs and lung cancer tissues had 87 overexpressed cDNAs. The combination of SSH and cDNA microarray is rapid and effective for screening and identification of differentially expressed genes in different samples. It may be potentially useful for diagnosis of lung cancer to further study the differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and other tumor tissues.

  20. High-content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries.

    Science.gov (United States)

    Strezoska, Žaklina; Perkett, Matthew R; Chou, Eldon T; Maksimova, Elena; Anderson, Emily M; McClelland, Shawn; Kelley, Melissa L; Vermeulen, Annaleen; Smith, Anja van Brabant

    2017-06-10

    The CRISPR-Cas9 system has been utilized for large-scale, loss-of-function screens mainly using lentiviral pooled formats and cell-survival phenotypic assays. Screening in an arrayed format expands the types of phenotypic readouts that can be used to now include high-content, morphology-based assays, and with the recent availability of synthetic crRNA libraries, new studies are emerging. Here, we use a cell cycle reporter cell line to perform an arrayed, synthetic crRNA:tracrRNA screen targeting 169 genes (>600 crRNAs) and used high content analysis (HCA) to identify genes that regulate the cell cycle. Seven parameters were used to classify cells into cell cycle categories and multiple parameters were combined using a new analysis technique to identify hits. Comprehensive hit follow-up experiments included target gene expression analysis, confirmation of DNA insertions/deletions, and validation with orthogonal reagents. Our results show that most hits had three or more independent crRNAs per gene that demonstrated a phenotype with consistent individual parameters, indicating that our screen produced high-confidence hits with low off-target effects and allowed us to identify hits with more subtle phenotypes. The results of our screen demonstrate the power of using arrayed, synthetic crRNAs for functional phenotypic screening using multiparameter HCA assays. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  1. Sustainable synthesis and automated deposition: an accessible discovery screening library of fragment-like purines.

    Science.gov (United States)

    Kamper, Christoph; Korpis, Katharina; Specker, Edgar; Anger, Lennart; Neuenschwander, Martin; Bednarski, Patrick J; Link, Andreas

    2012-08-01

    A sub-library of 88 information-rich lead-like purine derivatives were prepared and deposited in an open access academic screening facility. The rationale for the synthesis of these rigid low complexity structures was the privileged character of the purine heterocycle associated with its inherent probability of interactions with multiple adenine-related targets. Although generally expected to be weak binders in many assays, such fragment-like compounds are estimated to match diverse binding sites. It is suggested that heterocycles with many anchor points for hydrogen bonds can be anticipated to undergo very specific interactions to produce more negative enthalpies and thus provide superior starting points for lead optimization than compounds that owe their activity to entropic effects. The in vitro cytotoxicity of the small compounds on a panel of human cancer cell lines has been investigated and some of them showed marked unselective or selective toxicity. This data may be useful if these fragments are to be incorporated into drug-like structures via metabolically cleavable connections. The sub-library will be implemented as part of the ChemBioNet ( www.chembionet.info ) library, and it is open to screening campaigns of academic research groups striving for a fragment-based approach in their biological assays.

  2. Physicochemical Profiles of the Marketed Agrochemicals and Clues for Agrochemical Lead Discovery and Screening Library Development.

    Science.gov (United States)

    Rao, Hanbing; Huangfu, Changxin; Wang, Yanying; Wang, Xianxiang; Tang, Tiansheng; Zeng, Xianyin; Li, Zerong; Chen, Yuzong

    2015-05-01

    Combinatorial chemistry, high-throughput and virtual screening technologies have been extensively used for discovering agrochemical leads from chemical libraries. The knowledge of the physicochemical properties of the marketed agrochemicals is useful for guiding the design and selection of such libraries. Since the earlier profiling of marketed agrochemicals, the number and types of marketed agrochemicals have significantly increased. Recent studies have shown the change of some physicochemical properties of oral drugs with time. There is a need to also profile the physicochemical properties of the marketed agrochemicals. In this work, we analyzed the key physicochemical properties of 1751 marketed agrochemicals in comparison with the previously-analyzed herbicides and insecticides, 106 391 natural products and 57 548 diverse synthetic libraries compounds. Our study revealed the distribution profiles and evolution trend of different types of agrochemicals that in many respects are broadly similar to the reported profiles for oral drugs, with the most marked difference being that agrochemicals have a lower number of hydrogen bond donors. The derived distribution patterns provided the rule of thumb guidelines for selecting potential agrochemical leads and also provided clues for further improving the libraries for agrochemical lead discovery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Horse cDNA clones encoding two MHC class I genes

    Energy Technology Data Exchange (ETDEWEB)

    Barbis, D.P.; Maher, J.K.; Stanek, J.; Klaunberg, B.A.; Antczak, D.F.

    1994-12-31

    Two full-length clones encoding MHC class I genes were isolated by screening a horse cDNA library, using a probe encoding in human HLA-A2.2Y allele. The library was made in the pcDNA1 vector (Invitrogen, San Diego, CA), using mRNA from peripheral blood lymphocytes obtained from a Thoroughbred stallion (No. 0834) homozygous for a common horse MHC haplotype (ELA-A2, -B2, -D2; Antczak et al. 1984; Donaldson et al. 1988). The clones were sequenced, using SP6 and T7 universal primers and horse-specific oligonucleotides designed to extend previously determined sequences.

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

    Science.gov (United States)

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

    2015-12-01

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

  5. Directed evolution of DNA polymerases: construction and screening of DNA polymerase mutant libraries.

    Science.gov (United States)

    Gloeckner, Christian; Kranaster, Ramon; Marx, Andreas

    2010-06-01

    The protocols in this article describe the construction of a mutant DNA polymerase library using error-prone PCR (epPCR) as a method for gene randomization, followed by screening of the library using two different approaches. The examples described use an N-terminally truncated form of the thermostable DNA polymerase I of Thermus aquaticus (Taq DNA polymerase), namely Klentaq (KTQ), and protocols are included for the identification of variants with (1) increased DNA lesion-bypass ability and (2) enhanced selectivity for DNA match/mismatch recognition. The screening assays are based on double-stranded DNA detection (using SYBR Green I) which can be carried out using standard laboratory equipment. The described assays are designed for use in a 384-well plate format to increase screening throughput and reduce material costs. For improved accuracy and ease of liquid handling, the use of an automated liquid handling device is recommended. Curr. Protoc. Chem Biol. 2:89-109. © 2010 by John Wiley & Sons, Inc.

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

    Science.gov (United States)

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

    1993-01-01

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

  7. Construction, arraying, and high-density screening of large insert libraries of human chromosomes X and 21: their potential use as reference libraries.

    Science.gov (United States)

    Nizetić, D; Zehetner, G; Monaco, A P; Gellen, L; Young, B D; Lehrach, H

    1991-01-01

    We have constructed cosmid libraries from flow-sorted human chromosomes X and 21, each of which contains greater than 30 genome equivalents, and have developed systems allowing permanent storage of primary clones, easy screening of libraries in high-density filter formats, and the simultaneous generation of fingerprinting and mapping data on the same set of cosmid clones. Clones are picked into microtiter plate wells and stored at -70 degrees C. A semiautomatic robot system allows the generation of filter replicas containing up to 10,000 clones per membrane. Sets of membranes containing 15-20 chromosome equivalents of both chromosomes will be used for the construction of ordered clone libraries by hybridization fingerprinting protocols. In addition, multiple sets of two membranes containing 4 chromosome equivalents of the human X chromosome, and one membrane containing 3 chromosome equivalents of chromosome 21, have been distributed to other interested laboratories as part of a system of reference libraries. This system allows other groups easy access to the clones and offers an efficient protocol to combine results generated in different laboratories using these libraries. Here we describe the construction of the libraries and demonstrate the use of high-density screening filters in oligonucleotide probe hybridizations and the isolation of cosmids by hybridization with probes from the X chromosome. Images PMID:2014245

  8. Constructing and Screening a Metagenomic Library of a Cold and Alkaline Extreme Environment.

    Science.gov (United States)

    Glaring, Mikkel A; Vester, Jan K; Stougaard, Peter

    2017-01-01

    Natural cold or alkaline environments are common on Earth. A rare combination of these two extremes is found in the permanently cold (less than 6 °C) and alkaline (pH above 10) ikaite columns in the Ikka Fjord in Southern Greenland. Bioprospecting efforts have established the ikaite columns as a source of bacteria and enzymes adapted to these conditions. They have also highlighted the limitations of cultivation-based methods in this extreme environment and metagenomic approaches may provide access to novel extremophilic enzymes from the uncultured majority of bacteria. Here, we describe the construction and screening of a metagenomic library of the prokaryotic community inhabiting the ikaite columns.

  9. Constructing and screening a metagenomic library of a cold and alkaline extreme environment

    DEFF Research Database (Denmark)

    Glaring, Mikkel Andreas; Vester, Jan Kjølhede; Stougaard, Peter

    2017-01-01

    Natural cold or alkaline environments are common on Earth. A rare combination of these two extremes is found in the permanently cold (less than 6 °C) and alkaline (pH above 10) ikaite columns in the Ikka Fjord in Southern Greenland. Bioprospecting efforts have established the ikaite columns...... as a source of bacteria and enzymes adapted to these conditions. They have also highlighted the limitations of cultivation-based methods in this extreme environment and metagenomic approaches may provide access to novel extremophilic enzymes from the uncultured majority of bacteria. Here, we describe...... the construction and screening of a metagenomic library of the prokaryotic community inhabiting the ikaite columns....

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

    Science.gov (United States)

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

    2017-03-01

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

  11. An automated two-phase microfluidic system for kinetic analyses and the screening of compound libraries.

    Science.gov (United States)

    Clausell-Tormos, Jenifer; Griffiths, Andrew D; Merten, Christoph A

    2010-05-21

    Droplet-based microfluidic systems allow biological and chemical reactions to be performed on a drastically decreased scale. However, interfacing the outside world with such systems and generating high numbers of microdroplets of distinct chemical composition remain challenging. We describe here an automated system in which arrays of chemically distinct plugs are generated from microtiter plates. Each array can be split into multiple small-volume copies, thus allowing several screens of the same library. The system is fully compatible with further on-chip manipulation(s) and allows monitoring of individual plugs over time (e.g. for recording reaction kinetics). Hence the technology eliminates several bottlenecks of current droplet-based microfluidic systems and should open the way for (bio-)chemical and cell-based screens.

  12. Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening

    Science.gov (United States)

    Machutta, Carl A.; Kollmann, Christopher S.; Lind, Kenneth E.; Bai, Xiaopeng; Chan, Pan F.; Huang, Jianzhong; Ballell, Lluis; Belyanskaya, Svetlana; Besra, Gurdyal S.; Barros-Aguirre, David; Bates, Robert H.; Centrella, Paolo A.; Chang, Sandy S.; Chai, Jing; Choudhry, Anthony E.; Coffin, Aaron; Davie, Christopher P.; Deng, Hongfeng; Deng, Jianghe; Ding, Yun; Dodson, Jason W.; Fosbenner, David T.; Gao, Enoch N.; Graham, Taylor L.; Graybill, Todd L.; Ingraham, Karen; Johnson, Walter P.; King, Bryan W.; Kwiatkowski, Christopher R.; Lelièvre, Joël; Li, Yue; Liu, Xiaorong; Lu, Quinn; Lehr, Ruth; Mendoza-Losana, Alfonso; Martin, John; McCloskey, Lynn; McCormick, Patti; O'Keefe, Heather P.; O'Keeffe, Thomas; Pao, Christina; Phelps, Christopher B.; Qi, Hongwei; Rafferty, Keith; Scavello, Genaro S.; Steiginga, Matt S.; Sundersingh, Flora S.; Sweitzer, Sharon M.; Szewczuk, Lawrence M.; Taylor, Amy; Toh, May Fern; Wang, Juan; Wang, Minghui; Wilkins, Devan J.; Xia, Bing; Yao, Gang; Zhang, Jean; Zhou, Jingye; Donahue, Christine P.; Messer, Jeffrey A.; Holmes, David; Arico-Muendel, Christopher C.; Pope, Andrew J.; Gross, Jeffrey W.; Evindar, Ghotas

    2017-07-01

    The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.

  13. Isolation of xylose isomerases by sequence- and function-based screening from a soil metagenomic library

    Directory of Open Access Journals (Sweden)

    Parachin Nádia

    2011-05-01

    Full Text Available Abstract Background Xylose isomerase (XI catalyses the isomerisation of xylose to xylulose in bacteria and some fungi. Currently, only a limited number of XI genes have been functionally expressed in Saccharomyces cerevisiae, the microorganism of choice for lignocellulosic ethanol production. The objective of the present study was to search for novel XI genes in the vastly diverse microbial habitat present in soil. As the exploitation of microbial diversity is impaired by the ability to cultivate soil microorganisms under standard laboratory conditions, a metagenomic approach, consisting of total DNA extraction from a given environment followed by cloning of DNA into suitable vectors, was undertaken. Results A soil metagenomic library was constructed and two screening methods based on protein sequence similarity and enzyme activity were investigated to isolate novel XI encoding genes. These two screening approaches identified the xym1 and xym2 genes, respectively. Sequence and phylogenetic analyses revealed that the genes shared 67% similarity and belonged to different bacterial groups. When xym1 and xym2 were overexpressed in a xylA-deficient Escherichia coli strain, similar growth rates to those in which the Piromyces XI gene was expressed were obtained. However, expression in S. cerevisiae resulted in only one-fourth the growth rate of that obtained for the strain expressing the Piromyces XI gene. Conclusions For the first time, the screening of a soil metagenomic library in E. coli resulted in the successful isolation of two active XIs. However, the discrepancy between XI enzyme performance in E. coli and S. cerevisiae suggests that future screening for XI activity from soil should be pursued directly using yeast as a host.

  14. Identification of novel open reading frames from metagenomic libraries generated from extremophilic organisms: application of metagenomics and high throughput screening for novel enzyme isolation

    CSIR Research Space (South Africa)

    Visser, Daniel F

    2006-10-01

    Full Text Available South African mines. Genomic DNA was isolated from these biofilms, and various metagenomic libraries generated. These libraries were in turn screened for industrially important enzymes, in particular proteases and lipases. Resultant hits had plasmid DNA...

  15. 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.

  16. Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA.

    Science.gov (United States)

    Sakajo, S; Nakamura, K; Asahi, T

    1987-06-01

    A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.

  17. A New Age in Functional Genomics Using CRISPR/Cas9 in Arrayed Library Screening

    Directory of Open Access Journals (Sweden)

    Alexander eAgrotis

    2015-09-01

    Full Text Available CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9 to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

  18. Differential gene expression in gall midge susceptible rice genotypes revealed by suppressive subtraction hybridization (SSH) cDNA libraries and microarray analysis.

    Science.gov (United States)

    Rawat, Nidhi; Neeraja, Chiruvuri Naga; Nair, Suresh; Bentur, Jagadish S

    2012-12-01

    A major pest of rice, the Asian rice gall midge (Orseolia oryzae Wood-Mason), causes significant yield losses in the rice growing regions throughout Asia. Feeding by the larvae induces susceptible plants to produce nutritive tissue to support growth and development. In order to identify molecular signatures during compatible interactions, genome wide transcriptional profiling was performed using SSH library and microarray technology. Results revealed up-regulation of genes related to primary metabolism, nutrient relocation, cell organization and DNA synthesis. Concomitantly, defense, secondary metabolism and signaling genes were suppressed. Further, real-time PCR validation of a selected set of 20 genes, in three susceptible rice varieties (TN1, Kavya and Suraksha) during the interaction with the respective virulent gall midge biotypes, also revealed variation in gene expression in Kavya as compared to TN1 and Suraksha. These studies showed that virulent insects induced the plants to step up metabolism and transport nutrients to their feeding site and suppressed defense responses. But Kavya rice mounted an elevated defense response during early hours of virulent gall midge infestation, which was over-powered later, resulting in host plant susceptibility.

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

    Directory of Open Access Journals (Sweden)

    Min Lin

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

  20. High-throughput screen of drug repurposing library identifies inhibitors of Sarcocystis neurona growth

    Directory of Open Access Journals (Sweden)

    Gregory D. Bowden

    2018-04-01

    Full Text Available The apicomplexan parasite Sarcocystis neurona is the primary etiologic agent of equine protozoal myeloencephalitis (EPM, a serious neurologic disease of horses. Many horses in the U.S. are at risk of developing EPM; approximately 50% of all horses in the U.S. have been exposed to S. neurona and treatments for EPM are 60–70% effective. Advancement of treatment requires new technology to identify new drugs for EPM. To address this critical need, we developed, validated, and implemented a high-throughput screen to test 725 FDA-approved compounds from the NIH clinical collections library for anti-S. neurona activity. Our screen identified 18 compounds with confirmed inhibitory activity against S. neurona growth, including compounds active in the nM concentration range. Many identified inhibitory compounds have well-defined mechanisms of action, making them useful tools to study parasite biology in addition to being potential therapeutic agents. In comparing the activity of inhibitory compounds identified by our screen to that of other screens against other apicomplexan parasites, we found that most compounds (15/18; 83% have activity against one or more related apicomplexans. Interestingly, nearly half (44%; 8/18 of the inhibitory compounds have reported activity against dopamine receptors. We also found that dantrolene, a compound already formulated for horses with a peak plasma concentration of 37.8 ± 12.8 ng/ml after 500 mg dose, inhibits S. neurona parasites at low concentrations (0.065 μM [0.036–0.12; 95% CI] or 21.9 ng/ml [12.1–40.3; 95% CI]. These studies demonstrate the use of a new tool for discovering new chemotherapeutic agents for EPM and potentially providing new reagents to elucidate biologic pathways required for successful S. neurona infection. Keywords: Drug repurposing, High-throughput screen, Sarcocystis neurona, Equine protozoal myeloencephalitis

  1. 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.

  2. 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.

  3. Characterization of cDNA clones encoding rabbit and human serum paraoxonase: The mature protein retains its signal sequence

    Energy Technology Data Exchange (ETDEWEB)

    Hassett, C.; Richter, R.J.; Humbert, R.; Omiecinski, C.J.; Furlong, C.E. (Univ. of Washington, Seattle (United States)); Chapline, C.; Crabb, J.W. (W.Alton Jones Cell Science Center, Lake Placid, NY (United States))

    1991-10-22

    Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.

  4. Screening and hit evaluation of a chemical library against blood-stage Plasmodium falciparum.

    Science.gov (United States)

    Avery, Vicky M; Bashyam, Sridevi; Burrows, Jeremy N; Duffy, Sandra; Papadatos, George; Puthukkuti, Shyni; Sambandan, Yuvaraj; Singh, Shivendra; Spangenberg, Thomas; Waterson, David; Willis, Paul

    2014-05-27

    In view of the need to continuously feed the pipeline with new anti-malarial agents adapted to differentiated and more stringent target product profiles (e.g., new modes of action, transmission-blocking activity or long-duration chemo-protection), a chemical library consisting of more than 250,000 compounds has been evaluated in a blood-stage Plasmodium falciparum growth inhibition assay and further assessed for chemical diversity and novelty. The selection cascade used for the triaging of hits from the chemical library started with a robust three-step in vitro assay followed by an in silico analysis of the resulting confirmed hits. Upon reaching the predefined requirements for selectivity and potency, the set of hits was subjected to computational analysis to assess chemical properties and diversity. Furthermore, known marketed anti-malarial drugs were co-clustered acting as 'signposts' in the chemical space defined by the hits. Then, in cerebro evaluation of the chemical structures was performed to identify scaffolds that currently are or have been the focus of anti-malarial medicinal chemistry programmes. Next, prioritization according to relaxed physicochemical parameters took place, along with the search for structural analogues. Ultimately, synthesis of novel chemotypes with desired properties was performed and the resulting compounds were subsequently retested in a P. falciparum growth inhibition assay. This screening campaign led to a 1.25% primary hit rate, which decreased to 0.77% upon confirmatory repeat screening. With the predefined potency (EC₅₀  10) criteria, 178 compounds progressed to the next steps where chemical diversity, physicochemical properties and novelty assessment were taken into account. This resulted in the selection of 15 distinct chemical series. A selection cascade was applied to prioritize hits resulting from the screening of a medium-sized chemical library against blood-stage P. falciparum. Emphasis was placed on chemical

  5. Identification of four genomic loci highly related to casein-kinase-2-alpha cDNA and characterization of a casein kinase-2-alpha pseudogene within the mouse genome

    DEFF Research Database (Denmark)

    Boldyreff, B; Wehr, K; Hecht, R

    1992-01-01

    Using the coding region of the human CK-2 alpha cDNA as a probe for screening a genomic mouse library, positive clones representing four different genomic loci were isolated. Partial DNA sequences of these loci encompassing the first 120 nucleotides of the putative coding region are reported. One...

  6. A Clinical Drug Library Screen Identifies Tosufloxacin as Being Highly Active against Staphylococcus aureus Persisters

    Directory of Open Access Journals (Sweden)

    Hongxia Niu

    2015-07-01

    Full Text Available To identify effective compounds that are active against Staphylococcus aureus (S. aureus persisters, we screened a clinical drug library consisting of 1524 compounds and identified six drug candidates that had anti-persister activity: tosufloxacin, clinafloxacin, sarafloxacin, doxycycline, thiostrepton, and chlorosalicylanilide. Among them, tosufloxacin had the highest anti-persister activity, which could completely eradicate S. aureus persisters within 2 days in vitro. Clinafloxacin ranked the second with very few persisters surviving the drug exposure. Interestingly, we found that both tosufloxacin and trovafloxacin that had high activity against persisters contained at the N-1 position the 2,4-difluorophenyl group, which is absent in other less active quinolones and may be associated with the high anti-persister activity. Further studies are needed to evaluate tosufloxacin in animal models and to explain its unique activity against bacterial persisters. Our findings may have implications for improved treatment of persistent bacterial infections.

  7. Novel ZnO-binding peptides obtained by the screening of a phage display peptide library

    Energy Technology Data Exchange (ETDEWEB)

    Golec, Piotr [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Molecular Biology (affiliated with the University of Gdansk) (Poland); Karczewska-Golec, Joanna [University of Gdansk and Medical University of Gdansk, Laboratory of Molecular Bacteriology, Intercollegiate Faculty of Biotechnology (Poland); Los, Marcin; Wegrzyn, Grzegorz, E-mail: wegrzyn@biotech.univ.gda.pl [University of Gdansk, Department of Molecular Biology (Poland)

    2012-11-15

    Zinc oxide (ZnO) is a semiconductor compound with a potential for wide use in various applications, including biomaterials and biosensors, particularly as nanoparticles (the size range of ZnO nanoparticles is from 2 to 100 nm, with an average of about 35 nm). Here, we report isolation of novel ZnO-binding peptides, by screening of a phage display library. Interestingly, amino acid sequences of the ZnO-binding peptides reported in this paper and those described previously are significantly different. This suggests that there is a high variability in sequences of peptides which can bind particular inorganic molecules, indicating that different approaches may lead to discovery of different peptides of generally the same activity (e.g., binding of ZnO) but having various detailed properties, perhaps crucial under specific conditions of different applications.

  8. Cummings, Merrill, and Borrelli’s Inquiry into Small Screen Use by Academic Library Users: Timing is Everything

    Directory of Open Access Journals (Sweden)

    Catharine Bomhold

    2018-01-01

    Full Text Available A Review of: Cummings, J., Merrill, A., & Borrelli, S. (2010. The use of handheld mobile devices: Their impact and implications for library services. Library Hi Tech, (281, 22-40. https://doi.org/10.1108/07378831011026670 Abstract Objective – The authors undertook this study to understand the relatively new phenomenon of handheld computing and the use of small-screen devices among academic library users. They sought to determine if users would be inclined to search the online library catalogue on their devices and, by extension, if there would be a growing demand for small-screen compatible library services. Design – Online and paper surveys were used with both closed and open questions. Respondents included students, faculty, and staff at Washington State University (WSU. Setting – Washington State University Library, Pullman, Washington, United States of America. Subjects – The survey was open to any user of the Washington State University (Pullman Library. The 206 respondents included 126 (61.2% undergraduates, 26 (12.6% graduate or professional students, 32 (15.3% WSU employees, and 15 (7.3% faculty members. Methods – A survey was distributed both online and on paper. The online version used Surveymonkey.com and participation was solicited through various social media. It was open for three months during the Spring semester, 2007. The paper version was distributed to all library users on two days in June 2007. Eighty-four online and 122 paper responses were received. Main Results – Most of the respondents (58.4% who owned a personal digital assistant (PDA or Web-enabled cell phone (WECP indicated that they would search the library catalogue on a small-screen device. Responses to the open question “How would you use the OPAC [online public access catalogue] if it was available on a PDA or WECP?” were mixed, both positive and negative. The positive responders noted the possible time savings associated with the availability of

  9. Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

    International Nuclear Information System (INIS)

    Chao, S.; Chao, L.; Chao, J.

    1989-01-01

    A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

  10. A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy.

    Science.gov (United States)

    Chen, Robert; Rishi, Harneet S; Potapov, Vladimir; Yamada, Masaki R; Yeh, Vincent J; Chow, Thomas; Cheung, Celia L; Jones, Austin T; Johnson, Terry D; Keating, Amy E; DeLoache, William C; Dueber, John E

    2015-11-20

    Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8 × 8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms.

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

    Science.gov (United States)

    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.

  12. Toward performance-diverse small-molecule libraries for cell-based phenotypic screening using multiplexed high-dimensional profiling

    Science.gov (United States)

    Wawer, Mathias J.; Li, Kejie; Gustafsdottir, Sigrun M.; Ljosa, Vebjorn; Bodycombe, Nicole E.; Marton, Melissa A.; Sokolnicki, Katherine L.; Bray, Mark-Anthony; Kemp, Melissa M.; Winchester, Ellen; Taylor, Bradley; Grant, George B.; Hon, C. Suk-Yee; Duvall, Jeremy R.; Wilson, J. Anthony; Bittker, Joshua A.; Dančík, Vlado; Narayan, Rajiv; Subramanian, Aravind; Winckler, Wendy; Golub, Todd R.; Carpenter, Anne E.; Shamji, Alykhan F.; Schreiber, Stuart L.; Clemons, Paul A.

    2014-01-01

    High-throughput screening has become a mainstay of small-molecule probe and early drug discovery. The question of how to build and evolve efficient screening collections systematically for cell-based and biochemical screening is still unresolved. It is often assumed that chemical structure diversity leads to diverse biological performance of a library. Here, we confirm earlier results showing that this inference is not always valid and suggest instead using biological measurement diversity derived from multiplexed profiling in the construction of libraries with diverse assay performance patterns for cell-based screens. Rather than using results from tens or hundreds of completed assays, which is resource intensive and not easily extensible, we use high-dimensional image-based cell morphology and gene expression profiles. We piloted this approach using over 30,000 compounds. We show that small-molecule profiling can be used to select compound sets with high rates of activity and diverse biological performance. PMID:25024206

  13. Cloning and characterization of a female genital complex cDNA from the liver fluke Fasciola hepatica.

    Science.gov (United States)

    Zurita, M; Bieber, D; Ringold, G; Mansour, T E

    1987-01-01

    A cDNA clone whose RNA is abundant in the female genital complex of the liver fluke Fasciola hepatica has been isolated from a cDNA library in lambda gt10 by differential screening. The pattern of expression in different fluke tissues and at different stages of miracidium formation suggests that this gene is expressed in the F. hepatica vitelleria. The nucleotide sequence of the cloned cDNA was determined and the primary structure of the putative protein was deduced. The proposed protein is rich in glycine, lysine, and tyrosine and its overall amino acid composition agrees with that reported for the F. hepatica egg shell. The clone has homology with DNA from other trematodes; this homology is higher in organisms in which egg development is similar to that of F. hepatica and suggests that the protein is conserved in organisms in which miracidium formation occurs in fresh water. Images PMID:3470798

  14. A polystyrene binding target-unrelated peptide isolated in the screening of phage display library.

    Science.gov (United States)

    Bakhshinejad, Babak; Sadeghizadeh, Majid

    2016-11-01

    Phage display is a powerful methodology for the identification of peptide ligands binding to any desired target. However, the selection of target-unrelated peptides (TUPs) appears as a huge problem in the screening of phage display libraries through biopanning. The phage-displayed peptide TLHPAAD has been isolated both in our laboratory and by another reserach group on completely different screening targets prompting us to hypothesize that it may be a potential TUP. In the current study, we analyzed the binding characteristics and propagation rate of phage clone displaying TLHPAAD peptide (SW-TUP clone). The results of ELISA experiment and phage recovery assay provided strong support for the notion that SW-TUP phage binds to polystyrene with a significantly higher affinity than control phage clones. Furthermore, this polystyrene binding was demonstrated to occur in a concentration- and pH-dependent mode. Characterization of the propagation profile of phage clones within a specified time course revealed no statistically significant difference between the amplification rate of SW-TUP and control phages. Our findings lead us to the conclusion that SW-TUP phage clone with the displayed peptide TLHPAAD is not a true target binder and its selection in biopanning experiments results from its bidning affinity to the polystyrene surface of the solid phase. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

    Tomkinson, B.; Jonsson, A-K

    1991-01-01

    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

  16. A high-throughput screen for hyaluronic acid accumulation in recombinant Escherichia coli transformed by libraries of engineered sigma factors.

    Science.gov (United States)

    Yu, Huimin; Tyo, Keith; Alper, Hal; Klein-Marcuschamer, Daniel; Stephanopoulos, Gregory

    2008-11-01

    Hyaluronic acid (HA) is an important biomaterial with functional medical and cosmetic applications. As its synthesis has been recently reported in recombinant bacteria, it is of interest to develop a high throughput screening method for the rapid isolation of HA accumulating strains transformed by combinatorial libraries. Here we report a novel two-step screening strategy to select for better HA-producing recombinant Escherichia coli strains transformed by mutation libraries of rpoD and rpoS, coding for the sigma(D) and sigma(S) factors of the RNA polymerase, respectively. The first screen, based on translucent colony morphology identification, was used to qualitatively distinguish HA-producing strains on agar plates from non-HA producing strains that exhibit dense colony morphology. The second screen was based on the photometric measurement of an alcian blue staining solution that precipitates with HA, creating an inverse relationship between HA concentration and alcian blue absorbance. The color attenuation fitted a second-order polynomial between HA concentration and OD(540) absorbance. Using the alcian blue absorbance quantification, 74 translucent colonies from the HA-rpoD library and 78 translucent colonies from the HA-rpoS library were isolated and cultured for optimal strain selection. Three representative superior recombinants with high, medium and low increase of HA accumulation, respectively, were identified by the screen from the HA-rpoD and HA-rpoS mutant library. Further flask culture confirmed that results of the library screen were reliable and the superior recombinant D72 highly accumulated HA of 561.4 mg/L with a productivity of approximately 265 mg HA/g dry cell. Sequencing results showed that the mutant rpoD gene in D72 is in a truncated protein that lacks the conserved regions 3 and 4 of the sigma(D). Generally, this two-step high throughput screen presents a promising strategy for selecting superior HA-producing strains from large scale

  17. Isolation and characterization of a Coffea canephora ERF-like c-DNA

    African Journals Online (AJOL)

    A cDNA corresponding to an ERF gene has been isolated from a Coffea canephora fruit cDNA library. The cDNA was 1,317 nucleotides long and has an open reading frame of 987 bp. The predicted polypeptide showed a great similitude with equivalent proteins from others plant species. The binding domain shows 98.3% ...

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

    OpenAIRE

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

    1988-01-01

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

  19. 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.

  20. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Directory of Open Access Journals (Sweden)

    Han Wang

    2016-09-01

    Full Text Available Tetanus neurotoxin (TeNT produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  1. Nylon-3 copolymers that generate cell-adhesive surfaces identified by library screening.

    Science.gov (United States)

    Lee, Myung-Ryul; Stahl, Shannon S; Gellman, Samuel H; Masters, Kristyn S

    2009-11-25

    Polymers in the nylon-3 family contain subunits derived from beta-amino acids, which are linked to one another via amide bonds. Thus, the nylon-3 backbone is homologous to the alpha-amino acid-based backbone of proteins. This molecular-level homology suggests that nylon-3 materials might be intrinsically protein-mimetic. The experiments described here explore this prospect in the context of cell adhesion, with tissue engineering as a long-range goal. We have evaluated a small library of sequence-random nylon-3 copolymers for the ability to render surfaces attractive to NIH 3T3 fibroblast adhesion and spreading. Library screening was accomplished in a high-throughput, parallel mode via attachment of the copolymers in a two-dimensional array to a modified glass surface. Significant variations in fibroblast adhesion and spreading were observed as a function of nylon-3 subunit identity and proportion. Several of the nylon-3 copolymers supported cell adhesion and morphology that was comparable, or even superior, to that achieved on positive control substrates such as tissue culture polystyrene and collagen-coated glass. Moreover, studies conducted under serum-free conditions demonstrated that specific nylon-3 derivatives supported cell adhesion independently of serum protein adsorption. Although cell adhesion was diminished in the absence of serum, particular copolymers demonstrated an ability to support substantially greater cell adhesion than any of the other conditions, including the positive controls. The nylon-3 copolymers that were most effective at promoting adhesion to a modified glass surface proved also to be effective at promoting adhesion when attached to a PEG-based hydrogel, demonstrating the potential for these copolymers to be used in tissue engineering applications.

  2. Nylon-3 Co-Polymers that Generate Cell-Adhesive Surfaces Identified by Library Screening

    Science.gov (United States)

    Lee, Myung-Ryul; Stahl, Shannon S.; Gellman, Samuel H.; Masters, Kristyn S.

    2010-01-01

    Polymers in the nylon-3 family contain subunits derived from β-amino acids, which are linked to one another via amide bonds. Thus, the nylon-3 backbone is homologous to the α-amino acid-based backbone of proteins. This molecular-level homology suggests that nylon-3 materials might be intrinsically protein-mimetic. The experiments described here explore this prospect in the context of cell adhesion, with tissue engineering as a long-range goal. We have evaluated a small library of sequence-random nylon-3 copolymers for the ability to render surfaces attractive to NIH 3T3 fibroblast adhesion and spreading. Library screening was accomplished in a high-throughput, parallel mode via attachment of the copolymers in a two-dimensional array to a modified glass surface. Significant variations in fibroblast adhesion and spreading were observed as a function of nylon-3 subunit identity and proportion. Several of the nylon-3 copolymers supported cell adhesion and morphology that was comparable, or even superior, to that achieved on positive control substrates such as tissue culture polystyrene and collagen-coated glass. Moreover, studies conducted under serum-free conditions demonstrated that specific nylon-3 derivatives supported cell adhesion independently of serum protein adsorption. Although cell adhesion was diminished in the absence of serum, particular copolymers demonstrated an ability to support substantially greater cell adhesion than any of the other conditions, including the positive controls. The nylon-3 copolymers that were most effective at promoting adhesion to a modified glass surface proved also to be effective at promoting adhesion when attached to a PEG-based hydrogel, demonstrating the potential for these copolymers to be used in tissue engineering applications. PMID:19886604

  3. 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.

  4. Screening of cellular proteins that interact with the classical swine ...

    Indian Academy of Sciences (India)

    In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A's function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database ...

  5. 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.

  6. Process automation toward ultra-high-throughput screening of combinatorial one-bead-one-compound (OBOC) peptide libraries.

    Science.gov (United States)

    Cha, Junhoe; Lim, Jaehong; Zheng, Yiran; Tan, Sylvia; Ang, Yi Li; Oon, Jessica; Ang, Mei Wei; Ling, Jingjing; Bode, Marcus; Lee, Su Seong

    2012-06-01

    With an aim to develop peptide-based protein capture agents that can replace antibodies for in vitro diagnosis, an ultra-high-throughput screening strategy has been investigated by automating labor-intensive, time-consuming processes that are the construction of peptide libraries, sorting of positive beads, and peptide sequencing through analysis of tandem mass spectrometry data. Although instruments for automation, such as peptide synthesizers and automatic bead sorters, have been used in some groups, the overall process has not been well optimized to minimize time, cost, and efforts, as well as to maximize product quality and performance. Herein we suggest and explore several solutions to the existing problems with the automation of the key processes. The overall process optimization has been done successfully in orchestration with the technologies such as rapid cleavage of peptides from beads and semiautomatic peptide sequencing that we have developed previously. This optimization allowed one-round screening, from peptide library construction to peptide sequencing, to be completed within 4 to 5 days. We also successfully identified a 6-mer ligand for carcinoembryonic antigen-cell adhesion molecule 5 (CEACAM 5) through three-round screenings, including one-round screening of a focused library.

  7. Combining the AFLOW GIBBS and elastic libraries to efficiently and robustly screen thermomechanical properties of solids

    Science.gov (United States)

    Toher, Cormac; Oses, Corey; Plata, Jose J.; Hicks, David; Rose, Frisco; Levy, Ohad; de Jong, Maarten; Asta, Mark; Fornari, Marco; Buongiorno Nardelli, Marco; Curtarolo, Stefano

    2017-06-01

    Thorough characterization of the thermomechanical properties of materials requires difficult and time-consuming experiments. This severely limits the availability of data and is one of the main obstacles for the development of effective accelerated materials design strategies. The rapid screening of new potential materials requires highly integrated, sophisticated, and robust computational approaches. We tackled the challenge by developing an automated, integrated workflow with robust error-correction within the AFLOW framework which combines the newly developed "Automatic Elasticity Library" with the previously implemented GIBBS method. The first extracts the mechanical properties from automatic self-consistent stress-strain calculations, while the latter employs those mechanical properties to evaluate the thermodynamics within the Debye model. This new thermoelastic workflow is benchmarked against a set of 74 experimentally characterized systems to pinpoint a robust computational methodology for the evaluation of bulk and shear moduli, Poisson ratios, Debye temperatures, Grüneisen parameters, and thermal conductivities of a wide variety of materials. The effect of different choices of equations of state and exchange-correlation functionals is examined and the optimum combination of properties for the Leibfried-Schlömann prediction of thermal conductivity is identified, leading to improved agreement with experimental results than the GIBBS-only approach. The framework has been applied to the AFLOW.org data repositories to compute the thermoelastic properties of over 3500 unique materials. The results are now available online by using an expanded version of the REST-API described in the Appendix.

  8. Isolation and characterization of the murine alpha-L-iduronidase cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Clarke, L.A.; Zhang, H. Nasir, J. [Univ. of British Columbia, Vancouver (Canada)] [and others

    1994-09-01

    Mucopolysaccharidosis I (MPS I) are a group of disorders caused by deficiency of the lysosomal enzyme alpha-L-iduronidase. The characterization of the human gene and the identification of mutations underlying MPS I in humans has led to the delineation of the molecular basis of this disorder. Model systems are now needed for the evaluation and development of therapeutics for this disorder. Both canine and feline models for MPS type I have been described but only the canine gene has been isolated and characterized. We report here the cloning and expression of the murine alpha-L-iduronidase cDNA. The murine cDNA was obtained by screening a mouse liver cDNA library with a probe from the human cDNA. The full length murine cDNA is 3120 base pairs in length and thus is considerably larger than both the human and canine transcripts. The increase in size is due to a 1.2 kb 3{prime} untranslated region in the murine cDNA that contains a CA dinucleotide repeat. Within the coding region the murine cDNA shows sequences. At the protein level the murine protein shows 77% similarity with the human protein and 75% similarity with the canine protein. There are significant differences in both the start and stop sites with the murine protein 9 amino acids shorter at both the N terminal signal peptide region and the C terminus. Expression of the murine cDNA in COS-1 cells resulted in a 20 fold increase in intracellular alpha-L-iduronidase activity as well as the detection of considerable enzyme activity in the culture medium. Comparison of the reported missense mutations underlying MPS I in humans (A75T, H82P, R89Q, L218P, P533R, Q310X, T366P) has shown conservation of these amino acid residues in the murine protein. The isolation of the murine iduronidase cDNA will now allow for the development of a murine model for MPS I.

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

    Science.gov (United States)

    Wu, Yuanzhong; Kang, Tiebang

    2016-06-29

    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.

  10. ‘Function-first’ Lead Discovery: Mode of Action Profiling of Natural Product Libraries Using Image-Based Screening

    Science.gov (United States)

    Schulze, Christopher J.; Bray, Walter M.; Woerhmann, Marcos H.; Stuart, Joshua; Lokey, R. Scott; Linington, Roger G.

    2013-01-01

    Summary Cytological profiling is a high-content image-based screening technology that provides insight into the mode of action (MOA) for test compounds by directly measuring hundreds of phenotypic cellular features. We have extended this recently reported technology to the mechanistic characterization of unknown natural products libraries for the direct prediction of compound MOAs at the primary screening stage. By analyzing a training set of commercial compounds of known mechanism and comparing these profiles to those obtained from natural product library members, we have successfully annotated extracts based on mode of action, dereplicated known compounds based on biological similarity to the training set, and identified and predicted the MOA of a family of new iron siderophores. Coupled with traditional analytical techniques, cytological profiling provides a new avenue for the creation of ‘function-first’ platforms for natural products discovery. PMID:23438757

  11. Fast identification of selective resins for removal of genotoxic aminopyridine impurities via screening of molecularly imprinted polymer libraries.

    Science.gov (United States)

    Kecili, Rustem; Billing, Johan; Nivhede, David; Sellergren, Börje; Rees, Anthony; Yilmaz, Ecevit

    2014-04-25

    This study describes the identification and evaluation of molecularly imprinted polymers (MIPs) for the selective removal of potentially genotoxic aminopyridine impurities from pharmaceuticals. Screening experiments were performed using existing MIP resin libraries to identify resins selective towards those impurities in the presence of model pharmaceutical compounds. A hit resin with a considerable imprinting effect was found in the screening and upon further investigation, the resin was found to show a broad selectivity towards five different aminopyridines in the presence of the two model active pharmaceutical ingredients (APIs) piroxicam and tenoxicam. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2016-01-01

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

  13. 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.

  14. 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...

  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. A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil

    NARCIS (Netherlands)

    Cretoiu, M.S.; Berini, F; Kielak, A.M.; Marinelli, F.; Van Elsas, J.D.

    2015-01-01

    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

  17. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

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

    1987-01-01

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

  18. Isolation and subcloning of chitinase clone from chickpea genomic library.

    Science.gov (United States)

    Grover, A; Tyagi, A; Koundal, K R; Sharma, R P

    1996-06-01

    Chickpea genomic library constructed earlier in phage lambda (EMBL-3) was screened for the presence of chitinase clone using tobacco chitinase cDNA as a probe. Positive clones obtained by primary screening of plaques (2 x 10(6)) were ascertained by secondary and tertiary screening. Presence of chitinase insert in the positive clones obtained, was further confirmed by restricting phage DNA with Sal I and then doing southern with tobacco chitinase. The insert band was eluted out and subcloned in puc 19 plasmid.

  19. Target discovery screens using pooled shRNA libraries and next-generation sequencing: A model workflow and analytical algorithm.

    Directory of Open Access Journals (Sweden)

    Christiane Schaefer

    Full Text Available In the search for novel therapeutic targets, RNA interference screening has become a valuable tool. High-throughput technologies are now broadly accessible but their assay development from baseline remains resource-intensive and challenging. Focusing on this assay development process, we here describe a target discovery screen using pooled shRNA libraries and next-generation sequencing (NGS deconvolution in a cell line model of Ewing sarcoma. In a strategy designed for comparative and synthetic lethal studies, we screened for targets specific to the A673 Ewing sarcoma cell line. Methods, results and pitfalls are described for the entire multi-step screening procedure, from lentiviral shRNA delivery to bioinformatics analysis, illustrating a complete model workflow. We demonstrate that successful studies are feasible from the first assay performance and independent of specialized screening units. Furthermore, we show that a resource-saving screen depth of 100-fold average shRNA representation can suffice to generate reproducible target hits despite heterogeneity in the derived datasets. Because statistical analysis methods are debatable for such datasets, we created ProFED, an analysis package designed to facilitate descriptive data analysis and hit calling using an aim-oriented profile filtering approach. In its versatile design, this open-source online tool provides fast and easy analysis of shRNA and other count-based datasets to complement other analytical algorithms.

  20. cDNA expression library screening and identification of two novel antigens, ubiquitin and receptor for activated C kinase (RACK) homologue, of the fish parasite Trypanosoma carassii

    NARCIS (Netherlands)

    Ruszczyk, A.; Joerink, M.; Guldenaar, C.; Hermsen, G.J.; Savelkoul, H.F.J.; Wiegertjes, G.F.

    2008-01-01

    Trypanosoma carassii is a kinetoplastid parasite infecting cyprinid fish with a high prevalence in nature. Antibodies have been shown to play a protective role in the immune response against this parasite in common carp, Cyprinus carpio. To identify immunogenic and putative protective T. carassii

  1. Characteristics and Expression Profile of KRT71 Screened by Suppression Subtractive Hybridization cDNA Library in Curly Fleece Chinese Tan Sheep.

    Science.gov (United States)

    Kang, Xiaolong; Liu, Yufang; Zhang, Jibin; Xu, Qinqin; Liu, Chengkun; Fang, Meiying

    2017-07-01

    As an important commercial trait for sheep, curly fleece has a great economic impact on production costs and efficiency in sheep industry. To identify genes that are important for curly fleece formation in mammals, a suppression subtractive hybridization analysis was performed on the shoulder skin tissues exposed to two different growth stages of Chinese Tan sheep with different phenotypes (curly fleece and noncurling fleece). BLAST analysis identified 67 differentially expressed genes, of which 31 were expressed lower and 36 were expressed higher in lambs than in adult sheep. Differential expressions of seven randomly selected genes were verified using quantitative real-time polymerase chain reaction (qRT-PCR). KRT71 gene was selected for further study due to its high correlation with the curly hair phenotype in various mammal species. Semi-qPCR showed distinctively high expression of KRT71 in skin tissues. Moreover, qPCR result showed a significantly higher expression of KRT71 in curly fleece than noncurling Tan sheep. The luciferase assay and electrophoresis mobility shift assay showed that there were transcription factor binding sites in the promoter region of KRT71 related to the differential expression of KRT71 at the two growth stages of Tan sheep. Online bioinformation tools predicted MFZ1 as a transcriptional factor that regulates the expression of KRT71. These studies on KRT71 gene revealed some mechanisms underlying the relationship between the KRT71 gene and the curly fleece phenotype of Tan sheep.

  2. Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera

    Science.gov (United States)

    Hu, Yun-Fei; Zhao, Dun; Yu, Xing-Long; Hu, Yu-Li; Li, Run-Cheng; Ge, Meng; Xu, Tian-Qi; Liu, Xiao-Bo; Liao, Hua-Yuan

    2017-01-01

    Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species. PMID:28184219

  3. Construction and screening of vast libraries of natural product-like macrocyclic peptides using in vitro display technologies.

    Science.gov (United States)

    Bashiruddin, Nasir K; Suga, Hiroaki

    2015-02-01

    Macrocyclic structure and backbone N-methylation represent characteristic features of peptidic natural products, which play critical roles in their biological activity. Although natural products have been the traditional source of such peptides, recent developments in synthesizing natural product-like macrocyclic peptides using reconstituted translation systems have enabled us to construct vast trillion-member libraries of non-standard macrocyclic peptides. In addition, a method for displaying such libraries on their corresponding mRNA templates allows us to rapidly screen them for potent ligands against various drug targets. This review describes methodologies for the ribosomal synthesis of novel natural product-like macrocyclic peptides and their recent applications in the discovery of bioactive molecules using in vitro display technologies. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  4. On various metrics used for validation of predictive QSAR models with applications in virtual screening and focused library design.

    Science.gov (United States)

    Roy, Kunal; Mitra, Indrani

    2011-07-01

    Quantitative structure-activity relationships (QSARs) have important applications in drug discovery research, environmental fate modeling, property prediction, etc. Validation has been recognized as a very important step for QSAR model development. As one of the important objectives of QSAR modeling is to predict activity/property/toxicity of new chemicals falling within the domain of applicability of the developed models and QSARs are being used for regulatory decisions, checking reliability of the models and confidence of their predictions is a very important aspect, which can be judged during the validation process. One prime application of a statistically significant QSAR model is virtual screening for molecules with improved potency based on the pharmacophoric features and the descriptors appearing in the QSAR model. Validated QSAR models may also be utilized for design of focused libraries which may be subsequently screened for the selection of hits. The present review focuses on various metrics used for validation of predictive QSAR models together with an overview of the application of QSAR models in the fields of virtual screening and focused library design for diverse series of compounds with citation of some recent examples.

  5. Confocal nanoscanning, bead picking (CONA): PickoScreen microscopes for automated and quantitative screening of one-bead one-compound libraries.

    Science.gov (United States)

    Hintersteiner, Martin; Buehler, Christof; Uhl, Volker; Schmied, Mario; Müller, Jürgen; Kottig, Karsten; Auer, Manfred

    2009-01-01

    Solid phase combinatorial chemistry provides fast and cost-effective access to large bead based libraries with compound numbers easily exceeding tens of thousands of compounds. Incubating one-bead one-compound library beads with fluorescently labeled target proteins and identifying and isolating the beads which contain a bound target protein, potentially represents one of the most powerful generic primary high throughput screening formats. On-bead screening (OBS) based on this detection principle can be carried out with limited automation. Often hit bead detection, i.e. recognizing beads with a fluorescently labeled protein bound to the compound on the bead, relies on eye-inspection under a wide-field microscope. Using low resolution detection techniques, the identification of hit beads and their ranking is limited by a low fluorescence signal intensity and varying levels of the library beads' autofluorescence. To exploit the full potential of an OBS process, reliable methods for both automated quantitative detection of hit beads and their subsequent isolation are needed. In a joint collaborative effort with Evotec Technologies (now Perkin-Elmer Cellular Technologies Germany GmbH), we have built two confocal bead scanner and picker platforms PS02 and a high-speed variant PS04 dedicated to automated high resolution OBS. The PS0X instruments combine fully automated confocal large area scanning of a bead monolayer at the bottom of standard MTP plates with semiautomated isolation of individual hit beads via hydraulic-driven picker capillaries. The quantification of fluorescence intensities with high spatial resolution in the equatorial plane of each bead allows for a reliable discrimination between entirely bright autofluorescent beads and real hit beads which exhibit an increased fluorescence signal at the outer few micrometers of the bead. The achieved screening speed of up to 200,000 bead assayed in less than 7 h and the picking time of approximately 1 bead

  6. Coordinate regulation of stromelysin and collagenase genes determined with cDNA probes

    International Nuclear Information System (INIS)

    Frisch, S.M.; Clark, E.J.; Werb, Z.

    1987-01-01

    Secreted proteinases are required for tumor metastasis, angiogenesis, and tissue remodeling during wound healing and embryonic growth. Thus, the regulation of the genes of secreted proteinases may serve as an interesting model for growth-controlled genes in general. The authors studied the genes of the secreted proteinases stromelysin and collagenase by using molecularly cloned cDNAs from each proteinase. Stromelysin cDNA was cloned by differential screening of a total cDNA library from rabbit synovial cells treated with phorbol 12-myristate 13-acetate, which yielded a clone of 1.2 kilobase pairs; collagenase cDNA was obtained by cloning reverse transcripts of anti-collagenase-immunoadsorbed polysomal mRNA, which yielded a clone of 0.8 kilobase pairs. Stromelysin and collagenase mRNA species of 2.2 and 2.4 kilobases, respectively, were detected on hybridization blots of RNA from phorbol 12-myristate 13-acetate-treated but not untreated rabbit synovial cells. Expression of stromelysin mRNA was also induced in rabbit alveolar macrophages and rabbit brain capillary endothelial cells treated with phorbol 12-myristate 13-acetate. Stromelysin and collagenase mRNA were both induced by phorbol 12-myristate 13-acetate and cytochalasin B at a constant ratio of the two gene products; this suggest coordinate regulation. The fact that induction was blocked after inhibition of protein synthesis by cycloheximide implicates an indirect signal transduction pathway that requires new protein synthesis

  7. 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...

  8. 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

    2015-09-29

    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.

  9. Screening a random mutagenesis library of a fungal β-fructofuranosidase using FT-MIR ATR spectroscopy and multivariate analysis.

    Science.gov (United States)

    Trollope, K M; Nieuwoudt, H H; Görgens, J F; Volschenk, H

    2014-05-01

    Short-chain fructooligosaccharides (scFOS) are valuable health-promoting food additives. During the batch production of scFOS from sucrose the β-fructofuranosidase catalyst is subject to product inhibition by glucose. Engineering the enzyme for reduced sensitivity to glucose could improve product yields or process productivity while preserving the simple industrial batch design. Random mutagenesis is a useful technique for engineering proteins but should be coupled to a relevant high-throughput screen. Such a screen for sucrose and scFOS quantification remains elusive. This work presents the development of a screening method displaying potential high-throughput capacity for the evaluation of β-fructofuranosidase libraries using Fourier transform mid-infrared attenuated total reflectance (FT-MIR ATR) spectroscopy and multivariate analysis. A calibration model for the quantification of sucrose in enzyme assay samples ranged from 5 to 200 g/l and the standard error of prediction was below 13 g/l. A library of the Aspergillus japonicus fopA gene was generated by error prone PCR and screened in Saccharomyces cerevisiae. Using FT-MIR ATR spectroscopy, potential hits were identified as those variants that converted more sucrose in the presence of the glucose inhibitor than the parent. Subsequent analysis of reaction products generated by top performers using high-performance liquid chromatography identified a variant producing higher scFOS levels than the parent. At the peak difference in performance the variant produced 28 % more scFOS from the same amount of sucrose. This study highlights the application of FT-MIR ATR spectroscopy to a variant discovery pipeline in the directed evolution of a β-fructofuranosidase for enhanced scFOS production.

  10. 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

  11. 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

  12. Studying screen interactions long-term : the library as a case

    NARCIS (Netherlands)

    Kanis, Marije; Groen, Maarten; Meys, W.T.; 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

  13. Cloning of the human androgen receptor cDNA

    International Nuclear Information System (INIS)

    Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

    1988-01-01

    The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

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

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

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

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

    Science.gov (United States)

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

    2006-01-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-09-01

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

  18. Germacrene C synthase from Lycopersicon esculentum cv. VFNT cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase.

    Science.gov (United States)

    Colby, S M; Crock, J; Dowdle-Rizzo, B; Lemaux, P G; Croteau, R

    1998-03-03

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, beta-caryophyllene, alpha-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton delta-cadinene synthase (50% identity).

  19. Speeding cis-trans regulation discovery by phylogenomic analyses coupled with screenings of an arrayed library of Arabidopsis transcription factors.

    Directory of Open Access Journals (Sweden)

    Gabriel Castrillo

    Full Text Available Transcriptional regulation is an important mechanism underlying gene expression and has played a crucial role in evolution. The number, position and interactions between cis-elements and transcription factors (TFs determine the expression pattern of a gene. To identify functionally relevant cis-elements in gene promoters, a phylogenetic shadowing approach with a lipase gene (LIP1 was used. As a proof of concept, in silico analyses of several Brassicaceae LIP1 promoters identified a highly conserved sequence (LIP1 element that is sufficient to drive strong expression of a reporter gene in planta. A collection of ca. 1,200 Arabidopsis thaliana TF open reading frames (ORFs was arrayed in a 96-well format (RR library and a convenient mating based yeast one hybrid (Y1H screening procedure was established. We constructed an episomal plasmid (pTUY1H to clone the LIP1 element and used it as bait for Y1H screenings. A novel interaction with an HD-ZIP (AtML1 TF was identified and abolished by a 2 bp mutation in the LIP1 element. A role of this interaction in transcriptional regulation was confirmed in planta. In addition, we validated our strategy by reproducing the previously reported interaction between a MYB-CC (PHR1 TF, a central regulator of phosphate starvation responses, with a conserved promoter fragment (IPS1 element containing its cognate binding sequence. Finally, we established that the LIP1 and IPS1 elements were differentially bound by HD-ZIP and MYB-CC family members in agreement with their genetic redundancy in planta. In conclusion, combining in silico analyses of orthologous gene promoters with Y1H screening of the RR library represents a powerful approach to decipher cis- and trans-regulatory codes.

  20. Identification of tumor associated single-chain Fv by panning and screening antibody phage library using tumor cells

    Science.gov (United States)

    Nie, Yong-Zhan; He, Feng-Tian; Li, Zhi-Kui; Wu, Kai-Chun; Cao, Yun-Xin; Chen, Bao-Jun; Fan, Dai-Ming

    2002-01-01

    AIM: To study the feasibility of panning and screening phage-displaying recombinant single-chain variable fragment (ScFv) of anti-tumor monoclonal antibodies for fixed whole cells as the carriers of mAb-binding antigens. METHODS: The recombinant phage displaying libraries for anti-colorectal tumor mAb MC3Ab, MC5Ab and anti-gastric tumor mAb MGD1 was constructed. Panning and screening were carried out by means of modified fixation of colorectal and gastric tumor cells expressed the mAb-binding antigens. Concordance of binding specificity to tumor cells between phage clones and parent antibodies was analyzed. The phage of positive clones was identified with competitive ELISA, and infected by E. coli HB2151 to express soluble ScFv. RESULTS: The ratio of positive clones to MC3-ScF-MC5-ScFv and MGD1-ScFv were 60%, 24% and 30%. MC3-ScFv had Mr 32000 confirmed by Western blot. The specificity to antigen had no difference between 4 positive recombinant phage antibodies and MC3Ab. CONCLUSION: The modified process of fixing whole tumor cells is efficient, convenient and feasible to pan and screen the phage-displaying ScFv of anti-tumor monoclonal antibodies. PMID:12174367

  1. An automated high-throughput system for phenotypic screening of chemical libraries on C. elegans and parasitic nematodes.

    Science.gov (United States)

    Partridge, Frederick A; Brown, Anwen E; Buckingham, Steven D; Willis, Nicky J; Wynne, Graham M; Forman, Ruth; Else, Kathryn J; Morrison, Alison A; Matthews, Jacqueline B; Russell, Angela J; Lomas, David A; Sattelle, David B

    2017-12-02

    Parasitic nematodes infect hundreds of millions of people and farmed livestock. Further, plant parasitic nematodes result in major crop damage. The pipeline of therapeutic compounds is limited and parasite resistance to the existing anthelmintic compounds is a global threat. We have developed an INVertebrate Automated Phenotyping Platform (INVAPP) for high-throughput, plate-based chemical screening, and an algorithm (Paragon) which allows screening for compounds that have an effect on motility and development of parasitic worms. We have validated its utility by determining the efficacy of a panel of known anthelmintics against model and parasitic nematodes: Caenorhabditis elegans, Haemonchus contortus, Teladorsagia circumcincta, and Trichuris muris. We then applied the system to screen the Pathogen Box chemical library in a blinded fashion and identified compounds already known to have anthelmintic or anti-parasitic activity, including tolfenpyrad, auranofin, and mebendazole; and 14 compounds previously undescribed as anthelmintics, including benzoxaborole and isoxazole chemotypes. This system offers an effective, high-throughput system for the discovery of novel anthelmintics. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Yeast screens for host factors in positive-strand RNA virus replication based on a library of temperature-sensitive mutants.

    Science.gov (United States)

    Nawaz-ul-Rehman, Muhammad Shah; Reddisiva Prasanth, K; Baker, Jannine; Nagy, Peter D

    2013-02-01

    RNA viruses exploit host cells by altering cellular pathways, recruiting host factors, remodeling intracellular membranes and escaping host antiviral responses. Model hosts, such as Saccharomyces cerevisiae (yeast), are valuable to identify host factors involved in viral RNA replication. The many advantages of using yeast include the availability of various yeast mutant libraries, such as (i) single gene-deletion library; (ii) the essential gene library (yTHC); and (iii) the yeast ORF over-expression library. Here, we have used a novel temperature-sensitive (ts) mutant library of essential yeast genes to identify 118 host proteins affecting replication of Tomato bushy stunt virus, in yeast model host. Testing 787 ts mutants led to the identification of host factors, of which 72 proteins facilitated TBSV replication in yeast and 46 proteins were inhibitory. Altogether, ~85% of the identified proteins are novel host factors affecting tombusvirus replication. The ts mutant library screen also led to the identification of 17 essential genes, which have been documented before, thus confirming the importance of these genomic screens. Overall, we show the power of ts mutant library in identification of host factors for RNA virus replication. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Three human alcohol dehydrogenase subunits: cDNA structure and molecular and evolutionary divergence

    International Nuclear Information System (INIS)

    Ikuta, T.; Szeto, S.; Yoshida, A.

    1986-01-01

    Class I human alcohol dehydrogenase (ADH; alcohol:NAD + oxidoreductase, EC 1.1.1.1) consists of several homo- and heterodimers of α, β, and γ subunits that are governed by the ADH1, ADH2, and ADH3 loci. The authors previously cloned a full length of cDNA for the β subunit, and the complete sequence of 374 amino acid residues was established. cDNAs for the α and γ subunits were cloned and characterized. A human liver cDNA library, constructed in phage λgt11, was screened by using a synthetic oligonucleotide probe that was matched to the γ but not to the β sequence. Clone pUCADHγ21 and clone pUCADHα15L differed from β cDNA with respect to restriction sites and hybridization with the nucleotide probe. Clone pUCADHγ21 contained an insertion of 1.5 kilobase pairs (kbp) and encodes 374 amino acid residues compatible with the reported amino acid sequence of the γ subunit. Clone pUCADHα15L contained an insertion of 2.4 kbp and included nucleotide sequences that encode 374 amino acid residues for another subunit, the γ subunit. In addition, this clone contained the sequences that encode the COOH-terminal part of the β subunit at its extended 5' region. The amino acid sequences and coding regions of the cDNAs of the three subunits are very similar. A high degree of resemblance is observed also in their 3' noncoding regions. However, distinctive differences exist in the vicinity of the Zn-binding cysteine residue at position 46. Based on the cDNA sequences and the deduced amino acid sequences of the three subunits, their structural and evolutionary relationships are discussed

  4. Synthesis and evaluation of a series of 6-chloro-4-methylumbelliferyl glycosides as fluorogenic reagents for screening metagenomic libraries for glycosidase activity.

    Science.gov (United States)

    Chen, Hong-Ming; Armstrong, Zachary; Hallam, Steven J; Withers, Stephen G

    2016-02-08

    Screening of large enzyme libraries such as those derived from metagenomic sources requires sensitive substrates. Fluorogenic glycosides typically offer the best sensitivity but typically must be used in a stopped format to generate good signal. Use of fluorescent phenols of pKa libraries yielded a "hit rate" of 1 in 60. Hits were then readily deconvoluted with the individual substrates in a single plate to identify specific activities within each clone. The use of such a collection of substrates greatly accelerates the screening process. Copyright © 2015 Elsevier Ltd. All rights reserved.

  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)

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

    1988-01-01

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

  6. High-throughput screening for gene libraries expressing carbohydrate hydrolase activity

    NARCIS (Netherlands)

    Leemhuis, Hans; Euverink, Gert-Jan W.; Dijkhuizen, Lubbert

    2003-01-01

    A simple and fast method is described allowing screening of large number of Escherichia coli clones (4000 per day) for the presence of functional or improved carbohydrate hydrolase enzymes. The procedure is relatively cheap and has the advantage that carbohydrate degrading activity can be directly

  7. Semi-synthetic vNAR libraries screened against therapeutic antibodies primarily deliver anti-idiotypic binders.

    Science.gov (United States)

    Könning, Doreen; Rhiel, Laura; Empting, Martin; Grzeschik, Julius; Sellmann, Carolin; Schröter, Christian; Zielonka, Stefan; Dickgießer, Stephan; Pirzer, Thomas; Yanakieva, Desislava; Becker, Stefan; Kolmar, Harald

    2017-08-29

    Anti-idiotypic binders which specifically recognize the variable region of monoclonal antibodies have proven to be robust tools for pharmacokinetic studies of antibody therapeutics and for the development of cancer vaccines. In the present investigation, we focused on the identification of anti-idiotypic, shark-derived IgNAR antibody variable domains (vNARs) targeting the therapeutic antibodies matuzumab and cetuximab for the purpose of developing specific capturing ligands. Using yeast surface display and semi-synthetic, CDR3-randomized libraries, we identified several highly specific binders targeting both therapeutic antibodies in their corresponding variable region, without applying any counter selections during screening. Importantly, anti-idiotypic vNAR binders were not cross-reactive towards cetuximab or matuzumab, respectively, and comprised good target recognition in the presence of human and mouse serum. When coupled to magnetic beads, anti-idiotypic vNAR variants could be used as efficient capturing tools. Moreover, a two-step procedure involving vNAR-functionalized beads was employed for the enrichment of potentially bispecific cetuximab × matuzumab antibody constructs. In conclusion, semi-synthetic and CDR3-randomized vNAR libraries in combination with yeast display enable the fast and facile identification of anti-idiotypic vNAR domains targeting monoclonal antibodies primarily in an anti-idiotypic manner.

  8. Nematicidal protease genes screened from a soil metagenomic library to control Radopholus similis mediated by Pseudomonas fluorescens pf36.

    Science.gov (United States)

    Chen, Deqiang; Wang, Dongwei; Xu, Chunling; Chen, Chun; Li, Junyi; Wu, Wenjia; Huang, Xin; Xie, Hui

    2018-04-01

    Controlling Radopholus similis, an important phytopathogenic nematode, is a challenge worldwide. Herein, we constructed a metagenomic fosmid library from the rhizosphere soil of banana plants, and six clones with protease activity were obtained by functionally screening the library. Furthermore, subclones were constructed using the six clones, and three protease genes with nematicidal activity were identified: pase1, pase4, and pase6. The pase4 gene was successfully cloned and expressed, demonstrating that the protease PASE4 could effectively degrade R. similis tissues and result in nematode death. Additionally, we isolated a predominant R. similis-associated bacterium, Pseudomonas fluorescens (pf36), from 10 R. similis populations with different hosts. The pase4 gene was successfully introduced into the pf36 strain by vector transformation and conjugative transposition, and two genetically modified strains were obtained: p4MCS-pf36 and p4Tn5-pf36. p4MCS-pf36 had significantly higher protease expression and nematicidal activity (p < 0.05) than p4Tn5-pf36 in a microtiter plate assay, whereas p4Tn5-pf36 was superior to p4MCS-pf36 in terms of genetic stability and controlling R. similis in growth pot tests. This study confirmed that R. similis is inhibited by the associated bacterium pf36-mediated expression of nematicidal proteases. Herein, a novel approach is provided for the study and development of efficient, environmentally friendly, and sustainable biocontrol techniques against phytonematodes.

  9. Screening of a Combinatorial Library of Organic Polymers for the Solid-Phase Extraction of Patulin from Apple Juice.

    Science.gov (United States)

    Giovannoli, Cristina; Spano, Giulia; Di Nardo, Fabio; Anfossi, Laura; Baggiani, Claudio

    2017-05-20

    Patulin is a water-soluble mycotoxin produced by several species of fungi. Governmental bodies have placed it under scrutiny for its potential negative health effects, and maximum residue limits are fixed in specific food matrices to protect consumers' health. Confirmatory analysis of patulin in complex food matrices can be a difficult task, and sample clean-up treatments are frequently necessary before instrumental analyses. With the aim of simplifying the clean-up step, we prepared a 256-member combinatorial polymeric library based on 16 functional monomers, four cross-linkers and four different porogenic solvents. The library was screened for the binding towards patulin in different media (acetonitrile and citrate buffer at pH 3.2), with the goal of identifying polymer formulations with good binding properties towards the target compound. As a proof of concept, a methacrylic acid-co-pentaerithrytole tetraacrylate polymer prepared in chloroform was successfully used as a solid-phase extraction material for the clean-up and extraction of patulin from apple juice. Clean chromatographic patterns and acceptable recoveries were obtained for juice spiked with patulin at concentration levels of 25 (64 ± 12%), 50 (83 ± 5.6%) and 100 μg L -1 (76 ± 4.5%). The within-day and between-day reproducibility evaluated at a concentration level of 25 μg L -1 were 5.6 and 7.6%, respectively.

  10. Screening of a Combinatorial Library of Organic Polymers for the Solid-Phase Extraction of Patulin from Apple Juice

    Directory of Open Access Journals (Sweden)

    Cristina Giovannoli

    2017-05-01

    Full Text Available Patulin is a water-soluble mycotoxin produced by several species of fungi. Governmental bodies have placed it under scrutiny for its potential negative health effects, and maximum residue limits are fixed in specific food matrices to protect consumers’ health. Confirmatory analysis of patulin in complex food matrices can be a difficult task, and sample clean-up treatments are frequently necessary before instrumental analyses. With the aim of simplifying the clean-up step, we prepared a 256-member combinatorial polymeric library based on 16 functional monomers, four cross-linkers and four different porogenic solvents. The library was screened for the binding towards patulin in different media (acetonitrile and citrate buffer at pH 3.2, with the goal of identifying polymer formulations with good binding properties towards the target compound. As a proof of concept, a methacrylic acid-co-pentaerithrytole tetraacrylate polymer prepared in chloroform was successfully used as a solid-phase extraction material for the clean-up and extraction of patulin from apple juice. Clean chromatographic patterns and acceptable recoveries were obtained for juice spiked with patulin at concentration levels of 25 (64 ± 12%, 50 (83 ± 5.6% and 100 μg L−1 (76 ± 4.5%. The within-day and between-day reproducibility evaluated at a concentration level of 25 μg L−1 were 5.6 and 7.6%, respectively.

  11. Identification of three classes of heteroaromatic compounds with activity against intracellular Trypanosoma cruzi by chemical library screening.

    Directory of Open Access Journals (Sweden)

    Esther Bettiol

    Full Text Available The development of new drugs against Chagas disease is a priority since the currently available medicines have toxic effects, partial efficacy and are targeted against the acute phase of disease. At present, there is no drug to treat the chronic stage. In this study, we have optimized a whole cell-based assay for high throughput screening of compounds that inhibit infection of mammalian cells by Trypanosoma cruzi trypomastigotes. A 2000-compound chemical library was screened using a recombinant T. cruzi (Tulahuen strain expressing beta-galactosidase. Three hits were selected for their high activity against T. cruzi and low toxicity to host cells in vitro: PCH1, NT1 and CX1 (IC(50: 54, 190 and 23 nM, respectively. Each of these three compounds presents a different mechanism of action on intracellular proliferation of T. cruzi amastigotes. CX1 shows strong trypanocidal activity, an essential characteristic for the development of drugs against the chronic stage of Chagas disease where parasites are found intracellular in a quiescent stage. NT1 has a trypanostatic effect, while PCH1 affects parasite division. The three compounds also show high activity against intracellular T. cruzi from the Y strain and against the related kinetoplastid species Leishmania major and L. amazonensis. Characterization of the anti-T. cruzi activity of molecules chemically related to the three library hits allowed the selection of two compounds with IC(50 values of 2 nM (PCH6 and CX2. These values are approximately 100 times lower than those of the medicines used in patients against T. cruzi. These results provide new candidate molecules for the development of treatments against Chagas disease and leishmaniasis.

  12. A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection

    OpenAIRE

    Cole, Krystal; Roessler, Christian G.; Mulé, Elizabeth A.; Benson-Xu, Emma J.; Mullen, Jeffrey D.; Le, Benjamin A.; Tieman, Alanna M.; Birone, Claire; Brown, Maria; Hernandez, Jesus; Neff, Sherry; Williams, Daniel; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.

    2014-01-01

    High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handl...

  13. Biophysical Screening of a Focused Library for the Discovery of CYP121 Inhibitors as Novel Antimycobacterials.

    Science.gov (United States)

    Brengel, Christian; Thomann, Andreas; Schifrin, Alexander; Allegretta, Giuseppe; Kamal, Ahmed A M; Haupenthal, Jörg; Schnorr, Isabell; Cho, Sang Hyun; Franzblau, Scott G; Empting, Martin; Eberhard, Jens; Hartmann, Rolf W

    2017-10-09

    The development of novel antimycobacterial agents against Mycobacterium tuberculosis (Mtb) is urgently required due to the appearance of multidrug resistance (MDR) combined with complicated long-term treatment. CYP121 was shown to be a promising novel target for inhibition of mycobacterial growth. In this study, we describe the rational discovery of new CYP121 inhibitors by a systematic screening based on biophysical and microbiological methods. The best hits originating from only one structural class gave initial information about molecular motifs required for binding and activity. The initial screening procedure was followed by mode-of-action studies and further biological characterizations. The results demonstrate superior antimycobacterial efficacy and a decreased toxicity profile of our frontrunner compound relative to the reference compound econazole. Due to its low molecular weight, promising biological profile, and physicochemical properties, this compound is an excellent starting point for further rational optimization. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    International Nuclear Information System (INIS)

    Yang, Xu; Lazar, Iulia M

    2009-01-01

    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

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

    International Nuclear Information System (INIS)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E.; Flores, B.M.; Hagen, F.S.

    1990-01-01

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

  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. shRNA target prediction informed by comprehensive enquiry (SPICE): a supporting system for high-throughput screening of shRNA library.

    Science.gov (United States)

    Kamatuka, Kenta; Hattori, Masahiro; Sugiyama, Tomoyasu

    2016-12-01

    RNA interference (RNAi) screening is extensively used in the field of reverse genetics. RNAi libraries constructed using random oligonucleotides have made this technology affordable. However, the new methodology requires exploration of the RNAi target gene information after screening because the RNAi library includes non-natural sequences that are not found in genes. Here, we developed a web-based tool to support RNAi screening. The system performs short hairpin RNA (shRNA) target prediction that is informed by comprehensive enquiry (SPICE). SPICE automates several tasks that are laborious but indispensable to evaluate the shRNAs obtained by RNAi screening. SPICE has four main functions: (i) sequence identification of shRNA in the input sequence (the sequence might be obtained by sequencing clones in the RNAi library), (ii) searching the target genes in the database, (iii) demonstrating biological information obtained from the database, and (iv) preparation of search result files that can be utilized in a local personal computer (PC). Using this system, we demonstrated that genes targeted by random oligonucleotide-derived shRNAs were not different from those targeted by organism-specific shRNA. The system facilitates RNAi screening, which requires sequence analysis after screening. The SPICE web application is available at http://www.spice.sugysun.org/.

  18. Acetylcholinesterase immobilized on modified magnetic beads as a tool for screening a compound library

    International Nuclear Information System (INIS)

    Vanzolini, Kenia L.; Vieira, Lucas C. C.; Corrêa, Arlene G.; Cass, Quezia B.; Moaddel, Ruin

    2015-01-01

    Acetylcholinesterase (AChE) from Electrophorus electricus was immobilized on the surface of amino-modified magnetic beads (AChE-MB), and its activity evaluated by the quantification of acetylcholine hydrolysis. A reference mixture composed of AChE binders (galanthamine and a probe coumarin, K i  = 0.031 ± 0.010 μM) and non-binders (ketamine and propranolol) was used to probe the fishing assay. The performance of the bioconjugation assay was demonstrated with a library of 12 reference coumarins from which two ligands were directly identified by LC-MS/MS in a single assay, demonstrating the usefulness of this approach. (author)

  19. Virtual screening of combinatorial library of novel benzenesulfonamides on mycobacterial carbonic anhydrase II

    Directory of Open Access Journals (Sweden)

    Dikant F.

    2016-12-01

    Full Text Available Combinatorial library of novel benzenesulfonamides was docked (Schrodinger Glide into mycobacterial carbonic anhydrase (mtCA II and human (hCA II isoforms with an aim to find drug candidates with selective activity on mtCA II. The predicted selectivity was calculated based on optimized MM-GBSA free energies for ligand enzyme interactions. Selectivity, LogP (o/w and interaction energy were used to calculate the selection index which determined the subset of best scoring molecules selected for further evaluation. Structure-activity relationship was found for fragment subsets, showing us the possible way regarding how to influence lipophilicity without affecting ligand-enzyme binding properties.

  20. Microcystin-LR nanobody screening from an alpaca phage display nanobody library and its expression and application.

    Science.gov (United States)

    Xu, Chongxin; Yang, Ying; Liu, Liwen; Li, Jianhong; Liu, Xiaoqin; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Liu, Xianjin

    2018-04-30

    Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC 50 ) were 0.87, 1.17 and 1.47μg/L, their detection limits (IC 10 ) were 0.06, 0.08 and 0.12μg/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7-116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0-106.5%, coefficient of variations (CVs) were 3.4-10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple

  1. Construction of Rabbit Immune Antibody Libraries.

    Science.gov (United States)

    Nguyen, Thi Thu Ha; Lee, Jong Seo; Shim, Hyunbo

    2018-01-01

    Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

  2. A Novel Public Library-Based Sexually Transmitted Infection Screening Program for Younger High-Risk Groups in Omaha, Nebraska, USA.

    Science.gov (United States)

    Delair, Shirley F; Lyden, Elizabeth R; O'Keefe, Anne L; Simonsen, Kari A; Nared, Sherri R; Berthold, Elizabeth A; Watanabe-Galloway, Shinobu

    2016-04-01

    Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are the two most commonly reported sexually transmitted infections (STIs) in the United States (U.S.) and Douglas County, Nebraska has STI rates consistently above the U.S. average. The Douglas County Health Department (DCHD) developed an outreach CT and NG screening program in public libraries to address the problem beyond the traditional STI clinic setting. This study evaluates the effectiveness of the program and identifies factors predictive of CT and NG infections. A retrospective review of surveys of library patrons and DCHD traditional STI clinic clients who submitted urine tests for CT and NG from June 2010 through April 2014 was done. Chi square, Fisher exact, Student's t tests, univariate and multivariate logistic regression were conducted. A total of 977 library records and 4871 DCHD clinic records were reviewed. The percent positive was lower in the library than in the traditional clinic for CT (9.9 vs. 11.2 %) and NG (2.74 vs. 5.3 %) (p = 0.039 and p Library clients were more likely to be 19 years and younger (OR 6.14, 95 % CI: 5.0, 7.5), Black (OR 3.4, 95 % CI: 2.8, 4.1), and asymptomatic (OR 12.4, 95 % CI: 9.9, 15.5) compared to traditional clinic clients. The library STI screening program effectively reaches a younger, asymptomatic, and predominantly Black population compared to a traditional health department clinic site.

  3. 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.

  4. 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

    2014-11-07

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. A senescence rescue screen identifies BCL6 as an inhibitor of anti-proliferative p19ARF-p53 signaling

    NARCIS (Netherlands)

    Shvarts, A.; Brummelkamp, T.; Koh, E.; Daley, G.Q.; Bernards, R.A.

    2002-01-01

    Senescence limits the proliferative capacity of primary cells in culture. We describe here a genetic screen to identify genes that allow bypass of this checkpoint. Using retroviral cDNA expression libraries, we identify BCL6 as a potent inhibitor of senescence. BCL6 is frequently activated in

  6. Readiness and capacity of librarians in public libraries to implement a breast cancer outreach and screening campaign in medically underserved communities.

    Science.gov (United States)

    Goytia, Elliott J; Rapkin, Bruce; Weiss, Elisa S; Golub, David; Guzman, Vivian; O'Connor, Maureen

    2005-11-01

    Community-based partnerships are an important means of addressing cancer health disparities in medically underserved communities. Public libraries may be ideal partners in this effort. To assess the readiness and capacity of a public library system to implement cancer recruitment and outreach campaigns, 58 librarians in the Queens Borough Public Library System in New York completed self-administered questionnaires before and after a training on breast health, cancer, and screening. Results indicate that they are interested in participating in a cancer outreach campaign and feel it is a critical need in their community. Many librarians lacked the knowledge about cancer and cancer information resources needed to participate optimally. Nevertheless, librarians provide a cultural bridge to medically underserved communities. Partnering with a public library system to improve access to care has great potential, yet a number of challenges need to be overcome.

  7. Novel anti-campylobacter compounds identified using high throughput screening of a pre-selected enriched small molecules library

    Directory of Open Access Journals (Sweden)

    Anand eKumar

    2016-04-01

    Full Text Available 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 4,182 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 analogues of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.

  8. cDNA cloning of a major allergen from timothy grass (Phleum pratense) pollen; characterization of the recombinant Phl pV allergen

    NARCIS (Netherlands)

    Vrtala, S.; Sperr, W. R.; Reimitzer, I.; van Ree, R.; Laffer, S.; Müller, W. D.; Valent, P.; Lechner, K.; Rumpold, H.; Kraft, D.

    1993-01-01

    We isolated a cDNA encoding a major grass pollen allergen from a timothy grass (Phleum pratense) pollen expression cDNA library using allergic patients' IgE. The complete cDNA encoded an allergen that binds IgE from about 80% of grass pollen-allergic patients. Significant sequence homology was found

  9. A screen of the NIH Clinical Collection small molecule library identifies potential anti-coronavirus drugs.

    Science.gov (United States)

    Cao, Jianzhong; Forrest, J Craig; Zhang, Xuming

    2015-02-01

    With the recent emergence of Middle East Respiratory Syndrome coronavirus in humans and the outbreak of devastating porcine epidemic diarrhea coronavirus in swine, therapeutic intervention is urgently needed. However, anti-coronavirus drugs currently are not available. In an effort to assist rapid development of anti-coronavirus drugs, here we screened the NIH Clinical Collection in cell culture using a luciferase reporter-expressing recombinant murine coronavirus. Of the 727 compounds screened, 84 were found to have a significant anti-coronavirus effect. Further experiments revealed that 51 compounds blocked virus entry while 19 others inhibited viral replication. Additional validation studies with the top 3 inhibitors (hexachlorophene, nitazoxanide and homoharringtonine) demonstrated robust anti-coronavirus activities (a reduction of 6 to 8log10 in virus titer) with an IC50 ranging from 11nM to 1.2μM. Furthermore, homoharringtonine and hexachlorophene exhibited broad antiviral activity against diverse species of human and animal coronaviruses. Since the NIH Clinical Collection consists of compounds that have already been through clinical trials, these small molecule inhibitors have a great potential for rapid development as anti-coronavirus drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. 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.

  11. Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP LC-MS/MS system and library searching.

    Science.gov (United States)

    Dresen, S; Ferreirós, N; Gnann, H; Zimmermann, R; Weinmann, W

    2010-04-01

    The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a "scheduled" survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization-MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography-mass spectrometry, and liquid chromatography-diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).

  12. Isolation and characterization of cDNA clones for the gamma subunit of Xenopus fibrinogen, the product of a coordinately regulated gene family.

    Science.gov (United States)

    Bhattacharya, A; Shepard, A R; Moser, D R; Holland, L J

    1990-09-10

    Fibrinogen, the major structural protein involved in blood coagulation, is synthesized and secreted by the liver. In the frog Xenopus laevis, fibrinogen production is dramatically induced by glucocorticoids. The hormonal stimulation requires synthesis of three separate subunits, designated A alpha, B beta, and gamma. For investigation of the molecular mechanisms underlying this coordinate induction, we have isolated cDNA clones for the subunits of Xenopus fibrinogen. In this communication we describe the identification of clones for the gamma chain. Initially, a Xenopus liver cDNA library in pBR322 was screened with a rat gamma chain cDNA and a clone representing half of the 1600-base frog gamma mRNA was identified. This clone was shown to be complementary to gamma mRNA by hybrid selection of mRNA that translated in vitro into the gamma polypeptide. A clone about 1460 base pairs in length was then isolated from a Xenopus liver lambda gt10 cDNA library and subcloned into Bluescript SK-. This clone, designated X1 gamma 3, contains the entire 3'-end and lacks 38 bases at the 5'-end of gamma mRNA. The deduced amino acid sequence at the N-terminal is compatible with a signal peptide of 20-23 amino acids, in agreement with the calculated size of the frog gamma chain signal peptide. Following the signal sequence is a region of highly conserved amino acids that participate in disulfide bond formation critical for the maintenance of tertiary structure in mammalian fibrinogen. The gamma cDNA clone was used to measure gamma mRNA in purified Xenopus liver cells treated with glucocorticoids in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. 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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

    Jang Songhun; Zhou Fang; Xia Laixin; Zhao Wei; Cheng Hanhua; Zhou Rongjia

    2006-01-01

    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

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

    International Nuclear Information System (INIS)

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J.

    1988-01-01

    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 x 10 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

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

    Science.gov (United States)

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

    1992-04-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  18. 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.

  19. Increased mRNA expression of a laminin-binding protein in human colon carcinoma: Complete sequence of a full-length cDNA encoding the protein

    International Nuclear Information System (INIS)

    Yow, Hsiukang; Wong, Jau Min; Chen, Hai Shiene; Lee, C.; Steele, G.D. Jr.; Chen, Lanbo

    1988-01-01

    Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers the authors constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here they report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is ∼9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant and highly negatively charged

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  1. Isolation of an ATP synthase cDNA from Sinonovacula constricta ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... 2Ningbo City College of Vocational Technology, Ningbo, 315100 People's Republic of China. 3National Marine Environmental Monitoring Center. Dalian, 116023, People's ... The SMART cDNA library of S. constricta was constructed by our laboratory. Random sequencing of the library using T3 primer.

  2. 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.

  3. Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening

    Science.gov (United States)

    Ebert, Maximilian C. C. J. C.; Mugford, Paul F.; Pelletier, Joelle N.

    2017-01-01

    The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells. PMID:28178357

  4. Evaluation and optimization of virtual screening workflows with DEKOIS 2.0--a public library of challenging docking benchmark sets.

    Science.gov (United States)

    Bauer, Matthias R; Ibrahim, Tamer M; Vogel, Simon M; Boeckler, Frank M

    2013-06-24

    The application of molecular benchmarking sets helps to assess the actual performance of virtual screening (VS) workflows. To improve the efficiency of structure-based VS approaches, the selection and optimization of various parameters can be guided by benchmarking. With the DEKOIS 2.0 library, we aim to further extend and complement the collection of publicly available decoy sets. Based on BindingDB bioactivity data, we provide 81 new and structurally diverse benchmark sets for a wide variety of different target classes. To ensure a meaningful selection of ligands, we address several issues that can be found in bioactivity data. We have improved our previously introduced DEKOIS methodology with enhanced physicochemical matching, now including the consideration of molecular charges, as well as a more sophisticated elimination of latent actives in the decoy set (LADS). We evaluate the docking performance of Glide, GOLD, and AutoDock Vina with our data sets and highlight existing challenges for VS tools. All DEKOIS 2.0 benchmark sets will be made accessible at http://www.dekois.com.

  5. Library synthesis, screening, and discovery of modified Zinc(II)-Bis(dipicolylamine) probe for enhanced molecular imaging of cell death.

    Science.gov (United States)

    Plaunt, Adam J; Harmatys, Kara M; Wolter, William R; Suckow, Mark A; Smith, Bradley D

    2014-04-16

    Zinc(II)-bis(dipicolylamine) (Zn-BDPA) coordination complexes selectively target the surfaces of dead and dying mammalian cells, and they have promise as molecular probes for imaging cell death. A necessary step toward eventual clinical imaging applications is the development of next-generation Zn-BDPA complexes with enhanced affinity for the cell death membrane biomarker, phosphatidylserine (PS). This study employed an iterative cycle of library synthesis and screening, using a novel rapid equilibrium dialysis assay, to discover a modified Zn-BDPA structure with high and selective affinity for vesicles containing PS. The lead structure was converted into a deep-red fluorescent probe and its targeting and imaging performance was compared with an unmodified control Zn-BDPA probe. The evaluation process included a series of FRET-based vesicle titration studies, cell microscopy experiments, and rat tumor biodistribution measurements. In all cases, the modified probe exhibited comparatively higher affinity and selectivity for the target membranes of dead and dying cells. The results show that this next-generation deep-red fluorescent Zn-BDPA probe is well suited for preclinical molecular imaging of cell death in cell cultures and animal models. Furthermore, it should be possible to substitute the deep-red fluorophore with alternative reporter groups that enable clinically useful, deep-tissue imaging modalities, such as MRI and nuclear imaging.

  6. Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening.

    Directory of Open Access Journals (Sweden)

    Daniela Quaglia

    Full Text Available The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A, the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random. Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  8. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

    Directory of Open Access Journals (Sweden)

    Poustka Annemarie

    2004-06-01

    Full Text Available Abstract Background cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library. Results We describe a method to construct SMART full-length enriched cDNA libraries with large insert sizes. Sub-libraries were generated from size-fractionated cDNA with an average insert size of up to seven kb. The percentage of full-length clones was calculated for different size ranges from BLAST results of over 12,000 5'ESTs. Conclusions The presented technique is suitable to generate full-length enriched cDNA libraries with large average insert sizes in a straightforward and robust way. The representation of full-coding clones is high also for large cDNAs (70%, 4–10 kb, when high-quality starting mRNA is used.

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

    Directory of Open Access Journals (Sweden)

    KANAMURA Herminia Yohko

    1997-01-01

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

  10. 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)

    Zeng Jun; Liu Ciyi; Xie Wenhui; Hu Silong; Jin Muxiu

    2004-01-01

    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

  11. “Splicing up” drug discovery. Cell-Based Expression and Screening of Genetically-Encoded Libraries of Backbone Cyclized Polypeptides

    Science.gov (United States)

    Sancheti, Harshkumar; Camarero, Julio A.

    2012-01-01

    The present paper reviews the use of protein splicing for the biosynthesis of backbone cyclic polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to attenuate or inhibit cellular processes thus providing a new way for finding therapeutic agents. PMID:19628015

  12. Rapid discovery of potent α-fucosidase inhibitors by in situ screening of a library of (pyrrolidin-2-yl)triazoles.

    Science.gov (United States)

    Elías-Rodríguez, Pilar; Moreno-Clavijo, Elena; Carmona, Ana T; Moreno-Vargas, Antonio J; Robina, Inmaculada

    2014-08-21

    The synthesis of a small library of (pyrrolidin-2-yl)triazoles via copper catalysed cycloaddition of an alkynyl iminocyclopentitol and a set of commercial and synthetic azides has been achieved. The in situ screening for the activity towards α-fucosidase of the resulting triazoles has allowed the identification of one of the most potent and selective pyrrolidine derived inhibitors of this enzyme (Ki = 4 nM).

  13. Reducing Disparities in Cancer Screening and Prevention through Community-Based Participatory Research Partnerships with Local Libraries: A Comprehensive Dynamic Trial.

    Science.gov (United States)

    Rapkin, Bruce D; Weiss, Elisa; Lounsbury, David; Michel, Tamara; Gordon, Alexis; Erb-Downward, Jennifer; Sabino-Laughlin, Eilleen; Carpenter, Alison; Schwartz, Carolyn E; Bulone, Linda; Kemeny, Margaret

    2017-09-01

    Reduction of cancer-related disparities requires strategies that link medically underserved communities to preventive care. In this community-based participatory research project, a public library system brought together stakeholders to plan and undertake programs to address cancer screening and risk behavior. This study was implemented over 48 months in 20 large urban neighborhoods, selected to reach diverse communities disconnected from care. In each neighborhood, Cancer Action Councils were organized to conduct a comprehensive dynamic trial, an iterative process of program planning, implementation and evaluation. This process was phased into neighborhoods in random, stepped-wedge sequence. Population-level outcomes included self-reported screening adherence and smoking cessation, based on street intercept interviews. Event-history regressions (n = 9374) demonstrated that adherence outcomes were associated with program implementation, as were mediators such as awareness of screening programs and cancer information seeking. Findings varied by ethnicity, and were strongest among respondents born outside the U.S. or least engaged in care. This intervention impacted health behavior in diverse, underserved and vulnerable neighborhoods. It has been sustained as a routine library system program for several years after conclusion of grant support. In sum, participatory research with the public library system offers a flexible, scalable approach to reduce cancer health disparities. © Society for Community Research and Action 2017.

  14. 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

    2014-09-26

    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.

  15. 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

    2018-01-01

    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

  16. Screening a Phage Display Library for Two Novel OmpU-Binding Peptides with Adhesion Antagonistic Activity against Vibrio mimicus.

    Directory of Open Access Journals (Sweden)

    Lifang Qi

    Full Text Available Vibrio mimicus is a pathogen that causes ascites disease in fish. We have previously demonstrated that the outer membrane protein U (OmpU is an important adhesin in V. mimicus. Here eight specific OmpU-binding phage clones, which presented three different OmpU-binding peptides (designated P1, P2, P3, were screened from a commercially available phage displayed 12-mer peptide library using rOmpU protein as target. Then, synthetic OmpU-binding peptides were measured for their adhesion antagonistic activity and binding affinity via adhesion inhibition test and non-competitive ELISA, respectively. The results showed that after co-incubated with the mixture of rOmpU and P3, visible green fluorescence could be observed on the epithelioma papulosum cyprinidi (EPC cells surface; while the EPC cells co-incubated with the mixture of rOmpU and P1/P2 exhibited little green fluorescence. The average adhesion number of V. mimicus 04-14 isolate before and after treatment with peptide was 21.4 ± 1.5, 20.8 ± 0.8 (irrelevant peptide, 20.2 ± 0.5 (P3, 5.1 ± 0.7 (P1 and 3.4 ± 0.8 (P2, respectively. There was a significant decrease in the adhesive level of 04-14 isolate treated with P1/ P2 compared to the untreated isolate (p<0.01. The affinity constants of P1 and P2 were (6.17 ± 0.19 × 108 L/mol and (1.24 ± 0.56 × 109 L/mol, respectively. Furthermore, protective effects of P1 and P2 on grass carps challenged with V. mimicus were preliminary detected. It was found there was delayed death of fish in the groups treated with P1/P2, and the survival rate of challenged fish improved with the increase of the dose of adhesion antagonistic peptide. Taken together, two novel OmpU-binding peptides, which possessed adhesion antagonistic activity, high affinity and a certain degree of antibacterial activity against V. mimicus, were screened and identified.

  17. Screening a Phage Display Library for Two Novel OmpU-Binding Peptides with Adhesion Antagonistic Activity against Vibrio mimicus.

    Science.gov (United States)

    Qi, Lifang; Liu, Yan; Tao, Huizhu; Xiao, Ning; Li, Jinnian; Kong, Lingyan; Hou, Liting

    2016-01-01

    Vibrio mimicus is a pathogen that causes ascites disease in fish. We have previously demonstrated that the outer membrane protein U (OmpU) is an important adhesin in V. mimicus. Here eight specific OmpU-binding phage clones, which presented three different OmpU-binding peptides (designated P1, P2, P3), were screened from a commercially available phage displayed 12-mer peptide library using rOmpU protein as target. Then, synthetic OmpU-binding peptides were measured for their adhesion antagonistic activity and binding affinity via adhesion inhibition test and non-competitive ELISA, respectively. The results showed that after co-incubated with the mixture of rOmpU and P3, visible green fluorescence could be observed on the epithelioma papulosum cyprinidi (EPC) cells surface; while the EPC cells co-incubated with the mixture of rOmpU and P1/P2 exhibited little green fluorescence. The average adhesion number of V. mimicus 04-14 isolate before and after treatment with peptide was 21.4 ± 1.5, 20.8 ± 0.8 (irrelevant peptide), 20.2 ± 0.5 (P3), 5.1 ± 0.7 (P1) and 3.4 ± 0.8 (P2), respectively. There was a significant decrease in the adhesive level of 04-14 isolate treated with P1/ P2 compared to the untreated isolate (p<0.01). The affinity constants of P1 and P2 were (6.17 ± 0.19) × 108 L/mol and (1.24 ± 0.56) × 109 L/mol, respectively. Furthermore, protective effects of P1 and P2 on grass carps challenged with V. mimicus were preliminary detected. It was found there was delayed death of fish in the groups treated with P1/P2, and the survival rate of challenged fish improved with the increase of the dose of adhesion antagonistic peptide. Taken together, two novel OmpU-binding peptides, which possessed adhesion antagonistic activity, high affinity and a certain degree of antibacterial activity against V. mimicus, were screened and identified.

  18. Characterization of three small molecule inhibitors of enterovirus 71 identified from screening of a library of natural products.

    Science.gov (United States)

    Li, Guiming; Gao, Qianqian; Yuan, Shilin; Wang, Lili; Altmeyer, Ralf; Lan, Ke; Yin, Feifei; Zou, Gang

    2017-07-01

    Enterovirus 71 (EV-A71) is a major cause of hand, foot, and mouth disease (HFMD). Infection with EV-A71 is more often associated with neurological complications in children and is responsible for the majority of fatalities, but currently there is no approved antiviral therapy for treatment. Here, we identified auraptene, formononetin, and yangonin as effective inhibitors of EV-A71 infection in the low-micromolar range from screening of a natural product library. Among them, formononetin and yangonin selectively inhibited EV-A71 while auraptene could inhibit viruses within the enterovirus species A. Time of addition studies showed that all the three inhibitors inhibit both attachment and postattachment step of entry. We found mutations conferring the resistance to these inhibitors in the VP1 and VP4 capsid proteins and confirmed the target residues using a reverse genetic approach. Interestingly, auraptene- and formononetin-resistant viruses exhibit cross-resistance to other inhibitors while yangonin-resistant virus still remains susceptible to auraptene and formononetin. Moreover, auraptene and formononetin, but not yangonin protected EV-A71 against thermal inactivation, indicating a direct stabilizing effect of both compounds on virion capsid conformation. Finally, neither biochanin A (an analog of formononetin) nor DL-Kavain (an analog of yangonin) exhibited anti-EV-A71 activity, suggesting the structural elements required for anti-EV-A71 activity. Taken together, these compounds could become potential lead compounds for anti-EV-A71 drug development and also serve as tool compounds for studying virus entry. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2004-08-01

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

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

    DEFF Research Database (Denmark)

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

    1991-01-01

    A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C...

  1. cDNA cloning and mRNA expression of heat shock protein 70 gene ...

    African Journals Online (AJOL)

    In this study, the full-length heat shock protein 70 of Tegillarca granosa was cloned from cDNA library by rapid amplification of cDNA end (RACE). The open reading frame (ORF) of heat shock protein 70 was 1968 bp, and it encoded a protein of 655 amino acids with a predicted molecular weight of 71.48 kDa and an ...

  2. Simultaneous screening of targeted and non-targeted contaminants using an LC-QTOF-MS system and automated MS/MS library searching.

    Science.gov (United States)

    Herrera-Lopez, S; Hernando, M D; García-Calvo, E; Fernández-Alba, A R; Ulaszewska, M M

    2014-09-01

    Simultaneous high-resolution full-scan and tandem mass spectrometry (MS/MS) analysis using time of flight mass spectrometry brings an answer for increasing demand of retrospective and non-targeted data analysis. Such analysis combined with spectral library searching is a promising tool for targeted and untargeted screening of small molecules. Despite considerable extension of the panel of compounds of tandem mass spectral libraries, the heterogeneity of spectral data poses a major challenge against the effective usage of spectral libraries. Performance evaluation of available LC-MS/MS libraries will significantly increase credibility in the search results. The present work was aimed to evaluate fluctuation of MS/MS pattern, in the peak intensities distribution together with mass accuracy measurements, and in consequence, performance compliant with ion ratio and mass error criteria as principles in identification processes for targeted and untargeted contaminants at trace levels. Matrix effect and ultra-trace levels of concentration (from 50 ng l(-1) to 1000 ng l(-1) were evaluated as potential source of inaccuracy in the performance of spectral matching. Matrix-matched samples and real samples were screened for proof of applicability. By manual review of data and application of ion ratio and ppm error criteria, false negatives were obtained; this number diminished when in-house library was used, while with on-line MS/MS databases 100% of positive samples were found. In our experience, intensity of peaks across spectra was highly correlated to the concentration effect and matrix complexity. In turn, analysis of spectra acquired at trace concentrations and in different matrices results in better performance in providing correct and reliable identification. Copyright © 2014 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

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

    2008-06-01

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

  4. Succinyl-CoA:3-oxoacid transferase (SCOT): Human cDNA and genomic cloning and chromosomal mapping of the human gene

    Energy Technology Data Exchange (ETDEWEB)

    Kassovska-Bratinova, S.; Mitchell, G.A. [Hopital Ste-Justine, Montreal, Quebec (Canada); Duncan, A. [Kingston General Hospital, Kingston, Ontario (Canada)] [and others

    1994-09-01

    SCOT (EC 2.8.3.5) mediates the activation and utilization of ketone bodies in extrahepatic tissues, especially brain, heart and kidney. Children with hereditary SCOT deficiency have episodes of severe ketoacidosis. Using a partial human heart SCOT cDNA, hSCOT-G, we detected a single {approximately}3 Kb mRNA in human heart and leukocytes, but not in liver. The length of the mouse SCOT mRNA detected with hSCOT-G in muscle, heart, kidney and brain is {approximately}3 Kb. We mapped the human SCOT gene to chromosome 5p13 by in situ hybridization. To date we have isolated human heart cDNAs spanning 2.9 Kb and including a 1248 bp open reading frame. The 3{prime} nontranslated region of the human SCOT mRNA extends at least 1712 bp, in contrast to the 209 bp sequence reported for pig SCOT cDNA. In one heart cDNA clone we detected a 58 bp insertion 258 bp downstream from the stop codon. We performed RT-PCR using a 5{prime} degenerate-sequence primer designed from the pig SCOT leader peptide sequence and 3{prime} human-specific primers. We obtained a fragment of the expected 320 bp length which strongly hybridizes to an internal oligonucleotide and which we are now characterizing. Human genomic Southern blot analysis with a partial human cDNA as probe suggests that the length of the human SCOT gene is about 40 K. Using hSCOT-G as a probe, we screened a human leukocyte genomic library in EMBL-3 phage and isolated two genomic clones. One of them contains a processed pseudogene. The other contains at least two exons of the human SCOT gene spanning cDNA residues 431 to 734. These findings will be useful for mutation analysis in SCOT-deficient patients.

  5. Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

    Science.gov (United States)

    Colby, Sheila M.; Crock, John; Dowdle-Rizzo, Barbara; Lemaux, Peggy G.; Croteau, Rodney

    1998-01-01

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, β-caryophyllene, α-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton δ-cadinene synthase (50% identity). PMID:9482865

  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