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Sample records for full-length enriched cdna

  1. 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

  2. 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

  3. 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

  4. Large-scale sequencing based on full-length-enriched cDNA libraries in pigs: contribution to annotation of the pig genome draft sequence

    PubMed Central

    2012-01-01

    Background Along with the draft sequencing of the pig genome, which has been completed by an international consortium, collection of the nucleotide sequences of genes expressed in various tissues and determination of entire cDNA sequences are necessary for investigations of gene function. The sequences of expressed genes are also useful for genome annotation, which is important for isolating the genes responsible for particular traits. Results We performed a large-scale expressed sequence tag (EST) analysis in pigs by using 32 full-length-enriched cDNA libraries derived from 28 kinds of tissues and cells, including seven tissues (brain, cerebellum, colon, hypothalamus, inguinal lymph node, ovary, and spleen) derived from pigs that were cloned from a sow subjected to genome sequencing. We obtained more than 330,000 EST reads from the 5′-ends of the cDNA clones. Comparison with human and bovine gene catalogs revealed that the ESTs corresponded to at least 15,000 genes. cDNA clones representing contigs and singlets generated by assembly of the EST reads were subjected to full-length determination of inserts. We have finished sequencing 31,079 cDNA clones corresponding to more than 12,000 genes. Mapping of the sequences of these cDNA clones on the draft sequence of the pig genome has indicated that the clones are derived from about 15,000 independent loci on the pig genome. Conclusions ESTs and cDNA sequences derived from full-length-enriched libraries are valuable for annotation of the draft sequence of the pig genome. This information will also contribute to the exploration of promoter sequences on the genome and to molecular biology-based analyses in pigs. PMID:23150988

  5. 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

  6. 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

    2013-01-01

    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

  7. 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

  8. 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. PMID:24630959

  9. A drosophila full-length cDNA resource

    SciTech Connect

    Stapleton, Mark; Carlson, Joseph; Brokstein, Peter; Yu, Charles; Champe, Mark; George, Reed; Guarin, Hannibal; Kronmiller, Brent; Pacleb, Joanne; Park, Soo; Rubin, Gerald M.; Celniker, Susan E.

    2003-05-09

    Background: A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages. Results: We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40 percent of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing. Conclusions: We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.

  10. [Comparison of methods to construct a full-length cDNA library].

    PubMed

    Mao, Xin-Guo; Jing, Rui-Lian; Kong, Xiu-Ying; Zhao, Guang-Yao; Jia, Ji-Zeng

    2006-07-01

    The use of full-length cDNA libraries is an effective tool to obtain complete gene information in a high-efficiency, high-throughput manner, especially in organisms with huge genomes that are not amenable to whole genome sequencing. In this review, we outlined several methods of full-length cDNA library construction and compared their advantages and disadvantages based on their respective principles. Drawing on our own experience, we described the Cap-trapper method in detail, with an emphasis on its application in wheat full-length cDNA library construction as well as the determination of the ratio of full-length cDNA in a library. PMID:16825176

  11. 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

  12. [Construction and sequencing of full-length cDNA of peste des petits ruminants virus].

    PubMed

    Zhai, Jun-Jun; Dou, Yong-Xi; Zhang, Hai-Rui; Mao, Li; Meng, Xue-Lian; Luo, Xuo-Nong; Cai, Xue-Peng

    2010-07-01

    To develop a reverse genetics system of Peste des petits ruminants virus(PPRV), five pairs of oligonucleotide primers were designed on the basis of the full-length genomic sequence of PPRV Nigeria 75/ 1 strain. Using RT-PCR technique, five over-lapping cDNA fragments, designated as JF1, JF2, JF3, JF4 and JF5, respectively, were amplified, followed by cloning into pcDNA3.1(+)vector. An AscI restriction enzyme site and a T7 promoter sequence were introduced immediately upstream of 5'-end, while a PacI restriction enzyme site was engineered downstream of 3'-end. Using pok12 as a plasmid vector, the full-length cDNA clone pok12-PPRV of Nigeria 75/1 was assembled by connecting the five cDNA fragments via the unique restriction endonuclease site of PPRV genome. The resultant nucleotide sequence of the PPRV Nigeria 75/1 strain in the study was compared with other members of genus morbillivirus, and phylogenetic analysis was used to examine the evolutionary relationships. The results showed that PPRV Nigeria 75/ 1 was antigenically closely related to Rinderpest virus and Measles virus. Successful construction of full-length cDNA clone of PPRV Nigeria 75/1 strain lays the basis rescuing PPRV effectively and enables further research of PPRV at molecular level. PMID:20836386

  13. [Rapid construction of full-length MnSOD cDNA of chickens by one-step 3'RACE].

    PubMed

    Bu, You-Quan; Luo, Xu-Gang; Liu, Bin; Li, Su-Fen

    2004-07-01

    RACE (rapid amplification of cDNA ends) is a popular technique to rapidly obtain the full-length cDNA. After obtaining the 3' cDNA and 5' cDNA fragments with a overlapped region by 3' RACE and 5' RACE, the full-length cDNA could be generated by end-to-end PCR or subcloning. In this study, 3' RACE combined with touch-down PCR was successfully used for the rapid construction of full-length MnSOD cDNA of chickens. Compared with the conventional end-to-end PCR or subcloning, this method, called one-step 3' RACE, is fast, economical and highly specific. It especially fits the rapid construction of full-length cDNA by RACE method. PMID:15640053

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  15. Full-length cDNA cloning and structural characterization of preproinsulin in Alligator sinensis.

    PubMed

    Zhang, R; Zhang, S Z; Li, E; Wang, C; Wang, C L; Wu, X B

    2014-01-01

    Insulin is an important endocrine hormone that plays a critical physiological role in regulating metabolism and glucostasis in vertebrates. In this study, the complete cDNA of Alligator sinensis preproinsulin gene was cloned for the first time by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods; the amino acid sequence encoded and protein structure were analyzed. The full-length of preproinsulin cDNA sequence consists of 528 base pairs (bp), comprising a 34-bp 5'-untranslated region, a 170-bp 3'-untranslated region and an open reading frame that is 324 bp in length. The open reading frame encodes a 107-amino acid preproinsulin with a molecular weight of approximately 12,153.8 Da, theoretical isoelectric point of 5.68, aliphatic index of 92.06, and grand average of hydropathicity of -0.157, from which a signal peptide, a B-chain, a C-peptide, and an A-chain are derived. Online analysis suggested that the deduced preproinsulin amino acid sequence contains a transmembrane region, and that it has a signal peptide whose cleavage site occurs between alanine 24 and alanine 25. Comparative analysis of preproinsulin amino acid sequences indicated that the A-chain and B-chain sequences of preproinsulins are highly conserved between reptiles and birds, and that the preproinsulin amino acid sequence of Alligator sinensis shares 89% similarity to that of Chelonia mydas, but low similarity of 48-63% to those of mammals and fishes. The phylogenetic tree constructed using the neighbor-joining method revealed that preproinsulin of Alligator sinensis had high homology with reptiles and birds, such as Chelonia mydas, Gallus gallus, and Columba livia. PMID:25366775

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

    PubMed

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

    2014-02-01

    RT-PCR, 5'RACE, 3'RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities. PMID:24381103

  17. Isolation, characterization and functional analysis of full length p53 cDNA from Bubalus bubalis.

    PubMed

    Singh, Minu; Aggarwal, Suruchi; Mohanty, Ashok K; Mukhopadhyay, Tapas

    2015-09-01

    p53 plays a pivotal role in maintaining the genomic integrity of the cell and has an important role in cellular transformation. We isolated and cloned a full length p53 cDNA (Bp53) from water buffalo in expression vectors designed to generate tagged proteins with FLAG or GFP. Bp53 was found to be 1161 nucleotide long and codes for 386 amino acid residues with 79% homology with human p53 containing 393 amino acids. Although Bp53 has some inherent differences in amino acid composition in different functional domains as compared to human p53 but the total electrostatic charge of amino acids has been maintained. Bp53 cDNA was transiently transfected in a p53 null human NSCLC cell line and as expected, it was predominantly localized in the nucleus. Besides, Bp53 effectively transactivates a number of target genes similar to human p53 and exerts most of its anti-tumorigenic potential in culture as observed in clonogenic and cell viability assays. Like human p53 mutants, core domain mutant version of Bp53 was found to be mis-localized to cytoplasm with diminished tumor suppressor activity. However, Bp53 appeared to be more sensitive to mdm2 mediated degradation and as a result, this protein was less stable as compared to human p53. For the first time we have characterized a functionally efficient wild-type p53 from buffalo having lower stability than human p53 and thus, buffalo p53 could be used as a model system for further insight to the molecular basis of wild-type p53 instability. PMID:26003295

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A full-length cDNA library with complete genomic coverage is a powerful tool for functional genomic studies. We have constructed a full-length cDNA library from urediniospores of race PST-78 of Puccinia striiformis f. sp. tritici, the fungal pathogen causing wheat stripe rust. The full-length cDNA l...

  19. Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

    PubMed Central

    Polo, S; Ketner, G; Levis, R; Falgout, B

    1997-01-01

    The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript-derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts. PMID:9188607

  20. Giardia canis: ultrastructural analysis of G. canis trophozoites transfected with full length G. canis virus cDNA transcripts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Giardia canis virus (GCV) is a double-stranded RNA (dsRNA) virus of the family Totiviridae. In this study, the full-length cDNA of the G. canis virus was constructed in pPoly2/sfinot vector and RNA was transcribed in vitro. Virus-free G. canis trophozoites were transfected with in vitro transcribed ...

  1. Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.

    PubMed

    Zhang, Quan; Liu, Long; Zhu, Feng; Ning, ZhongHua; Hincke, Maxwell T; Yang, Ning; Hou, ZhuoCheng

    2014-01-01

    Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length

  2. An efficient full-length cDNA amplification strategy based on bioinformatics technology and multiplexed PCR methods.

    PubMed

    Chen, Nan; Wang, Wei-Min; Wang, Huan-Ling

    2016-01-01

    A novel strategy for amplification full-length cDNA and promoter sequences has been developed using bioinformatics technology and multiplexed PCR methods in this study. The amplification of 3' ends of cDNA is performed according to the modified classic 3' RACE techniques, therein the more efficient and effective oligo(dT)-anchor primer with hairpin structure is specially designed. For the amplification of 5' ends of cDNA, two or three-round TAIL-PCR or touch-down PCR using arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers is performed until the 5' sequence of multi-assembled fragment reaches the exon1 region identified by aligning this fragment to reference genome database. Then another TAIL-PCR or touch-down PCR using genomic DNA as template is conducted to obtain the remaining 5' and promoter sequences. The 5' end sites of cDNA are predicted by aligning finally assembled fragment to homologous reference genes of other species, and screening the relative locations of common characteristic cis-elements in silico on promoter. The putative 5' ends are further validated by primers corresponding to these predicted sites in cDNAs. This method is suitable for researchers to isolate limited full-length cDNA sequences due to its operability, inexpensiveness, efficiency and speediness. PMID:26758040

  3. An efficient full-length cDNA amplification strategy based on bioinformatics technology and multiplexed PCR methods

    PubMed Central

    Chen, Nan; Wang, Wei-Min; Wang, Huan-Ling

    2016-01-01

    A novel strategy for amplification full-length cDNA and promoter sequences has been developed using bioinformatics technology and multiplexed PCR methods in this study. The amplification of 3′ ends of cDNA is performed according to the modified classic 3′ RACE techniques, therein the more efficient and effective oligo(dT)-anchor primer with hairpin structure is specially designed. For the amplification of 5′ ends of cDNA, two or three-round TAIL-PCR or touch-down PCR using arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers is performed until the 5′ sequence of multi-assembled fragment reaches the exon1 region identified by aligning this fragment to reference genome database. Then another TAIL-PCR or touch-down PCR using genomic DNA as template is conducted to obtain the remaining 5′ and promoter sequences. The 5′ end sites of cDNA are predicted by aligning finally assembled fragment to homologous reference genes of other species, and screening the relative locations of common characteristic cis-elements in silico on promoter. The putative 5′ ends are further validated by primers corresponding to these predicted sites in cDNAs. This method is suitable for researchers to isolate limited full-length cDNA sequences due to its operability, inexpensiveness, efficiency and speediness. PMID:26758040

  4. Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

    PubMed

    Gao, Ruimin; Niu, Shengniao; Dai, Weifang; Kitajima, Elliot; Wong, Sek-Man

    2016-10-01

    A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone. PMID:27139727

  5. [Full-length cDNA cloning of flavonol synthase genes of Carthamus tinctorius and construction plant expression vector].

    PubMed

    Yang, Wen-ting; Liu, Xiu-ming; Wan, Qiu; Yao, Na; Wang, Nan; Zhang, Xue-meng; Jiao, Zhong-da; Li, Hai-yan; Li, Xiao-kun

    2015-02-01

    Flavonol synthase (FLS) is one of the key enzymes in flavonoids metabolic pathways. In this study, middle sequence was obtained from Carthamus tinctorius transcriptome sequencing results. Full-length cDNAs of FLS was cloned from petals of C. tinctorius to FLS by using RT-PCR and RACE technology. Its full-length cDNA was 1,201 bp, with an open reading frame of 1,101 bp and 336 encoded amino acids. The phylogenetic analysis showed that, FLS gene encoded amino acids in C. tinctorius were highly homologous with amino acids in congeneric Compositae species, especially Rudbeckia laciniata. The pBASTA-FLS plant expression vector was successfully built by the molecular biology method, which lays a foundation for further studying biology functions of the gene and biosynthesis mechanism of flavonoids. PMID:26137682

  6. 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

  7. Penalized likelihood for sparse contingency tables with an application to full-length cDNA libraries

    PubMed Central

    Dahinden, Corinne; Parmigiani, Giovanni; Emerick, Mark C; Bühlmann, Peter

    2007-01-01

    Background The joint analysis of several categorical variables is a common task in many areas of biology, and is becoming central to systems biology investigations whose goal is to identify potentially complex interaction among variables belonging to a network. Interactions of arbitrary complexity are traditionally modeled in statistics by log-linear models. It is challenging to extend these to the high dimensional and potentially sparse data arising in computational biology. An important example, which provides the motivation for this article, is the analysis of so-called full-length cDNA libraries of alternatively spliced genes, where we investigate relationships among the presence of various exons in transcript species. Results We develop methods to perform model selection and parameter estimation in log-linear models for the analysis of sparse contingency tables, to study the interaction of two or more factors. Maximum Likelihood estimation of log-linear model coefficients might not be appropriate because of the presence of zeros in the table's cells, and new methods are required. We propose a computationally efficient ℓ1-penalization approach extending the Lasso algorithm to this context, and compare it to other procedures in a simulation study. We then illustrate these algorithms on contingency tables arising from full-length cDNA libraries. Conclusion We propose regularization methods that can be used successfully to detect complex interaction patterns among categorical variables in a broad range of biological problems involving categorical variables. PMID:18072965

  8. Full-Length cDNA, Prokaryotic Expression, and Antimicrobial Activity of UuHb-F-I from Urechis unicinctus

    PubMed Central

    Niu, Rongli; Chen, Xiang

    2016-01-01

    Hemoglobin, which widely exists in all vertebrates and in some invertebrates, is possibly a precursor of antimicrobial peptides (AMPs). However, AMPs in the hemoglobin of invertebrates have been rarely investigated. This study is the first to report the full-length cDNA, prokaryotic expression, and antimicrobial activity of UuHb-F-I from Urechis unicinctus. The full-length cDNA sequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids. MALDI-TOF-MS suggested that the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 15,168.01 Da, and its N-terminal amino acid sequence was MGLTGAQIDAIK. rUuHb-F-I exhibited different antimicrobial activities against microorganisms. The lowest minimum inhibitory concentration against Micrococcus luteus was 2.78–4.63 μM. Our results may help elucidate the immune defense mechanism of U. unicinctus and may provide insights into new AMPs in drug discovery. PMID:27471730

  9. Full-Length cDNA, Prokaryotic Expression, and Antimicrobial Activity of UuHb-F-I from Urechis unicinctus.

    PubMed

    Niu, Rongli; Chen, Xiang

    2016-01-01

    Hemoglobin, which widely exists in all vertebrates and in some invertebrates, is possibly a precursor of antimicrobial peptides (AMPs). However, AMPs in the hemoglobin of invertebrates have been rarely investigated. This study is the first to report the full-length cDNA, prokaryotic expression, and antimicrobial activity of UuHb-F-I from Urechis unicinctus. The full-length cDNA sequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids. MALDI-TOF-MS suggested that the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 15,168.01 Da, and its N-terminal amino acid sequence was MGLTGAQIDAIK. rUuHb-F-I exhibited different antimicrobial activities against microorganisms. The lowest minimum inhibitory concentration against Micrococcus luteus was 2.78-4.63 μM. Our results may help elucidate the immune defense mechanism of U. unicinctus and may provide insights into new AMPs in drug discovery. PMID:27471730

  10. Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries.

    PubMed

    Liu, Nan-Nan; Liu, Zeng-Shan; Lu, Shi-Ying; Hu, Pan; Li, Yan-Song; Feng, Xiao-Li; Zhang, Shou-Yin; Wang, Nan; Meng, Qing-Feng; Yang, Yong-Jie; Tang, Feng; Xu, Yun-Ming; Zhang, Wen-Hui; Guo, Xing; Chen, Xiao-Feng; Zhou, Yu; Ren, Hong-Lin

    2015-04-15

    Peroxiredoxin 6 (Prdx6), an important antioxidant enzyme that can eliminate reactive oxygen species (ROS) to maintain homeostasis, is a bifunctional protein that possesses the activities of both glutathione peroxidase and phospholipase A2. In this study, a novel full-length Prdx6 cDNA (OaPrdx6) was cloned from Sheep (Ovis aries) using rapid amplification of cDNA ends (RACE). The full-length cDNA of OaPrdx6 was 1753bp containing a 5'-untranslated region (UTR) of 93bp, a 3'-UTR of 985bp with a poly(A) tail, and an open reading frame (ORF) of 675bp encoding a protein of 224 amino acid residues with a predicted molecular weight of 25.07kDa. The recombinant protein OaPrdx6 was expressed and purified, and its DNA protection activity was identified. In order to analyze the Prdx6 protein expression in tissues from O. aries, monoclonal antibodies against OaPrdx6 were prepared. Western blotting results indicated that OaPrdx6 protein could be detected in heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node and white blood cells, and the highest expression was found in lung while the lowest expression in muscle. Compared to the normal sheep group, the mRNA transcription level of Prdx6 in buffy coat was up-regulated in the group infected with a virulent field strain of Brucella melitensis, and down-regulated in the group inoculated with a vaccine strain S2 of brucellosis. The results indicated that Prdx6 was likely to be involved in the host immune responses against Brucella infection, and probably regarded as a molecular biomarker for distinguishing between animals infected with virulent Brucella infection and those inoculated with vaccine against brucellosis. PMID:25712755

  11. Isolation and characterization of a full length cDNA for dentatorubral-pallidoluysian atrophy (DRPLA) gene

    SciTech Connect

    Oyake, M.; Onodera, O.; Ikeuchi, T.

    1994-09-01

    Hereditary dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant spinocerebellar degeneration characterized by anticipation and variable combination of symptoms including myoclonus, epilepsy, cerebellar ataxia, choleoathetosis, and dementia. Recently, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of a B37 gene on chromosome 12. To characterize functions of the DRPLA gene product, we isolated several cDNA clones for the DRPLA gene from human adult and fetus brain cDNA libraries, using an oligonucleotide flanking the CAG repeat. The cDNA spans 4247 bp in length and there is only an open reading frame coding for 986 amino acids. The CAG repeat, which is expanded in DRPLA, is located 291 bp downstream from the initiation methionine and encodes a polyglutamine tract. The deduced amino acid sequence from amino acids residues 582 to 707 has a high homology to published human hippocampus derived expressed sequence (M78755) located at chromosome 1p (63.8% identity), and 3{prime}-untranslated region of the DRPLA cDNA revealed homology to the mouse small nuclear RNA U7 gene (X54165). Northern blot analysis revealed a 4.7 knt transcript which is widely expressed in various tissues including heart, lung, kidney, placenta, skeletal muscle, and brain. In human adult brain, the transcript was broadly expressed including amygdala, caudate nucleus, corpus callosum, hippocampus, hypothalamus, substantia nigra, subthalamic nucleus and thalamus, and was not specific to the dentatorubral-pallidoluysian system. The availability of a full length cDNA will be highly useful for analyzing the pathogenesis of this unique neurodegenerative disease as well as for analyzing other CAG repeat related neurodegenerative diseases.

  12. Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA.

    PubMed Central

    Zibert, A; Maass, G; Strebel, K; Falk, M M; Beck, E

    1990-01-01

    A full-length cDNA plasmid of foot-and-mouth disease virus has been constructed. RNA synthesized in vitro by means of a bacteriophage SP6 promoter inserted in front of the cDNA led to the production of infectious particles upon transfection of BHK-21 cells. These particles were also found to be highly infectious for primary bovine kidney cells as well as for baby mice. The difficulty in cloning the foot-and-mouth disease virus cytidyl tract in Escherichia coli was circumvented by joining two separate cloned parts, representing the S and L fragments of the genome, and, in a second step, inserting a dC-dG homopolymer. Homopolymeric sequences of up to 25 cytidyl residues did not lead to the production of virus. Replicons containing poly(C) tracts long enough to permit virus replication were first established in yeast cells. One of these constructs could also be maintained in E. coli and was used to produce infectious RNA in vitro. The length of the poly(C) sequence in this cDNA plasmid was 32 nucleotides. However, the poly(C) tracts of two recombinant viruses found in transfected BHK-21 cells were 60 and 80 nucleotides long, respectively. Possible mechanisms leading to the enlargement of the poly(C) tract during virus replication are discussed. Images PMID:2159523

  13. Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries.

    PubMed

    Liu, Nan-Nan; Liu, Zeng-Shan; Hu, Pan; Zhang, Ying; Lu, Shi-Ying; Li, Yan-Song; Yang, Yong-Jie; Zhang, Dong-Song; Zhou, Yu; Ren, Hong-Lin

    2016-01-01

    Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5'-untranslated region (UTR) of 24 bp, a 3'-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future. PMID:27483239

  14. Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries

    PubMed Central

    Liu, Nan-Nan; Liu, Zeng-Shan; Hu, Pan; Zhang, Ying; Lu, Shi-Ying; Li, Yan-Song; Yang, Yong-Jie; Zhang, Dong-Song; Zhou, Yu; Ren, Hong-Lin

    2016-01-01

    Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5′-untranslated region (UTR) of 24 bp, a 3′-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future. PMID:27483239

  15. Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

    SciTech Connect

    Imanishi, Tadashi; Itoh, Takeshi; Suzuki, Yutaka; O'Donovan, Claire; Fukuchi, Satoshi; Koyanagi, Kanako O.; Barrero, Roberto A.; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Yura, Kei; Miyazaki, Satoru; Ikeo, Kazuho; Homma, Keiichi; Kasprzyk, Arek; Nishikawa, Tetsuo; Hirakawa, Mika; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Ashurst, Jennifer; Jia, Libin; Nakao, Mitsuteru; Thomas, Michael A.; Mulder, Nicola; Karavidopoulou, Youla; Jin, Lihua; Kim, Sangsoo; Yasuda, Tomohiro; Lenhard, Boris; Eveno, Eric; Suzuki, Yoshiyuki; Yamasaki, Chisato; Takeda, Jun-ichi; Gough, Craig; Hilton, Phillip; Fujii, Yasuyuki; Sakai, Hiroaki; Tanaka, Susumu; Amid, Clara; Bellgard, Matthew; de Fatima Bonaldo, Maria; Bono Hidemasa; Bromberg, Susan K.; Brookes, Anthony J.; Bruford, Elspeth; Carninci Piero; Chelala, Claude; Couillault, Christine; de Souza, Sandro J.; Debily, Marie-Anne; Devignes, Marie-Dominique; Dubchak, Inna; Endo, Toshinori; Estreicher, Anne; Eyras, Eduardo; Fukami-Kobayashi, Kaoru; Gopinath, Gopal R.; Graudens, Esther; Hahn, Yoonsoo; Han, Michael; Han, Ze-Guang; Hanada, Kousuke; Hanaoka, Hideki; Harada, Erimi; Hashimoto, Katsuyuki; Hinz, Ursula; Hirai, Momoki; Hishiki, Teruyoshi; Hopkinson, Ian; Imbeaud, Sandrine; Inoko, Hidetoshi; Kanapin, Alexander; Kaneko, Yayoi; Kasukawa, Takeya; Kelso, Janet; Kersey, Paul; Kikuno Reiko; Kimura, Kouichi; Korn, Bernhard; Kuryshev, Vladimir; Makalowska, Izabela; Makino Takashi; Mano, Shuhei; Mariage-Samson, Regine; Mashima, Jun; Matsuda, Hideo; Mewes, Hans-Werner; Minoshima, Shinsei; Nagai, Keiichi; Nagasaki, Hideki; Nagata, Naoki; Nigam, Rajni; Ogasawara, Osamu; Ohara, Osamu; Ohtsubo, Masafumi; Okada, Norihiro; Okido, Toshihisa; Oota, Satoshi; Ota, Motonori; Ota, Toshio; Otsuki, Tetsuji; Piatier-Tonneau, Dominique; Poustka, Annemarie; Ren, Shuang-Xi; Saitou, Naruya; Sakai, Katsunaga; Sakamoto, Shigetaka; Sakate, Ryuichi; Schupp, Ingo; Servant, Florence; Sherry, Stephen; Shiba Rie; et al.

    2004-01-15

    The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4 percent of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5 percent of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for nonprotein-coding RNA

  16. 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. PMID:22516182

  17. 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

  18. 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

    2013-09-01

    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

  19. 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

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

    SciTech Connect

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

    2005-10-31

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

  1. Computer-Based Methods for the Mouse Full-Length cDNA Encyclopedia: Real-Time Sequence Clustering for Construction of a Nonredundant cDNA Library

    PubMed Central

    Konno, Hideaki; Fukunishi, Yoshifumi; Shibata, Kazuhiro; Itoh, Masayoshi; Carninci, Piero; Sugahara, Yuichi; Hayashizaki, Yoshihide

    2001-01-01

    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/). [The sequence data described in this paper have been submitted to the DDBJ data library under accession nos. AV00011–AV175734, AV204013–AV382295, and BB561685–BB609425.] PMID:11157791

  2. Identification and expression analysis of a full-length cDNA encoding a Kandelia candel tonoplast intrinsic protein.

    PubMed

    Huang, Wei; Fang, Xiao-Dong; Lin, Qi-Fen; Li, Guan-Yi; Zhao, Wen-Ming

    2003-03-01

    corresponding to the 5' end of this gene was obtained using the GSP2 primer. Two primers that flank the putative open reading frame (ORF) were designed to obtain the cDNA containing the complete ORF by RACE PCR reaction. The full-length cDNA of KCTIP1, containing a 756 bp open reading frame (ORF), was approximately 1.1 kb; the start codon was located at the nucleotides of 99-101 and stop codon at the nucleotides of 855-857 followed by a poly (A) tail. The KCTIP1 cDNA sequence in this research was released in GenBank with accession number AF521135. Using ExPASy Proteomics tools provided by EMBL, the isoelectric point and MWt of KCTIP1 are estimated as 5.77 and 26.3 kD respectively. Transmembrane prediction analysis revealed the deduced KCTIP1 protein sequence contains six transmembrane regions at amino acid residues of 20 - 42, 57 - 79, 86 - 108, 113 - 135, 142 - 164 and 217 - 239. Two highly conserved asparagine-proline-alanine (NPA) motifs were located at 85 - 87 and 199 - 201 amino acid residues respectively. KCTIP1 is also predicted to contain the Cys residue (Cys 118) that are shown to confer Hg-sensitivity in Arabidopsis gamma-TIP and delta-TIP. Similarity analysis showed that KCTIP1 shared 77% - 79% amino acid sequence identity with the TIPs from Vitis berlandieri, Brassica oleracea and Arabidopsis thaliana. Expression analyses indicated that KCTIP1 had different expression among species of Mangroves. Expressions of KCTIP1 in Kandelia candel, Rhizophora apoculata and Ceriops tagal were suppressed by salt, and were insensitive to salt stress in unknown species of Mangroves. Previous studied showed that salt conditions might result in large and rapid changes in extracellular water potential and serious disturbance to the cytoplasm. In order to compensate for this imbalance, the relative contribution of water channels to flow across the root could thus vary. K. candel is a species that is native to intertial zone of tropical and subtropical coast and is well-adapted to salt

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

    PubMed Central

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

    2014-01-01

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

  4. Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a De Novo-Reference Hybrid Assembly

    PubMed Central

    Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka

    2010-01-01

    Background Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. Methodology We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence ∼800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. Conclusions The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only ∼US$3 per clone, demonstrating a significant advantage over previous approaches. PMID:20479877

  5. Identification of genes expressed in human CD34+ hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning

    PubMed Central

    Mao, Mao; Fu, Gang; Wu, Ji-Sheng; Zhang, Qing-Hua; Zhou, Jun; Kan, Li-Xin; Huang, Qiu-Hua; He, Kai-Li; Gu, Bai-Wei; Han, Ze-Guang; Shen, Yu; Gu, Jian; Yu, Ya-Ping; Xu, Shu-Hua; Wang, Ya-Xin; Chen, Sai-Juan; Chen, Zhu

    1998-01-01

    Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34+ HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34+ cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2,603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5′ ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation. PMID:9653160

  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. Identification and isolation of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from guarana (Paullinia cupana).

    PubMed

    Figueirêdo, L C; Faria-Campos, A C; Astolfi-Filho, S; Azevedo, J L

    2011-01-01

    The current intense production of biological data, generated by sequencing techniques, has created an ever-growing volume of unanalyzed data. We reevaluated data produced by the guarana (Paullinia cupana) transcriptome sequencing project to identify cDNA clones with complete coding sequences (full-length clones) and complete sequences of genes of biotechnological interest, contributing to the knowledge of biological characteristics of this organism. We analyzed 15,490 ESTs of guarana in search of clones with complete coding regions. A total of 12,402 sequences were analyzed using BLAST, and 4697 full-length clones were identified, responsible for the production of 2297 different proteins. Eighty-four clones were identified as full-length for N-methyltransferase and 18 were sequenced in both directions to obtain the complete genome sequence, and confirm the search made in silico for full-length clones. Phylogenetic analyses were made with the complete genome sequences of three clones, which showed only 0.017% dissimilarity; these are phylogenetically close to the caffeine synthase of Theobroma cacao. The search for full-length clones allowed the identification of numerous clones that had the complete coding region, demonstrating this to be an efficient and useful tool in the process of biological data mining. The sequencing of the complete coding region of identified full-length clones corroborated the data from the in silico search, strengthening its efficiency and utility. PMID:21732283

  8. Development of an agroinoculation system for full-length and GFP-tagged cDNA clones of cucumber green mottle mosaic virus.

    PubMed

    Zheng, Hongying; Xiao, Caili; Han, Kelei; Peng, Jiejun; Lin, Lin; Lu, Yuwen; Xie, Li; Wu, Xiaohua; Xu, Pei; Li, Guojing; Chen, Jianping; Yan, Fei

    2015-11-01

    The complete 6243-nucleotide sequence of a cucumber green mottle mosaic virus (CGMMV) isolate from bottle gourd in Zhejiang province, China, was determined. A full-length cDNA clone of this isolate was constructed by inserting the cDNA between the 35S promoter and the ribozyme in the binary plasmid pCB301-CH. A suspension of an Agrobacterium tumefaciens EHA105 clone carrying this construct was highly infectious in Nicotiana benthamiana and bottle gourd. Another infectious clone containing the green fluorescence protein (GFP) reporter gene was also successfully constructed. This study is the first report of the efficient use of agroinoculation for generating CGMMV infections. PMID:26323263

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

    PubMed

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

    2015-12-01

    Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure. PMID:26633465

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

    PubMed Central

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

    2015-01-01

    Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure. PMID:26633465

  11. Characterisation of a new infectious full-length cDNA clone of BVDV genotype 2 and generation of virus mutants.

    PubMed

    Mischkale, Katrin; Reimann, Ilona; Zemke, J; König, P; Beer, Martin

    2010-04-21

    Based on their genomic sequences, two genotypes of Bovine viral diarrhea virus (BVDV) can be differentiated, BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). The complete genomic sequence of the highly virulent BVDV-2 strain 890 was cloned as cDNA to establish the infectious cDNA clone p890FL. In vitro-synthesised full-length RNA of p890FL was transfected into bovine cells and infectious virus could be recovered (v890FL). In vitro, recombinant v890FL showed similar growth characteristics as wild type virus v890WT. However, infection experiments in calves revealed an attenuation of recombinant v890FL in comparison to the parental isolate. Both leukocytopenia and fever were less pronounced in v890FL-infected calves. Nevertheless, viremia and virus shedding were comparable between recombinant and parental BVDV 890. Furthermore, mutants with partial deletions of the genomic region encoding for the autoprotease N(pro) (p890DeltaN(pro)) or the capsid protein (p890DeltaC) were constructed and characterised. In order to generate pseudovirions, replicon p890DeltaC was efficiently trans-complemented on a helper cell line. In summary, the newly developed construct p890FL represents the first infectious full-length cDNA clone for the BVDV-2 strain 890 and offers a useful tool for further studies on the pathogenesis of BVDV-2 and the development of novel recombinant BVDV-2 specific vaccine candidates. PMID:19875251

  12. Development of three full-length infectious cDNA clones of distinct brassica yellows virus genotypes for agrobacterium-mediated inoculation.

    PubMed

    Zhang, Xiao-Yan; Dong, Shu-Wei; Xiang, Hai-Ying; Chen, Xiang-Ru; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

    2015-02-01

    Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes. PMID:25499296

  13. Construction and analysis of infectious transcripts synthesized from full-length cDNA clones of both genomic RNAs of pea early browning virus.

    PubMed

    MacFarlane, S A; Wallis, C V; Taylor, S C; Goulden, M G; Wood, K R; Davies, J W

    1991-05-01

    Full-length cDNA clones of both RNAs of pea early browning virus have been constructed. Synthetic transcripts derived in vitro from these clones are infectious when inoculated onto plants. Electron microscopy revealed the presence of virions in transcript-inoculated plants, and both purified RNA and virions isolated from such plants could be used to infect other plants. Transcripts of RNA1 alone were able to replicate and spread systemically which is a characteristic of members of the tobravirus group of plant viruses. PMID:2024460

  14. Molecular and biological characterization of highly infectious transcripts from full-length cDNA clones of broad bean wilt virus 1.

    PubMed

    Ferriol, Inmaculada; Ambrós, Silvia; da Silva, Dorivaldo M; Falk, Bryce W; Rubio, Luis

    2016-06-01

    Broad bean wilt virus 1 (BBWV-1), genus Fabavirus, has a genome composed of two single-stranded positive-sense RNAs of ∼5.8 (RNA1) and 3.4kb (RNA2). Full-length cDNA clones of both genomic RNAs (pBenR1 and pBenR2) from BBWV-1 isolate Ben were constructed under the control of the T7 promoter. In vitro derived capped transcripts were infectious in Nicotiana benthamiana, Chenopodium quinoa and Vicia faba plants. The biological activity of viral transcripts was not affected by extra bases at the 5'-terminus introduced during in vitro transcription. Virions derived from the infectious cDNA clones displayed similar viral infectivity and accumulation, as well as symptom induction as the wild-type BBWV-1 isolate. PMID:26951858

  15. Characterization of a full-length infectious cDNA clone and a GFP reporter derivative of the oncolytic picornavirus SVV-001.

    PubMed

    Poirier, John T; Reddy, P Seshidhar; Idamakanti, Neeraja; Li, Shawn S; Stump, Kristine L; Burroughs, Kevin D; Hallenbeck, Paul L; Rudin, Charles M

    2012-12-01

    Seneca Valley virus (SVV-001) is an oncolytic picornavirus with selective tropism for a subset of human cancers with neuroendocrine differentiation. To characterize further the specificity of SVV-001 and its patterns and kinetics of intratumoral spread, bacterial plasmids encoding a cDNA clone of the full-length wild-type virus and a derivative virus expressing GFP were generated. The full-length cDNA of the SVV-001 RNA genome was cloned into a bacterial plasmid under the control of the T7 core promoter sequence to create an infectious cDNA clone, pNTX-09. A GFP reporter virus cDNA clone, pNTX-11, was then generated by cloning a fusion protein of GFP and the 2A protein from foot-and-mouth disease virus immediately following the native SVV-001 2A sequence. Recombinant GFP-expressing reporter virus, SVV-GFP, was rescued from cells transfected with in vitro RNA transcripts from pNTX-11 and propagated in cell culture. The proliferation kinetics of SVV-001 and SVV-GFP were indistinguishable. The SVV-GFP reporter virus was used to determine that a subpopulation of permissive cells is present in small-cell lung cancer cell lines previously thought to lack permissivity to SVV-001. Finally, it was shown that SVV-GFP administered to tumour-bearing animals homes in to and infects tumours whilst having no detectable tropism for normal mouse tissues at 1×10(11) viral particles kg(-1), a dose equivalent to that administered in ongoing clinical trials. These infectious clones will be of substantial value in further characterizing the biology of this virus and as a backbone for the generation of additional oncolytic derivatives. PMID:22971818

  16. Completion of Kunjin virus RNA sequence and recovery of an infectious RNA transcribed from stably cloned full-length cDNA.

    PubMed Central

    Khromykh, A A; Westaway, E G

    1994-01-01

    Completion of the Kunjin virus (KUN) RNA sequence showed that it is the longest flavivirus sequence reported (11,022 bases), commencing with a 5' noncoding region of 96 bases. The 3' noncoding sequence of 624 nucleotides included a unique insertion sequence of 46 bases adjacent to the stop codon, but otherwise it had properties similar to those of RNAs of closely related flaviviruses. A full-length KUN cDNA clone which could be stably propagated in Escherichia coli DH5 alpha was constructed; SP6 polymerase RNA transcripts from amplified cDNA were infectious when transfected into BHK-21 cells. A mutational change abolishing the BamHI restriction site at position 4049, leading to a conservative amino acid change of Arg-175 to Lys in the NS2A protein, was introduced into the cDNA during construction and was retained in the recovered virus. Extra terminal nucleotides introduced during cloning of the cDNA were shown to be present in the in vitro RNA transcripts but absent in the RNA of recovered virus. Although recovered virus differed from the parental KUN by a smaller plaque phenotype and delayed growth rate in BHK-21 cells and mice, it was very similar as assessed by several other criteria, such as peak titer during growth in cells, infectivity titer in cells and in mice, rate of adsorption and penetration in cells, replication at 39 degrees C, and neurovirulence after intraperitoneal injection in mice. The KUN stably cloned cDNA will provide a useful basis for future studies in defining and characterizing functional roles of all the gene products. Images PMID:8207832

  17. Expression of a full-length cDNA for the human MDR1 gene confers resistance to colchicine, doxorubicin, and vinblastine

    SciTech Connect

    Ueda, K.; Cardarelli, C.; Gottesman, M.M.; Pastan, I.

    1987-05-01

    Intrinsic and acquired multidrug resistance (MDR) is an important problem in cancer therapy. MDR in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or doxorubicin (former generic name adriamycin) is associated with overexpression of the MDR1 gene, which encodes P-glycoprotein. The authors previously have isolated an overlapping set of cDNA clones for the human MDR1 gene from multidrug-resistant KB cells. Here they report the construction of a full-length cDNA for the human MDR1 gene and show that this reconstructed cDNA, when inserted into a retroviral expression vector containing the long terminal repeats of Moloney leukemia virus or Harvey sarcoma virus, functions in mouse NIH 3T3 and human KB cells to confer the complete multidrug-resistance phenotype. These results suggest that the human MDR1 gene may be used as a positive selectable marker to introduce genes into human cells and to transform human cells to multidrug resistance without introducing nonhuman antigens.

  18. Insights from computational analysis of full-length β-ketoacyl-[ACP] synthase-II cDNA isolated from American and African oil palms

    PubMed Central

    Bhore, Subhash J.; Cha, Thye S.; Amelia, Kassim; Shah, Farida H.

    2014-01-01

    Background: Palm oil derived from fruits (mesocarp) of African oil palm (Elaeis guineensis Jacq. Tenera) and American oil palm (E. oleifera) is important for food industry. Due to high yield, Elaeis guineensis (Tenera) is cultivated on commercial scale, though its oil contains high (~54%) level of saturated fatty acids. The rate-limiting activity of beta-ketoacyl-[ACP] synthase-II (KAS-II) is considered mainly responsible for the high (44%) level of palmitic acid (C16:0) in the oil obtained from E. guineensis. Objective: The objective of this study was to annotate KAS-II cDNA isolated from American and African oil palms. Materials and Methods: The full-length E. oleifera KAS-II (EoKAS-II) cDNA clone was isolated using random method of gene isolation. Whereas, the E. guineensis KAS-II (EgTKAS-II) cDNA was isolated using reverse transcriptase polymerase chain reaction (RT-PCR) technique; and missing ends were obtained by employing 5’and 3’ RACE technique. Results: The results show that EoKAS-II and EgTKAS-II open reading frames (ORFs) are of 1689 and 1721 bp in length, respectively. Further analysis of the both EoKAS-II and EgTKAS-II predicted protein illustrates that they contains conserved domains for ‘KAS-I and II’, ‘elongating’ condensing enzymes, ‘condensing enzymes super-family’, and ‘3-oxoacyl-[ACP] synthase II’. The predicted protein sequences shows 95% similarity with each other. Consecutively, the three active sites (Cys, His, and His) were identified in both proteins. However, difference in positions of two active Histidine (His) residues was noticed. Conclusion: These insights may serve as the foundation in understanding the variable activity of KAS-II in American and African oil palms; and cDNA clones could be useful in the genetic engineering of oil palms. PMID:24678202

  19. 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

  20. Agroinoculation of a full-length cDNA clone of cotton leafroll dwarf virus (CLRDV) results in systemic infection in cotton and the model plant Nicotiana benthamiana.

    PubMed

    Delfosse, Verónica C; Casse, María F; Agrofoglio, Yamila C; Kresic, Iván Bonacic; Hopp, Horacio E; Ziegler-Graff, Véronique; Distéfano, Ana J

    2013-07-01

    Cotton blue disease is the most important viral disease of cotton in the southern part of South America. Its etiological agent, cotton leafroll dwarf virus (CLRDV), is specifically transmitted to host plants by the aphid vector (Aphis gossypii) and any attempt to perform mechanical inoculations of this virus into its host has failed. This limitation has held back the study of this virus and the disease it causes. In this study, a full-length cDNA of CLRDV was constructed and expressed in vivo under the control of cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system for the cloned cDNA construct of CLRDV was developed. Northern and immunoblot analyses showed that after several weeks the replicon of CLRDV delivered by Agrobacterium tumefaciens in Gossypium hirsutum plants gave rise to a systemic infection and typical blue disease symptoms correlated to the presence of viral RNA and P3 capsid protein. We also demonstrated that the virus that accumulated in the agroinfected plants was transmissible by the vector A. gossypii. This result confirms the production of biologically active transmissible virions. In addition, the clone was infectious in Nicotiana benthamiana plants which developed interveinal chlorosis three weeks postinoculation and CLRDV was detected both in the inoculated and systemic leaves. Attempts to agroinfect Arabidopsis thaliana plants were irregularly successful. Although no symptoms were observed, the P3 capsid protein as well as the genomic and subgenomic RNAs were irregularly detected in systemic leaves of some agroinfiltrated plants. The inefficient infection rate infers that A. thaliana is a poor host for CLRDV. This is the first report on the construction of a biologically-active infectious full-length clone of a cotton RNA virus showing successful agroinfection of host and non-host plants. The system herein developed will be useful to study CLRDV viral functions and plant-virus interactions using a reverse

  1. [Rescue of bovine Asia 1 serotype foot-and-mouth disease virus from a full-length cDNA clone].

    PubMed

    Li, Shuang; Zhang, Runxiang; Song, Ge; Gao, Mingchun; Liu, Xiangtao; Wang, Junwei

    2009-11-01

    After sequencing the Asia 1 foot-and-mouth disease virus (FMDV) (As01 strain), we amplified the two fragments covering the whole genome by overlapping PCR and long PCR. The 5' fragment was 1.8 kb in length including 15Cs, and the 3' fragment was 6.7 kb in length. The two fragments were cloned into the pBluescript SK vector to construct recombinant plasmid pBSAs carrying the full-length cDNA of FMDV As01 strain. The RNA transcript was synthesized in vitro using T7 polymerase and transfected into BHK-21 cells. We observed the typical CPE caused by rescued FMDV. The harvested virus was confirmed to be Asia 1 FMDV by RT-PCR, indirect immunofluorescence assay (IFA) and electron microscope observation. The rescued virus showed a similar pathogenicity in suckling mouse (LD50) compared to its wild-type virus. The infectious cDNA clone of the FMDV As01 strain laid a new ground for further investigation of FMDV virulence determinants and development of novel vaccines against FMD. PMID:20222458

  2. Full-Length Venom Protein cDNA Sequences from Venom-Derived mRNA: Exploring Compositional Variation and Adaptive Multigene Evolution

    PubMed Central

    Modahl, Cassandra M.; Mackessy, Stephen P.

    2016-01-01

    Envenomation of humans by snakes is a complex and continuously evolving medical emergency, and treatment is made that much more difficult by the diverse biochemical composition of many venoms. Venomous snakes and their venoms also provide models for the study of molecular evolutionary processes leading to adaptation and genotype-phenotype relationships. To compare venom complexity and protein sequences, venom gland transcriptomes are assembled, which usually requires the sacrifice of snakes for tissue. However, toxin transcripts are also present in venoms, offering the possibility of obtaining cDNA sequences directly from venom. This study provides evidence that unknown full-length venom protein transcripts can be obtained from the venoms of multiple species from all major venomous snake families. These unknown venom protein cDNAs are obtained by the use of primers designed from conserved signal peptide sequences within each venom protein superfamily. This technique was used to assemble a partial venom gland transcriptome for the Middle American Rattlesnake (Crotalus simus tzabcan) by amplifying sequences for phospholipases A2, serine proteases, C-lectins, and metalloproteinases from within venom. Phospholipase A2 sequences were also recovered from the venoms of several rattlesnakes and an elapid snake (Pseudechis porphyriacus), and three-finger toxin sequences were recovered from multiple rear-fanged snake species, demonstrating that the three major clades of advanced snakes (Elapidae, Viperidae, Colubridae) have stable mRNA present in their venoms. These cDNA sequences from venom were then used to explore potential activities derived from protein sequence similarities and evolutionary histories within these large multigene superfamilies. Venom-derived sequences can also be used to aid in characterizing venoms that lack proteomic profiles and identify sequence characteristics indicating specific envenomation profiles. This approach, requiring only venom, provides

  3. Full-Length Venom Protein cDNA Sequences from Venom-Derived mRNA: Exploring Compositional Variation and Adaptive Multigene Evolution.

    PubMed

    Modahl, Cassandra M; Mackessy, Stephen P

    2016-06-01

    Envenomation of humans by snakes is a complex and continuously evolving medical emergency, and treatment is made that much more difficult by the diverse biochemical composition of many venoms. Venomous snakes and their venoms also provide models for the study of molecular evolutionary processes leading to adaptation and genotype-phenotype relationships. To compare venom complexity and protein sequences, venom gland transcriptomes are assembled, which usually requires the sacrifice of snakes for tissue. However, toxin transcripts are also present in venoms, offering the possibility of obtaining cDNA sequences directly from venom. This study provides evidence that unknown full-length venom protein transcripts can be obtained from the venoms of multiple species from all major venomous snake families. These unknown venom protein cDNAs are obtained by the use of primers designed from conserved signal peptide sequences within each venom protein superfamily. This technique was used to assemble a partial venom gland transcriptome for the Middle American Rattlesnake (Crotalus simus tzabcan) by amplifying sequences for phospholipases A2, serine proteases, C-lectins, and metalloproteinases from within venom. Phospholipase A2 sequences were also recovered from the venoms of several rattlesnakes and an elapid snake (Pseudechis porphyriacus), and three-finger toxin sequences were recovered from multiple rear-fanged snake species, demonstrating that the three major clades of advanced snakes (Elapidae, Viperidae, Colubridae) have stable mRNA present in their venoms. These cDNA sequences from venom were then used to explore potential activities derived from protein sequence similarities and evolutionary histories within these large multigene superfamilies. Venom-derived sequences can also be used to aid in characterizing venoms that lack proteomic profiles and identify sequence characteristics indicating specific envenomation profiles. This approach, requiring only venom, provides

  4. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    NASA Astrophysics Data System (ADS)

    Qi, Fei; Guo, Huarong; Wang, Jian

    2008-02-01

    Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  5. Cloning, expression and protective immunity evaluation of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum.

    PubMed

    Yu, JunLong; Wang, ShiPing; Li, WenKai; Dai, Gan; Xu, ShaoRui; He, Zhuo; Peng, XianChu; Zhou, SongHua; Liu, XueQin

    2007-04-01

    1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3' and 5' ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2 pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (positive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also suggested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity. PMID:17447029

  6. Complete genome sequence and construction of infectious full-length cDNA clones of tobacco ringspot Nepovirus, a viral pathogen causing bud blight in soybean.

    PubMed

    Zhao, Fumei; Hwang, Un Sun; Lim, Seungmo; Yoo, Ran Hee; Igori, Davaajargal; Lee, Su-Heon; Lim, Hyoun-Sub; Moon, Jae Sun

    2015-08-01

    Tobacco ringspot virus (TRSV, genus Nepovirus), causes severe diseases in soybean and tobacco plants. TRSV-induced bud blight disease significantly reduced both the yield and quality of soybeans. The function of the encoded viral gene product involved in TRSV infection was unclear due to the limitation of reverse genetics studies on the viral genome. Here, we represent the successful construction of infectious full-length cDNA clones of TRSV genome (RNA1 and RNA2). The cDNAs of TRSV RNA1 and RNA2 were cloned into the binary vector pPZP211 immediately downstream of a double cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator. Seven days after agrobacterium-mediated co-inoculation of these two constructs, Nicotiana benthamiana plants developed a systemic infection with necrotic ringspot symptoms and weak stunting of the leaves, similar to that induced by natural TRSV. The systemic infection was confirmed by transmission electron microscopy and Western blot analysis. Simultaneously, soybean, tomato, and Arabidopsis ecotype Estland were mechanically inoculated with sap prepared from TRSV-agroinfiltrated N. benthamiana leaves, showing typical symptoms of bud blight, necrotic spots, and lethal systemic necrosis, respectively. The system developed herein will be an appealing way to determine TRSV viral gene functions and study host-TRSV interactions. PMID:26159876

  7. 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.

  8. 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.

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

    SciTech Connect

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

    1987-04-01

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

  10. Expression of active iron regulatory factor from a full-length human cDNA by in vitro transcription/translation.

    PubMed Central

    Hirling, H; Emery-Goodman, A; Thompson, N; Neupert, B; Seiser, C; Kühn, L C

    1992-01-01

    Iron regulatory factor (IRF), also called iron responsive element-binding protein (IRE-BP), is a cytoplasmic RNA-binding protein which regulates post-transcriptionally transferrin receptor mRNA stability and ferritin mRNA translation. By using the polymerase chain reaction (PCR) and the sequence published by Rouault et al. (1990) a probe was derived which permitted the isolation of three human IRF cDNA clones. Hybridization to genomic DNA and mRNA, as well as sequencing data indicated a single copy gene of about 40 kb specifying a 4.0 kb mRNA that translates into a protein of 98,400 dalton. By in vitro transcription of a assembled IRF cDNA coupled to in vitro translation in a wheat germ extract, we obtained full sized IRF that bound specifically to a human ferritin IRE. In vitro translated IRF retained sensitivity to sulfhydryl oxidation by diamide and could be reactivated by beta-mercaptoethanol in the same way as native placental IRF. An IRF deletion mutant shortened by 132 amino acids at the COOH-terminus was no longer able to bind to an IRE, indicating that this region of the protein plays a role in RNA recognition. Placental IRF has previously been shown to migrate as a doublet on SDS-polyacrylamide gels. After V8 protease digestion the heterogeneity was located in a 65/70 kDa NH2-terminal doublet. The liberated 31 kDa COOH-terminal polypeptide was found to be homogeneous by amino acid sequencing supporting the conclusion of a single IRF gene. Images PMID:1738601

  11. Molecular cloning of a full-length cDNA for dentatorubral-pallidoluysian atrophy and regional expressions of the expanded alleles in the CNS

    SciTech Connect

    Onodera, Osamu; Oyake, Mutsuo; Takano, Hiroki

    1995-11-01

    Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by genetic anticipation and variable combinations of symptoms including myoclonus, epilepsy, cerebellar ataxia, choreoathetosis, and dementia. Recently, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of a gene on the short arm of chromosome 12. We determined the consensus DRPLA cDNA sequence containing the complete coding region for 1,185 amino acids. The CAG repeat, which is expanded in DRPLA, is located 1,462 bp downstream from the putative methionine initiation codon and encodes a poly-glutamine tract. Although poly-serine and proline tracts exist near the CAG repeats, these poly-serine or proline tracts did not show any polymorphisms, which is in strong contrast to the high heterogeneity in the length of the CAG repeat. Northern blot analysis revealed a 4.7-kb transcript that is widely expressed in various tissues including heart, lung, kidney, placenta, skeletal muscle, and brain. Reverse transcription-PCR analysis revealed that the expanded alleles are transcribed to levels comparable to those of normal alleles. These results indicate that there is no difference in transcriptional efficiency between expanded and normal alleles. Furthermore, mRNA from cerebellar hemispheres of DRPLA patients showed smaller sizes of CAG repeats compared with other regions of the brain, which reflects somatic mosaicism of the expanded alleles of the DRPLA gene. 49 refs., 6 figs.

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

    PubMed

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

    2014-12-01

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

  13. Express Sequence Tag Analysis - Identification of Anseriformes Trypsin Genes from Full-Length cDNA Library of the Duck (Anas platyrhynchos) and Characterization of Their Structure and Function.

    PubMed

    Yu, Haining; Cai, Shasha; Gao, Jiuxiang; Wang, Chen; Qiao, Xue; Wang, Hui; Feng, Lan; Wang, Yipeng

    2016-02-01

    Trypsins are key proteins important in animal protein digestion by breaking down the peptide bonds on the carboxyl side of lysine and arginine residues, hence it has been used widely in various biotechnological processes. In the current study, a full-length cDNA library with capacity of 5·10(5) CFU/ml from the duck (Anas platyrhynchos) was constructed. Using express sequence tag (EST) sequencing, genes coding two trypsins were identified and two full-length trypsin cDNAs were then obtained by rapid-amplification of cDNA end (RACE)-PCR. Using Blast, they were classified into the trypsin I and II subfamilies, but both encoded a signal peptide, an activation peptide, and a 223-a.a. mature protein located in the C-terminus. The two deduced mature proteins were designated as trypsin-IAP and trypsin-IIAP, and their theoretical isoelectric points (pI) and molecular weights (MW) were 7.99/23466.4 Da and 4.65/24066.0 Da, respectively. Molecular characterizations of genes were further performed by detailed bioinformatics analysis. Phylogenetic analysis revealed that trypsin-IIAP has an evolution pattern distinct from trypsin-IAP, suggesting its evolutionary advantage. Then the duck trypsin-IIAP was expressed in an Escherichia coli system, and its kinetic parameters were measured. The three dimensional structures of trypsin-IAP and trypsin-IIAP were predicted by homology modeling, and the conserved residues required for functionality were identified. Two loops controlling the specificity of the trypsin and the substrate-binding pocket represented in the model are almost identical in primary sequences and backbone tertiary structures of the trypsin families. PMID:27260395

  14. Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA.

    PubMed

    Bascomb, N F; Turner, K J; Schmidt, R R

    1986-06-01

    A specific polysome immunoadsorption procedure, employing soluble rabbit anti-NADP-GDH IgG and sheep anti-rabbit IgG covalently-linked to an insoluble cellulose matrix, was used to immunoselect polysomes translating mRNA for a chloroplastic ammonium-inducible NADP-GDH in fully induced cells of Chlorella sorokiniana. The immunoselected polysomes were dissociated, and the NADP-GDH mRNA was recovered by oligo (dT)cellulose chromatography. The translatable NADP-GDH mRNA was estimated to be 0.07 and 90% of the total polysomal poly(A)(+)RNA before and after immunoselection of the polysomes, respectively. The immunoadsorption procedure resulted in an 83% recovery and 1,291-fold purification of translatable NADP-GDH mRNA. In vitro translation of the immunoselected poly(A)(+)RNA yielded a single radioactive protein (on sodium dodecyl sufate polyacrylamide gels) with a molecular weight of 58,500, i.e. size of the putative precursor-protein of the NADP-GDH subunit in the holoenzyme in fully induced cells. The purified NADP-GDH mRNA was used for synthesis of a high proportion of nearly full-length single-stranded cDNA and double-stranded cDNA molecules. PMID:16664850

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

    PubMed

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

    2016-06-01

    A full-length infectious cDNA clone of the genome of a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan was constructed and designated pSVL-f02. Transfection of Cos-7 or IBRS-2 cells with this clone allowed the recovery of infectious virus. The recovered virus had the same in vitro characterization as the parental virus with regard to antigenicity in neutralization and indirect immunofluorescence tests, plaque size and one-step growth. Pigs were experimentally infected with the parental virus or the recombinant virus recovered from pSVL-f02 transfected cells. There were no significant differences in clinical signs or antibody responses between the two groups, and virus isolation and viral RNA detection from clinical samples were similar. Virus recovered from transfected cells therefore retained the in vitro characteristics and the in vivo pathogenicity of their parental strain. This cDNA clone should be a valuable tool to analyze determinants of pathogenicity and mechanisms of virus replication, and to develop genetically engineered vaccines against foot-and-mouth disease virus. PMID:27234555

  16. RiceFOX: A Database of Arabidopsis Mutant Lines Overexpressing Rice Full-Length cDNA that Contains a Wide Range of Trait Information to Facilitate Analysis of Gene Function

    PubMed Central

    Sakurai, Tetsuya; Kondou, Youichi; Akiyama, Kenji; Kurotani, Atsushi; Higuchi, Mieko; Ichikawa, Takanari; Kuroda, Hirofumi; Kusano, Miyako; Mori, Masaki; Saitou, Tsutomu; Sakakibara, Hitoshi; Sugano, Shoji; Suzuki, Makoto; Takahashi, Hideki; Takahashi, Shinya; Takatsuji, Hiroshi; Yokotani, Naoki; Yoshizumi, Takeshi; Saito, Kazuki; Shinozaki, Kazuo; Oda, Kenji; Hirochika, Hirohiko; Matsui, Minami

    2011-01-01

    Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named ‘RiceFOX’. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/. PMID:21186176

  17. Technology development for gene discovery and full-length sequencing

    SciTech Connect

    Marcelo Bento Soares

    2004-07-19

    In previous years, with support from the U.S. Department of Energy, we developed methods for construction of normalized and subtracted cDNA libraries, and constructed hundreds of high-quality libraries for production of Expressed Sequence Tags (ESTs). Our clones were made widely available to the scientific community through the IMAGE Consortium, and millions of ESTs were produced from our libraries either by collaborators or by our own sequencing laboratory at the University of Iowa. During this grant period, we focused on (1) the development of a method for preferential cloning of tissue-specific and/or rare transcripts, (2) its utilization to expedite EST-based gene discovery for the NIH Mouse Brain Molecular Anatomy Project, (3) further development and optimization of a method for construction of full-length-enriched cDNA libraries, and (4) modification of a plasmid vector to maximize efficiency of full-length cDNA sequencing by the transposon-mediated approach. It is noteworthy that the technology developed for preferential cloning of rare mRNAs enabled identification of over 2,000 mouse transcripts differentially expressed in the hippocampus. In addition, the method that we optimized for construction of full-length-enriched cDNA libraries was successfully utilized for the production of approximately fifty libraries from the developing mouse nervous system, from which over 2,500 full-ORF-containing cDNAs have been identified and accurately sequenced in their entirety either by our group or by the NIH-Mammalian Gene Collection Program Sequencing Team.

  18. A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region.

    PubMed

    Fang, Ying; Rowland, Raymond R R; Roof, Michael; Lunney, Joan K; Christopher-Hennings, Jane; Nelson, Eric A

    2006-12-01

    The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States. PMID:16971421

  19. Identification of Habitat-Specific Biomes of Aquatic Fungal Communities Using a Comprehensive Nearly Full-Length 18S rRNA Dataset Enriched with Contextual Data.

    PubMed

    Panzer, Katrin; Yilmaz, Pelin; Weiß, Michael; Reich, Lothar; Richter, Michael; Wiese, Jutta; Schmaljohann, Rolf; Labes, Antje; Imhoff, Johannes F; Glöckner, Frank Oliver; Reich, Marlis

    2015-01-01

    Molecular diversity surveys have demonstrated that aquatic fungi are highly diverse, and that they play fundamental ecological roles in aquatic systems. Unfortunately, comparative studies of aquatic fungal communities are few and far between, due to the scarcity of adequate datasets. We combined all publicly available fungal 18S ribosomal RNA (rRNA) gene sequences with new sequence data from a marine fungi culture collection. We further enriched this dataset by adding validated contextual data. Specifically, we included data on the habitat type of the samples assigning fungal taxa to ten different habitat categories. This dataset has been created with the intention to serve as a valuable reference dataset for aquatic fungi including a phylogenetic reference tree. The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases.The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of

  20. Identification of Habitat-Specific Biomes of Aquatic Fungal Communities Using a Comprehensive Nearly Full-Length 18S rRNA Dataset Enriched with Contextual Data

    PubMed Central

    Panzer, Katrin; Yilmaz, Pelin; Weiß, Michael; Reich, Lothar; Richter, Michael; Wiese, Jutta; Schmaljohann, Rolf; Labes, Antje; Imhoff, Johannes F.; Glöckner, Frank Oliver; Reich, Marlis

    2015-01-01

    Molecular diversity surveys have demonstrated that aquatic fungi are highly diverse, and that they play fundamental ecological roles in aquatic systems. Unfortunately, comparative studies of aquatic fungal communities are few and far between, due to the scarcity of adequate datasets. We combined all publicly available fungal 18S ribosomal RNA (rRNA) gene sequences with new sequence data from a marine fungi culture collection. We further enriched this dataset by adding validated contextual data. Specifically, we included data on the habitat type of the samples assigning fungal taxa to ten different habitat categories. This dataset has been created with the intention to serve as a valuable reference dataset for aquatic fungi including a phylogenetic reference tree. The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases.The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of

  1. Analysis of 4,664 high-quality sequence-finished poplar full-length cDNA clones and their utility for the discovery of genes responding to insect feeding

    PubMed Central

    Ralph, Steven G; Chun, Hye Jung E; Cooper, Dawn; Kirkpatrick, Robert; Kolosova, Natalia; Gunter, Lee; Tuskan, Gerald A; Douglas, Carl J; Holt, Robert A; Jones, Steven JM; Marra, Marco A; Bohlmann, Jörg

    2008-01-01

    Background The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. Results As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL)-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa × P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones for genes that were

  2. A Full-length cDNA Infectious Clone of North American Type 1 Porcine Reproductive and Respiratory Syndrome Virus: Expression of Green Fluorescent Protein in the Nsp2 Region

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The recent emergence of a unique group of North American Type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the U.S. presents new disease control problems for a swine industry that has already been seriously impacted by North American Type 2 PRRSV. In this study, a full-length cDN...

  3. A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development

    PubMed Central

    Tsetsarkin, Konstantin A.; Kenney, Heather; Chen, Rubing; Liu, Guangping; Manukyan, Hasmik; Whitehead, Stephen S.; Laassri, Majid

    2016-01-01

    ABSTRACT An arthropod-borne virus, Zika virus (ZIKV), has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics. PMID:27555311

  4. Infectious full-length clones of Calibrachoa Mottle Virus (CbMV)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Full-length cDNA clones derived from genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) of Calibrachoa mottle virus (CbMV) were constructed under the control of the T7 RNA promoter and ligated into plasmid pUC-18. The capped and uncapped in vitro transcripts, synthesized from full length genomic cDNA...

  5. Isolation of full-length RNA from a thermophilic cyanobacterium.

    PubMed

    Luo, X Z; Stevens, S E

    1997-11-01

    Isolation of full-length mRNA without degradation is critical in the study of in vivo gene regulation and transcription, cDNA synthesis and reverse transcription (RT)-PCR. It is particularly difficult to isolate full-length mRNA from thermophiles, which have higher turnover rates of mRNA degradation. Mastigocladus laminosus is a thermophilic heterocystous cyanobacterium. The assay of M. laminosus cell lysates showed that RNase activity was high and was resistant to the conventional guanidine thiocyanate and 2-mercaptoethanol denaturation methods. The mRNA isolated by several conventional methods was completely degraded. A method was developed to purify full-length mRNA by a combination of fast cooling, vanadyl-ribonucleoside-complex inhibition, phenol-chloroform-isoamyl alcohol extraction, lithium chloride precipitation and the lysing of cells with the French Press. This method produced high-quality, full-length mRNA in high yield. Purified mRNA was suitable for Northern blotting, cDNA synthesis and RT-PCR. This method could be applicable to other thermophiles in which the RNase activity is high and/or is resistant to guanidine thiocyanate. PMID:9383558

  6. Full-length minor ampullate spidroin gene sequence.

    PubMed

    Chen, Gefei; Liu, Xiangqin; Zhang, Yunlong; Lin, Senzhu; Yang, Zijiang; Johansson, Jan; Rising, Anna; Meng, Qing

    2012-01-01

    Spider silk includes seven protein based fibers and glue-like substances produced by glands in the spider's abdomen. Minor ampullate silk is used to make the auxiliary spiral of the orb-web and also for wrapping prey, has a high tensile strength and does not supercontract in water. So far, only partial cDNA sequences have been obtained for minor ampullate spidroins (MiSps). Here we describe the first MiSp full-length gene sequence from the spider species Araneus ventricosus, using a multidimensional PCR approach. Comparative analysis of the sequence reveals regulatory elements, as well as unique spidroin gene and protein architecture including the presence of an unusually large intron. The spliced full-length transcript of MiSp gene is 5440 bp in size and encodes 1766 amino acid residues organized into conserved nonrepetitive N- and C-terminal domains and a central predominantly repetitive region composed of four units that are iterated in a non regular manner. The repeats are more conserved within A. ventricosus MiSp than compared to repeats from homologous proteins, and are interrupted by two nonrepetitive spacer regions, which have 100% identity even at the nucleotide level. PMID:23251707

  7. Full-Length Minor Ampullate Spidroin Gene Sequence

    PubMed Central

    Chen, Gefei; Liu, Xiangqin; Zhang, Yunlong; Lin, Senzhu; Yang, Zijiang; Johansson, Jan; Rising, Anna; Meng, Qing

    2012-01-01

    Spider silk includes seven protein based fibers and glue-like substances produced by glands in the spider's abdomen. Minor ampullate silk is used to make the auxiliary spiral of the orb-web and also for wrapping prey, has a high tensile strength and does not supercontract in water. So far, only partial cDNA sequences have been obtained for minor ampullate spidroins (MiSps). Here we describe the first MiSp full-length gene sequence from the spider species Araneus ventricosus, using a multidimensional PCR approach. Comparative analysis of the sequence reveals regulatory elements, as well as unique spidroin gene and protein architecture including the presence of an unusually large intron. The spliced full-length transcript of MiSp gene is 5440 bp in size and encodes 1766 amino acid residues organized into conserved nonrepetitive N- and C-terminal domains and a central predominantly repetitive region composed of four units that are iterated in a non regular manner. The repeats are more conserved within A. ventricosus MiSp than compared to repeats from homologous proteins, and are interrupted by two nonrepetitive spacer regions, which have 100% identity even at the nucleotide level. PMID:23251707

  8. Full-length transcriptome analysis of human retina-derived cell lines ARPE-19 and Y79 using the vector-capping method.

    PubMed

    Oshikawa, Mio; Tsutsui, Chihiro; Ikegami, Tomoko; Fuchida, Yuki; Matsubara, Maki; Toyama, Shigeru; Usami, Ron; Ohtoko, Kuniyo; Kato, Seishi

    2011-08-01

    PURPOSE. To collect an entire set of full-length cDNA clones derived from human retina-derived cell lines and to identify full-length transcripts for retinal preferentially expressed genes. METHODS. The full-length cDNA libraries were constructed from a retinoblastoma cell line, Y79, and a retinal pigment epithelium cell line, ARPE-19, using the vector-capping method, which generates a genuine full-length cDNA. By single-pass sequencing of the 5'-end of cDNA clones and subsequent mapping to the human genome, the authors determined their transcriptional start sites and annotated the cDNA clones. RESULTS. Of the 23,616 clones isolated from Y79-derived cDNA libraries, 19,229 full-length cDNA clones were identified and classified into 4808 genes, including genes of >10 kbp. Of the 7067 genes obtained from the Y79 and ARPE-19 libraries, the authors selected 72 genes that were preferentially expressed in the eye, of which 131 clones corresponding to 57 genes were fully sequenced. As a result, we discovered many variants that were produced by different transcriptional start sites, alternative splicing, and alternative polyadenylation. CONCLUSIONS. The bias-free, full-length cDNA libraries constructed using the vector-capping method were shown to be useful for collecting an entire set of full-length cDNA clones for these retinal cell lines. Full-length transcriptome analysis of these cDNA libraries revealed that there were, unexpectedly, many transcript variants for each gene, indicating that obtaining the full-length cDNA for each variant is indispensable for analyzing its function. The full-length cDNA clones (approximately 80,000 clones each for ARPE-19 and Y79) will be useful as a resource for investigating the human retina. PMID:21697133

  9. Full-length infectious clone of a low passage dengue virus serotype 2 from Brazil

    PubMed Central

    Santos, Jefferson José da Silva; Magalhães, Tereza; Silva, José Valter Joaquim; da Silva, Andréa Nazaré Monteiro Rangel; Cordeiro, Marli Tenório; Gil, Laura Helena Vega Gonzales

    2015-01-01

    Full-length dengue virus (DENV) cDNA clones are an invaluable tool for many studies, including those on the development of attenuated or chimeric vaccines and on host-virus interactions. Furthermore, the importance of low passage DENV infectious clones should be highlighted, as these may harbour critical and unique strain-specific viral components from field-circulating isolates. The successful construction of a functional Brazilian low passage DENV serotype 2 full-length clone through homologous recombination reported here supports the use of a strategy that has been shown to be highly useful by our group for the development of flavivirus infectious clones and replicons. PMID:26200712

  10. Management of full-length complete ureteral avulsion

    PubMed Central

    Tang, Kaifa; Sun, Fa; Tian, Yuan; Zhao, Yili

    2016-01-01

    ABSTRACT Introduction Complete ureteral avulsion is one of the most serious complications of ureteroscopy. The aim of this report was to look for a good solution to full-length complete ureteral avulsion. Case presentation A 40-year-old man underwent ureteroscopic management. Full-length complete avulsion of ureter occurred during ureteroscopy. Pyeloureterostomy plus greater omentum investment outside the avulsed ureter and ureterovesical anastomosis were performed 6 hours after ureteral avulsion. The patient was followed-up during 34 months. Double-J tube was removed at 3 months after operation. Twenty three months after the first operation, the patient developed hydronephrosis because of a new ureter upside stone, then rigid ureteroscopy and holmium laser lithotripsy were used successfully. Conclusion Pyeloureterostomy plus greater omentum investment outside the avulsed ureter and ureterovesical anastomosis may be a good choice for full-length complete ureteral avulsion. PMID:27136483

  11. Full-length fuel rod behavior under severe accident conditions

    SciTech Connect

    Lombardo, N J; Lanning, D D; Panisko, F E

    1992-12-01

    This document presents an assessment of the severe accident phenomena observed from four Full-Length High-Temperature (FLHT) tests that were performed by the Pacific Northwest Laboratory (PNL) in the National Research Universal (NRU) reactor at Chalk River, Ontario, Canada. These tests were conducted for the US Nuclear Regulatory Commission (NRC) as part of the Severe Accident Research Program. The objectives of the test were to simulate conditions and provide information on the behavior of full-length fuel rods during hypothetical, small-break, loss-of-coolant severe accidents, in commercial light water reactors.

  12. Sequencing and Analysis of Full-Length cDNAs, 5′-ESTs and 3′-ESTs from a Cartilaginous Fish, the Elephant Shark (Callorhinchus milii)

    PubMed Central

    Tan, Yue Ying; Kodzius, Rimantas; Tay, Boon-Hui; Tay, Alice; Brenner, Sydney; Venkatesh, Byrappa

    2012-01-01

    Cartilaginous fishes are the most ancient group of living jawed vertebrates (gnathostomes) and are, therefore, an important reference group for understanding the evolution of vertebrates. The elephant shark (Callorhinchus milii), a holocephalan cartilaginous fish, has been identified as a model cartilaginous fish genome because of its compact genome (∼910 Mb) and a genome project has been initiated to obtain its whole genome sequence. In this study, we have generated and sequenced full-length enriched cDNA libraries of the elephant shark using the ‘oligo-capping’ method and Sanger sequencing. A total of 6,778 full-length protein-coding cDNA and 10,701 full-length noncoding cDNA were sequenced from six tissues (gills, intestine, kidney, liver, spleen, and testis) of the elephant shark. Analysis of their polyadenylation signals showed that polyadenylation usage in elephant shark is similar to that in mammals. Furthermore, both coding and noncoding transcripts of the elephant shark use the same proportion of canonical polyadenylation sites. Besides BLASTX searches, protein-coding transcripts were annotated by Gene Ontology, InterPro domain, and KEGG pathway analyses. By comparing elephant shark genes to bony vertebrate genes, we identified several ancient genes present in elephant shark but differentially lost in tetrapods or teleosts. Only ∼6% of elephant shark noncoding cDNA showed similarity to known noncoding RNAs (ncRNAs). The rest are either highly divergent ncRNAs or novel ncRNAs. In addition to full-length transcripts, 30,375 5′-ESTs and 41,317 3′-ESTs were sequenced and annotated. The clones and transcripts generated in this study are valuable resources for annotating transcription start sites, exon-intron boundaries, and UTRs of genes in the elephant shark genome, and for the functional characterization of protein sequences. These resources will also be useful for annotating genes in other cartilaginous fishes whose genomes have been targeted for

  13. Recovering full-length viral genomes from metagenomes

    PubMed Central

    Smits, Saskia L.; Bodewes, Rogier; Ruiz-González, Aritz; Baumgärtner, Wolfgang; Koopmans, Marion P.; Osterhaus, Albert D. M. E.; Schürch, Anita C.

    2015-01-01

    Infectious disease metagenomics is driven by the question: “what is causing the disease?” in contrast to classical metagenome studies which are guided by “what is out there?” In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral genome from a metagenomic sample. However, retrieval of a full-length genome of a divergent virus is technically challenging and can be time-consuming and costly. Here we discuss different assembly and fragment linkage strategies such as iterative assembly, motif searches, k-mer frequency profiling, coverage profile binning, and other strategies used to recover genomes of potential viral pathogens in a timely and cost-effective manner. PMID:26483782

  14. The Human Transcript Database: A Catalogue of Full Length cDNA Inserts

    SciTech Connect

    Bouckk John; Michael McLeod; Kim Worley; Richard Gibbs

    1999-09-10

    The BCM Search Launcher provided improved access to web-based sequence analysis services during the granting period and beyond. The Search Launcher web site grouped analysis procedures by function and provided default parameters that provided reasonable search results for most applications. For instance, most queries were automatically masked for repeat sequences prior to sequence database searches to avoid spurious matches. In addition to the web-based access and arrangements that were made using the functions easier, the BCM Search Launcher provided unique value-added applications like the BEAUTY sequence database search tool that combined information about protein domains and sequence database search results to give an enhanced, more complete picture of the reliability and relative value of the information reported. This enhanced search tool made evaluating search results more straight-forward and consistent. Some of the favorite features of the web site are the sequence utilities and the batch client functionality that allows processing of multiple samples from the command line interface. One measure of the success of the BCM Search Launcher is the number of sites that have adopted the models first developed on the site. The graphic display on the BLAST search from the NCBI web site is one such outgrowth, as is the display of protein domain search results within BLAST search results, and the design of the Biology Workbench application. The logs of usage and comments from users confirm the great utility of this resource.

  15. Renal Agenesis with Full Length Ipsilateral Refluxing Ureter.

    PubMed

    Pal, Dilip Kumar; Chandra, Vipin; Banerjee, Manju

    2016-01-01

    Unilateral renal agenesis with vesicoureteral reflux in the ipsilateral full length ureter is a rare phenomenon. Herein we report a case of 10-year old boy who presented with recurrent urinary tract infections. No renal tissue was identified on left side in various imaging studies. Micturating cystourethrogram (MCUG) showed left sided refluxing and blind ending ureter. Left ureterectomy was done because of recurrent UTI in the refluxing system. PMID:27170916

  16. Renal Agenesis with Full Length Ipsilateral Refluxing Ureter

    PubMed Central

    Chandra, Vipin; Banerjee, Manju

    2016-01-01

    Unilateral renal agenesis with vesicoureteral reflux in the ipsilateral full length ureter is a rare phenomenon. Herein we report a case of 10-year old boy who presented with recurrent urinary tract infections. No renal tissue was identified on left side in various imaging studies. Micturating cystourethrogram (MCUG) showed left sided refluxing and blind ending ureter. Left ureterectomy was done because of recurrent UTI in the refluxing system. PMID:27170916

  17. Structural photoactivation of a full-length bacterial phytochrome

    PubMed Central

    Björling, Alexander; Berntsson, Oskar; Lehtivuori, Heli; Takala, Heikki; Hughes, Ashley J.; Panman, Matthijs; Hoernke, Maria; Niebling, Stephan; Henry, Léocadie; Henning, Robert; Kosheleva, Irina; Chukharev, Vladimir; Tkachenko, Nikolai V.; Menzel, Andreas; Newby, Gemma; Khakhulin, Dmitry; Wulff, Michael; Ihalainen, Janne A.; Westenhoff, Sebastian

    2016-01-01

    Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro- and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes. PMID:27536728

  18. Conformational states of the full-length glucagon receptor

    NASA Astrophysics Data System (ADS)

    Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

    2015-07-01

    Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism.

  19. Conformational states of the full-length glucagon receptor

    PubMed Central

    Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

    2015-01-01

    Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism. PMID:26227798

  20. Development of a full-length human protein production pipeline

    PubMed Central

    Saul, Justin; Petritis, Brianne; Sau, Sujay; Rauf, Femina; Gaskin, Michael; Ober-Reynolds, Benjamin; Mineyev, Irina; Magee, Mitch; Chaput, John; Qiu, Ji; LaBaer, Joshua

    2014-01-01

    There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high-throughput (HT) methods, we transferred the genes of 31 full-length proteins into three expression vectors, and expressed the collection as N-terminal HaloTag fusion proteins in Escherichia coli and two commercial cell-free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip® GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full-length human proteins in these three expression systems. PMID:24806540

  1. Simulations of The Dalles Dam Proposed Full Length Spillwall

    SciTech Connect

    Rakowski, Cynthia L.; Perkins, William A.; Richmond, Marshall C.; Serkowski, John A.

    2008-02-25

    This report presents results of a computational fluid dynamics (CFD) modeling study to evaluatethe impacts of a full-length spillwall at The Dalles Dam. The full-length spillwall is being designed and evaluated as a structural means to improve tailrace egress and thus survival of juvenile fish passing through the spillway. During the course of this study, a full-length spillwall at Bays 6/7 and 8/9 were considered. The U.S. Army Corps of Engineers (USACE) has proposed extending the spillwall constructed in the stilling basin between spillway Bays 6 and 7 about 590 ft farther downstream. It is believed that the extension of the spillwall will improve egress conditions for downstream juvenile salmonids by moving them more rapidly into the thalweg of the river hence reducing their exposure to predators. A numerical model was created, validated, and applied the The Dalles Dam tailrace. The models were designed to assess impacts to flow, tailrace egress, navigation, and adult salmon passage of a proposed spill wall extension. The more extensive model validation undertaken in this study greatly improved our confidence in the numerical model to represent the flow conditions in The Dalles tailrace. This study used these validated CFD models to simulate the potential impacts of a spillwall extension for The Dalles Dam tailrace for two locations. We determined the following: (1)The construction of an extended wall (between Bays 6/7) will not adversely impact entering or exiting the navigation lock. Impact should be less if a wall were constructed between Bays 8/9. (2)The construction of a wall between Bays 6/7 will increase the water surface elevation between the wall and the Washington shore. Although the increased water surface elevation would be beneficial to adult upstream migrants in that it decreases velocities on the approach to the adult ladder, the increased flow depth would enhance dissolved gas production, impacting potential operations of the project because of

  2. Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

    NASA Astrophysics Data System (ADS)

    Hamid, Nur Athirah Abd; Ismail, Ismanizan

    2013-11-01

    Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

  3. Molecular cloning and characterization of the full-length Hsp90 gene from Matricaria recutita.

    PubMed

    Ling, S P; Su, S S; Zhang, H M; Zhang, X S; Liu, X Y; Pan, G F; Yuan, Y

    2014-01-01

    Heat shock protein 90 (Hsp90) is one of the most abundant and conserved chaperone proteins and plays important roles in plant growth and responses to environmental stimuli. However, little is known regarding the sequence and function of Hsp90s in Matricaria recutita. In the present study, we cloned the full-length cDNA sequence of the hsp90 gene from this species. Using rapid amplification of cDNA ends technologies with 2 degenerate primers that were designed based on the hsp90 gene sequence from other members of Asteraceae, we isolated and characterized an Hsp90 homolog gene from M. recutita (Mr-Hsp90). The full-length Mr-hsp90 cDNA sequence, containing 2097 base pairs, encodes a protein of 698 amino acids. Based on amino acid sequence identity, Mr-Hsp90 showed high similarity to other cloned Hsp90 proteins. The Mr-Hsp90 protein was closely clustered with the Lactuca sativa in a phylogenetic tree. These results indicate that the cloned sequence of Mr-Hsp90 is a member of the Hsp90 family, which is reported for the first time in M. recutita. Next, we conducted a salt stress experiment to determine the protein's function under salt stress conditions. Survival of chamomile seedlings subjected to heat-shock pretreatment was significantly increased compared with groups that had not undergone heat-shock pretreatment in a salt stress environment. This indicates that Mr-Hsp90 plays an important role in the salt resistance of chamomile seedlings. PMID:25526220

  4. Quantifying elongation rhythm during full-length protein synthesis.

    PubMed

    Rosenblum, Gabriel; Chen, Chunlai; Kaur, Jaskiran; Cui, Xiaonan; Zhang, Haibo; Asahara, Haruichi; Chong, Shaorong; Smilansky, Zeev; Goldman, Yale E; Cooperman, Barry S

    2013-07-31

    Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification. PMID:23822614

  5. Analysis and Optimization of "Full-Length" Diodes

    SciTech Connect

    Schock, Alfred

    2012-01-19

    A method of analyzing the axial variation of the heat generation rate, temperature, voltage, current density and emitter heat flux in a thermionic converter is described. The method is particularly useful for the case of "long" diodes, each extending over the full length of the reactor core. For a given diode geometry and fuel distribution, the analysis combines a nuclear solution of the axial fission density profile with the iterative solution of four differential equations representing the thermal, electrical, and thermionic interactions within the diode. The digital computer program developed to solve these equations can also perform a design optimization with respect to lead resistance, load voltage, and emitter thickness, for a specified maximum emitter temperature. Typical results are presented, and the use of this analysis for predicting the diode operating characteristics is illustrated.

  6. Full length talin stimulates integrin activation and axon regeneration

    PubMed Central

    Tan, Chin Lik; Kwok, Jessica C.F.; Heller, Janosch P.D.; Zhao, Rongrong; Eva, Richard; Fawcett, James W.

    2015-01-01

    Integrin function is regulated by activation involving conformational changes that modulate ligand-binding affinity and downstream signaling. Activation is regulated through inside-out signaling which is controlled by many signaling pathways via a final common pathway through kindlin and talin, which bind to the intracellular tail of beta integrins. Previous studies have shown that the axon growth inhibitory molecules NogoA and chondroitin sulfate proteoglycans (CSPGs) inactivate integrins. Overexpressing kindlin-1 in dorsal root ganglion (DRG) neurons activates integrins, enabling their axons to overcome inhibitory molecules in the environment, and promoting regeneration in vivo following dorsal root crush. Other studies have indicated that expression of the talin head alone or with kindlin can enhance integrin activation. Here, using adult rat DRG neurons, we investigate the effects of overexpressing various forms of talin on axon growth and integrin signaling. We found that overexpression of the talin head activated axonal integrins but inhibited downstream signaling via FAK, and did not promote axon growth. Similarly, co-expression of the talin head and kindlin-1 prevented the growth-promoting effect of kindlin-1, suggesting that the talin head acts as a form of dominant negative for integrin function. Using full-length talin constructs in PC12 cells we observed that neurite growth was enhanced by the expression of wild-type talin and more so by two ‘activated’ forms of talin produced by point mutation (on laminin and aggrecan–laminin substrates). Nevertheless, co-expression of full-length talin with kindlin did not promote neurite growth more than either molecule alone. In vivo, we find that talin is present in PNS axons (sciatic nerve), and also in CNS axons of the corticospinal tract. PMID:25771432

  7. Crystal Structure of a Full-Length [beta]-Catenin

    SciTech Connect

    Xing, Yi; Takemaru, Ken-Ichi; Liu, Jing; Berndt, Jason D.; Zheng, Jie J.; Moon, Randall T.; Xu, Wenqing

    2008-08-19

    {beta}-catenin plays essential roles in cell adhesion and Wnt signaling, while deregulation of {beta}-catenin is associated with multiple diseases including cancers. Here, we report the crystal structures of full-length zebrafish {beta}-catenin and a human {beta}-catenin fragment that contains both the armadillo repeat and the C-terminal domains. Our structures reveal that the N-terminal region of the C-terminal domain, a key component of the C-terminal transactivation domain, forms a long {alpha} helix that packs on the C-terminal end of the armadillo repeat domain, and thus forms part of the {beta}-catenin superhelical core. The existence of this helix redefines our view of interactions of {beta}-catenin with some of its critical partners, including ICAT and Chibby, which may form extensive interactions with this C-terminal domain {alpha} helix. Our crystallographic and NMR studies also suggest that the unstructured N-terminal and C-terminal tails interact with the ordered armadillo repeat domain in a dynamic and variable manner.

  8. Massive Collection of Full-Length Complementary DNA Clones and Microarray Analyses:. Keys to Rice Transcriptome Analysis

    NASA Astrophysics Data System (ADS)

    Kikuchi, Shoshi

    2009-02-01

    Completion of the high-precision genome sequence analysis of rice led to the collection of about 35,000 full-length cDNA clones and the determination of their complete sequences. Mapping of these full-length cDNA sequences has given us information on (1) the number of genes expressed in the rice genome; (2) the start and end positions and exon-intron structures of rice genes; (3) alternative transcripts; (4) possible encoded proteins; (5) non-protein-coding (np) RNAs; (6) the density of gene localization on the chromosome; (7) setting the parameters of gene prediction programs; and (8) the construction of a microarray system that monitors global gene expression. Manual curation for rice gene annotation by using mapping information on full-length cDNA and EST assemblies has revealed about 32,000 expressed genes in the rice genome. Analysis of major gene families, such as those encoding membrane transport proteins (pumps, ion channels, and secondary transporters), along with the evolution from bacteria to higher animals and plants, reveals how gene numbers have increased through adaptation to circumstances. Family-based gene annotation also gives us a new way of comparing organisms. Massive amounts of data on gene expression under many kinds of physiological conditions are being accumulated in rice oligoarrays (22K and 44K) based on full-length cDNA sequences. Cluster analyses of genes that have the same promoter cis-elements, that have similar expression profiles, or that encode enzymes in the same metabolic pathways or signal transduction cascades give us clues to understanding the networks of gene expression in rice. As a tool for that purpose, we recently developed "RiCES", a tool for searching for cis-elements in the promoter regions of clustered genes.

  9. Crystal Structure of a Full-Length Autotransporter

    SciTech Connect

    van den Berg, B.

    2010-01-01

    The autotransporter (AT) secretion mechanism is the most common mechanism for the secretion of virulence factors across the outer membrane (OM) from pathogenic Gram-negative bacteria. In addition, ATs have attracted biotechnological and biomedical interest for protein display on bacterial cell surfaces. Despite their importance, the mechanism by which passenger domains of ATs pass the OM is still unclear. The classical view is that the {beta}-barrel domain provides the conduit through which the unfolded passenger moves, with the energy provided by vectorial folding of the {beta}-strand-rich passenger on the extracellular side of the OM. We present here the first structure of a full-length AT, the esterase EstA from Pseudomonas aeruginosa, at a resolution of 2.5 {angstrom}. EstA has a relatively narrow, 12-stranded {beta}-barrel that is covalently attached to the passenger domain via a long, curved helix that occupies the lumen of the {beta}-barrel. The passenger has a structure that is dramatically different from that of other known passengers, with a globular fold that is dominated by {alpha}-helices and loops. The arrangement of secondary-structure elements suggests that the passenger can fold sequentially, providing the driving force for passenger translocation. The esterase active-site residues are located at the apical surface of the passenger, at the entrance of a large hydrophobic pocket that contains a bound detergent molecule that likely mimics substrate. The EstA structure provides insight into AT mechanism and will facilitate the design of fusion proteins for cell surface display.

  10. Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle.

    PubMed

    Escobar, Helena; Schöwel, Verena; Spuler, Simone; Marg, Andreas; Izsvák, Zsuzsanna

    2016-01-01

    Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysf(prmd) Prkdc(scid)/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy. PMID:26784637

  11. Two methods for full-length RNA sequencing for low quantities of cells and single cells

    PubMed Central

    Pan, Xinghua; Durrett, Russell E.; Zhu, Haiying; Tanaka, Yoshiaki; Li, Yumei; Zi, Xiaoyuan; Marjani, Sadie L.; Euskirchen, Ghia; Ma, Chao; LaMotte, Robert H.; Park, In-Hyun; Snyder, Michael P.; Mason, Christopher E.; Weissman, Sherman M.

    2013-01-01

    The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3′ biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling. PMID:23267071

  12. [Isolation, identification and full-length genome sequence analysis of encephalomyocarditis virus from local aardvarks].

    PubMed

    Chang, Hong-Tao; Liu, Hui-Min; He, Xiu-Yuan; Zhao, Jun; Chen, Lu; Wang, Xin-Wei; Yang, Xia; Yao, Hui-Xia; Wang, Chuan-Qing

    2014-07-01

    Encephalomyocarditis virus (EMCV) is a natural epidemic zoonotic pathogen. However, no reports have been published regarding the isolation, identification and full-length genome of EMCV from a local aardvark population. In present study, an EMCV isolate HNXX13 was isolated from aardvarks named Huainan-pig in Henan Province. The systematic identification, full-length genome sequencing and molecular characteristic analysis of the isolate HNXX13 were conducted. The result showed that the isolate was spherical with a diameter of 24-30 nm, neither heat- nor acid-resistant, sensitive to trypsin, insensitive to chloroform, not protected by bivalent cationic, and the specific fluorescence was observed in the cytoplasm of BHK-21 cells infected with the isolate by using indirect fluorescence assay. The full-length genome of EMCV HNXX13 generated a 7 725bp sequence (GenBank: F771002), with 81.0%-99.9% nucleotide identity to reference strains from different animals, and 99.5% with a Chinese reference strain isolated earlier from a commercial pig herd. The phylogenetic tree based on the full-length genome and ORF sequences identified that all EMCV strains were divided into three groups G1, G2 and G3, and strain HNXX13 belonging to the G1 group with other Chinese reference strains. The result also identified that this EMCV infection could cause severe clinical signs in a local aardvark population, and enriches the molecular epidemiological data of EMCV in China. Regional differences exist in EMCV genome and transmission is limited within a certain area. However, the cross-infection and transmission of EMCV between aardvark and mice appears most likely. Mutations have occurred in some amino acids of EMCV strain HNXX13 during the transmission in local aardvark herd and these mutations might make the virus easier to infect the aardvark. PMID:25272589

  13. Molecular cloning and properties of a full-length putative thyroid hormone receptor coactivator.

    PubMed

    Takeshita, A; Yen, P M; Misiti, S; Cardona, G R; Liu, Y; Chin, W W

    1996-08-01

    Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. The conserved carboxy-terminal region of the ligand-binding domain (AF-2) has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivator(s). Using bacterially-expressed TR as a probe, far-Western-based expression cDNA library screening identified cDNAs that encode, in part, the recently reported partial steroid receptor coactivator-1 (SRC-1) sequence. Additional work, including 5' RACE, has characterized a full-length cDNA that encodes a approximately 160 kD protein as a putative thyroid hormone receptor coactivator (F-SRC-1). In vitro binding studies show that F-SRC-1 binds to a variety of nuclear hormone receptors in a ligand-dependent manner, along with TBP and TFIIB, suggesting that F-SRC-1 may play a role as a bridging molecule between nuclear hormone receptors and general transcription factors. Interestingly, AF-2 mutants also retain ligand-dependent interaction with F-SRC-1. Although F-SRC-1 recognizes the ligand-induced conformational changes of nuclear hormone receptors, our observations suggest that F-SRC-1 may bind directly with subregion(s) in nuclear hormone receptors other than the AF-2 region. PMID:8754792

  14. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    PubMed

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury. PMID:26345909

  15. The Drosophila gene collection: Identification of putative full-length cDNAs for 70 percent of D. melanogaster genes

    SciTech Connect

    Stapleton, Mark; Liao, Guochun; Brokstein, Peter; Hong, Ling; Carninci, Piero; Shiraki, Toshiyuki; Hayashizaki, Yoshihide; Champe, Mark; Pacleb, Joanne; Wan, Ken; Yu, Charles; Carlson, Joe; George, Reed; Celniker, Susan; Rubin, Gerald M.

    2002-08-12

    Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5prime expressed sequence tags (EST) from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to {approx}40 percent of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remaining genes, we have generated an additional 157,835 5prime ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22hr embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70 percent of the predicted genes in Drosophila.

  16. Full-Length GB Virus C (Hepatitis G Virus) RNA Transcripts Are Infectious in Primary CD4-Positive T Cells

    PubMed Central

    Xiang, Jinhua; Wünschmann, Sabina; Schmidt, Warren; Shao, Jianqiang; Stapleton, Jack T.

    2000-01-01

    GB virus C (GBV-C or hepatitis G virus) is a recently described flavivirus which frequently leads to chronic viremia in humans. Although GBV-C is associated with acute posttransfusion hepatitis, it is not clear if the virus is pathogenic for humans. We constructed a full-length cDNA from the plasma of a person with chronic GBV-C viremia. Peripheral blood mononuclear cells (PBMCs) transfected with full-length RNA transcripts from this GBV-C clone resulted in viral replication. This was demonstrated by serial passage of virus from cell culture supernatants, detection of increasing concentrations of positive- and negative-sense GBV-C RNA over time, and the detection of the GBV-C E2 antigen by confocal microscopy. In addition, two types of GBV-C particles were identified in cell lysates; these particles had buoyant densities of 1.06 and 1.12 to 1.17 g/ml in sucrose gradients. PBMCs sorted for expression of CD4 contained 100-fold-more GBV-C RNA than CD4-negative cells. Taken together, these data demonstrate that RNA transcripts from GBV-C full-length cDNA are infectious in primary CD4-positive T cells. In contrast, RNA transcripts from an infectious hepatitis C virus clone did not replicate in the same cell culture system. Infectious RNA transcripts from GBV-C cDNA should prove useful for studying viral replication and may allow identification of differences between GBV-C and hepatitis C virus cultivation in vitro. PMID:10982359

  17. High-quality full-length immunoglobulin profiling with unique molecular barcoding.

    PubMed

    Turchaninova, M A; Davydov, A; Britanova, O V; Shugay, M; Bikos, V; Egorov, E S; Kirgizova, V I; Merzlyak, E M; Staroverov, D B; Bolotin, D A; Mamedov, I Z; Izraelson, M; Logacheva, M D; Kladova, O; Plevova, K; Pospisilova, S; Chudakov, D M

    2016-09-01

    High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d. PMID:27490633

  18. Functional and expression analyses of transcripts based on full-length cDNAs of Sorghum bicolor

    PubMed Central

    Shimada, Setsuko; Makita, Yuko; Kuriyama-Kondou, Tomoko; Kawashima, Mika; Mochizuki, Yoshiki; Hirakawa, Hideki; Sato, Shusei; Toyoda, Tetsuro; Matsui, Minami

    2015-01-01

    Sorghum bicolor is one of the most important crops for food and bioethanol production. Its small diploid genome and resistance to environmental stress make sorghum an attractive model for studying the functional genomics of the Saccharinae and other C4 grasses. We analyzed the domain-based functional annotation of the cDNAs using the gene ontology (GO) categories for molecular function to characterize all the genes cloned in the full-length cDNA library of sorghum. The sorghum cDNA library successfully captured a wide range of cDNA-encoded proteins with various functions. To characterize the protein function of newly identified cDNAs, a search of their deduced domains and comparative analyses in the Oryza sativa and Zea mays genomes were carried out. Furthermore, genes on the sense strand corresponding to antisense transcripts were classified based on the GO of molecular function. To add more information about these genes, we have analyzed the expression profiles using RNA-Seq of three tissues (spikelet, seed and stem) during the starch-filling phase. We performed functional analysis of tissue-specific genes and expression analysis of genes of starch biosynthesis enzymes. This functional analysis of sorghum full-length cDNAs and the transcriptome information will facilitate further analysis of the Saccharinae and grass families. PMID:26546227

  19. Functional and expression analyses of transcripts based on full-length cDNAs of Sorghum bicolor.

    PubMed

    Shimada, Setsuko; Makita, Yuko; Kuriyama-Kondou, Tomoko; Kawashima, Mika; Mochizuki, Yoshiki; Hirakawa, Hideki; Sato, Shusei; Toyoda, Tetsuro; Matsui, Minami

    2015-12-01

    Sorghum bicolor is one of the most important crops for food and bioethanol production. Its small diploid genome and resistance to environmental stress make sorghum an attractive model for studying the functional genomics of the Saccharinae and other C4 grasses. We analyzed the domain-based functional annotation of the cDNAs using the gene ontology (GO) categories for molecular function to characterize all the genes cloned in the full-length cDNA library of sorghum. The sorghum cDNA library successfully captured a wide range of cDNA-encoded proteins with various functions. To characterize the protein function of newly identified cDNAs, a search of their deduced domains and comparative analyses in the Oryza sativa and Zea mays genomes were carried out. Furthermore, genes on the sense strand corresponding to antisense transcripts were classified based on the GO of molecular function. To add more information about these genes, we have analyzed the expression profiles using RNA-Seq of three tissues (spikelet, seed and stem) during the starch-filling phase. We performed functional analysis of tissue-specific genes and expression analysis of genes of starch biosynthesis enzymes. This functional analysis of sorghum full-length cDNAs and the transcriptome information will facilitate further analysis of the Saccharinae and grass families. PMID:26546227

  20. Identification and Functional Analyses of 11 769 Full-length Human cDNAs Focused on Alternative Splicing

    PubMed Central

    Wakamatsu, Ai; Kimura, Kouichi; Yamamoto, Jun-ichi; Nishikawa, Tetsuo; Nomura, Nobuo; Sugano, Sumio; Isogai, Takao

    2009-01-01

    We analyzed diversity of mRNA produced as a result of alternative splicing in order to evaluate gene function. First, we predicted the number of human genes transcribed into protein-coding mRNAs by using the sequence information of full-length cDNAs and 5′-ESTs and obtained 23 241 of such human genes. Next, using these genes, we analyzed the mRNA diversity and consequently sequenced and identified 11 769 human full-length cDNAs whose predicted open reading frames were different from other known full-length cDNAs. Especially, 30% of the cDNAs we identified contained variation in the transcription start site (TSS). Our analysis, which particularly focused on multiple variable first exons (FEVs) formed due to the alternative utilization of TSSs, led to the identification of 261 FEVs expressed in the tissue-specific manner. Quantification of the expression profiles of 13 genes by real-time PCR analysis further confirmed the tissue-specific expression of FEVs, e.g. OXR1 had specific TSS in brain and tumor tissues, and so on. Finally, based on the results of our mRNA diversity analysis, we have created the FLJ Human cDNA Database. From our result, it has been understood mechanisms that one gene produces suitable protein-coding transcripts responding to the situation and the environment. PMID:19880432

  1. 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

  2. Analysis of 4,664 high-quality sequence-finished poplar full-length

    SciTech Connect

    Ralph, S.; Gunter, Lee E; Tuskan, Gerald A; Douglas, Carl; Holt, Robert A.; Jones, Steven; Marra, Marco; Bohlmann, J.

    2008-01-01

    The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL)-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa x P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones for genes that were differentially expressed in

  3. Responses of Nontransformed Human Hepatocytes to Conditional Expression of Full-Length Hepatitis C Virus Open Reading Frame

    PubMed Central

    Tang, Weiliang; Lázaro, Catherine A.; Campbell, Jean S.; Parks, W. Tony; Katze, Michael G.; Fausto, Nelson

    2007-01-01

    Hepatitis C virus (HCV) is a major cause of chronic hepatitis that can lead to cirrhosis and hepatocellular carcinoma. To study the effects of HCV protein expression on host cells, we established conditional expression of the full-length open reading frame (ORF) of an infectious cDNA clone of HCV (genotype 1a, H77 strain) in the nontransformed human hepatocyte line cell HH4 using the ecdysone receptor regulatory system. Treatment with the ecdysone analog ponasterone-A induced tightly regulated and dose-dependent full-length HCV ORF expression and properly processed HCV proteins. HCV Core, NS3, and NS5A colocalized in perinuclear regions and associated with the early endosomal protein EEA1. HCV ORF expression caused marked growth inhibition, increased intracellular reactive oxygen species, up-regulation of glutamate-l-cysteine ligase activity, increased glutathione level, and activation of nuclear factor κB. Although it was not directly cytotoxic, HCV ORF expression sensitized HH4 cells to Fas at certain concentrations but not to tumor necrosis factor-related apoptosis-inducing ligand. HCV ORF expression in HH4 cells up-regulated genes involved in innate immune response/inflammation and oxidative stress responses and down-regulated cell growth-related genes. Expression of HCV ORF in host cells may contribute to HCV pathogenesis by producing oxidative stress and increasing the expression of genes related to the innate immune response and inflammation. PMID:17991716

  4. Retrotransposon mdg3 of Drosophila: General structure and functional domains of the full-length copy

    SciTech Connect

    Avedisov, S.N.; Ilyin, Yu.V.

    1995-09-01

    A full-length copy of the transposable element mdg3 from the plasmid clone Dm38 of Drosophila melanogaster was obtained by screening the DNA library of the cell culture 67J25D. Previous work demonstrated that only full-length copies of mdg3 (5.5 kb) are amplified in this culture, whereas the number of deleted copies probably has not changed since the cell line was established. We sequenced the full-length copy of mdg3 from cDm38 by the method described by Sanger. 10 refs., 2 figs., 2 tabs.

  5. 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

  6. Fabrication and Testing of Full-Length Single-Cell Externally Fueled Converters for Thermionic Reactors

    SciTech Connect

    Schock, Alfred

    1995-08-01

    Paper presented at the 29th IECEC in Monterey, CA in August 1994. The present paper describes the fabrication and testing of full-length prototypcial converters, both unfueled and fueled, and presents parametric results of electrically heated tests.

  7. [Cloning of full-length coding sequence of tree shrew CD4 and prediction of its molecular characteristics].

    PubMed

    Tian, Wei-Wei; Gao, Yue-Dong; Guo, Yan; Huang, Jing-Fei; Xiao, Chang; Li, Zuo-Sheng; Zhang, Hua-Tang

    2012-02-01

    The tree shrews, as an ideal animal model receiving extensive attentions to human disease research, demands essential research tools, in particular cellular markers and monoclonal antibodies for immunological studies. In this paper, a 1 365 bp of the full-length CD4 cDNA encoding sequence was cloned from total RNA in peripheral blood of tree shrews, the sequence completes two unknown fragment gaps of tree shrews predicted CD4 cDNA in the GenBank database, and its molecular characteristics were analyzed compared with other mammals by using biology software such as Clustal W2.0 and so forth. The results showed that the extracellular and intracellular domains of tree shrews CD4 amino acid sequence are conserved. The tree shrews CD4 amino acid sequence showed a close genetic relationship with Homo sapiens and Macaca mulatta. Most regions of the tree shrews CD4 molecule surface showed positive charges as humans. However, compared with CD4 extracellular domain D1 of human, CD4 D1 surface of tree shrews showed more negative charges, and more two N-glycosylation sites, which may affect antibody binding. This study provides a theoretical basis for the preparation and functional studies of CD4 monoclonal antibody. PMID:22345010

  8. In vitro translation of the full-length RNA transcript of figwort mosaic virus (Caulimovirus).

    PubMed

    Ranu, R S; Gowda, S; Scholthof, H; Wu, F C; Shepherd, R J

    1996-01-01

    The circular DNA genome of FMV consists of seven tandemly arranged genes placed successively on a full-length RNA transcript that spans the entire circular viral genome. This transcript is a tentative mRNA for at least five of the six major conserved genes of this virus (genes I-V) that are positioned on this transcript. The sixth major gene (gene VI) is expressed as a separate monocistronic transcript. A long 5'-nontranslated leader (598 nucleotides), a small nonconserved gene (VII), and a short intergenic region (57 nucleotides) precede the five major conserved genes (I through V) on the full-length transcript. A reporter gene (CAT), as a separate cistron or fused in-frame, to viral cistrons in various downstream positions in cloned versions of the viral genome was used in a transcription vector to generate artificial full-length transcripts of FMV. When these mRNAs were translated in vitro (rabbit reticulocyte lysate system), the reporter gene was translated efficiently in all positions. Translation of internal native viral gene positioned on the full-length transcript of FMV was also determined (the gene VI product). These observations suggest that the full-length FMV transcript functions as a polycistronic mRNA in plants. Results are best explained on the basis of translational coupling/relay race model. PMID:8882638

  9. Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses.

    PubMed Central

    Lie, Y S; Penuel, E M; Low, M A; Nguyen, T P; Mangahas, J O; Anderson, K P; Petropoulos, C J

    1994-01-01

    We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons. Images PMID:7966574

  10. Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses.

    PubMed

    Lie, Y S; Penuel, E M; Low, M A; Nguyen, T P; Mangahas, J O; Anderson, K P; Petropoulos, C J

    1994-12-01

    We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons. PMID:7966574

  11. Nanoscale insights into full-length prion protein aggregation on model lipid membranes.

    PubMed

    Pan, Yangang; Wang, Bin; Zhang, Tong; Zhang, Yanan; Wang, Hongda; Xu, Bingqian

    2016-06-30

    The aggregates of the full-length human recombinant prion protein (PrP) (23-231) on model membranes were investigated by combining the atomic force microscopy (AFM) measurements and theoretical calculations at pH 5.0, showing the great effect of PrP concentration on its supramolecular assemblies on the lipid bilayer. PMID:27284592

  12. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

    PubMed Central

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  13. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

    PubMed

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

    2016-01-29

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  14. The structure of full-length LysR-type transcriptional regulators. Modeling of the full-length OxyR transcription factor dimer

    PubMed Central

    Zaim, Jolanta; Kierzek, Andrzej M.

    2003-01-01

    The LysR-type transcriptional regulators (LTTRs) comprise the largest family of prokaryotic transcription factors. These proteins are composed of an N-terminal DNA binding domain (DBD) and a C-terminal cofactor binding domain. To date, no structure of the DBD has been solved. According to the SUPERFAMILY and MODBASE databases, a reliable homology model of LTTR DBDs may be built using the structure of the Escherichia coli ModE transcription factor, containing a winged helix– turn–helix (HTH) motif, as a template. The remote, but statistically significant, sequence similarity between ModE and LTTR DBDs and an alignment generated using SUPERFAMILY and MODBASE methods was independently confirmed by alignment of sequence profiles representing ModE and LTTR family DBDs. Using the crystal structure of the E.coli OxyR C-terminal domain and the DBD alignments we constructed a structural model of the full-length dimer of this LTTR family member and used it to investigate the mode of protein–DNA interaction. We also applied the model to interpret, in a structural context, the results of numerous biochemical studies of mutated LTTRs. A comparison of the LTTR DBD model with the structures of other HTH proteins also provides insights into the interaction of LTTRs with the C-terminal domain of the RNA polymerase α subunit. PMID:12595552

  15. Development of a full-length cDNA-derived enterovirus A71 vaccine candidate using reverse genetics technology.

    PubMed

    Yang, Ya-Ting; Chow, Yen-Hung; Hsiao, Kuang-Nan; Hu, Kai-Chieh; Chiang, Jen-Ron; Wu, Suh-Chin; Chong, Pele; Liu, Chia-Chyi

    2016-08-01

    Enterovirus A71 (EV-A71) is responsible for epidemics of hand, foot and mouth disease (HFMD) in young children. To circumvent difficulties in obtaining clinical enterovirus isolates that might be contaminated with other viruses, a platform technology was developed to quickly generate vaccine virus strains based on the published enterovirus genomic sequences. A recombinant plasmid containing the full-length infectious cDNA clone of EV-A71 vaccine strain E59 was directly generated after transfecting the recombinant plasmid into Vero, RD or HEK293A cells, and phenotypic characteristics similar to the parental strain were observed. The cDNA-derived infectious EV-A71 virus grown in Vero cells produced relatively stable virus titers in both T-flasks and microcarrier culture systems. To evaluate the genetic stability of the cDNA-derived EV-A71 viruses, the immunodominant structural proteins, VP1 and VP2, of the recombinant EV-A71 viruses were sequenced and analyzed. The cDNA-derived EV-A71 virus showed weak pathogenicity in a human SCARB2 mouse model. These results show the successful generation of a recombinant virus derived from a published viral genomic sequence that demonstrated good genetic stability and viral yields, which could represent an efficient and safe vaccine strain for cGMP-grade manufacturing. PMID:27387826

  16. Loss of GATA-1 full length as a cause of Diamond-Blackfan anemia phenotype.

    PubMed

    Parrella, Sara; Aspesi, Anna; Quarello, Paola; Garelli, Emanuela; Pavesi, Elisa; Carando, Adriana; Nardi, Margherita; Ellis, Steven R; Ramenghi, Ugo; Dianzani, Irma

    2014-07-01

    Mutations in the hematopoietic transcription factor GATA-1 alter the proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2 interfere with the synthesis of the full-length isoform of GATA-1 and lead to the production of a shortened isoform, GATA-1s. These mutations have been found in patients with Diamond-Blackfan anemia (DBA), a congenital erythroid aplasia typically caused by mutations in genes encoding ribosomal proteins. We sequenced GATA-1 in 23 patients that were negative for mutations in the most frequently mutated DBA genes. One patient showed a c.2T > C mutation in the initiation codon leading to the loss of the full-length GATA-1 isoform. PMID:24453067

  17. Structure of the full-length TRPV2 channel by cryo-EM

    NASA Astrophysics Data System (ADS)

    Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

    2016-03-01

    Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a `minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ~5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

  18. Synaptonemal complex extension from clustered telomeres mediates full-length chromosome pairing in Schmidtea mediterranea

    PubMed Central

    Xiang, Youbin; Miller, Danny E.; Ross, Eric J.; Sánchez Alvarado, Alejandro; Hawley, R. Scott

    2014-01-01

    In the 1920s, József Gelei proposed that chromosome pairing in flatworms resulted from the formation of a telomere bouquet followed by the extension of synapsis from telomeres at the base of the bouquet, thus facilitating homolog pairing in a processive manner. A modern interpretation of Gelei’s model postulates that the synaptonemal complex (SC) is nucleated close to the telomeres and then extends progressively along the full length of chromosome arms. We used the easily visible meiotic chromosomes, a well-characterized genome, and RNAi in the sexual biotype of the planarian Schmidtea mediterranea to test that hypothesis. By identifying and characterizing S. mediterranea homologs of genes encoding synaptonemal complex protein 1 (SYCP1), the topoisomerase-like protein SPO11, and RAD51, a key player in homologous recombination, we confirmed that SC formation begins near the telomeres and progresses along chromosome arms during zygotene. Although distal regions pair at the time of bouquet formation, pairing of a unique interstitial locus is not observed until the formation of full-length SC at pachytene. Moreover, neither full extension of the SC nor homologous pairing is dependent on the formation of double-strand breaks. These findings validate Gelei’s speculation that full-length pairing of homologous chromosomes is mediated by the extension of the SC formed near the telomeres. S. mediterranea thus becomes the first organism described (to our knowledge) that forms a canonical telomere bouquet but does not require double-strand breaks for synapsis between homologous chromosomes. However, the initiation of SC formation at the base of the telomere bouquet, which then is followed by full-length homologous pairing in planarian spermatocytes, is not observed in other species and may not be conserved. PMID:25404302

  19. Full-length high-temperature severe fuel damage test No. 2. Final safety analysis

    SciTech Connect

    Hesson, G.M.; Lombardo, N.J.; Pilger, J.P.; Rausch, W.N.; King, L.L.; Hurley, D.E.; Parchen, L.J.; Panisko, F.E.

    1993-09-01

    Hazardous conditions associated with performing the Full-Length High- Temperature (FLHT). Severe Fuel Damage Test No. 2 experiment have been analyzed. Major hazards that could cause harm or damage are (1) radioactive fission products, (2) radiation fields, (3) reactivity changes, (4) hydrogen generation, (5) materials at high temperature, (6) steam explosion, and (7) steam pressure pulse. As a result of this analysis, it is concluded that with proper precautions the FLHT- 2 test can be safely conducted.

  20. Detection and Full-Length Genome Characterization of Novel Canine Vesiviruses

    PubMed Central

    Pinto, Pierfrancesco; Lorusso, Eleonora; Di Martino, Barbara; Wang, Qiuhong; Larocca, Vittorio; Cavalli, Alessandra; Camero, Michele; Decaro, Nicola; Bányai, Krisztián; Saif, Linda J.; Buonavoglia, Canio

    2015-01-01

    Vesiviruses have been detected in several animal species and as accidental contaminants of cells. We detected vesiviruses in asymptomatic kennel dogs (64.8%) and symptomatic (1.1%) and asymptomatic (3.5%) household dogs in Italy. The full-length genome of 1 strain, Bari/212/07/ITA, shared 89%–90% nt identity with vesiviruses previously detected in contaminated cells. PMID:26196075

  1. Phylogenetic Analysis of Near Full-Length HIV Type 1 Genomic Sequences from 21 Korean Individuals

    PubMed Central

    Kim, Jung-Eun; Foley, Brian T.

    2013-01-01

    Abstract The Korean subclade of subtype B (KSB) is the most prevalent HIV-1 strain found in Korea. To date, only two near full-length HIV-1 sequences from Korean patients have been reported. Here, we analyzed a total of 24 near full-length genomes of HIV-1 strains that were isolated from 17 antiretroviral therapy (ART)-naive patients and four ART-exposed patients. Proviral DNA from peripheral blood mononuclear cells was PCR amplified and directly sequenced. Phylogenetic analyses were used to classify viruses from 19 patients as KSB, from one patient as subtype B, from one patient as subtype D, and three viruses from one patient as CRF02_AG. All KSB viruses demonstrated TAAAA instead of TATAA at the TATA box in the LTR. Of the 19 KSB patients, their sequence identities at the nucleotide level ranged from 89.8% to 97.1% from the lowest env gene to the highest pol gene. Other than the CRF02_AG viruses, no recombination events were noted in any of the 19 KSB patients, which is consistent with our previous studies on the pol, vif, and nef genes. Except for one strain, all of the strains were classified as non-syncytium-inducing strains. This is the first report to describe near full-length KSB. PMID:23199052

  2. Crystallization and X-ray structure of full-length recombinant human butyrylcholinesterase

    SciTech Connect

    Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana; Asojo, Oluwatoyin A.

    2007-09-01

    The first crystals and the 2.8 Å X-ray structure of full-length recombinant human butyrylcholinesterase are reported. Human butyrylcholinesterase (BChE) has been shown to function as an endogenous scavenger of diverse poisons. BChE is a 340 kDa tetrameric glycoprotein that is present in human serum at a concentration of 5 mg l{sup −1}. The well documented therapeutic effects of BChE on cocaine toxicity and organophosphorus agent poisoning has increased the need for effective methods of producing recombinant therapeutic BChE. In order to be therapeutically useful, BChE must have a long circulatory residence time or associate as tetramers. Full-length recombinant BChE produced in Chinese hamster ovary (CHO) cells or human embryonic kidney cells has been shown to associate as monomers, with a shorter circulatory residence time than the naturally occurring tetrameric serum protein. Based on the preceding observation as well as the need to develop novel methodologies to facilitate the mass production of therapeutic recombinant BChE, studies have been initiated to determine the structural basis of tetramer formation. Towards these ends, full-length monomeric recombinant BChE has been crystallized for the first time. A 2.8 Å X-ray structure was solved in space group P42{sub 1}2, with unit-cell parameters a = b = 156, c = 146 Å.

  3. Cognitive flexibility deficits in a mouse model for the absence of full-length dystrophin.

    PubMed

    Remmelink, E; Aartsma-Rus, A; Smit, A B; Verhage, M; Loos, M; van Putten, M

    2016-07-01

    Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full-length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full-length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full-length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients. PMID:27220066

  4. Full-length genomic characterizations of two canine parvoviruses prevalent in Northwest China.

    PubMed

    Han, Shi-Chong; Guo, Hui-Chen; Sun, Shi-Qi; Shu, Long; Wei, Yan-Quan; Sun, De-Hui; Cao, Sui-Zhong; Peng, Guang-Neng; Liu, Xiang-Tao

    2015-05-01

    Canine parvovirus (CPV) can cause acute hemorrhagic diarrhea and fatal myocarditis in young dogs. Currently, most studies have focused on the evolution of the VP2 gene, whereas the full-length genome of CPV has been rarely reported. In this study, the whole genomes of CPV-LZ1 and CPV-LZ2 strains prevalent in Northwest China were determined and analyzed in comparison with those of the reference CPVs. The genome sequences of both LZ strains consisted of 5053 nucleotides. CPV-LZ1 and CPV-LZ2 strains were designated as new CPV-2a and CPV-2b, respectively. Sequence alignment analysis results revealed that these two new strains underwent specific unique variations during the process of local adaption. The left non-translated regions of these strains formed a Y-shaped hairpin structure, whereas the right non-translated regions lacked the reiteration of DNA sequence. A phylogenetic tree constructed from 33 whole coding regions of CPVs showed a strong spatial clustering, and these two strains belonged to the Chinese strain cluster lineage. This study provides a method to obtain the full-length genome of CPV. The isolation and characterization of these viruses adds incrementally to the knowledge of the full-length genome of CPV. The results from this study also provide insight into the molecular epidemiology and genetic diversity of the CPV field isolates from Northwest China and can be useful in preventing and controlling CPV infection in this region. PMID:25690604

  5. GST-His purification: a two-step affinity purification protocol yielding full-length purified proteins.

    PubMed

    Maity, Ranjan; Pauty, Joris; Krietsch, Jana; Buisson, Rémi; Genois, Marie-Michelle; Masson, Jean-Yves

    2013-01-01

    Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest. PMID:24193370

  6. The full-length form of the Drosophila amyloid precursor protein is involved in memory formation.

    PubMed

    Bourdet, Isabelle; Preat, Thomas; Goguel, Valérie

    2015-01-21

    The APP plays a central role in AD, a pathology that first manifests as a memory decline. Understanding the role of APP in normal cognition is fundamental in understanding the progression of AD, and mammalian studies have pointed to a role of secreted APPα in memory. In Drosophila, we recently showed that APPL, the fly APP ortholog, is required for associative memory. In the present study, we aimed to characterize which form of APPL is involved in this process. We show that expression of a secreted-APPL form in the mushroom bodies, the center for olfactory memory, is able to rescue the memory deficit caused by APPL partial loss of function. We next assessed the impact on memory of the Drosophila α-secretase kuzbanian (KUZ), the enzyme initiating the nonamyloidogenic pathway that produces secreted APPLα. Strikingly, KUZ overexpression not only failed to rescue the memory deficit caused by APPL loss of function, it exacerbated this deficit. We further show that in addition to an increase in secreted-APPL forms, KUZ overexpression caused a decrease of membrane-bound full-length species that could explain the memory deficit. Indeed, we observed that transient expression of a constitutive membrane-bound mutant APPL form is sufficient to rescue the memory deficit caused by APPL reduction, revealing for the first time a role of full-length APPL in memory formation. Our data demonstrate that, in addition to secreted APPL, the noncleaved form is involved in memory, raising the possibility that secreted and full-length APPL act together in memory processes. PMID:25609621

  7. Performance of initial full-length RHIC (Relativistic Heavy Ion Collider) dipoles

    SciTech Connect

    Dahl, P.; Cottingham, J.; Garber, M.; Ghosh, A.; Goodzeit, C.; Greene, A.; Herrera, J.; Kahn, S.; Kelly, E.; Morgan, G.

    1987-01-01

    The first four full-length (9.7 m) R and D dipoles for the proposed Relativistic Heavy Ion Collider (RHIC) have been successfully tested. The magnets reached a quench plateau of approximately 4.5 T with very reasonable training - a field level comfortably above the design field of 3.45 T required for operation with beams of 100 GeV/amu gold nuclei. Measured field multipoles are considered to be quite acceptable for this series of R and D magnets.

  8. Effects of Full-Length Kisspeptin Administration on Follicular Development in Japanese Black Beef Cows

    PubMed Central

    NANIWA, Yousuke; NAKATSUKASA, Keisuke; SETSUDA, Shohei; OISHI, Shinya; FUJII, Nobutaka; MATSUDA, Fuko; UENOYAMA, Yoshihisa; TSUKAMURA, Hiroko; MAEDA, Kei-ichiro; OHKURA, Satoshi

    2013-01-01

    Abstract Kisspeptin is a key molecule that stimulates gonadotropin secretion via release of gonadotropin-releasing hormone (GnRH). In the present study, our aim was to investigate whether kisspeptin has stimulatory effects on follicular development via GnRH/gonadotropin secretion in cows. Japanese Black beef cows were intravenously injected with full-length bovine kisspeptin [Kp-53 (0.2 or 2 nmol/kg)] or vehicle 5 days after they exhibited standing estrus (Day 0). In cows injected with Kp-53 at 2 nmol/kg, the follicular sizes of the first dominant follicles increased on Day 6 and thereafter. Ovulation of the first dominant follicle occurred in 1 out of 4 cows treated with Kp-53 at 2 nmol/kg. Injection of Kp-53 at 2 nmol/kg increased the concentration of plasma luteinizing hormone (LH) but not follicle-stimulating hormone, over a 4-h period following injection in all cows. The present study suggests that administration of full-length kisspeptin causes LH secretion, which is sustained for a few hours, and it is capable of stimulating follicular development and/or ovulation. PMID:24107742

  9. Structure of the full-length TRPV2 channel by cryo-EM

    PubMed Central

    Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

    2016-01-01

    Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a ‘minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2–6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels. PMID:27021073

  10. Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes.

    PubMed

    Alfano, Massimo; Cinque, Paola; Giusti, Guido; Proietti, Silvia; Nebuloni, Manuela; Danese, Silvio; D'Alessio, Silvia; Genua, Marco; Portale, Federica; Lo Porto, Manuela; Singhal, Pravin C; Rastaldi, Maria Pia; Saleem, Moin A; Mavilio, Domenico; Mikulak, Joanna

    2015-01-01

    Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms' tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur(-/-) mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR. PMID:26380915

  11. Structural Organization of a Full-Length Gp130/LIF-R Cytokine Receptor Transmembrane Complex

    SciTech Connect

    Skiniotis, G.; Lupardus, P.J.; Martick, M.; Walz, T.; Garcia, K.C.

    2009-05-26

    gp130 is a shared receptor for at least nine cytokines, and can signal either as a homodimer, or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-R{alpha}). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6R{alpha} hexameric complex, CNTF/CNTF-R{alpha} heterodimerizes gp130 and LIF-R via non-cooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic (EM) analysis of the full-length gp130/LIF-R/CNTF-R{alpha}/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the 'tall' class of gp130-family cytokine receptor complexes including LIF, IL-27, IL-12, and others.

  12. Full-length genomic characterization and molecular evolution of canine parvovirus in China.

    PubMed

    Zhou, Ling; Tang, Qinghai; Shi, Lijun; Kong, Miaomiao; Liang, Lin; Mao, Qianqian; Bu, Bin; Yao, Lunguang; Zhao, Kai; Cui, Shangjin; Leal, Élcio

    2016-06-01

    Canine parvovirus type 2 (CPV-2) can cause acute haemorrhagic enteritis in dogs and myocarditis in puppies. This disease has become one of the most serious infectious diseases of dogs. During 2014 in China, there were many cases of acute infectious diarrhoea in dogs. Some faecal samples were negative for the CPV-2 antigen based on a colloidal gold test strip but were positive based on PCR, and a viral strain was isolated from one such sample. The cytopathic effect on susceptible cells and the results of the immunoperoxidase monolayer assay, PCR, and sequencing indicated that the pathogen was CPV-2. The strain was named CPV-NY-14, and the full-length genome was sequenced and analysed. A maximum likelihood tree was constructed using the full-length genome and all available CPV-2 genomes. New strains have replaced the original strain in Taiwan and Italy, although the CPV-2a strain is still predominant there. However, CPV-2a still causes many cases of acute infectious diarrhoea in dogs in China. PMID:27038801

  13. Full-length high-temperature severe fuel damage test No. 5

    SciTech Connect

    Lanning, D.D.; Lombardo, N.J.; Hensley, W.K.; Fitzsimmons, D.E.; Panisko, F.E.; Hartwell, J.K.

    1993-09-01

    This report describes and presents data from a severe fuel damage test that was conducted in the National Research Universal (NRU) reactor at Chalk River Nuclear Laboratories (CRNL), Ontario, Canada. The test, designated FLHT-5, was the fourth in a series of full-length high-temperature (FLHT) tests on light-water reactor fuel. The tests were designed and performed by staff from the US Department of Energy`s Pacific Northwest Laboratory (PNL), operated by Battelle Memorial Institute. The test operation and test results are described in this report. The fuel bundle in the FLHT-5 experiment included 10 unirradiated full-length pressurized-water reactor (PWR) rods, 1 irradiated PWR rod and 1 dummy gamma thermometer. The fuel rods were subjected to a very low coolant flow while operating at low fission power. This caused coolant boilaway, rod dryout and overheating to temperatures above 2600 K, severe fuel rod damage, hydrogen generation, and fission product release. The test assembly and its effluent path were extensively instrumented to record temperatures, pressures, flow rates, hydrogen evolution, and fission product release during the boilaway/heatup transient. Post-test gamma scanning of the upper plenum indicated significant iodine and cesium release and deposition. Both stack gas activity and on-line gamma spectrometer data indicated significant ({approximately}50%) release of noble fission gases. Post-test visual examination of one side of the fuel bundle revealed no massive relocation and flow blockage; however, rundown of molten cladding was evident.

  14. Design, fabrication, and testing of an external fuel (UO2), full-length thermionic converter

    NASA Technical Reports Server (NTRS)

    Schock, A.; Raab, B.

    1971-01-01

    The development of a full-length external-fuel thermionic converter for in-pile testing is described. The development program includes out-of-pile performance testing of the fully fueled-converter, using RF-induction heating, before its installation in the in-pile test capsule. The external-fuel converter is cylindrical in shape, and consists of an inner, centrally cooled collector, and an outer emitter surrounded by nuclear fuel. The term full-length denotes that the converter is long enough to extend over the full height of the reactor core. Thus, the converter is not a scaled-down test device, but a full-scale fuel element of the thermionic reactor. The external-fuel converter concept permits a number of different design options, particularly with respect to the fuel composition and shape, and the collector cooling arrangement. The converter described was developed for the Jet Propulsion Laboratory, and is based on their concept for a thermionic reactor with uninsulated collector cooling as previously described. The converter is double-ended, with through-flow cooling, and with ceramic seals and emitter and collector power take-offs at both ends. The design uses a revolver-shaped tungsten emitter body, with the central emitter hole surrounded by six peripheral fuel holes loaded with cylindrical UO2 pellets.

  15. Full-length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells.

    PubMed

    Meregalli, Mirella; Navarro, Claire; Sitzia, Clementina; Farini, Andrea; Montani, Erica; Wein, Nicolas; Razini, Paola; Beley, Cyriaque; Cassinelli, Letizia; Parolini, Daniele; Belicchi, Marzia; Parazzoli, Dario; Garcia, Luis; Torrente, Yvan

    2013-12-01

    The protein dysferlin is abundantly expressed in skeletal and cardiac muscles, where its main function is membrane repair. Mutations in the dysferlin gene are involved in two autosomal recessive muscular dystrophies: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Development of effective therapies remains a great challenge. Strategies to repair the dysferlin gene by skipping mutated exons, using antisense oligonucleotides (AONs), may be suitable only for a subset of mutations, while cell and gene therapy can be extended to all mutations. AON-treated blood-derived CD133+ stem cells isolated from patients with Miyoshi myopathy led to partial dysferlin reconstitution in vitro but failed to express dysferlin after intramuscular transplantation into scid/blAJ dysferlin null mice. We thus extended these experiments producing the full-length dysferlin mediated by a lentiviral vector in blood-derived CD133+ stem cells isolated from the same patients. Transplantation of engineered blood-derived CD133+ stem cells into scid/blAJ mice resulted in sufficient dysferlin expression to correct functional deficits in skeletal muscle membrane repair. Our data suggest for the first time that lentivirus-mediated delivery of full-length dysferlin in stem cells isolated from Miyoshi myopathy patients could represent an alternative therapeutic approach for treatment of dysferlinopathies. PMID:24028392

  16. Mechanism of activation gating in the full-length KcsA K[superscript +] channel

    SciTech Connect

    Uysal, Serdar; Cuello, Luis G.; Cortes, D. Marien; Koide, Shohei; Kossiakoff, Anthony A.; Perozo, Eduardo

    2012-10-25

    Using a constitutively active channel mutant, we solved the structure of full-length KcsA in the open conformation at 3.9 {angstrom}. The structure reveals that the activation gate expands about 20 {angstrom}, exerting a strain on the bulge helices in the C-terminal domain and generating side windows large enough to accommodate hydrated K{sup +} ions. Functional and spectroscopic analysis of the gating transition provides direct insight into the allosteric coupling between the activation gate and the selectivity filter. We show that the movement of the inner gate helix is transmitted to the C-terminus as a straightforward expansion, leading to an upward movement and the insertion of the top third of the bulge helix into the membrane. We suggest that by limiting the extent to which the inner gate can open, the cytoplasmic domain also modulates the level of inactivation occurring at the selectivity filter.

  17. Full-length apolipoprotein E protects against the neurotoxicity of an apoE-related peptide

    PubMed Central

    Crutcher, K.A.; Lilley, H.N.; Anthony, S. R.; Zhou, W.; Narayanaswami, V.

    2009-01-01

    Apolipoprotein E was found to protect against the neurotoxic effects of a dimeric peptide derived from the receptor-binding region of this protein (residues 141–149). Both apoE3 and apoE4 conferred protection but the major N-terminal fragment of each isoform did not. Nor was significant protection provided by bovine serum albumin or apoA-I. Full-length apoE3 and apoE4 also inhibited the uptake of a fluorescent-labeled derivative of the peptide, suggesting that the mechanism of inhibition might involve competition for cell surface receptors/proteoglycans that mediate endocytosis and/or signaling pathways. These results might bear on the question of the role of apoE in neuronal degeneration, such as occurs in Alzheimer’s disease where apoE4 confers a significantly greater risk of pathology. PMID:19836363

  18. On the Minimum Weight of Simple Full-Length Array LDPC Codes

    NASA Astrophysics Data System (ADS)

    Sugiyama, Kenji; Kaji, Yuichi

    We investigate the minimum weights of simple full-length array LDPC codes (SFA-LDPC codes). The SFA-LDPC codes are a subclass of LDPC codes, and constructed algebraically according to two integer parameters p and j. Mittelholzer and Yang et al. have studied the minimum weights of SFA-LDPC codes, but the exact minimum weights of the codes are not known except for some small p and j. In this paper, we show that the minimum weights of the SFA-LDPC codes with j=4 and j=5 are upper-bounded by 10 and 12, respectively, independent from the prime number p. By combining the results with Yang's lower-bound limits, we can conclude that the minimum weights of the SFA-LDPC codes with j=4 and p>7 are exactly 10 and those of the SFA-LDPC codes with j=5 are 10 or 12.

  19. The first detection and full-length genome sequence of porcine deltacoronavirus isolated in Lao PDR.

    PubMed

    Lorsirigool, Athip; Saeng-Chuto, Kepalee; Temeeyasen, Gun; Madapong, Adthakorn; Tripipat, Thitima; Wegner, Matthew; Tuntituvanont, Angkana; Intrakamhaeng, Manakant; Nilubol, Dachrit

    2016-10-01

    Porcine deltacoronavirus (PDCoV) has been reported in many countries, including Hong Kong, the United States, South Korea, China and Thailand. In January 2016, clinical diarrhea similar to that of porcine epidemic diarrhea virus (PEDV) with a lower mortality rate was reported on a swine farm in Lao PDR. Intestine samples were collected from 3-day-old pigs with clinical diarrhea and assayed for the presence of swine enteric coronaviruses. The PCR results were positive for PDCoV but negative for PEDV and TGEV. A phylogenetic tree demonstrated that PDCoV from Lao PDR was grouped separately from PDCoV isolates from China and the USA, but was more closely related to the Chinese isolates than to the US isolates. The full-length genome sequence of the novel PDCoV isolate P1_16_BTL_0116 was determined. PMID:27424024

  20. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

    PubMed Central

    Robinson, Michael-Paul; Ke, Na; Lobstein, Julie; Peterson, Cristen; Szkodny, Alana; Mansell, Thomas J.; Tuckey, Corinna; Riggs, Paul D.; Colussi, Paul A.; Noren, Christopher J.; Taron, Christopher H.; DeLisa, Matthew P.; Berkmen, Mehmet

    2015-01-01

    Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named ‘cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. PMID:26311203

  1. Cocrystallization studies of full-length recombinant butyrylcholinesterase (BChE) with cocaine

    SciTech Connect

    Asojo, Oluwatoyin Ajibola; Asojo, Oluyomi Adebola; Ngamelue, Michelle N.; Homma, Kohei; Lockridge, Oksana

    2011-09-16

    Human butyrylcholinesterase (BChE; EC 3.1.1.8) is a 340 kDa tetrameric glycoprotein that is present in human serum at about 5 mg l{sup -1} and has well documented therapeutic effects on cocaine toxicity. BChE holds promise as a therapeutic that reduces and finally eliminates the rewarding effects of cocaine, thus weaning an addict from the drug. There have been extensive computational studies of cocaine hydrolysis by BChE. Since there are no reported structures of BChE with cocaine or any of the hydrolysis products, full-length monomeric recombinant wild-type BChE was cocrystallized with cocaine. The refined 3 {angstrom} resolution structure appears to retain the hydrolysis product benzoic acid in sufficient proximity to form a hydrogen bond to the active-site Ser198.

  2. Generation of a Mouse Full-length Balancer with Versatile Cassette-shuttling Selection Strategy

    PubMed Central

    Ye, Zhisheng; Sun, Lei; Li, Rongbo; Han, Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

    2016-01-01

    Balancer chromosomes are important tools for a variety of genetic manipulations in lower model organisms, owing to their ability to suppress recombination. In mouse, however, such effort has not been accomplished, mostly due to the size of the chromosomes and the complexity of multiple step chromosomal engineering. We developed an effective and versatile cassette-shuttling selection (CASS) strategy involving only two selection markers to achieve the sequential production of multiple large inversions along the chromosome. Using this strategy, we successfully generated the first full-length balancer in mice and showed that Balancer 17M-GFP can efficiently suppress recombination. Our study has not only generated a useful genetic resource, but also provided a strategy for constructing mammalian balancer chromosomes. PMID:27489495

  3. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.

    PubMed

    Arturo, Emilia C; Gupta, Kushol; Héroux, Annie; Stith, Linda; Cross, Penelope J; Parker, Emily J; Loll, Patrick J; Jaffe, Eileen K

    2016-03-01

    Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate. PMID:26884182

  4. Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq

    PubMed Central

    Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

    2015-01-01

    Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

  5. Production, purification and characterization of recombinant, full-length human claudin-1.

    PubMed

    Bonander, Nicklas; Jamshad, Mohammed; Oberthür, Dominik; Clare, Michelle; Barwell, James; Hu, Ke; Farquhar, Michelle J; Stamataki, Zania; Harris, Helen J; Dierks, Karsten; Dafforn, Timothy R; Betzel, Christian; McKeating, Jane A; Bill, Roslyn M

    2013-01-01

    The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81. PMID:23704991

  6. Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq.

    PubMed

    Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

    2015-01-01

    Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

  7. Fabrication and Testing of Full-Length Single-Cell Externally Fueled Converters for Thermionic Reactors

    SciTech Connect

    Schock, Alfred

    1994-06-01

    The preceding paper described designs and analyses of thermionic reactors employing full-core-length single-cell converters with their heated emitters located on the outside of their internally cooled collectors, and it presented results of detailed parametric analyses which illustrate the benefits of this unconventional design. The present paper describes the fabrication and testing of full-length prototypical converters, both unfueled and fueled, and presents parametric results of electrically heated tests. The unfueled converter tests demonstrated the practicality of operating such long converters without shorting across a 0.3-mm interelectrode gap. They produced a measured peak output of 751 watts(e) from a single diode and a peak efficiency of 15.4%. The fueled converter tests measured the parametric performance of prototypic UO(subscript 2)-fueled converters designed for subsequent in-pile testing. They employed revolver-shaped tungsten elements with a central emitter hole surrounded by six fuel chambers. The full-length converters were heated by a water-cooled RF-induction coil inside an ion-pumped vacuum chamber. This required development of high-vacuum coaxial RF feedthroughs. In-pile test rules required multiple containment of the UO (subscript 2)-fuel, which complicated the fabrication of the test article and required successful development of techniques for welding tungsten and other refractory components. The test measured a peak power output of 530 watts(e) or 7.1 watts/cm (superscript 2) at an efficiency of 11.5%. There are three copies in the file. Cross-Reference a copy FSC-ESD-217-94-529 in the ESD files with a CID #8574.

  8. Methionine oxidation induces amyloid fibril formation by full-length apolipoprotein A-I

    PubMed Central

    Wong, Yuan Qi; Binger, Katrina J.; Howlett, Geoffrey J.; Griffin, Michael D. W.

    2010-01-01

    Apolipoprotein A-I (apoA-I) is the major protein component of HDL, where it plays an important role in cholesterol transport. The deposition of apoA-I derived amyloid is associated with various hereditary systemic amyloidoses and atherosclerosis; however, very little is known about the mechanism of apoA-I amyloid formation. Methionine residues in apoA-I are oxidized via several mechanisms in vivo to form methionine sulfoxide (MetO), and significant levels of methionine oxidized apoA-I (MetO-apoA-I) are present in normal human serum. We investigated the effect of methionine oxidation on the structure, stability, and aggregation of full-length, lipid-free apoA-I. Circular dichrosim spectroscopy showed that oxidation of all three methionine residues in apoA-I caused partial unfolding of the protein and decreased its thermal stability, reducing the melting temperature (Tm) from 58.7 °C for native apoA-I to 48.2 °C for MetO-apoA-I. Analytical ultracentrifugation revealed that methionine oxidation inhibited the native self association of apoA-I to form dimers and tetramers. Incubation of MetO-apoA-I for extended periods resulted in aggregation of the protein, and these aggregates bound Thioflavin T and Congo Red. Inspection of the aggregates by electron microscopy revealed fibrillar structures with a ribbon-like morphology, widths of approximately 11 nm, and lengths of up to several microns. X-ray fibre diffraction studies of the fibrils revealed a diffraction pattern with orthogonal peaks at spacings of 4.64 Å and 9.92 Å, indicating a cross-β amyloid structure. This systematic study of fibril formation by full-length apoA-I represents the first demonstration that methionine oxidation can induce amyloid fibril formation. PMID:20133843

  9. Characterization of full-length recombinant human Proteoglycan 4 as an ocular surface boundary lubricant.

    PubMed

    Samsom, Michael L; Morrison, Sheila; Masala, Nemanja; Sullivan, Benjamin D; Sullivan, David A; Sheardown, Heather; Schmidt, Tannin A

    2014-10-01

    Proteoglycan 4 (PRG4, or lubricin) is a lubricating mucin-like glycoprotein recently discovered at the ocular surface, where it functions as a boundary lubricant and appears to play a protective role. Recent technological advances have enabled abundant expression of full-length recombinant human PRG4 (rhPRG4). The objectives of this study were to 1) biochemically characterize the gross structure and glycosylations of full-length rhPRG4, and 2) assess the ocular surface boundary lubricating ability of rhPRG4 at both human cornea-eyelid and human cornea-polydimethylsiloxane (PDMS) biointerfaces. rhPRG4 expressed by a Chinese hamster ovary cell line was characterized and compared to native bovine PRG4 by SDS-PAGE western blotting, and protein identity was assessed by tandem mass spectrometry (MS/MS). Human corneas were articulated against PDMS or human eyelids, at effective sliding velocities of 0.3-30 mm/s under physiological loads of ∼15 kPa, to assess and compare the ocular lubricating ability of rhPRG4 to PRG4. Samples were tested serially in PRG4, rhPRG4 (both 300 μg/ml), then saline. Western blotting indicated that rhPRG4 had immunoreactivity at the appropriate apparent molecular weight, and possessed O-linked glycosylation consistent with that of PRG4. rhPRG4 protein identity was confirmed by MS/MS. Both PRG4 and rhPRG4 significantly, and similarly, reduced friction compared to saline at both human cornea - PDMS and human cornea-eyelid biointerfaces. In conclusion, the rhPRG4 studied here demonstrated appropriate higher order structure, O-linked glycosylations, and ocular surface boundary lubricating. Purified rhPRG4 may have clinical utility as a topical treatment of dry eye disease or contact lens biomaterial coating to promote more comfortable wear. PMID:24997456

  10. A Glutamine/Asparagine-Rich Fragment of Gln3, but not the Full-Length Protein, Aggregates in Saccharomyces cerevisiae.

    PubMed

    Antonets, K S; Sargsyan, H M; Nizhnikov, A A

    2016-04-01

    The amino acid sequence of protein Gln3 in yeast Saccharomyces cerevisiae has a region enriched with Gln (Q) and Asn (N) residues. In this study, we analyzed the effects of overexpression of Gln3 and its Q/N-rich fragment fused with yellow fluorescent protein (YFP). Being overexpressed, full-length Gln3-YFP does not form aggregates, inhibits vegetative growth, and demonstrates nuclear localization, while the Q/N-rich fragment (Gln3QN) fused with YFP forms aggregates that do not colocalize with the nucleus and do not affect growth of the cells. Although detergent-resistant aggregates of Gln3QN are formed in the absence of yeast prions, the aggregation of Gln3QN significantly increases in the presence of [PIN(+)] prion, while in the presence of two prions, [PSI(+)] and [PIN(+)], the percentage of cells with Gln3QN aggregates is significantly lower than in the strain bearing only [PIN(+)]. Data on colocalization demonstrate that this effect is mediated by interaction between Gln3QN aggregates and [PSI(+)] and [PIN(+)] prions. PMID:27293098

  11. Construction of phosphorylation interaction networks by text mining of full-length articles using the eFIP system

    PubMed Central

    Tudor, Catalina O.; Ross, Karen E.; Li, Gang; Vijay-Shanker, K.; Wu, Cathy H.; Arighi, Cecilia N.

    2015-01-01

    Protein phosphorylation is a reversible post-translational modification where a protein kinase adds a phosphate group to a protein, potentially regulating its function, localization and/or activity. Phosphorylation can affect protein–protein interactions (PPIs), abolishing interaction with previous binding partners or enabling new interactions. Extracting phosphorylation information coupled with PPI information from the scientific literature will facilitate the creation of phosphorylation interaction networks of kinases, substrates and interacting partners, toward knowledge discovery of functional outcomes of protein phosphorylation. Increasingly, PPI databases are interested in capturing the phosphorylation state of interacting partners. We have previously developed the eFIP (Extracting Functional Impact of Phosphorylation) text mining system, which identifies phosphorylated proteins and phosphorylation-dependent PPIs. In this work, we present several enhancements for the eFIP system: (i) text mining for full-length articles from the PubMed Central open-access collection; (ii) the integration of the RLIMS-P 2.0 system for the extraction of phosphorylation events with kinase, substrate and site information; (iii) the extension of the PPI module with new trigger words/phrases describing interactions and (iv) the addition of the iSimp tool for sentence simplification to aid in the matching of syntactic patterns. We enhance the website functionality to: (i) support searches based on protein roles (kinases, substrates, interacting partners) or using keywords; (ii) link protein entities to their corresponding UniProt identifiers if mapped and (iii) support visual exploration of phosphorylation interaction networks using Cytoscape. The evaluation of eFIP on full-length articles achieved 92.4% precision, 76.5% recall and 83.7% F-measure on 100 article sections. To demonstrate eFIP for knowledge extraction and discovery, we constructed phosphorylation

  12. Varicella-zoster virus (VZV) transcription during latency in human ganglia: detection of transcripts mapping to genes 21, 29, 62, and 63 in a cDNA library enriched for VZV RNA.

    PubMed Central

    Cohrs, R J; Barbour, M; Gilden, D H

    1996-01-01

    Information on the extent of virus DNA transcription and translation in infected tissue is crucial to an understanding of herpesvirus latency. To detect low-abundance latent varicella-zoster virus (VZV) transcripts, poly(A)+ RNA extracted from latently infected human trigeminal ganglia was enriched for VZV transcripts by hybridization to biotinylated VZV DNA. After hybridization, the RNA-DNA hybrid was isolated by binding to avidin-coated beads and extensively washed, and the RNA was released by heat denaturation. A lambda-based cDNA library was then constructed from the enriched RNA. PCR and DNA sequencing of DNA extracted from the cDNA library revealed the presence of VZV genes 21, 29, 62, and 63, but not VZV genes 4, 10, 40, 51, and 61, in the enriched cDNA library. These findings confirm the detection of VZV gene 29 and 62 transcripts on Northern (RNA) blots prepared from latently infected human ganglia (J.L. Meier, R.P. Holman, K.D. Croen, J.E. Smialek, and S.E. Straus, Virology 193:193-200, 1993) and the presence of VZV gene 21 transcripts in a cDNA library from mRNA of latently infected ganglia (R.J. Cohrs, K. Srock, M.B. Barbour, G. Owens, R. Mahalingam, M.E. Devlin, M. Wellish and D.H. Gilden, J. Virol. 68:7900-7908,1994) and also reveal, for the first time, the presence of VZV gene 63 RNA in latently infected human ganglia. PMID:8627753

  13. How to obtain a non-digital Full-length lower limb Radiographs using regular X-ray plates

    PubMed Central

    Parihar, Mangal; Singh, Tarandeep; Shah, Lalitkumar

    2011-01-01

    Full-length hip-to-ankle radiographs are an important part of planning and execution of any realignment procedure of the lower limb. This technical note describes a simple and inexpensive technique to obtain plain, standing, full-length hip-to-ankle radiograph using regular x-ray plates.

  14. Factors Influencing the Production of MFSV Full-Length Clone: Maize Fine Streak Virus Proteins in Drosophila S2 Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize fine streak virus (MFSV) is negative-sense RNA virus member of the genus Nucleorhabdovirus. Our goal is to determine whether Drosophila S2 cells can support the production of a full-length clone of MFSV. We have previously demonstrated that the full-length MFSV nucleoprotein (N) and phosphopro...

  15. Effect of systemically increasing human full-length Klotho on glucose metabolism in db/db mice.

    PubMed

    Forsberg, E A; Olauson, H; Larsson, T; Catrina, S B

    2016-03-01

    The metabolic effects of antiaging Klotho were previously investigated in vivo by genetic manipulation. We have here studied the metabolic effect of physiologic levels of circulating full length Klotho in db/db mice. Increasing the full-length human Klotho levels has a positive effect on blood glucose through increasing insulin secretion. PMID:26806457

  16. Structure and Dynamics of Full Length HIV-1 Capsid Protein in Solution

    PubMed Central

    Deshmukh, Lalit; Schwieters, Charles D.; Grishaev, Alexander; Ghirlando, Rodolfo; Baber, James L.; Clore, G. Marius

    2013-01-01

    The HIV-1 capsid protein plays a crucial role in viral infectivity, assembling into a cone that encloses the viral RNA. In the mature virion, the N-terminal domain of the capsid protein forms hexameric and pentameric rings, while C-terminal domain homodimers connect adjacent N-terminal domain rings to one another. Structures of disulfide-linked hexamer and pentamer assemblies, as well as structures of the isolated domains have been solved previously. The dimer configuration in C-terminal domain constructs differs in solution (residues 144–231) and crystal (residues 146–231) structures by ~30°, and it has been postulated that the former connects the hexamers while the latter links pentamers to hexamers. Here we study the structure and dynamics of full-length capsid protein in solution, comprising a mixture of monomeric and dimeric forms in dynamic equilibrium, using ensemble simulated annealing driven by experimental NMR residual dipolar couplings and X-ray scattering data. The complexity of the system necessitated the development of a novel computational framework that should be generally applicable to many other challenging systems that currently escape structural characterization by standard application of mainstream techniques of structural biology. We show that the orientation of the C-terminal domains in dimeric full-length capsid and isolated C-terminal domain constructs is the same in solution, and obtain a quantitative description of the conformational space sampled by the N-terminal domain relative to the C-terminal domain on the nano- to millisecond time-scale. The positional distribution of the N-terminal domain relative to the C-terminal domain is large and modulated by the oligomerization state of the C-terminal domain. We also show that a model of the hexamer/pentamer assembly can be readily generated with a single configuration of the C-terminal domain dimer, and that capsid assembly likely proceeds via conformational selection of sparsely

  17. Single-Molecule Studies of Unlabeled Full-Length p53 Protein Binding to DNA.

    PubMed

    Nuttall, Philippa; Lee, Kidan; Ciccarella, Pietro; Carminati, Marco; Ferrari, Giorgio; Kim, Ki-Bum; Albrecht, Tim

    2016-03-10

    p53 is an antitumor protein that plays an important role in apoptosis, preserving genomic stability and preventing angiogenesis, and it has been implicated in a large number of human cancers. For this reason it is an interesting target for both fundamental studies, such as the mechanism of interaction with DNA, and applications in biosensing. Here, we report a comprehensive study of label-free, full length p53 (flp53) and its interaction with engineered double-stranded DNA in vitro, at the single-molecule level, using atomic force microscopy (AFM) imaging and solid-state nanopore sensing. AFM data show that dimeric and tetrameric p53 bind to the DNA in a sequence-specific manner, confirming previously reported relative binding affinities. The statistical significance is tested using both the Grubbs test and stochastic simulations. For the first time, ultralow noise solid-state nanopore sensors are employed for the successful differentiation between bare DNA and p53/DNA complexes. Furthermore, translocation statistics reflect the binding affinities of different DNA sequences, in accordance with AFM data. Our results thus highlight the potential of solid-state nanopore sensors for single-molecule biosensing, especially when labeling is either not possible or at least not a viable option. PMID:26855037

  18. Improving the diffraction of full-length human selenomethionyl metavinculin crystals by streak-seeding

    SciTech Connect

    Rangarajan, Erumbi S.; Izard, Tina

    2012-06-28

    Metavinculin is an alternatively spliced isoform of vinculin that has a 68-residue insert in its tail domain (1134 total residues) and is exclusively expressed in cardiac and smooth muscle tissue, where it plays important roles in myocyte adhesion complexes. Mutations in the metavinculin-specific insert are associated with dilated cardiomyopathy (DCM) in man. Crystals of a DCM-associated mutant of full-length selenomethionine-labeled metavinculin grown by hanging-drop vapor diffusion diffracted poorly and were highly sensitive to radiation, preventing the collection of a complete X-ray diffraction data set at the highest possible resolution. Streak-seeding markedly improved the stability, crystal-growth rate and diffraction quality of DCM-associated mutant metavinculin crystals, allowing complete data collection to 3.9 {angstrom} resolution. These crystals belonged to space group P4{sub 3}2{sub 1}2, with two molecules in the asymmetric unit and unit-cell parameters a = b = 170, c = 211 {angstrom}, {alpha} = {beta} = {gamma} = 90{sup o}.

  19. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

    PubMed Central

    Bezem, Maria T.; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I.

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

  20. Complete sequencing and characterization of 21,243 full-length human cDNAs.

    PubMed

    Ota, Toshio; Suzuki, Yutaka; Nishikawa, Tetsuo; Otsuki, Tetsuji; Sugiyama, Tomoyasu; Irie, Ryotaro; Wakamatsu, Ai; Hayashi, Koji; Sato, Hiroyuki; Nagai, Keiichi; Kimura, Kouichi; Makita, Hiroshi; Sekine, Mitsuo; Obayashi, Masaya; Nishi, Tatsunari; Shibahara, Toshikazu; Tanaka, Toshihiro; Ishii, Shizuko; Yamamoto, Jun-ichi; Saito, Kaoru; Kawai, Yuri; Isono, Yuko; Nakamura, Yoshitaka; Nagahari, Kenji; Murakami, Katsuhiko; Yasuda, Tomohiro; Iwayanagi, Takao; Wagatsuma, Masako; Shiratori, Akiko; Sudo, Hiroaki; Hosoiri, Takehiko; Kaku, Yoshiko; Kodaira, Hiroyo; Kondo, Hiroshi; Sugawara, Masanori; Takahashi, Makiko; Kanda, Katsuhiro; Yokoi, Takahide; Furuya, Takako; Kikkawa, Emiko; Omura, Yuhi; Abe, Kumi; Kamihara, Kumiko; Katsuta, Naoko; Sato, Kazuomi; Tanikawa, Machiko; Yamazaki, Makoto; Ninomiya, Ken; Ishibashi, Tadashi; Yamashita, Hiromichi; Murakawa, Katsuji; Fujimori, Kiyoshi; Tanai, Hiroyuki; Kimata, Manabu; Watanabe, Motoji; Hiraoka, Susumu; Chiba, Yoshiyuki; Ishida, Shinichi; Ono, Yukio; Takiguchi, Sumiyo; Watanabe, Susumu; Yosida, Makoto; Hotuta, Tomoko; Kusano, Junko; Kanehori, Keiichi; Takahashi-Fujii, Asako; Hara, Hiroto; Tanase, Tomo-o; Nomura, Yoshiko; Togiya, Sakae; Komai, Fukuyo; Hara, Reiko; Takeuchi, Kazuha; Arita, Miho; Imose, Nobuyuki; Musashino, Kaoru; Yuuki, Hisatsugu; Oshima, Atsushi; Sasaki, Naokazu; Aotsuka, Satoshi; Yoshikawa, Yoko; Matsunawa, Hiroshi; Ichihara, Tatsuo; Shiohata, Namiko; Sano, Sanae; Moriya, Shogo; Momiyama, Hiroko; Satoh, Noriko; Takami, Sachiko; Terashima, Yuko; Suzuki, Osamu; Nakagawa, Satoshi; Senoh, Akihiro; Mizoguchi, Hiroshi; Goto, Yoshihiro; Shimizu, Fumio; Wakebe, Hirokazu; Hishigaki, Haretsugu; Watanabe, Takeshi; Sugiyama, Akio; Takemoto, Makoto; Kawakami, Bunsei; Yamazaki, Masaaki; Watanabe, Koji; Kumagai, Ayako; Itakura, Shoko; Fukuzumi, Yasuhito; Fujimori, Yoshifumi; Komiyama, Megumi; Tashiro, Hiroyuki; Tanigami, Akira; Fujiwara, Tsutomu; Ono, Toshihide; Yamada, Katsue; Fujii, Yuka; Ozaki, Kouichi; Hirao, Maasa; Ohmori, Yoshihiro; Kawabata, Ayako; Hikiji, Takeshi; Kobatake, Naoko; Inagaki, Hiromi; Ikema, Yasuko; Okamoto, Sachiko; Okitani, Rie; Kawakami, Takuma; Noguchi, Saori; Itoh, Tomoko; Shigeta, Keiko; Senba, Tadashi; Matsumura, Kyoka; Nakajima, Yoshie; Mizuno, Takae; Morinaga, Misato; Sasaki, Masahide; Togashi, Takushi; Oyama, Masaaki; Hata, Hiroko; Watanabe, Manabu; Komatsu, Takami; Mizushima-Sugano, Junko; Satoh, Tadashi; Shirai, Yuko; Takahashi, Yukiko; Nakagawa, Kiyomi; Okumura, Koji; Nagase, Takahiro; Nomura, Nobuo; Kikuchi, Hisashi; Masuho, Yasuhiko; Yamashita, Riu; Nakai, Kenta; Yada, Tetsushi; Nakamura, Yusuke; Ohara, Osamu; Isogai, Takao; Sugano, Sumio

    2004-01-01

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs. PMID:14702039

  1. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain.

    PubMed

    Sükösd, Zsuzsanna; Andersen, Ebbe S; Seemann, Stefan E; Jensen, Mads Krogh; Hansen, Mathias; Gorodkin, Jan; Kjems, Jørgen

    2015-12-01

    A distance constrained secondary structural model of the ≈10 kb RNA genome of the HIV-1 has been predicted but higher-order structures, involving long distance interactions, are currently unknown. We present the first global RNA secondary structure model for the HIV-1 genome, which integrates both comparative structure analysis and information from experimental data in a full-length prediction without distance constraints. Besides recovering known structural elements, we predict several novel structural elements that are conserved in HIV-1 evolution. Our results also indicate that the structure of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping protein-coding regions the COS is supported by a particular high frequency of compensatory base changes, suggesting functional importance for this element. This new structural element potentially organizes the whole genome into three major domains protruding from a conserved core structure with potential roles in replication and evolution for the virus. PMID:26476446

  2. Recombinant production and film properties of full-length hornet silk proteins.

    PubMed

    Kambe, Yusuke; Sutherland, Tara D; Kameda, Tsunenori

    2014-08-01

    Full-length versions of the four main components of silk cocoons of Vespa simillima hornets, Vssilk1-4, were produced as recombinant proteins in Escherichia coli. In shake flasks, the recombinant Vssilk proteins yielded 160-330mg recombinant proteinl(-1). Films generated from solutions of single Vssilk proteins had a secondary structure similar to that of films generated from native hornet silk. The films made from individual recombinant hornet silk proteins had similar or enhanced mechanical performance compared with films generated from native hornet silk, possibly reflecting the homogeneity of the recombinant proteins. The pH-dependent changes in zeta (ζ) potential of each Vssilk film were measured, and isoelectric points (pI) of Vssilk1-4 were determined as 8.9, 9.1, 5.0 and 4.2, respectively. The pI of native hornet silk, a combination of the four Vssilk proteins, was 4.7, a value similar to that of Bombyx mori silkworm silk. Films generated from Vssilk1 and 2 had net positive charge under physiological conditions and showed significantly higher cell adhesion activity. It is proposed that recombinant hornet silk is a valuable new material with potential for cell culture applications. PMID:24862540

  3. Comprehensive Analysis of the Green-to-Blue Photoconversion of Full-Length Cyanobacteriochrome Tlr0924

    PubMed Central

    Hardman, Samantha J.O.; Hauck, Anna F.E.; Clark, Ian P.; Heyes, Derren J.; Scrutton, Nigel S.

    2014-01-01

    Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of central importance in biological light-activated signaling mechanisms. These photoreceptors are known to reversibly convert between two states in a photoinitiated process that involves a basic E/Z isomerization of the bilin chromophore and, in certain cases, the breakage of a thioether linkage to a conserved cysteine residue in the bulk protein structure. The exact details and timescales of the reactions involved in these photoconversions have not been conclusively shown. The cyanobacteriochrome Tlr0924 contains phycocyanobilin and phycoviolobilin chromophores, both of which photoconvert between two species: blue-absorbing and green-absorbing, and blue-absorbing and red-absorbing, respectively. Here, we followed the complete green-to-blue photoconversion process of the phycoviolobilin chromophore in the full-length form of Tlr0924 over timescales ranging from femtoseconds to seconds. Using a combination of time-resolved visible and mid-infrared transient absorption spectroscopy and cryotrapping techniques, we showed that after photoisomerization, which occurs with a lifetime of 3.6 ps, the phycoviolobilin twists or distorts slightly with a lifetime of 5.3 μs. The final step, the formation of the thioether linkage with the protein, occurs with a lifetime of 23.6 ms. PMID:25418104

  4. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme.

    PubMed

    Bezem, Maria T; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

  5. Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes

    PubMed Central

    Ayoub, Nadia A.; Garb, Jessica E.; Tinghitella, Robin M.; Collin, Matthew A.; Hayashi, Cheryl Y.

    2007-01-01

    Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers. PMID:17565367

  6. The full-length transcripts and promoter analysis of intergenic microRNAs in Drosophila melanogaster.

    PubMed

    Qian, Jinjun; Zhang, Zan; Liang, Jingdong; Ge, Qiongqiong; Duan, Xuchu; Ma, Fei; Li, Fei

    2011-05-01

    MicroRNA (miRNA) transcription is still not well understood until now. To increase the miRNA abundance, we stimulated miRNA transcription with CuSO(4) and knocked down Drosha enzyme using dsRNA in Drosophila S2 cells. The full length transcripts of bantam, miR-276a and miR-277, the 5'-end of miR-8, the 3'-end of miR-2b and miR-10 were obtained. We also conducted a series of miRNA promoter analysis to prove the reliability of RACE results. Luciferase-reporter assays proved that both bantam and miR-276a promoters successfully drove the expressions of downstream luciferase genes. The promoter activities were impaired by introducing one or multiple mutations at predicted transcription factor binding sites. Chromatin immunoprecipitation analysis confirmed that hypophosphorylated RNA polymerase II and transcription factor c-Myc physically bind at miRNA promoter. RNA interference of transcription factors Mad and Prd led to down-expression of bantam, miR-277 and miR-2b but not miR-276a, whereas RNAi of Dorsal had the opposite effect. PMID:21333734

  7. Functional Characterization of a Full Length Pregnane X Receptor, Expression in vivo, and Identification of PXR Alleles in Zebrafish (Danio rerio)

    PubMed Central

    Bainy, Afonso C. D.; Kubota, Akira; Goldstone, Jared V.; Lille-Langøy, Roger; Karchner, Sibel I.; Celander, Malin C.; Hahn, Mark E.; Goksøyr, Anders

    2013-01-01

    The pregnane X receptor (PXR) (nuclear receptor NR1I2) is a ligand activated transcription factor, mediating responses to diverse xenobiotic and endogenous chemicals. The properties of PXR in fish are not fully understood. Here we report on cloning and characterization of full-length PXR of zebrafish, Danio rerio, and pxr expression in vivo. Initial efforts gave a cDNA encoding a 430 amino acid protein identified as zebrafish pxr by phylogenetic and synteny analysis. The sequence of the cloned Pxr DNA binding domain was highly conserved, with 74% identity to human PXR, while the ligand-binding domain (LBD) of the cloned sequence was only 44% identical to human PXR-LBD. Sequence variation among clones in the initial effort prompted sequencing of multiple clones from a single fish. There were two prominent variants, one sequence with S183, Y218 and H383 and the other with I183, C218 and N383, which we designate as alleles pxr*1 (nr1i2*1) and pxr*2 (nr1i2*2), respectively. In COS-7 cells co-transfected with a PXR-responsive reporter gene, the full-length Pxr*1 (the more common variant) was activated by known PXR agonists clotrimazole and pregnenolone 16α-carbonitrile but to a lesser extent than the full-length human PXR. Activation of full-length Pxr*1 was only 10% of that with the Pxr*1 LBD. Quantitative real time PCR analysis showed prominent expression of pxr in liver and eye, as well as brain and intestine of adult zebrafish. The pxr was expressed in heart and kidney at levels similar to that in intestine. The expression of pxr in liver was weakly induced by ligands for mammalian PXR or CAR (NR1I3). The results establish a foundation for PXR studies in this vertebrate model. PXR allelic variation and the differences between the full-length PXR and the LBD in reporter assays have implications for assessing the action of PXR ligands in zebrafish. PMID:24121122

  8. Correlation of the level of full-length CFTR transcript with pulmonary phenotype in patients carrying R117H and 1342-1,-2delAG mutations

    SciTech Connect

    Hamosh, A.; Cutting, G.R.; Oates, R.; Amos, J.

    1994-09-01

    The R117H mutation occurs on two chromosome backgrounds, one associated with a 7 thymidine tract (7T-R11H) in the splice-acceptor site of intron 8, the other with a 5 thymidine tract (5T-R117H). We examined exon 9 splicing efficiency in 5 patients of genotype R117H/{delta}F508 and one carrying 1342-1,-2delAG{delta}F508, an obligate exon 9 slice site mutation. Four patients carried R117H on a 7T background -- three adult men with congenital bilateral absence of the vas deferens and one adolescent female with pancreatitis and borderline sweat chloride concentration. The patient with R117H on a 5T background had pancreatic sufficient CF (PS-CF). The 1342-1,-2delAG patient has classic pancreatic insufficient CF (PI-CF). cDNA was synthesized from total RNA extracted from nasal epithlial cells and analyzed for CFTR splicing by 35 cycle PCR using primers in exon 7 and 11. The quantity of full length transcript derived from the R117H or {delta}F508 alleles was assessed by allele-specific oligonucleotide hybridization. While 91.4% of transcript from the 5T-R117H allele was full-length, only 42.2% of CFTR transcript from the 5T-R117H allele was full length. Since CBAVD patients have no lung disease and PS-CF patients do, this indicates that the threshold of developing CF lung disease is crossed when the amount of CFTR transcript bearing R117H is reduced by half. Interestingly, 17.1% of transcript derived from the 1342-1,-2delAG allele (or 8.6% of total CFTR transcript) was normal and full length. This suggests that up to 9% of full length wild-type CFTR transcript may be inadequate to escape the lung disease of CF and that a 9 thymidine tract followed by AAC (the result of the AG deletion) can be used as a splice donor with 2-9% efficiency.

  9. Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB

    PubMed Central

    Itoh, Yuzuru; Sekine, Shun-ichi; Yokoyama, Shigeyuki

    2015-01-01

    Selenocysteine (Sec), the 21st amino acid in translation, uses its specific tRNA (tRNASec) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNASec (Sec-tRNASec) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1–3), followed by four winged-helix domains (WHD1–4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNASec is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNASec, SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNASec allows SelB to specifically recognize tRNASec and characteristically place it at the ribosomal A-site. PMID:26304550

  10. Purification and Activity Testing of the Full-Length YycFGHI Proteins of Staphylococcus aureus

    PubMed Central

    Türck, Michael; Bierbaum, Gabriele

    2012-01-01

    Background The YycFG two-component regulatory system (TCS) of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in Gram-positive bacteria with a low G+C-content. YycG (WalK/VicK) is a sensor histidine-kinase and YycF (WalR/VicR) is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance. Methodology/Principal Findings Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF) only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposome-model indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment. Conclusions/Significance The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies and for identification of all signals received by the YycFGHI system. PMID:22276191

  11. Full-Length Semaphorin-3C Is an Inhibitor of Tumor Lymphangiogenesis and Metastasis.

    PubMed

    Mumblat, Yelena; Kessler, Ofra; Ilan, Neta; Neufeld, Gera

    2015-06-01

    Semaphorins play important regulatory roles in diverse processes such as axon guidance, angiogenesis, and immune responses. We find that semaphorin-3C (sema3C) induces the collapse of the cytoskeleton of lymphatic endothelial cells (LEC) in a neuropilin-2-, plexin-D1-, and plexin-A1-dependent manner, while most other semaphorins, including antiangiogenic semaphorins such as sema3A do not. Sema3C is cleaved, like other class-3 semaphorins, by furin-like pro-protein convertases (FPPC). Cleaved sema3C (p65-Sema3C) was unable to induce the collapse of the cytoskeleton of LEC. FPPC are strongly upregulated in tumor cells. In order to examine the effects of full-length sema3C on tumor progression, we therefore generated an active point mutated furin cleavage-resistant sema3C (FR-sema3C). FR-sema3C inhibited potently proliferation of LEC and to a lesser extent proliferation of human umbilical vein-derived endothelial cells. FR-sema3C also inhibited VEGF-C-induced phosphorylation of VEGFR-3, ERK1/2, and AKT. Expression of recombinant FR-sema3C in metastatic, triple-negative LM2-4 breast cancer cells did not affect their migration or proliferation in vitro. However, tumors derived from FR-sema3C-expressing LM2-4 cells implanted in mammary fat pads developed at a slower rate, contained a lower concentration of blood vessels and lymph vessels, and metastasized much less effectively to lymph nodes. Interestingly, p65-Sema3C, but not FR-sema3C, rendered A549 lung cancer cells resistant to serum deprivation, suggesting that previously reported protumorigenic activities of sema3C may be due to p65-Sema3C produced by tumor cells. Our observations suggest that FR-sema3C may be further developed into a novel antitumorigenic drug. PMID:25808871

  12. Modelling the structure of full-length Epstein-Barr virus nuclear antigen 1.

    PubMed

    Hussain, Mushtaq; Gatherer, Derek; Wilson, Joanna B

    2014-12-01

    Epstein-Barr virus is a clinically important human virus associated with several cancers and is the etiologic agent of infectious mononucleosis. The viral nuclear antigen-1 (EBNA1) is central to the replication and propagation of the viral genome and likely contributes to tumourigenesis. We have compared EBNA1 homologues from other primate lymphocryptoviruses and found that the central glycine/alanine repeat (GAr) domain as well as predicted cellular protein (USP7 and CK2) binding sites are present in homologues in the Old World primates, but not the marmoset, suggesting that these motifs may have co-evolved. Using the resolved structure of the C-terminal one-third of EBNA1 (homodimerization and DNA binding domain), we have gone on to develop monomeric and dimeric models in silico of the full-length protein. The C-terminal domain is predicted to be structurally highly similar between homologues, indicating conserved function. Zinc could be stably incorporated into the model, bonding with two N-terminal cysteines predicted to facilitate multimerisation. The GAr contains secondary structural elements in the models, while the protein binding regions are unstructured, irrespective of the prediction approach used and sequence origin. These intrinsically disordered regions may facilitate the diversity observed in partner interactions. We hypothesize that the structured GAr could mask the disordered regions, thereby protecting the protein from default degradation. In the dimer conformation, the C-terminal tails of each monomer wrap around a proline-rich protruding loop of the partner monomer, providing dimer stability, a feature which could be exploited in therapeutic design. PMID:25011696

  13. Cognitive defects are reversible in inducible mice expressing pro-aggregant full-length human Tau

    PubMed Central

    Sydow, Astrid; Hofmann, Anne; Wu, Dan; Messing, Lars; Balschun, Detlef; D'Hooge, Rudi; Mandelkow, Eva-Maria

    2016-01-01

    Neurofibrillary lesions of abnormal Tau are hallmarks of Alzheimer´s disease and frontotemporal dementias. Our regulatable (Tet-OFF) mouse models of tauopathy express variants of human full-length Tau in the forebrain (CaMKIIα promoter) either with mutation ΔK280 (pro-aggregant) or ΔK280/I277P/I308P (anti-aggregant). Co-expression of luciferase enables in vivo quantification of gene expression by bioluminescence imaging. Pro-aggregant mice develop synapse loss and Tau pathology including missorting, phosphorylation and early pretangle formation, whereas anti-aggregant mice do not. We correlated hippocampal Tau pathology with learning/memory performance and synaptic plasticity. Pro-aggregant mice at 16 months of gene expression exhibited severe cognitive deficits in Morris water-maze and in passive-avoidance paradigms, whereas anti-aggregant mice were comparable to controls. Cognitive impairment of pro-aggregant mice was accompanied by loss of hippocampal LTP in CA1 and CA3 areas and by a reduction of synaptic proteins and dendritic spines, although no neuronal loss was observed. Remarkably, memory and LTP recovered when pro-aggregant Tau was switched-OFF for ∼4 months, Tau phosphorylation and missorting were reversed, and synapses recovered. Moreover soluble and insoluble pro-aggregant hTau40 disappeared while insoluble mouse Tau was still present. This study links early Tau pathology without neurofibrillary tangles and neuronal death to cognitive decline and synaptic dysfunction. It demonstrates that Tau-induced impairments are reversible after switching-OFF pro-aggregant Tau. Therefore our mouse model may mimic an early phase of AD when the hippocampus does not yet suffer from irreversible cell death but cognitive deficits are already striking. It offers potential to evaluate drugs with regard to learning and memory performance. PMID:22532069

  14. Structural Influences: Cholesterol, Drug, and Proton Binding to Full-Length Influenza A M2 Protein.

    PubMed

    Ekanayake, E Vindana; Fu, Riqiang; Cross, Timothy A

    2016-03-29

    The structure and functions of the M2 protein from Influenza A are sensitive to pH, cholesterol, and the antiinfluenza drug Amantadine. This is a tetrameric membrane protein of 97 amino-acid residues that has multiple functions, among them as a proton-selective channel and facilitator of viral budding, replacing the need for the ESCRT proteins that other viruses utilize. Here, various amino-acid-specific-labeled samples of the full-length protein were prepared and mixed, so that only interresidue (13)C-(13)C cross peaks between two differently labeled proteins representing interhelical interactions are observed. This channel is activated at slightly acidic pH values in the endosome when the His(37) residues in the middle of the transmembrane domain take on a +2 or +3 charged state. Changes observed here in interhelical distances in the N-terminus can be accounted for by modest structural changes, and no significant changes in structure were detected in the C-terminal portion of the channel upon activation of the channel. Amantadine, which blocks proton conductance by binding in the aqueous pore near the N-terminus, however, significantly modifies the tetrameric structure on the opposite side of the membrane. The interactions between the juxtamembrane amphipathic helix of one monomer and its neighboring monomer observed in the absence of drug are disrupted in its presence. However, the addition of cholesterol prevents this structural disruption. In fact, strong interactions are observed between cholesterol and residues in the amphipathic helix, accounting for cholesterol binding adjacent to a native palmitoylation site and near to an interhelix crevice that is typical of cholesterol binding sites. The resultant stabilization of the amphipathic helix deep in the bilayer interface facilitates the bilayer curvature that is essential for viral budding. PMID:27028648

  15. Identification and quantification of full-length BK channel variants in the developing mouse cochlea.

    PubMed

    Sakai, Yoshihisa; Harvey, Margaret; Sokolowski, Bernd

    2011-11-01

    Maxi-K(+) (BK) channel diversity is attributed to alternative splicing in the kcnma1 gene. The resultant variants manifest themselves in different cell types, tissues, and functions, such as excitation, metabolism, and signaling. Immunoelectron microscopy revealed immunogold particle labeling of BK in apical and basal regions of inner and outer hair cells, respectively. Additional labeling occurs in Deiters' cells and the inner mitochondrial membrane. Identification of full-length sequences reveals 27 BK variants from embryonic and postnatal mouse inner ear, per classification by tail motif, VYR, DEC, and ERL, and by exon usage. Three predicted start codons are found encoding MAN, MSS, and MDA, of which MDA shows the greatest expression through all stages in development, whereas MAN is undetectable. Complex splice sites occur between exons 9 and 10 and between 21 and 23. Spliced-in/out exons between 8 and 10 reveal a short fragment composed of exons 8 + 10, detectable on postnatal day (PD) 14 and PD30, and a longer fragment composed of exons 8 + 9 + 10 that is upregulated on embryonic day (ED) 14. Spliced-in exons 22 or 23 are expressed on ED14 but decrease over time; however, exon 22 increases again on PD34. Using tail-specific primers, qRT-PCR from ED14, PD4, -14, and -30 shows that BK-VYR and -ERL dominate expression on ED14, whereas DEC dominates after birth in all cochlear regions. The localization of BK and the changes in expression of its exons and tail types, by alternative splicing during development, may contribute to cochlear organization, acquisition of hearing, and intracellular signaling. PMID:21800349

  16. Sequence Variability, Gene Structure, and Expression of Full-Length Human Endogenous Retrovirus H

    PubMed Central

    Jern, Patric; Sperber, Göran O.; Ahlsén, Göran; Blomberg, Jonas

    2005-01-01

    Recently, we identified and classified 926 human endogenous retrovirus H (HERV-H)-like proviruses in the human genome. In this paper, we used the information to, in silico, reconstruct a putative ancestral HERV-H. A calculated consensus sequence was nearly open in all genes. A few manual adjustments resulted in a putative 9-kb HERV-H provirus with open reading frames (ORFs) in gag, pro, pol, and env. Long terminal repeats (LTRs) differed by 1.1%, indicating proximity to an integration event. The gag ORF was extended upstream of the normal myristylation start site. There was a long leader (including a “pre-gag” ORF) region positioned like the N terminus of murine leukemia virus (MLV) “glyco-Gag,” potentially encoding a proline- and serine-rich domain remotely similar to MLV pp12. Another ORF, starting inside the 5′ LTR, had no obvious similarity to known protein domains. Unlike other hitherto described gammaretroviruses, the reconstructed Gag had two zinc finger motifs. Alternative splicing of sequences related to the HERV-H consensus was confirmed using dbEST data. env transcripts were most prevalent in colon tumors, but also in normal testis. We found no evidence for full length env transcripts in the dbEST. HERV-H had a markedly skewed nucleotide composition, disfavoring guanine and favoring cytidine. We conclude that the HERV-H consensus shared a gene arrangement common to gammaretroviruses with gag separated by stop codon from pro-pol in the same reading frame, while env resides in another reading frame. There was also alternative splicing. HERV-H consensus yielded new insights in gammaretroviral evolution and will be useful as a model in studies on expression and function. PMID:15858016

  17. Mapping Full-Length Porcine Endogenous Retroviruses in a Large White Pig

    PubMed Central

    Herring, C.; Quinn, G.; Bower, R.; Parsons, N.; Logan, N. A.; Brawley, A.; Elsome, K.; Whittam, A.; Fernandez-Suarez, X. M.; Cunningham, D.; Onions, D.; Langford, G.; Scobie, L.

    2001-01-01

    Xenotransplantation may bridge the widening gap between the shortage of donor organs and the increasing number of patients waiting for transplantation. However, a major safety issue is the potential cross-species transmission of porcine endogenous retroviruses (PERV). This problem could be resolved if it is possible to produce pigs that do not contain replication-competent copies of this virus. In order to determine the feasibility of this, we have determined the number of potentially replication-competent full-length PERV proviruses and obtained data on their integration sites within the porcine genome. We have screened genomic DNA libraries from a Large White pig for potentially intact proviruses. We identified six unique PERV B proviruses that were apparently intact in all three genes, while the majority of isolated proviruses were defective in one or more genes. No intact PERV A proviruses were found in this pig, despite the identification of multiple defective A proviruses. Genotyping of 30 unrelated pigs for these unique proviruses showed a heterogeneous distribution. Two proviruses were uncommon, present in 7 of 30 and 3 of 30 pigs, while three were each present in 24 of 30 pigs, and one was present in 30 of 30 animals examined. Our data indicate that few PERV proviruses in Large White pigs are capable of productive infection and suggest that many could be removed by selective breeding. Further studies are required to determine if all potentially functional proviruses could be removed by breeding or whether gene knockout techniques will be required to remove the residuum. PMID:11711616

  18. Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB.

    PubMed

    Itoh, Yuzuru; Sekine, Shun-Ichi; Yokoyama, Shigeyuki

    2015-10-15

    Selenocysteine (Sec), the 21(st) amino acid in translation, uses its specific tRNA (tRNA(Sec)) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNA(Sec) (Sec-tRNA(Sec)) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1-3), followed by four winged-helix domains (WHD1-4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNA(Sec) is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNA(Sec), SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNA(Sec) allows SelB to specifically recognize tRNA(Sec) and characteristically place it at the ribosomal A-site. PMID:26304550

  19. Expression, Purification, and Identification of Associated Proteins of the Full-length hCDK12/CyclinK Complex*

    PubMed Central

    Bartkowiak, Bartlomiej; Greenleaf, Arno L.

    2015-01-01

    The coupling of transcription and associated processes has been shown to be dependent on the RNA polymerase II (RNAPII) C-terminal repeat domain (CTD) and the phosphorylation of the heptad repeats of which it is composed (consensus sequence Y1S2P3T4S5P6S7). Two primary S2 position CTD kinases have been identified in higher eukaryotes: P-TEFb and CDK12/CyclinK. The more recently discovered CDK12 appears to act at the 3′-end of the transcription unit and has been identified as a tumor suppressor for ovarian cancer; however much is still unknown about the in vivo roles of CDK12/CyclinK. In an effort to further characterize these roles we have purified to near homogeneity and characterized, full-length, active, human CDK12/CyclinK, and identified hCDK12-associated proteins via mass spectrometry. We find that employing a “2A” peptide-linked multicistronic construct containing CDK12 and CyclinK results in the efficient production of active, recombinant enzyme in the baculovirus/Sf9 expression system. Using GST-CTD fusion protein substrates we find that CDK12/CyclinK prefers a substrate with unmodified repeats or one that mimics prephosphorylation at the S7 position of the CTD; also the enzyme is sensitive to the inhibitor flavopiridol at higher concentrations. Identification of CDK12-associating proteins reveals a strong enrichment for RNA-processing factors suggesting that CDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors. PMID:25429106

  20. A novel copper(II) coordination at His186 in full-length murine prion protein

    SciTech Connect

    Watanabe, Yasuko; Hiraoka, Wakako; Igarashi, Manabu; Ito, Kimihito; Shimoyama, Yuhei; Horiuchi, Motohiro; Yamamori, Tohru; Yasui, Hironobu; Kuwabara, Mikinori; Inagaki, Fuyuhiko; Inanami, Osamu

    2010-04-09

    To explore Cu(II) ion coordination by His{sup 186} in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP{sup C}.

  1. Soluble expression, purification and characterization of the full length IS2 Transposase

    PubMed Central

    2011-01-01

    Background The two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein. Results A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common

  2. High avidity antibodies to full-length VAR2CSA correlate with absence of placental malaria.

    PubMed

    Tutterrow, Yeung Lo; Salanti, Ali; Avril, Marion; Smith, Joseph D; Pagano, Ian S; Ako, Simon; Fogako, Josephine; Leke, Rose G F; Taylor, Diane Wallace

    2012-01-01

    VAR2CSA mediates sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, increasing the risk of poor pregnancy outcomes. Naturally acquired antibodies (Ab) to placental parasites at delivery have been associated with improved pregnancy outcomes, but Ab levels and how early in pregnancy Ab must be present in order to eliminate placental parasites before delivery remains unknown. Antibodies to individual Duffy-binding like domains of VAR2CSA have been studied, but the domains lack many of the conformational epitopes present in full-length VAR2CSA (FV2). Thus, the purpose of this study was to describe the acquisition of Ab to FV2 in women residing in high and low transmission areas and determine how Ab levels during pregnancy correlate with clearance of placental parasites. Plasma samples collected monthly throughout pregnancy from pregnant women living in high and low transmission areas in Cameroon were evaluated for Ab to FV2 and the proportion of high avidity Ab (i.e., Ab that remain bound in the presence of 3M NH(4)SCN) was assessed. Ab levels and proportion of high avidity Ab were compared between women with placental malaria (PM(+)) and those without (PM(-)) at delivery. Results showed that PM(-) women had significantly higher Ab levels (p = 0.0047) and proportion of high avidity Ab (p = 0.0009) than PM(+) women throughout pregnancy. Specifically, women with moderate to high Ab levels (>5,000 MFI) and those with ≥ 35% high avidity Ab at 5-6 months were found to have 2.3 (95% CI, 1.0-4.9) and 7.6-fold (p = 0.0013, 95% CI: 1.2-50.0) reduced risk of placental malaria, respectively. These data show that high levels of Ab to FV2, particularly those with high avidity for FV2, produced by mid-pregnancy are important in clearing parasites from the placenta. Both high Ab levels and proportion of high avidity Ab to FV2 may serve as correlates of protection for assessing immunity against placental malaria. PMID:22761948

  3. Identification of cDNAs encoding viper venom hyaluronidases: cross-generic sequence conservation of full-length and unusually short variant transcripts.

    PubMed

    Harrison, Robert A; Ibison, Frances; Wilbraham, Davina; Wagstaff, Simon C

    2007-05-01

    The immobilisation of prey by snakes is most efficiently achieved by the rapid dissemination of venom from its site of injection into the blood stream. Hyaluronidase is a common component of snake venoms and has been termed the "venom spreading factor". In the absence of nucleotide or protein sequence data to confirm the functional identity of this venom component, we interrogated a venom gland EST database for the saw-scaled viper, Echis ocellatus (Nigeria), using the gene ontology (GO) term "carbohydrate metabolism". A single hyalurononglucosaminadase-activity matching sequence (EOC00242) was found and used to design PCR primers to acquire the full-length cDNA sequence. Although very different from the bee venom and mammalian hyaluronidase sequences, the E. ocellatus sequence retained all the catalytic, positional and structural residues that characterise this class of carbohydrate metabolising hydrolases. An extraordinarily high level of sequence identity (>95%) was observed in analogous venom gland cDNA sequences isolated (by PCR) from another saw-scaled viper species, E. pyramidum leakeyi (Kenya), and from the sahara horned viper, Cerastes cerastes cerastes (Egypt) and the puff adder, Bitis arietans (Nigeria). Smaller amplicons, lacking hyaluronidase catalytic residues because of 768 bp or 855 bp central deletions, appear to encode either truncated peptides without hyaluronidase activity, or are non-translated transcripts because they lack consensus translation initiating motifs. PMID:17210232

  4. Effects of irradiating adult mdx mice before full-length dystrophin cDNA transfer on host anti-dystrophin immunity.

    PubMed

    Eghtesad, S; Zheng, H; Nakai, H; Epperly, M W; Clemens, P R

    2010-09-01

    Duchenne muscular dystrophy is a fatal, genetic disorder in which dystrophin-deficient muscle progressively degenerates, for which dystrophin gene transfer could provide effective treatment. The host immune response to dystrophin, however, is an obstacle to therapeutic gene expression. Understanding the dystrophin-induced host immune response will facilitate the discovery of strategies to prolong expression of recombinant dystrophin in dystrophic muscle. Using whole-body irradiation of the dystrophic mdx mouse before gene transfer, we temporally removed the immune system; a 600 rad dose removed peripheral immune cells, which were restored by self-reconstitution, and a 900 rad dose removed central and peripheral immune cells, which were restored by adoptive transfer of bone marrow from a syngeneic, dystrophin-normal donor. The anti-dystrophin humoral response was delayed and dystrophin expression was partially preserved in irradiated, vector-treated mice. Nonirradiated, vector-treated control mice lost muscle dystrophin expression completely, had an earlier anti-dystrophin humoral response and demonstrated muscle fibers focally surrounded with T cells. We conclude that dystrophin gene transfer induced anti-dystrophin humoral immunity and cell-mediated responses that were significantly diminished and delayed by temporal removal of the host central or peripheral immune cells. Furthermore, manipulation of central immunity altered the pattern of regulatory T cells in muscle. PMID:20827278

  5. Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A

    PubMed Central

    Beall, Anne; Yount, Boyd; Lin, Chun-Ming; Hou, Yixuan; Wang, Qiuhong; Saif, Linda

    2016-01-01

    ABSTRACT Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic alphacoronavirus. In the United States, highly virulent PEDV strains cause between 80 and 100% mortality in suckling piglets and are rapidly transmitted between animals and farms. To study the genetic factors that regulate pathogenesis and transmission, we developed a molecular clone of PEDV strain PC22A. The infectious-clone-derived PEDV (icPEDV) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo. Importantly, recombinant PEDV was rapidly transmitted to uninoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. Using reverse genetics, we removed open reading frame 3 (ORF3) and replaced this region with a red fluorescent protein (RFP) gene to generate icPEDV-ΔORF3-RFP. icPEDV-ΔORF3-RFP replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. However, the diarrheic scores in icPEDV-ΔORF3-RFP-infected pigs were lower than those in wild-type-virus- or icPEDV-infected pigs, and the virus formed smaller plaques than those of PC22A. Together, these data describe the development of a robust reverse-genetics platform for identifying genetic factors that regulate pathogenic outcomes and transmission efficiency in vivo, providing key infrastructural developments for developing and evaluating the efficacy of live attenuated vaccines and therapeutics in a clinical setting. PMID:26733065

  6. 78 FR 13071 - Guidance for Industry: Implementation of an Acceptable Full-Length and Abbreviated Donor History...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-26

    ... (76 FR 44013), FDA announced the availability of the draft guidance of the same title dated July 2011...- Length and Abbreviated Donor History Questionnaires and Accompanying Materials for Use in Screening... ``Guidance for Industry: Implementation of an Acceptable Full-Length and Abbreviated Donor...

  7. Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase from Escherichia coli

    SciTech Connect

    Weidner, Annett; Neumann, Piotr; Wille, Georg; Stubbs, Milton T.; Tittmann, Kai

    2008-03-01

    The peripheral membrane flavoprotein pyruvate oxidase from E. coli has been crystallized in the full-length form and as a proteolytically activated truncation variant lacking the last 23 amino acids at the C-terminus. The thiamine diphosphate- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from Escherichia coli (EcPOX) has been crystallized in the full-length form and as a proteolytically activated C-terminal truncation variant which lacks the last 23 amino acids (Δ23 EcPOX). Crystals were grown by the hanging-drop vapour-diffusion method using either protamine sulfate (full-length EcPOX) or 2-methyl-2,4-pentanediol (Δ23 EcPOX) as precipitants. Native data sets were collected at a X-ray home source to a resolution of 2.9 Å. The two forms of EcPOX crystallize in different space groups. Whereas full-length EcPOX crystallizes in the tetragonal space group P4{sub 3}2{sub 1}2 with two monomers per asymmetric unit, the crystals of Δ23 EcPOX belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and contain 12 monomers per asymmetric unit.

  8. Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics

    PubMed Central

    2010-01-01

    and aid in tomato functional genomics and molecular breeding. Full-length cDNA sequences and their annotations are provided in the database KaFTom http://www.pgb.kazusa.or.jp/kaftom/ via the website of the National Bioresource Project Tomato http://tomato.nbrp.jp. PMID:20350329

  9. Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation

    PubMed Central

    Wang, Jin; Gines, Silvia; MacDonald, Marcy E; Gusella, James F

    2005-01-01

    Background Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1–171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 < 15 μM, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant HdhQ111/Q111 striatal cells. Conclusions At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused

  10. Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study

    PubMed Central

    2011-01-01

    Background Approaches to simplify and streamline the construction of full-length infectious cDNA clones (FL-cDNAs) are needed. Among desirable improvements are the ability to use total nucleic acids (TNA) extracts from infected hosts (to bypass viral purification limitations) for the direct one-step amplification of large FL-cDNAs, the possibility to inoculate plants with uncloned FL-cDNAs and the simplified cloning of these large molecules. Results Using the 7.55 kb genome of Apple chlorotic leaf spot trichovirus (ACLSV) approaches allowing the rapid generation from TNA extracts of FL-cDNAs under the control of the T7 promoter and the successful inoculation of plants using in vitro transcripts obtained from these uncloned amplification products have been developed. We also show that the yeast homologous recombination system permits efficient cloning of FL-cDNAs and the simultaneous one-step tailoring of a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector allowing efficient inoculation of both herbaceous and woody host plants by agroinfiltration. Conclusions The fast and efficient strategies described here should have broad applications, in particular for the study of "difficult" plant viruses, such as those infecting woody hosts, and potentially for other, non plant-infecting viral agents. PMID:22040379

  11. Caspase 3 inactivates biologically active full length interleukin-33 as a classical cytokine but does not prohibit nuclear translocation

    SciTech Connect

    Ali, Shafaqat; Nguyen, Dang Quan; Falk, Werner; Martin, Michael Uwe

    2010-01-15

    IL-33 is a member of the IL-1 family of cytokines with dual function which either activates cells via the IL-33 receptor in a paracrine fashion or translocates to the nucleus to regulate gene transcription in an intracrine manner. We show that full length murine IL-33 is active as a cytokine and that it is not processed by caspase 1 to mature IL-33 but instead cleaved by caspase 3 at aa175 to yield two products which are both unable to bind to the IL-33 receptor. Full length IL-33 and its N-terminal caspase 3 breakdown product, however, translocate to the nucleus. Finally, bioactive IL-33 is not released by cells constitutively or after activation. This suggests that IL-33 is not a classical cytokine but exerts its function in the nucleus of intact cells and only activates others cells via its receptor as an alarm mediator after destruction of the producing cell.

  12. Increased proteolytic processing of full-length Gli2 transcription factor reduces the Hedgehog pathway activity in vivo

    PubMed Central

    Li, Juan; Wang, Chengbing; Pan, Yong; Bai, Zengliang; Wang, Baolin

    2011-01-01

    The proteolytic processing of Gli2 and Gli3 full-length transcription factors into repressors is a key step of the regulation in Hedgehog (Hh) signaling. The differential Gli2 and Gli3 processing is controlled by the processing determinant domain or PDD, but its significance is not clear. We generated a Gli2 mutant allele, Gli23PDD, in which the Gli3PDD substitutes for the Gli2PDD. As expected, Gli23PDD is processed more efficiently and at the different position as compared to Gli2, indicating that PDD also determines the extent and site of Gli2 and Gli3 processing in vivo. The increase in levels of the Gli2 repressor in Gli23PDD mutant reduces the Hh pathway activity. Gli23PDD processing is still regulated by Hh signaling. These results indicate that the proper balance between the Gli2 full-length activator and repressor is essential for Hh signaling. PMID:21337666

  13. RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

    PubMed

    Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

    2016-02-01

    During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks. PMID:26611227

  14. Kinetics of Hedgehog-Dependent Full-Length Gli3 Accumulation in Primary Cilia and Subsequent Degradation ▿ † ‡

    PubMed Central

    Wen, Xiaohui; Lai, Cary K.; Evangelista, Marie; Hongo, Jo-Anne; de Sauvage, Frederic J.; Scales, Suzie J.

    2010-01-01

    Hedgehog (Hh) signaling in vertebrates depends on intraflagellar transport (IFT) within primary cilia. The Hh receptor Patched is found in cilia in the absence of Hh and is replaced by the signal transducer Smoothened within an hour of Hh stimulation. By generating antibodies capable of detecting endogenous pathway transcription factors Gli2 and Gli3, we monitored their kinetics of accumulation in cilia upon Hh stimulation. Localization occurs within minutes of Hh addition, making it the fastest reported readout of pathway activity, which permits more precise temporal and spatial localization of Hh signaling events. We show that the species of Gli3 that accumulates at cilium tips is full-length and likely not protein kinase A phosphorylated. We also confirmed that phosphorylation and βTrCP/Cul1 are required for endogenous Gli3 processing and that this is inhibited by Hh. Surprisingly, however, Hh-dependent inhibition of processing does not lead to accumulation of full-length Gli3, but instead renders it labile, leading to its proteasomal degradation via the SPOP/Cul3 complex. In fact, full-length Gli3 disappears with faster kinetics than the Gli3 repressor, the latter not requiring SPOP/Cul3 or βTrCP/Cul1. This may contribute to the increased Gli3 activator/repressor ratios found in IFT mutants. PMID:20154143

  15. A systematic approach for testing expression of human full-length proteins in cell-free expression systems

    PubMed Central

    Langlais, Claudia; Guilleaume, Birgit; Wermke, Nadja; Scheuermann, Tina; Ebert, Lars; LaBaer, Joshua; Korn, Bernhard

    2007-01-01

    Background The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems. Results In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%. Conclusion We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield. PMID:17915018

  16. Species-Specific Expression of Full-Length and Alternatively Spliced Variant Forms of CDK5RAP2.

    PubMed

    Park, John S Y; Lee, Marie-Katrina; Kang, SungMyung; Jin, Yan; Fu, Songbin; Rosales, Jesusa L; Lee, Ki-Young

    2015-01-01

    CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2) or an alternatively spliced variant form (hCDK5RAP2-V1) that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species. PMID:26550838

  17. Species-Specific Expression of Full-Length and Alternatively Spliced Variant Forms of CDK5RAP2

    PubMed Central

    Park, John S. Y.; Lee, Marie-Katrina; Kang, SungMyung; Jin, Yan; Fu, Songbin; Rosales, Jesusa L.; Lee, Ki-Young

    2015-01-01

    CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2) or an alternatively spliced variant form (hCDK5RAP2-V1) that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species. PMID:26550838

  18. Identification of 48 full-length MHC-DAB functional alleles in miiuy croaker and evidence for positive selection.

    PubMed

    Liu, Jiang; Sun, Yueyan; Xu, Tianjun

    2016-07-01

    Major histocompatibility complex (MHC) molecules play a vital role in the immune response and are a highly polymorphic gene superfamily in vertebrates. As the molecular marker associated with polymorphism and disease susceptibility/resistance, the polymorphism of MHC genes has been investigated in many tetrapods and teleosts. Most studies were focused on the polymorphism of the second exon, which encodes the peptide-binding region (PBR) in the α1- or β1-domain, but few studies have examined the full-length coding region. To comprehensive investigate the polymorphism of MHC gene, we identified 48 full-length miiuy croaker (Miichthys miiuy) MHC class IIB (Mimi-DAB) functional alleles from 26 miiuy croaker individuals. All of the alleles encode 34 amino acid sequences, and a high level of polymorphism was detected in Mimi-DAB alleles. The rate of non-synonymous substitutions (dN) occurred at a significantly higher frequency than that of synonymous substitutions (dS) in the PBR, and this result suggests that balancing selection maintains polymorphisms at the Mimi-DAB locus. Phylogenetic analysis based on the full-length and exon 2 sequences of Mimi-DAB alleles both showed that the Mimi-DAB alleles were clustered into two major groups. A total of 19 positive selected sites were identified on the Mimi-DAB alleles after testing for positive selection, and 14 sites were predicted to be associated with antigen-binding sites, which suggests that most of selected sites are significant for disease resistance. The polymorphism of Mimi-DAB alleles provides an important resource for analyzing the association between the polymorphism of MHC gene and disease susceptibility/resistance, and for researching the molecular selective breeding of miiuy croaker with enhanced disease resistance. PMID:27164216

  19. The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding*

    PubMed Central

    Lu, Bin; Kiessling, Volker; Tamm, Lukas K.; Cafiso, David S.

    2014-01-01

    Synaptotagmin 1 (Syt1) is the calcium sensor for synchronous neurotransmitter release. The two C2 domains of Syt1, which may mediate fusion by bridging the vesicle and plasma membranes, are connected to the vesicle membrane by a 60-residue linker. Here, we use site-directed spin labeling and a novel total internal reflection fluorescence vesicle binding assay to characterize the juxtamembrane linker and to test the ability of reconstituted full-length Syt1 to interact with opposing membrane surfaces. EPR spectroscopy demonstrates that the majority of the linker interacts with the membrane interface, thereby limiting the extension of the C2A and C2B domains into the cytoplasm. Pulse dipolar EPR spectroscopy provides evidence that purified full-length Syt1 is oligomerized in the membrane, and mutagenesis indicates that a glycine zipper/GXXXG motif within the linker helps mediate oligomerization. The total internal reflection fluorescence-based vesicle binding assay demonstrates that full-length Syt1 that is reconstituted into supported lipid bilayers will capture vesicles containing negatively charged lipid in a Ca2+-dependent manner. Moreover, the rate of vesicle capture increases with Syt1 density, and mutations in the GXXXG motif that inhibit oligomerization of Syt1 reduce the rate of vesicle capture. This work demonstrates that modifications within the 60-residue linker modulate both the oligomerization of Syt1 and its ability to interact with opposing bilayers. In addition to controlling its activity, the oligomerization of Syt1 may play a role in organizing proteins within the active zone of membrane fusion. PMID:24973220

  20. Binding of Full-Length HIV-1 gp120 to CD4 Induces Structural Reorientation around the gp120 Core

    SciTech Connect

    Ashish,F.; Garg, R.; Anguita, J.; Krueger, J.

    2006-01-01

    Small-angle x-ray scattering data on the unliganded full-length fully glycosylated HIV-1 gp120, the soluble CD4 (domains 1-2) receptor and their complex in solution are presented. Ab initio structure restorations using these data provides the first look at the envelope shape for the unliganded and the complexed gp120 molecule. Fitting known crystal structures of the unliganded SIV and the complexed HIV gp120 core regions within our resultant shape constraints reveals movement of the V3 loop upon binding.

  1. 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.

  2. Purification, crystallization and preliminary crystallographic analysis of the full-length cystathionine β-synthase from Apis mellifera

    PubMed Central

    Oyenarte, Iker; Majtan, Tomas; Ereño, June; Corral-Rodríguez, María Angeles; Klaudiny, Jaroslav; Majtan, Juraj; Kraus, Jan P.; Martínez-Cruz, Luis Alfonso

    2012-01-01

    Cystathionine β-synthase (CBS) is a pyridoxal-5′-phosphate-dependent enzyme that catalyzes the first step of the transsulfuration pathway, namely the condensation of serine with homocysteine to form cystathionine. Mutations in the CBS gene are the single most common cause of hereditary homocystinuria, a multisystemic disease affecting to various extents the vasculature, connective tissues and central nervous system. At present, the crystal structure of CBS from Drosophila melanogaster is the only available structure of the full-length enzyme. Here we describe a cloning, overexpression, purification and preliminary crystallographic analysis of a full-length CBS from Apis mellifera (AmCBS) which maintains 51 and 46% sequence identity with its Drosophila and human homologs, respectively. The AmCBS yielded crystals belonging to space group P212121, with unit-cell parameters a = 85.90, b = 95.87, c = 180.33 Å. Diffraction data were collected to a resolution of 3.0 Å. The crystal structure contained two molecules in the asymmetric unit which presumably correspond to the dimeric species observed in solution. PMID:23143241

  3. Near full-length genomic characterization of a HIV type 1 BC recombinant strain from Manipur, India.

    PubMed

    Sarkar, Roni; Sarkar, Kamalesh; Singh, N Brajachand; Singh, Y Manihar; Chakrabarti, Sekhar

    2012-10-01

    Genetic complexity of HIV-1 is brought about by recombination between HIV-1 subtypes which leads to the development of epidemiologically significant founder strains. In the present study, the near full-length genome sequence of an HIV-1 isolate from an injecting drug user of Manipur (India) was determined, which evidenced the presence of a novel HIV-1 BC recombinant strain. Near full-length genome was amplified by polymerase chain reaction using primer walking approach. The recombination break points were detected using bootscan and simplot analyses. This isolate exhibited a mosaic structure consisting of subtype C backbone with subtype B insertions at the upstream of pol gene (3026-3259) and the downstream of env gene which spanned till the nef gene (8183-8961). Phylogenetic relationships determined with neighbor-joining trees, revealed that the subtype C sequences clustered with sequences from Indian subtype C HIV-1 strains, and the subtype B sequences clustered with HIV-1 subtype B strains from Thailand. This finding may create a complex scenario of HIV-1 epidemic among the injecting drug users of Manipur in near future. PMID:22710995

  4. Characterization of 40 full-length MHC class IIA functional alleles in miiuy croaker: Polymorphism and positive selection.

    PubMed

    Xu, Tianjun; Liu, Jiang; Sun, Yueyan; Zhu, Zhihuang; Liu, Tianxing

    2016-02-01

    The major histocompatibility complex is a highly polymorphic gene superfamily in vertebrates that plays an important role in adaptive immune response. In the present study, we identified 40 full-length miiuy croaker MHC class IIA (Mimi-DAA) functional alleles from 26 miiuy croaker individuals and found that the alleles encode 30 amino acid sequences. A high level of polymorphism in Mimi-DAA was detected in miiuy croaker. The rate of non-synonymous substitutions (d(N)) occurred at a significantly higher frequency than that of synonymous substitutions (d(S)) in the peptide-binding region (PBR) and non-PBR. This result suggests that balancing selection maintains polymorphisms at the Mimi-DAA locus. Phylogenetic analysis based on the full-length sequences showed that the Mimi-DAA alleles clustered into three groups. However, the phylogenetic tree constructed using the exon 2 sequences indicated that the Mimi-DAA alleles clustered into two groups. A total of 22 positively selected sites were identified on the Mimi-DAA alleles after testing for positive selection, and five sites were predicted to be associated with the binding of peptide antigen, suggesting that a few selected residues may play a significant role in immune function. PMID:26598111

  5. Predicting the strength of UP-elements and full-length E. coli σE promoters

    PubMed Central

    Rhodius, Virgil A.; Mutalik, Vivek K.; Gross, Carol A.

    2012-01-01

    Predicting the location and strength of promoters from genomic sequence requires accurate sequenced-based promoter models. We present the first model of a full-length bacterial promoter, encompassing both upstream sequences (UP-elements) and core promoter modules, based on a set of 60 promoters dependent on σE, an alternative ECF-type σ factor. UP-element contribution, best described by the length and frequency of A- and T-tracts, in combination with a PWM-based core promoter model, accurately predicted promoter strength both in vivo and in vitro. This model also distinguished active from weak/inactive promoters. Systematic examination of promoter strength as a function of RNA polymerase (RNAP) concentration revealed that UP-element contribution varied with RNAP availability and that the σE regulon is comprised of two promoter types, one of which is active only at high concentrations of RNAP. Distinct promoter types may be a general mechanism for increasing the regulatory capacity of the ECF group of alternative σ's. Our findings provide important insights into the sequence requirements for the strength and function of full-length promoters and establish guidelines for promoter prediction and for forward engineering promoters of specific strengths. PMID:22156164

  6. Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

    PubMed Central

    Anbouhi, Mahdi Habibi; Baraz, Aida Feiz; Bouzari, Saeid; Abolhassani, Mohsen; Khanahmad, Hossein; Golkar, Majid; Aghasadeghi, Mohammad Reza; Behdani, Mahdi; Najafabadi, Ali Jahanian; Shokrgozar, Mohammad Ali

    2012-01-01

    Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. Methods: In this study, we expressed the extra membrane loop of hCD20 (exCD20) consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a(+) expression vector. The desired protein was expressed in fusion with thioredoxin and 6× His tag in E. coli Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. Results: We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin (exCD20) can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies. PMID:23023212

  7. Global Identification of the Full-Length Transcripts and Alternative Splicing Related to Phenolic Acid Biosynthetic Genes in Salvia miltiorrhiza

    PubMed Central

    Xu, Zhichao; Luo, Hongmei; Ji, Aijia; Zhang, Xin; Song, Jingyuan; Chen, Shilin

    2016-01-01

    Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza. PMID:26904067

  8. [Rapid site-directed mutagenesis on full-length plasmid DNA by using designed restriction enzyme assisted mutagenesis].

    PubMed

    Zhang, Baozhong; Ran, Duoliang; Zhang, Xin; An, Xiaoping; Shan, Yunzhu; Zhou, Yusen; Tong, Yigang

    2009-02-01

    To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes. PMID:19459340

  9. Full-length sequence analysis of chloroquine resistance transporter gene in Plasmodium falciparum isolates from Sabah, Malaysia.

    PubMed

    Tan, Lii Lian; Lau, Tiek Ying; Timothy, William; Prabakaran, Dhanaraj

    2014-01-01

    Chloroquine resistance (CQR) in falciparum malaria was identified to be associated with several mutations in the chloroquine resistance transporter gene (pfcrt) that encodes the transmembrane transporter in digestive vacuole membrane of the parasite. This study aimed to investigate the point mutations across the full-length pfcrt in Plasmodium falciparum isolates in Sabah, Malaysia. A total of 31 P. falciparum positive samples collected from Keningau, Kota Kinabalu, and Kudat, Sabah, were analyzed. pfcrt was PCR amplified and cloned prior to sequence analysis. This study showed that all the previously described 10 point mutations associated with CQR at codons 72, 74, 75, 76, 97, 220, 271, 326, 356, and 371 were found with different prevalence. Besides, two novel point mutations, I166V and H273N, were identified with 22.5% and 19.3%, respectively. Three haplotypes, namely, CVMNK (29%), CVIET (3.2%), and SVMNT (67.7%), were identified. High prevalence of SVMNT among P. falciparum isolates from Sabah showed that these isolates are closer to the P. falciparum isolates from Papua New Guinea rather than to the more proximal Southeast Asian CVIET haplotype. Full-length analysis of pfcrt showed that chloroquine resistant P. falciparum in Sabah is still prevalent despite the withdrawal of chloroquine usage since 1979. PMID:25574497

  10. Predicting the strength of UP-elements and full-length E. coli σE promoters.

    PubMed

    Rhodius, Virgil A; Mutalik, Vivek K; Gross, Carol A

    2012-04-01

    Predicting the location and strength of promoters from genomic sequence requires accurate sequenced-based promoter models. We present the first model of a full-length bacterial promoter, encompassing both upstream sequences (UP-elements) and core promoter modules, based on a set of 60 promoters dependent on σ(E), an alternative ECF-type σ factor. UP-element contribution, best described by the length and frequency of A- and T-tracts, in combination with a PWM-based core promoter model, accurately predicted promoter strength both in vivo and in vitro. This model also distinguished active from weak/inactive promoters. Systematic examination of promoter strength as a function of RNA polymerase (RNAP) concentration revealed that UP-element contribution varied with RNAP availability and that the σ(E) regulon is comprised of two promoter types, one of which is active only at high concentrations of RNAP. Distinct promoter types may be a general mechanism for increasing the regulatory capacity of the ECF group of alternative σ's. Our findings provide important insights into the sequence requirements for the strength and function of full-length promoters and establish guidelines for promoter prediction and for forward engineering promoters of specific strengths. PMID:22156164

  11. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing

    PubMed Central

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  12. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing.

    PubMed

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  13. Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis

    PubMed Central

    Foolen, Jasper; Shiu, Jau-Ye; Mitsi, Maria; Zhang, Yang; Chen, Christopher S.; Vogel, Viola

    2016-01-01

    Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. ‘compact and adsorbed to collagen’ versus ‘extended and fibrillar’ fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin’s contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen

  14. Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis.

    PubMed

    Foolen, Jasper; Shiu, Jau-Ye; Mitsi, Maria; Zhang, Yang; Chen, Christopher S; Vogel, Viola

    2016-01-01

    Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. 'compact and adsorbed to collagen' versus 'extended and fibrillar' fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin's contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen decoration of

  15. Modular structure of the full-length DNA gyrase B subunit revealed by small-angle X-ray scattering.

    PubMed

    Costenaro, Lionel; Grossmann, J Günter; Ebel, Christine; Maxwell, Anthony

    2007-03-01

    DNA gyrase, the only topoisomerase able to introduce negative supercoils into DNA, is essential for bacterial transcription and replication; absent from humans, it is a successful target for antibacterials. From biophysical experiments in solution, we report a structural model at approximately 12-15 A resolution of the full-length B subunit (GyrB). Analytical ultracentrifugation shows that GyrB is mainly a nonglobular monomer. Ab initio modeling of small-angle X-ray scattering data for GyrB consistently yields a "tadpole"-like envelope. It allows us to propose an organization of GyrB into three domains-ATPase, Toprim, and Tail-based on their crystallographic and modeled structures. Our study reveals the modular organization of GyrB and points out its potential flexibility, needed during the gyrase catalytic cycle. It provides important insights into the supercoiling mechanism by gyrase and suggests new lines of research. PMID:17355868

  16. Molecular characterization of full-length MLV-related endogenous retrovirus ChiRV1 from the chicken, Gallus gallus.

    PubMed

    Borysenko, Leonid; Stepanets, Volodymir; Rynditch, Alla V

    2008-06-20

    We report the first full-length sequence of an endogenous retrovirus from the genome of domestic chicken, that is not related to the Avian leukemia viruses (ALV). This retrovirus, designated ChiRV1, clusters with Murine leukemia virus (MLV)-related retroviruses and hence is the first complete gammaretrovirus from the genome of a bird. Nevertheless it is not related to exogenous MLV-related retroviruses infecting chicken. The provirus is 9133 bp long and contains 90%-identical LTRs as well as reading frames for the gag, pol and env genes, interrupted by in-frame stop codons. Expression analysis showed that ChiRV1 is a transcribed provirus. Screening of the chicken genome database revealed 100 ChiRV1-related sequences that are grouped into three classes based upon LTR alignment and subsequent phylogenetic analysis. PMID:18440041

  17. Internalization of the Extracellular Full-Length Tau Inside Neuro2A and Cortical Cells Is Enhanced by Phosphorylation.

    PubMed

    Wauters, Mathilde; Wattiez, Ruddy; Ris, Laurence

    2016-01-01

    Tau protein is mainly intracellular. However, several studies have demonstrated that full-length Tau can be released into the interstitial fluid of the brain. The physiological or pathological function of this extracellular Tau remains unknown. Moreover, as evidence suggests, extracellular Tau aggregates can be internalized by neurons, seeding Tau aggregation. However, much less is known about small species of Tau. In this study, we hypothesized that the status of phosphorylation could alter the internalization of recombinant Tau in Neuro2A and cortical cells. Our preliminary results revealed that the highly phosphorylated form of Tau entered the cells ten times more easily than a low phosphorylated one. This suggests that hyperphosphorylated Tau protein could spread between neurons in pathological conditions such as Alzheimer's disease. PMID:27548242

  18. Characterization of full-length HIV-1 CRF17_BF genomes and comparison to the prototype CRF12_BF strains.

    PubMed

    Aulicino, Paula C; Gómez-Carrillo, Manuel; Bello, Gonzalo; Rocco, Carlos; Mangano, Andrea; Carr, Jean; Sen, Luisa; Foley, Brian

    2012-03-01

    The aim of this work is to characterize the full-length intersubtype recombinant structure of the HIV-1 Circulating Recombinant Form CRF17_BF. A single genome of CRF17_BF was originally described in 2001 as being largely similar to CRF12_BF. Since then, more genomes of CRF17_BF have been sequenced but not adequately described in publications. Here we describe CRF17_BF as a genuine CRF, and analyze its recombination pattern based on bootscan analyses, subtype signature patterns, and phylogenetic reconstruction of subtype-delimited segments. We show that CRF17_BF can be distinguished from CRF12_BF in several regions of the genome, including vpu, pol, env and nef. A complete and accurate characterization and description of recombination breakpoints in CRFs is required for a proper surveillance of HIV-1 genotypes, and important for epidemiological purposes. PMID:22266022

  19. Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab.

    PubMed

    Scapin, Giovanna; Yang, Xiaoyu; Prosise, Winifred W; McCoy, Mark; Reichert, Paul; Johnston, Jennifer M; Kashi, Ramesh S; Strickland, Corey

    2015-12-01

    Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. We report the structure of the human full-length IgG4 S228P anti-PD1 antibody pembrolizumab, solved to 2.3-Å resolution. Pembrolizumab is a compact molecule, consistent with the presence of a short hinge region. The Fc domain is glycosylated at the CH2 domain on both chains, but one CH2 domain is rotated 120° with respect to the conformation observed in all reported structures to date, and its glycan chain faces the solvent. We speculate that this new conformation is driven by the shorter hinge. The structure suggests a role for the S228P mutation in preventing the IgG4 arm exchange. In addition, this unusual Fc conformation suggests possible structural diversity between IgG subclasses and shows that use of isolated antibody fragments could mask potentially important interactions, owing to molecular flexibility. PMID:26595420

  20. Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection.

    PubMed

    Yang, Zhi-Tao; Huang, Su-Yuan; Chen, Li; Liu, Feng; Cai, Xiao-Hui; Guo, Yang-Fan; Wang, Ming-Jie; Han, Yue; Yu, De-Min; Jiang, Jie-Hong; Zhang, Dong-Hua; Gong, Qi-Ming; Zhang, Guo-Qing; Zang, Guo-Qing; Lu, Zhong-Hua; Huang, Li-Hua; Zhang, Xin-Xin

    2015-07-01

    Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T- and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated. PMID:25926495

  1. A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

    SciTech Connect

    Pierce, Anson; Wei, Rochelle; Halade, Dipti; Yoo, Si-Eun; Ran, Qitao; Richardson, Arlan

    2010-11-05

    Research highlights: {yields} Development of mouse overexpressing native human HSF1 in all tissues including CNS. {yields} HSF1 overexpression enhances heat shock response at whole-animal and cellular level. {yields} HSF1 overexpression protects from polyglutamine toxicity and favors aggresomes. {yields} HSF1 overexpression enhances proteostasis at the whole-animal and cellular level. -- Abstract: The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenic mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1{sup +/0}) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1{sup +/0} mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1{sup +/0} cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1{sup +/0} cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.

  2. Short-Read Assembly of Full-Length 16S Amplicons Reveals Bacterial Diversity in Subsurface Sediments

    PubMed Central

    Miller, Christopher S.; Handley, Kim M.; Wrighton, Kelly C.; Frischkorn, Kyle R.; Thomas, Brian C.; Banfield, Jillian F.

    2013-01-01

    In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries), or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing). Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the “long tail” of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change. PMID:23405248

  3. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    PubMed

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences. PMID:25523504

  4. Conditional expression of full-length humanized anti-prion protein antibodies in Chinese hamster ovary cells.

    PubMed

    Mueller, Daniel A; Heinig, Lars; Ramljak, Sanja; Krueger, Astrid; Schulte, Reiner; Wrede, Arne; Stuke, Andreas W

    2010-12-01

    Because of their high antigen specificity and metabolic stability, genetically engineered human monoclonal antibodies are on the way to becoming one of the most promising medical diagnostics and therapeutics. In order to establish an in vitro system capable of producing such biosimilar antibodies, we used human constant chain sequences to design the novel human antibody expressing vector cassette pMAB-ABX. A bidirectional tetracycline (tet)-controllable promotor was used for harmonized expression of immunoglobulin type G (IgG) heavy and light chains. As an example we used anti-prion protein (anti-PrP) IgGs. Therefore, the variable heavy (V(H)) and light chain (V(L)) sequences of anti-PrP antibodies, previously generated in our laboratory by DNA immunization of prion protein knock-out mice, were isolated from murine hybridoma cell lines and inserted into pMAB-ABX vector. After transfection of Chinese hamster ovary (CHO) cells, a number of stable antibody producing cell clones were selected. One cell line (pMAB-ABX-13F10/3B5) stably expressing the recombinant humanized antibody (rechuAb) 13F10/3B5 was selected for detailed characterization by Western blot, immunofluorescence, and flow cytometric analyses. The full-length recombinant humanized IgG antibody showed a high level of expression in the cytoplasm. In conclusion, the new cell system described here is a suitable tool to produce functional intact full-length humanized IgG antibodies. PMID:21087094

  5. Near-Full-Length Genome Sequences of a Novel HIV-1 Circulating Recombinant Form, CRF01_AE/B'/C (CRF78_cpx), in Yunnan, China.

    PubMed

    Song, Yindi; Feng, Yue; Miao, Zhijiang; Wang, Binghui; Yang, Ming; Zhang, A-Mei; Liu, Li; Xia, Xueshan

    2016-06-01

    We report a novel HIV circulating recombinant form (CRF78_cpx) composed mainly of CRF01_AE with inserts from subtypes B and C identified from three epidemiologically unlinked individuals in Yunnan province, China. Two of the subjects are heterosexual men and one is a male intravenous drug user. Sequencing and analyzing the near-full-length genome of these three isolates (YNTC88, YNTC19, and YNTC35) revealed identical recombination breakpoints in all three viruses, but considerable genetic diversity between them, across the genomes, indicating that this is not a newly created CRF, only newly detected. CRF78_cpx differs from previously documented CRF01-AE/B'/C forms in its distinct backbone, inserted fragment size, and breakpoints, and is not related to other described recombinants in the region such as CRF07_BC or CRF65_cpx (also composed of CRF01_AE, B', and C). Our present findings further enrich the diversity of the prevalent HIV-1 CRFs in Yunnan, which is considered as an epicenter of HIV-1 infections in China. PMID:26885715

  6. A full-length Plasmodium falciparum recombinant circumsporozoite protein expressed by Pseudomonas fluorescens platform as a malaria vaccine candidate.

    PubMed

    Noe, Amy R; Espinosa, Diego; Li, Xiangming; Coelho-Dos-Reis, Jordana G A; Funakoshi, Ryota; Giardina, Steve; Jin, Hongfan; Retallack, Diane M; Haverstock, Ryan; Allen, Jeffrey R; Vedvick, Thomas S; Fox, Christopher B; Reed, Steven G; Ayala, Ramses; Roberts, Brian; Winram, Scott B; Sacci, John; Tsuji, Moriya; Zavala, Fidel; Gutierrez, Gabriel M

    2014-01-01

    The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy

  7. Improved yields of full-length functional human FGF1 can be achieved using the methylotrophic yeast Pichia pastoris.

    PubMed

    Fantoni, Adele; Bill, Roslyn M; Gustafsson, Lena; Hedfalk, Kristina

    2007-03-01

    We have produced human fibroblast growth factor 1 (hFGF1) in the methylotrophic yeast Pichia pastoris in order to obtain the large amounts of active protein required for subsequent functional and structural characterization. Four constructs were made to examine both intracellular and secreted expression, with variations in the location of the His6 tag at either end of the peptide. hFGF1 could be produced from all four constructs in shake flasks, but production was optimized by growing only the highest-yielding of these strains, which produced hFGF1 intracellularly, under tightly controlled conditions in a 3 L fermentor. One hundred and eight milligrams of pure protein was achieved per liter culture (corresponding to 0.68 mg of protein per gram of wet cells), the function of which was verified using NIH 3T3 cell cultures. This is a 30-fold improvement over previously reported yields of full-length hFGF1. PMID:17134911

  8. Computational Analysis of Full-length cDNAs Reveals Frequent Coupling Between Transcriptional and Splicing Programs

    PubMed Central

    Chern, Tzu-Ming; Paul, Nicodeme; van Nimwegen, Erik; Zavolan, Mihaela

    2008-01-01

    High-throughput sequencing studies revealed that the majority of human and mouse multi-exon genes have multiple splice forms. High-density oligonucleotide array-based measurements have further established that many exons are expressed in a tissue-specific manner. The mechanisms underlying the tissue-dependent expression of most alternative exons remain, however, to be understood. In this study, we focus on one possible mechanism, namely the coupling of (tissue specific) transcription regulation with alternative splicing. We analyzed the FANTOM3 and H-Invitational datasets of full-length mouse and human cDNAs, respectively, and found that in transcription units with multiple start sites, the inclusion of at least 15% and possibly up to 30% of the ‘cassette’ exons correlates with the use of specific transcription start sites (TSS). The vast majority of TSS-associated exons are conserved between human and mouse, yet the conservation is weaker when compared with TSS-independent exons. Additionally, the currently available data only support a weak correlation between the probabilities of TSS association of orthologous exons. Our analysis thus suggests frequent coupling of transcriptional and splicing programs, and provides a large dataset of exons on which the molecular basis of this coupling can be further studied. PMID:18276623

  9. Virus recovery and full-length sequence analysis of atypical bovine pestivirus Th/04_KhonKaen.

    PubMed

    Liu, Lihong; Kampa, Jaruwan; Belák, Sándor; Baule, Claudia

    2009-07-01

    Phylogenetic analysis of recently identified "atypical" bovine pestiviruses, performed based on different gene regions, has revealed unclear relationships with other established species, therefore, their phylogenetic position could not be determined so far. In this study, the atypical pestivirus Th/04_KhonKaen was recovered from serum of a naturally infected calf and the complete genome sequence was determined and analysed, as means to define its position. The viral genome is 12,337 nucleotides (nt) long, and comprises a 5'-UTR of 383 nt, a 3'-UTR of 254 nt and an open reading frame of 11,700 nt, without duplication of viral sequences or insertions of cellular sequences. The phylogenetic analyses of the full-length sequence, performed by Neighbor-joining, Maximum likelihood, and the Bayesian approach, unanimously placed Th/04_KhonKaen in a single lineage, distinct from the established pestivirus species, and close to bovine viral diarrhea virus types 1 and 2. Furthermore, Th/04_KhonKaen and two previously reported atypical pestiviruses D32/00_'HoBi' and CH-KaHo/cont formed a well-supported monophyletic clade in trees based on the complete N(pro) and E2 gene regions. The finding provides conclusive classification of the Th/04_KhonKaen virus and confirms the standing of the "atypical" bovine pestiviruses as a novel pestivirus species. PMID:19349128

  10. Aminoglycosides Restore Full-length Type VII Collagen by Overcoming Premature Termination Codons: Therapeutic Implications for Dystrophic Epidermolysis Bullosa

    PubMed Central

    Cogan, Jon; Weinstein, Jacqueline; Wang, Xinyi; Hou, Yingping; Martin, Sabrina; South, Andrew P; Woodley, David T; Chen, Mei

    2014-01-01

    Patients with recessive dystrophic epidermolysis bullosa (RDEB) have severe, incurable skin fragility, blistering, and multiple skin wounds due to mutations in the gene encoding type VII collagen (C7), the major component of anchoring fibrils mediating epidermal-dermal adherence. Nearly 10–25% of RDEB patients carry nonsense mutations leading to premature stop codons (PTCs) that result in truncated C7. In this study, we evaluated the feasibility of using aminoglycosides to suppress PTCs and induce C7 expression in two RDEB keratinocyte cell lines (Q251X/Q251X and R578X/R906) and two primary RDEB fibroblasts (R578X/R578X and R163X/R1683X). Incubation of these cells with aminoglycosides (geneticin, gentamicin, and paromomycin) resulted in the synthesis and secretion of a full-length C7 in a dose-dependent and sustained manner. Importantly, aminoglycoside-induced C7 reversed the abnormal RDEB cell phenotype and incorporated into the dermal-epidermal junction of skin equivalents. We further demonstrated the general utility of aminoglycoside-mediated readthrough in 293 cells transiently transfected with expression vectors encoding 22 different RDEB nonsense mutations. This is the first study demonstrating that aminoglycosides can induce PTC readthrough and restore functional C7 in RDEB caused by nonsense mutations. Therefore, aminoglycosides may have therapeutic potential for RDEB patients and other inherited skin diseases caused by nonsense mutations. PMID:25155989