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Sample records for enriched cdna library

  1. 3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

    PubMed

    Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

    2008-09-01

    We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

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

    PubMed Central

    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. PMID:27327613

  3. A phloem-enriched cDNA library from Ricinus: insights into phloem function.

    PubMed

    Doering-Saad, C; Newbury, H J; Couldridge, C E; Bale, J S; Pritchard, J

    2006-01-01

    The aim of this study was to identify genes that are expressed in the phloem. Increased knowledge of phloem regulation will contribute to our understanding of its many roles, from transport of solutes to information about interactions with pathogens. A cDNA library constructed from phloem-enriched sap exuding from cut Ricinus communis (L.) hypocotyls was sequenced. To assess contamination from other tissues, two libraries were constructed: one using the first 15 min of exudation and the other from sap collected after 120 min of exudation had elapsed. Of 1012 clones sequenced, 158 unique transcripts were identified. The presence of marker molecules such as profilin, the low occurrence of chloroplast-related mRNAs, and the sieve element localization of constituent mRNA using in situ hybridization were consistent with a phloem origin of the sap. Functional analysis of the cDNAs revealed classifications including ribosomal function, interaction with the environment, transport, DNA/RNA binding, and protein turnover. An analysis of the closest Arabidopsis thaliana (L.) homologue for each clone indicated that genes involved in cell localization, protein synthesis, tissue localization, organ localization, organ differentiation, and cell fate were represented at twice the level occurring in the whole Arabidopsis genome. The transcripts found in this phloem-enriched library are discussed in the context of phloem function and the relationship between the companion cell and sieve element.

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

    PubMed Central

    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

  5. Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili

    PubMed Central

    Futamura, Norihiro; Totoki, Yasushi; Toyoda, Atsushi; Igasaki, Tomohiro; Nanjo, Tokihiko; Seki, Motoaki; Sakaki, Yoshiyuki; Mari, Adriano; Shinozaki, Kazuo; Shinohara, Kenji

    2008-01-01

    Background Cryptomeria japonica D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of C. japonica should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages. Results We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis. An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as DEF/GLO/GGM13-, AG-, AGL6-, TM3- and TM8-like MIKCC genes and type I MADS-box genes. Conclusion Our full-length enriched cDNA library derived from C. japonica male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in C. japonica. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species. PMID:18691438

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

    PubMed

    Clepet, Christian; Joobeur, Tarek; Zheng, Yi; Jublot, Delphine; Huang, Mingyun; Truniger, Veronica; Boualem, Adnane; Hernandez-Gonzalez, Maria Elena; Dolcet-Sanjuan, Ramon; Portnoy, Vitaly; Mascarell-Creus, Albert; Caño-Delgado, Ana I; Katzir, Nurit; Bendahmane, Abdelhafid; Giovannoni, James J; Aranda, Miguel A; Garcia-Mas, Jordi; Fei, Zhangjun

    2011-05-20

    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. 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 longer than many other dicot plants. Codon

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

    PubMed Central

    2011-01-01

    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 longer than many other dicot

  8. Full-length enriched cDNA libraries and ORFeome analysis of sugarcane hybrid and ancestor genotypes.

    PubMed

    Nishiyama, Milton Yutaka; Ferreira, Savio Siqueira; Tang, Pei-Zhong; Becker, Scott; Pörtner-Taliana, Antje; Souza, Glaucia Mendes

    2014-01-01

    Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between Saccharum officinarum and Saccharum spontaneum. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100-130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for Saccharum officinarum, Saccharum spontaneum, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation.

  9. Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes

    PubMed Central

    Becker, Scott; Pörtner-Taliana, Antje; Souza, Glaucia Mendes

    2014-01-01

    Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between Saccharum officinarum and Saccharum spontaneum. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100–130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for Saccharum officinarum, Saccharum spontaneum, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation. PMID:25222706

  10. Normalized cDNA libraries

    DOEpatents

    Soares, M.B.; Efstratiadis, A.

    1997-06-10

    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{prime} 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. 4 figs.

  11. Normalized cDNA libraries

    DOEpatents

    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.

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

    PubMed

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

    2003-09-30

    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. Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4). The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60%) did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences putatively related to responses to important

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

    PubMed Central

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

    2003-01-01

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

  14. 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

    PubMed Central

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

    2017-01-01

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

  15. 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.

    PubMed

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

    2017-02-03

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

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

    PubMed Central

    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

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    Kim, Tae-Hun; Kim, Nam-Soon; Lim, Dajeong; Lee, Kyung-Tai; Oh, Jung-Hwa; Park, Hye-Sook; Jang, Gil-Won; Kim, Hyung-Yong; Jeon, Mina; Choi, Bong-Hwan; Lee, Hae-Young; Chung, HY; Kim, Heebal

    2006-01-01

    Background Genome research in farm animals will expand our basic knowledge of the genetic control of complex traits, and the results will be applied in the livestock industry to improve meat quality and productivity, as well as to reduce the incidence of disease. A combination of quantitative trait locus mapping and microarray analysis is a useful approach to reduce the overall effort needed to identify genes associated with quantitative traits of interest. Results We constructed a full-length enriched cDNA library from porcine backfat tissue. The estimated average size of the cDNA inserts was 1.7 kb, and the cDNA fullness ratio was 70%. In total, we deposited 16,110 high-quality sequences in the dbEST division of GenBank (accession numbers: DT319652-DT335761). For all the expressed sequence tags (ESTs), approximately 10.9 Mb of porcine sequence were generated with an average length of 674 bp per EST (range: 200–952 bp). Clustering and assembly of these ESTs resulted in a total of 5,008 unique sequences with 1,776 contigs (35.46%) and 3,232 singleton (65.54%) ESTs. From a total of 5,008 unique sequences, 3,154 (62.98%) were similar to other sequences, and 1,854 (37.02%) were identified as having no hit or low identity (<95%) and 60% coverage in The Institute for Genomic Research (TIGR) gene index of Sus scrofa. Gene ontology (GO) annotation of unique sequences showed that approximately 31.7, 32.3, and 30.8% were assigned molecular function, biological process, and cellular component GO terms, respectively. A total of 1,854 putative novel transcripts resulted after comparison and filtering with the TIGR SsGI; these included a large percentage of singletons (80.64%) and a small proportion of contigs (13.36%). Conclusion The sequence data generated in this study will provide valuable information for studying expression profiles using EST-based microarrays and assist in the condensation of current pig TCs into clusters representing longer stretches of cDNA sequences

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

    PubMed Central

    2011-01-01

    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 other oilseed plants

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

    PubMed

    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

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

    PubMed Central

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

    2012-01-01

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

  3. 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.

    PubMed

    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.

  4. Work Enrichment for Academic Libraries.

    ERIC Educational Resources Information Center

    Martell, Charles; Untawale, Mercedes

    1983-01-01

    Explores important quality of work life strategy--job redesign--and discusses job enlargement and job enrichment. A case study of academic library personnel demonstrates how introduction of automated systems at University of California, Berkeley led to restructuring and enrichment of jobs. References and list of selected resources are appended.…

  5. Method for construction of normalized cDNA libraries

    DOEpatents

    Soares, M.B.; Efstratiadis, A.

    1996-01-09

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form. The method comprises: (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. 4 figs.

  6. Method for construction of normalized cDNA libraries

    DOEpatents

    Soares, Marcelo B.; Efstratiadis, Argiris

    1996-01-01

    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.

  7. Method for construction of normalized cDNA libraries

    DOEpatents

    Soares, M.B.; Efstratiadis, A.

    1998-11-03

    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. 19 figs.

  8. Method for construction of normalized cDNA libraries

    DOEpatents

    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.

  9. Constructing and detecting a cDNA library for mites.

    PubMed

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

    PubMed

    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.

  11. Preparation of cDNA libraries from vascular cells.

    PubMed

    Lieb, M E; Taubman, M B

    1999-01-01

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

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

    PubMed

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

    2015-04-01

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

  13. Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction.

    PubMed

    Zhu, Y Y; Machleder, E M; Chenchik, A; Li, R; Siebert, P D

    2001-04-01

    Here, we describe a fast, simple method for constructing full-length cDNA libraries using SMART technology. This novel procedure uses the template-switching activity of Moloney murine leukemia virus (MMLV) reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, three cycles of PCR are performed using a modified oligo(dT) primer and an anchor primer to enrich the cDNA population for full-length sequences. Starting with 1 microgram human skeletal muscle poly(A)+ RNA, a cDNA library was constructed that contained 3 x 10(6) independent clones with an average insert size of 2 kb. Sequence analysis of 172 randomly selected clones showed that 77% of cDNA clones corresponding to known genes contained intact open reading frames. The average length of complete open reading frames was 2.4 kb. Furthermore, 86% of the full-length clones retained longer 5' UTR sequences than the longest 5' end deposited in the GenBank database. cDNA libraries generated using this method will be useful for accelerating the collection of mRNA 5' end sequence information, which is currently very limited in GenBank.

  14. Procedure for normalization of cDNA libraries

    DOEpatents

    Bonaldo, M.D.; Soares, M.B.

    1997-12-30

    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. 1 fig.

  15. Procedure for normalization of cDNA libraries

    DOEpatents

    Bonaldo, Maria DeFatima; Soares, Marcelo Bento

    1997-01-01

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

  16. High-Throughput Plasmid cDNA Library Screening

    SciTech Connect

    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.

  17. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    EPA Science Inventory

    A Bioinformatic Strategy to Rapidly Characterize cDNA Libraries

    G. 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...

  18. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    EPA Science Inventory

    A Bioinformatic Strategy to Rapidly Characterize cDNA Libraries

    G. 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...

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

    NASA Astrophysics Data System (ADS)

    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.

  20. 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

    PubMed Central

    Pang, Chaoyou; Fan, Shuli; Song, Meizhen; Yu, Shuxun

    2013-01-01

    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 assembly and annotation

  1. Construction and characterization of a normalized cDNA library.

    PubMed

    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.

  2. Construction and characterization of a normalized cDNA library

    SciTech Connect

    Soares, M.B.; De Fatima Bonaldo, M.; Jelene, P.; Su, L.; Lawton, L.; Efstratiadis, A.

    1994-09-27

    The authors 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, they 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 C{sub 0}t, 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.

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

    PubMed

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

    2014-01-01

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

  4. Generation of Arabidopsis mutants by heterologous expression of a full length cDNA library from tomato fruits

    USDA-ARS?s Scientific Manuscript database

    Heterologous expression of cDNA libraries in Arabidopsis and other plants has been used for gene identifications. To identify functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with ...

  5. Enriching screening libraries with bioactive fragment space.

    PubMed

    Zhang, Na; Zhao, Hongtao

    2016-08-01

    By deconvoluting 238,073 bioactive molecules in the ChEMBL library into extended Murcko ring systems, we identified a set of 2245 ring systems present in at least 10 molecules. These ring systems belong to 2221 clusters by ECFP4 fingerprints with a minimum intracluster similarity of 0.8. Their overlap with ring systems in commercial libraries was further quantified. Our findings suggest that success of a small fragment library is driven by the convergence of effective coverage of bioactive ring systems (e.g., 10% coverage by 1000 fragments vs. 40% by 2million HTS compounds), high enrichment of bioactive ring systems, and low molecular complexity enhancing the probability of a match with the protein targets. Reconciling with the previous studies, bioactive ring systems are underrepresented in screening libraries. As such, we propose a library of virtual fragments with key functionalities via fragmentation of bioactive molecules. Its utility is exemplified by a prospective application on protein kinase CK2, resulting in the discovery of a series of novel inhibitors with the most potent compound having an IC50 of 0.5μM and a ligand efficiency of 0.41kcal/mol per heavy atom. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed

    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.

  7. An improved method for construction of directionally cloned cDNA libraries from microdissected cells.

    PubMed

    Peterson, L A; Brown, M R; Carlisle, A J; Kohn, E C; Liotta, L A; Emmert-Buck, M R; Krizman, D B

    1998-12-01

    Here, we developed an improved method for constructing microdissected cDNA libraries, based on strand-switching properties of reverse transcriptase, followed by PCR amplification with primers to mediate unidirectional insert cloning. Using RNA from microdissected ovarian carcinoma cells, we constructed a cDNA library consisting of 1.3 x 10(6) unidirectional recombinants with an average insert size of 500 bp. Single-pass sequencing of 100 clones with the T7 primer revealed 89 inserts derived from known genes, anonymous expressed sequence tags (ESTs), or novel sequences. Among these clones were known genes and ESTs previously found in cDNA libraries from bulk ovarian tissue RNA, sequences seen for the first time in an ovarian-derived library, and novel sequences not previously seen in any cDNA library. These results demonstrate a methodology for constructing quality cDNA libraries that are cloned in a unidirectional fashion, are complex and diverse, and reflect the tissue of origin.

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

    PubMed

    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.

  9. Computer-based methods for the mouse full-length cDNA encyclopedia: real-time sequence clustering for construction of a nonredundant cDNA library.

    PubMed

    Konno, H; Fukunishi, Y; Shibata, K; Itoh, M; Carninci, P; Sugahara, Y; Hayashizaki, Y

    2001-02-01

    We developed computer-based methods for constructing a nonredundant mouse full-length cDNA library. Our cDNA library construction process comprises assessment of library quality, sequencing the 3' ends of inserts and clustering, and completing a re-array to generate a nonredundant library from a redundant one. After the cDNA libraries are generated, we sequence the 5' ends of the inserts to check the quality of the library; then we determine the sequencing priority of each library. Selected libraries undergo large-scale sequencing of the 3' ends of the inserts and clustering of the tag sequences. After clustering, the nonredundant library is constructed from the original libraries, which have redundant clones. All libraries, plates, clones, sequences, and clusters are uniquely identified, and all information is saved in the database according to this identifier. At press time, our system has been in place for the past two years; we have clustered 939,725 3' end sequences into 127,385 groups from 227 cDNA libraries/sublibraries (see http://genome.gse.riken.go.jp/).

  10. Display of a Maize cDNA library on baculovirus infected insect cells

    PubMed Central

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

    2008-01-01

    Background 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. Results 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 × 105 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. Conclusion 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. PMID:18700036

  11. Construction of a representative cDNA library from prostatic intraepithelial neoplasia.

    PubMed

    Krizman, D B; Chuaqui, R F; Meltzer, P S; Trent, J M; Duray, P H; Linehan, W M; Liotta, L A; Emmert-Buck, M R

    1996-12-01

    We report the construction of a plasmid-based cDNA library made from microdissected cells derived from prostatic intraepithelial neoplasia. Total RNA was extracted and converted to blunt-ended, double-stranded cDNA by oligo(dT)-mediated reverse transcription followed by linker addition. A linker-specific primer with UDG-compatible ends was used to amplify the cDNA and the resulting PCR product was subcloned. A total of 154 clones were sequenced and results indicated that 81.5% of the clones derived from either known genes, anonymous expressed sequence tags, or novel transcripts with very little redundancy of screened clones. These results demonstrate the feasibility of constructing complex representative cDNA libraries from specific microdissected cell populations that represent microscopic precursor stages of cancer progression. This method should facilitate identification of transcripts specifically expressed in cells of a distinct histological origin and tumorigenic stage.

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

    PubMed Central

    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. PMID:25056322

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

    PubMed Central

    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

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

    PubMed

    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.

  15. Generation of expressed sequence tags from a normalized porcine skeletal muscle cDNA library.

    PubMed

    Yao, Jianbo; Coussens, Paul M; Saama, Peter; Suchyta, Steven; Ernst, Catherine W

    2002-11-01

    Recent developments in microarray technologies permit scientists to analyze expression of thousands of genes simultaneously in diverse biological systems. In an effort to provide integrated resources for application of microarray technologies to studies of skeletal muscle growth and development in swine, we have constructed a normalized cDNA library from porcine skeletal muscle. The effectiveness of normalization was evaluated by DNA sequencing of clones randomly picked from the library before and after normalization, and also by Southern blot hybridization using probes representing abundant transcripts. Our data suggests that the normalization procedure successfully reduced the highly abundant cDNA species in the normalized library. To date, a total of 782 EST (expressed sequence tag) sequences have been generated from this normalized library (687 ESTs) and the original library (95 ESTs). The sequence information of these ESTs plus their BLAST results has been made available through a web accessible database (http://nbfgc.msu.edu). Cluster analysis of the data indicates that a total of 742 unique sequences are present in this collection. BLASTN search of the 742 EST sequences against the public database (dbEST) revealed that 139 had no significant matches (E-value > 10(-15)) to porcine ESTs already entered in the database, suggesting the possibility of their specific expression in porcine skeletal muscle. Generation of non-redundant ESTs from this library will allow us to construct cDNA microarrays for identification of gene expression changes that regulate muscle growth and affect meat quality in swine.

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

    PubMed

    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.

  17. Contamination of cDNA libraries and expressed sequence-tags databases

    SciTech Connect

    Dean, M.; Allikmets, R.

    1995-11-01

    Partially sequenced cDNAs, or expressed sequence tags (ESTs), are claimed to represent an efficient strategy for characterizing an organism`s genes. By necessity, these sequences are incompletely characterized, and examples of contamination of cDNA libraries with sequences from other species have been described. It has been suggested that a Human T-cell cDNA library (Clontech HL1963g) is contaminated by sequences from yeast (Saccharomyces cerevisiae) and an unknown bacterium. We are characterizing human ESTs that represent new members of the ATP-binding cassette transporter super-family. In examining human ESTs generated from the T-cell library, we have encountered one gene that was in fact a yeast sequence (Genbank Z15214 = SSH2 locus) and several genes that do not hybridize to human DNA or RNA. PCR primers from these sequences failed to amplify a product from human, yeast, or Escherichia coli DNA but did produce a product from a Clontech kidney cDNA library (HL1123a). To determine the source of the contamination, we amplified a conserved segment of the 16S rDNA (following a suggestion from Dr. C. Savakis) from the kidney library. The sequence of this product was nearly identical to that of the bacterium Leuconostoc lactis (300 of 304 bp). Leuconostoc species are commonly found in dairy products, fruits, vegetables, and wine and are nonpathogenic to humans. 6 refs., 1 fig.

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

    PubMed

    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.

  19. Construction of cDNA libraries from microdissected benign and malignant thyroid tissue.

    PubMed

    Kaserer, Klaus; Knezevic, Vladimir; Pichlhöfer, Bettina; Scheuba, Christian; Passler, Christian; Worth, Jennifer; Niederle, Bruno; Krizman, David

    2002-12-01

    cDNA libraries were constructed from thyroid epithelial cells gained by laser capture microdissection for gene expression analysis of the progression of thyroid cancer. Six histologically diverse thyroid tissue specimens were used. A mean of 93 ng of total RNA was gained per tissue sample from a mean estimated number of 25,000 microdissected cells per sample. Analysis of randomly selected clones from six libraries showed an average insert size of 600 (range, 300-1500) bp. Preliminary sequencing of clones selected from the six libraries indicates a range of 46% to 62% known genes per library, 4% to 25% anonymous expressed sequence tags per library, and 15% to 43% novel expressed sequence tags per library. Thyroglobulin was found in normal thyroid epithelium and follicular thyroid adenoma, whereas calcitonin precursor transcripts were found in medullary thyroid carcinoma. We demonstrate production of high-quality cDNA libraries of microdissected tissue of the thyroid, which should prove useful for gene expression analysis of human thyroid tumors.

  20. Construction and Evaluation of cDNA Libraries for Large-Scale Expressed Sequence Tag Sequencing in Wheat (Triticum aestivum L.)

    PubMed Central

    Zhang, D.; Choi, D. W.; Wanamaker, S.; Fenton, R. D.; Chin, A.; Malatrasi, M.; Turuspekov, Y.; Walia, H.; Akhunov, E. D.; Kianian, P.; Otto, C.; Simons, K.; Deal, K. R.; Echenique, V.; Stamova, B.; Ross, K.; Butler, G. E.; Strader, L.; Verhey, S. D.; Johnson, R.; Altenbach, S.; Kothari, K.; Tanaka, C.; Shah, M. M.; Laudencia-Chingcuanco, D.; Han, P.; Miller, R. E.; Crossman, C. C.; Chao, S.; Lazo, G. R.; Klueva, N.; Gustafson, J. P.; Kianian, S. F.; Dubcovsky, J.; Walker-Simmons, M. K.; Gill, K. S.; Dvořák, J.; Anderson, O. D.; Sorrells, M. E.; McGuire, P. E.; Qualset, C. O.; Nguyen, H. T.; Close, T. J.

    2004-01-01

    A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 “unigenes.” Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature. PMID:15514038

  1. Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families.

    PubMed

    Sim, G K; Kafatos, F C; Jones, C W; Koehler, M D; Efstratiadis, A; Maniatis, T

    1979-12-01

    A cDNA library has been constructed from an RNA preparation highly enriched in silkmoth chorion mRNAs. Many distinct clones have been identified from this library using a stepwise procedure: scoring for infrequent hexanucleotide restriction enzyme recognition sequences; detailed characterization with restriction enzymes that recognize relatively frequent tetranucleotide sequences; probing the arrangement of the corresponding sequences in chromosomal DNA by the Southern procedure; and detailed cross-hybridization analysis. Unique clones, as well as two classes of distinct but related clones, were revealed by hybridization. The cross-hybridization analysis was greatly facilitated by a newly developed, semiquantitative dot hybridization procedure. The same procedure made it feasible to conveniently estimate the relative abundance of several different sequences in an mRNA mixture. Cloned sequences which scored as relatively abundant in total chorion mRNA were tested with stage-specific chorion mRNA at a very stringent criterion of hybridization. They were thus characterized as early, middle or late sequences with respect to development. The characterized cDNA clones can now be used as probes for studying the evolution, chromosomal organization and regulated developmental expression of the chorion multigene families.

  2. Optimized Construction of SSR-enriched Libraries

    USDA-ARS?s Scientific Manuscript database

    We have developed a more efficient method to construct simple sequence repeat (SSR) libraries. We have designed and tested several new adapter. We changed the typical blunt-end ligation of adapters for the more effective sticky-end ligation. We optimized temperature, enzymes and length of each st...

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:25607527

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

    SciTech Connect

    Rasmussen, K

    2005-08-29

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

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

    PubMed Central

    Naoumkina, Marina; Torres-Jerez, Ivone; Allen, Stacy; He, Ji; Zhao, Patrick X; Dixon, Richard A; May, Gregory D

    2007-01-01

    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. PMID:18034910

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

    PubMed

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

    1999-01-01

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

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

    PubMed

    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.

  9. RT-Bst: an integrated approach for reverse transcription and enrichment of cDNA from viral RNA.

    PubMed

    Kabir, M S; Clements, M O; Kimmitt, P T

    2015-01-01

    The synthesis of cDNA from RNA is challenging due to the inefficiency of reverse transcription (RT). In order to address this, an RT-Bst method was developed for sequential RT of RNA and Bst DNA polymerase amplification for enrichment of cDNA in a single-tube reaction. Using genomic RNA from bacteriophage MS2, the yield of cDNA produced by RT alone and RT-Bst were compared by analysis of polymerase chain reaction (PCR)-amplified products. A superior performance was observed when amplifying MS2 cDNA with random primers following RT-Bst compared to RT alone, indicating greater quantities of cDNA were present after RT-Bst. RT-Bst was also compared with RT alone for their relative ability to produce sufficient cDNA to amplify eight target regions spanning the respiratory syncytial virus (RSV) genome. Six out of eight targets were amplified consistently by PCR subsequent to RT-Bst amplification, whereas only three out of eight targets could be amplified after RT alone. The RSV sequences were selectively amplified using RSV-specific primers from a mixed template containing an excess of MS2 RNA without amplifying MS2 sequences. This suggests that RT-Bst can be used to amplify RNA sequences non-specifically using random primers and specifically using sequence-specific primers, and enhances the yield of cDNA when compared to RT alone.

  10. Enriching Peptide Libraries for Binding Affinity and Specificity Through Computationally Directed Library Design.

    PubMed

    Foight, Glenna Wink; Chen, T Scott; Richman, Daniel; Keating, Amy E

    2017-01-01

    Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model.

  11. In-Frame cDNA Library Combined with Protein Complementation Assay Identifies ARL11-Binding Partners

    PubMed Central

    Lee, Sangkyou; Lee, Ilkyun; Jung, Yoonsuh; McConkey, David; Czerniak, Bogdan

    2012-01-01

    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. PMID:23272234

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

    PubMed

    Lee, Sangkyou; Lee, Ilkyun; Jung, Yoonsuh; McConkey, David; Czerniak, Bogdan

    2012-01-01

    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.

  13. Study on construction of cDNA libraries from rat normal liver and regeneration liver with smart technique.

    PubMed

    Jun-Tang, Lin; Cong-Rui, Wang; Pramanik, Jogenananda; Hui-Yong, Zhang; Hui-Gen, Feng; Bao-Sheng, Yang; Yu-Chang, L I; Cun-Shuan, Xu

    2004-07-01

    The aim of the study is to construct cDNA libraries from the normal liver and regeneration liver of rat by SMART (switching mechanism at 5' end of RNA transcript) technique and analyze their quality. The total RNA was separated from the normal liver and regeneration liver of rat and the frist-strand cDNA was synthesized through reverse transcription by a modified oligo (dT) primer (contained sfi IB site) while the SMART oligonucleotide (contained sfi IA site) was utilized as a template so that the first-strand cDNA could be extended over the 5' end of mRNA. The double-strand cDNA was amplified by LD-PCR (long-distance PCR) with the above two primers and then digested by sfi I (IA & IB) restriction, enzyme. After cDNA size fractionation through Chroma Spin 400 column, the double-strand cDNA was ligated into the sfi I-digested lambda TripIEx2 vector and then the recombinant DNA was packagedin vitro. The unamplified rat normal liver cDNA library consists of 1.3×10(7) pfu/ml, and regeneration liver cDNA library consists of 1.6×10(7) pfu/ml in which the percentage of recombinant clones both are about 100%. Through testing, the high quality cDNA libraries containing full-length cDNA of rat normal liver and regeneration liver have been constructed. The titer of the amplified cDNA library is 4.5×10(10) pfu/ml and 3.6×10(10) pfu/ml. the average exogenous inserts of the recombinants both are about 1.5 kb. These results show that the normal liver and regeneration liver of rat cDNA libraries both have an excellent quality and lay solid foundation to study liver functions and the mechanism of liver regeneration.

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

    DOEpatents

    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.

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

    DOEpatents

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

    1998-12-08

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods. 25 figs.

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

    PubMed

    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.

  17. Novel antimicrobial peptides identified from an endoparasitic wasp cDNA library.

    PubMed

    Shen, Xiaojing; Ye, Gongyin; Cheng, Xiongying; Yu, Chunyan; Yao, Hongwei; Hu, Cui

    2010-01-01

    We screened an endoparasitic wasp (Pteromalus puparum) cDNA library for DNA sequences having antimicrobial activity using a vital dye exclusion assay. Two dozens of clones were isolated that inhibited the growth of host Escherichia coli cells due to expression of the cloned genes. Three peptides (PP13, PP102 and PP113) were synthesized chemically based on the amino acid sequences deduced from these clones and assayed for their antimicrobial activity. These peptides have net positive charges and are active against both Gram-negative and -positive bacteria, but are not active against fungi tested. Their hemolytic activity on human red blood cells was measured, and no hemolytic activity was observed after 1-h incubation at a concentration of 62.5 microM or below. A Blast search indicated that the three peptides have not been previously characterized as antimicrobial peptides (AMPs). Salt-dependency studies revealed that the biocidal activity of these peptides against E. coli decreased with increasing concentration of NaCl. Transmission electron microscopic (TEM) examination of PP13-treated E. coli cells showed extensive damage of cell membranes. The CD spectroscopy studies noted that the enhanced alpha-helical characteristics of PP13 strongly contribute to its higher antimicrobial properties. These results demonstrate the feasibility to identify novel AMPs by screening the expressional cDNA library.

  18. Primary Analysis of the Expressed Sequence Tags in a Pentastomid Nymph cDNA Library

    PubMed Central

    Yuan, Zhongying; Yin, Jianhai; Zang, Wei; Xu, Yuxin; Lu, Weiyuan; Wang, Yanjuan; Wang, Ying; Cao, Jianping

    2013-01-01

    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. PMID:23437150

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

    PubMed Central

    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

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

    SciTech Connect

    Kandpal, R.P.; Kandpal, G.; Weissman, S.M. )

    1994-01-04

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

  1. Isolation of SMA candidate genes from a YAC contig by direct selection of cDNA clones from normalized cDNA libraries

    SciTech Connect

    Ross, B.M.; Bonaldo, M.F.; Vitale, E.

    1994-09-01

    A YAC contig has been constructed across the spinal muscular atrophy (SMA) region of chromosome 5 (5q11-13). Further definition by pedigree analysis has yielded a minimal genetic region of 400 kb. For isolation of candidate genes in this region, the following cDNA selection method was hybridized to directionally cloned normalized (Cot 1 DNA-preannealed) cDNA libraries in the form of single-stranded circles. The libraries used were constructed from SMA infant brain and normal fetal liver+spleen. Hybridizing circles were eluted off the filter, partially converted into duplexes and electroporated into bacteria. The selected clones were then sequentially hybridized with a human Cot 1 DNA probe (BRL), and a probe made from the corresponding YAC DNA. Clones that hybridized only to the YAC DNA probe were verified to map to the critical region by genomic Southern analyses. Ten different cDNA clones have been isolated by this procedure so far. Three of them have been definitively mapped back to the region. Four of the ten clones are now completely sequenced. One clone shows sequence homology to a transcriptional initiation factor; another has homology to a prokaryotic attachment site sequence for the lipid moiety of membrane lipoproteins. Two clones show no homology to sequences represented in the public databases. We are continuing the full characterization of the cDNA clones as candidates for the SMA gene.

  2. Construction and characterization of a normalized cDNA library from the river snail Bellamya aeruginosa after exposure to copper.

    PubMed

    Li, Zi-Cheng; An, Li-Hui; Fu, Qing; Liu, Ying; Zhang, Lei; Chen, Hao; Zhao, Xing-Ru; Wang, Li-Jing; Zheng, Bing-Hui; Zhang, Lin-Bo

    2012-01-01

    The construction of a normalized cDNA library is a popular tool for identifying novel biomarkers for monitoring environmental pollution. In the present study, a normalized cDNA library was constructed from the river snail Bellamya aeruginosa after exposure to Cu(2+) by using the SMART technique. The titer of the cDNA library was 1.78 × 10(6) pfu/ml, with a recombinant efficiency of 95.8%. In addition, from 6,000 randomly selected and sequenced clones, 5,473 high-quality ESTs were identified. After processing the sequences, 3,961 unigenes representing 897 contigs and 3,064 singlets were obtained with 27.6% redundancy. Analysis of expressed sequenced tags using COG and GO annotation and KEGG pathway data showed that a large group of genes related to growth and development, signal transduction, and defense mechanisms were present in the cDNA library. Based on our findings, this normalized cDNA library will provide a valuable resource for further research on functional genes and ecotoxicology in B. aeruginosa.

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

    SciTech Connect

    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.

  4. Construction of a normalized cDNA library by introduction of a semi-solid mRNA-cDNA hybridization system.

    PubMed Central

    Sasaki, Y F; Ayusawa, D; Oishi, M

    1994-01-01

    We report a novel procedure to construct a normalized (equalized) cDNA library. By introduction of the highly efficient self-hybridization system between a whole mRNA population and their corresponding cDNA immobilized on latex beads, which involves relatively simple manipulations, we were able to generate an mRNA population in which the copy number of abundant species was reduced while that of rare species was enriched. In a typical experiment, after several cycles of self-hybridization on the beads, the ratio of the most to the least abundant marker mRNA species dropped by a factor of 300 (from 10,000 to 30) while the complexity and length of mRNAs in the population remained unchanged. The procedure should provide a potent tool for the expression cloning of cDNA and also facilitate the construction of whole cDNA catalogs from specific tissues (or cell types) from higher organisms. Images PMID:8152931

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

    PubMed

    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.

  6. [Construction and significance of directional expression cDNA library from myeloid leukemia cell line U937].

    PubMed

    Chen, Gang; Zhang, Wang-Gang; Fu, Jie; Cao, Xing-Mei; Zhao, Wan-Hong; Zhao, Ai-Zhi; Han, Yue-Heng; Li, Fu-Yang; Liu, Xin-Ping; Yao, Li-Bo

    2003-08-01

    To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted. The first and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated. Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl-S400 spin column, the fragments longer than 400 bp were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the U937 cell line cDNA library consisting of 2.87 x 10(6) recombinant bacteriophages was constructed. The average size of exogenous insert in the recombinants was about 1.7 kb. It is concluded that the constructed cDNA library can be used to screen target clones.

  7. 2058 Expressed sequence tags (ESTs) from a human fetal lung cDNA library

    SciTech Connect

    Kazunori, Sudo |; Katsuya Chinen; Yusuke Nakamura

    1994-11-15

    ESTs (expressed sequence tags) provide complementary resources for structural and functional analyses of the human genome. The authors have performed single-pass sequencing of 2058 randomly selected, directionally cloned cDNAs isolated from a fetal-lung cDNA library constructed with oligo (dT) primers. Computer analyses of the 5{prime}-end sequences revealed that 60.4% of the clones were considered to be identical to previously reported human genes or ESTs; 9.0% of them showed significant homology to known genes in human, other mammals, or lower organisms; 30.6% showed no homology to any genes or DNA sequences in the public database. These data and reagents will be useful for future investigations of gene expression during prenatal development of human lung. 11 refs., 1 fig., 2 tabs.

  8. A genomic analysis of Histomonas meleagridis through sequencing of a cDNA library.

    PubMed

    Klodnicki, M E; McDougald, L R; Beckstead, R B

    2013-04-01

    Histomonas meleagridis, a flagellated protozoan of the Order Trichomonadida, is the causative agent of blackhead disease in gallinaceous birds. Few genes have been identified in this organism; thus, little is known regarding the molecular basis for its metabolism, virulence, and antigenicity. To identify new genes, a cDNA library derived from a lab strain of H. meleagridis was sequenced and annotated. Data obtained from these experiments identified 3,425 H. meleagridis genes. Analysis of the data allowed the identification of 81 genes coding for putative hydrogenosomal proteins and was used to determine the codon usage frequency. Sequence information also identified bacteria that are cultured with H. meleagridis. Future analysis of these data should provide valuable molecular insights into H. meleagridis and provide the platform for molecular studies aimed at understanding the pathogenesis of blackhead disease.

  9. Preparation of a cDNA library from the hagfish slime gland

    SciTech Connect

    Gadbois, D.M.; Salo, W.L.; Ann, D.K.; Downing, S.W.; Carlson, D.M.

    1986-05-01

    The slime glands of hagfish have two major cell types, gland thread cells (GTCs) and gland mucous cells (GMCs), both of which upon contact with water contribute to an abundant viscous mucus. GTCs are unique specialized cells in which 60-80% of the mature GTC volume is occupied by a single tapered thread about 60 cm long formed by bundling keratin intermediate filaments (IFs). Each IF is composed of three 63.5 kD protein subunits (..cap alpha.., ..beta.., ..gamma..). Poly (A/sup +/) mRNA was isolated from GTCs and translated by the rabbit reticulocyte system with (/sup 35/S) Met. Analysis of the translation products showed a major labeled protein at 65 kD. A cDNA library prepared from the poly (A/sup +/) mRNAs was screened with /sup 32/P-labeled IF probes from human epidermis. Several clones containing cross-hybridizing inserts were identified.

  10. Screening of novel malaria DNA vaccine candidates using full-length cDNA library.

    PubMed

    Shibui, Akiko; Nakae, Susumu; Watanabe, Junichi; Sato, Yoshitaka; Tolba, Mohammed E M; Doi, Junko; Shiibashi, Takashi; Nogami, Sadao; Sugano, Sumio; Hozumi, Nobumichi

    2013-11-01

    No licensed malaria vaccine exists, in spite of intensive development efforts. We have been investigating development of a DNA vaccine to prevent malaria infection. To date, we have established a full-length cDNA expression library from the erythrocytic-stage murine malaria parasite, Plasmodium berghei. We found that immunization of mice with combined 2000 clones significantly prolonged survival after challenge infection and that splenocytes from the immunized mice showed parasite-specific cytokine production. We determined the 5'-end one-pass sequence of these clones and mapped a draft genomic sequence for P. berghei for use in screening vaccine candidates for efficacy. In this study, we annotated these cDNA clones by comparing them with the genomic sequence of Plasmodium falciparum. We then divided them into several subsets based on their characteristics and examined their protective effects against malaria infection. Consequently, we selected 104 clones that strongly induced specific IgG production and decreased the mortality rate in the early phase. Most of these 104 clones coded for unknown proteins. The results suggest that these clones represent potential novel malaria vaccine candidates.

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

    PubMed

    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.

  12. Expressed sequence tags from a NaCl-treated Suaeda salsa cDNA library.

    PubMed

    Zhang, L; Ma, X L; Zhang, Q; Ma, C L; Wang, P P; Sun, Y F; Zhao, Y X; Zhang, H

    2001-04-18

    Past efforts to improve plant tolerance to osmotic stress have had limited success owing to the genetic complexity of stress responses. The first step towards cataloging and categorizing genetically complex abotic stress responses is the rapid discovery of genes by the large-scale partial sequencing of randomly selected cDNA clones or expressed sequence tags (ESTs). Suaeda salsa, which can survive seawater-level salinity, is a favorite halophytic model for salt tolerant research. We constructed a NaCl-treated cDNA library of Suaeda salsa and sequenced 1048 randomly selected clones, out of which 1016 clones produced readable sequences (773 showed homology to previously identified genes, 227 matched unknown protein coding regions, 16 anomalous sequences or sequences of bacterial origin were excluded from further analysis). By sequence analysis we identified 492 unique clones: 315 showed homology to previously identified genes, 177 matched unknown protein coding regions (101 of which have been found before in other organisms and 76 are completely novel). All our EST data are available on the Internet. We believe that our dbEST and the associated DNA materials will be a useful source to scientists engaging in stress-tolerance study.

  13. Isolation of chicken alpha ENaC splice variants from a cochlear cDNA library.

    PubMed

    Killick, R; Richardson, G

    1997-01-03

    Three splice variants of the alpha subunit of the amiloride-sensitive epithelial sodium channel (alpha ENaC) have been isolated from a chicken cochlear cDNA library. A PCR product, generated from the cochlear library using degenerate primers to regions of homology between the rat alpha ENaC and the degenerin Mec-4, was used as a probe. The three splice variant cDNAs with sizes of 2321, 3399 and 3845 bp correspond to transcripts of 2.5, 3.5 and 3.9 kb as detected by Northern blot analysis. The 3399 bp clone differs from the 2321 clone solely by the addition of 1079 bases in the 3'-non-coding region. Both these cDNAs code for an identical predicted protein of 637 amino acids which has 68% similarity to the rat alpha ENaC, and is probably the chicken homologue of alpha ENaC. The third cDNA of 3845 bp is similar to the 3399 bp clone but includes two exons within the open reading frame. The first of these exons introduces a premature stop codon resulting in a truncated predicted protein of 434 amino acids. Northern blot analysis shows expression of the 2.5 and 3.5 kb transcripts in cochlea and colon, the 2.5 kb transcript in cartilage, whilst the 3.9 kb transcript is only detected in cochlea. No expression is detected in brain, liver, and heart nor, most notably, in lung or kidney.

  14. Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae)

    USDA-ARS?s Scientific Manuscript database

    Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding. cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens ‘Vardar Valley’ and seque...

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

    PubMed

    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.

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

    PubMed Central

    BILIC, I.; LEBERL, M.; HESS, M.

    2010-01-01

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

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

    PubMed

    Bilic, I; Leberl, M; Hess, M

    2009-04-01

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

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

    PubMed Central

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

    2011-01-01

    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. PMID:21340003

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

    PubMed

    Rong, Mingqiang; Yang, Shilong; Wen, Bo; Mo, Guoxiang; Kang, Di; Liu, Jie; Lin, Zhilong; Jiang, Wenbin; Li, Bowen; Du, Chaoqin; Yang, Shuanjuan; Jiang, Hui; Feng, Qiang; Xu, Xun; Wang, Jun; Lai, Ren

    2015-01-30

    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 were more complicated than any other animal toxins and even exhibited large differences in homologues. Meanwhile, a large number of variants generated by alternative cleavage sites were detected by mass spectra. Odd number of cystein (3, 5, 7) found in the mature peptides were seldom seen in peptide toxins. In additional, two novel cysteine frameworks (C-C-C-CCC, C-C-C-C-CC-CC) were identified from 16 different cysteine frameworks from centipede peptides. Only 29 precursors have clear targets, while others may provide a potential diversity function for centipede. These findings highlight the extensive diversity of centipede toxins and provide powerful tools to understand the capture and defense weapon of centipede. Peptide toxins from venomous animal have attracted increasing attentions due to their extraordinary chemical and pharmacological diversity. Centipedes are one of the most used Chinese traditional medicines, but little was known about the active components. The venom of Scolopendra subspinipes mutilans L. Koch is first deeply analyzed in this work and most of peptides were never discovered before. Interestingly, the number and arrangement of cysteine showed a larger different to known peptide toxins such spider or scorpion toxins. Moreover, only 29 peptides from this centipede venom were identified with known function. It suggested that our work not only important to

  20. Analyzing serial cDNA libraries revealed reactive oxygen species and gibberellins signaling pathways in the salt response of Upland cotton (Gossypium hirsutum L.).

    PubMed

    Shi, Gongyao; Guo, Xiaoyan; Guo, Jinyan; Liu, Laihua; Hua, Jinping

    2015-06-01

    By comparing series full-length cDNA libraries stressed and control, the dynamic process of salt stress response in Upland cotton was studied, and reactive oxygen species and gibberellins signaling pathways were proposed. The Upland cotton is the most important fiber plant with highly salt tolerance. However, the molecular mechanism underlying salt tolerance in domesticated cotton was unclear. Here, seven full-length cDNA libraries were constructed for seedling roots of Upland cotton 'Zhong G 5' at 0, 3, 12 and 48 h after the treatment of control or 150 mM NaCl stress. About 3300 colonies in each library were selected robotically for 5'-end pyrosequencing, resulting in 20,358 expressed sequence tags (ESTs) totally. And 8516 uniESTs were then assembled, including 2914 contigs and 5602 singletons, and explored for Gene Ontology (GO) function. GO comparison between serial stress libraries and control reflected the growth regulation, stimulus response, signal transduction and biology regulation processes were conducted dynamically in response to salt stress. MYB, MYB-related, WRKY, bHLH, GRAS and ERF families of transcription factors were significantly enriched in the early response. 65 differentially expressed genes (DEGs), mainly associated with reactive oxygen species (ROS) scavenging, gibberellins (GAs) metabolism, signal transduction, transcription regulation, stress response and transmembrane transport, were identified and confirmed by quantitative real-time PCR. Overexpression of selected DEGs increased tolerance against salt stress in transgenic yeast. Results in this study supported that a ROS-GAs interacting signaling pathway of salt stress response was activated in Upland cotton. Our results provided valuable gene resources for further investigation of the molecular mechanism of salinity tolerance.

  1. Development of SSR markers derived from SSR-enriched genomic library of eggplant (Solanum melongena L.).

    PubMed

    Nunome, Tsukasa; Negoro, Satomi; Kono, Izumi; Kanamori, Hiroyuki; Miyatake, Koji; Yamaguchi, Hirotaka; Ohyama, Akio; Fukuoka, Hiroyuki

    2009-10-01

    Eggplant (Solanum melongena L.), also known as aubergine or brinjal, is an important vegetable in many countries. Few useful molecular markers have been reported for eggplant. We constructed simple sequence repeat (SSR)-enriched genomic libraries in order to develop SSR markers, and sequenced more than 14,000 clones. From these sequences, we designed 2,265 primer pairs to flank SSR motifs. We identified 1,054 SSR markers from amplification of 1,399 randomly selected primer pairs. The markers have an average polymorphic information content of 0.27 among eight lines of S. melongena. Of the 1,054 SSR markers, 214 segregated in an intraspecific mapping population. We constructed cDNA libraries from several eggplant tissues and obtained 6,144 expressed sequence tag (EST) sequences. From these sequences, we designed 209 primer pairs, 7 of which segregated in the mapping population. On the basis of the segregation data, we constructed a linkage map, and mapped the 236 segregating markers to 14 linkage groups. The linkage map spans a total length of 959.1 cM, with an average marker distance of 4.3 cM. The markers should be a useful resource for qualitative and quantitative trait mapping and for marker-assisted selection in eggplant breeding.

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

    PubMed

    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.

  3. Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening

    SciTech Connect

    Robertson, N.G.; Gutierrez-Espeleta, G.A.; Bieber, F.R. |

    1994-09-01

    We used a combination of subtractive hybridization and differential screening strategies to identify genes that may function normally in hearing and, when mutated, result in deafness. A human fetal cochlear (membranous labyrinth) cDNA library was subtracted against total human fetal brain RNAs by an avidin-biotin-based procedure to enrich for cochlear transcripts. Subtracted cochlear clones were differentially screened with {sup 32}P-labeled total cochlear and total brain cDNA probes. Sequence analysis of clones that hybridized more intensely with cochlear than with brain cDNA probes revealed some previously characterized genes, including mitochondrial sequences, collagen type I {alpha}-2 (COL1A2), collagen type II {alpha}-1 (COL2A1), collagen type III {alpha}-1 (COL3A1), spermidine/spermine N{sup 1}-acetyltransferase (SAT), osteonectin (SPARC), and peripheral myelin protein 22 (PMP22). Also identified were clones that are potential novel cochlear genes. Northern blots of cochlear and brain RNAs probed with COL1A2, COL2A1, COL3A1, SAT, SPARC, PMP22, and a novel sequence, designated Coch-5B2, confirm results of the subtractive procedure by showing preferential cochlear expression. A number of these genes serve structural or regulatory functions in extracellular matrix or neural conduction; defects in some of these genes are associated with disorders involving hearing loss. Partial sequence analysis of Coch-5B2 reveals a von Willebrand factor type A-like domain in this cDNA. To assess the cochlear specificity of Coch-5B2, a Northern blot panel of 14 human fetal tissue RNAs was probed with Coch-5B2, showing differential expression of this novel gene in the cochlea. 68 refs., 3 figs.

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

    PubMed Central

    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

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

    PubMed Central

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

    2010-01-01

    It is well known that the nutritional quality of the American oil-palm (Elaeis oleifera) mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we constructed a cDNA library and generated expressed sequence tags (3,205) from the mesocarp tissue of the American oil-palm. We continued to annotate each of these cDNAs after submitting to GenBank/DDBJ/EMBL. A rough analysis turned our attention to the beta-carotene hydroxylase (Chyb) enzyme encoding cDNA. Then, we completed the full sequencing of cDNA clone for its both strands using M13 forward and reverse primers. The full nucleotide and protein sequence was further analyzed and annotated using various Bioinformatics tools. The analysis results showed the presence of fatty acid hydroxylase superfamily domain in the protein sequence. The multiple sequence alignment of selected Chyb amino acid sequences from other plant species and algal members with E. oleifera Chyb using ClustalW and its phylogenetic analysis suggest that Chyb from monocotyledonous plant species, Lilium hubrid, Crocus sativus and Zea mays are the most evolutionary related with E. oleifera Chyb. This study reports the annotation of E. oleifera Chyb. Abbreviations ESTs - expressed sequence tags, EoChyb - Elaeis oleifera beta-carotene hydroxylase, MC - main cluster PMID:21364789

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

    PubMed

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

    2010-09-20

    It is well known that the nutritional quality of the American oil-palm (Elaeis oleifera) mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we constructed a cDNA library and generated expressed sequence tags (3,205) from the mesocarp tissue of the American oil-palm. We continued to annotate each of these cDNAs after submitting to GenBank/DDBJ/EMBL. A rough analysis turned our attention to the beta-carotene hydroxylase (Chyb) enzyme encoding cDNA. Then, we completed the full sequencing of cDNA clone for its both strands using M13 forward and reverse primers. The full nucleotide and protein sequence was further analyzed and annotated using various Bioinformatics tools. The analysis results showed the presence of fatty acid hydroxylase superfamily domain in the protein sequence. The multiple sequence alignment of selected Chyb amino acid sequences from other plant species and algal members with E. oleifera Chyb using ClustalW and its phylogenetic analysis suggest that Chyb from monocotyledonous plant species, Lilium hubrid, Crocus sativus and Zea mays are the most evolutionary related with E. oleifera Chyb. This study reports the annotation of E. oleifera Chyb. ESTs - expressed sequence tags, EoChyb - Elaeis oleifera beta-carotene hydroxylase, MC - main cluster.

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

    PubMed

    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.

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

    PubMed Central

    2011-01-01

    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-genome sequences. In addition the

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

    PubMed Central

    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. PMID:27579677

  10. A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector.

    PubMed Central

    Rayner, J R; Gonda, T J

    1994-01-01

    cDNA expression cloning is a powerful method for the rescue and identification of genes that are able to confer a readily identifiable phenotype on specific cell types. Retroviral vectors provide several advantages over DNA-mediated gene transfer for the introduction of expression libraries into eukaryotic cells since they can be used to express genes in a wide range of cell types, including those that form important experimental systems such as the hemopoietic system. We describe here a straightforward and efficient method for generating expression libraries by using a murine retroviral vector. Essentially, the method involves the directional cloning of cDNA into the retroviral vector and the generation of pools of stable ecotropic virus producing cells from this DNA. The cells so derived constitute the library, and the virus they yield is used to infect appropriate target cells for subsequent functional screening. We have demonstrated the feasibility of this procedure by constructing several large retroviral libraries (10(5) to 10(6) individual clones) and then using one of these libraries to isolate cDNAs for interleukin-3 and granulocyte-macrophage colony-stimulating factor on the basis of the ability of these factors to confer autonomous growth on the factor-dependent hemopoietic cell line FDC-P1. Moreover, the frequency at which these factor-independent clones were isolated approximated the frequency at which they were represented in the original plasmid library. These results suggest that expression cloning with retroviruses is a practical and efficient procedure and should be a valuable method for the isolation of important regulatory genes. Images PMID:8289827

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

    PubMed

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

    2003-04-02

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

  12. Screening of substrate peptide sequences for tissue-type transglutaminase (TGase 2) using T7 phage cDNA library.

    PubMed

    Sugimura, Yoshiaki; Yamashita, Hiroyuki; Hitomi, Kiyotaka

    2011-03-01

    Transglutaminase (TGase) is a family of enzymes that catalyzes cross-linking reaction between glutamine- and lysine residue of substrate proteins in several mammalian biological events. Substrate proteins for TGase and their physiological relevance have been still in research, continuously expanding. In this study, we have established a novel screening system that enables identification of cDNA sequence encoding favorable primary structure as a substrate for tissue-type transglutaminase (TGase 2), a multifunctional and ubiquitously expressing isozyme. By the screening, we identified several T7 phage clones that displayed substrate peptides for TGase 2 as a translated product from human brain cDNA library. Among the selected clones, the C-terminal region of IKAP, IkappaB kinase complex associated protein, appeared as a highly reactive substrate sequence for TGase 2. This system will open possibility of rapid identification of substrate sequences for transglutaminases at a genetic level.

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

    PubMed Central

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

    2007-01-01

    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 the Institute for Genome

  14. Thymus cDNA library survey uncovers novel features of immune molecules in Chinese giant salamander Andrias davidianus.

    PubMed

    Zhu, Rong; Chen, Zhong-Yuan; Wang, Jun; Yuan, Jiang-Di; Liao, Xiang-Yong; Gui, Jian-Fang; Zhang, Qi-Ya

    2014-10-01

    A ranavirus-induced thymus cDNA library was constructed from Chinese giant salamander, the largest extant amphibian species. Among the 137 putative immune-related genes derived from this library, these molecules received particular focus: immunoglobulin heavy chains (IgM, IgD, and IgY), IFN-inducible protein 6 (IFI6), and T cell receptor beta chain (TCRβ). Several unusual features were uncovered: IgD displays a structure pattern distinct from those described for other amphibians by having only four constant domains plus a hinge region. A unique IgY form (IgY(ΔFc)), previously undescribed in amphibians, is present in serum. Alternative splicing is observed to generate IgH diversification. IFI6 is newly-identified in amphibians, which occurs in two forms divergent in subcelluar distribution and antiviral activity. TCRβ immunoscope profile follows the typical vertebrate pattern, implying a polyclonal T cell repertoire. Collectively, the pioneering survey of ranavirus-induced thymus cDNA library from Chinese giant salamander reveals immune components and characteristics in this primitive amphibian.

  15. Generation and analysis of expressed sequence tags from Trypanosoma cruzi trypomastigote and amastigote cDNA libraries.

    PubMed

    Agüero, Fernán; Abdellah, Karim Ben; Tekiel, Valeria; Sánchez, Daniel O; González, Antonio

    2004-08-01

    We have generated 2771 expressed sequence tags (ESTs) from two cDNA libraries of Trypanosoma cruzi CL-Brener. The libraries were constructed from trypomastigote and amastigotes, using a spliced leader primer to synthesize the cDNA second strand, thus selecting for full-length cDNAs. Since the libraries were not normalized nor pre-screened, we compared the representation of transcripts between the two using a statistical test and identify a subset of transcripts that show apparent differential representation. A non-redundant set of 1619 reconstructed transcripts was generated by sequence clustering. This dataset was used to perform similarity searches against protein and nucleotide databases. Based on these searches, 339 sequences could be assigned a putative identity. One thousand one-hundred and sixteen sequences in the non-redundant clustered dataset (68.8%) are new expression tags, not represented in the T. cruzi epimastigote ESTs that are in the public databases. Additional information is provided online at http://genoma.unsam.edu.ar/projects/tram. To the best of our knowledge these are the first ESTs reported for the life cycle stages of T. cruzi that occur in the vertebrate host.

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

    PubMed

    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.

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

    PubMed

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

    2013-02-15

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

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

    PubMed

    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.

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

    PubMed

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

    2002-05-01

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

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

    NASA Astrophysics Data System (ADS)

    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. A microarray method for identifying tumor antigens by screening a tumor cDNA expression library against cancer sera

    PubMed Central

    Whittemore, Kurt; Sykes, Kathryn

    2013-01-01

    The immune system responds to tumor cells. The challenge has been how to effectively use these responses to treat or protect against cancer. Toward the goal of developing a cancer vaccine, we are pursuing methodologies for the discovery and testing of useful antigens. We present an array-based approach for discovering these B cell antigens by directly screening for specific host-sera reactivity to lysates from tumor-derived cDNA expression libraries. Several cancer-specific antigens were identified, and these are currently being validated as potential candidates. PMID:23851590

  2. A microarray method for identifying tumor antigens by screening a tumor cDNA expression library against cancer sera.

    PubMed

    Whittemore, Kurt; Sykes, Kathryn

    2013-10-01

    The immune system responds to tumor cells. The challenge has been how to effectively use these responses to treat or protect against cancer. Toward the goal of developing a cancer vaccine, we are pursuing methodologies for the discovery and testing of useful antigens. We present an array-based approach for discovering these B cell antigens by directly screening for specific host-sera reactivity to lysates from tumor-derived cDNA expression libraries. Several cancer-specific antigens were identified, and these are currently being validated as potential candidates.

  3. Representative cDNA libraries and their utility in gene expression profiling.

    PubMed

    Endege, W O; Steinmann, K E; Boardman, L A; Thibodeau, S N; Schlegel, R

    1999-03-01

    An increasing interest in gene expression profiles in human diseases has led to the use of microdissected tumors and biopsies in gene discovery approaches. Since many of these clinical samples yield extremely small amounts of RNA, reproducible methods are needed to amplify this RNA while maintaining the original message profile. Using the SMART cDNA Synthesis Method, we show that high-, medium- and low-abundance transcripts can be amplified in a representative fashion and that the resulting cDNA can also be used as a complex probe to confirm gene expression differences identified by other techniques.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  6. Construction of a full-length cDNA library of Solen grandis dunker and identification of defense- and immune-related genes

    NASA Astrophysics Data System (ADS)

    Sun, Guohua; Liu, Xiangquan; Ren, Lihua; Yang, Jianmin; Wei, Xiumei; Yang, Jialong

    2013-11-01

    The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the `switching mechanism at the 5'-end of the RNA transcript' (SMART) technique. Total RNA was isolated from the immune-relevant tissues, gills and hemocytes, using the Trizol reagent, and cDNA fragments were digested with Sfi I before being ligated to the pBluescript II SK* vector. The cDNA library had a titer of 1048 cfu μL-1 and a storage capacity of 1.05×106 cfu. Approximately 98% of the clones in the library were recombinants, and the fragment lengths of insert cDNA ranged from 0.8 kb to 3.0 kb. A total of 2038 expressed sequence tags were successfully sequenced and clustered into 965 unigenes. BLASTN analysis showed that 240 sequences were highly similar to the known genes (E-value < 1e -5; percent identity >80%), accounting for 25% of the total unigenes. According to the Gene Ontology, these unigenes were related to several biological processes, including cell structure, signal transport, protein synthesis, transcription, energy metabolism, and immunity. Fifteen of the identified sequences were related to defense and immunity. The full-length cDNA sequence of HSC70 was obtained. The cDNA library of S. grandis provided a useful resource for future researches of functional genomics related to stress tolerance, immunity, and other physiological activities.

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

    PubMed

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

    2015-01-01

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

  8. Enriching Critical Thinking and Language Learning with Educational Digital Libraries

    ERIC Educational Resources Information Center

    Lu, Hsin-lin

    2012-01-01

    As the amount of information available in online digital libraries increases exponentially, questions arise concerning the most productive way to use that information to advance learning. Applying the earlier information seeking theories advocated by Kelly (1963), Taylor (1968), and Belkin (1980) to the digital libraries experience, Carol Kuhlthau…

  9. Enriching Critical Thinking and Language Learning with Educational Digital Libraries

    ERIC Educational Resources Information Center

    Lu, Hsin-lin

    2012-01-01

    As the amount of information available in online digital libraries increases exponentially, questions arise concerning the most productive way to use that information to advance learning. Applying the earlier information seeking theories advocated by Kelly (1963), Taylor (1968), and Belkin (1980) to the digital libraries experience, Carol Kuhlthau…

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

    PubMed

    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.

  11. In silico Analysis of 2085 Clones from a Normalized Rat Vestibular Periphery 3′ cDNA Library

    PubMed Central

    Roche, Joseph P.; Cioffi, Joseph A.; Kwitek, Anne E.; Erbe, Christy B.; Popper, Paul

    2005-01-01

    The inserts from 2400 cDNA clones isolated from a normalized Rattus norvegicus vestibular periphery cDNA library were sequenced and characterized. The Wackym-Soares vestibular 3′ cDNA library was constructed from the saccular and utricular maculae, the ampullae of all three semicircular canals and Scarpa's ganglia containing the somata of the primary afferent neurons, microdissected from 104 male and female rats. The inserts from 2400 randomly selected clones were sequenced from the 5′ end. Each sequence was analyzed using the BLAST algorithm compared to the Genbank nonredundant, rat genome, mouse genome and human genome databases to search for high homology alignments. Of the initial 2400 clones, 315 (13%) were found to be of poor quality and did not yield useful information, and therefore were eliminated from the analysis. Of the remaining 2085 sequences, 918 (44%) were found to represent 758 unique genes having useful annotations that were identified in databases within the public domain or in the published literature; these sequences were designated as known characterized sequences. 1141 sequences (55%) aligned with 1011 unique sequences had no useful annotations and were designated as known but uncharacterized sequences. Of the remaining 26 sequences (1%), 24 aligned with rat genomic sequences, but none matched previously described rat expressed sequence tags or mRNAs. No significant alignment to the rat or human genomic sequences could be found for the remaining 2 sequences. Of the 2085 sequences analyzed, 86% were singletons. The known, characterized sequences were analyzed with the FatiGO online data-mining tool (http://fatigo.bioinfo.cnio.es/) to identify level 5 biological process gene ontology (GO) terms for each alignment and to group alignments with similar or identical GO terms. Numerous genes were identified that have not been previously shown to be expressed in the vestibular system. Further characterization of the novel cDNA sequences may lead

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

    PubMed

    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.

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

    PubMed

    Wang, Jie; Xia, Yuxian

    2010-08-01

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

  14. Cryptosporidium parvum: identification of a new surface adhesion protein on sporozoite and oocyst by screening of a phage-display cDNA library.

    PubMed

    Yao, Longquan; Yin, Jigang; Zhang, Xichen; Liu, Quan; Li, Jianhua; Chen, Lifeng; Zhao, Yueping; Gong, Pengtao; Liu, Chengwu

    2007-04-01

    Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host interaction and the molecular mechanisms involved in the pathogenesis are unknown. In this study we described a novel phage display method to identify surface adhesion proteins of C. parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum expressed on the surface of T7 phage was screened with intestinal epithelial cells (IECs) from the newborn Cryptosporidium-free Holstein calves. Proteins that selectively and specifically bound to IECs were then enriched using a multi-step panning procedure. Two proteins of C. parvum were selected, one was previously reported (p23), which was an important surface adhesion protein; the other was a novel surface adherence protein (CP12). Sequence analysis showed that CP12 has a N-terminal signal peptide, a transmembrane region, a N-glycosylation site, a casein kinase II phosphorylation site and two N-myristoylation sites. Immunofluorescence assay (IFA) using antibody specific for rCP12 demonstrated that the antibody can specifically bind the surface of sporozoite and oocyst, especially apical region of sporozoite. The surface localization of CP12 and its involvement in the host-parasite interaction suggest that it may serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.

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

    PubMed

    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.

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

    PubMed

    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.

  17. Epstein-barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells

    SciTech Connect

    Margolskee, R.F.; Kavathas, P.; Berg, P.

    1988-07-01

    Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromcying B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastodi cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10/sup 6/ to 10/sup 7/ independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBP-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthing-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2. Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10/sup 6/ total clones or one clone of EBO-pcD-Leu-2 per 2 x 10/sup 4/ total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.

  18. Microsatellite markers isolated from the wild medicinal plant Centella asiatica (Apiaceae) from an enriched genomic library.

    PubMed

    Rakotondralambo, Soaharin'ny Ony Raoseta; Lussert, Alexandra; Rivallan, Ronan; Danthu, Pascal; Noyer, Jean-Louis; Baurens, Franc-Christophe

    2012-04-01

    Microsatellite markers for Centella asiatica, an important medicinal herb, were developed and characterized to promote genetic and molecular studies. A GA/GT-enriched genomic library was constructed from an accession from Madagascar. Roughly 75% of the 768 clones of the enriched library contained microsatellites. Eighty sequences containing microsatellites were obtained from 96 positive clones. Specific primers were designed for 20 loci, and 17 of them displayed polymorphism when screened across 17 C. asiatica accessions, with an average of 4.3 alleles per locus. The observed and expected heterozygosity values averaged 0.114 and 0.379, respectively. This is the first report constructing an enriched genomic library and identifying microsatellite markers from C. asiatica. These 17 polymorphic microsatellite markers are a useful resource for this plant, applicable for diversity studies, pedigree analyses, and genetic mapping.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

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

    PubMed

    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.

  2. Sequence analysis of a rainbow trout cDNA library and creation of a gene index.

    PubMed

    Rexroad, C E; Lee, Y; Keele, J W; Karamycheva, S; Brown, G; Koop, B; Gahr, S A; Palti, Y; Quackenbush, J

    2003-01-01

    Expressed sequence tag (EST) projects have produced extremely valuable resources for identifying genes affecting phenotypes of interest. A large-scale EST sequencing project for rainbow trout was initiated to identify and functionally annotate as many unique transcripts as possible. Over 45,000 5' ESTs were obtained by sequencing clones from a single normalized library constructed using mRNA from six tissues. The production of this sequence data and creation of a rainbow trout Gene Index eliminating redundancy and providing annotation for these sequences will facilitate research in this species.

  3. Developing new microsatellite markers in walnut (Juglans regia L.) from Juglans nigra genomic GA enriched library

    Treesearch

    Hayat Topcu; Nergiz Coban; Keith Woeste; Mehmet Sutyemez; Salih. Kafkas

    2015-01-01

    We attempted to develop new polymorphic SSR primer pairs in walnut using sequences derived from Juglans nigra L. genomic enriched library with GA repeat. The designed 94 SSR primer pairs were subjected to gradient PCR in 12 walnut cultivars to determine their optimum annealing temperatures and to determine whether they produce bands. Then, the...

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

    PubMed

    Junttila, Sini; Lim, Kean-Jin; Rudd, Stephen

    2009-10-05

    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. 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. 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.

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

    PubMed Central

    Junttila, Sini; Lim, Kean-Jin; Rudd, Stephen

    2009-01-01

    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. PMID:19804636

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

    PubMed Central

    2009-01-01

    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 and wheat defense genes

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

    PubMed

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

    2016-01-01

    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 knowledge of

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

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

    2015-01-01

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

  12. DNA sequence-based "bar codes" for tracking the origins of expressed sequence tags from a maize cDNA library constructed using multiple mRNA sources.

    PubMed

    Qiu, Fang; Guo, Ling; Wen, Tsui-Jung; Liu, Feng; Ashlock, Daniel A; Schnable, Patrick S

    2003-10-01

    To enhance gene discovery, expressed sequence tag (EST) projects often make use of cDNA libraries produced using diverse mixtures of mRNAs. As such, expression data are lost because the origins of the resulting ESTs cannot be determined. Alternatively, multiple libraries can be prepared, each from a more restricted source of mRNAs. Although this approach allows the origins of ESTs to be determined, it requires the production of multiple libraries. A hybrid approach is reported here. A cDNA library was prepared using 21 different pools of maize (Zea mays) mRNAs. DNA sequence "bar codes" were added during first-strand cDNA synthesis to uniquely identify the mRNA source pool from which individual cDNAs were derived. Using a decoding algorithm that included error correction, it was possible to identify the source mRNA pool of more than 97% of the ESTs. The frequency at which a bar code is represented in an EST contig should be proportional to the abundance of the corresponding mRNA in the source pool. Consistent with this, all ESTs derived from several genes (zein and adh1) that are known to be exclusively expressed in kernels or preferentially expressed under anaerobic conditions, respectively, were exclusively tagged with bar codes associated with mRNA pools prepared from kernel and anaerobically treated seedlings, respectively. Hence, by allowing for the retention of expression data, the bar coding of cDNA libraries can enhance the value of EST projects.

  13. cDNA library construction for next-generation sequencing to determine the transcriptional landscape of Legionella pneumophila.

    PubMed

    Sahr, Tobias; Buchrieser, Carmen

    2013-01-01

    The adaptation of Legionella pneumophila to the different conditions it encounters in the environment and in the host is governed by a complex regulatory system. Current knowledge of these regulatory networks and the transcriptome responses of L. pneumophila is mainly based on microarray analysis and limited to transcriptional products of annotated protein-coding genes. The application of the Next-Generation Sequencing (NGS) technology allows now genome-wide strand-specific sequencing and accurate determination of all expressed regions of the genome to reveal the complete transcriptional network and the dynamic interplay of specific regulators on a genome-wide level. NGS-based techniques promote deeper understanding of the global transcriptional organization of L. pneumophila by identifying transcription start sites (TSS), alternative TSS and operon organization, noncoding RNAs, antisense RNAs, and 5'-/3'-untranslated regions. In this chapter we describe the construction of cDNA libraries for (1) RNA deep sequencing (RNA-seq) and (2) TSS mapping using the Illumina technology.

  14. Identification of Toxoplasma gondii in-vivo induced antigens by cDNA library immunoscreening with chronic toxoplasmosis sera.

    PubMed

    Amerizadeh, Atefeh; Idris, Zulkarnain Md; Khoo, Boon Yin; Kotresha, Dupadahalli; Yunus, Muhammad Hafiznur; Karim, Izzati Zahidah Abdul; Saadatnia, Geita; Teh, Ai Ying; Noordin, Rahmah

    2013-01-01

    Toxoplasmosis is an infection caused by the parasite Toxoplasma gondii. Chronically-infected individuals with a compromised immune system are at risk for reactivation of the disease. In-vivo induced antigen technology (IVIAT) is a promising method for the identification of antigens expressed in-vivo. The aim of the present study was to apply IVIAT to identify antigens which are expressed in-vivo during T. gondii infection using sera from individuals with chronic toxoplasmosis. Forty serum samples were pooled, pre-adsorped against three different preparations of antigens, from each in-vitro grown T. gondii and Escherichia coli XLBlue MRF', and then used to screen a T. gondii cDNA expression library. Sequencing of DNA inserts from positive clones showed eight open reading frames with high homology to T. gondii genes. Expression analysis using quantitative real-time PCR showed that SAG1-related sequence 3 (SRS3) and two hypothetical genes were up-regulated in-vivo relative to their expression levels in-vitro. These three proteins also showed high sensitivity and specificity when tested with individual serum samples. Five other proteins namely M16 domain peptidase, microneme protein, elongation factor 1-alpha, pre-mRNA-splicing factor and small nuclear ribonucleoprotein F had lower RNA expression in-vivo as compared to in-vitro. SRS3 and the two hypothetical proteins warrant further investigation into their roles in the pathogenesis of toxoplasmosis.

  15. A transcriptional survey of the cDNA library of Macrolampis sp2 firefly lanterns (Coleoptera: Lampyridae).

    PubMed

    Viviani, Vadim R; Carmargo, Isabela A; Amaral, Danilo T

    2013-03-01

    The biochemistry of firefly bioluminescence is well understood; however, the molecular physiology of the lanterns is still poorly studied, especially the biosynthesis and origin of beetle luciferin which are almost unknown. Using a cDNA library previously constructed from Macrolampis sp2 lanterns, we randomly selected and sequenced 572 cDNAs in order to have a first transcriptional profile of the most represented messages found in the lanterns and therefore to better understand their molecular physiology. As expected, high percentage of the gene products (~22%) displayed high similarity with Coleoptera genome products. About 7% represented mitochondrial genes, including several copies of cytochrome oxidase, which are also expected for this tissue. Luciferase genes were especially abundant, representing ca 2% of the products. Gene products involved with cysteine and sulfur metabolism such as the cystathionine β-lyase and the S-adenosylmethionine synthetase were abundant. Noteworthy, an abundance of proteins involved with hormone metabolism was found, suggesting a possible link between bioluminescence and hormone metabolism.

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

    PubMed

    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.

  17. Gene discovery in the finger leather coral Sinularia notanda by construction and sequencing of a normalized cDNA library.

    PubMed

    Kim, Jae-Woo; Kim, Seong Ho; Jung, Min-Min; Kim, Heung Soo; Han, Seock-Jung; Moon, Tae Seok; Kim, Bong-Seok; Nam, Bo-Hye; Park, Chan-Il

    2015-02-01

    The transplantation of coral fragments is one of methods that restore coral communities. To form coral colonies, the fragmented corals initiated skeletal extension from the cut-edge of fragment then success the settlement. In order to understand the molecular events underlying fragment adhesion and settlement, we constructed a normalized cDNA library and generated and annotated expressed sequence tags (ESTs) from the fragmented adult polyps of soft coral Sinularia notanda. We generated 3251 high-quality ESTs with an average length of 580 bp and the EST cluster and assembly analyses produced 2796 unigenes, including 2487 singletons and 309 contigs. Of the known genes, 55 genes were sel ected to be involved in polyp fragment adhesion and settlement based on Gene Ontology (GO) classification. Notably, two EST clones were identified to show homology with galaxin gene which was demonstrated as coral specific calcifying protein of organic matrix. These EST sequences can provide utility as molecular markers in molecular and genetic studies of S. notanda and other soft coral. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Characterization of in planta-induced rust genes isolated from a haustorium-specific cDNA library.

    PubMed

    Hahn, M; Mendgen, K

    1997-05-01

    Rust fungi are plant parasites that depend on living host tissue for growth. For invasion of leaves, dikaryotic urediospores differentiate germ tubes and infection structures that penetrate through stomata. Biotrophic growth occurs by intercellular mycelia that form haustoria within host cells. A cDNA library was constructed from haustoria isolated from broad bean leaves infected by Uromyces fabae. Differential screening revealed that a high proportion (19%) of the haustorial cDNAs are specifically expressed in planta but are not expressed, or are much weaker, in germlings or infection structures produced in vitro. A total of 31 different in planta-induced genes (PIGs) were identified. Some of the PIGs are highly expressed in haustoria. The PIGs are single or low copy number genes in the rust genome. A variety of developmentally regulated expression patterns of PIG mRNAs were observed. Sequence analysis of PIG cDNAs revealed similarities to genes encoding proteins involved in amino acid transport, thiamine biosynthesis, short-chain dehydrogenases, metallothioneins, cytochrome P-450 monooxygenases, and peptidyl-prolyl isomerases.

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

    PubMed

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

    2013-12-01

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

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

    PubMed

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

    2015-10-19

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

  1. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

    PubMed Central

    Wellenreuther, Ruth; Schupp, Ingo; Poustka, Annemarie; Wiemann, Stefan

    2004-01-01

    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. PMID:15198809

  2. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones.

    PubMed

    Wellenreuther, Ruth; Schupp, Ingo; Poustka, Annemarie; Wiemann, Stefan

    2004-06-15

    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. 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. 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.

  3. TcTASV: A Novel Protein Family in Trypanosoma cruzi Identified from a Subtractive Trypomastigote cDNA Library

    PubMed Central

    Agüero, Fernán; Bernabó, Guillermo; Sánchez, Daniel O.; Tekiel, Valeria

    2010-01-01

    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 (lineage

  4. Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library.

    PubMed

    Naimuddin, Mohammed; Kitamura, Koichirou; Kinoshita, Yasunori; Honda-Takahashi, Yoko; Murakami, Marina; Ito, Masato; Yamamoto, Kenji; Hanada, Kazunori; Husimi, Yuzuru; Nishigaki, Koichi

    2007-01-01

    A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating 'SF-link'. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC(50) of around 30 nM, a K(d) of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed.

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

    PubMed

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

    2009-01-01

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

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

    PubMed

    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.

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

    PubMed

    He, Quanze; Duan, Zhigui; Yu, Ying; Liu, Zhen; Liu, Zhonghua; Liang, Songping

    2013-01-01

    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.

  8. The Venom Gland Transcriptome of Latrodectus tredecimguttatus Revealed by Deep Sequencing and cDNA Library Analysis

    PubMed Central

    He, Quanze; Duan, Zhigui; Yu, Ying; Liu, Zhen; Liu, Zhonghua; Liang, Songping

    2013-01-01

    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. PMID:24312294

  9. Construction of Trypanosoma brucei Illumina RNA-Seq libraries enriched for transcript ends.

    PubMed

    Kolev, Nikolay G; Ullu, Elisabetta; Tschudi, Christian

    2015-01-01

    High-throughput RNA sequencing (RNA-Seq) has quickly occupied center stage in the repertoire of available tools for transcriptomics. Among many advantages, the single-nucleotide resolution of this powerful approach allows mapping on a genome-wide scale of splice junctions and polyadenylation sites, and thus, the precise definition of mature transcript boundaries. This greatly facilitated the transcriptome annotation of the human pathogen Trypanosoma brucei, a protozoan organism in which all mRNA molecules are matured by spliced leader (SL) trans-splicing from longer polycistronic precursors. The protocols described here for the generation of three types of libraries for Illumina RNA-Seq, 5'-SL enriched, 5'-triphosphate-end enriched, and 3'-poly(A) enriched, enabled the discovery of an unprecedented heterogeneity of pre-mRNA-processing sites, a large number of novel coding and noncoding transcripts from previously unannotated genes, and quantify the cellular abundance of RNA molecules. The method for producing 5'-triphosphate-end-enriched libraries was instrumental for obtaining evidence that transcription initiation by RNA polymerase II in trypanosomes is bidirectional and biosynthesis of mRNA precursors is primed not only at the beginning of unidirectional gene clusters, but also at specific internal sites.

  10. Sequencing and comparative genomic analysis of 1227 Felis catus cDNA sequences enriched for developmental, clinical and nutritional phenotypes

    PubMed Central

    2012-01-01

    Background The feline genome is valuable to the veterinary and model organism genomics communities because the cat is an obligate carnivore and a model for endangered felids. The initial public release of the Felis catus genome assembly provided a framework for investigating the genomic basis of feline biology. However, the entire set of protein coding genes has not been elucidated. Results We identified and characterized 1227 protein coding feline sequences, of which 913 map to public sequences and 314 are novel. These sequences have been deposited into NCBI's genbank database and complement public genomic resources by providing additional protein coding sequences that fill in some of the gaps in the feline genome assembly. Through functional and comparative genomic analyses, we gained an understanding of the role of these sequences in feline development, nutrition and health. Specifically, we identified 104 orthologs of human genes associated with Mendelian disorders. We detected negative selection within sequences with gene ontology annotations associated with intracellular trafficking, cytoskeleton and muscle functions. We detected relatively less negative selection on protein sequences encoding extracellular networks, apoptotic pathways and mitochondrial gene ontology annotations. Additionally, we characterized feline cDNA sequences that have mouse orthologs associated with clinical, nutritional and developmental phenotypes. Together, this analysis provides an overview of the value of our cDNA sequences and enhances our understanding of how the feline genome is similar to, and different from other mammalian genomes. Conclusions The cDNA sequences reported here expand existing feline genomic resources by providing high-quality sequences annotated with comparative genomic information providing functional, clinical, nutritional and orthologous gene information. PMID:22257742

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

    PubMed

    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.

  12. RecA-assisted rapid enrichment of specific clones from model DNA libraries.

    PubMed

    Teintze, M; Arzimanoglou, I I; Lovelace, C I; Xu, Z J; Rigas, B

    1995-06-26

    An approach to library screening is being developed, in which the desired clone is "fished" out of a mixture of all the recombinants in a library with a RecA-coated probe. In the current embodiment of this method, we used as a probe the (+) strand of an M13 phage containing a fragment of the human albumin gene and a (dA)49 stretch. We screened a library of two plasmids, one containing the same albumin fragment as the probe, and one heterologous to the probe in 50-100 fold molar excess. The plasmids were linearized. Probe and library were reacted in the presence of RecA, the mixture was loaded onto an oligo(dT) column, which retained the probe-target complex by base-pairing to the dAs of the probe, the uncaptured plasmids were washed, and the probe-target complex was released from the column, religated and propagated into E. coli. Recovery of the homologous target was 15-28%, and enrichment for the homologous plasmid was 200 to 400-fold. This approach may provide a general method for expedited DNA library screening.

  13. Analysis of clones from a human cartilage cDNA library provides insight into chondrocyte gene expression and identifies novel candidate genes for the osteochondrodysplasias.

    PubMed

    Krakow, Deborah; Sebald, Eiman T; Pogue, Robert; Rimoin, Lauren P; King, Lily; Cohn, Daniel H

    2003-05-01

    To begin to define the gene expression pattern in fetal cartilage and to identify uncharacterized candidate genes for the osteochondrodysplasias, we analyzed clones from a fetal cartilage cDNA library. Sequence analysis of 420 cDNA clones identified 210 clones derived from established genes but, for many of them, expression in cartilage had not been previously reported. Among the established genes were 14 genes known to produce skeletal abnormalities in either humans or mice when mutated. Thirty-two uncharacterized genes and their respective chromosomal positions were also identified. To further understand the expression profile of these genes in fetal cartilage, we constructed a cDNA microarray utilizing the clones. The microarray was used to determine which genes had higher expression in cartilage as compared with dedifferentiated, cultured chondrocytes. Many of the established genes, as well as five of the uncharacterized genes, had increased expression in cartilage, suggesting an important role for these genes in the differentiated state of chondrocytes. These data provide new candidate genes for the osteochondrodysplasias and demonstrate the usefulness of cartilage cDNA microarrays in expanding our understanding of the complexity of fetal cartilage gene expression.

  14. Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens.

    PubMed

    Machuka, J; Bashiardes, S; Ruben, E; Spooner, K; Cuming, A; Knight, C; Cove, D

    1999-04-01

    Partial cDNA sequencing was used to obtain 169 expressed sequence tags (ESTs) in the moss, Physcomitrella patens. The source of ESTs was a random cDNA library constructed from 7 day-old protonemata following treatment with 10(-4) M abscisic acid (ABA). Analysis of the ESTs identified 69% with homology to known sequences, 61% of which had significant homology to sequences of plant origin. More importantly, at least 11 ESTs had significant similarities to genes which are implicated in plant stress-responses, including responses which may involve ABA. These included a cDNA associated with desiccation tolerance, two heat shock protein genes, one cold acclimation protein cDNA and five others that may be involved in either oxidative or chemical stress or both, i.e., Zn/Cu-superoxide dismutase, NADPH protochlorophyllide oxidoreductase (PorB), selenium binding protein, glutathione peroxidase and glutathione S transferase. Analysis of codon usage between P. patens and seed plants indicated that although mosses and higher plants are to a large extent similar, minor variations also exists that may represent the distinctiveness of each group.

  15. Enrichment of chemical libraries docked to protein conformational ensembles and application to aldehyde dehydrogenase 2.

    PubMed

    Wang, Bo; Buchman, Cameron D; Li, Liwei; Hurley, Thomas D; Meroueh, Samy O

    2014-07-28

    Molecular recognition is a complex process that involves a large ensemble of structures of the receptor and ligand. Yet, most structure-based virtual screening is carried out on a single structure typically from X-ray crystallography. Explicit-solvent molecular dynamics (MD) simulations offer an opportunity to sample multiple conformational states of a protein. Here we evaluate our recently developed scoring method SVMSP in its ability to enrich chemical libraries docked to MD structures of seven proteins from the Directory of Useful Decoys (DUD). SVMSP is a target-specific rescoring method that combines machine learning with statistical potentials. We find that enrichment power as measured by the area under the ROC curve (ROC-AUC) is not affected by increasing the number of MD structures. Among individual MD snapshots, many exhibited enrichment that was significantly better than the crystal structure, but no correlation between enrichment and structural deviation from crystal structure was found. We followed an innovative approach by training SVMSP scoring models using MD structures (SVMSPMD). The resulting models were applied to two difficult cases (p38 and CDK2) for which enrichment was not better than random. We found remarkable increase in enrichment power, particularly for p38, where the ROC-AUC increased by 0.30 to 0.85. Finally, we explored approaches for a priori identification of MD snapshots with high enrichment power from an MD simulation in the absence of active compounds. We found that the use of randomly selected compounds docked to the target of interest using SVMSP led to notable enrichment for EGFR and Src MD snapshots. SVMSP rescoring of protein-compound MD structures was applied for the search of small-molecule inhibitors of the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2). Rank-ordering of a commercial library of 50 000 compounds docked to MD structures of ALDH2 led to five small-molecule inhibitors. Four compounds had IC50s below 5

  16. Enrichment of Chemical Libraries Docked to Protein Conformational Ensembles and Application to Aldehyde Dehydrogenase 2

    PubMed Central

    2015-01-01

    Molecular recognition is a complex process that involves a large ensemble of structures of the receptor and ligand. Yet, most structure-based virtual screening is carried out on a single structure typically from X-ray crystallography. Explicit-solvent molecular dynamics (MD) simulations offer an opportunity to sample multiple conformational states of a protein. Here we evaluate our recently developed scoring method SVMSP in its ability to enrich chemical libraries docked to MD structures of seven proteins from the Directory of Useful Decoys (DUD). SVMSP is a target-specific rescoring method that combines machine learning with statistical potentials. We find that enrichment power as measured by the area under the ROC curve (ROC-AUC) is not affected by increasing the number of MD structures. Among individual MD snapshots, many exhibited enrichment that was significantly better than the crystal structure, but no correlation between enrichment and structural deviation from crystal structure was found. We followed an innovative approach by training SVMSP scoring models using MD structures (SVMSPMD). The resulting models were applied to two difficult cases (p38 and CDK2) for which enrichment was not better than random. We found remarkable increase in enrichment power, particularly for p38, where the ROC-AUC increased by 0.30 to 0.85. Finally, we explored approaches for a priori identification of MD snapshots with high enrichment power from an MD simulation in the absence of active compounds. We found that the use of randomly selected compounds docked to the target of interest using SVMSP led to notable enrichment for EGFR and Src MD snapshots. SVMSP rescoring of protein–compound MD structures was applied for the search of small-molecule inhibitors of the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2). Rank-ordering of a commercial library of 50 000 compounds docked to MD structures of ALDH2 led to five small-molecule inhibitors. Four compounds had IC50s below

  17. An ordered Arabidopsis thaliana mitochondrial cDNA library on high-density filters allows rapid systematic analysis of plant gene expression: a pilot study.

    PubMed

    Giegé, P; Konthur, Z; Walter, G; Brennicke, A

    1998-09-01

    The availability of the complete sequence of a genome allows a systematic analysis of its expression. Gene-specific variations of transcription levels and phenomena such as transcript processing and RNA editing require large numbers of clones to be examined. For the completely sequenced mitochondrial genome of Arabidopsis thaliana we adapted robot technology to identify and characterize expressed genes. A cDNA library of about 50,000 clones was constructed, robot-ordered into 384-well microtitre plates and spotted onto high-density filter membranes. These filters permit the isolation of large numbers of specific cDNA clones in a single hybridization step. The cox1, cox2 and cox3 genes were used to evaluate the feasibility and efficiency of this approach. A cluster of RNA editing sites observed outside the cox3 coding region identifies a novel reading frame orf95 in higher plants with significant similarity to a subunit of respiratory chain complex II.

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

    PubMed

    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.

  19. Constructing gene-enriched plant genomic libraries using methylation filtration technology.

    PubMed

    Rabinowicz, Pablo D

    2003-01-01

    Full genome sequencing in higher plants is a very difficult task, because their genomes are often very large and repetitive. For this reason, gene targeted partial genomic sequencing becomes a realistic option. The method reported here is a simple approach to generate gene-enriched plant genomic libraries called methylation filtration. This technique takes advantage of the fact that repetitive DNA is heavily methylated and genes are hypomethylated. Then, by simply using an Escherichia coli host strain harboring a wild-type modified cytosine restriction (McrBC) system, which cuts DNA containing methylcytosine, repetitive DNA is eliminated from these genomic libraries, while low copy DNA (i.e., genes) is recovered. To prevent cloning significant proportions of organelle DNA, a crude nuclear preparation must be performed prior to purifying genomic DNA. Adaptor-mediated cloning and DNA size fractionation are necessary for optimal results.

  20. Sequence evaluation of four pooled-tissue normalized bovine cDNA libraries and construction of a gene index for cattle.

    PubMed

    Smith, T P; Grosse, W M; Freking, B A; Roberts, A J; Stone, R T; Casas, E; Wray, J E; White, J; Cho, J; Fahrenkrug, S C; Bennett, G L; Heaton, M P; Laegreid, W W; Rohrer, G A; Chitko-McKown, C G; Pertea, G; Holt, I; Karamycheva, S; Liang, F; Quackenbush, J; Keele, J W

    2001-04-01

    An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained. Target tissues included those with highest likelihood to have impact on production parameters of animal health, growth, reproductive efficiency, and carcass merit. Success of normalization and inter- and intralibrary redundancy were assessed by collecting 6000-23,000 sequences from each of the libraries (68,520 total sequences deposited in GenBank). Sequence comparison and assembly of these sequences was performed in combination with 56,500 other bovine EST sequences present in the GenBank dbEST database to construct a cattle Gene Index (available from The Institute for Genomic Research at http://www.tigr.org/tdb/tgi.shtml). The 124,381 bovine ESTs present in GenBank at the time of the analysis form 16,740 assemblies that are listed and annotated on the Web site. Analysis of individual library sequence data indicates that the pooled-tissue approach was highly effective in preparing libraries for efficient deep sequencing.

  1. A modified enrichment method to construct microsatellite library from plateau pika genome (Ochotona curzoniae).

    PubMed

    Geng, Jianing; Li, Kexin; Zhang, Yanming; Hu, Songnian

    2010-03-01

    A microsatellite-enriched library of plateau pika (Ochotona curzoniae) was constructed according to the strong affinity between biotin and streptavidin. Firstly, genomic DNA was fragmented by ultrasonication, which is a major improvement over traditional methods. Linker-ligated DNA fragments were hybridized with biotinylated microsatellite probes, and then were subjected to streptavidin-coated magnetic beads. PCR amplification was performed to obtain double-stranded DNA fragments containing microsatellites. Ligation and transformation were carried out by using the pGEM-T Vector System I and Escherichia coli DH10B competent cells. Sequencing results showed that 80.2% of clones contained microsatellite repeat motif. Several modifications make this protocol time-efficient and technically easier than the traditional ones; particularly, composition and relative abundance of microsatellite repeats in plateau pika genome were truly represented through the optimized PCR conditions. This method has also been successfully applied to construct microsatellite-enriched genomic libraries of Chinese hamster (Cricetulus griseus) and small abalone [Haliotis diversicolor (Reeve)] with high rates of positive clones, demonstrating its feasibility and stability. 2010 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

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

    SciTech Connect

    Cool, D.E.; Tonks, N.K.; Charbonneau, H.; Walsh, K.A.; Fischer, E.H.; Krebs, E.G. )

    1989-07-01

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

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

    PubMed Central

    Cool, D E; Tonks, N K; Charbonneau, H; Walsh, K A; Fischer, E H; Krebs, E G

    1989-01-01

    A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). One positive clone was isolated and the nucleotide sequence was determined. It contained 1305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3' untranslated end, although a poly(A)+ tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues (Mr, 48,400). This was supported by the synthesis of a Mr 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low Mr PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes. Images PMID:2546150

  4. Molecular Cloning of Rat and Porcine Retina-derived POU Domain Factor 1 (POU6F2) from a Pituitary cDNA Library

    PubMed Central

    YOSHIDA, Saishu; UEHARU, Hiroki; HIGUCHI, Masashi; HORIGUCHI, Kotaro; NISHIMURA, Naoto; SHIBUYA, Shiori; MITSUISHI, Hideo; KATO, Takako; KATO, Yukio

    2014-01-01

    Homeobox transcription factors are known to play crucial roles in the anterior lobe of the pituitary gland. During molecular cloning with the Yeast One-Hybrid System using a 5’-upstream region of the porcine Fshβ as a bait sequence, we have cloned a cDNA encoding a partial sequence of the retina-derived POU domain factor 1 (RPF1) from the porcine pituitary cDNA library and confirmed its specific binding to the bait sequence. In situ hybridization was performed to examine localization of Rpf1 and showed that this gene is expressed in the stem/progenitor cells of the rat pituitary primordium as well as the diencephalon and retina. In addition, real-time PCR demonstrated that Rpf1 transcripts are abundant in early embryonic periods but that this is followed by a decrease during pituitary development, indicating that this factor plays a role in differentiating cells of the pituitary. The transcriptional activity of RPF1 for genes of Prop1, Prrx1 and Prrx2, which were characterized as genes participating in the pituitary stem/progenitor cells by our group, was then examined with full-length cDNA obtained from the rat pituitary. RPF1 showed regulatory activity for Prop1 and Prrx2, but not for Prrx1. These results indicate the involvement of this retina-derived factor in pituitary development. PMID:24804940

  5. Cloning of a protein binding to the most proximal Pit-1 binding element of prolactin gene from human pituitary cDNA library.

    PubMed

    Fumoto, Mariko; Okimura, Yasuhiko; Sakagami, Yoshio; Iguchi, Genzo; Kishimoto, Masahiko; Takahashi, Yutaka; Kaji, Hidesuke; Chihara, Kazuo

    2003-09-30

    A human pituitary cDNA library was screened using a yeast one-hybrid system to find a factor binding Pit-1 binding elements in the PRL gene other than Pit-1. Beside colonies containing Pit-1 or Oct-1 cDNA, three colonies contained mPOU cDNA, a member of the POU protein family. Immunohistochemical analysis showed mPOU-like immunoreactivity was present in human PRL-producing pituitary tumors but not in non-functioning pituitary tumors. Mobility shift analysis revealed that mPOU bound to Pit-1 binding elements of the PRL gene, 1P and 3P. mPOU activated the expression of 0.6 k PRL and 7x1P reporter genes in the presence of Pit-1 and cAMP, although it did not enhance Pit-1-induced expression of 7x3P reporter gene. These findings suggest that mPOU is involved in the activation of the PRL gene by cAMP through 1P in the presence of Pit-1.

  6. Preparation of a high-quality cDNA library from a single-cell quantity of mRNA using chum-RNA.

    PubMed

    Nojima, Hiroshi; Tougan, Takahiro

    2011-01-01

    Unlike exponential amplification using polymerase chain reaction (PCR), linear RNA amplification using T7 RNA polymerase is advantageous for genome-wide analysis of gene expression and for cDNA library preparation from single-cell quantities of RNA. However, the use of RNA polymerase requires a large amount of RNA, as the optimum concentration of the substrate (mRNA), or the Michaelis constant (K(m)), is one millionfold higher than the single-cell amount of mRNA. To circumvent this K(m) problem, we designed a small mRNA-like dummy molecule, termed chum-RNA, which can be easily removed after the completion of the reaction. Chum-RNA allowed the preparation of a high-quality cDNA library from single-cell quantities of RNA after four rounds of T7-based linear amplification, without using PCR amplification. The use of chum-RNA may also facilitate quantitative reverse-transcription (qRT)-PCR from small quantities of substrate.

  7. Analysis of dormant bud (Banjhi) specific transcriptome of tea (Camellia sinensis (L.) O. Kuntze) from cDNA library revealed dormancy-related genes.

    PubMed

    Thirugnanasambantham, Krishnaraj; Prabu, Gajjeraman; Palanisamy, Senthilkumar; Chandrabose, Suresh Ramraj Subhas; Mandal, Abul Kalam Azad

    2013-02-01

    Bud dormancy is of ecological and economical interest due to its impact on tea (Camellia sinensis (L.) O. Kuntze) plant growth and yield. Growth regulation associated with dormancy is an essential element in plant's life cycle that leads to changes in expression of large number of genes. In order to identify and provide a picture of the transcriptome profile, cDNA library was constructed from dormant bud (banjhi) of tea. Sequence and gene ontology analysis of 3,500 clones, in many cases, enabled their functional categorization concerning the bud growth. Based on the cDNA library data, the putative role of identified genes from tea is discussed in relation to growth and dormancy, which includes morphogenesis, cellular differentiation, tropism, cell cycle, signaling, and various metabolic pathways. There was a higher representation of unknown processes such as unknown molecular functions (65.80 %), unknown biological processes (62.46 %), and unknown cellular components (67.42 %). However, these unknown transcripts represented a novel component of transcripts in tea plant bud growth and/or dormancy development. The identified transcripts and expressed sequence tags provides a valuable public resource and preliminary insights into the molecular mechanisms of bud dormancy regulation. Further, the findings will be the target of future expression experiments, particularly for further identification of dormancy-related genes in this species.

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

    PubMed Central

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

    2010-01-01

    The micropylar endosperm is a major regulator of seed germination in endospermic species, to which the close Brassicaceae relatives Arabidopsis thaliana and Lepidium sativum (cress) belong. Cress seeds are about 20 times larger than the seeds of Arabidopsis. This advantage was used to construct a tissue-specific subtractive cDNA library of transcripts that are up-regulated late in the germination process specifically in the micropylar endosperm of cress seeds. The library showed that a number of transcripts known to be up-regulated late during germination are up-regulated in the micropylar endosperm cap. Detailed germination kinetics of SALK lines carrying insertions in genes present in our library showed that the identified transcripts do indeed play roles during germination. Three peroxidases were present in the library. These peroxidases were identified as orthologues of Arabidopsis AtAPX01, AtPrx16, and AtPrxIIE. The corresponding SALK lines displayed significant germination phenotypes. Their transcripts were quantified in specific cress seed tissues during germination in the presence and absence of ABA and they were found to be regulated in a tissue-specific manner. Peroxidase activity, and particularly its regulation by ABA, also differed between radicles and micropylar endosperm caps. Possible implications of this tissue-specificity are discussed. PMID:19884228

  9. Expressed sequence tags from normalized cDNA libraries prepared from gill and hypodermal tissues of the blue crab, Callinectes sapidus.

    PubMed

    Coblentz, Francie E; Towle, David W; Shafer, Thomas H

    2006-06-01

    Expressed sequence tags (ESTs) were produced from two normalized cDNA libraries from the blue crab, Callinectes sapidus. The gill library represented pooled RNA from respiratory and transporting gills after acclimation to either high or low salinity. The hypodermis library was from arthrodial and dorsal tissue from both pre- and post-molt crabs. Random clones were single-pass sequenced from the 5'-ends, resulting in 11,761 high quality ESTs averaging 652 bases. All the ESTs were assembled using Paracel Transcript Assembler software, producing 2176 potential transcripts-883 contigs and 1293 singlets. Of these, 1235 (56.7%) were sequenced only from the gill library, while 578 (26.6%) were exclusively hypodermal. There were 363 contigs containing ESTs from both tissues (16.7% of the putative transcripts). All contigs and singlets were compared to the public protein database using BLASTx, and descriptions of the three most similar proteins for each were recorded. Additional annotations included an Interpro analysis of protein domains and a listing of Gene Ontology (GO) categories inferred from similar proteins in GO-annotated databases. All sequences are available on a web page (http://firedev.bear.uncw.edu:8080/shaferlab/). The annotations can be searched, and BLAST alignment of user-inputted sequences against the putative transcripts is possible. In addition, the ESTs have been submitted to GenBank.

  10. 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

    NASA Astrophysics Data System (ADS)

    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.

  11. A subtracted cDNA library identifies genes up-regulated during PHOT1-mediated early step of de-etiolation in tomato (Solanum lycopersicum L.).

    PubMed

    Hloušková, Petra; Bergougnoux, Véronique

    2016-04-18

    De-etiolation is the switch from skoto- to photomorphogenesis, enabling the heterotrophic etiolated seedling to develop into an autotrophic plant. Upon exposure to blue light (BL), reduction of hypocotyl growth rate occurs in two phases: a rapid inhibition mediated by phototropin 1 (PHOT1) within the first 30-40 min of illumination, followed by the cryptochrome 1 (CRY1)-controlled establishment of the steady-state growth rate. Although some information is available for CRY1-mediated de-etiolation, less attention has been given to the PHOT1 phase of de-etiolation. We generated a subtracted cDNA library using the suppression subtractive hybridization method to investigate the molecular mechanisms of BL-induced de-etiolation in tomato (Solanum lycopersicum L.), an economically important crop. We focused our interest on the first 30 min following the exposure to BL when PHOT1 is required to induce the process. Our library generated 152 expressed sequence tags that were found to be rapidly accumulated upon exposure to BL and consequently potentially regulated by PHOT1. Annotation revealed that biological functions such as modification of chromatin structure, cell wall modification, and transcription/translation comprise an important part of events contributing to the establishment of photomorphogenesis in young tomato seedlings. Our conclusions based on bioinformatics data were supported by qRT-PCR analyses the specific investigation of V-H(+)-ATPase during de-etiolation in tomato. Our study provides the first report dealing with understanding the PHOT1-mediated phase of de-etiolation. Using subtractive cDNA library, we were able to identify important regulatory mechanisms. The profound induction of transcription/translation, as well as modification of chromatin structure, is relevant in regard to the fact that the entry into photomorphogenesis is based on a deep reprograming of the cell. Also, we postulated that BL restrains the cell expansion by the rapid modification

  12. Construction and Analysis of cDNA Libraries from the Antennae of Batocera horsfieldi and Expression Pattern of Putative Odorant Binding Proteins

    PubMed Central

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

    2014-01-01

    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 real-time 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 Minus- C 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. PMID:25373204

  13. 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.

    PubMed

    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

  14. Halogen-Enriched Fragment Libraries as Leads for Drug Rescue of Mutant p53

    PubMed Central

    2012-01-01

    The destabilizing p53 cancer mutation Y220C creates a druggable surface crevice. We developed a strategy exploiting halogen bonding for lead discovery to stabilize the mutant with small molecules. We designed halogen-enriched fragment libraries (HEFLibs) as starting points to complement classical approaches. From screening of HEFLibs and subsequent structure-guided design, we developed substituted 2-(aminomethyl)-4-ethynyl-6-iodophenols as p53-Y220C stabilizers. Crystal structures of their complexes highlight two key features: (i) a central scaffold with a robust binding mode anchored by halogen bonding of an iodine with a main-chain carbonyl and (ii) an acetylene linker, enabling the targeting of an additional subsite in the crevice. The best binders showed induction of apoptosis in a human cancer cell line with homozygous Y220C mutation. Our structural and biophysical data suggest a more widespread applicability of HEFLibs in drug discovery. PMID:22439615

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

    PubMed

    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

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

    PubMed Central

    Loit, Evelin; Melnyk, Charles W; MacFarlane, Amanda J; Scott, Fraser W; Altosaar, Illimar

    2009-01-01

    Background Exposure to dietary wheat proteins in genetically susceptible individuals has been associated with increased risk for the development of Type 1 diabetes (T1D). Recently, a wheat protein encoded by cDNA WP5212 has been shown to be antigenic in mice, rats and humans with autoimmune T1D. To investigate the genomic origin of the identified wheat protein cDNA, a hexaploid wheat genomic library from Glenlea cultivar was screened. Results Three unique wheat globulin genes, Glo-3A, Glo3-B and Glo-3C, were identified. We describe the genomic structure of these genes and their expression pattern in wheat seeds. The Glo-3A gene shared 99% identity with the cDNA of WP5212 at the nucleotide and deduced amino acid level, indicating that we have identified the gene(s) encoding wheat protein WP5212. Southern analysis revealed the presence of multiple copies of Glo-3-like sequences in all wheat samples, including hexaploid, tetraploid and diploid species wheat seed. Aleurone and embryo tissue specificity of WP5212 gene expression, suggested by promoter region analysis, which demonstrated an absence of endosperm specific cis elements, was confirmed by immunofluorescence microscopy using anti-WP5212 antibodies. Conclusion Taken together, the results indicate that a diverse group of globulins exists in wheat, some of which could be associated with the pathogenesis of T1D in some susceptible individuals. These data expand our knowledge of specific wheat globulins and will enable further elucidation of their role in wheat biology and human health. PMID:19615078

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

    PubMed Central

    Seong, Eun Soo; Yoo, Ji Hye; Choi, Jae Hoo; Kim, Chang Heum; Jeon, Mi Ran; Kang, Byeong Ju; Lee, Jae Geun; Choi, Seon Kang; Ghimire, Bimal Kumar; Yu, Chang Yeon

    2015-01-01

    Perilla frutescens is valuable as a medicinal plant as well as a natural medicine and functional food. However, comparative genomics analyses of P. frutescens are limited due to a lack of gene annotations and characterization. A full-length cDNA library from P. frutescens leaves was constructed to identify functional gene clusters and probable EST-SSR markers via analysis of 1,056 expressed sequence tags. Unigene assembly was performed using basic local alignment search tool (BLAST) homology searches and annotated Gene Ontology (GO). A total of 18 simple sequence repeats (SSRs) were designed as primer pairs. This study is the first to report comparative genomics and EST-SSR markers from P. frutescens will help gene discovery and provide an important source for functional genomics and molecular genetic research in this interesting medicinal plant. PMID:26664999

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Bacchetti De Gregoris, Tristano; Borra, Marco; Biffali, Elio; Bekel, Thomas; Burgess, J Grant; Kirby, Richard R; Clare, Anthony S

    2009-01-01

    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. PMID:19552808

  20. A Functional Yeast Survival Screen of Tumor-Derived cDNA Libraries Designed to Identify Anti-Apoptotic Mammalian Oncogenes

    PubMed Central

    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. PMID:23717670

  1. Halogen-enriched fragment libraries as chemical probes for harnessing halogen bonding in fragment-based lead discovery.

    PubMed

    Zimmermann, Markus O; Lange, Andreas; Wilcken, Rainer; Cieslik, Markus B; Exner, Thomas E; Joerger, Andreas C; Koch, Pierre; Boeckler, Frank M

    2014-04-01

    Halogen bonding has recently experienced a renaissance, gaining increased recognition as a useful molecular interaction in the life sciences. Halogen bonds are favorable, fairly directional interactions between an electropositive region on the halogen (the σ-hole) and a number of different nucleophilic interaction partners. Some aspects of halogen bonding are not yet understood well enough to take full advantage of its potential in drug discovery. We describe and present the concept of halogen-enriched fragment libraries. These libraries consist of unique chemical probes, facilitating the identification of favorable halogen bonds by sharing the advantages of classical fragment-based screening. Besides providing insights into the nature and applicability of halogen bonding, halogen-enriched fragment libraries provide smart starting points for hit-to-lead evolution.

  2. X chromosome cDNA microarray screening identifies a functional PLP2 promoter polymorphism enriched in patients with X-linked mental retardation

    PubMed Central

    Zhang, Lilei; Jie, Chunfa; Obie, Cassandra; Abidi, Fatima; Schwartz, Charles E.; Stevenson, Roger E.; Valle, David; Wang, Tao

    2007-01-01

    X-linked Mental Retardation (XLMR) occurs in 1 in 600 males and is highly genetically heterogeneous. We used a novel human X chromosome cDNA microarray (XCA) to survey the expression profile of X-linked genes in lymphoblasts of XLMR males. Genes with altered expression verified by Northern blot and/or quantitative PCR were considered candidates. To validate this approach, we documented the expected changes of expression in samples from a patient with a known X chromosome microdeletion and from patients with multiple copies of the X chromosome. We used our XCA to survey lymphoblast RNA samples from 43 unrelated XLMR males and found 15 genes with significant (≥1.5-fold) reduction in expression in at least one proband. Of these, subsequent analysis confirmed altered expression in 12. We followed up one, PLP2, at Xp11.23, which exhibits approximately fourfold decreased expression in two patients. Sequencing analysis in both patients revealed a promoter variant, −113C>A, that alters the core-binding site of the transcription factor ELK1. We showed that PLP2-(−113C>A) is sufficient to cause reduced expression using a luciferase reporter system and is enriched in a cohort of males with probable XLMR (14 of 239, 5.85%) as compared to normal males (9 of 577, 1.56%) (χ2 = 11.07, P < 0.001). PLP2 is expressed abundantly in the pyramidal cells of hippocampus and granular cells of the cerebellum in the brain. We conclude that our XCA screening is an efficient strategy to identify genes that show significant changes in transcript abundance as candidate genes for XLMR. PMID:17416750

  3. Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

    PubMed Central

    Carpenter, Meredith L.; Buenrostro, Jason D.; Valdiosera, Cristina; Schroeder, Hannes; Allentoft, Morten E.; Sikora, Martin; Rasmussen, Morten; Gravel, Simon; Guillén, Sonia; Nekhrizov, Georgi; Leshtakov, Krasimir; Dimitrova, Diana; Theodossiev, Nikola; Pettener, Davide; Luiselli, Donata; Sandoval, Karla; Moreno-Estrada, Andrés; Li, Yingrui; Wang, Jun; Gilbert, M. Thomas P.; Willerslev, Eske; Greenleaf, William J.; Bustamante, Carlos D.

    2013-01-01

    Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples. PMID:24568772

  4. Pulling out the 1%: whole-genome capture for the targeted enrichment of ancient DNA sequencing libraries.

    PubMed

    Carpenter, Meredith L; Buenrostro, Jason D; Valdiosera, Cristina; Schroeder, Hannes; Allentoft, Morten E; Sikora, Martin; Rasmussen, Morten; Gravel, Simon; Guillén, Sonia; Nekhrizov, Georgi; Leshtakov, Krasimir; Dimitrova, Diana; Theodossiev, Nikola; Pettener, Davide; Luiselli, Donata; Sandoval, Karla; Moreno-Estrada, Andrés; Li, Yingrui; Wang, Jun; Gilbert, M Thomas P; Willerslev, Eske; Greenleaf, William J; Bustamante, Carlos D

    2013-11-07

    Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062-147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217-73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next-Generation Sequencing.

    PubMed

    Marosy, Beth A; Craig, Brian D; Hetrick, Kurt N; Witmer, P Dane; Ling, Hua; Griffith, Sean M; Myers, Benjamin; Ostrander, Elaine A; Stanford, Janet L; Brody, Lawrence C; Doheny, Kimberly F

    2017-01-11

    This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  7. Designing Focused Chemical Libraries Enriched in Protein-Protein Interaction Inhibitors using Machine-Learning Methods

    PubMed Central

    Reynès, Christelle; Host, Hélène; Camproux, Anne-Claude; Laconde, Guillaume; Leroux, Florence; Mazars, Anne; Deprez, Benoit; Fahraeus, Robin; Villoutreix, Bruno O.; Sperandio, Olivier

    2010-01-01

    Protein-protein interactions (PPIs) may represent one of the next major classes of therapeutic targets. So far, only a minute fraction of the estimated 650,000 PPIs that comprise the human interactome are known with a tiny number of complexes being drugged. Such intricate biological systems cannot be cost-efficiently tackled using conventional high-throughput screening methods. Rather, time has come for designing new strategies that will maximize the chance for hit identification through a rationalization of the PPI inhibitor chemical space and the design of PPI-focused compound libraries (global or target-specific). Here, we train machine-learning-based models, mainly decision trees, using a dataset of known PPI inhibitors and of regular drugs in order to determine a global physico-chemical profile for putative PPI inhibitors. This statistical analysis unravels two important molecular descriptors for PPI inhibitors characterizing specific molecular shapes and the presence of a privileged number of aromatic bonds. The best model has been transposed into a computer program, PPI-HitProfiler, that can output from any drug-like compound collection a focused chemical library enriched in putative PPI inhibitors. Our PPI inhibitor profiler is challenged on the experimental screening results of 11 different PPIs among which the p53/MDM2 interaction screened within our own CDithem platform, that in addition to the validation of our concept led to the identification of 4 novel p53/MDM2 inhibitors. Collectively, our tool shows a robust behavior on the 11 experimental datasets by correctly profiling 70% of the experimentally identified hits while removing 52% of the inactive compounds from the initial compound collections. We strongly believe that this new tool can be used as a global PPI inhibitor profiler prior to screening assays to reduce the size of the compound collections to be experimentally screened while keeping most of the true PPI inhibitors. PPI-HitProfiler is

  8. Screening of a minimal enriched P450 BM3 mutant library for hydroxylation of cyclic and acyclic alkanes.

    PubMed

    Weber, Evelyne; Seifert, Alexander; Antonovici, Mihaela; Geinitz, Christopher; Pleiss, Jürgen; Urlacher, Vlada B

    2011-01-21

    A minimal enriched P450 BM3 library was screened for the ability to oxidize inert cyclic and acyclic alkanes. The F87A/A328V mutant was found to effectively hydroxylate cyclooctane, cyclodecane and cyclododecane. F87V/A328F with high activity towards cyclooctane hydroxylated acyclic n-octane to 2-(R)-octanol (46% ee) with high regioselectivity (92%).

  9. BiNChE: a web tool and library for chemical enrichment analysis based on the ChEBI ontology.

    PubMed

    Moreno, Pablo; Beisken, Stephan; Harsha, Bhavana; Muthukrishnan, Venkatesh; Tudose, Ilinca; Dekker, Adriano; Dornfeldt, Stefanie; Taruttis, Franziska; Grosse, Ivo; Hastings, Janna; Neumann, Steffen; Steinbeck, Christoph

    2015-02-21

    Ontology-based enrichment analysis aids in the interpretation and understanding of large-scale biological data. Ontologies are hierarchies of biologically relevant groupings. Using ontology annotations, which link ontology classes to biological entities, enrichment analysis methods assess whether there is a significant over or under representation of entities for ontology classes. While many tools exist that run enrichment analysis for protein sets annotated with the Gene Ontology, there are only a few that can be used for small molecules enrichment analysis. We describe BiNChE, an enrichment analysis tool for small molecules based on the ChEBI Ontology. BiNChE displays an interactive graph that can be exported as a high-resolution image or in network formats. The tool provides plain, weighted and fragment analysis based on either the ChEBI Role Ontology or the ChEBI Structural Ontology. BiNChE aids in the exploration of large sets of small molecules produced within Metabolomics or other Systems Biology research contexts. The open-source tool provides easy and highly interactive web access to enrichment analysis with the ChEBI ontology tool and is additionally available as a standalone library.

  10. DNA Sequence-Based “Bar Codes” for Tracking the Origins of Expressed Sequence Tags from a Maize cDNA Library Constructed Using Multiple mRNA Sources1

    PubMed Central

    Qiu, Fang; Guo, Ling; Wen, Tsui-Jung; Liu, Feng; Ashlock, Daniel A.; Schnable, Patrick S.

    2003-01-01

    To enhance gene discovery, expressed sequence tag (EST) projects often make use of cDNA libraries produced using diverse mixtures of mRNAs. As such, expression data are lost because the origins of the resulting ESTs cannot be determined. Alternatively, multiple libraries can be prepared, each from a more restricted source of mRNAs. Although this approach allows the origins of ESTs to be determined, it requires the production of multiple libraries. A hybrid approach is reported here. A cDNA library was prepared using 21 different pools of maize (Zea mays) mRNAs. DNA sequence “bar codes” were added during first-strand cDNA synthesis to uniquely identify the mRNA source pool from which individual cDNAs were derived. Using a decoding algorithm that included error correction, it was possible to identify the source mRNA pool of more than 97% of the ESTs. The frequency at which a bar code is represented in an EST contig should be proportional to the abundance of the corresponding mRNA in the source pool. Consistent with this, all ESTs derived from several genes (zein and adh1) that are known to be exclusively expressed in kernels or preferentially expressed under anaerobic conditions, respectively, were exclusively tagged with bar codes associated with mRNA pools prepared from kernel and anaerobically treated seedlings, respectively. Hence, by allowing for the retention of expression data, the bar coding of cDNA libraries can enhance the value of EST projects. PMID:14555776

  11. Xendorphin B1, a novel opioid-like peptide determined from a Xenopus laevis brain cDNA library, produces opioid antinociception after spinal administration in amphibians

    PubMed Central

    Stevens, Craig W.; Tóth, Géza; Borsodi, Anna; Benyhe, Sándor

    2007-01-01

    Prodynorphins (PDYNs) from the African clawed frog (Xenopus leavis), originally described as ‘proxendorphins’, are novel members of the family of opioid-like precursor polypeptides and were recently discovered based on polymerase chain reaction (PCR) isolates from a Xenopus brain cDNA library. This amphibian prodynorphin was found in two isoforms, XenPDYN-A and XenPDYN-B, consisting of 247 and 279 amino acids, respectively. Each prepropeptide contains five potential opioid-like peptides, collectively named xendorphins. One of these, xendorphin B1 (XenPDYN-B sequence 96–111: YGGFIRKPDKYKFLNA), is a hexadecapeptide that displaced [3H]naloxone and the radiolabelled kappa opioid, [3H]dynorphin A (1–17), with nanomolar affinity from rat brain membranes. Using the acetic acid pain test, the present study examined the antinociceptive effects of spinally administered xendorphin B1 in amphibians. Xendorphin B1 produced a long-lasting and dose-dependent antinociceptive effect in the Northern grass frog (Rana pipiens) with an ED50 value of 44.5 nmol/frog. The antinociceptive effects of xendorphin B1 were significantly blocked by pretreatment with the non-selective opioid antagonist, naltrexone. This is the first report of the in vivo characterization of a non-mammalian prodynorphin-derived peptide and suggests that xendorphin peptides may play a role in the modulation of noxious information in vertebrates. PMID:17292806

  12. Analysis of Rickettsia typhi-infected and uninfected cat flea (Ctenocephalides felis) midgut cDNA libraries: deciphering molecular pathways involved in host response to R. typhi infection

    PubMed Central

    Dreher-Lesnick, S. M.; Ceraul, S. M.; Lesnick, S. C.; Gillespie, J. J.; Anderson, J. M.; Jochim, R. C.; Valenzuela, J. G.; Azad, A. F.

    2011-01-01

    Murine typhus is a flea-borne febrile illness that is caused by the obligate intracellular bacterium, Rickettsia typhi. The cat flea, Ctenocephalides felis, acquires R. typhi by imbibing a bloodmeal from a rickettsemic vertebrate host. To explore which transcripts are expressed in the midgut in response to challenge with R. typhi, cDNA libraries of R. typhi-infected and uninfected midguts of C. felis were constructed. In this study, we examined midgut transcript levels for select C. felis serine proteases, GTPases and defence response genes, all thought to be involved in the fleas response to feeding or infection. An increase in gene expression was observed for the serine protease inhibitors and vesicular trafficking proteins in response to feeding. In addition, R. typhi infection resulted in an increase in gene expression for the chymotrypsin and rab5 that we studied. Interestingly, R. typhi infection had little effect on expression of any of the defence response genes that we studied. We are unsure as to the physiological significance of these gene expression profiles and are currently investigating their potential roles as it pertains to R. typhi infection. To our knowledge, this is the first report of differential expression of flea transcripts in response to infection with R. typhi. PMID:20017753

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

    SciTech Connect

    Teitz, T.; Naiman, T.; Avissar, S.S.; Bar, S.; Okayama, H.; Canaani, D.

    1987-12-01

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

  14. Mapping of an autoreactive epitope within glutamate decarboxylase using a diabetes-associated human monoclonal autoantibody and an epitope cDNA library.

    PubMed

    Richter, W; Northemann, W; Müller, M; Böhm, B O

    1996-04-01

    Glutamate decarboxylase (GAD65) is a major autoantigen in insulin-dependent diabetes (IDDM) and the neurological disorder Stiff-Man-Syndrome (SMS). We derived a human monoclonal autoantibody (MICA 2) from peripheral blood of a patient newly diagnosed with IDDM, which reacted with GAD65 in Western blots. This indicated that a linear epitope is recognized by MICA 2. Using an epitope cDNA library we mapped the MICA 2 epitope to a contiguous stretch of 26 amino acids (506-531) in the C-terminus of GAD65. Neither blocking experiments with synthetic peptides nor analysis of overlapping decapeptides expressed as fusion proteins allowed us to further narrow down the epitope to the typical size of linear epitopes of 6-8 amino acids. We suggest that a miniconformational epitope provided by amino acids 506-531 is recognized by MICA 2, which withstands SDS gel electrophoresis without destruction or partially refolds during the Western blot procedure. A sequence homology with human heat shock protein 60 (HSP60) maps to this region of GAD65 but no cross-reactivity of MICA 2 with HSP60 occurred. Our data demonstrate that reactivity of an antibody in Western blots does not necessarily define a classic linear epitope of 6-8 amino acids and describe a new autoreactive epitope in GAD65 different from those reported for sera from patients with SMS.

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

    PubMed

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

    2014-11-01

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

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

    PubMed

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

    2016-02-01

    Biological production of fatty acid (FA)-derived products has gained increasing attention to replace petroleum-based fuels and chemicals. FA biosynthesis is highly regulated, and usually it is challenging to design rational engineering strategies. In addition, the conventional 'one sample at a time' method for lipid determination is time consuming and laborious, and it is difficult to screen large numbers of samples. Here, a method for detecting free FAs in viable cells using Nile red staining was developed for use in large-scale screening. Following optimization of the method, it was used for screening a cDNA library from the oleaginous yeast Yarrowia lipolytica for identification of genes/enzymes that were able to enhance free FA accumulation in Saccharomyces cerevisiae. Several novel enzymes resulting in increasing FA accumulation were discovered. These targets include a GPI anchor protein, malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel method for high-throughput evaluation of the content of free FAs, but also give new insight into how enzymes from Y. lipolytica may increase the production of fatty acids in S. cerevisiae. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

    PubMed

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

    2013-01-14

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

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

    PubMed

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

    2011-11-01

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

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

    PubMed

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

    2012-11-01

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

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

    PubMed Central

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

    PubMed Central

    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

  2. Isolation and characterization of rice cesium transporter genes from a rice-transporter-enriched yeast expression library.

    PubMed

    Yamaki, Tomohiro; Otani, Masahiro; Ono, Kohei; Mimura, Takuro; Oda, Koshiro; Minamii, Takeshi; Matsumoto, Shingo; Matsuo, Yuzy; Kawamukai, Makoto; Akihiro, Takashi

    2017-03-28

    A considerable portion of agricultural land in central-east Japan has been contaminated by radioactive material, particularly radioactive Cs, due to the industrial accident at the Fukushima Daiichi nuclear power plant. Understanding the mechanism of absorption, translocation, and accumulation of Cs(+) in plants will greatly assist in developing approaches to help reduce the radioactive contamination of agricultural products. At present, however, little is known regarding the Cs(+) transporters in rice. A transporter-enriched yeast expression library was constructed and the library was screened for Cs(+) transporter genes. The 1452 full length cDNAs encoding transporter genes were obtained from the Rice Genome Resource Center and 1358 clones of these transporter genes were successively subcloned into yeast expression vectors; which were then transferred into yeast. Using this library, both positive and negative selection screens can be performed, which have not been previously possible. The constructed library is an excellent tool for the isolation of novel transporter genes. This library was screened for clones that were sensitive to Cs(+) using a SD-Gal medium containing either 30 or 70 mM CsCl; resulting in the isolation of thirteen Cs(+) sensitive clones. (137) Cs absorption experiments were conducted and confirmed that all of the identified clones were able to absorb (137) Cs. Three potassium transporters, two ABC transporters, and one NRAMP transporter were among the thirteen identified clones.

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

    PubMed

    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.

  4. Construction of a micro-library enriched with genomic replication origins of carrot somatic embryos by laser microdissection.

    PubMed

    Murata, Natsuko; Masuda, Kiyoshi; Nishiyama, Ryutaro; Nomura, Koji

    2005-06-01

    In this paper, we describe an effective method for constructing a micro-library enriched with chromosomal DNA replication origins. Carrot (Daucus carota L.) somatic embryos at early globular stage were incubated for 15 min in the presence of bromodeoxyuridine (BrdU) to pulse label newly synthesized DNA strands. Nuclei were isolated from the cells, and the DNA was extracted on microscopic slides. DNA fibers spread on slides were visualized using anti-BrdU and FITC-conjugated secondary antibodies. DNA regions where BrdU was incorporated were clearly visualized under a fluorescent microscope as dots on DNA fibers. Regions of DNA fiber containing many fluorescent dots should contain replicons in them. DNA fibers showing many fluorescence dots, or replicons were easily cut and collected using a laser microdissection system equipped with a pulse laser beam. DNA fragments containing many replicons were able to be collected with an efficiency of 20-30 DNA fragments per 1 h. Using degenerate oligonucleotide primed PCR, fragments were randomly amplified from the microdissected fragments, and subcloned to construct a micro-library. This is the first report of the application of a laser microdissection technique for constructing a micro-library enriched with replication origins of chromosomal DNA, although there were some reports on laser microdissection of chromosomes. The simple procedure established here should open up a new application of laser optics.

  5. 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

    PubMed Central

    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

  6. Schistosoma japonicum: screening of cercariae cDNA library by specific single-chain antibody against SIEA26-28 ku and immunization experiment of the recombinant plasmids containing the selected genes.

    PubMed

    Gao, Dong-mei; Wang, Shi-ping; He, Zhuo; Fung, Ming-chiu; Liu, Ming-she; Yu, Lu-xin; Chen, Xiu-chun

    2010-06-01

    To obtain the gene encoding SIEA26-28 ku, which has been proven to be a potential anti-schistosomiasis vaccine candidate, screening Schistosoma japonicum (Sj) cercariae cDNA library with soluble specific single-chain antibody (SIEA26-28 ku-scFv) was performed. A large amount of specific single-chain antibody was harvested through construction of recombinant expression vector pET32a/scFv. The protein was purified and characterized. By using this protein (PET32a-scFv) as a probe, S. japonicum cercariae cDNA library was screened. Two strong positive clones were selected, and their eukaryotic recombinant plasmids were constructed. These genes were named as S. japonicum ribosomal protein S4 (SjRPS4) and S. japonicum ribosomal protein L7 (SjRPL7), respectively. Experiments of mice showed that both SjRPS4 and SjRPL7 DNA vaccines could induce significant immunoprotection. Result of these experiments further proved that the specific single-chain antibody is a very valuable tool in screening of cDNA library to get the corresponding molecules.

  7. Focused chemical libraries--design and enrichment: an example of protein-protein interaction chemical space.

    PubMed

    Zhang, Xu; Betzi, Stéphane; Morelli, Xavier; Roche, Philippe

    2014-07-01

    One of the many obstacles in the development of new drugs lies in the limited number of therapeutic targets and in the quality of screening collections of compounds. In this review, we present general strategies for building target-focused chemical libraries with a particular emphasis on protein-protein interactions (PPIs). We describe the chemical spaces spanned by nine commercially available PPI-focused libraries and compare them to our 2P2I3D academic library, dedicated to orthosteric PPI modulators. We show that although PPI-focused libraries have been designed using different strategies, they share common subspaces. PPI inhibitors are larger and more hydrophobic than standard drugs; however, an effort has been made to improve the drug-likeness of focused chemical libraries dedicated to this challenging class of targets.

  8. An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries

    PubMed Central

    Habermann, Bianca; Bebin, Anne-Gaelle; Herklotz, Stephan; Volkmer, Michael; Eckelt, Kay; Pehlke, Kerstin; Epperlein, Hans Henning; Schackert, Hans Konrad; Wiebe, Glenis; Tanaka, Elly M

    2004-01-01

    Background The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. Results Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. Conclusions Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online. PMID:15345051

  9. A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits.

    PubMed

    Karamitros, Timokratis; Magiorkinis, Gkikas

    2015-12-15

    The enrichment of targeted regions within complex next generation sequencing libraries commonly uses biotinylated baits to capture the desired sequences. This method results in high read coverage over the targets and their flanking regions. Oxford Nanopore Technologies recently released an USB3.0-interfaced sequencer, the MinION. To date no particular method for enriching MinION libraries has been standardized. Here, using biotinylated PCR-generated baits in a novel approach, we describe a simple and efficient way for multiplexed enrichment of MinION libraries, overcoming technical limitations related with the chemistry of the sequencing-adapters and the length of the DNA fragments. Using Phage Lambda and Escherichia coli as models we selectively enrich for specific targets, significantly increasing the corresponding read-coverage, eliminating unwanted regions. We show that by capturing genomic fragments, which contain the target sequences, we recover reads extending targeted regions and thus can be used for the determination of potentially unknown flanking sequences. By pooling enriched libraries derived from two distinct E. coli strains and analyzing them in parallel, we demonstrate the efficiency of this method in multiplexed format. Crucially we evaluated the optimal bait size for large fragment libraries and we describe for the first time a standardized method for target enrichment in MinION platform.

  10. EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. I. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia.

    PubMed

    Cho, Eun-Min; Liu, Li; Farmerie, William; Keyhani, Nemat O

    2006-09-01

    The entomopathogenic fungus Beauveria (Cordyceps) bassiana holds much promise as a pest biological control agent. B. bassiana produces at least three in vitro single cell infectious propagules, including aerial conidia, vegetative cells termed blastospores and submerged conidia, that display different morphological, biochemical and virulence properties. Populations of aerial conidia, blastospores and submerged conidia were produced on agar plates, rich liquid broth cultures and under conditions of nutrient limitation in submerged cultures, respectively. cDNA libraries were generated from mRNA isolated from each B. bassiana cell type and approximately 2,500 5' end sequences were determined from each library. Sequences derived from aerial conidia clustered into 284 contigs and 963 singlets, with those derived from blastospores and submerged conidia forming 327 contigs with 788 singlets, and 303 contigs and 1,079 contigs, respectively. Almost half (40-45 %) of the sequences in each library displayed either no significant similarity (e value >10(-4)) or similarity to hypothetical proteins found in the NCBI database. The expressed sequence tag dataset also included sequences representing a significant portion of proteins in cellular metabolism, information storage and processing, transport and cell processes, including cell division and posttranslational modifications. Transcripts encoding a diverse array of pathogenicity-related genes, including proteases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites, were also identified. Comparative analysis between the libraries identified 2,416 unique sequences, of which 20-30 % were unique to each library, and only approximately 6 % of the sequences were shared between all three libraries. The unique and divergent representation of the B. bassiana transcriptome in the cDNA libraries from each cell type suggests robust differential gene expression profiles in response to environmental

  11. Complementary DNA libraries: an overview.

    PubMed

    Ying, Shao-Yao

    2004-07-01

    The generation of complete and full-length cDNA libraries for potential functional assays of specific gene sequences is essential for most molecules in biotechnology and biomedical research. The field of cDNA library generation has changed rapidly in the past 10 yr. This review presents an overview of the method available for the basic information of generating cDNA libraries, including the definition of the cDNA library, different kinds of cDNA libraries, difference between methods for cDNA library generation using conventional approaches and a novel strategy, and the quality of cDNA libraries. It is anticipated that the high-quality cDNA libraries so generated would facilitate studies involving genechips and the microarray, differential display, subtractive hybridization, gene cloning, and peptide library generation.

  12. Raman spectroscopy detects phenotypic differences among Escherichia coli enriched for 1-butanol tolerance using a metagenomic DNA library.

    PubMed

    Freedman, Benjamin G; Zu, Theresah N K; Wallace, Robert S; Senger, Ryan S

    2016-07-01

    Advances in Raman spectroscopy are enabling more comprehensive measurement of microbial cell chemical composition. Advantages include results returned in near real-time and minimal sample preparation. In this research, Raman spectroscopy is used to analyze E. coli with engineered solvent tolerance, which is a multi-genic trait associated with complex and uncharacterized phenotypes that are of value to industrial microbiology. To generate solvent tolerant phenotypes, E. coli transformed with DNA libraries are serially enriched in the presence of 0.9% (v/v) and 1.1% (v/v) 1-butanol. DNA libraries are created using degenerate oligonucleotide primed PCR (DOP-PCR) from the genomic DNA of E. coli, Clostridium acetobutylicum ATCC 824, and the metagenome of a stream bank soil sample, which contained DNA from 72 different phyla. DOP-PCR enabled high efficiency library cloning (with no DNA shearing or end-polishing) and the inclusion un-culturable organisms. Nine strains with improved tolerance are analyzed by Raman spectroscopy and vastly different solvent-tolerant phenotypes are characterized. Common among these are improved membrane rigidity from increasing the fraction of unsaturated fatty acids at the expense of cyclopropane fatty acids. Raman spectroscopy offers the ability to monitor cell phenotype changes in near real-time and is adaptable to high-throughput screening, making it relevant to metabolic engineering. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

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

    2010-05-26

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

  14. Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains

    PubMed Central

    Nelson, William; Luo, Meizhong; Ma, Jianxin; Estep, Matt; Estill, James; He, Ruifeng; Talag, Jayson; Sisneros, Nicholas; Kudrna, David; Kim, HyeRan; Ammiraju, Jetty SS; Collura, Kristi; Bharti, Arvind K; Messing, Joachim; Wing, Rod A; SanMiguel, Phillip; Bennetzen, Jeffrey L; Soderlund, Carol

    2008-01-01

    Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR) and methylation spanning linker libraries (MSLL). These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig), while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%). These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of epigenetic boundaries are barely

  15. Effective DNA fragmentation technique for simple sequence repeat detection with a microsatellite-enriched library and high-throughput sequencing.

    PubMed

    Tanaka, Keisuke; Ohtake, Rumi; Yoshida, Saki; Shinohara, Takashi

    2017-04-01

    Two different techniques for genomic DNA fragmentation before microsatellite-enriched library construction-restriction enzyme (NlaIII and MseI) digestion and sonication-were compared to examine their effects on simple sequence repeat (SSR) detection using high-throughput sequencing. Tens of thousands of SSR regions from 5 species of the plant family Myrtaceae were detected when the output of individual samples was >1 million paired-end reads. Comparison of the two DNA fragmentation techniques showed that restriction enzyme digestion was superior to sonication for identification of heterozygous genotypes, whereas sonication was superior for detection of various SSR flanking regions with both species-specific and common characteristics. Therefore, choosing the most suitable DNA fragmentation method depends on the type of analysis that is planned.

  16. Fidelity by design: Yoctoreactor and binder trap enrichment for small-molecule DNA-encoded libraries and drug discovery.

    PubMed

    Blakskjaer, Peter; Heitner, Tara; Hansen, Nils Jakob Vest

    2015-06-01

    DNA-encoded small-molecule library (DEL) technology allows vast drug-like small molecule libraries to be efficiently synthesized in a combinatorial fashion and screened in a single tube method for binding, with an assay readout empowered by advances in next generation sequencing technology. This approach has increasingly been applied as a viable technology for the identification of small-molecule modulators to protein targets and as precursors to drugs in the past decade. Several strategies for producing and for screening DELs have been devised by both academic and industrial institutions. This review highlights some of the most significant and recent strategies along with important results. A special focus on the production of high fidelity DEL technologies with the ability to eliminate screening noise and false positives is included: using a DNA junction called the Yoctoreactor, building blocks (BBs) are spatially confined at the center of the junction facilitating both the chemical reaction between BBs and encoding of the synthetic route. A screening method, known as binder trap enrichment, permits DELs to be screened robustly in a homogeneous manner delivering clean data sets and potent hits for even the most challenging targets.

  17. Library+

    ERIC Educational Resources Information Center

    Merrill, Alex

    2011-01-01

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

  18. Validation of SCALE 4. 0 -- CSAS25 module and the 27-group ENDF/B-IV cross-section library for low-enriched uranium systems

    SciTech Connect

    Jordan, W.C.

    1993-02-01

    A version of KENO V.a and the 27-group library in SCALE-4.0 were validated for use in evaluating the nuclear criticality safety of low-enriched uranium systems. A total of 59 critical systems were analyzed. A statistical analysis of the results was performed, and subcritical acceptanced criteria are established.

  19. Validation of SCALE 4.0 -- CSAS25 module and the 27-group ENDF/B-IV cross-section library for low-enriched uranium systems

    SciTech Connect

    Jordan, W.C.

    1993-02-01

    A version of KENO V.a and the 27-group library in SCALE-4.0 were validated for use in evaluating the nuclear criticality safety of low-enriched uranium systems. A total of 59 critical systems were analyzed. A statistical analysis of the results was performed, and subcritical acceptanced criteria are established.

  20. Genomics of hybrid poplar (Populus trichocarpax deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full-length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of insect-induced defences in poplar.

    PubMed

    Ralph, Steven; Oddy, Claire; Cooper, Dawn; Yueh, Hesther; Jancsik, Sharon; Kolosova, Natalia; Philippe, Ryan N; Aeschliman, Dana; White, Rick; Huber, Dezene; Ritland, Carol E; Benoit, François; Rigby, Tracey; Nantel, André; Butterfield, Yaron S N; Kirkpatrick, Robert; Chun, Elizabeth; Liu, Jerry; Palmquist, Diana; Wynhoven, Brian; Stott, Jeffrey; Yang, George; Barber, Sarah; Holt, Robert A; Siddiqui, Asim; Jones, Steven J M; Marra, Marco A; Ellis, Brian E; Douglas, Carl J; Ritland, Kermit; Bohlmann, Jörg

    2006-04-01

    As part of a genomics strategy to characterize inducible defences against insect herbivory in poplar, we developed a comprehensive suite of functional genomics resources including cDNA libraries, expressed sequence tags (ESTs) and a cDNA microarray platform. These resources are designed to complement the existing poplar genome sequence and poplar (Populus spp.) ESTs by focusing on herbivore- and elicitor-treated tissues and incorporating normalization methods to capture rare transcripts. From a set of 15 standard, normalized or full-length cDNA libraries, we generated 139,007 3'- or 5'-end sequenced ESTs, representing more than one-third of the c. 385,000 publicly available Populus ESTs. Clustering and assembly of 107,519 3'-end ESTs resulted in 14,451 contigs and 20,560 singletons, altogether representing 35,011 putative unique transcripts, or potentially more than three-quarters of the predicted c. 45,000 genes in the poplar genome. Using this EST resource, we developed a cDNA microarray containing 15,496 unique genes, which was utilized to monitor gene expression in poplar leaves in response to herbivory by forest tent caterpillars (Malacosoma disstria). After 24 h of feeding, 1191 genes were classified as up-regulated, compared to only 537 down-regulated. Functional classification of this induced gene set revealed genes with roles in plant defence (e.g. endochitinases, Kunitz protease inhibitors), octadecanoid and ethylene signalling (e.g. lipoxygenase, allene oxide synthase, 1-aminocyclopropane-1-carboxylate oxidase), transport (e.g. ABC proteins, calreticulin), secondary metabolism [e.g. polyphenol oxidase, isoflavone reductase, (-)-germacrene D synthase] and transcriptional regulation [e.g. leucine-rich repeat transmembrane kinase, several transcription factor classes (zinc finger C3H type, AP2/EREBP, WRKY, bHLH)]. This study provides the first genome-scale approach to characterize insect-induced defences in a woody perennial providing a solid platform for

  1. cDNA cloning and sequencing of tarantula hemocyanin subunits.

    PubMed

    Voit, R; Feldmaier-Fuchs, G

    1990-01-01

    Tarantula heart cDNA libraries were screened with synthetic oligonucleotide probes deduced from the highly conserved amino acid sequences of the two copper-binding sites, copper A and copper B, found in chelicerate hemocyanins. Positive cDNA clones could be obtained and four different cDNA types were characterized.

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  4. Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries.

    PubMed

    Leethanakul, C; Patel, V; Gillespie, J; Shillitoe, E; Kellman, R M; Ensley, J F; Limwongse, V; Emmert-Buck, M R; Krizman, D B; Gutkind, J S

    2000-09-01

    Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world affecting the oral cavity, salivary glands, larynx and pharynx. Utilizing tissue from patients with HNSCC, we sought to systematically identify and catalog genes expressed in HNSCC progression. Here, we demonstrate the successful use of laser capture microdissection for procuring pure populations of cells from patient tissue sets comprised of oral squamous cell carcinomas (OSCCs) and matching normal tissue. From the estimated 5000 cells procured for each sample, we were able to extract total RNA (14.7-18.6 ng) of sufficient quality to transcribe GAPDH by reverse transcriptase-polymerase chain reaction (RT-PCR). The RNA was used for the synthesis of blunt-ended, double-strand complementary DNAs (cDNAs) by oligo (dT)-mediated reverse transcription, followed by addition of linkers. Primers specific for these linkers with uracil deglycosylase-compatible ends were used to amplify these cDNAs by PCR and the product was subcloned into the pAMP10 cloning vector. Ninety-six clones from each of six libraries were randomly sequenced and results indicated that 76-96% of the inserts represent either anonymous expressed sequence tags (ESTs) (25-48%), known genes (9-29%) or novel sequences (27-51%), respectively, with very little redundancy. These results demonstrate that high quality, representative cDNA libraries can be generated from microdissected OSCC tissue. Furthermore, these finding suggest the existence of at least 132 novel genes expressed in our cDNA libraries, which may have a role in the pathogenesis of HNSCC, and may represent novel markers for early detection as well as targets for pharmacological intervention in this disease.

  5. Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library*

    PubMed Central

    Kiefer, Jonathan D.; Srinivas, Raja R.; Lobner, Elisabeth; Tisdale, Alison W.; Mehta, Naveen K.; Yang, Nicole J.; Tidor, Bruce; Wittrup, K. Dane

    2016-01-01

    The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation. PMID:27582495

  6. Isolation of cDNA and genomic DNA clones encoding type II collagen.

    PubMed Central

    Young, M F; Vogeli, G; Nunez, A M; Fernandez, M P; Sullivan, M; Sobel, M E

    1984-01-01

    A cDNA library constructed from total chick embryo RNA was screened with an enriched fraction of type II collagen mRNA. Two overlapping cDNA clones were characterized and shown to encode the COOH propeptide of type II collagen. In addition, a type II collagen clone was isolated from a Charon 4A library of chick genomic fragments. Definitive identification of the clones was based on DNA sequence analysis. The 3' end of the type II collagen gene appears to be similar to that of other interstitial collagen genes. Northern hybridization data indicates that there is a marked decrease in type II collagen mRNA levels in chondrocytes treated with the dedifferentiating agent 5-bromodeoxyuridine. The major type II collagen mRNA species is 5300 bases long, similar to that of other interstitial collagen RNAs. Images PMID:6203098

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

    PubMed

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

    2009-06-24

    In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 x 10(5) cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST) analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4%) were known genes but only five from A. pernyi, 37 (21.2%) were known ESTs without function annotation, and 41 (23.4%) were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151) was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination.

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

    PubMed Central

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

    2009-01-01

    In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 × 105 cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST) analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4%) were known genes but only five from A. pernyi, 37 (21.2%) were known ESTs without function annotation, and 41 (23.4%) were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151) was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination. PMID:19564928

  9. 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.

    PubMed

    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.

  10. Selective enrichment of environmental DNA libraries for genes encoding nonribosomal peptides and polyketides by phosphopantetheine transferase-dependent complementation of siderophore biosynthesis

    PubMed Central

    Charlop-Powers, Zachary; Banik, Jacob J.; Owen, Jeremy G.; Craig, Jeffrey W.; Brady, Sean F.

    2012-01-01

    The cloning of DNA directly from environmental samples provides a means to functionally access biosynthetic gene clusters present in the genomes of the large fraction of bacteria that remains recalcitrant to growth in the laboratory. Herein we demonstrate a method by which complementation of phosphopantetheine transferase deletion mutants can be used to restore siderophore biosynthesis and to therefore selectively enrich eDNA libraries for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene sequences to unprecedented levels. The common use of NRPS/PKS-derived siderophores across bacterial taxa makes this method generalizable and should allow for the facile selective enrichment of NRPS/PKS-containing biosynthetic gene clusters from large environmental DNA libraries using a wide variety of phylogenetically diverse bacterial hosts. PMID:23072412

  11. Selective enrichment of environmental DNA libraries for genes encoding nonribosomal peptides and polyketides by phosphopantetheine transferase-dependent complementation of siderophore biosynthesis.

    PubMed

    Charlop-Powers, Zachary; Banik, Jacob J; Owen, Jeremy G; Craig, Jeffrey W; Brady, Sean F

    2013-01-18

    The cloning of DNA directly from environmental samples provides a means to functionally access biosynthetic gene clusters present in the genomes of the large fraction of bacteria that remains recalcitrant to growth in the laboratory. Herein, we demonstrate a method by which complementation of phosphopantetheine transferase deletion mutants can be used to restore siderophore biosynthesis and to therefore selectively enrich eDNA libraries for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene sequences to unprecedented levels. The common use of NRPS/PKS-derived siderophores across bacterial taxa makes this method generalizable and should allow for the facile selective enrichment of NRPS/PKS-containing biosynthetic gene clusters from large environmental DNA libraries using a wide variety of phylogenetically diverse bacterial hosts.

  12. The Role of Libraries in the Mutual Enrichment of the National Cultures of the Peoples of the Soviet Union

    ERIC Educational Resources Information Center

    Fonotov, G. P.

    1978-01-01

    Traces literacy and library developments throughout the USSR; the focus is on publications in the various languages of the USSR, and on the role of the libraries in the dissemination of such materials. (Author)

  13. Exon-Enriched Libraries Reveal Large Genic Differences Between Aedes aegypti from Senegal, West Africa, and Populations Outside Africa

    PubMed Central

    Dickson, Laura B.; Campbell, Corey L.; Juneja, Punita; Jiggins, Francis M.; Sylla, Massamba; Black, William C.

    2016-01-01

    Aedes aegypti is one of the most studied mosquito species, and the principal vector of several arboviruses pathogenic to humans. Recently failure to oviposit, low fecundity, and poor egg-to-adult survival were observed when Ae. aegypti from Senegal (SenAae) West Africa were crossed with Ae. aegypti (Aaa) from outside of Africa, and in SenAae intercrosses. Fluorescent in situ hybridization analyses indicated rearrangements on chromosome 1, and pericentric inversions on chromosomes 2 and 3. Herein, high throughput sequencing (HTS) of exon-enriched libraries was used to compare chromosome-wide genetic diversity among Aaa collections from rural Thailand and Mexico, a sylvatic collection from southeastern Senegal (PK10), and an urban collection from western Senegal (Kaolack). Sex-specific polymorphisms were analyzed in Thailand and PK10 to assess genetic differences between sexes. Expected heterozygosity was greatest in SenAae. FST distributions of 15,735 genes among all six pairwise comparisons of the four collections indicated that Mexican and Thailand collections are genetically similar, while FST distributions between PK10 and Kaolack were distinct. All four comparisons of SenAae with Aaa indicated extreme differentiation. FST was uniform between sexes across all chromosomes in Thailand, but were different, especially on the sex autosome 1, in PK10. These patterns correlate with the reproductive isolation noted earlier. We hypothesize that cryptic Ae. aegypti taxa may exist in West Africa, and the large genic differences between Aaa and SenAae detected in the present study have accumulated over a long period following the evolution of chromosome rearrangements in allopatric populations that subsequently cause reproductive isolation when these populations became sympatric. PMID:28007834

  14. Exon-Enriched Libraries Reveal Large Genic Differences Between Aedes aegypti from Senegal, West Africa, and Populations Outside Africa.

    PubMed

    Dickson, Laura B; Campbell, Corey L; Juneja, Punita; Jiggins, Francis M; Sylla, Massamba; Black, William C

    2017-02-09

    Aedes aegypti is one of the most studied mosquito species, and the principal vector of several arboviruses pathogenic to humans. Recently failure to oviposit, low fecundity, and poor egg-to-adult survival were observed when Ae. aegypti from Senegal (SenAae) West Africa were crossed with Ae. aegypti (Aaa) from outside of Africa, and in SenAae intercrosses. Fluorescent in situ hybridization analyses indicated rearrangements on chromosome 1, and pericentric inversions on chromosomes 2 and 3. Herein, high throughput sequencing (HTS) of exon-enriched libraries was used to compare chromosome-wide genetic diversity among Aaa collections from rural Thailand and Mexico, a sylvatic collection from southeastern Senegal (PK10), and an urban collection from western Senegal (Kaolack). Sex-specific polymorphisms were analyzed in Thailand and PK10 to assess genetic differences between sexes. Expected heterozygosity was greatest in SenAae FST distributions of 15,735 genes among all six pairwise comparisons of the four collections indicated that Mexican and Thailand collections are genetically similar, while FST distributions between PK10 and Kaolack were distinct. All four comparisons of SenAae with Aaa indicated extreme differentiation. FST was uniform between sexes across all chromosomes in Thailand, but were different, especially on the sex autosome 1, in PK10. These patterns correlate with the reproductive isolation noted earlier. We hypothesize that cryptic Ae. aegypti taxa may exist in West Africa, and the large genic differences between Aaa and SenAae detected in the present study have accumulated over a long period following the evolution of chromosome rearrangements in allopatric populations that subsequently cause reproductive isolation when these populations became sympatric.

  15. A support vector machines approach for virtual screening of active compounds of single and multiple mechanisms from large libraries at an improved hit-rate and enrichment factor.

    PubMed

    Han, L Y; Ma, X H; Lin, H H; Jia, J; Zhu, F; Xue, Y; Li, Z R; Cao, Z W; Ji, Z L; Chen, Y Z

    2008-06-01

    Support vector machines (SVM) and other machine-learning (ML) methods have been explored as ligand-based virtual screening (VS) tools for facilitating lead discovery. While exhibiting good hit selection performance, in screening large compound libraries, these methods tend to produce lower hit-rate than those of the best performing VS tools, partly because their training-sets contain limited spectrum of inactive compounds. We tested whether the performance of SVM can be improved by using training-sets of diverse inactive compounds. In retrospective database screening of active compounds of single mechanism (HIV protease inhibitors, DHFR inhibitors, dopamine antagonists) and multiple mechanisms (CNS active agents) from large libraries of 2.986 million compounds, the yields, hit-rates, and enrichment factors of our SVM models are 52.4-78.0%, 4.7-73.8%, and 214-10,543, respectively, compared to those of 62-95%, 0.65-35%, and 20-1200 by structure-based VS and 55-81%, 0.2-0.7%, and 110-795 by other ligand-based VS tools in screening libraries of >or=1 million compounds. The hit-rates are comparable and the enrichment factors are substantially better than the best results of other VS tools. 24.3-87.6% of the predicted hits are outside the known hit families. SVM appears to be potentially useful for facilitating lead discovery in VS of large compound libraries.

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

    SciTech Connect

    Mitchell, S.C.; Bocskai, D.; Cao, Y.

    1997-12-31

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

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

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

    Travis, G.H.; Sutcliffe, J.G.

    1988-03-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA.

  20. A cDNA clone for 3-carene synthase from Salvia stenophylla.

    PubMed

    Hoelscher, Dirk J; Williams, David C; Wildung, Mark R; Croteau, Rodney

    2003-04-01

    The essential oil of Salvia stenophylla contains (+)-3-carene as the principal monoterpene component. Using an enriched cDNA library prepared from mRNA isolated from S. stenophylla peltate glandular trichomes, and a homology-based cloning strategy, a full-length cDNA was isolated that encoded a preprotein of 69.7 kDa which resembled a monoterpene synthase in sequence. Heterologous expression of the gene in Escherichia coli provided a soluble recombinant enzyme capable of catalyzing the divalent metal ion-dependent conversion of geranyl diphosphate to (+)-3-carene and to lesser amounts of limonene, myrcene, 4-carene and beta-phellandrene. This multiple-product synthase is responsible for the production of all of the essential oil monoterpenes of S. stenophylla.

  1. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, N.V.; Broekaert, W.F.; Namhai Chua; Kush, A.

    1993-02-16

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids.

  2. A High Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution.

    PubMed

    Naimuddin, Mohammed; Kubo, Tai

    2016-02-08

    We describe a high performance platform based on cDNA display technology by developing a new modified puromycin linker-oligonucleotide. The linker consists of four major characteristics: a "ligation site" for hybridization and ligation of mRNA by T4 RNA ligase, a "puromycin arm" for covalent linkage of the protein, a "polyadenosine site" for a longer puromycin arm and purification of protein fusions (optional) using oligo-dT matrices, and a "reverse transcription site" for the formation of stable cDNA protein fusions whose cDNA is covalently linked to its encoded protein. The linker was synthesized by a novel branching strategy and provided >8-fold higher yield than previous linkers. This linker enables rapid and highly efficient ligation of mRNA (>90%) and synthesis of protein fusions (∼ 50-95%) in various cell-free expression systems. Overall, this new cDNA display method provides 10-200 fold higher end-usage fusions than previous methods and benefits higher diversity libraries crucial for directed protein/peptide evolution. With the increased efficiency, this system was able to reduce the time for one selection cycle to <8 h and is potentially amenable to high-throughput systems. We demonstrate the efficiency of this system for higher throughput selections of various biomolecular interactions and achieved 30-40-fold enrichment per selection cycle. Furthermore, a 4-fold higher enrichment of Flag-tag was obtained from a doped mixture compared with that of the previous cDNA display method. A three-finger protein library was evolved to isolate superior nanomolar range binding candidates for vascular endothelial growth factor. This method is expected to provide a beneficial impact to accelerated drug discovery and proteome analysis.

  3. Intravenous infusion of phage-displayed antibody library in human cancer patients: enrichment and cancer-specificity of tumor-homing phage-antibodies.

    PubMed

    Shukla, Girja S; Krag, David N; Peletskaya, Elena N; Pero, Stephanie C; Sun, Yu-Jing; Carman, Chelsea L; McCahill, Laurence E; Roland, Thomas A

    2013-08-01

    Phage display is a powerful method for target discovery and selection of ligands for cancer treatment and diagnosis. Our goal was to select tumor-binding antibodies in cancer patients. Eligibility criteria included absence of preexisting anti-phage-antibodies and a Stage IV cancer status. All patients were intravenously administered 1 × 10(11) TUs/kg of an scFv library 1 to 4 h before surgical resection of their tumors. No significant adverse events related to the phage library infusion were observed. Phage were successfully recovered from all tumors. Individual clones from each patient were assessed for binding to the tumor from which clones were recovered. Multiple tumor-binding phage-antibodies were identified. Soluble scFv antibodies were produced from the phage clones showing higher tumor binding. The tumor-homing phage-antibodies and derived soluble scFvs were found to bind varying numbers (0-5) of 8 tested normal human tissues (breast, cervix, colon, kidney, liver, spleen, skin, and uterus). The clones that showed high tumor-specificity were found to bind corresponding tumors from other patients also. Clone enrichment was observed based on tumor binding and DNA sequence data. Clone sequences of multiple variable regions showed significant matches to certain cancer-related antibodies. One of the clones (07-2,355) that was found to share a 12-amino-acid-long motif with a reported IL-17A antibody was further studied for competitive binding for possible antigen target identification. We conclude that these outcomes support the safety and utility of phage display library panning in cancer patients for ligand selection and target discovery for cancer treatment and diagnosis.

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

    PubMed

    Rinaldy, A R; Steiner, M S

    1999-11-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  7. Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy

    SciTech Connect

    Mangin, M.; Webb, A.C.; Dreyer, B.E.; Posillico, J.T.; Ikeda, K.; Weir, E.C.; Stewart, A.F.; Bander, N.H.; Milstone, L.; Barton, D.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)/sup +/ 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 lambdagt10. 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. Novel HIV-1 Knockdown Targets Identified by an Enriched Kinases/Phosphatases shRNA Library Using a Long-Term Iterative Screen in Jurkat T-Cells

    PubMed Central

    Rato, Sylvie; Maia, Sara; Brito, Paula M.; Resende, Leonor; Pereira, Carina F.; Moita, Catarina; Freitas, Rui P.; Moniz-Pereira, José; Hacohen, Nir; Moita, Luis Ferreira; Goncalves, Joao

    2010-01-01

    HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies. PMID:20174665

  9. High-Affinity DNA Aptamer Generation Targeting von Willebrand Factor A1-Domain by Genetic Alphabet Expansion for Systematic Evolution of Ligands by Exponential Enrichment Using Two Types of Libraries Composed of Five Different Bases.

    PubMed

    Matsunaga, Ken-Ichiro; Kimoto, Michiko; Hirao, Ichiro

    2017-01-11

    The novel evolutionary engineering method ExSELEX (genetic alphabet expansion for systematic evolution of ligands by exponential enrichment) provides high-affinity DNA aptamers that specifically bind to target molecules, by introducing an artificial hydrophobic base analogue as a fifth component into DNA aptamers. Here, we present a newer version of ExSELEX, using a library with completely randomized sequences consisting of five components: four natural bases and one unnatural hydrophobic base, 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). In contrast to the limited number of Ds-containing sequence combinations in our previous library, the increased complexity of the new randomized library could improve the success rates of high-affinity aptamer generation. To this end, we developed a sequencing method for each clone in the enriched library after several rounds of selection. Using the improved library, we generated a Ds-containing DNA aptamer targeting von Willebrand factor A1-domain (vWF) with significantly higher affinity (KD = 75 pM), relative to those generated by the initial version of ExSELEX, as well as that of the known DNA aptamer consisting of only the natural bases. In addition, the Ds-containing DNA aptamer was stabilized by introducing a mini-hairpin DNA resistant to nucleases, without any loss of affinity (KD = 61 pM). This new version is expected to consistently produce high-affinity DNA aptamers.

  10. Enrichment of open reading frames presented on bacteriophage M13 using hyperphage.

    PubMed

    Hust, Michael; Meysing, Maren; Schirrmann, Thomas; Selke, Martin; Meens, Jochen; Gerlach, Gerald-F; Dübel, Stefan

    2006-09-01

    The enrichment of open reading frames (ORFs) from large gene libraries and the presentation of the corresponding polypeptides on filamentous phage M13 (phage display) is frequently used to identify binding partners of unknown ORFs. In particular phage display is a valuable tool for the identification of pathogen-related antigens and a first step for the development of new diagnostics and therapeutics. Here, we introduce a significant improvement of phage-based ORF enrichment by using Hyperphage, a helperphage with a truncated gIII. The methods allow both the enrichment of ORFs from cDNA libraries and the display of the corresponding polypeptides on phage, thus combining ORF enrichment with a screening for binding in one step without any further subcloning steps. We demonstrated the benefits of the method by isolating the sequences encoding two predicted immunogenic epitopes of the outer membrane protein D encoding gene (ompD) of Salmonella typhimurium. Here, we showed that when using a mixture of three constructs with only one containing an ORF solely this correct construct could be reisolated in phage particles. Further; both epitopes were detected by enzyme-linked immunosorbent assay (ELISA), demonstrating correct translation of fusion proteins. Furthermore, the enrichment system was evaluated by the enrichment of ORFs from total cDNA of lymphocytes. Here, we could show that 60% of the phage contained ORFs, which is an increase of an order of magnitude compared with conventional phage expression system. Together these data show that the Hyperphage-based enrichment system significantly improves the enrichment of ORFs and directly allows the display of the corresponding polypeptide on bacteriophage M13.

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

    PubMed Central

    Sakurai, Tetsuya; Plata, Germán; Rodríguez-Zapata, Fausto; Seki, Motoaki; Salcedo, Andrés; Toyoda, Atsushi; Ishiwata, Atsushi; Tohme, Joe; Sakaki, Yoshiyuki; Shinozaki, Kazuo; Ishitani, Manabu

    2007-01-01

    Background Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs). Results The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features. Conclusion The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome. PMID:18096061

  12. Insights into corn genes derived from large-scale cDNA sequencing.

    PubMed

    Alexandrov, Nickolai N; Brover, Vyacheslav V; Freidin, Stanislav; Troukhan, Maxim E; Tatarinova, Tatiana V; Zhang, Hongyu; Swaller, Timothy J; Lu, Yu-Ping; Bouck, John; Flavell, Richard B; Feldmann, Kenneth A

    2009-01-01

    We present a large portion of the transcriptome of Zea mays, including ESTs representing 484,032 cDNA clones from 53 libraries and 36,565 fully sequenced cDNA clones, out of which 31,552 clones are non-redundant. These and other previously sequenced transcripts have been aligned with available genome sequences and have provided new insights into the characteristics of gene structures and promoters within this major crop species. We found that although the average number of introns per gene is about the same in corn and Arabidopsis, corn genes have more alternatively spliced isoforms. Examination of the nucleotide composition of coding regions reveals that corn genes, as well as genes of other Poaceae (Grass family), can be divided into two classes according to the GC content at the third position in the amino acid encoding codons. Many of the transcripts that have lower GC content at the third position have dicot homologs but the high GC content transcripts tend to be more specific to the grasses. The high GC content class is also enriched with intronless genes. Together this suggests that an identifiable class of genes in plants is associated with the Poaceae divergence. Furthermore, because many of these genes appear to be derived from ancestral genes that do not contain introns, this evolutionary divergence may be the result of horizontal gene transfer from species not only with different codon usage but possibly that did not have introns, perhaps outside of the plant kingdom. By comparing the cDNAs described herein with the non-redundant set of corn mRNAs in GenBank, we estimate that there are about 50,000 different protein coding genes in Zea. All of the sequence data from this study have been submitted to DDBJ/GenBank/EMBL under accession numbers EU940701-EU977132 (FLI cDNA) and FK944382-FL482108 (EST).

  13. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1993-02-16

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a pu GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon.

  14. Characterization of rainbow trout gonad, brain and gill deep cDNA repertoires using a Roche 454-Titanium sequencing approach.

    PubMed

    Le Cam, Aurélie; Bobe, Julien; Bouchez, Olivier; Cabau, Cédric; Kah, Olivier; Klopp, Christophe; Lareyre, Jean-Jacques; Le Guen, Isabelle; Lluch, Jérôme; Montfort, Jérôme; Moreews, Francois; Nicol, Barbara; Prunet, Patrick; Rescan, Pierre-Yves; Servili, Arianna; Guiguen, Yann

    2012-05-25

    Rainbow trout, Oncorhynchus mykiss, is an important aquaculture species worldwide and, in addition to being of commercial interest, it is also a research model organism of considerable scientific importance. Because of the lack of a whole genome sequence in that species, transcriptomic analyses of this species have often been hindered. Using next-generation sequencing (NGS) technologies, we sought to fill these informational gaps. Here, using Roche 454-Titanium technology, we provide new tissue-specific cDNA repertoires from several rainbow trout tissues. Non-normalized cDNA libraries were constructed from testis, ovary, brain and gill rainbow trout tissue samples, and these different libraries were sequenced in 10 separate half-runs of 454-Titanium. Overall, we produced a total of 3million quality sequences with an average size of 328bp, representing more than 1Gb of expressed sequence information. These sequences have been combined with all publicly available rainbow trout sequences, resulting in a total of 242,187 clusters of putative transcript groups and 22,373 singletons. To identify the predominantly expressed genes in different tissues of interest, we developed a Digital Differential Display (DDD) approach. This approach allowed us to characterize the genes that are predominantly expressed within each tissue of interest. Of these genes, some were already known to be tissue-specific, thereby validating our approach. Many others, however, were novel candidates, demonstrating the usefulness of our strategy and of such tissue-specific resources. This new sequence information, acquired using NGS 454-Titanium technology, deeply enriched our current knowledge of the expressed genes in rainbow trout through the identification of an increased number of tissue-specific sequences. This identification allowed a precise cDNA tissue repertoire to be characterized in several important rainbow trout tissues. The rainbow trout contig browser can be accessed at the following

  15. cDNA cloning of the bovine low density lipoprotein receptor: feedback regulation of a receptor mRNA.

    PubMed Central

    Russell, D W; Yamamoto, T; Schneider, W J; Slaughter, C J; Brown, M S; Goldstein, J L

    1983-01-01

    The low density lipoprotein (LDL) receptor belongs to a class of migrant cell surface proteins that mediate endocytosis of macromolecular ligands. No cDNAs for this class of proteins have been isolated to date. In the current paper, we report the isolation of a cDNA clone for the LDL receptor from a bovine adrenal cDNA library. The library was constructed by the Okayama-Berg method from poly(A)+ RNA that had been enriched in receptor mRNA by immunopurification of polysomes. Mixtures of synthetic oligonucleotides encoding the amino acid sequence of two neighboring regions of a single cyanogen bromide fragment were used as hybridization probes to identify a recombinant plasmid containing the LDL receptor cDNA. This plasmid, designated pLDLR-1, contains a 2.8-kilobase (kb) insert that includes a sequence which corresponds to the known amino acid sequence of a 36-residue cyanogen bromide fragment of the receptor. pLDLR-1 hybridized to a mRNA of approximately equal to 5.5 kb in the bovine adrenal gland. This mRNA, like the receptor protein, was 9-fold more abundant in bovine adrenal than in bovine liver. pLDLR-1 cross-hybridized to a mRNA of approximately equal to 5.5 kb in cultured human epidermoid carcinoma A-431 cells. This mRNA was markedly reduced in amount when sterols were added to the culture medium, an observation that explains the previously observed feedback regulation of LDL receptor protein. Southern blot analysis of bovine genomic DNA with 32P-labeled pLDLR-1 revealed a simple pattern of hybridization, consistent with a single-copy gene containing introns. Images PMID:6143315

  16. Enriching the Catalog

    ERIC Educational Resources Information Center

    Tennant, Roy

    2004-01-01

    After decades of costly and time-consuming effort, nearly all libraries have completed the retrospective conversion of their card catalogs to electronic form. However, bibliographic systems still are really not much more than card catalogs on wheels. Enriched content that Amazon.com takes for granted--such as digitized tables of contents, cover…

  17. Enriching the Catalog

    ERIC Educational Resources Information Center

    Tennant, Roy

    2004-01-01

    After decades of costly and time-consuming effort, nearly all libraries have completed the retrospective conversion of their card catalogs to electronic form. However, bibliographic systems still are really not much more than card catalogs on wheels. Enriched content that Amazon.com takes for granted--such as digitized tables of contents, cover…

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

    PubMed

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

    1984-12-10

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

  19. Comparison of sequence of cDNA clone with other genomic and cDNA sequences for human C-reactive protein

    SciTech Connect

    Tenchini, M.L.; Bossi, E.; Marchetti, L.; Malcovati, M. ); Lorenzetti, R. )

    1992-04-01

    A clone for C-reactive protein (CRP) has been isolated from a human liver cDNA library; this clone harbors a plasmid, pC81, which has an insert of 1631 bp. When compared to genomic and cDNA sequences published to date now, pC81 has revealed homologies and differences that might help to clarify the structure of this gene and the presence of allelic variants in man.

  20. Transcriptome and full-length cDNA resources for the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major insect pest of pine forests.

    PubMed

    Keeling, Christopher I; Henderson, Hannah; Li, Maria; Yuen, Mack; Clark, Erin L; Fraser, Jordie D; Huber, Dezene P W; Liao, Nancy Y; Docking, T Roderick; Birol, Inanc; Chan, Simon K; Taylor, Greg A; Palmquist, Diana; Jones, Steven J M; Bohlmann, Joerg

    2012-08-01

    Bark beetles (Coleoptera: Curculionidae: Scolytinae) are major insect pests of many woody plants around the world. The mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins, is a significant historical pest of western North American pine forests. It is currently devastating pine forests in western North America--particularly in British Columbia, Canada--and is beginning to expand its host range eastward into the Canadian boreal forest, which extends to the Atlantic coast of North America. Limited genomic resources are available for this and other bark beetle pests, restricting the use of genomics-based information to help monitor, predict, and manage the spread of these insects. To overcome these limitations, we generated comprehensive transcriptome resources from fourteen full-length enriched cDNA libraries through paired-end Sanger sequencing of 100,000 cDNA clones, and single-end Roche 454 pyrosequencing of three of these cDNA libraries. Hybrid de novo assembly of the 3.4 million sequences resulted in 20,571 isotigs in 14,410 isogroups and 246,848 singletons. In addition, over 2300 non-redundant full-length cDNA clones putatively containing complete open reading frames, including 47 cytochrome P450s, were sequenced fully to high quality. This first large-scale genomics resource for bark beetles provides the relevant sequence information for gene discovery; functional and population genomics; comparative analyses; and for future efforts to annotate the MPB genome. These resources permit the study of this beetle at the molecular level and will inform research in other Dendroctonus spp. and more generally in the Curculionidae and other Coleoptera. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

    PubMed Central

    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

  2. Development of a highly efficient expression cDNA cloning system: application to oncogene isolation.

    PubMed Central

    Miki, T; Fleming, T P; Crescenzi, M; Molloy, C J; Blam, S B; Reynolds, S H; Aaronson, S A

    1991-01-01

    We developed an expression cDNA cloning system capable of generating high-complexity libraries with unidirectionally inserted cDNA fragments and allowing efficient plasmid rescue. As an application of this system, a cDNA library was constructed from an NIH 3T3 transformant induced by mouse hepatocellular carcinoma DNA. Transfection of NIH 3T3 cells by the library DNA led to the detection of several transformed foci from which identical plasmids with transforming ability could be rescued. Structure and sequence analysis of the cDNA clones revealed that the oncogene was created by recombinational events involving an unknown gene and the mouse homologue of the B-raf protooncogene. Detection of the same genetic rearrangement in independent primary transformants implied that generation of the oncogene occurred within the tumor rather than during DNA transfection or cDNA library construction. The high frequency at which clones were identified and the large sizes of some of the transforming cDNA inserts isolated suggest wide applicability of this mammalian expression cloning system for isolating cDNAs of biologic interest. Images PMID:2052597

  3. Cloning and expression of human tyrosine aminotransferase cDNA.

    PubMed

    Séralini, G E; Luu-Thé, V; Labrie, F

    1995-01-02

    Complementary DNA clones encoding human tyrosine aminotransferase (TAT) were isolated by screening a normal adult woman liver lambda gt11 library with rat TAT cDNA. The largest isolated cDNA is 2051 bp long (EMBL accession number X55675). This cDNA was subcloned downstream of the cytomegalovirus promoter in the pCMV vector for transfection into human cervical carcinoma HeLa cells. Expression of the TAT cDNA resulted in the synthesis of a protein with a molecular mass of approximately 50 kDa, as assessed by Western analysis, a value which is in close agreement with the predicted molecular weight of 50,399, for a deduced sequence of 454 amino acids. The expressed protein catalyzed specifically the conversion of L-[14C]tyrosine into p-[14C]hydroxyphenylpyruvate. The availability of a functional TAT cDNA provides a useful tool for detailed study of the structure-function relationship of the enzyme and its mutated derivatives.

  4. Identification and analysis of safener-inducible expressed sequence tags in Populus using a cDNA microarray.

    PubMed

    Rishi, A S; Munir, Shirin; Kapur, Vivek; Nelson, Neil D; Goyal, Arun

    2004-12-01

    Safeners are the chemicals used to protect plants from detrimental effects of herbicides, but their mode of action at the molecular level is not well understood. As an initial step towards understanding the molecular mechanism of safener action in trees, homologous genes in hybrid poplar (Populus nigra x Populus maximowiczii) that were induced by a safener were identified. We here describe the identification of differentially expressed genes in Populus that are induced by Concep-III, a herbicide safener. Expressed sequence tags (ESTs) enriched for transcriptionally induced genes were isolated by suppressive subtractive hybridization (SSH). The SSH library cDNA inserts were used to construct a cDNA microarray for high-throughput validation of the up-regulated expression of safener-induced genes. Single-pass and partial sequences of 1,344 safener-induced ESTs were assembled into 418 singletons and 328 clusters, but the putative functions of almost 53% of the ESTs are not known. Genes encoding proteins involved in all three different phases of safener action, viz., oxidation, conjugation, and sequestration, were found in the SSH library. Almost 75% of genes that showed greater than 2-fold expression upon safener treatment were redundant in the SSH library. The expression pattern for selected genes was validated by reverse transcription-polymerase chain reaction. A few safener-induced genes that were not previously reported to be induced by safeners, but which may have a role in herbicide metabolism, were identified. The newly identified genes could have potential for application in genetic engineering of plants for herbicide detoxification and tolerance.

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

    SciTech Connect

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

    1989-11-01

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-K{sub 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.

  6. A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing

    PubMed Central

    Bent, Zachary W.; Poorey, Kunal; LaBauve, Annette E.; Hamblin, Rachelle; Williams, Kelly P.

    2016-01-01

    When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich for pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000–3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs. PMID:28002481

  7. A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing

    DOE PAGES

    Bent, Zachary W.; Poorey, Kunal; LaBauve, Annette E.; ...

    2016-12-21

    When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich formore » pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000–3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs.« less

  8. Diversity of microbial eukaryotes in sediment at a deep-sea methane cold seep: surveys of ribosomal DNA libraries from raw sediment samples and two enrichment cultures.

    PubMed

    Takishita, Kiyotaka; Yubuki, Naoji; Kakizoe, Natsuki; Inagaki, Yuji; Maruyama, Tadashi

    2007-07-01

    Recent culture-independent surveys of eukaryotic small-subunit ribosomal DNA (SSU rDNA) from many environments have unveiled unexpectedly high diversity of microbial eukaryotes (microeukaryotes) at various taxonomic levels. However, such surveys were most probably biased by various technical difficulties, resulting in underestimation of microeukaryotic diversity. In the present study on oxygen-depleted sediment from a deep-sea methane cold seep of Sagami Bay, Japan, we surveyed the diversity of eukaryotic rDNA in raw sediment samples and in two enrichment cultures. More than half of all clones recovered from the raw sediment samples were of the basidiomycetous fungus Cryptococcus curvatus. Among other clones, phylotypes of eukaryotic parasites, such as Apicomplexa, Ichthyosporea, and Phytomyxea, were identified. On the other hand, we observed a marked difference in phylotype composition in the enrichment samples. Several phylotypes belonging to heterotrophic stramenopiles were frequently found in one enrichment culture, while a phylotype of Excavata previously detected at a deep-sea hydrothermal vent dominated the other. We successfully established a clonal culture of this excavate flagellate. Since these phylotypes were not identified in the raw sediment samples, the approach incorporating a cultivation step successfully found at least a fraction of the "hidden" microeukaryotic diversity in the environment examined.

  9. cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA–protein fusions

    PubMed Central

    Yamaguchi, Junichi; Naimuddin, Mohammed; Biyani, Manish; Sasaki, Toru; Machida, Masayuki; Kubo, Tai; Funatsu, Takashi; Husimi, Yuzuru; Nemoto, Naoto

    2009-01-01

    We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA–protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a ‘ligation site’ for T4 RNA ligase, a ‘biotin site’ for solid-phase handling, a ‘reverse transcription primer site’ for the efficient and rapid conversion from an unstable mRNA–protein fusion (mRNA display) to a stable mRNA/cDNA–protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a ‘restriction enzyme site’ for the release of a complex from the solid support. This enables not only stabilizing mRNA–protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications. PMID:19528071

  10. Cloning and expression of a cDNA encoding a Vorticella convallaria spasmin: an EF-hand calcium-binding protein.

    PubMed

    Maciejewski, J J; Vacchiano, E J; McCutcheon, S M; Buhse, H E

    1999-01-01

    The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.

  11. Sequencing and analysis of 10,967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis reveals post-tetraploidization transcriptome remodeling.

    PubMed

    Morin, Ryan D; Chang, Elbert; Petrescu, Anca; Liao, Nancy; Griffith, Malachi; Chow, William; Kirkpatrick, Robert; Butterfield, Yaron S; Young, Alice C; Stott, Jeffrey; Barber, Sarah; Babakaiff, Ryan; Dickson, Mark C; Matsuo, Corey; Wong, David; Yang, George S; Smailus, Duane E; Wetherby, Keith D; Kwong, Peggy N; Grimwood, Jane; Brinkley, Charles P; Brown-John, Mabel; Reddix-Dugue, Natalie D; Mayo, Michael; Schmutz, Jeremy; Beland, Jaclyn; Park, Morgan; Gibson, Susan; Olson, Teika; Bouffard, Gerard G; Tsai, Miranda; Featherstone, Ruth; Chand, Steve; Siddiqui, Asim S; Jang, Wonhee; Lee, Ed; Klein, Steven L; Blakesley, Robert W; Zeeberg, Barry R; Narasimhan, Sudarshan; Weinstein, John N; Pennacchio, Christa Prange; Myers, Richard M; Green, Eric D; Wagner, Lukas; Gerhard, Daniela S; Marra, Marco A; Jones, Steven J M; Holt, Robert A

    2006-06-01

    Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection Initiative. Here we present 10,967 full ORF verified cDNA clones (8049 from X. laevis and 2918 from X. tropicalis) as a community resource. Because the genome of X. laevis, but not X. tropicalis, has undergone allotetraploidization, comparison of coding sequences from these two clawed (pipid) frogs provides a unique angle for exploring the molecular evolution of duplicate genes. Within our clone set, we have identified 445 gene trios, each comprised of an allotetraploidization-derived X. laevis gene pair and their shared X. tropicalis ortholog. Pairwise dN/dS, comparisons within trios show strong evidence for purifying selection acting on all three members. However, dN/dS ratios between X. laevis gene pairs are elevated relative to their X. tropicalis ortholog. This difference is highly significant and indicates an overall relaxation of selective pressures on duplicated gene pairs. We have found that the paralogs that have been lost since the tetraploidization event are enriched for several molecular functions, but have found no such enrichment in the extant paralogs. Approximately 14% of the paralogous pairs analyzed here also show differential expression indicative of subfunctionalization.

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

    PubMed

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

    2011-09-01

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

  13. Isolation of a cDNA clone for human threonyl-tRNA synthetase

    SciTech Connect

    Kontis, K.J.; Arfin, S.M.

    1989-05-01

    A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA /lambda/gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.

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

    PubMed

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

    1992-09-01

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

  15. Molecular cloning and characterization of pigeon (Columba liva) ubiquitin and ubiquitin-conjugating enzyme genes from pituitary gland library.

    PubMed

    Gao, Peng-fei; Cao, Guo-qing; Zhao, Hui-ting; Zhang, Gui-xian; Jiang, Yu-suo; Wang, Qin-de

    2009-01-01

    In the study of the regulation of incubation, broodiness and laying performance in pigeons (Columba liva), a cDNA library, which was enriched with full-length brooding-related genes, was constructed by SMART LD-PCR techniques using the pituitary glands of incubating White King pigeons. The titers of optimal primary libraries were 1.54x10(6) pfu/mL and 1.80x10(6) pfu/mL and the titers of amplified libraries were 1.89x10(8) pfu/mL and 2.32x10(9 )pfu/mL. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. A positive clone was sequenced and named ubiquitin based on the highly similar from other species. The fragment has the four initial codons of ATG, a termination codon of TAA and a signal sequence of AATAAA for adding the poly-A tail. The open reading frame of 918bp encodes 305 amino acids (NCBI accession number is EU981283). Recombinant pigeon ubiquitin protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pET28a(+) expression vector containing the DNA sequence encoding mature pigeon ubiquitin. The molecular weight of expressed protein is the same as predicted size of approximately 35kD. To improve the efficiency of cloning full-length cDNA, strategies of RACE combined with cDNA library were used. The length of pigeons ubiquitin-conjugating enzyme gene obtained was 1263 bp containing a complete open reading frame of 435 bp that encodes 144 aa (NCBI accession number is EU914824). The results of this study not only provide a starting point for further study of ubiquitin function in pigeon species, but also provide a starting point for investigating the brooding mechanisms of pigeons.

  16. Sequence of the cDNA encoding an actin homolog in the crayfish Procambarus clarkii.

    PubMed

    Kang, W K; Naya, Y

    1993-11-15

    A cDNA library was constructed by using mRNAs purified from crayfish (Procambarus clarkii) muscle. Using a homology search of the nucleotide (nt) sequences, a clone of the library was found to encode a protein homologous to actin (Act). The insert fragment of this cDNA clone was 1072 nt in length. The amino acid sequence deduced from the nt sequence showed significant similarity to Act of various organisms as follows: 88.1% to Drosophila melanogaster, 88.2% to silk worm, 87.3% to brine shrimp, 86.3% to rat, and 86.3% to human (% identity).

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

    PubMed

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

    2016-05-12

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

  18. Human liver apolipoprotein B-100 cDNA: complete nucleic acid and derived amino acid sequence.

    PubMed Central

    Law, S W; Grant, S M; Higuchi, K; Hospattankar, A; Lackner, K; Lee, N; Brewer, H B

    1986-01-01

    Human apolipoprotein B-100 (apoB-100), the ligand on low density lipoproteins that interacts with the low density lipoprotein receptor and initiates receptor-mediated endocytosis and low density lipoprotein catabolism, has been cloned, and the complete nucleic acid and derived amino acid sequences have been determined. ApoB-100 cDNAs were isolated from normal human liver cDNA libraries utilizing immunoscreening as well as filter hybridization with radiolabeled apoB-100 oligodeoxynucleotides. The apoB-100 mRNA is 14.1 kilobases long encoding a mature apoB-100 protein of 4536 amino acids with a calculated amino acid molecular weight of 512,723. ApoB-100 contains 20 potential glycosylation sites, and 12 of a total of 25 cysteine residues are located in the amino-terminal region of the apolipoprotein providing a potential globular structure of the amino terminus of the protein. ApoB-100 contains relatively few regions of amphipathic helices, but compared to other human apolipoproteins it is enriched in beta-structure. The delineation of the entire human apoB-100 sequence will now permit a detailed analysis of the conformation of the protein, the low density lipoprotein receptor binding domain(s), and the structural relationship between apoB-100 and apoB-48 and will provide the basis for the study of genetic defects in apoB-100 in patients with dyslipoproteinemias. PMID:3464946

  19. Losing Libraries, Saving Libraries

    ERIC Educational Resources Information Center

    Miller, Rebecca

    2010-01-01

    This summer, as public libraries continued to get budget hit after budget hit across the country, several readers asked for a comprehensive picture of the ravages of the recession on library service. In partnership with 2010 Movers & Shakers Laura Solomon and Mandy Knapp, Ohio librarians who bought the Losing Libraries domain name,…

  20. Losing Libraries, Saving Libraries

    ERIC Educational Resources Information Center

    Miller, Rebecca

    2010-01-01

    This summer, as public libraries continued to get budget hit after budget hit across the country, several readers asked for a comprehensive picture of the ravages of the recession on library service. In partnership with 2010 Movers & Shakers Laura Solomon and Mandy Knapp, Ohio librarians who bought the Losing Libraries domain name,…

  1. Targeting a Complex Transcriptome: The Construction of the Mouse Full-Length cDNA Encyclopedia

    PubMed Central

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

    2003-01-01

    We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3′-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5′ end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5′-end clusters identify regions that are potential promoters for 8637 known genes and 5′-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete. PMID:12819125

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

    PubMed

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

    2003-06-01

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

  3. Reexamining Content-Enriched Access: Its Effect on Usage and Discovery

    ERIC Educational Resources Information Center

    Tosaka, Yuji; Weng, Cathy

    2011-01-01

    Content-enriched metadata in bibliographic records is considered helpful to library users in identifying and selecting library materials for their needs. The paper presents a study, using circulation data from a medium-sized academic library, of the effect of content-enriched records on library materials usage. The study also examines OPAC search…

  4. Reexamining Content-Enriched Access: Its Effect on Usage and Discovery

    ERIC Educational Resources Information Center

    Tosaka, Yuji; Weng, Cathy

    2011-01-01

    Content-enriched metadata in bibliographic records is considered helpful to library users in identifying and selecting library materials for their needs. The paper presents a study, using circulation data from a medium-sized academic library, of the effect of content-enriched records on library materials usage. The study also examines OPAC search…

  5. Development of novel simple sequence repeat markers in bitter gourd (Momordica charantia L.) through enriched genomic libraries and their utilization in analysis of genetic diversity and cross-species transferability.

    PubMed

    Saxena, Swati; Singh, Archana; Archak, Sunil; Behera, Tushar K; John, Joseph K; Meshram, Sudhir U; Gaikwad, Ambika B

    2015-01-01

    Microsatellite or simple sequence repeat (SSR) markers are the preferred markers for genetic analyses of crop plants. The availability of a limited number of such markers in bitter gourd (Momordica charantia L.) necessitates the development and characterization of more SSR markers. These were developed from genomic libraries enriched for three dinucleotide, five trinucleotide, and two tetranucleotide core repeat motifs. Employing the strategy of polymerase chain reaction-based screening, the number of clones to be sequenced was reduced by 81 % and 93.7 % of the sequenced clones contained in microsatellite repeats. Unique primer-pairs were designed for 160 microsatellite loci, and amplicons of expected length were obtained for 151 loci (94.4 %). Evaluation of diversity in 54 bitter gourd accessions at 51 loci indicated that 20 % of the loci were polymorphic with the polymorphic information content values ranging from 0.13 to 0.77. Fifteen Indian varieties were clearly distinguished indicative of the usefulness of the developed markers. Markers at 40 loci (78.4 %) were transferable to six species, viz. Momordica cymbalaria, Momordica subangulata subsp. renigera, Momordica balsamina, Momordica dioca, Momordica cochinchinesis, and Momordica sahyadrica. The microsatellite markers reported will be useful in various genetic and molecular genetic studies in bitter gourd, a cucurbit of immense nutritive, medicinal, and economic importance.

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

    USDA-ARS?s Scientific Manuscript database

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

  7. A model organism for new gene discovery by cDNA sequencing

    SciTech Connect

    El-Saved, N.M.; Donelson, J.E.; Alarcon, C.M.

    1994-09-01

    One method of new gene discovery is single pass sequencing of cDNAs to identify expressed sequence tags (ESTs). Model organisms can have biological properties which makes their use advantageous over studies with humans. One such model organism with advantages for cDNA sequencing is the African trypanosome T. brucei rhodesiense. This organism has the same 40 nucleotide sequence (splice leader sequence) on the 5{prime} end of all mRNAs. We have constructed a 5{prime} cDNA library by priming off the splice leader sequence and have begun sequencing this cDNA library. To date, over nearly 500 such cDNA expressed sequence tags (ESTs) have been examined. Forty-three percent of the sequences sampled from the trypanosome cDNA library have significant similarities to sequences already in the protein and translated nucleic acid databases. Among these are cDNA sequences which encode previously reported T. brucej proteins such as the VSG, tubulin, calflagin, etc., and proteins previously identified in other trypanosomatids. Other cDNAs display significant similarities to genes in unrelated organisms encoding several ribosomal proteins, metabolic enzymes, GTP binding proteins, transcription factors, cyclophillin, nucleosomal histones, histone H1, and a macrophage stress protein, among others. The 57% of the cDNAs that are not similar to sequences currently in the databases likely encode both trypanosome-specific proteins and housekeeping proteins shared with other eukaryotes. These cDNA ESTs provide new avenues of research for exploring both the biochemistry and the genome organization of this parasite, as well as a resource for identifying the 5{prime} sequence of novel genes likely to have homology to genes expressed in other organisms.

  8. Cloning of cDNA encoding steroid 11. beta. -hydroxylase (P450c11)

    SciTech Connect

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-10-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11..beta..-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage lambda vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11..beta..-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia.

  9. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 12 figs.

  10. Brain cDNA clone for human cholinesterase

    SciTech Connect

    McTiernan, C.; Adkins, S.; Chatonnet, A.; Vaughan, T.A.; Bartels, C.F.; Kott, M.; Rosenberry, T.L.; La Du, B.N.; Lockridge, O.

    1987-10-01

    A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase. Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum. The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase rather than acetylcholinesterase. It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes coded for cholinesterase.

  11. CDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  12. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  13. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 11 figures.

  14. cDNA encoding a polypeptide including a hevein sequence

    SciTech Connect

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  15. Creating Library Spaces: Libraries 2040.

    ERIC Educational Resources Information Center

    Bruijnzeels, Rob

    This paper suggests that by 2004, the traditional public libraries will have ceased to exist and new, attractive future libraries will have taken their place. The Libraries 2040 project of the Netherlands is initiating seven different libraries of the future. The Brabant library is the "ultimate library of the future" for the Dutch…

  16. Gene enrichment in plant genomic shotgun libraries.

    PubMed

    Rabinowicz, Pablo D; McCombie, W Richard; Martienssen, Robert A

    2003-04-01

    The Arabidopsis genome (about 130 Mbp) has been completely sequenced; whereas a draft sequence of the rice genome (about 430 Mbp) is now available and the sequencing of this genome will be completed in the near future. The much larger genomes of several important crop species, such as wheat (about 16,000 Mbp) or maize (about 2500 Mbp), may not be fully sequenced with current technology. Instead, sequencing-analysis strategies are being developed to obtain sequencing and mapping information selectively for the genic fraction (gene space) of complex plant genomes.

  17. Methods for Selecting Phage Display Antibody Libraries.

    PubMed

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

    2016-01-01

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

  18. Project ENRICH.

    ERIC Educational Resources Information Center

    Gwaley, Elizabeth; And Others

    Project ENRICH was conceived in Beaver County, Pennsylvania, to: (1) identify preschool children with learning disabilities, and (2) to develop a program geared to the remediation of the learning disabilities within a school year, while allowing the child to be enrolled in a regular class situation for the following school year. Through…

  19. Job Enrichment

    ERIC Educational Resources Information Center

    Sanders, Rick

    1970-01-01

    Job enrichment means giving people more decision-making power, more responsibility, more grasp of the totality of the job, and a sense of their own importance in the company. This article presents evidence of the successful working of this approach (Donnelly Mirrors), and the lack of success with an opposing approach (General Motors). (NL)

  20. Cell cycle regulated synthesis of stable mouse thymidine kinase mRNA is mediated by a sequence within the cDNA.

    PubMed Central

    Hofbauer, R; Müllner, E; Seiser, C; Wintersberger, E

    1987-01-01

    The cDNA for mouse thymidine kinase (TK) was isolated from a cDNA library in lambda-gt11 and sequenced. It was used as a probe to follow the time course of TK mRNA expression in growth stimulated mouse fibroblasts. Linked to the HSV-TK promoter the cDNA was able to transform LTK-cells to the TK+ phenotype. The transformed cells expressed the TK mRNA and enzyme activity in a growth dependent fashion suggesting that the regulatory element is localized on the cDNA. Images PMID:3822814

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

    PubMed

    Shen

    1999-01-01

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

  2. Ezrin and osteonectin, two proteins associated with cell shape and growth, are enriched in the locus coeruleus.

    PubMed

    Bergson, C; Zhao, H; Saijoh, K; Duman, R S; Nestler, E J

    1993-02-01

    In an attempt to characterize proteins which are enriched or specifically expressed in the locus coeruleus (LC), a region of the brain which plays a critical role in opiate dependence and withdrawal, we have screened a bovine LC cDNA library with an LC minus cerebellum subtracted cDNA probe and isolated several (38) positively hybridizing clones. DNA sequence analysis revealed that two of the clones encoded ezrin and osteonectin, proteins normally associated with cell growth and morphology in peripheral tissues. Regional northern blots from bovine brain and in situ hybridization studies in rat show that ezrin is expressed at high levels in the LC with only low levels detectable in other brain regions. Osteonectin is also abundant in the LC, but in contrast to ezrin, is expressed at high levels in the dorsal raphe and substantia nigra. The results indicate that two proteins, ezrin and osteonectin, are highly enriched in the LC. This raises the possibility that these polypeptides, which play a role in the morphological response of some non-neuronal cell types to growth factors and to other intracellular signals, may also regulate these properties in noradrenergic and other selected neurons in the brain.

  3. Library Computing.

    ERIC Educational Resources Information Center

    Library Journal, 1985

    1985-01-01

    This special supplement to "Library Journal" and "School Library Journal" includes articles on technological dependency, promise of computers for reluctant readers, copyright and database downloading, access to neighborhood of Mister Rogers, library acquisitions, circulating personal computers, "microcomputeritis,"…

  4. Molecular cloning and nucleotide sequence of rat lingual lipase cDNA.

    PubMed Central

    Docherty, A J; Bodmer, M W; Angal, S; Verger, R; Riviere, C; Lowe, P A; Lyons, A; Emtage, J S; Harris, T J

    1985-01-01

    Purified rat lingual lipase (EC3113), a glycoprotein of approximate molecular weight 52,000, was used to generate polyclonal antibodies which were able to recognise the denatured and deglycosylated enzyme. These immunoglobulins were used to screen a cDNA library prepared from mRNA isolated from the serous glands of rat tongue cloned in E. coli expression vectors. An almost full length cDNA clone was isolated and the nucleotide and predicted amino acid sequence obtained. Comparison with the N-terminal amino acid sequence of the purified enzyme confirmed the identity of the cDNA and indicated that there was a hydrophobic signal sequence of 18 residues. The amino acid sequence of mature rat lingual lipase consists of 377 residues and shares little homology with porcine pancreatic lipase apart from a short region containing a serine residue at an analogous position to the ser 152 of the porcine enzyme. Images PMID:3839077

  5. Isolation and nucleotide sequence of a cDNA clone encoding rat mitochondrial malate dehydrogenase.

    PubMed Central

    Grant, P M; Tellam, J; May, V L; Strauss, A W

    1986-01-01

    We have determined the complete sequence of the rat mitochondrial malate dehydrogenase (mMDH) precursor derived from nucleotide sequence of the cDNA. A single synthetic oligodeoxynucleotide probe was used to screen a rat atrial cDNA library constructed in lambda gt10. A 1.2 kb full-length cDNA clone provided the first complete amino acid sequence of pre-mMDH. The 1014 nucleotide-long open reading frame encodes the 314 residue long mature mMDH protein and a 24 amino acid NH2-terminal extension which directs mitochondrial import and is cleaved from the precursor after import to generate mature mMDH. The amino acid composition of the transit peptide is polar and basic. The pre-mMDH transit peptide shows marked homology with those of two other enzymes targeted to the rat mitochondrial matrix. Images PMID:3755817

  6. Isolation and characterization of human defensin cDNA clones

    SciTech Connect

    Daher, K.A.; Lehrer, R.I.; Ganz, T.; Kronenberg, M. )

    1988-10-01

    Four clones that encode defensins, a group of microbicidal and cytotoxic peptides made by neutrophils, were isolated from an HL-60 human promyelocytic leukemia cDNA library. Analysis of these clones indicated that the defensins are made as precursor proteins, which must be cleaved to yield the mature peptides. Defensin mRNA was detected in normal bone marrow cells, but not in normal peripheral blood leukocytes. Defensin transcripts were also found in the peripheral leukocytes of some leukemia patients and in some lung and intestine tissues. Defensin mRNA content was augmented by treatment of HL-60 cells with dimethyl sulfoxide. These results define important aspects of the mechanism of synthesis and the tissue-specific expression of a major group of neutrophil granule proteins.

  7. Strategy for the design of custom cDNA microarrays.

    PubMed

    Lorenz, Matthias G O; Cortes, Lizette M; Lorenz, Juergen J; Liu, Edison T

    2003-06-01

    DNA microarrays are valuable but expensive tools for expression profiling of cells, tissues, and organs. The design of custom microarrays leads to cost reduction without necessarily compromising their biological value. Here we present a strategy for designing custom cDNA microarrays and constructed a microarray for mouse immunology research (ImmunoChip). The strategy used interrogates expressed sequence tag databases available in the public domain but overcomes many of the problems encountered. Immunologically relevant clusters were selected based on the expression of expressed sequence tags in relevant libraries. Selected clusters were organized in modules, and the best representative clones were identified. When tested, this microarray was found to have minimal clone identity errors or phage contamination and identified molecular signatures of lymphoid cell lines. Our proposed design of custom microarrays avoids probe redundancy, allows the organization of the chip to optimize chip production, and reduces microarray production costs. The strategy described is also useful for the design of oligonucleotide microarrays.

  8. Human somatostatin I: sequence of the cDNA.

    PubMed Central

    Shen, L P; Pictet, R L; Rutter, W J

    1982-01-01

    RNA has been isolated from a human pancreatic somatostatinoma and used to prepare a cDNA library. After prescreening, clones containing somatostatin I sequences were identified by hybridization with an anglerfish somatostatin I-cloned cDNA probe. From the nucleotide sequence of two of these clones, we have deduced an essentially full-length mRNA sequence, including the preprosomatostatin coding region, 105 nucleotides from the 5' untranslated region and the complete 150-nucleotide 3' untranslated region. The coding region predicts a 116-amino acid precursor protein (Mr, 12.727) that contains somatostatin-14 and -28 at its COOH terminus. The predicted amino acid sequence of human somatostatin-28 is identical to that of somatostatin-28 isolated from the porcine and ovine species. A comparison of the amino acid sequences of human and anglerfish preprosomatostatin I indicated that the COOH-terminal region encoding somatostatin-14 and the adjacent 6 amino acids are highly conserved, whereas the remainder of the molecule, including the signal peptide region, is more divergent. However, many of the amino acid differences found in the pro region of the human and anglerfish proteins are conservative changes. This suggests that the propeptides have a similar secondary structure, which in turn may imply a biological function for this region of the molecule. Images PMID:6126875

  9. cDNA sequences of two apolipoproteins from lamprey

    SciTech Connect

    Pontes, M.; Xu, X.; Graham, D.; Riley, M.; Doolittle, R.F.

    1987-03-24

    The messages for two small but abundant apolipoproteins found in lamprey blood plasma were cloned with the aid of oligonucleotide probes based on amino-terminal sequences. In both cases, numerous clones were identified in a lamprey liver cDNA library, consistent with the great abundance of these proteins in lamprey blood. One of the cDNAs (LAL1) has a coding region of 105 amino acids that corresponds to a 21-residue signal peptide, a putative 8-residue propeptide, and the 76-residue mature protein found in blood. The other cDNA (LAL2) codes for a total of 191 residues, the first 23 of which constitute a signal peptide. The two proteins, which occur in the high-density lipoprotein fraction of ultracentrifuged plasma, have amino acid compositions similar to those of apolipoproteins found in mammalian blood; computer analysis indicates that the sequences are largely helix-permissive. When the sequences were searched against an amino acid sequence data base, rat apolipoprotein IV was the best matching candidate in both cases. Although a reasonable alignment can be made with that sequence and LAL1, definitive assignment of the two lamprey proteins to typical mammalian classes cannot be made at this point.

  10. Expression cloning of the murine interferon gamma receptor cDNA.

    PubMed

    Munro, S; Maniatis, T

    1989-12-01

    A cDNA encoding a receptor for murine interferon gamma (IFN-gamma) was isolated from an expression library made from murine thymocytes. The clone was identified by transfecting the library into monkey COS cells and probing the transfected monolayer with radiolabeled murine IFN-gamma. Cells expressing the receptor were identified by autoradiography and plasmids encoding the receptor were directly rescued from those cells producing a positive signal. A partial cDNA so obtained was used to isolate a full-length cDNA from mouse L929 cells by conventional means. When this cDNA was expressed in COS cells it produced a specific binding site for murine IFN-gamma with an affinity constant similar to that of the receptor found on L929 cells. The predicted amino acid sequence of the murine IFN-gamma receptor shows homology to that previously reported for the human IFN-gamma receptor. However, although the two proteins are clearly related, they show less than 60% identity in both the putative extracellular domain and the intracellular domain.

  11. Isolation and characterization of a cDNA clone encoding wheat germ agglutinin

    SciTech Connect

    Raikhel, N.V.; Wilkins, T.A.

    1987-10-01

    Two sets of synthetic oligonucleotides coding for amino acids in the amino- and carboxyl-terminal portions of wheat germ agglutinin were synthesized and used as hybridization probes to screen cDNA libraries derived from developing embryos of tetraploid wheat. The nucleotide sequence for a cDNA clone recovered from the cDNA library was determined by dideoxynucleotide chain-termination sequencing in vector M13. The amino acid sequence deduced from the DNA sequence indicated that this cDNA clone (pNVR1) encodes isolectin 3 of wheat germ agglutinin. Comparison of the deduced amino acid sequence of clone pNVR1 with published sequences indicates isolectin 3 differs from isolectins 1 and 2 by 10 and 8 amino acid changes, respectively. In addition, the protein encoded by pNVR1 extends 15 amino acids beyond the carboxyl terminus of the published amino acid sequence for isolectins 1 and 2 and includes a potential site for N-linked glycosylation. Utilizing the insert of pNVR1 as a hybridization probe, the authors have demonstrated that the expression of genes for wheat germ agglutinin is modulated by exogenous abscisic acid. Striking homology is observed between wheat germ agglutinin and chitinase, both of which are proteins that bind chitin.

  12. Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material.

    PubMed

    Langevin, Stanley A; Bent, Zachary W; Solberg, Owen D; Curtis, Deanna J; Lane, Pamela D; Williams, Kelly P; Schoeniger, Joseph S; Sinha, Anupama; Lane, Todd W; Branda, Steven S

    2013-04-01

    Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

  13. Library 2000.

    ERIC Educational Resources Information Center

    Drake, Miriam A.

    In fall 1984, the Georgia Institute of Technology administration and library staff began planning for Library 2000, a project aimed at creating a showcase library to demonstrate the application of the latest information technology in an academic and research environment. The purposes of Library 2000 include: increasing awareness of students,…

  14. Library Buildings.

    ERIC Educational Resources Information Center

    Manley, Will; And Others

    1989-01-01

    The innovative designs of three libraries are described: the Tempe (Arizona) Public Library, which emphasizes services for children and students; an underground library at Park College, Missouri; and a public library located in the Vancouver (Washington) Mall. The fourth article describes the work going on to restore the Los Angeles (California)…

  15. Library Computing.

    ERIC Educational Resources Information Center

    Dayall, Susan A.; And Others

    1987-01-01

    Six articles on computers in libraries discuss training librarians and staff to use new software; appropriate technology; system upgrades of the Research Libraries Group's information system; pre-IBM PC microcomputers; multiuser systems for small to medium-sized libraries; and a library user's view of the traditional card catalog. (EM)

  16. Special Libraries.

    ERIC Educational Resources Information Center

    Foskett, D. J.

    The Special Library is distinguished from other libraries as being a library serving a particular group of readers, who have an existence as a group outside of their readership of the library, and whose members direct at least some of their activities towards a common purpose. Thus, the special librarian's first and major responsibility is to know…

  17. Isolation and mapping of human chromosome 21 cDNA: Progress in constructing a chromosome 21 expression map

    SciTech Connect

    Jan-Fang Cheng; Boyartchuk, V.; Zhu Y.

    1994-09-01

    We have isolated 175 cDNA clones from a fetal brain library by direct cDNA selection using genomic DNA isolated from pools of human chromosome 21 (HC21) cosmids. DNA sequences have revealed that 16 of these cDNA clones contain overlapping sequences. Of the other 159 cDNA sequences, 10 match previously identified HC21 genes, and 9 match previously determined cDNA sequences, including the Wilms tumor related transcript (QM), the human testican cDNA, the mammalian calponin cDNA, and 6 anonymous expressed sequence tags. All isolated cDNAs were hybridized to their corresponding cosmids, which suggests that they originated from HC21. We have localized 92 cDNA clones to previously reported HC21q YACs. The remaining unmapped cDNAs contain either sequences not included in the isolated HC21q YACs or sequences that hybridize to yeast DNA. The cDNAs not included in the YACs should be useful in isolating new YACs to bridge the gaps. PCR primers were derived from 4 novel cDNA sequences that had been mapped to the YACs in the suspected Down syndrome region and used in RT-PCR analysis. All 4 primer sequences amplified RNA fragments with the expected sizes, suggesting that these sequences could be used for expression analysis. The construction of a chromosome 21 cDNA map not only is important in the refinement of physical maps, but also will identify a set of genes in the disease regions for detailed characterization. 30 refs., 2 figs., 2 tabs.

  18. Horse cDNA clones encoding two MHC class I genes

    SciTech Connect

    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.

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

    SciTech Connect

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

    1991-01-15

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

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

    PubMed

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

    1993-04-15

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

  1. Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor.

    PubMed Central

    Weiser, W Y; Temple, P A; Witek-Giannotti, J S; Remold, H G; Clark, S C; David, J R

    1989-01-01

    A cDNA encoding a human macrophage migration inhibitory factor (MIF) was isolated, through functional expression cloning in COS-1 cells, from a cDNA library prepared from a lectin-stimulated T-cell hybridoma, T-CEMB. The 115-amino acid polypeptide encoded by the MIF cDNA (p7-1) was effectively released from the transfected COS-1 cells and yielded readily detectable MIF activity in the culture supernatant despite the apparent lack of a classical protein secretory sequence. Insertional mutational analysis and elution of MIF activity from polyacrylamide gel slices demonstrated that the Mr 12,000 protein with MIF activity released by the COS-1 cells is encoded by p7-1. The p7-1 cDNA hybridized with a 700-base mRNA expressed by Con-A-stimulated lymphocytes but not unstimulated lymphocytes. The availability of the MIF cDNA clone and recombinant MIF will facilitate the analysis of the role of this lymphokine in cell-mediated immunity, immunoregulation, and inflammation. Images PMID:2552447

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

    SciTech Connect

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

    1987-01-01

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

  3. cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity

    SciTech Connect

    Taira, M.; Yoshida, T.; Miyagawa, K.; Sakamoto, H.; Terada, M.; Sugimura, T.

    1987-05-01

    The hst gene was originally identified as a transforming gene in DNAs from human stomach cancers and from a noncancerous portion of stomach mucosa by DNA-mediated transfection assay using NIH3T3 cells. cDNA clones of hst were isolated from the cDNA library constructed from poly(A)/sup +/ RNA of a secondary transformant induced by the DNA from a stomach cancer. The sequence analysis of the hst cDNA revealed the presence of two open reading frames. When this cDNA was inserted into an expression vector containing the simian virus 40 promoter, it efficiently induced the transformation of NIH3T3 cells upon transfection. It was found that one of the reading frames, which coded for 206 amino acids, was responsible for the transforming activity.

  4. Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation

    PubMed Central

    Ji, Ping; Jaworski, Elizabeth; Xia, Zheng; Li, Wei

    2017-01-01

    Abstract The recent emergence of alternative polyadenylation (APA) as an engine driving transcriptomic diversity has stimulated the development of sequencing methodologies designed to assess genome-wide polyadenylation events. The goal of these approaches is to enrich, partition, capture and ultimately sequence poly(A) site junctions. However, these methods often require poly(A) enrichment, 3΄ linker ligation steps, and RNA fragmentation, which can necessitate higher levels of starting RNA, increase experimental error and potentially introduce bias. We recently reported a click-chemistry based method for generating RNAseq libraries called ‘ClickSeq’. Here, we adapt this method to direct the cDNA synthesis specifically toward the 3΄UTR/poly(A) tail junction of cellular RNA. With this novel approach, we demonstrate sensitive and specific enrichment for poly(A) site junctions without the need for complex sample preparation, fragmentation or purification. Poly(A)-ClickSeq (PAC-seq) is therefore a simple procedure that generates high-quality RNA-seq poly(A) libraries. As a proof-of-principle, we utilized PAC-seq to explore the poly(A) landscape of both human and Drosophila cells in culture and observed outstanding overlap with existing poly(A) databases and also identified previously unannotated poly(A) sites. Moreover, we utilize PAC-seq to quantify and analyze APA events regulated by CFIm25 illustrating how this technology can be harnessed to identify alternatively polyadenylated RNA. PMID:28449108

  5. Cloning of the rat ErbB3 cDNA and characterization of the recombinant protein.

    PubMed

    Hellyer, N J; Kim, H H; Greaves, C H; Sierke, S L; Koland, J G

    1995-11-20

    Three cDNA fragments that encoded all but the extreme N terminus of the rat ErbB3 protein were cloned by low-stringency screening of a rat liver cDNA library with a human ERBB3 probe. The remaining 5'-end of the cDNA was generated by a reverse transcription-polymerase chain reaction method, and a single full-length rat ErbB3 cDNA was assembled. A comparison of the deduced amino acid (aa) sequences of human and rat ErbB3 was made, and the effects of certain aa substitutions in the putative protein tyrosine kinase domain were considered. The rat ErbB3 cDNA was subsequently expressed in cultured NIH-3T3 mouse fibroblasts, in which a high level of approx. 180-kDa recombinant ErbB3 (re-ErbB3) was generated. The rat re-ErbB3 produced in transfected fibroblasts was responsive to the polypeptide, heregulin, a known ligand for ErbB3. Challenge of transfected fibroblasts with heregulin stimulated the phosphorylation of rat re-ErbB3 on Tyr residues and promoted its association with the p85 subunit of phosphatidylinositol 3-kinase. Together, these results indicate that a fully functional rat ErbB3 cDNA has been isolated, and that fibroblast cells expressing this cDNA will be suitable for investigations of the signal transduction mechanism of ErbB3.

  6. Enchytraeus albidus Microarray: Enrichment, Design, Annotation and Database (EnchyBASE)

    PubMed Central

    Novais, Sara C.; Arrais, Joel; Lopes, Pedro; Vandenbrouck, Tine; De Coen, Wim; Roelofs, Dick; Soares, Amadeu M. V. M.; Amorim, Mónica J. B.

    2012-01-01

    Enchytraeus albidus (Oligochaeta) is an ecologically relevant species used as standard test organisms for risk assessment. Effects of stressors in this species are commonly determined at the population level using reproduction and survival as endpoints. The assessment of transcriptomic responses can be very useful e.g. to understand underlying mechanisms of toxicity with gene expression fingerprinting. In the present paper the following is being addressed: 1) development of suppressive subtractive hybridization (SSH) libraries enriched for differentially expressed genes after metal and pesticide exposures; 2) sequencing and characterization of all generated cDNA inserts; 3) development of a publicly available genomic database on E. albidus. A total of 2100 Expressed Sequence Tags (ESTs) were isolated, sequenced and assembled into 1124 clusters (947 singletons and 177 contigs). From these sequences, 41% matched known proteins in GenBank (BLASTX, e-value≤10-5) and 37% had at least one Gene Ontology (GO) term assigned. In total, 5.5% of the sequences were assigned to a metabolic pathway, based on KEGG. With this new sequencing information, an Agilent custom oligonucleotide microarray was designed, representing a potential tool for transcriptomic studies. EnchyBASE (http://bioinformatics.ua.pt/enchybase/) was developed as a web freely available database containing genomic information on E. albidus and will be further extended in the near future for other enchytraeid species. The database so far includes all ESTs generated for E. albidus from three cDNA libraries. This information can be downloaded and applied in functional genomics and transcription studies. PMID:22558086

  7. Special Libraries

    ERIC Educational Resources Information Center

    Lavendel, Giuliana

    1977-01-01

    Discusses problems involved in maintaining special scientific or engineering libraries, including budget problems, remote storage locations, rental computer retrieval systems, protecting trade secrets, and establishing a magnetic tape library. (MLH)

  8. Library Buildings

    ERIC Educational Resources Information Center

    Allen, Walter C.

    1976-01-01

    Examines a century of library architecture in relation to the changing perceptions of library functions, the development of building techniques and materials, fluctuating esthetic fashions and sometimes wildly erratic economic climates. (Author)

  9. Induction of mosquitocidal activity in mice immunized with Anopheles gambiae midgut cDNA.

    PubMed

    Foy, B D; Magalhaes, T; Injera, W E; Sutherland, I; Devenport, M; Thanawastien, A; Ripley, D; Cárdenas-Freytag, L; Beier, J C

    2003-04-01

    Vaccines that induce mosquito-killing (mosquitocidal) activity could substantially reduce the transmission of certain mosquito-borne diseases, especially vaccines against African malaria vectors, such as the mosquito Anopheles gambiae. To generate and characterize antimosquito immunity we immunized groups of mice with two individual A. gambiae midgut cDNAs, Ag-Aper1 (a secreted peritrophic matrix protein) and AgMuc1 (a midgut-bound mucin), and an A. gambiae midgut cDNA library from blood-fed mosquitoes. We observed significantly increased mortality among mosquitoes that fed on either the AgMuc1- or the cDNA library-immunized mice compared to that of controls, but no differences were observed among those fed on Ag-Aper1-immunized mice. Analysis of the humoral and cellular immune responses from mice showed that the induced mosquitocidal effect was associated with immune profiles characterized by elevated tumor necrosis factor alpha and gamma interferon cytokine levels and very low antibody titers. Furthermore, an additional immunization of cDNA library-immunized mice with midgut protein shifted immunity toward a Th2-type immune response, characterized by elevated antibody titers and high interleukin-5 and interleukin-10 cytokine levels; importantly, mosquitoes feeding on these mice exhibited no undue mortality. Finally, when immune sera was ingested by mosquitoes through a membrane feeder, no effect on mosquito mortality was observed, indicating that serum factors alone were not responsible for the mosquitocidal effect. Our results demonstrate that mosquitocidal immunity in mice can be consistently generated by midgut cDNA immunization and suggest this cDNA-induced mosquitocidal immunity is cell mediated.

  10. Isolation and characterization of the murine alpha-L-iduronidase cDNA

    SciTech Connect

    Clarke, L.A.; Zhang, H. Nasir, J.

    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.

  11. Library Skills.

    ERIC Educational Resources Information Center

    Paul, Karin; Kuhlthau, Carol C.; Branch, Jennifer L.; Solowan, Diane Galloway; Case, Roland; Abilock, Debbie; Eisenberg, Michael B.; Koechlin, Carol; Zwaan, Sandi; Hughes, Sandra; Low, Ann; Litch, Margaret; Lowry, Cindy; Irvine, Linda; Stimson, Margaret; Schlarb, Irene; Wilson, Janet; Warriner, Emily; Parsons, Les; Luongo-Orlando, Katherine; Hamilton, Donald

    2003-01-01

    Includes 19 articles that address issues related to library skills and Canadian school libraries. Topics include information literacy; inquiry learning; critical thinking and electronic research; collaborative inquiry; information skills and the Big 6 approach to problem solving; student use of online databases; library skills; Internet accuracy;…

  12. Library Skills.

    ERIC Educational Resources Information Center

    Paul, Karin; Kuhlthau, Carol C.; Branch, Jennifer L.; Solowan, Diane Galloway; Case, Roland; Abilock, Debbie; Eisenberg, Michael B.; Koechlin, Carol; Zwaan, Sandi; Hughes, Sandra; Low, Ann; Litch, Margaret; Lowry, Cindy; Irvine, Linda; Stimson, Margaret; Schlarb, Irene; Wilson, Janet; Warriner, Emily; Parsons, Les; Luongo-Orlando, Katherine; Hamilton, Donald

    2003-01-01

    Includes 19 articles that address issues related to library skills and Canadian school libraries. Topics include information literacy; inquiry learning; critical thinking and electronic research; collaborative inquiry; information skills and the Big 6 approach to problem solving; student use of online databases; library skills; Internet accuracy;…

  13. Library Computing.

    ERIC Educational Resources Information Center

    Goodgion, Laurel; And Others

    1986-01-01

    Eight articles in special supplement to "Library Journal" and "School Library Journal" cover a computer program called "Byte into Books"; microcomputers and the small library; creating databases with students; online searching with a microcomputer; quality automation software; Meckler Publishing Company's…

  14. Library Computing

    ERIC Educational Resources Information Center

    Library Computing, 1985

    1985-01-01

    Special supplement to "Library Journal" and "School Library Journal" covers topics of interest to school, public, academic, and special libraries planning for automation: microcomputer use, readings in automation, online searching, databases of microcomputer software, public access to microcomputers, circulation, creating a…

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

    SciTech Connect

    Hassett, C.; Richter, R.J.; Humbert, R.; Omiecinski, C.J.; Furlong, C.E. ); Chapline, C.; Crabb, J.W. )

    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.

  16. Isolation and sequencing of the cDNA of a novel cytochrome P450 from rat oesophagus.

    PubMed

    Brookman-Amissah, N; Mackay, A G; Swann, P F

    2001-01-01

    RT-PCR was used to find whether cytochromes P450 of the 2A, 2B and 2E sub-families are expressed in the rat oesophagus. This showed that this tissue expresses a previously unknown member of the CYP2B sub-family, now designated CYP2B21. Using a combination of 5'- and 3'-RACE (rapid amplification of cDNA ends) and library screening, the cDNA was amplified and sequenced. The cDNA sequence (GenBank accession no. AF159245) covers the whole of the coding region and the whole of the 3'-untranslated region (UTR), but only 17 nt of the 5'-UTR. The DNA sequence has strong similarity to those of CYP2B1 and CYP2B2, with the derived amino acid sequence being 84 and 83% identical, respectively. The ease with which this cDNA was found in the cDNA library suggests that CYP2B21 is a major P450 of the oesophagus. The catalytic activity of this new CYP2B is not yet known, but as previous authors have reported that other members of this sub-family (CYP2B1 or 2B2) metabolize the selective oesophageal carcinogen N:-nitrosomethylbutylamine with the chemical selectivity necessary for carcinogenesis, i.e. they preferentially hydroxylate the alpha-carbon of the butyl chain, this new CYP2B may be the nitrosamine-activating enzyme of the oesophagus.

  17. Rapid plasmid library screening using RecA-coated biotinylated probes.

    PubMed

    Rigas, B; Welcher, A A; Ward, D C; Weissman, S M

    1986-12-01

    A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.

  18. Isolation and preliminary characterisation of cDNA clones representing mRNAs associated with tumour progression and metastasis in colorectal cancer.

    PubMed Central

    Elvin, P.; Kerr, I. B.; McArdle, C. S.; Birnie, G. D.

    1988-01-01

    We have constructed cDNA libraries from the poly(A)+ RNA of normal colonic mucosa and a liver metastasis from a colonic adenocarcinoma. Differential screening of these libraries using 32P-labelled cDNAs transcribed from poly(A)+ RNAs isolated from specimens of four normal colonic mucosae, five adenocarcinomas, and three liver metastases by Grunstein-Hogness and dot-blot hybridization has identified a number of recombinant cDNA clones homologous to mRNAs that appear to differ significantly in abundance between normal and neoplastic colon and metastases. These cDNA clones, and others identified in the libraries, may be of considerable importance both as diagnostic tools and in defining the phenotypic changes associated with tumour progression and metastasis. Images Figure 2 Figure 3 Figure 4 PMID:2450556

  19. Molecular cloning and chromosomal localization of a novel human tracheo-bronchial mucin cDNA containing tandemly repeated sequences of 48 base pairs.

    PubMed

    Porchet, N; Nguyen, V C; Dufosse, J; Audie, J P; Guyonnet-Duperat, V; Gross, M S; Denis, C; Degand, P; Bernheim, A; Aubert, J P

    1991-03-15

    A lambda gt11 cDNA library constructed from human tracheo-bronchial mucosa was screened with a polyclonal antiserum raised to chemically deglycosylated pronase glycopeptides from human bronchial mucins. Out of 20 positives clones, one partial cDNA clone was isolated and allowed to map a novel human tracheo-bronchial mucin gene. It contains 48 nucleotide tandem repeats quite perfectly identical which encodes a protein containing about 50% of hydroxy amino-acids. This clone hybridized to polydisperse messages produced by human tracheo-bronchial and human colonic mucosae. The gene (proposed name MUC 4) from which cDNA is derived maps to chromosome 3.

  20. Chromosomal localization of a human cDNA containing a DIDS binding domain and demonstrating high homology to yeast omnipotent suppressor 45.

    PubMed

    Grenett, H E; Eipers, P G; Kidd, V J; Bounelis, P; Fuller, G M

    1992-01-01

    We recently have identified a full-length cDNA (TB3-1) from a human adenocarcinoma cell line T84 cDNA library that encodes a 47.8-kDa protein. TB3-1 shares identity with the putative yeast translation termination factor omnipotent suppressor 45. Using human-mouse somatic cell panel analysis, a family of sequences with high homology to the TB3-1 cDNA clone were localized to human chromosomes 5, 6, 7, and X. Southern analysis of a panel of mammalian and chicken genomic DNA demonstrates that TB3-1 is well conserved in higher vertebrates.

  1. Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

    SciTech Connect

    Chen, J.; Varner, J.E.

    1985-07-01

    Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.

  2. Automated Workflow for Preparation of cDNA for Cap Analysis of Gene Expression on a Single Molecule Sequencer

    PubMed Central

    Nagao-Sato, Sayaka; Saijo, Eri; Lassmann, Timo; Kanamori-Katayama, Mutsumi; Kaiho, Ai; Lizio, Marina; Kawaji, Hideya; Carninci, Piero; Forrest, Alistair R. R.; Hayashizaki, Yoshihide

    2012-01-01

    Background Cap analysis of gene expression (CAGE) is a 5′ sequence tag technology to globally determine transcriptional starting sites in the genome and their expression levels and has most recently been adapted to the HeliScope single molecule sequencer. Despite significant simplifications in the CAGE protocol, it has until now been a labour intensive protocol. Methodology In this study we set out to adapt the protocol to a robotic workflow, which would increase throughput and reduce handling. The automated CAGE cDNA preparation system we present here can prepare 96 ‘HeliScope ready’ CAGE cDNA libraries in 8 days, as opposed to 6 weeks by a manual operator.We compare the results obtained using the same RNA in manual libraries and across multiple automation batches to assess reproducibility. Conclusions We show that the sequencing was highly reproducible and comparable to manual libraries with an 8 fold increase in productivity. The automated CAGE cDNA preparation system can prepare 96 CAGE sequencing samples simultaneously. Finally we discuss how the system could be used for CAGE on Illumina/SOLiD platforms, RNA-seq and full-length cDNA generation. PMID:22303458

  3. Assembly, Verification, and Initial Annotation of the NIA Mouse 7.4K cDNA Clone Set

    PubMed Central

    VanBuren, Vincent; Piao, Yulan; Dudekula, Dawood B.; Qian, Yong; Carter, Mark G.; Martin, Patrick R.; Stagg, Carole A.; Bassey, Uwem C.; Aiba, Kazuhiro; Hamatani, Toshio; Kargul, George J.; Luo, Amber G.; Kelso, Janet; Hide, Winston; Ko, Minoru S.H.

    2002-01-01

    A set of 7407 cDNA clones (NIA mouse 7.4K) was assembled from >20 cDNA libraries constructed mainly from early mouse embryos, including several stem cell libraries. The clone set was assembled from embryonic and newborn organ libraries consisting of ∼120,000 cDNA clones, which were initially re-arrayed into a set of ∼11,000 unique cDNA clones. A set of tubes was constructed from the racks in this set to prevent contamination and potential mishandling errors in all further re-arrays. Sequences from this set (11K) were analyzed further for quality and clone identity, and high-quality clones with verified identity were re-arrayed into the final set (7.4K). The set is freely available, and a corresponding database was built to provide comprehensive annotation for those clones with known identity or homology, and has been made available through an extensive Web site that includes many link-outs to external databases and analysis servers. [The sequence data from this study have been submitted to GenBank under accession nos. BQ550036–BQ563104.] PMID:12466305

  4. Large Scale Full-Length cDNA Sequencing Reveals a Unique Genomic Landscape in a Lepidopteran Model Insect, Bombyx mori

    PubMed Central

    Suetsugu, Yoshitaka; Futahashi, Ryo; Kanamori, Hiroyuki; Kadono-Okuda, Keiko; Sasanuma, Shun-ichi; Narukawa, Junko; Ajimura, Masahiro; Jouraku, Akiya; Namiki, Nobukazu; Shimomura, Michihiko; Sezutsu, Hideki; Osanai-Futahashi, Mizuko; Suzuki, Masataka G; Daimon, Takaaki; Shinoda, Tetsuro; Taniai, Kiyoko; Asaoka, Kiyoshi; Niwa, Ryusuke; Kawaoka, Shinpei; Katsuma, Susumu; Tamura, Toshiki; Noda, Hiroaki; Kasahara, Masahiro; Sugano, Sumio; Suzuki, Yutaka; Fujiwara, Haruhiko; Kataoka, Hiroshi; Arunkumar, Kallare P.; Tomar, Archana; Nagaraju, Javaregowda; Goldsmith, Marian R.; Feng, Qili; Xia, Qingyou; Yamamoto, Kimiko; Shimada, Toru; Mita, Kazuei

    2013-01-01

    The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes. PMID:23821615

  5. Large scale full-length cDNA sequencing reveals a unique genomic landscape in a lepidopteran model insect, Bombyx mori.

    PubMed

    Suetsugu, Yoshitaka; Futahashi, Ryo; Kanamori, Hiroyuki; Kadono-Okuda, Keiko; Sasanuma, Shun-ichi; Narukawa, Junko; Ajimura, Masahiro; Jouraku, Akiya; Namiki, Nobukazu; Shimomura, Michihiko; Sezutsu, Hideki; Osanai-Futahashi, Mizuko; Suzuki, Masataka G; Daimon, Takaaki; Shinoda, Tetsuro; Taniai, Kiyoko; Asaoka, Kiyoshi; Niwa, Ryusuke; Kawaoka, Shinpei; Katsuma, Susumu; Tamura, Toshiki; Noda, Hiroaki; Kasahara, Masahiro; Sugano, Sumio; Suzuki, Yutaka; Fujiwara, Haruhiko; Kataoka, Hiroshi; Arunkumar, Kallare P; Tomar, Archana; Nagaraju, Javaregowda; Goldsmith, Marian R; Feng, Qili; Xia, Qingyou; Yamamoto, Kimiko; Shimada, Toru; Mita, Kazuei

    2013-09-04

    The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.

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

    SciTech Connect

    Chen, H.; Leipprandt, J.R.; Traviss, C.E.

    1994-09-01

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

  7. A cDNA clone encoding human cAMP-dependent protein kinase catalytic subunit C. alpha

    SciTech Connect

    Maldonado, F.; Hanks, S.K. )

    1988-08-25

    The authors have determined the nucleotide sequence from both complementary strands of a human cDNA coding for cAMP-dependent protein kinase catalytic subunit type {alpha} (cAPK-C{alpha}). This cDNA was one of many protein kinase cDNAs isolated from a HeLa cell library by screening with oligonucleotide probes designed to recognize target sequences encoding highly conserved segments within the catalytic domains. The deduced human cAPK-C{alpha} amino acid sequence of 350 residues differs from the bovine and murine sequences at 3 and 7 positions, respectively.

  8. Prokaryotic suppression subtractive hybridization PCR cDNA subtraction, a targeted method to identify differentially expressed genes.

    PubMed

    De Long, Susan K; Kinney, Kerry A; Kirisits, Mary Jo

    2008-01-01

    Molecular biology tools can be used to monitor and optimize biological treatment systems, but the application of nucleic acid-based tools has been hindered by the lack of available sequences for environmentally relevant biodegradation genes. The objective of our work was to extend an existing molecular method for eukaryotes to prokaryotes, allowing us to rapidly identify differentially expressed genes for subsequent sequencing. Suppression subtractive hybridization (SSH) PCR cDNA subtraction is a technique that can be used to identify genes that are expressed under specific conditions (e.g., growth on a given pollutant). While excellent methods for eukaryotic SSH PCR cDNA subtraction are available, to our knowledge, no methods previously existed for prokaryotes. This work describes our methodology for prokaryotic SSH PCR cDNA subtraction, which we validated using a model system: Pseudomonas putida mt-2 degrading toluene. cDNA from P. putida mt-2 grown on toluene (model pollutant) or acetate (control substrate) was subjected to our prokaryotic SSH PCR cDNA subtraction protocol to generate subtraction clone libraries. Over 90% of the sequenced clones contained gene fragments encoding toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were sequenced. Based on these results, prokaryotic SSH PCR cDNA subtraction shows promise as a targeted method for gene identification.

  9. The National Cryptologic Museum Library

    DTIC Science & Technology

    2010-09-01

    Intelligence Service, books were collected wherever they could be found regardless of age or language . Thus the library has many rare and hard-to-find items...enriched last spring by the aquisition of the personal collection of the late Louis Kruh, a nationally known collector and colleague of Kahn. Among the

  10. Cloning and characterization of a cDNA encoding an 18.0-kDa class-I low-molecular-weight heat-shock protein from rice.

    PubMed

    Lee, Y L; Chang, P F; Yeh, K W; Jinn, T L; Kung, C C; Lin, W C; Chen, Y M; Lin, C Y

    1995-11-20

    A novel cDNA clone, Oshp18.0 cDNA, encoding a rice (Oryza sativa L. cv. Tainong 67) 18.0-kDa heat-shock protein (HSP), was isolated from a cDNA library of heat-shocked rice seedlings by use of the rice HSP cDNA, Oshsp17.3 cDNA, as a probe. The sequence showed that Oshsp18.0 cDNA contains a 749-bp insert encoding an ORF of 160 amino acids, with a predicted molecular mass of 18.0 kDa and a pI of 7.3. Sequence comparison reveals that Oshsp18.0 cDNA is highly homologous to other low-molecular-weight (LMW) HSP cDNAs. Also, the results of hybrid-selected in vitro translation clearly establish that Oshsp18.0 cDNA is the rice 18.0-kDa LMW HSP-encoding cDNA clone. The recombinant Oshsp18.0 fusion protein produced in Escherichia coli was of the size predicted, and was recognized by the class-I rice 16.9-kDa HSP antiserum. The results suggest that Oshsp18.0 cDNA is an 18.0-kDa class-I LMW HSP- encoding cDNA clone from rice.

  11. Genomic and cDNA actin sequences from a virulent strain of Entamoeba histolytica.

    PubMed Central

    Edman, U; Meza, I; Agabian, N

    1987-01-01

    Invasiveness of Entamoeba histolytica strains that cause acute amoebiasis is characterized by aggressive behavior associated with cell motility and actin function. Analysis of actin genes from E. histolytica was initiated by devising methods for the isolation of biologically active nucleic acids, which allowed the preparation of cDNA and genomic DNA libraries. E. histolytica actin-encoding cDNAs and genomic clones have been isolated from libraries prepared from the virulent HM1:IMSS strain using a heterologous actin probe. Nucleotide sequence analysis of three independent cDNA clones and one genomic clone reveals a highly unusual codon bias and the absence of intervening sequences in E. histolytica actin. The coding sequence of the genomic clone is identical to that of two of the three cDNA clones. These represent at least two distinct mRNAs differing only by five silent changes in the protein coding sequence. Multiple genomic copies of the actin gene can be detected by Southern hybridization. E. histolytica actin exhibits a higher degree of homology to cytoplasmic than to muscle actin. Although the protein has been shown not to bind DNase I, the inferred amino acid sequence indicates conservation of all residues implied to participate in this binding. Images PMID:2883657

  12. Identification of a human cDNA with high homology to yeast omnipotent suppressor 45.

    PubMed

    Grenett, H E; Bounelis, P; Fuller, G M

    1992-01-15

    Omnipotent suppression is a well-established phenomenon in yeast and bacteria in which nonsense mutations are misread. Wild-type (wt) suppressors are presumed to be involved in ensuring the fidelity of translation. We report a human homolog to wt yeast omnipotent suppressor 45 which shares 63% identity at the nucleotide level in the area of open reading frame (ORF) and 73% similarity at the amino acid (aa) level. The aa sequence of the human protein was deduced from a 2.3-kb cDNA (TB3-1) isolated from an adenocarcinoma T84 cell line cDNA library. The cDNA contains an ORF of 1284 bp which encodes a 47.8-kDa protein. Two transcripts for the clone were identified (2.6 and 4.0 kb) in a variety of human cell types. The strong structural similarity to yeast omnipotent suppressor 45, and its widespread expression suggest that this cDNA may play a role in the accurate recognition of nonsense codons in mammalian cells.

  13. Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning

    SciTech Connect

    Pierce, R.A.; Deak, S.B.; Stolle, C.A.; Boyd, C.D. )

    1990-10-01

    A {lambda}gt11 library constructed from poly(A{plus}) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first, screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3{prime}-untranslated region. A subsequent screen using a 5{prime} rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin CDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.

  14. Cloning and characterization of the lectin cDNA clones from onion, shallot and leek.

    PubMed

    Van Damme, E J; Smeets, K; Engelborghs, I; Aelbers, H; Balzarini, J; Pusztai, A; van Leuven, F; Goldstein, I J; Peumans, W J

    1993-10-01

    Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells. cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level. Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5-13 kDa) after post-translational modifications. Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.

  15. Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes

    SciTech Connect

    Chung, T.D.Y.; Drake, F.H.; Tan, K.B.; Per, S.R.; Crooke, S.T.; Mirabelli, C.K. )

    1989-12-01

    Several DNA topoisomerase II partial cDNA clones obtained from a human Raji-HN2 cDNA library were sequenced and two classes of nucleotide sequences were found. One member of the first class, SP1, was identical to an internal fragment of human HeLa cell Topo II cDNA described earlier. A member of the second class, SP11, shared extensive nucleotide (75%) and predicted peptide (92%) sequence similarities with the first two-thirds of HeLa Topo II. Each class of cDNAs hybridized to unique, nonoverlapping restriction enzyme fragments of genomic DNA from several human cell lines. Synthetic 24-mer oligonucleotide probes specific for each cDNA class hybridized to 6.5-kilobase mRNAs; furthermore, hybridization of probe specific for one class was not blocked by probe specific for the other. Antibodies raised against a synthetic SP1-encoded dodecapeptide specifically recognized the 170-kDa form of Topo II, while antibodies raised against the corresponding SP11-encoded dodecapeptide, or a second unique SP11-encoded tridecapeptide, selectively recognized the 180-kDa form of Topo II. These data provide genetic and immunochemical evidence for two Topo II isozymes.

  16. Isolation and characterization of a cDNA for human MEP/Cathepsin L

    SciTech Connect

    Gal, S.; Gottesman, M.M.

    1987-05-01

    The major excreted acid protease from malignantly transformed mouse cells (MEP) has similar enzymatic properties to Cathepsin L. They have cloned and sequenced the mouse cDNA for this protein. In this work they report the isolation and characterization of a cDNA for human MEP. A 1.6 kb cDNA from an Okayama-Berg library made from mRNA from SV-40 transformed human fibroblasts was isolated by cross-hybridization at low stringency to the mouse cDNA. Sequence analysis shows that the cloned DNA is more than 70% homologous to the mouse DNA within the protein coding region, non-coding 3' and 5' regions being less homologous than this. The deduced amino acid sequence from the single open reading frame is 98% homologous with the partial protein sequence published for human Cathepsin L and 70% homologous to mouse MEP. Northern blots show that mouse and human cells make a similar size message, about 1.8 kb. A Northern blot of human tissues shows the highest levels of MEP/Cathepsin L mRNA in liver, stomach, ovary and small bowel, with lower levels in other tissues. The isolation of this human clone for MEP/Cathepsin L will allow them to determine whether expression of this protease is correlated with tumorigenicity in human cells as it is in the mouse system.

  17. Simple, directional cDNA cloning for in situ transcript hybridization screens.

    PubMed

    Tamme, R; Mills, K; Rainbird, B; Nornes, S; Lardelli, M

    2001-10-01

    Here, we describe a suppression PCR-based method for directional cloning of randomly primed cDNAs from small quantities of tissue. Synthesis of the first cDNA strand is conducted on oligonucleotide-coated magnetic beads. Synthesis of the second strand is accomplished using nonspecifically primed suppression PCR. This method is used to synthesize a cDNA library from zebrafish embryos at 6-9 h after fertilization. The sequencing of the clones and their use in an in situ hybridization screen to detect restricted patterns of gene transcription in zebrafish embryos showed that this method allows the rapid identification of genes that are important for development and genes that are expressed at levels undetectable by whole-mount in situ transcript hybridization. The random priming of cDNA alleviates the problems encountered in the identification of zebrafish genes from poly(dT)-primed cDNA clones caused by the long 3' UTRs frequently found in transcripts from this organism.

  18. Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

    PubMed Central

    Knutzon, Deborah S.; Bleibaum, Janice L.; Nelsen, Janet; Kridl, Jean C.; Thompson, Gregory A.

    1992-01-01

    Oleoyl-acyl carrier protein (18:1-ACP) thioesterase has been partially purified from developing safflower (Carthamus tinctorius) seeds. Protein species with molecular masses of 34 and 40 kD associated with thioesterase activity were identified and partially sequenced. Analysis of amino-terminal and internal cyanogen bromide peptide sequences revealed no differences in the primary structure of the two species. Amino acid sequence was used to design degenerate oligonucleotides for primers in a polymerase chain reaction (PCR) using safflower embryo cDNA as a template. A 380-base pair PCR product was used to isolate two classes of cDNA clones, designated 2-1 and 5-2, from the embryo cDNA library. Clone 2-1 encodes a 389-amino acid protein including a 60-amino acid transit peptide, and contains all of the protein sequence determined from the 34- and 40-kD proteins. Clone 5-2 encodes a 385-amino acid protein with 80% identity to that encoded by 2-1. Expression of the two safflower cDNA clones in Escherichia coli resulted in a 50- to 100-fold increase in the level of 18:1-ACP thioesterase activity. Both thioesterases are most active on 18:1-ACP; however, the enzyme encoded by 5-2 shows less discrimination against saturated 16- and 18-carbon acyl-ACP substrates. Images Figure 1 Figure 4 PMID:16653193

  19. Aspartylglucosaminuria: cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease.

    PubMed Central

    Ikonen, E; Baumann, M; Grön, K; Syvänen, A C; Enomaa, N; Halila, R; Aula, P; Peltonen, L

    1991-01-01

    We have isolated a 2.1 kb cDNA which encodes human aspartylglucosaminidase (AGA, E.C. 3.5.1.26). The activity of this lysosomal enzyme is deficient in aspartylglucosaminuria (AGU), a recessively inherited lysosomal accumulation disease resulting in severe mental retardation. The polypeptide chain deduced from the AGA cDNA consists of 346 amino acids, has two potential N-glycosylation sites and 11 cysteine residues. Transient expression of this cDNA in COS-1 cells resulted in increased expression of immunoprecipitable AGA protein. Direct sequencing of amplified AGA cDNA from an AGU patient revealed a G----C transition resulting in the substitution of cysteine 163 with serine. This mutation was subsequently found in all the 20 analyzed Finnish AGU patients, in the heterozygous form in all 53 carriers and in none of 67 control individuals, suggesting that it represents the major AGU causing mutation enriched in this isolated population. Since the mutation produces a change in the predicted flexibility of the AGA polypeptide chain and removes an intramolecular S-S bridge, it most probably explains the deficient enzyme activity found in cells and tissues of AGU patients. Images PMID:1703489

  20. Lectin cDNA and transgenic plants derived therefrom

    DOEpatents

    Raikhel, Natasha V.

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  1. Molecular cloning of rat brain Na,K-ATPase alpha-subunit cDNA.

    PubMed Central

    Schneider, J W; Mercer, R W; Caplan, M; Emanuel, J R; Sweadner, K J; Benz, E J; Levenson, R

    1985-01-01

    We have isolated a cDNA clone for the rat brain Na,K-ATPase alpha subunit. A lambda gt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, lambda rb5, containing a 1200-base-pair cDNA insert expressed a beta-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the alpha subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of lambda rb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney greater than brain greater than liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to lambda rb5 cDNA were amplified approximately 40-fold. Analysis of the lambda rb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase alpha-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the lambda rb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase alpha subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase. Images PMID:2994074

  2. TRAV gene usage in pig T-cell receptor alpha cDNA.

    PubMed

    Yamamoto, Ryuji; Uenishi, Hirohide; Hatsuse, Hiromi; Sato, Eimei; Awata, Takashi; Yasue, Hiroshi; Takagaki, Yohtaroh

    2005-05-01

    Pig (Sus scrofa) TRA clones were isolated from cDNA libraries of total RNA from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes of a 5-month-old Clawn strain pig. Among 103 complete TRA cDNA clones from both sources, 33 different TRAV genes were identified. By comparing their sequence identities against one another, these pig TRAV genes were grouped into 20 subgroups, including 13 subgroups, each containing only a single member. All of these pig subgroups gave corresponding human and mouse functional counterparts, suggesting their functional commonality. An exception was the Va01 gene segment, which lacked a functional human counterpart. The present report provides groundwork for studies on pig TRA expression.

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

    PubMed

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

    1990-09-25

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

  4. Molecular cloning of cDNA for rat liver gap junction protein

    PubMed Central

    1986-01-01

    An affinity-purified antibody directed against the 27-kD protein associated with isolated rat liver gap junctions was produced. Light and electron microscopic immunocytochemistry showed that this antigen was localized specifically to the cytoplasmic surfaces of gap junctions. The antibody was used to select cDNA from a rat liver library in the expression vector lambda gt11. The largest cDNA selected contained 1,494 bp and coded for a protein with a calculated molecular mass of 32,007 daltons. Northern blot analysis indicated that brain, kidney, and stomach express an mRNA with similar size and homology to that expressed in liver, but that heart and lens express differently sized, less homologous mRNA. PMID:3013898

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

    SciTech Connect

    Barbry, P.; Chassande, O.; Champigny, G.; Lingueglia, E.; Frelin, C.; Lazdunski, M. ); Champe, M.; Munemitsu, S.; Ullrich, A. ); Maes, P.; Tartar, A. Institut Pasteur de Lille )

    1990-10-01

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

  6. cDNA cloning and sequence analysis of human pancreatic procarboxypeptidase A1.

    PubMed Central

    Catasús, L; Villegas, V; Pascual, R; Avilés, F X; Wicker-Planquart, C; Puigserver, A

    1992-01-01

    Using polyclonal antibodies raised against human pancreatic procarboxypeptidases, a full-length cDNA coding for an A-type proenzyme was isolated from a lambda gt11 human pancreatic library. This cDNA contains standard 3' and 5' flanking regions, a poly(A)+ tail and a central region of 1260 nucleotides coding for a protein of 419 amino acids. On the basis of sequence comparisons, the human protein was classified as a procarboxypeptidase A1 which is very similar to the previously described A1 forms from rat and bovine pancreatic glands. The presence of the amino acid sequences assumed to be of importance for the zymogen inhibition by its activation segment, primarily on the basis of the recently reported crystal structure of the B form, further supports the proposed classification. PMID:1417781

  7. Characterization of a cDNA from Beta maritima that confers nickel tolerance in yeast.

    PubMed

    Bozdag, Gonensin O; Kaya, Alaattin; Koc, Ahmet; Noll, Gundula A; Prüfer, Dirk; Karakaya, Hüseyin Caglar

    2014-04-01

    Nickel is an essential micronutrient due to its involvement in many enzymatic reactions as a cofactor. However, excess of this element is toxic to biological systems. Here, we constructed a cDNA library from Beta maritima and screened it in the yeast system to identify genes that confer resistance to toxic levels of nickel. A cDNA clone (NIC6), which encodes for a putative membrane protein with unknown function, was found to help yeast cells to tolerate toxic levels of nickel. A GFP fused form of Nic6 protein was localized to multivesicular structures in tobacco epidermal cells. Thus, our results suggest a possible role of Nic6 in nickel and intracellular ion homeostasis. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. [Cloning of cDNA fragments related to adventitious root formation from mango cotyledon section].

    PubMed

    Xiao, Jie-Ning; Huang, Xue-Lin; Zhang, Yi-Shun; Li, Yin; Li, Xiao-Ju

    2004-04-01

    Two cut surfaces of mango cotyledon (distal and proximal cut surfaces) showed different capability of adventitious root formation, only proximal cut surface could be induced to form the roots and the distal cut surface did not. cDNA fragments related to adventitious root formation from the cut sections were isolated with suppressive subtractive hybridization. The forward substracted cDNA library was constructed using the cDNAs of distal (non-rooting) cut surface as driver and the cDNAs of proximal (rooting) cut surface as tester. Six positive clones were obtained by Virtual Northern blots. In this study, the putative up-regulated genes showed by sequence analysis were reported in mango for the first time, the deduced proteins among the positive clones were homologous to transporters, transcriptional regulators and enzymes.

  9. Libraries program

    USGS Publications Warehouse

    2011-01-01

    The U.S. Congress authorized a library for the U.S. Geological Survey (USGS) in 1879. The library was formally established in 1882 with the naming of the first librarian and began with a staff of three and a collection of 1,400 books. Today, the USGS Libraries Program is one of the world's largest Earth and natural science repositories and a resource of national significance used by researchers and the public worldwide.

  10. cDNA cloning and characterization of a human sperm antigen (SPAG6) with homology to the product of the Chlamydomonas PF16 locus.

    PubMed

    Neilson, L I; Schneider, P A; Van Deerlin, P G; Kiriakidou, M; Driscoll, D A; Pellegrini, M C; Millinder, S; Yamamoto, K K; French, C K; Strauss, J F

    1999-09-15

    Serum from an infertile male with high-titer anti-sperm antibodies was used to identify a novel human sperm antigen by screening of a testis expression library. The clone, initially designated Repro-SA-1 (HUGO-approved symbol SPAG6), was found to encode a sequence highly enriched in testis. The deduced amino acid sequence of the full-length cDNA revealed striking homology to the product of the Chlamydomonas reinhardtii PF16 locus, which encodes a protein localized to the central pair of the flagellar axoneme. The human gene encodes 1.8- and 2.8-kb mRNAs highly expressed in testis but not in prostate, ovary, spleen, thymus, small intestine, colon, peripheral blood leukocytes, heart, brain, placenta, liver, muscle, kidney, and pancreas. The gene was mapped to chromosome 10p11.2-p12. Antibodies raised against SPAG6 sequences localized the protein to the tails of permeabilized human sperm. Both the Chlamydomonas protein and SPAG6 contain eight contiguous armadillo repeats, which place them in a family of proteins known to mediate protein-protein interactions. The cloning of the human homologue of the Chlamydomonas PF16 locus provides a new avenue to explore the role of the axoneme central pair in human sperm function. Copyright 1999 Academic Press.

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

    ERIC Educational Resources Information Center

    Lance, Keith Curry; Lyons, Ray

    2009-01-01

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

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

    ERIC Educational Resources Information Center

    Lance, Keith Curry; Lyons, Ray

    2009-01-01

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

  13. Fabrication of high quality cDNA microarray using a small amount of cDNA.

    PubMed

    Park, Chan Hee; Jeong, Ha Jin; Jung, Jae Jun; Lee, Gui Yeon; Kim, Sang-Chul; Kim, Tae Soo; Yang, Sang Hwa; Chung, Hyun Cheol; Rha, Sun Young

    2004-05-01

    DNA microarray technology has become an essential part of biological research. It enables the genome-scale analysis of gene expression in various types of model systems. Manufacturing high quality cDNA microarrays of microdeposition type depends on some key factors including a printing device, spotting pins, glass slides, spotting solution, and humidity during spotting. UsingEthe Microgrid II TAS model printing device, this study defined the optimal conditions for producing high density, high quality cDNA microarrays with the least amount of cDNA product. It was observed that aminosilane-modified slides were superior to other types of surface modified-slides. A humidity of 30+/-3% in a closed environment and the overnight drying of the spotted slides gave the best conditions for arraying. In addition, the cDNA dissolved in 30% DMSO gave the optimal conditions for spotting compared to the 1X ArrayIt, 3X SSC and 50% DMSO. Lastly, cDNA in the concentration range of 100-300 ng/ micro l was determined to be best for arraying and post-processing. Currently, the printing system in this study yields reproducible 9000 spots with a spot size 150 mm diameter, and a 200 nm spot spacing.

  14. Molecular cloning and characterization of a novel human protein phosphatase 2C cDNA (PP2C epsilon*).

    PubMed

    Jin, Feng; Ji, Chaoneng; Liu, Lingfeng; Dai, Jianfeng; Gu, Shaohua; Sun, Xianfei; Xie, Yi; Mao, Yumin

    2004-09-01

    We have isolated a novel cDNA from the human fetal brain cDNA library with homology to the Mg2+ -dependent serine/threonine protein phosphatase 2C (PP2C) family. The cDNA is 3055 bp in length, and the predicted coding region encodes a 360-amino-acid protein, which shows 99% identity to the PP2C epsilon from rat and mouse. Then we term it human PP2C epsilon gene. The gene is mapped to chromosome 3q26.1 and contains 4 exons. RT-PCR analysis shows that the PP2C epsilon is widely expressed in human tissues and the expression levels in heart, placenta, lung, liver, kidney, and pancreas are relatively high.

  15. Isolation and characterization of a cDNA clone for a harvest-induced asparagine synthetase from Asparagus officinalis L.

    PubMed Central

    Davies, K M; King, G A

    1993-01-01

    A full-length cDNA clone (pTIP27) encoding asparagine synthetase (AS; EC 6.3.5.4) was isolated from a cDNA library prepared from the tip section (apex to 30 mm) of Asparagus officinalis L. spears. The cDNA clone encodes an mRNA of 1978 bp, giving a derived protein of 66.5 kD molecular mass. The derived amino acid sequence is 81% homologous to AS from Pisum sativum. Only low levels of transcript for AS could be detected in growing spears, roots, or ferns. However, AS mRNA levels began to increase in the tips of harvested spears after 2 h at 20 degrees C, and in the other sections of the spear after 4 h, suggesting that all sections of the spear were responding to the same postharvest signal. The results are discussed in relation to metabolic changes occurring in harvested spears. PMID:7904077

  16. Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L. 1

    PubMed Central

    Sánchez, Federico; Campos, Francisco; Padilla, Jaime; Bonneville, Jean-Marc; Enríquez, Consuelo; Caput, Daniel

    1987-01-01

    Nodule-specific uricase (uricase II) from Phaseolus vulgaris L. was purified to homogeneity by chromatographic methods. Purification data indicated that uricase II is approximately 2% of the total soluble protein from mature nodules. Specific antiserum was raised and used to determine the developmental expression and for immunoselection of polysomes. Uricase II was antigenically detected early in nodule development, 2 to 3 days before nitrogen fixation. Uricase-encoding cDNA clones were isolated by hybridizing a nodule-specific pUC9 cDNA library with labeled mRNA from immunoselected polysomes and a 35,000 molecular weight uricase II-encoding cDNA from soybean. An homologous clone (pNF-UR07) was used to assess the expression pattern of the specific transcript during development. Northern-blot analysis indicated that uricase II mRNA is exclusively expressed in nodule tissue. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16665575

  17. Brain-specific expression of MAP2 detected using a cloned cDNA probe

    PubMed Central

    1986-01-01

    We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low

  18. Development of a prostate cDNA microarray and statistical gene expression analysis package.

    PubMed

    Carlisle, A J; Prabhu, V V; Elkahloun, A; Hudson, J; Trent, J M; Linehan, W M; Williams, E D; Emmert-Buck, M R; Liotta, L A; Munson, P J; Krizman, D B

    2000-05-01

    A cDNA microarray comprising 5184 different cDNAs spotted onto nylon membrane filters was developed for prostate gene expression studies. The clones used for arraying were identified by cluster analysis of > 35 000 prostate cDNA library-derived expressed sequence tags (ESTs) present in the dbEST database maintained by the National Center for Biotechnology Information. Total RNA from two cell lines, prostate line 8.4 and melanoma line UACC903, was used to make radiolabeled probe for filter hybridizations. The absolute intensity of each individual cDNA spot was determined by phosphorimager scanning and evaluated by a bioinformatics package developed specifically for analysis of cDNA microarray experimentation. Results indicated 89% of the genes showed intensity levels above background in prostate cells compared with only 28% in melanoma cells. Replicate probe preparations yielded results with correlation values ranging from r = 0.90 to 0.93 and coefficient of variation ranging from 16 to 28%. Findings indicate that among others, the keratin 5 and vimentin genes were differentially expressed between these two divergent cell lines. Follow-up northern blot analysis verified these two expression changes, thereby demonstrating the reliability of this system. We report the development of a cDNA microarray system that is sensitive and reliable, demonstrates a low degree of variability, and is capable of determining verifiable gene expression differences between two distinct human cell lines. This system will prove useful for differential gene expression analysis in prostate-derived cells and tissue.

  19. Flavin reductase: sequence of cDNA from bovine liver and tissue distribution.

    PubMed Central

    Quandt, K S; Hultquist, D E

    1994-01-01

    Flavin reductase catalyzes electron transfer from reduced pyridine nucleotides to methylene blue or riboflavin, and this catalysis is the basis of the therapeutic use of methylene blue or riboflavin in the treatment of methemoglobinemia. A cDNA for a mammalian flavin reductase has been isolated and sequenced. Degenerate oligonucleotides, with sequences based on amino acid sequences of peptides derived from bovine erythrocyte flavin reductase, were used as primers in PCR to selectively amplify a partial cDNA that encodes the bovine reductase. The template used in the PCR was first strand cDNA synthesized from bovine liver total RNA using oligo(dT) primers. A PCR product was used as a specific probe to screen a bovine liver cDNA library. The sequence determined from two overlapping clones contains an open reading frame of 621 nucleotides and encodes 206 amino acids. The amino acid sequence deduced from the bovine liver flavin reductase cDNA matches the amino acid sequences determined for erythrocyte reductase-derived peptides, and the predicted molecular mass of 22,001 Da for the liver reductase agrees well with the molecular mass of 21,994 Da determined for the erythrocyte reductase by electrospray mass spectrometry. The amino acid sequence at the N terminus of the reductase has homology to sequences of pyridine nucleotide-dependent enzymes, and the predicted secondary structure, beta alpha beta, resembles the common nucleotide-binding structural motif. RNA blot analysis indicates a single 1-kilobase reductase transcript in human heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle. Images PMID:7937764

  20. Sanfilippo syndrome type B: cDNA and gene encoding human {alpha}-N-acetylglucosaminidase

    SciTech Connect

    Zhao, H.G.; Lopez, R.; Rennecker, J.

    1994-09-01

    Deficiency of the lysosomal enzyme {alpha}-N-acetlyglucosaminidase underlies the type B Sanfilippo syndrome (MPS III B), a mucopolysaccharide storage disease with profound neurologic deterioration. We are acquiring tools to study the molecular basis of the disorder. The enzyme was purified from bovine testis; after ConA-, DEAE- and phenyl-Sepharose chromatography, it was subjected to SDS-PAGE without preheating. Of two bands of activity detected on the gel, 170 kDa and 87 kDa, the larger one, which coincided with a well-defined Coomassie blue band, was selected for sequence analysis. Degenerate 17-base oligonucleotides, corresponding to the ends of an internal 23 amino acid sequence, were used for RT-PCR of RNA from human fibroblasts. A 41-mer was synthesized from the sequence of the RT-PCR product and used to screen a human testis cDNA library. A number of cDNA inserts were isolated, all lacking the 5{prime} end and none longer than 1.7 kb. An additional 300 bp segment has been obtained by RACE. The cDNA sequence accounts for 9 of 11 peptides, allowing for species difference. Northern analysis of fibroblast RNA with a 1.5 kb cDNA probe showed the presence of a 3 kb mRNA; marked deficiency of this mRNA in two MPS III B fibroblast lines confirmed the authenticity of the cloned cDNA. While no homologous amino acid sequence has been found in a search of GenBank, the nucleotide sequence (interrupted by 4 introns) is present in a flanking region upstream of an unrelated gene on chromosome 17q11-21 (human 17{beta}-hydroxysteroid dehydrogenase). This must therefore be the chromosomal locus of the {alpha}-N-acetylglucosaminidase gene and of MPS III B.

  1. cDNA cloning of the immunoglobulin heavy chain binding protein.

    PubMed Central

    Haas, I G; Meo, T

    1988-01-01

    A cDNA library was constructed from size-fractionated poly(A)+ RNA prepared from a murine pre-B-cell hybridoma expressing high levels of immunoglobulin heavy chain binding protein (BiP) and mu heavy chains. Transformed bacterial colonies were screened for recombinant plasmids containing cDNA coding for BiP by hybrid-selected mRNA translation. A clone, pMBiP, containing a 736-base-pair insert was shown to encode the protein. Translation in vitro of hybridoma mRNA selected by hybridization to the pMBiP cDNA yielded a single polypeptide of BiP-like size. The authenticity of this mRNA was verified by comparing the peptides obtained by the limited proteolysis of its in vitro translation product with those obtained from the in vivo produced BiP. Likewise, the authenticity of the cDNA insert was verified by an RNase A protection assay of heteroduplex molecules obtained by annealing a uniformly labeled single-strand copy of the cDNA clone with the same mRNA selected by hybridization and tested by translation. The nucleotide sequence of this clone enabled us to deduce the carboxyl-terminal 142 amino acids of BiP and to establish its kinship with the 70-kDa heat shock protein family. The finding of a single copy of the BiP gene in DNA blots of mouse and rat implies that the BiP-related RNA transcripts constitutively expressed in various murine tissues and cell lines are indeed products of the same gene. These findings imply that BiP plays a more general role than previously anticipated on the basis of the discovery of its association with immunoglobulin heavy chains. Images PMID:2895472

  2. Cloning and characterization of Taenia saginata paramyosin cDNA.

    PubMed

    Ferrer, Elizabeth; Moyano, Eva; Benitez, Laura; González, Luis Miguel; Bryce, Denise; Foster-Cuevas, Mildred; Dávila, Iris; Cortéz, Maria Milagros; Harrison, Leslie J S; Parkhouse, R Michael E; Gárate, Teresa

    2003-09-01

    A lambdaZAP-express cDNA library of Taenia saginata metacestodes was constructed. Antibody screening yielded a clone with an insert of 3,408 bp, an open reading frame of 2,589 bp, a deduced sequence of 863 amino acid and a molecular mass of 98.89 kDa. Alignments of the predicted amino acid sequence showed identity with paramyosins from several species: 98.8% with Taenia solium, 96.3% with Echinococcus.granulosus and about 70% with Schistosoma spp. The insert was expressed and purified. A collagen binding assay was performed which showed that T. saginata GST-paramyosin retained this property in a dose-dependent manner. Problems were encountered due to high backgrounds in serological assays in the homologous T. saginata system. However, the recombinant paramyosin was recognized by antibodies present in 31.6% of sera from T. solium seropositive cysticercosis patients and 100% of the sera from acute cysticercosis patients. The immunodominant epitope was the carboxyl-terminal fragment of the molecule.

  3. Library Research.

    ERIC Educational Resources Information Center

    Wright, Nancy Kirkpatrick

    This workbook, designed for a Library Research course at Yavapai College, provides 15 lessons in advanced library reference skills. Each lesson provides explanatory text and reinforcement exercises. After Lesson I introduces specialized dictionaries and encyclopedias (e.g., for foreign languages, medicine, music, economics, social sciences, and…

  4. Privatizing Libraries

    ERIC Educational Resources Information Center

    Jerrard, Jane; Bolt, Nancy; Strege, Karen

    2012-01-01

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

  5. Library Research.

    ERIC Educational Resources Information Center

    Wright, Nancy Kirkpatrick

    This workbook, designed for a Library Research course at Yavapai College, provides 15 lessons in advanced library reference skills. Each lesson provides explanatory text and reinforcement exercises. After Lesson I introduces specialized dictionaries and encyclopedias (e.g., for foreign languages, medicine, music, economics, social sciences, and…

  6. Privatizing Libraries

    ERIC Educational Resources Information Center

    Jerrard, Jane; Bolt, Nancy; Strege, Karen

    2012-01-01

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

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

    PubMed

    Zhernakov, Aleksandr; Rotter, Björn; Winter, Peter; Borisov, Alexey; Tikhonovich, Igor; Zhukov, Vladimir

    2017-03-01

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

  8. Human cyclooxygenase-2 cDNA.

    PubMed Central

    Hla, T; Neilson, K

    1992-01-01

    Cyclooxygenase (Cox), also known as prostaglandin (PG) H synthase (EC 1.14.99.1), catalyzes the rate-limiting step in the formation of inflammatory PGs. A major regulatory step in PG biosynthesis is at the level of Cox: growth factors, cytokines, and tumor promoters induce Cox activity. We have cloned the second form of the Cox gene (Cox-2) from human umbilical vein endothelial cells (HUVEC). The cDNA encodes a polypeptide of 604 amino acids that is 61% identical to the previously isolated human Cox-1 polypeptide. In vitro translation of the human (h)Cox-2 transcript in rabbit reticulocyte lysates resulted in the synthesis of a 70-kDa protein that is immunoprecipitated by antiserum to ovine Cox. Expression of the hCox-2 open reading frame in Cos-7 monkey kidney cells results in the elaboration of cyclooxygenase activity. hCox-2 cDNA hybridizes to a 4.5-kilobase mRNA species in HUVEC, whereas the hCox-1 cDNA hybridizes to 3- and 5.3-kilobase species. Both Cox-1 and Cox-2 mRNAs are expressed in HUVEC, vascular smooth muscle cells, monocytes, and fibroblasts. Cox-2 mRNA was preferentially induced by phorbol 12-myristate 13-acetate and lipopolysaccharide in human endothelial cells and monocytes. Together, these data demonstrate that the Cox enzyme is encoded by at least two genes that are expressed and differentially regulated in a variety of cell types. High-level induction of the hCox-2 transcript in mesenchymal-derived inflammatory cells suggests a role in inflammatory conditions. Images PMID:1380156

  9. Beyond Job Enrichment to Employment Enrichment

    ERIC Educational Resources Information Center

    Werther, William B., Jr.

    1975-01-01

    Employment enrichment views the total work environment confronting employees as a system consisting of two overlapping areas: worker-job and worker-organization subsystems. Job enrichment has improved the worker-job subsystem. The focus of this article is on methods of improving the worker-organization relationship. (Author/JB)

  10. GLANET: genomic loci annotation and enrichment tool.

    PubMed

    Otlu, Burçak; Firtina, Can; Keles, Sündüz; Tastan, Oznur

    2017-09-15

    Genomic studies identify genomic loci representing genetic variations, transcription factor (TF) occupancy, or histone modification through next generation sequencing (NGS) technologies. Interpreting these loci requires evaluating them with known genomic and epigenomic annotations. We present GLANET as a comprehensive annotation and enrichment analysis tool which implements a sampling-based enrichment test that accounts for GC content and/or mappability biases, jointly or separately. GLANET annotates and performs enrichment analysis on these loci with a rich library. We introduce and perform novel data-driven computational experiments for assessing the power and Type-I error of its enrichment procedure which show that GLANET has attained high statistical power and well-controlled Type-I error rate. As a key feature, users can easily extend its library with new gene sets and genomic intervals. Other key features include assessment of impact of single nucleotide variants (SNPs) on TF binding sites and regulation based pathway enrichment analysis. GLANET can be run using its GUI or on command line. GLANET's source code is available at https://github.com/burcakotlu/GLANET . Tutorials are provided at https://glanet.readthedocs.org . burcak@ceng.metu.edu.tr or oznur.tastan@cs.bilkent.edu.tr. Supplementary data are available at Bioinformatics online.

  11. Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus.

    PubMed

    Todaka, Nemuri; Moriya, Shigeharu; Saita, Kanako; Hondo, Tomoko; Kiuchi, Isao; Takasu, Hirotoshi; Ohkuma, Moriya; Piero, Carninci; Hayashizaki, Yoshihide; Kudo, Toshiaki

    2007-03-01

    To clarify the lignocellulolytic process of the lower termite symbiotic protistan system, we constructed a cDNA library from an as yet uncultivated symbiotic protist community of the lower termite Reticulitermes speratus. The library was constructed by the biotinylated CAP trapper method and analyzed by one-pass sequencing. Phylogenetic analysis of actin orthologs confirmed that the resulting library reflected the intact organismal and mRNA composition of the symbiotic system. The contents of the library included abundant numbers of lignocellulolytic genes of the glycosyl hydrolase family orthologs (families 3, 5, 7, 8, 10, 11, 26, 43, 45 and 62). Our results clearly indicated that a multiple family of glycosyl hydrolase enzymes was involved in the protistan cellulose degradation system. The data also suggested that the most extensively expressed enzyme was glycosyl hydrolase family 7, a cellobiohydrolase ortholog. This family of enzymes enables the degradation of crystalline cellulose, the principal component of wood biomass.

  12. Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component of human. cap alpha. -ketoacid dehydrogenase complexes

    SciTech Connect

    Pons, G.; Raefsky-Estrin, C.; Carothers, D.J.; Pepin, R.A.; Javed, A.A.; Jesse, B.W.; Ganapathi, M.K.; Samols, D.; Patel, M.S.

    1988-03-01

    cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (>98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.

  13. The Status, Quality, and Expansion of the NIH Full-Length cDNA Project: The Mammalian Gene Collection (MGC)

    PubMed Central

    2004-01-01

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5′-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline. PMID:15489334

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

    PubMed Central

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

    2016-01-01

    To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules. PMID:27554526

  15. Molecular cloning and expression of a cDNA encoding an olfactory-specific mouse phenol sulphotransferase.

    PubMed Central

    Tamura, H O; Harada, Y; Miyawaki, A; Mikoshiba, K; Matsui, M

    1998-01-01

    Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme, PSTg. In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum. The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide. Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1. The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme. These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT-PCR). PMID:9560327

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

    SciTech Connect

    Her, Chengtao; Weinshilboum, R.M.; Kaur, G.P.

    1997-05-01

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

  17. Isolation and characterization of a cDNA clone encoding testis protamine Z1 from the dog-fish Scylliorhinus caniculus.

    PubMed

    Berlot-Picard, F; Vodjdani, G; Doly, J

    1987-06-15

    A clone containing a 445-bp cDNA insert was isolated from a cDNA library synthesized from dog-fish testes mRNA. The nucleotide sequence was determined and corresponded to a 50-amino-acid protein. The known five-amino-acid N-terminal sequence corresponded exactly to our deduced amino acid sequence. After in vitro transcription of this cDNA using SP6 RNA polymerase, the translated polypeptide comigrated with the Z1 scylliorhinine marker. Analysis of the cDNA 3' flanking region of our Scylliorhinus protamine Z1 revealed an inverted repeat sequence, an ACAA motif and a CAGGAAAGA box known as regulatory signals for transcription termination in histone genes. In addition, sequences homologous to the simian virus (SV 40) and polyoma virus core enhancer elements were identified in the 5' and 3' flanking regions.

  18. Trehalase from male accessory gland of an insect, Tenebrio molitor. cDNA sequencing and developmental profile of the gene expression.

    PubMed Central

    Takiguchi, M; Niimi, T; Su, Z H; Yaginuma, T

    1992-01-01

    A cDNA of alpha alpha-trehalase (EC 3.2.1.28) from a cDNA library of male bean-shaped accessory gland of the mealworm beetle, Tenebrio molitor, has been isolated by the homology screening approach. Sequence analysis of the cDNA (1830 bp) revealed that the cDNA encoded a protein of 555 amino acids with a calculated M(r) of 64457. The deduced amino acid sequence had significant similarities to rabbit small intestine and Escherichia coli trehalases. Northern blotting and semi-quantitative PCR analyses revealed that a trehalase transcript with about 2.0 kb was abundant in bean-shaped accessory glands. In the glands, the amount of trehalase transcript increased from 1 to 2 days after adult ecdysis. These tissue- and stage-specific gene expressions of trehalase corresponded to the tissue- and stage-specificity of trehalase activity. Images Fig. 2. Fig. 3. Fig. 4 PMID:1445264

  19. [The Presidential Libraries.

    ERIC Educational Resources Information Center

    Webb, John

    There are seven Presidential libraries in various states of existence, from quite active to proposed: (1) Franklin D. Roosevelt Library, (2) Harry S. Truman Library, (3) Herbert Hoover Library, (4) Dwight D. Eisenhower Library, (5) John F. Kennedy Memorial Library (6) Lyndon B. Johnson Library and (7) Rutherford B. Hayes Memorial Library. Each…

  20. Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

    DTIC Science & Technology

    2013-01-01

    Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries PRINCIPAL INVESTIGATOR: Daniel J. Lindner, M.D., Ph.D...YYYY) 2013 2. REPORT TYPE Final 3. DATES COVERED (From - To) 201 31 2012 4. TITLE AND SUBTITLE Complementation of Myelodysplastic Syndrome Clones...vitro and engrafted in the marrow of SG3, but not NSG mice. 15. SUBJECT TERMS Myelodysplastic syndrome , lentivirus, cDNA libraries

  1. A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep

    PubMed Central

    Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon

    2014-01-01

    The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes. PMID:24447069

  2. Academic Libraries

    ERIC Educational Resources Information Center

    Library Journal, 1970

    1970-01-01

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

  3. Digital Libraries.

    ERIC Educational Resources Information Center

    Fox, Edward A.; Urs, Shalini R.

    2002-01-01

    Provides an overview of digital libraries research, practice, and literature. Highlights include new technologies; redefining roles; historical background; trends; creating digital content, including conversion; metadata; organizing digital resources; services; access; information retrieval; searching; natural language processing; visualization;…

  4. Digital Libraries.

    ERIC Educational Resources Information Center

    Fox, Edward A.; Urs, Shalini R.

    2002-01-01

    Provides an overview of digital libraries research, practice, and literature. Highlights include new technologies; redefining roles; historical background; trends; creating digital content, including conversion; metadata; organizing digital resources; services; access; information retrieval; searching; natural language processing; visualization;…

  5. Presidential Libraries

    ERIC Educational Resources Information Center

    Cole, Garold L.

    1972-01-01

    Presented are a description of the history of the presidential papers in the United States, the development and functions of the presidential library system, and a brief look at future developments. (39 references) (Author)

  6. Callpath Library

    SciTech Connect

    Gamblin, T.

    2013-11-09

    The "Callpath Library" is a software abstraction layer over a number of stack tracing utilities. It allows tool develoopers to conveniently represent and mNipulate call paths gathered fro U. Wisconsin's Stackwalker API and GNU Backtrace.

  7. Derived enriched uranium market

    SciTech Connect

    Rutkowski, E.

    1996-12-01

    The potential impact on the uranium market of highly enriched uranium from nuclear weapons dismantling in the Russian Federation and the USA is analyzed. Uranium supply, conversion, and enrichment factors are outlined for each country; inventories are also listed. The enrichment component and conversion components are expected to cause little disruption to uranium markets. The uranium component of Russian derived enriched uranium hexafluoride is unresolved; US legislation places constraints on its introduction into the US market.

  8. An in vitro polysome display system for identifying ligands from very large peptide libraries.

    PubMed Central

    Mattheakis, L C; Bhatt, R R; Dower, W J

    1994-01-01

    We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes. A pool of DNA sequences encoding 10(12) random decapeptides was incubated in an Escherichia coli S30 coupled transcription/translation system. Polysomes were isolated and screened by affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B. The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection. A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA. After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody. Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM. The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems. Images PMID:7522328

  9. Cloning and characterization of human liver cDNA encoding a protein S precursor.

    PubMed Central

    Hoskins, J; Norman, D K; Beckmann, R J; Long, G L

    1987-01-01

    Human liver cDNA encoding a protein S precursor was isolated from two cDNA libraries by two different techniques. Based upon the frequency of positive clones, the abundance of mRNA for protein S is approximately 0.01%. Blot hybridization of electrophoretically fractionated poly(A)+ RNA revealed a major mRNA approximately 4 kilobases long and two minor forms of approximately 3.1 and approximately equal to 2.6 kilobases. One of the cDNA clones contains a segment encoding a 676 amino acid protein S precursor, as well as 108 and 1132 nucleotides of 5' and 3' noncoding sequence, respectively, plus a poly(A) region at the 3' end. The cDNAs are adenosine plus thymidine-rich (60%) except for the 5' noncoding region, where 78% of the nucleotides are guanosine or cytosine. The protein precursor consists of a 41 amino acid "leader" peptide followed by 635 amino acids corresponding to mature protein S. Comparison of the mature protein region with homologous vitamin K-dependent plasma proteins shows that it is composed of the following domains: an amino-terminal gamma-carboxyglutamic acid-rich region of 37 amino acids; a 36 amino acid linker region rich in hydroxy amino acids; four epidermal growth factor-like segments, each approximately 45 amino acids long; and a 387 amino acid carboxyl-terminal domain of unrecognized structure and unknown function. Images PMID:3467362

  10. Chitinase cDNA cloning and mRNA induction by fungal elicitor, wounding, and infection.

    PubMed

    Hedrick, S A; Bell, J N; Boller, T; Lamb, C J

    1988-01-01

    Chitinase, which catalyzes the hydrolysis of beta-1,4 N-acetylglucosamine linkages of the fungal cell wall polymer chitin, is a component of the inducible defenses of plants. We show that chitinase synthesis is stimulated in bean (Phaseolus vulgaris L.) cell suspension cultures treated with fungal cell wall elicitors and in hypocotyls in response to infection with the fungus Colletotrichum lindemuthianum. Chitinase cDNA clones were isolated by antibody screening of a lambdagt11 cDNA library containing sequences complementary to poly A(+) RNA from elicited cells. The identity of these clones was confirmed by nucleotide sequence analysis and comparison of the deduced amino acid sequence with that determined for the amino-terminal sequence of bean chitinase. Elicitor causes a very rapid activation of chitinase transcription with a 10-fold stimulation after 5 minutes and 30-fold increase within 20 minutes. This leads to a marked, transient accumulation of chitinase transcripts with maximum levels 2 hours after elicitor treatment, concomitant with the phase of rapid enzyme synthesis. Chitinase transcripts also markedly accumulate in wounded and infected hypocotyls. Chitinase cDNA sequences hybridize to several genomic fragments suggesting there are several chitinase genes in the bean genome.

  11. A cDNA clone highly expressed in ripe banana fruit shows homology to pectate lyases.

    PubMed

    Dominguez-Puigjaner, E; LLop, I; Vendrell, M; Prat, S

    1997-07-01

    A cDNA clone (Ban17), encoding a protein homologous to pectate lyase, has been isolated from a cDNA library from climacteric banana fruit by means of differential screening. Northern analysis showed that Ban17 mRNA is first detected in early climacteric fruit, reaches a steady-state maximum at the climacteric peak, and declines thereafter in overripe fruit. Accumulation of the Ban17 transcript can be induced in green banana fruit by exogenous application of ethylene. The demonstrates that expression of this gene is under hormonal control, its induction being regulated by the rapid increase in ethylene production at the onset of ripening. The deduced amino acid sequence derived from the Ban17 cDNA shares significant identity with pectate lyases from pollen and plant pathogenic bacteria of the genus Erwinia. Similarity to bacterial pectate lyases that were proven to break down the pectic substances of the plant cell wall suggest that Ban17 might play a role in the loss of mesocarp firmness during fruit ripening.

  12. cDNA encoding a polypeptide including a hev ein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  13. Cloning and expression of a putative transferrin cDNA of the spruce budworm, Choristoneura fumiferana.

    PubMed

    Ampasala, D R; Zheng, S-C; Retnakaran, A; Krell, P J; Arif, B M; Feng, Q-L

    2004-05-01

    A spruce budworm (Choristoneura fumiferana) transferrin cDNA (CfTf) was isolated and cloned from a cDNA library that was constructed using mRNA from fifth to sixth instar larvae. CfTf cDNA encoded a predicted protein of 681 amino acids with a molecular mass of approximately 76 kDa. CfTf shared 72% and 74% identities at the amino acid level with transferrins of Manduca sexta and Bombyx mori, respectively. Like other transferrins, CfTf retains most of the N-terminal, iron-binding amino acid residues. Northern blot analyses indicated that CfTf mRNA was present at high levels after ecdysis, but that the expression level was low prior to ecdysis at the fourth-sixth instar stages. The highest level of CfTf expression was detected in the fat body. Relatively low levels of expression were detected in the epidermis and no expression was found in the midgut. Expression of CfTf mRNA could be induced by bacteria but not fungi. Expression of CfTf mRNA was suppressed by iron load.

  14. Complete cDNA and derived amino acid sequence of human factor V

    SciTech Connect

    Jenny, R.J.; Pittman, D.D.; Toole, J.J.; Kriz, R.W.; Aldape, R.A.; Hewick, R.M.; Kaufman, R.J.; Mann, K.G.

    1987-07-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A)tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approx. 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approx. 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approx. 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

  15. Molecular cloning, sequencing and expression of cDNA encoding human trehalase.

    PubMed

    Ishihara, R; Taketani, S; Sasai-Takedatsu, M; Kino, M; Tokunaga, R; Kobayashi, Y

    1997-11-20

    A complete cDNA clone encoding human trehalase, a glycoprotein of brush-border membranes, has been isolated from a human kidney library. The cDNA encodes a protein of 583 amino acids with a calculated molecular weight of 66,595. Human enzyme contains a typical cleavable signal peptide at amino terminus, five potential glycosylation sites, and a hydrophobic region at carboxyl terminus where the protein is anchored to plasma membranes via glycosylphosphatidylinositol. The deduced amino acid sequence of the human enzyme showed similarity to sequences of the enzyme from rabbit, silk worm, Tenebrio molitor, Escherichia coli and yeast. Northern blots revealed that human trehalase mRNA of approx. 2.0 kb was found mainly in the kidney, liver and small intestine. Expression of the recombinant trehalase in E. coli provided a high level of the enzyme activity. The isolation and expression of cDNA for human trehalase should facilitate studies of the structure of the gene, as well as a basis for a better understanding of the catalytic mechanism.

  16. cDNA sequence and chromosomal localization of human enterokinase, the proteolytic activator of trypsinogen.

    PubMed

    Kitamoto, Y; Veile, R A; Donis-Keller, H; Sadler, J E

    1995-04-11

    Enterokinase is a serine protease of the duodenal brush border membrane that cleaves trypsinogen and produces active trypsin, thereby leading to the activation of many pancreatic digestive enzymes. Overlapping cDNA clones that encode the complete human enterokinase amino acid sequence were isolated from a human intestine cDNA library. Starting from the first ATG codon, the composite 3696 nt cDNA sequence contains an open reading frame of 3057 nt that encodes a 784 amino acid heavy chain followed by a 235 amino acid light chain; the two chains are linked by at least one disulfide bond. The heavy chain contains a potential N-terminal myristoylation site, a potential signal anchor sequence near the amino terminus, and six structural motifs that are found in otherwise unrelated proteins. These domains resemble motifs of the LDL receptor (two copies), complement component Clr (two copies), the metalloprotease meprin (one copy), and the macrophage scavenger receptor (one copy). The enterokinase light chain is homologous to the trypsin-like serine proteinases. These structural features are conserved among human, bovine, and porcine enterokinase. By Northern blotting, a 4.4 kb enterokinase mRNA was detected only in small intestine. The enterokinase gene was localized to human chromosome 21q21 by fluorescence in situ hybridization.

  17. Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

    DTIC Science & Technology

    2011-07-01

    cDNA expression library. MDS bone marrow cells from 5q- (pictured at right), mono 7 / 7q-, trisomy 8, and del 20q were transfected at an moi of 0.1...home to and engraft within the bone-marrow endosteal region. Nat Biotechnol 25:1315- 21 . 3. Ito, M., H. Hiramatsu, K. Kobayashi, K. Suzue, M

  18. A majority of Ig H chain cDNA of normal human adult blood lymphocytes resembles cDNA for fetal Ig and natural autoantibodies

    SciTech Connect

    Huang, C.; Stollar, B.D. )

    1993-11-15

    Certain Ig V[sub H] gene segments, with few or no mutations, recur frequently in natural autoantibodies, fetal antibodies, and products of B cell tumors. The goal of this study was to determine whether similar Ig gene segment usage occurs in normal human adult PBL. Extending previous analyses, 105 randomly picked H chain V region clones of representative cDNA libraries from PBL were sequenced. Clones were from: IgM and IgG libraries from one RNA sample of a normal adult; a second IgM library from the same subject 11 mo later; and one IgM library from a second subject. Although some clones had clear evidence of mutation, 48 of 77 IgM clones (62%) shared 99% or more identity with known germline V[sub H] segments, and most of these had no mutations in the CDR3 portion of the J[sub H] segment. Certain V[sub H] gene segments, expressed in autoantibodies and fetal antibodies, occurred at high frequency in these libraries. Fourteen of the clones with 99% identity to known V[sub H] segments had CDR3 segments identical to portions of known germline D[sub H] gene sequences; two such clones had no N nucleotides at the V[sub H] D[sub H] or D[sub H]J[sub H] junctions. IgG-encoding sequences had more mutations than IgM-encoding sequences. J[sub H] and D[sub H] usage was not random. The circulating B cell population may represent a distinct compartment, with a large proportion of cells similar to those of the fetal and natural autoantibody repertoire. Polyreactive Ig products of these circulating cells may serve a screening function, binding and delivering diverse Ag to secondary lymphoid tissues where more highly selective antibodies are formed to foreign or Self-Ag. 40 refs., 2 figs., 6 tabs.

  19. Communities of purple sulfur bacteria in a Baltic Sea coastal lagoon analyzed by puf LM gene libraries and the impact of temperature and NaCl concentration in experimental enrichment cultures.

    PubMed

    Tank, Marcus; Blümel, Martina; Imhoff, Johannes F

    2011-12-01

    Shallow coastal waters, where phototrophic purple sulfur bacteria (PSB) regularly form massive blooms, are subjected to massive diurnal and event-driven changes of physicochemical conditions including temperature and salinity. To analyze the ability of PSB to cope with these environmental factors and to compete in complex communities we have studied changes of the environmental community of PSB of a Baltic Sea lagoon under experimental enrichment conditions with controlled variation of temperature and NaCl concentration. For the first time, changes within a community of PSB were specifically analyzed using the photosynthetic reaction center genes pufL and M by RFLP and cloning experiments. The most abundant PSB phylotypes in the habitat were found along the NaCl gradient from freshwater conditions up to 7.5% NaCl. They were accompanied by smaller numbers of purple nonsulfur bacteria and aerobic anoxygenic phototrophic bacteria. Major components of the PSB community of the brackish lagoon were affiliated to PSB genera and species known as marine, halophilic or salt-tolerant, including species of M arichromatium, H alochromatium, T hiorhodococcus, A llochromatium, T hiocapsa, T hiorhodovibrio, and T hiohalocapsa. A dramatic shift occurred at elevated temperatures of 41 and 44°C when M arichromatium gracile became most prominent which was not detected at lower temperatures.

  20. Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains.

    PubMed

    Schmidt, Frederike; Müller, Miryam; Prox, Johannes; Arnold, Philipp; Schönherr, Caroline; Tredup, Claudia; Minder, Petra; Ebsen, Henriette; Janssen, Ottmar; Annaert, Wim; Pietrzik, Claus; Schmidt-Arras, Dirk; Sterchi, Erwin E; Becker-Pauly, Christoph

    2016-09-01

    Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. As several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified as present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified.

  1. Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains.

    PubMed

    Schmidt, Frederike; Müller, Miryam; Prox, Johannes; Arnold, Philipp; Schönherr, Caroline; Tredup, Claudia; Minder, Petra; Ebsen, Henriette; Janssen, Ottmar; Annaert, Wim; Pietrzik, Claus; Schmidt-Arras, Dirk; Sterchi, Erwin E; Becker-Pauly, Christoph

    2016-05-14

    Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It was shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. Since several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified being present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified.

  2. Characterization of full-length sequenced cDNA inserts (FLIcs) from Atlantic salmon (Salmo salar)

    PubMed Central

    Andreassen, Rune; Lunner, Sigbjørn; Høyheim, Bjørn

    2009-01-01

    Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs) are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP), the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91%) of the transcripts were annotated using Gene Ontology (GO) terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS). The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS). This suggests that the remaining cDNA

  3. Enrichment by hybridisation of long DNA fragments for Nanopore sequencing

    PubMed Central

    Eckert, Sabine E.; Chan, Jackie Z.-M.; Houniet, Darren; Breuer, Judy

    2016-01-01

    Enrichment of DNA by hybridisation is an important tool which enables users to gather target-focused next-generation sequence data in an economical fashion. Current in-solution methods capture short fragments of around 200–300 nt, potentially missing key structural information such as recombination or translocations often found in viral or bacterial pathogens. The increasing use of long-read third-generation sequencers requires methods and protocols to be adapted for their specific requirements. Here, we present a variation of the traditional bait–capture approach which can selectively enrich large fragments of DNA or cDNA from specific bacterial and viral pathogens, for sequencing on long-read sequencers. We enriched cDNA from cultured influenza virus A, human cytomegalovirus (HCMV) and genomic DNA from two strains of Mycobacterium tuberculosis (M. tb) from a background of cell line or spiked human DNA. We sequenced the enriched samples on the Oxford Nanopore MinION™ and the Illumina MiSeq platform and present an evaluation of the method, together with analysis of the sequence data. We found that unenriched influenza A and HCMV samples had no reads matching the target organism due to the high background of DNA from the cell line used to culture the pathogen. In contrast, enriched samples sequenced on the MinION™ platform had 57 % and 99 % best-quality on-target reads respectively. PMID:28785419

  4. Nucleotide sequence of a cloned cDNA for proopiomelanocortin precursor of chum salmon, Onchorynchus keta.

    PubMed Central

    Soma, G I; Kitahara, N; Nishizawa, T; Nanami, H; Kotake, C; Okazaki, H; Andoh, T

    1984-01-01

    We have isolated a cDNA clone encoding salmon proopiomelanocortin precursor. Polyadenylated RNA was isolated from pituitary neurointermediate lobes and used to construct a cDNA library. The library was screened with 17 mer of oligodeoxyribonucleotides specific for the hexapeptide sequence in salmon beta-endorphin I, Phe-Met-Lys-Pro-Tyr-Thr at positions 4-9 excluding the third nucleotide. One positive clone, pSSM17 containing an insert of 1303 base pairs (bp) was characterized. Sequence determination revealed that it possessed sequences covering the entire regions encoding ACTH and beta-lipotropin and that the mRNA had the same overall organization as those of other mammalian species, i.e., the following peptide hormones were arranged in order from 5' upstream, ACTH including alpha-melanotropin and corticotropin-like intermediate lobe peptide, beta-lipotropin including gamma-lipotropin, beta-melanotropin and beta-endorphin. Amino acid sequences for putative salmon ACTH, beta-, and gamma-lipotropin were predicted. Comparison of the salmon mRNA sequence with those of mammals showed that the regions of alpha- and beta-MSH are relatively homologous, but other regions are much less so, especially in the 3' nontranslated region where it is much longer and completely heterologous. Images PMID:6095185

  5. Hypoxically inducible barley lactate dehydrogenase: cDNA cloning and molecular analysis

    SciTech Connect

    Hondred, D. ); Hanson, A.D. Univ. de Montreal, Quebec )

    1990-09-01

    In the roots of barley and other cereals, hypoxia induces a set of five isozymes of L-lactate dehydrogenase (LDH; (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). Biochemical and genetic data indicate that the five LDH isozymes are tetramers that arise from random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH were isolated from a {lambda}gt11 cDNA library derived from hypoxically treated barley roots. The library was screened with antiserum raised against barley LDH purified {approx}3,000-fold by an improved three-step procedure. Immunopositive clones were rescreened with a cDNA probe synthesized by the polymerase chain reaction using primers modeled from the amino acid sequences of two tryptic LDH peptides. Two types of LDH clones were found. Nucleotide sequence analysis of one representative insert of each type (respectively, 1,305 and 1,166 base pairs) revealed open reading framed encoding 10 peptide fragments of LDH. The 1,305-base-pair insert included the entire coding region of a 356-residue LDH monomer. The nucleotide sequences of the two LDH cDNAs were 92% identical in the coding region, but highly divergent in the 3{prime} noncoding region, and thus probably correspond to the two postulated Ldh loci. The deduced amino acid sequences of the two barley LDHs were 96% identical to each other and very similar to those from vertebrate and bacterial LDHs. RNA blot hybridization showed a single mRNA band of 1.5 kilobases whose level rose about 8-fold in roots during hypoxic induction, as did the level of translatable LDH message.

  6. Spruce budworm (Choristoneura fumiferana) juvenile hormone esterase: hormonal regulation, developmental expression and cDNA cloning.

    PubMed

    Feng, Q L; Ladd, T R; Tomkins, B L; Sundaram, M; Sohi, S S; Retnakaran, A; Davey, K G; Palli, S R

    1999-02-25

    We have used the differential display of mRNAs technique to identify Choristoneura fumiferana genes that are induced by juvenile hormone I (JH I). Of the six PCR products identified, one bound to a 2.8-kb mRNA from CF-203 cells whose abundance increased when the cells were grown in the presence of JH I. The same 2.8-kb mRNA decreased to undetectable levels when the CF-203 cells were grown in the presence of 20-hydroxyecdysone (20E). The PCR fragment probe also detected a 2.8-kb mRNA in the C. fumiferana larval tissues. This 2.8-kb mRNA was present on the first day of the first, third, fourth, fifth and sixth larval and pupal stadia, but was conspicuously absent on the first day of the second larval stadium, as well as during the intermolt periods of the first to fifth instar larval stages. In the sixth instar larvae the 2.8-kb mRNA was detected in the fat body, epidermis and midgut during the intermolt period. The PCR fragment was used as a probe to screen a cDNA library. The deduced amino acid sequence of this 2.8-kb cDNA clone showed similarity with the deduced amino acid sequences of Heliothis virescens juvenile hormone esterases (HvJHE). The deduced amino acid sequence of the cDNA clone contained all five functional motifs that are present in most of esterases, proteases and lipases. The cDNA clone was expressed in the baculovirus expression system, producing a protein that showed JHE activity.

  7. Cloning of the cDNA for the human. beta. /sub 1/-adrenergic receptor

    SciTech Connect

    Frielle, T.; Collins, S.; Daniel, K.W.; Caron, M.G.; Lefkowitz, R.J.; Kobilka, B.K.

    1987-11-01

    Screening of a human placenta lambdagt11 library has led to the isolation of the cDNA for the human ..beta../sub 1/-adrenergic receptor (..beta../sub 1/AR). Used as the probe was the human genomic clone termed G-21. This clone, which contains an intronless gene for a putative receptor, was previously isolated by virtue of its cross hybridization with the human ..beta../sub 2/-adrenergic receptor (..beta../sub 2/AR). The 2.4-kilobase cDNA for the human ..beta../sub 1/AR encodes a protein of 477 amino acid residues that is 69% homologous with the avian ..beta..AR but only 54% homologous with the human ..beta../sub 2/AR. This suggests that the avian gene encoding ..beta..AR and the human gene encoding ..beta../sub 1/AR evolved from a common ancestral gene. RNA blot analysis indicates a message of 2.5 kilobases in rat tissues, with a pattern of tissue distribution consistent with ..beta../sub 1/AR binding. This pattern is quite distinct from the pattern obtained when the ..beta../sub 2/AR cDNA is used as a probe. Expression of receptor protein in Xenopus laevis oocytes conveys adenylate cyclase responsiveness to catecholamines with a typical ..beta../sub 1/AR specificity. This contrasts with the typical ..beta../sub 2/ subtype specificity observed when the human ..beta../sub 2/AR cDNA is expressed in this system. Mammalian ..beta../sub 1/AR and ..beta../sub 2/AR are thus products of distinct genes, both of which are apparently related to the putative G-21 receptor.

  8. Isolation of cDNA clones coding for the beta subunit of human beta-hexosaminidase.

    PubMed Central

    O'Dowd, B F; Quan, F; Willard, H F; Lamhonwah, A M; Korneluk, R G; Lowden, J A; Gravel, R A; Mahuran, D J

    1985-01-01

    The major forms of beta-hexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) occur as multimers of alpha and beta chains--hexosaminidase A (alpha beta a beta b) and hexosaminidase B 2(beta a beta b). To facilitate the investigation of beta-chain biosynthesis and the nature of mutation in Sandhoff disease, a human hexosaminidase beta-chain cDNA clone was isolated. Hexosaminidase B (10 mg) was treated with CNBr, five peptide fragments were isolated by reverse-phase HPLC, and their amino acid sequences were determined. One of these contained a string of six amino acids from which an oligonucleotide probe was defined. The simian virus 40-transformed human fibroblast cDNA library of Okayama and Berg was screened by colony hybridization with the radiolabeled probe. Thirteen probe-binding clones were selected out of 50,000 clones screened. Four of these designated pHex were shown to be identical at their 3' ends by restriction enzyme mapping, differing only in their 5' extensions (1.4-1.7 kilobases). The nucleotide sequence of a 174-base-pair segment contained the deduced amino acid sequence of two of the five CNBr peptides, indicating that the pHex clones encode the beta subunit of hexosaminidase. In addition, pHex cDNA was found homologous to multiple bands in digests of genomic human DNA totaling 43 kilobases (kb), all of which were mapped to chromosome 5 in somatic cell hybrids, as expected of the HEXB gene. The pHex cDNA also hybridized to a 2.2-kilobase RNA that apparently codes for the pre-beta-polypeptide of hexosaminidase. This RNA species was absent in the fibroblasts of one of three patients with Sandhoff disease examined. We anticipate that these clones will be of value to diagnosis and carrier detection of Sandhoff disease in affected families. Images PMID:2579389

  9. America's Star Libraries

    ERIC Educational Resources Information Center

    Lyons, Ray; Lance, Keith Curry

    2009-01-01

    "Library Journal"'s new national rating of public libraries, the "LJ" Index of Public Library Service, identifies 256 "star" libraries. It rates 7,115 public libraries. The top libraries in each group get five, four, or three Michelin guide-like stars. All included libraries, stars or not, can use their scores to learn from their peers and improve…

  10. Personal Virtual Libraries

    ERIC Educational Resources Information Center

    Pappas, Marjorie L.

    2004-01-01

    Virtual libraries are becoming more and more common. Most states have a virtual library. A growing number of public libraries have a virtual presence on the Web. Virtual libraries are a growing addition to school library media collections. The next logical step would be personal virtual libraries. A personal virtual library (PVL) is a collection…

  11. America's Star Libraries

    ERIC Educational Resources Information Center

    Lyons, Ray; Lance, Keith Curry

    2009-01-01

    "Library Journal"'s new national rating of public libraries, the "LJ" Index of Public Library Service, identifies 256 "star" libraries. It rates 7,115 public libraries. The top libraries in each group get five, four, or three Michelin guide-like stars. All included libraries, stars or not, can use their scores to learn from their peers and improve…

  12. Personal Virtual Libraries

    ERIC Educational Resources Information Center

    Pappas, Marjorie L.

    2004-01-01

    Virtual libraries are becoming more and more common. Most states have a virtual library. A growing number of public libraries have a virtual presence on the Web. Virtual libraries are a growing addition to school library media collections. The next logical step would be personal virtual libraries. A personal virtual library (PVL) is a collection…

  13. Molecular cloning and characterization of a new cDNA sequence encoding a venom peptide from the centipede Scolopendra subspinipes mutilans.

    PubMed

    Liu, Wanhong; Luo, Feng; He, Jing; Cao, Zhijian; Miao, Lixia

    2012-01-01

    Many studies have been performed on venomous peptides derived from animals. However, little of this research has focused on peptides from centipede venoms. Here, a venom gland cDNA library was successfully constructed for the centipede Scolopendra subspinipes mutilans. A new cDNA encoding the precursor of a venom peptide, named SsmTx, was cloned from the venomous gland cDNA library of the centipede S. subspinipes mutilans. The full-length SsmTx cDNA sequence is 465 nt, including a 249 nt ORF, a 45 nt 5' UTR and a 171 nt 3' UTR. There is a signal tail AATAAA 31 nt upstream of the poly (A) tail. The precursor nucleotide sequence of SsmTx encodes a signal peptide of 25 residues and a mature peptide of 57 residues, which is bridged by two pairs of disulfide bonds. SsmTx displays a unique cysteine motif that is completely different from that of other venomous animal toxins. This is the first reported cDNA sequence encoding a venom peptide from the centipede S. subspinipes mutilans.

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

    SciTech Connect

    Tebbs, R.S.; Tucker, J.D.; Hwang, M.

    1995-07-03

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

  15. cDNA clones of the auxin-binding protein from corn coleoptiles (Zea mays L.): isolation and characterization by immunological methods.

    PubMed

    Tillmann, U; Viola, G; Kayser, B; Siemeister, G; Hesse, T; Palme, K; Löbler, M; Klämbt, D

    1989-09-01

    An auxin-binding protein (ABP) cDNA clone was selected from a lambda gt11 cDNA library from corn coleoptiles with highly purified IgGanti ABP. The sequence of 794 bp contains an open reading frame (ORF) of 603 bp, coding for a 22 kd protein. There are indications of a signal peptide of 38 amino acids (von Heijne, G. 1983, Eur. J. Biochem., 133, 17-21). A N-glycosylation site can be deduced and a C-terminal KDEL amino acid sequence is detected. An EcoRI fragment containing the beginning portion of the cDNA with about three quarters of the ORF was used to select cDNA clones from an independently produced lambda gt11 cDNA library of corn coleoptiles. Northern blot analysis with in vitro transcribed biotinylated RNA showed a single band of not more than 850 bases. The full-length in vitro transcript directed the in vitro synthesis of a protein which is precipitated by IgGanti ABP. Rabbit antibodies raised against a fusion protein detect the ABP as a double band on Western blots. Only the smaller of the two ABP bands is labeled by two different KDEL-specific IgG preparations.

  16. Infectious Maize rayado fino virus from cloned cDNA

    USDA-ARS?s Scientific Manuscript database

    Maize rayado fino virus (MRFV) is the type member of the marafiviruses within the family Tymoviridae. A cDNA clone from which infectious RNA can be transcribed was produced from a US isolate of MRFV (MRFV-US). Infectivity of transcripts derived from cDNA clones was demonstrated by infection of mai...

  17. Two distinct factor-binding DNA elements in cardiac myosin light chain 2 gene are essential for repression of its expression in skeletal muscle. Isolation of a cDNA clone for repressor protein Nished.

    PubMed

    Dhar, M; Mascareno, E M; Siddiqui, M A

    1997-07-18

    The expression of the cardiac myosin light chain 2 (MLC2) gene is repressed in skeletal muscle as a result of the negative regulation of its transcription. Two regulatory elements, the cardiac specific sequence (CSS) located upstream (-360 base pairs) and a downstream negative modulatory sequence (NMS), which function in concert with each other, are required for repression of the MLC2 promoter activity in skeletal muscle. Individually, CSS and NMS have no effect. Transient transfection analysis with recombinant plasmids indicated that CSS- and NMS-mediated repression of transcription is position- and orientation-dependent and is transferable to heterologous promoters. A minimal conserved motif, GAAG/CTTC, present in both CSS and NMS, is responsible for repression as the mutation in the core CTTC sequence alone was sufficient to abrogate its repressor activity. The DNA binding assay by gel mobility shift analysis revealed that one of the two complexes, CSSBP2, is significantly enriched in embryonic skeletal muscle relative to cardiac muscle. In extracts from adult skeletal muscle, where the cardiac MLC2 expression is suppressed, both complexes, CSSBP1 and CSSBP2, were present, whereas the cardiac muscle extracts contained CSSBP1 alone, suggesting that the protein(s) in the CSSBP2 complex accounts for the negative regulation of cardiac MLC2 in skeletal muscle. A partial cDNA clone (Nished) specific for the candidate repressor factor was isolated by expression screening of the skeletal muscle cDNA library by multimerized CSS-DNA as probe. The recombinant Nished protein binds to the CSS-DNA, but not to DeltaCSS-DNA where the core CTTC sequence was mutated. The amino acid sequence of Nished showed a significant structural similarity to the sequence of transcription factor "runt," a known repressor of gap and pair-rule gene expression in Drosophila.

  18. Underground Libraries.

    ERIC Educational Resources Information Center

    Fuhlrott, Rolf

    1986-01-01

    Discussion of underground buildings constructed primarily during last two decades for various reasons (energy conservation, density of environment, preservation of landscape and historic buildings) notes advantages, disadvantages, and psychological and design considerations. Examples of underground libraries, built mainly in United States, are…

  19. Public Libraries

    ERIC Educational Resources Information Center

    Library Journal, 1972

    1972-01-01

    Building data is given for the following public libraries: New York, New York; Blue Island, Illinois; Corte Madera, California; Muskogee, Oklahoma: Charlotte, North Carolina; Washington, D.C.; Houston, Texas; Albermarle, North Carolina; Spokane, Washington; and Hemet, California. (Author/NH)

  20. Combinatorial synthesis of deuterium-enriched (S)-oxybutynin.

    PubMed

    Li, Feng; Jiang, Wenfeng; Czarnik, Anthony W; Li, Wenbao

    2016-08-01

    The concept of deuterium enrichment has gained more attention due to its advantages in the studies of clinical pharmacokinetics and metabolic profiles. In addition, it is cost and time efficient to develop deuterium-enriched drugs. Herein we built a combinatorial library of deuterated (S)-oxybutynins which all 8 D-compounds were characterized by MS, [Formula: see text] NMR and [Formula: see text]C NMR.

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

    SciTech Connect

    Goei, V.L.; Capossela, A.; Gruen, J.R.; Parimoo, S.; Chu, T.W. )

    1994-02-01

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

  2. Cloning and sequencing of the medium-chain S-acyl fatty acid synthetase thioester hydrolase cDNA from rat mammary gland.

    PubMed Central

    Naggert, J; Williams, B; Cashman, D P; Smith, S

    1987-01-01

    cDNA clones coding for the medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II) from rat mammary gland were identified in a bacteriophage lambda gt11 library and their nucleotide sequences were determined. The predicted coding region spans 263 amino acid residues and includes a sequence identical with that of a peptide derived from the enzyme active site. The rat thioesterase II cDNA sequence exhibits homology with that of a thioesterase found in duck uropygial glands. Images Fig. 3. PMID:3632637

  3. Heterogeneity of rat type I 5 alpha-reductase cDNA: cloning, expression and regulation by pituitary implants and dihydrotestosterone.

    PubMed

    Lopez-Solache, I; Luu-The, V; Séralini, G E; Labrie, F

    1996-03-01

    Primer extension analysis reveals the presence of different forms of mRNA species for rat type I 5 alpha-reductase. Using a 5 alpha-reductase cDNA probe to screen the rat liver lambda gt11 cDNA library, we isolated cDNA clones that have 4 additional amino acids in the NH2-terminal region as compared with the previously reported sequence for rat type I 5 alpha-reductase. These four additional amino acids elongate the rat type I 5 alpha-reductase amino acid sequence to 259 amino acids, the same number as in human type I 5 alpha-reductase, with which it shares 60% identity. Expression of the long and short rat type I 5 alpha-reductase by transfection in human adrenal adenocarcinoma cells, SW-13 cells, indicated that the long cDNA encoded a protein with a higher affinity for the substrate than the short cDNA. To determine the effect of pituitary hormones and dihydrotestosterone (DHT), the mRNA levels in the livers of rats treated with pituitary implants, hypophysectomized, castrated, and castrated coupled with DHT treatment were quantified by dot-blot hybridization assay using rat type I 5 alpha-reductase cDNA as probes. The results demonstrated that rat type I 5 alpha-reductase mRNA is stimulated by pituitary hormones and castration but is decreased by DHT and hypophysectomy.

  4. Cloning and expression of the cDNA encoding human fumarylacetoacetate hydrolase, the enzyme deficient in hereditary tyrosinemia: assignment of the gene to chromosome 15.

    PubMed Central

    Phaneuf, D; Labelle, Y; Bérubé, D; Arden, K; Cavenee, W; Gagné, R; Tanguay, R M

    1991-01-01

    Type 1 hereditary tyrosinemia (HT) is an autosomal recessive disease characterized by a deficiency of the enzyme fumarylacetoacetate hydrolase (FAH; E.C.3.7.1.2). We have isolated human FAH cDNA clones by screening a liver cDNA expression library using specific antibodies and plaque hybridization with a rat FAH cDNA probe. A 1,477-bp cDNA was sequenced and shown to code for FAH by an in vitro transcription-translation assay and sequence homology with tryptic fragments of purified FAH. Transient expression of this FAH cDNA in transfected CV-1 mammalian cells resulted in the synthesis of an immunoreactive protein comigrating with purified human liver FAH on SDS-PAGE and having enzymatic activity as shown by the hydrolysis of the natural substrate fumarylacetoacetate. This indicates that the single polypeptide chain encoded by the FAH gene contains all the genetic information required for functional activity, suggesting that the dimer found in vivo is a homodimer. The human FAH cDNA was used as a probe to determine the gene's chromosomal localization using somatic cell hybrids and in situ hybridization. The human FAH gene maps to the long arm of chromosome 15 in the region q23-q25. Images Figure 1 Figure 3 Figure 4 Figure 6 Figure 8 PMID:1998338

  5. Tissue-specific expression and cDNA cloning of small nuclear ribonucleoprotein-associated polypeptide N

    SciTech Connect

    McAllister, G.; Amara, S.G.; Lerner, M.R. )

    1988-07-01

    Sera from some patients with systemic lupus erythematosus and other autoimmune diseases have antibodies against nuclear antigens. An example is anti-Sm sera, which recognize proteins associated with small nuclear RNA molecules (small nuclear ribonucleoprotein (snRNP) particles). In this paper anti-Sm sera were used to probe immunoblots of various rat tissues. A previously unidentified M{sub r} 28,000 polypeptide was recognized by these anti-Sm sera. This polypeptide, referred to as N, is expressed in a tissue-specific manner, being most abundant in rat brain, less so in heart, and undetectable in the other tissues examined. Immunoprecipitation experiments using antibodies directed against the cap structure of small nuclear RNAs have demonstrated that N is a snRNP-associated polypeptide. Anti-Sm serum was also used to isolate a partial cDNA clone ({lambda}rb91) from a rat brain phage {lambda}gt11 cDNA expression library. A longer cDNA clone was obtained by rescreening the library with {lambda}rb91. In vitro transcription and subsequent translation of this subcloned, longer insert (pGMA2) resulted in a protein product with the same electrophoretic and immunological properties as N, confirming that pGMA2 encodes N. The tissue distribution of N and the involvement of snRNP particles in nuclear pre-mRNA processing may imply a role for N in tissue-specific pre-mRNA splicing.

  6. Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee.

    PubMed

    Whitfield, Charles W; Band, Mark R; Bonaldo, Maria F; Kumar, Charu G; Liu, Lei; Pardinas, Jose R; Robertson, Hugh M; Soares, M Bento; Robinson, Gene E

    2002-04-01

    To accelerate the molecular analysis of behavior in the honey bee (Apis mellifera), we created expressed sequence tag (EST) and cDNA microarray resources for the bee brain. Over 20,000 cDNA clones were partially sequenced from a normalized (and subsequently subtracted) library generated from adult A. mellifera brains. These sequences were processed to identify 15,311 high-quality ESTs representing 8912 putative transcripts. Putative transcripts were functionally annotated (using the Gene Ontology classification system) based on matching gene sequences in Drosophila melanogaster. The brain ESTs represent a broad range of molecular functions and biological processes, with neurobiological classifications particularly well represented. Roughly half of Drosophila genes currently implicated in synaptic transmission and/or behavior are represented in the Apis EST set. Of Apis sequences with open reading frames of at least 450 bp, 24% are highly diverged with no matches to known protein sequences. Additionally, over 100 Apis transcript sequences conserved with other organisms appear to have been lost from the Drosophila genome. DNA microarrays were fabricated with over 7000 EST cDNA clones putatively representing different transcripts. Using probe derived from single bee brain mRNA, microarrays detected gene expression for 90% of Apis cDNAs two standard deviations greater than exogenous control cDNAs. [The sequence data described in this paper have been submitted to Genbank data library under accession nos. BI502708-BI517278. The sequences are also available at http://titan.biotec.uiuc.edu/bee/honeybee_project.htm.

  7. An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones

    PubMed Central

    Butterfield, Yaron S. N.; Marra, Marco A.; Asano, Jennifer K.; Chan, Susanna Y.; Guin, Ranabir; Krzywinski, Martin I.; Lee, Soo Sen; MacDonald, Kim W. K.; Mathewson, Carrie A.; Olson, Teika E.; Pandoh, Pawan K.; Prabhu, Anna-Liisa; Schnerch, Angelique; Skalska, Ursula; Smailus, Duane E.; Stott, Jeff M.; Tsai, Miranda I.; Yang, George S.; Zuyderduyn, Scott D.; Schein, Jacqueline E.; Jones, Steven J. M.

    2002-01-01

    We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites. PMID:12034834

  8. The mining of pearl formation genes in pearl oyster Pinctada fucata by cDNA suppression subtractive hybridization.

    PubMed

    Wang, Ning; Kinoshita, Shigeharu; Nomura, Naoko; Riho, Chihiro; Maeyama, Kaoru; Nagai, Kiyohito; Watabe, Shugo

    2012-04-01

    Recent researches revealed the regional preference of biomineralization gene transcription in the pearl oyster Pinctada fucata: it transcribed mainly the genes responsible for nacre secretion in mantle pallial, whereas the ones regulating calcite shells expressed in mantle edge. This study took use of this character and constructed the forward and reverse suppression subtractive hybridization (SSH) cDNA libraries. A total of 669 cDNA clones were sequenced and 360 expressed sequence tags (ESTs) greater than 100 bp were generated. Functional annotation associated 95 ESTs with specific functions, and 79 among them were identified from P. fucata at the first time. In the forward SSH cDNA library, it recognized mass amount of nacre protein genes, biomineralization genes dominantly expressed in the mantle pallial, calcium-ion-binding genes, and other biomineralization-related genes important for pearl formation. Real-time PCR showed that all the examined genes were distributed in oyster mantle tissues with a consistence to the SSH design. The detection of their RNA transcripts in pearl sac confirmed that the identified genes were certainly involved in pearl formation. Therefore, the data from this work will initiate a new round of pearl formation gene study and shed new insights into molluscan biomineralization.

  9. Minnesota Zoological Garden Library.

    ERIC Educational Resources Information Center

    Norell, Angela

    1988-01-01

    Describes the history and functions of the Minnesota Zoological Garden library. Topics covered include the library collections; library services, including online search capabilities; and the various groups of users served by the library. (three references) (CLB)

  10. Cloning and sequence analysis of an Ophiophagus hannah cDNA encoding a precursor of two natriuretic peptide domains.

    PubMed

    Lei, Weiwei; Zhang, Yong; Yu, Guoyu; Jiang, Ping; He, Yingying; Lee, Wenhui; Zhang, Yun

    2011-04-01

    The king cobra (Ophiophagus hannah) is the largest venomous snake. Despite the components are mainly neurotoxins, the venom contains several proteins affecting blood system. Natriuretic peptide (NP), one of the important components of snake venoms, could cause local vasodilatation and a promoted capillary permeability facilitating a rapid diffusion of other toxins into the prey tissues. Due to the low abundance, it is hard to purify the snake venom NPs. The cDNA cloning of the NPs become a useful approach. In this study, a 957 bp natriuretic peptide-encoding cDNA clone was isolated from an O. hannah venom gland cDNA library. The open-reading frame of the cDNA encodes a 210-amino acid residues precursor protein named Oh-NP. Oh-NP has a typical signal peptide sequence of 26 amino acid residues. Surprisingly, Oh-NP has two typical NP domains which consist of the typical sequence of 17-residue loop of CFGXXDRIGC, so it is an unusual NP precursor. These two NP domains share high amino acid sequence identity. In addition, there are two homologous peptides of unknown function within the Oh-NP precursor. To our knowledge, Oh-NP is the first protein precursor containing two NP domains. It might belong to another subclass of snake venom NPs. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli

    SciTech Connect

    Wiktorowicz, J.E.; Whitman, J.M.

    1986-05-01

    Pooled antisera against homogeneous, glutaraldehyde cross-linked hexosaminidase (hex) A was adsorbed with E. coli lysate insolubilized on Sepharose 4B. Aliquots of a human liver lambdagtll cDNA library (50,000-100,000 pfu) were plated on E. coli Y1090. Expression of cloned cDNA, after sufficient plaque growth at 42/sup 0/, was accomplished by induction with isopropylthiogalactoside soaked nitrocellulose filters. Identification of hex cDNA clones was performed by incubation of the filters with purified antisera. Protein A labelled with I-125 was used to develop the reactive plaques. Positive plaques, identified by autoradiography, were picked, replated at a lower density, and rescreened. This was repeated several more times until all plaques yielded positive signals. Identification of the clones as containing ..cap alpha.. or ..beta.. cDNA was accomplished by replating the purified phage and rescreening the plaques with anti-hex B antiserum preadsorbed with E. coli lysate. According to this protocol several hex ..cap alpha.. clones have been identified. While these clones generate ..beta..-galactosidase: hex ..cap alpha.. fusion proteins, these findings suggest that in the future it may be possible to obtain large quantities of unmodified hex ..cap alpha.. and ..beta.. polypeptides from E. coli for the study of the structural and enzymatic properties of these polypeptides and for diagnostic purposes in the GM2 gangliosidoses.

  12. Isolation of cDNA clones specifying the fourth component of mouse complement and its isotype, sex-limited protein.

    PubMed Central

    Nonaka, M; Takahashi, M; Natsuume-Sakai, S; Nonaka, M; Tanaka, S; Shimizu, A; Honjo, T

    1984-01-01

    cDNA clones specific for the fourth component of mouse complement (C4) and its hormonally regulated isotype, sex-linked protein (Slp), were isolated using as a probe a 20-mer synthetic oligonucleotide corresponding to a known sequence of human C4 cDNA. Two types of clones, one specific for C4 (pFC4/10, with a 3.7 kilobase insert) and one specific for Slp (pFSlp/1, with a 4.7 kilobase insert), were isolated from liver cDNA libraries constructed from the Slp-producing FM mouse strain. The cDNA inserts of these clones shared 70% of the restriction sites determined. Only one type of clone was isolated from the Slp-negative DBA/1 strain; this type showed restriction maps indistinguishable from that of pFC4/10. pFC4/10 and pFSlp/1 displayed extensive homology: 94% nucleotide homology and 89% derived amino acid homology in the C4a region and 92% nucleotide homology and 89% derived amino acid homology in the thiol-ester region. An Arg-Gln-Lys-Arg sequence in the beta-alpha junction and a Cys-Ala-Glu-Gln sequence in the thiol-ester site were identified for both proteins. A remarkable divergency between C4 and Slp sequences was recognized in the region immediately following the C4a sequence. PMID:6208559

  13. Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-)

    SciTech Connect

    Hirono, A.; Beutler, E. )

    1988-06-01

    Glucose-6-phosphate dehydrogenase A(-) is a common variant in Blacks that causes sensitivity to drug- and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3{prime} end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C{sup 33} {yields} G, G{sup 202} {yields} A, and A{sup 376} {yields} G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The findings of the same mutation in G6PD A(-) as is found in G6PD A(+) strongly suggests that the G6PD A(-) mutation arose in an individual with G6PD A(+), adding another mutation that causes the in vivo instability of this enzyme protein.

  14. Expressed Sequence Tags With cDNA Termini: Previously Overlooked Resources for Gene Annotation and Transcriptome Exploration in Chlamydomonas reinhardtii

    PubMed Central

    Liang, Chun; Liu, Yuansheng; Liu, Lin; Davis, Adam C.; Shen, Yingjia; Li, Qingshun Quinn

    2008-01-01

    Many of Chlamydomonas reinhardtii expressed sequence tags (ESTs) in GenBank dbEST and community EST assemblies were either over- or undertrimmed in terms of their cDNA termini, which are defined as the diagnostic sequence elements that delineate 3′/5′ ends of mRNA transcripts. Overtrimming represents a loss of directional, positional, and structural information of transcript ends whereas undertrimming causes unclean spurious sequences retained in ESTs that exert deleterious impacts on downstream EST-based applications. We examined 309,278 raw EST sequencing trace files of C. reinhardtii and found that only 57% had cDNA termini that matched the expected structures specified in their cDNA library constructions while satisfying our minimum length requirement for their final clean sequences. Using GMAP, 156,963 individual ESTs were mapped to the genome successfully, with their in silico-verified cDNA termini anchored to the genome. Our data analysis suggested strong macro- and microheterogeneity of 3′/5′ end positions of individual transcripts derived from the same genes in C. reinhardtii. This work annotating differential ends of individual transcripts in the draft genome presents the research community with a new stream of data that will facilitate accurate determination of gene structures, genome annotation, and exploration of the transcriptome and mRNA metabolism in C. reinhardtii. PMID:18493042

  15. Molecular cloning and expression in Escherichia coli of the cDNA coding for rat lipocortin I (calpactin II).

    PubMed

    Shimizu, Y; Takabayashi, E; Yano, S Y; Shimizu, N; Yamada, K; Gushima, H

    1988-05-15

    Lipocortins (LC) are a family of proteins that were initially described to be induced by glucocorticosteroids and to inhibit phospholipase A2 (PLA2). Using oligodeoxynucleotide probes corresponding to partial amino acid (aa) sequences of rat lipocortin I (LCI), we have isolated a cDNA clone for rat LCI from a cDNA library prepared from poly(A)+RNA of peritoneal cells of dexamethasone-treated rat. The cDNA insert (1355 bp) had an open reading frame of 1038 bp that encoded a 346-aa polypeptide (Mr 38,784). The nucleotide sequence and the amino acid sequence deduced from it showed high homology with the reported sequences of human LCI. A plasmid containing the trc promoter and cDNA sequence for 346 aa residues of the rat LCI was constructed and expressed in Escherichia coli. Antibody to human LCI crossreacted with the recombinant rat LCI, and the recombinant protein had characteristics of natural rat LCI including PLA2 inhibitory activity in vitro.

  16. Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends.

    PubMed

    Graham, A; Hopkins, B; Powell, S J; Danks, P; Briggs, I

    1991-05-31

    Functional cDNA clones for human NK-2 receptor were isolated from human lung RNA using a polymerase chain reaction (PCR) based method (RACE-PCR). In this method the cDNA was isolated as 5' end and 3'-end fragments; the entire cDNA was obtained by RNA-PCR. The sequence derived was 398 amino acids in length encoding an open-reading frame that was highly homologous to both the bovine and rat NK-2 receptor. The entire human cDNA sequence was cloned into a mammalian expression vector and mRNA was synthesised by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesised mRNA. The most potent of the three tachykinin peptides tested was neurokinin A. We have screened a human cosmid library and isolated a clone which contains the entire NK-2 receptor gene. The gene contains five exons and we have determined the complete sequence of the exons and the intron-exon junctions.

  17. Metadata Harvesting in Regional Digital Libraries in the PIONIER Network

    ERIC Educational Resources Information Center

    Mazurek, Cezary; Stroinski, Maciej; Werla, Marcin; Weglarz, Jan

    2006-01-01

    Purpose: The paper aims to present the concept of the functionality of metadata harvesting for regional digital libraries, based on the OAI-PMH protocol. This functionality is a part of regional digital libraries platform created in Poland. The platform was required to reach one of main objectives of the Polish PIONIER Programme--to enrich the…

  18. Metadata Harvesting in Regional Digital Libraries in the PIONIER Network

    ERIC Educational Resources Information Center

    Mazurek, Cezary; Stroinski, Maciej; Werla, Marcin; Weglarz, Jan

    2006-01-01

    Purpose: The paper aims to present the concept of the functionality of metadata harvesting for regional digital libraries, based on the OAI-PMH protocol. This functionality is a part of regional digital libraries platform created in Poland. The platform was required to reach one of main objectives of the Polish PIONIER Programme--to enrich the…

  19. Enrichment through Creative Arts.

    ERIC Educational Resources Information Center

    Krause, Claire S.

    The CREST (Creative Resources Enriching Student Talents) Project, an enrichment approach for elementary gifted, talented, and creative students, is described. The project is explained to incorporate an interdisciplinary approach to instruction in art and science using resources within the community. Chapter 1 outlines the project philosophy,…

  20. Symposium on Presidential Libraries.

    ERIC Educational Resources Information Center

    Relyea, Harold C.; And Others

    1994-01-01

    Includes five articles that discuss presidential libraries. Highlights include an overview of the development of the federal presidential library system; the Ronald Reagan library; the Richard Nixon library archives; access at the Gerald Ford library; and computerizing the Jimmy Carter library. (LRW)

  1. Turkish Libraries: Historical Context.

    ERIC Educational Resources Information Center

    Cakin, Irfan

    1984-01-01

    Summary of the development of libraries in Turkey highlights the existence of libraries in the ninth century, the Shamssaddin Altunaba Medrese library in Konya, libraries established during the Ottoman era, reports to reform libraries (1869-70, 1909), and reports and library developments attributed to the Republican Era beginning in 1923. (EJS)

  2. The Sister Libraries Programs.

    ERIC Educational Resources Information Center

    Sager, Don

    2000-01-01

    These articles describe the Sister Libraries program, part of the White House Millennium project, sponsored by the National Commission on Libraries and Information Science and the American Library Association to build relationships with libraries in other cultures. Discusses projects with public and school libraries and library development in…

  3. Turkish Libraries: Historical Context.

    ERIC Educational Resources Information Center

    Cakin, Irfan

    1984-01-01

    Summary of the development of libraries in Turkey highlights the existence of libraries in the ninth century, the Shamssaddin Altunaba Medrese library in Konya, libraries established during the Ottoman era, reports to reform libraries (1869-70, 1909), and reports and library developments attributed to the Republican Era beginning in 1923. (EJS)

  4. Library Research and Statistics.

    ERIC Educational Resources Information Center

    Lynch, Mary Jo; St. Lifer, Evan; Halstead, Kent; Fox, Bette-Lee; Miller, Marilyn L.; Shontz, Marilyn L.

    2001-01-01

    These nine articles discuss research and statistics on libraries and librarianship, including libraries in the United States, Canada, and Mexico; acquisition expenditures in public, academic, special, and government libraries; price indexes; state rankings of public library data; library buildings; expenditures in school library media centers; and…

  5. Principles and application of antibody libraries for infectious diseases.

    PubMed

    Lim, Bee Nar; Tye, Gee Jun; Choong, Yee Siew; Ong, Eugene Boon Beng; Ismail, Asma; Lim, Theam Soon

    2014-12-01

    Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

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

    PubMed Central

    Amelia, Kassim; Singh, Jasvin; Shah, Farida Habib; Bhore, Subhash J.

    2015-01-01

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

  7. Identification, characterization, and sequence analysis of a cDNA encoding a phosphoprotein of human herpesvirus 6.

    PubMed Central

    Chang, C K; Balachandran, N

    1991-01-01

    Human herpesvirus 6 (HHV-6)-specific monoclonal antibody (Mab) 9A5D12 reacted with the nucleus of HHV-6 strain GS-infected cells and immunoprecipitated a phosphorylated polypeptide with an approximate size of 41 kDa, designated HHV-6 P41. A 110-kDa polypeptide was also immunoprecipitated by the MAb. These polypeptides were synthesized early in infection, and the synthesis was greatly reduced by phosphonoacetic acid. Polypeptides with identical sizes were recognized by the MAb from cells infected with an additional eight HHV-6 strains. A 2.1-kb cDNA insert was identified from an HHV-6(GS) cDNA library constructed in the lambda gt11 expression system by using MAb 9A5D12. This cDNA insert hybridized specifically with viral DNA from HHV-6 strains GS and Z-29 and with two predominant transcripts with approximate sizes of 2.5 and 1.2 kb from infected cells. The reactivity of the MAb with a fusion protein expressed in the prokaryotic vector suggested that the cDNA encodes a 62- to 66-kDa protein. Analysis of the nucleotide sequence of the cDNA insert revealed a 623-amino-acid-residue single open reading frame of 1,871 nucleotides, with an open 5' end. The predicted polypeptide is highly basic and contains a long stretch of highly hydrophobic residues localized to the carboxy terminus. The amino-terminal half of the predicted HHV-6 protein from the cDNA shows significant homology with the UL44 gene product of human cytomegalovirus, coding for the ICP36 family of early-late-class phosphoproteins. Two TATA boxes are located at nucleotide positions 668 and 722 of the cDNA. In vitro translation of RNA transcribed in vitro from the cDNA resulted in the synthesis of a 41-kDa polypeptide only. This polypeptide was readily immunoprecipitated by MAb 9A5D12, and its partial peptide map was identical to that of the 41-kDa polypeptide detected in infected cells. Together, these results indicate that the HHV-6 P41 is encoded within a gene coding for a larger protein. Images PMID

  8. Comparison of latent and nominal rabbit Ig VHa1 allotype cDNA sequences.

    PubMed

    McCormack, W T; Dhanarajan, P; Roux, K H

    1988-09-15

    The genetic basis for the expression of a latent VH allotype in the rabbit was investigated. VH region cDNA libraries were produced from spleen mRNA derived from a homozygous a2a2 rabbit expressing an induced latent VHa1 allotype and, for comparison, from a normal homozygus a1a1 rabbit expressing nominal VHa1 allotype. The deduced amino acid sequences of the nominal VHa1 cDNA were concordant with previously published VHa1 protein sequences. A comparison of two complete VH-DH-JH and six partial VHa1 sequences reveals highly conserved sequence within VH framework regions (FR) and considerable diversity in complementarity-determining regions and D region sequences. Two functional JH genes or alleles are evident. Amino acid sequencing of the N-terminal 15 residues of pooled affinity-purified latent VHa1 H chain showed complete sequence identity with the nominal VHa1 sequences. Possible latent VHa1-encoding cDNA clones, derived from the a2a2 rabbit, were selected by hybridization with oligonucleotide probes corresponding to the VHa1 allotype-associated segments of the first and third framework regions (FR1 and FR3). cDNA sequence analysis reveals that the 5' untranslated regions of nominal and latent VHa1 cDNA were virtually identical to each other and to previously reported sequences associated with VHa2 and VHa-negative genes. Moreover, some latent VHa1 genes encode FR1 segments that are essentially homologous to the corresponding segment of a nominal VHa1 allotype. In contrast, other putative latent genes display blocks of VHa1 sequence in either FR1 or FR3 that are flanked by blocks of sequence identical to other rabbit VH genes (i.e., VHa2 or VHa-negative). These composite sequences may be directly encoded by composite germ-line VH genes or may be the products of somatically generated recombination or gene conversion between genes encoding latent and nominal allotypes. The data do not support the hypothesis that latent genes are the result of extensive modification

  9. Now's the Time: Online Library Orientations

    ERIC Educational Resources Information Center

    Farrell, Sandy L.; Driver, Carol; Weathers, Anita

    2011-01-01

    Increasingly at West Kentucky Community and Technical College (WKCTC), English 101, English 102, and Enrichment 091 are taught online, allowing students more work availability and time with family, while decreasing commute time and expense. In order to meet the library orientation needs of these online students and provide other options for…

  10. Now's the Time: Online Library Orientations

    ERIC Educational Resources Information Center

    Farrell, Sandy L.; Driver, Carol; Weathers, Anita

    2011-01-01

    Increasingly at West Kentucky Community and Technical College (WKCTC), English 101, English 102, and Enrichment 091 are taught online, allowing students more work availability and time with family, while decreasing commute time and expense. In order to meet the library orientation needs of these online students and provide other options for…

  11. Isolation of a cDNA clone encoding the amino-terminal region of human apolipoprotein B

    SciTech Connect