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Sample records for virus cdna constructs

  1. Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector.

    Science.gov (United States)

    Sun, Kai; Zhao, Danyang; Liu, Yong; Huang, Changjun; Zhang, Wei; Li, Zhenghe

    2017-11-07

    The availability of infectious full-length clone is indispensable for reverse genetics studies of virus biology, pathology and construction of viral vectors. However, for RNA viruses with large genome sizes or those exhibiting inherent cloning difficulties, procedure to generate biologically active circular DNA (cDNA) clones can be time-consuming or technically challenging. Here we have constructed a yeast- Escherichia coli - Agrobacterium shuttle vector that enables highly efficient homologous recombination in yeast for assembly of Agrobacterium compatible plant virus clones. Using this vector, we show that infectious cDNA clones of a plant negative-stranded RNA virus, sonchus yellow net rhabdovirus, can be rapidly assembled. In addition, one-step assembly of infectious clones of potato virus Y in yeast, either with or without intron, was readily achieved from as many as eight overlapping DNA fragments. More importantly, the recovered yeast plasmids can be transformed directly into Agrobacterium for inoculation, thereby obviating the E. coli cloning steps and associated toxicity issues. This method is rapid, highly efficient and cost-effective and should be readily applicable to a broad range of plant viruses.

  2. Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector

    Directory of Open Access Journals (Sweden)

    Kai Sun

    2017-11-01

    Full Text Available The availability of infectious full-length clone is indispensable for reverse genetics studies of virus biology, pathology and construction of viral vectors. However, for RNA viruses with large genome sizes or those exhibiting inherent cloning difficulties, procedure to generate biologically active complementary DNA (cDNA clones can be time-consuming or technically challenging. Here we have constructed a yeast-Escherichia coli-Agrobacterium shuttle vector that enables highly efficient homologous recombination in yeast for assembly of Agrobacterium compatible plant virus clones. Using this vector, we show that infectious cDNA clones of a plant negative-stranded RNA virus, sonchus yellow net rhabdovirus, can be rapidly assembled. In addition, one-step assembly of infectious clones of potato virus Y in yeast, either with or without intron, was readily achieved from as many as eight overlapping DNA fragments. More importantly, the recovered yeast plasmids can be transformed directly into Agrobacterium for inoculation, thereby obviating the E. coli cloning steps and associated toxicity issues. This method is rapid, highly efficient and cost-effective and should be readily applicable to a broad range of plant viruses.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Decai Tuo

    2015-12-01

    Full Text Available 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.

  5. Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus (GALV-Nf) and its biological activity in Nicotiana benthamiana and grapevine plants.

    Science.gov (United States)

    Lovato, Arianna; Faoro, Franco; Gambino, Giorgio; Maffi, Dario; Bracale, Marcella; Polverari, Annalisa; Santi, Luca

    2014-11-03

    Grapevine Algerian latent virus (GALV) is a tombusvirus first isolated in 1989 from an Algerian grapevine (Vitis spp.) plant and more recently from water samples and commercial nipplefruit and statice plants. No further reports of natural GALV infections in grapevine have been published in the last two decades, and artificial inoculations of grapevine plants have not been reported. We developed and tested a synthetic GALV construct for the inoculation of Nicotiana benthamiana plants and different grapevine genotypes to investigate the ability of this virus to infect and spread systemically in different hosts. We carried out a phylogenetic analysis of all known GALV sequences and an epidemiological survey of grapevine samples to detect the virus. A GALV-Nf clone under the control of the T7 promoter was chemically synthesized based on the full-length sequence of the nipplefruit isolate GALV-Nf, the only available sequence at the time the project was conceived, and the infectious transcripts were tested in N. benthamiana plants. A GALV-Nf-based binary vector was then developed for the agroinoculation of N. benthamiana and grapevine plants. Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species. Sequence analysis showed that the GALV coat protein is highly conserved among diverse isolates. The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples. The agroinoculation of N. benthamiana and grapevine plants with the GALV-Nf binary vector promoted efficient infections, as revealed by serological and molecular analysis. The GALV-Nf infection of grapevine plants was characterized in more detail by inoculating different cultivars, revealing distinct patterns of symptom development. Ultrastructural changes induced by GALV-Nf in N. benthamiana were similar to those induced by tombusviruses in

  6. Construction of an Infectious cDNA Clone of Aichi Virus (a New Member of the Family Picornaviridae) and Mutational Analysis of a Stem-Loop Structure at the 5′ End of the Genome

    Science.gov (United States)

    Sasaki, Jun; Kusuhara, Yasuhiro; Maeno, Yoshimasa; Kobayashi, Nobumichi; Yamashita, Teruo; Sakae, Kenji; Takeda, Naokazu; Taniguchi, Koki

    2001-01-01

    Aichi virus is the type species of a new genus, Kobuvirus, of the family Picornaviridae. In this study, we constructed a full-length cDNA clone of Aichi virus whose in vitro transcripts were infectious to Vero cells. During construction of the infectious cDNA clone, a novel sequence of 32 nucleotides was identified at the 5′ end of the genome. Computer-assisted prediction of the secondary structure of the 5′ end of the genome, including the novel sequence, suggested the formation of a stable stem-loop structure consisting of 42 nucleotides. The function of this stem-loop in virus replication was investigated using various site-directed mutants derived from the infectious cDNA clone. Our data indicated that correct folding of the stem-loop at the 5′ end of the positive strand, but not at the 3′ end of the negative strand, is critical for viral RNA replication. The primary sequence in the lower part of the stem was also suggested to be crucial for RNA replication. In contrast, nucleotide changes in the loop segment did not so severely reduce the efficiency of virus replication. A double mutant, in which both nucleotide stretches of the middle part of the stem were replaced by their complementary nucleotides, had efficient RNA replication and translation abilities but was unable to produce viruses. These results indicate that the stem-loop at the 5′ end of the Aichi virus genome is an element involved in both viral RNA replication and production of infectious virus particles. PMID:11483747

  7. Construction of an infectious cDNA clone of genotype 1 avian hepatitis E virus: characterization of its pathogenicity in broiler breeders and demonstration of its utility in studying the role of the hypervariable region in virus replication.

    Science.gov (United States)

    Park, Soo-Jeong; Lee, Byung-Woo; Moon, Hyun-Woo; Sung, Haan Woo; Yoon, Byung-Il; Meng, Xiang-Jin; Kwon, Hyuk Moo

    2015-05-01

    A full-length infectious cDNA clone of the genotype 1 Korean avian hepatitis E virus (avian HEV) (pT11-aHEV-K) was constructed and its infectivity and pathogenicity were investigated in leghorn male hepatoma (LMH) chicken cells and broiler breeders. We demonstrated that capped RNA transcripts from the pT11-aHEV-K clone were translation competent when transfected into LMH cells and infectious when injected intrahepatically into the livers of chickens. Gross and microscopic pathological lesions underpinned the avian HEV infection and helped characterize its pathogenicity in broiler breeder chickens. The avian HEV genome contains a hypervariable region (HVR) in ORF1. To demonstrate the utility of the avian HEV infectious clone, several mutants with various deletions in and beyond the known HVR were derived from the pT11-aHEV-K clone. The HVR-deletion mutants were replication competent in LMH cells, although the deletion mutants extending beyond the known HVR were non-viable. By using the pT11-aHEV-K infectious clone as the backbone, an avian HEV luciferase reporter replicon and HVR-deletion mutant replicons were also generated. The luciferase assay results of the reporter replicon and its mutants support the data obtained from the infectious clone and its derived mutants. To further determine the effect of HVR deletion on virus replication, the capped RNA transcripts from the wild-type pT11-aHEV-K clone and its mutants were injected intrahepatically into chickens. The HVR-deletion mutants that were translation competent in LMH cells displayed in chickens an attenuation phenotype of avian HEV infectivity, suggesting that the avian HEV HVR is important in modulating the virus infectivity and pathogenicity. © 2015 The Authors.

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

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    Page Jonathan E

    2008-01-01

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

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

    Science.gov (United States)

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

    2001-04-01

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

  10. Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA.

    Science.gov (United States)

    Hurrelbrink, R J; Nestorowicz, A; McMinn, P C

    1999-12-01

    An infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was constructed by stably inserting genome-length cDNA into the low-copy-number plasmid vector pMC18. Designated pMVE-1-51, the clone consisted of genome-length cDNA of MVE-1-51 under the control of a T7 RNA polymerase promoter. The clone was constructed by using existing components of a cDNA library, in addition to cDNA of the 3' terminus derived by RT-PCR of poly(A)-tailed viral RNA. Upon comparison with other flavivirus sequences, the previously undetermined sequence of the 3' UTR was found to contain elements conserved throughout the genus FLAVIVIRUS: RNA transcribed from pMVE-1-51 and subsequently transfected into BHK-21 cells generated infectious virus. The plaque morphology, replication kinetics and antigenic profile of clone-derived virus (CDV-1-51) was similar to the parental virus in vitro. Furthermore, the virulence properties of CDV-1-51 and MVE-1-51 (LD(50) values and mortality profiles) were found to be identical in vivo in the mouse model. Through site-directed mutagenesis, the infectious clone should serve as a valuable tool for investigating the molecular determinants of virulence in MVE virus.

  11. Constructing computer virus phylogenies

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    Goldberg, L.A. [Warwick Univ., Coventry (United Kingdom) Dept. of Computer Science; Goldberg, P.W. [Aston Univ., Birmingham (United Kingdom) Dept. of Applied Mathematics; Phillips, C.A. [Sandia National Labs., Albuquerque, NM (United States); Sorkin, G.B. [International Business Machines Corp., Yorktown Heights, NY (United States). Thomas J. Watson Research Center

    1996-03-01

    There has been much recent algorithmic work on the problem of reconstructing the evolutionary history of biological species. Computer virus specialists are interested in finding the evolutionary history of computer viruses--a virus is often written using code fragments from one or more other viruses, which are its immediate ancestors. A phylogeny for a collection of computer viruses is a directed acyclic graph whose nodes are the viruses and whose edges map ancestors to descendants and satisfy the property that each code fragment is ``invented`` only once. To provide a simple explanation for the data, we consider the problem of constructing such a phylogeny with a minimal number of edges. In general, this optimization problem cannot be solved in quasi-polynomial time unless NQP=QP; we present positive and negative results for associated approximated problems. When tree solutions exist, they can be constructed and randomly sampled in polynomial time.

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

    African Journals Online (AJOL)

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

  13. Toward a whole cDNA catalog: construction of an equalized cDNA library from mouse embryos.

    Science.gov (United States)

    Takahashi, N; Ko, M S

    1994-09-01

    The construction of a cDNA library containing all genes without redundancy is one of the major technical challenges for biology. Toward this goal, we have developed an equalized (normalized) cDNA library from mRNA pools derived from mouse embryos that cover the entire period of mouse ontogenesis. Colony hybridization analyses with 11 genes showed the reduction of abundance variation from at least 6000-fold in the unequalized S-library to approximately 33-fold in the EIII-library, which was constructed after three cycles of equalization procedure. Limiting dilution PCR analyses with 26 tissue-specific genes showed the reduction of abundance variation from at least approximately 1,000,000-fold in the S-cDNA mixture to approximately 100-fold in the EIII-cDNA mixture. Based on these analyses, we estimate that at least 15,000 independent cDNA clones are included with little redundancy in the EIII-cDNA library. This will be a useful resource for mouse biology as well as the mouse genome project.

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

    African Journals Online (AJOL)

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

  15. Cloning of the complete infectious cDNA of the plum pox virus strain PPV-Rec.

    Science.gov (United States)

    Predajňa, L; Nagyová, A; Glasa, M; Subr, Z W

    2012-01-01

    Plum pox virus (PPV) is the causal agent of Sharka, considered to be the most detrimental viral disease of Prunus spp. worldwide. So far, several PPV strains have been recognized, three of them (PPV-D, PPV-M, and PPV-Rec) having shown serious economic impact in the European area. Infectious cDNA clones of plant RNA viruses are excellent tools for functional studies of viral genomes. Preparation and use of PPV-D and PPV-M infectious clones have been previously reported. Here we describe the construction of an infectious cDNA clone of the strain PPV-Rec (isolate BOR-3) by the strategy involving the subsequent exchanges of homologous BOR-3 genome parts in the backbone of the previously prepared PPV-D infectious construct. The infectivity of each intermediate chimeric cDNA as well as that of the final construct (pIC-PPV-Rec) was confirmed by biolistic transfection of Nicotiana benthamiana plants. Complete sequence of the cloned viral BOR-3 cDNA revealed 0.14% of difference at the nucleotide level compared to original BOR-3 sequence, resulting in four amino acid changes. This slight inequality was related to the population heterogeneity of the initial BOR-3 isolate; no difference in the amino acid sequence resulted from the cloning steps performed. inter-strain chimera; biolistics; genome sequence.

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

    OpenAIRE

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

    2003-01-01

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

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

    Science.gov (United States)

    Bei, Zhu-chun; Wang, Jing-yan

    2004-06-01

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

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

    Science.gov (United States)

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

    2002-09-01

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

  19. A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector.

    Science.gov (United States)

    Junqueira, Bruna Rayane Teodoro; Nicolini, Cícero; Lucinda, Natalia; Orílio, Anelise Franco; Nagata, Tatsuya

    2014-03-01

    Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

    Science.gov (United States)

    Yoon, Ju-Yeon; Cho, In-Sook; Choi, Gug-Seoun; Choi, Seung-Kook

    2014-01-01

    Chrysanthemum stunt viroid (CSVd), a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1) were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants. PMID:25288987

  1. Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

    Directory of Open Access Journals (Sweden)

    Ju-Yeon Yoon

    2014-03-01

    Full Text Available Chrysanthemum stunt viroid (CSVd, a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1 were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants.

  2. Complete nucleotide sequences and construction of full-length infectious cDNA clones of Cucumber green mottle virus (CGMMV) in a versatile newly developed binary vector including both 35S and T7 promoters

    Science.gov (United States)

    Seed-transmitted viruses have caused significant damage to watermelon crops in Korea in recent years, with Cucumber green mottle mosaic virus (CGMMV) infection widespread as a result of infected seed lots. To determine the likely origin of CGMMV infection, we collected CGMMV isolates from watermelon...

  3. Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus

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    Meng, Baozhong, E-mail: bmeng@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Venkataraman, Srividhya; Li, Caihong; Wang, Weizhou [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Dayan-Glick, Cathy; Mawassi, Munir [The Plant Pathology Department-The Virology Unit, Plant Protection Institute, Agricultural Research Organization, The Volcani Center, Bet-Dagan 50250 (Israel)

    2013-01-20

    Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35 S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.

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

    African Journals Online (AJOL)

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

  5. Construction of a full-length cDNA library and analysis of expressed ...

    African Journals Online (AJOL)

    sunny t

    2015-06-10

    Jun 10, 2015 ... Biotechnol. 16:95-104. Talon M, Gmitter FG Jr. (2008). Citrus genomics. Int. J. Plant Genomics. 2008:528361. Tran T, Vu TQC, Abdullah MP, Omar H, Noroozi M, Ky H, Napis S. (2011). Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus.

  6. A cDNA clone of tomato mosaic virus is infectious in plants.

    Science.gov (United States)

    Weber, H; Haeckel, P; Pfitzner, A J

    1992-01-01

    A cDNA clone of tomato mosaic virus (ToMV) genomic RNA was fused to the cauliflower mosaic virus 35S RNA promoter and the nopaline synthase gene polyadenylation signal. The transcriptional initiation site of the 35S RNA promoter was altered by in vitro mutagenesis so that the resulting transcripts start at the first nucleotide of the ToMV sequence. In addition, 12 nucleotides were inserted in the 5' untranslated region of the ToMV genome. This plasmid, pSLN, was used to inoculate several host plants of ToMV. Among five plant species tested, only Chenopodium quinoa accumulated large amounts of viral particles. The infectivities and systemic movements of the resulting viruses were the same as those of virus preparations obtained from a ToMV infection of C. quinoa. Primer extension analyses revealed that the 5' end of the viral genomic RNA was identical to those of RNAs isolated from virus progeny of an infection with T7 transcripts analogous to pSLN. Moreover, the insertion in the 5' untranslated region of the viral genome was stably maintained through several systemic passages of the virus. Thus, inoculation of plants with a plasmid containing a cDNA clone of an RNA virus under the control of a eukaryotic promoter seems to be a convenient alternative to the generation of in vitro transcripts and should facilitate the analysis of viral mutants generated at the DNA level. Images PMID:1583735

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

    Science.gov (United States)

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

    2011-02-01

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

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

    Science.gov (United States)

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

    2003-11-01

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

  9. Chimeric cDNA Sequences from Citrus tristeza virus Confer RNA Silencing-Mediated Resistance in Transgenic Nicotiana benthamiana Plants.

    Science.gov (United States)

    Roy, Gourgopal; Sudarshana, Mysore R; Ullman, Diane E; Ding, Shou-Wei; Dandekar, Abhaya M; Falk, Bryce W

    2006-08-01

    ABSTRACT RNA silencing has been shown to be an important mechanism for conferring resistance in transgenic, virus-resistant plants. We used this approach to evaluate resistance in Nicotiana benthamiana plants transformed with chimeric coding and noncoding sequences from Citrus tristeza virus (CTV). Several independent transgenic plant lines were generated, using two constructs (pCTV1 and pCTV2) designed to produce self-complementary transcripts. The pCTV1 contained cDNA sequences from the CTV capsid protein (CP), p20, and 3' untranslated region (UTR); and pCTV2 contained CP, p23, and 3' UTR sequences. Heterologous recombinant Potato virus X (PVX) containing either homologous or heterologous CTV sequences was used to challenge plants and resistance was evaluated phenotypically and validated with reverse-transcriptase polymerase chain reaction and northern hybridization analysis. Transgenic plants (T1 generation) for each construct showed resistance to recombinant PVX constructs used for challenge experiments when PVX contained p20 or UTR (for CTV1 plants), or p23 or UTR (for CTV2 plants). However, no resistance was seen when plants were challenged with PVX containing the CTV CP. T2 generation plants also showed resistance even when challenged with PVX containing the cognate CTV sequences obtained from heterologous CTV isolates. The presence of transgene-specific small interfering RNAs in the resistant CTV1 and CTV2 plants indicated that resistance was mediated by post-transcriptional gene silencing.

  10. Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik

    2013-01-01

    Background Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described....... This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. Results A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable...... rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed...

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

    Science.gov (United States)

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

    2010-12-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2007-11-01

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

  14. KARAKTERISTIK SEKUEN cDNA PENGKODE GEN ANTI VIRUS DARI UDANG WINDU, Penaeus monodon

    Directory of Open Access Journals (Sweden)

    Andi Parenrengi

    2016-11-01

    Full Text Available Transgenesis pada ikan merupakan sebuah teknik modern yang berpotensi besar dalam menghasilkan organisme yang memiliki karakter lebih baik melalui rekombinan DNA gen target termasuk gen anti virus dalam peningkatan resistensi pada udang. Gen anti virus PmAV (Penaeus monodon Anti Viral gene merupakan salah satu gen pengkode anti virus yang berasal dari spesies krustase. Penelitian ini dilakukan untuk mengetahui karakteristik gen anti virus yang diisolasi dari udang windu, Penaeus monodon. Isolasi gen anti virus menggunakan metode Polymerase Chain Reaction (PCR dan selanjutnya dipurifikasi untuk sekuensing. Data yang dihasilkan dianalisis dengan program Genetyx Versi 7 dan basic local alignment search tool (BLAST. Hasil penelitian menunjukkan bahwa gen anti virus PmAV yang berhasil diisolasi dari cDNA udang windu dengan panjang sekuen 520 bp yang mengkodekan 170 asam amino. BLAST-N menunjukkan tingkat similaritas yang sangat tinggi (100% dengan gen anti virus yang ada di GeneBank. Komposisi asam amino penyusun gen anti virus yang paling besar adalah serin (10,00%, sedangkan yang terkecil adalah asam amino prolin dan lisin masing-masing 1,76%. Analisis sekuen gen dan deduksi asam amino (BLAST-P memperlihatkan adanya C-type lectin-like domain (CTLD yang memiliki kemiripan dengan gen C-type lectin yang diisolasi dari beberapa spesies krustase. Transgenic fish technology is a potential modern technique in producing better character organism through DNA recombinant of target genes including anti viral gene for improvement of shrimp immunity. PmAV (Penaeus monodon Anti Viral gene is one of anti viral genes isolated from crustacean species. The research was conducted to analyze the characteristics anti viral gene isolated from tiger prawn, Penaeus monodon. Anti viral gene was isolated using Polymerase Chain Reaction (PCR technique and then purified for sequencing. Data obtained were analyzed using Genetyx Version 7 software and basic local alignment

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2010-07-01

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

  17. Rescue of a genotype 4 human hepatitis E virus from cloned cDNA and characterization of intergenotypic chimeric viruses in cultured human liver cells and in pigs

    Science.gov (United States)

    Córdoba, Laura; Feagins, Alicia R.; Opriessnig, Tanja; Cossaboom, Caitlin M.; Dryman, Barbara A.; Huang, Yao-Wei

    2012-01-01

    Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 are zoonotic, infecting both humans and pigs. This report describes, for the first time, the successful rescue of infectious HEV in vitro and in vivo from cloned cDNA of a genotype 4 human HEV (strain TW6196E). The complete genomic sequence of the TW6196E virus was determined and a full-length cDNA clone (pHEV-4TW) was assembled. Capped RNA transcripts from the pHEV-4TW clone were replication competent in Huh7 cells and infectious in HepG2/C3A cells. Pigs inoculated intrahepatically with capped RNA transcripts from pHEV-4TW developed an active infection, as evidenced by faecal virus shedding and seroconversion, indicating the successful rescue of infectious genotype 4 HEV and cross-species infection of pigs by a genotype 4 human HEV. To demonstrate the utility of the genotype 4 HEV infectious clone and to evaluate the potential viral determinant(s) for species tropism, four intergenotypic chimeric clones were constructed by swapping various genomic regions between genotypes 1 and 4, and genotypes 1 and 3. All four chimeric clones were replication competent in Huh7 cells, but only the two chimeras with sequences swapped between genotypes 1 and 4 human HEVs produced viruses capable of infecting HepG2/C3A cells. None of the four chimeras was able to establish a robust infection in pigs. The availability of a genotype 4 HEV infectious clone affords an opportunity to delineate the molecular mechanisms of HEV cross-species infection in the future. PMID:22837416

  18. Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi

    NARCIS (Netherlands)

    Gemeren, I.A. van; Musters, W.; Hondel, C.A.M.J.J. van den; Verrips, C.T.

    1995-01-01

    A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the

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

    Science.gov (United States)

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

    2003-07-01

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

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

    Science.gov (United States)

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

    2006-02-28

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

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

    Science.gov (United States)

    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.

  2. IS-98-ST1 West Nile virus derived from an infectious cDNA clone retains neuroinvasiveness and neurovirulence properties of the original virus.

    Directory of Open Access Journals (Sweden)

    Céline Bahuon

    Full Text Available Infectious clones of West Nile virus (WNV have previously been generated and used to decipher the role of viral proteins in WNV virulence. The majority of molecular clones obtained to date have been derived from North American, Australian, or African isolates. Here, we describe the construction of an infectious cDNA clone of a Mediterranean WNV strain, IS-98-ST1. We characterized the biological properties of the recovered recombinant virus in cell culture and in mice. The growth kinetics of recombinant and parental WNV were similar in Vero cells. Moreover, the phenotype of recombinant and parental WNV was indistinguishable as regards viremia, viral load in the brain, and mortality in susceptible and resistant mice. Finally, the pathobiology of the infectious clone was examined in embryonated chicken eggs. The capacity of different WNV strains to replicate in embryonated chicken eggs closely paralleled their ability to replicate in mice, suggesting that inoculation of embryonated chicken eggs could provide a practical in vivo model for the study of WNV pathogenesis. In conclusion, the IS-98-ST1 infectious clone will allow assessment of the impact of selected mutations and novel genomic changes appearing in emerging European strains pathogenicity and endemic or epidemic potential. This will be invaluable in the context of an increasing number of outbreaks and enhanced severity of infections in the Mediterranean basin and Eastern Europe.

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

    Science.gov (United States)

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

    2004-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kaur Jatinder

    2010-05-01

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

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

    OpenAIRE

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

    1980-01-01

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

  6. An Infectious cDNA Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and Antiviral Inhibitors.

    Science.gov (United States)

    Shan, Chao; Xie, Xuping; Muruato, Antonio E; Rossi, Shannan L; Roundy, Christopher M; Azar, Sasha R; Yang, Yujiao; Tesh, Robert B; Bourne, Nigel; Barrett, Alan D; Vasilakis, Nikos; Weaver, Scott C; Shi, Pei-Yong

    2016-06-08

    The Asian lineage of Zika virus (ZIKV) has recently caused epidemics and severe disease. Unraveling the mechanisms causing increased viral transmissibility and disease severity requires experimental systems. We report an infectious cDNA clone of ZIKV that was generated using a clinical isolate of the Asian lineage. The cDNA clone-derived RNA is infectious in cells, generating recombinant ZIKV. The recombinant virus is virulent in established ZIKV mouse models, leading to neurological signs relevant to human disease. Additionally, recombinant ZIKV is infectious for Aedes aegypti and thus provides a means to examine virus transmission. The infectious cDNA clone was further used to generate a luciferase ZIKV that exhibited sensitivity to a panflavivirus inhibitor, highlighting its potential utility for antiviral screening. This ZIKV reverse genetic system, together with mouse and mosquito infection models, may help identify viral determinants of human virulence and mosquito transmission as well as inform vaccine and therapeutic strategies. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Full-length enriched multistage cDNA library construction covering ...

    African Journals Online (AJOL)

    DR TONUKARI NYEROVWO

    2012-04-10

    Apr 10, 2012 ... Flowering is an important event in the life cycle of all flowering plants. Current knowledge ... developmental cycles between vegetative and reproductive growth periods are .... phages from female floral buds cDNA library (M, DL2000 marker; 1 to 12, PCR products of different λ phages from male and female ...

  8. Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

    Directory of Open Access Journals (Sweden)

    Sandra Arenhart

    Full Text Available Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3. The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

  9. Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs.

    Science.gov (United States)

    Huang, Y W; Haqshenas, G; Kasorndorkbua, C; Halbur, P G; Emerson, S U; Meng, X J

    2005-02-01

    Swine hepatitis E virus (swine HEV), the first animal strain of HEV to be isolated, is a zoonotic agent. We report here the construction and in vitro and in vivo characterizations of infectious cDNA clones of swine HEV. Eight overlapping fragments spanning the entire genome were amplified by reverse transcription-PCR and assembled into a full-length cDNA clone, clone C, which contained 14 mutations compared to the consensus sequence of swine HEV. RNA transcripts from clone C were not infectious, as determined by intrahepatic inoculation into pigs and by in vitro transfection of Huh7 cells. Multiple site-based site-directed mutagenesis was performed to generate three new cDNA clones (pSHEV-1, pSHEV-2, and pSHEV-3) which differed from each other. The transfection of capped RNA transcripts into human liver Huh7 cells resulted in the synthesis of both ORF2 capsid and ORF3 proteins, indicating that the cDNA clones were replication competent. Each of the three clones resulted in active swine HEV infections after the intrahepatic inoculation of pigs with capped RNA transcripts. The patterns of seroconversion, viremia, and fecal virus shedding for pigs inoculated with RNA transcripts from clones pSHEV-2 and pSHEV-3 were similar to each other and to those for pigs inoculated with wild-type swine HEV, suggesting that the nucleotide differences between these two cDNA clones were not critical for replication. Pigs inoculated with RNA transcripts from clone pSHEV-1, which contained three nonsilent mutations in the ORF2 capsid gene, had a delayed appearance of seroconversion and fecal virus shedding and had undetectable viremia. The availability of these infectious cDNA clones affords us an opportunity to understand the mechanisms of cross-species infection by constructing chimeric human and swine HEVs.

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

    Directory of Open Access Journals (Sweden)

    Zhou Sun

    2012-05-01

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

  11. In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts

    Directory of Open Access Journals (Sweden)

    Carolyn A. Owen

    2016-06-01

    Full Text Available Cucurbit yellow stunting disorder virus (CYSDV, a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ comprising the 5′- and 3′-UTRs plus the 3′-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants.

  12. In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts.

    Science.gov (United States)

    Owen, Carolyn A; Moukarzel, Romy; Huang, Xiao; Kassem, Mona A; Eliasco, Eleonora; Aranda, Miguel A; Coutts, Robert H A; Livieratos, Ioannis C

    2016-06-14

    Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5'- and 3'-UTRs plus the 3'-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants.

  13. Construction of an infectious cDNA clone of foot-and-mouth disease ...

    Indian Academy of Sciences (India)

    The immunogenicity of an oil-adjuvant vaccine prepared using the inactivated recombinant virus was tested in guinea pigs and cattle. Neutralizing antibodies were produced in both vaccinated guinea pigs and cattle. Vaccinated animals were protected on challenge. The results demonstrated that the recombinant virus was ...

  14. Construction of an infectious cDNA clone of foot-and-mouth disease ...

    Indian Academy of Sciences (India)

    Prakash

    et al 1990; Lopez de Quinto and Martinez-Salas 1997). The 5′ UTR of cardioviruses and aphthoviruses contains a homopolymeric poly (C) tract that separates ...... Bachrach H L, Callis J J, Hess W R and Patty R E 1957 A plaque assay for foot-and-mouth disease virus and kinetics of virus reproduction; Virology 4 224−236.

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

    Science.gov (United States)

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

    2009-02-01

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

  16. Yoctomole electrochemical genosensing of Ebola virus cDNA by rolling circle and circle to circle amplification.

    Science.gov (United States)

    Carinelli, S; Kühnemund, M; Nilsson, M; Pividori, M I

    2017-07-15

    This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. A single-plasmid reverse genetics system for the rescue of non-segmented negative-strand RNA viruses from cloned full-length cDNA

    NARCIS (Netherlands)

    Peeters, Ben; Leeuw, de Olav

    2017-01-01

    Reverse genetics systems for non-segmented negative-strand RNA viruses rely on co-transfection of a plasmid containing the full-length viral cDNA and helper plasmids encoding essential viral replication proteins. Here, a system is presented in which virus can be rescued from a single plasmid

  18. Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Dräger, Carolin

    2015-01-01

    RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high...... the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral......-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from...

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

    Science.gov (United States)

    Hou, Q; Chen, K; Shan, Z

    2015-01-01

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

  20. Construction of a full-length cDNA library and analysis of expressed ...

    African Journals Online (AJOL)

    White jute (Corchorus capsularis L.) is recognized as an important industrial raw material fibre crop owing to its elite characters. However, little information is known about its molecular basis and genomics. In this study, a complementary DNA library of white jute was constructed and expressed sequence tags (ESTs) were ...

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

    Directory of Open Access Journals (Sweden)

    Chen Xianming

    2007-06-01

    Full Text Available Abstract Background Puccinia striiformis is a plant pathogenic fungus causing stripe rust, one of the most important diseases on cereal crops and grasses worldwide. However, little is know about its genome and genes involved in the biology and pathogenicity of the pathogen. We initiated the functional genomic research of the fungus by constructing a full-length cDNA and determined functions of the first group of genes by sequence comparison of cDNA clones to genes reported in other fungi. Results A full-length cDNA library, consisting of 42,240 clones with an average cDNA insert of 1.9 kb, was constructed using urediniospores of race PST-78 of P. striiformis f. sp. tritici. From 196 sequenced cDNA clones, we determined functions of 73 clones (37.2%. In addition, 36 clones (18.4% had significant homology to hypothetical proteins, 37 clones (18.9% had some homology to genes in other fungi, and the remaining 50 clones (25.5% did not produce any hits. From the 73 clones with functions, we identified 51 different genes encoding protein products that are involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism, and virulence/infection. Conclusion The full-length cDNA library is useful in identifying functional genes of P. striiformis.

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

    Science.gov (United States)

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

    2014-08-01

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

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

    Science.gov (United States)

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

    2011-03-01

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

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

    Science.gov (United States)

    Guan, Yunyan; Huang, Liangmin; He, Maoxian

    2011-05-01

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

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Science.gov (United States)

    He, Tao; Jia, Jing Fen

    2008-12-01

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

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

    Science.gov (United States)

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

    2007-06-20

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

  8. Genomics of hepatitis B virus-related hepatocellular carcinoma and adjacent noncancerous tissues with cDNA microarray.

    Science.gov (United States)

    Huang, Yu-kun; Fan, Xue-gong; Qiu, Fu; Wang, Zhi-ming

    2011-07-05

    Hepatocellular carcinoma (HCC) is a common primary cancer frequently associated with hepatitis B virus (HBV) infection. However, whether these identified genes are particularly associated with HBV-related HCC remains unknown. The aim of this study was to investigate the differential gene expression between HBV-related HCC tissues and adjacent noncancerous tissues. cDNA microarray was used to detect the differential gene expression profile in the HBV-related HCC tissues and adjacent noncancerous tissues, and reverse transcription-polymerase chain reaction (RT-PCR) was performed to verify the differential expression of candidate genes obtained from cDNA microarray experiment. In this study, 1369 genes or expressed sequence tags (ESTs) including 121 genes or ESTs with at least two-fold expression alterations between cancerous and noncancerous tissues were identified. Special AT-rich sequence binding protein 1 (SATB-1) expression was positive in 73% (16/22) of cancerous tissues and negative (0/22) in all noncancerous tissues of HBV-related HCC patients. Transmembrane 4 superfamily member 1 (TM4SF-1) expression was positive in 86% (19/22) of cancerous tissues and negative (0/22) in all noncancerous tissues. Suppression of tumorigenicity 14 (ST-14) expression was positive in 73% (16/22) of noncancerous tissues in patients with HBV-related HCC and negative in all HCC tissues (0/22). This study provided the gene expression profile of HBV-related HCC and presented differential expression patterns of SATB-1, TM4SF-1 and ST-14 between cancerous and noncancerous tissues in patients with HBV-related HCC.

  9. Full-length enriched cDNA library construction from tissues related to energy metabolism in pigs.

    Science.gov (United States)

    Lee, Kyung-Tai; Byun, Mi-Jeong; Lim, Dajeong; Kang, Kyung-Soo; Kim, Nam-Soon; Oh, Jung-Hwa; Chung, Chung-Soo; Park, Hae-Suk; Shin, Younhee; Kim, Tae-Hun

    2009-12-31

    Genome sequencing of the pig is being accelerated because of its importance as an evolutionary and biomedical model animal as well as a major livestock animal. However, information on expressed porcine genes is insufficient to allow annotation and use of the genomic information. A series of expressed sequence tags of 5' ends of five full-length enriched cDNA libraries (SUSFLECKs) were functionally characterized. SUSFLECKs were constructed from porcine abdominal fat, induced fat cells, loin muscle, liver, and pituitary gland, and were composed of non-normalized and normalized libraries. A total of 55,658 ESTs that were sequenced once from the 5' ends of clones were produced and assembled into 17,684 unique sequences with 7,736 contigs and 9,948 singletons. In Gene Ontology analysis, two significant biological process leaf nodes were found: gluconeogenesis and translation elongation. In functional domain analysis based on the Pfam database, the beta transducin repeat domain of WD40 protein was the most frequently occurring domain. Twelve genes, including SLC25A6, EEF1G, EEF1A1, COX1, ACTA1, SLA, and ANXA2, were significantly more abundant in fat tissues than in loin muscle, liver, and pituitary gland in the SUSFLECKs. These characteristics of SUSFLECKs determined by EST analysis can provide important insight to discover the functional pathways in gene networks and to expand our understanding of energy metabolism in the pig.

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Science.gov (United States)

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

    2015-10-19

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

  12. Expression of a synthetic rust fungal virus cDNA in yeast.

    Science.gov (United States)

    Cooper, Bret; Campbell, Kimberly B; Garrett, Wesley M

    2016-01-01

    Mycoviruses are viruses that infect fungi. Recently, mycovirus-like RNAs were sequenced from the fungus Phakopsora pachyrhizi, the causal agent of soybean rust. One of the RNAs appeared to represent a novel mycovirus and was designated Phakopsora pachyrhizi virus 2383 (PpV2383). The genome of PpV2383 resembles Saccharomyces cerevisiae virus L-A, a double-stranded (ds) RNA mycovirus of yeast. PpV2383 encodes two major, overlapping open reading frames with similarity to gag (capsid protein) and pol (RNA-dependent RNA polymerase), and a -1 ribosomal frameshift is necessary for the translation of a gag-pol fusion protein. Phylogenetic analysis of pol relates PpV2383 to members of the family Totiviridae, including L-A. Because the obligate biotrophic nature of P. pachyrhizi makes it genetically intractable for in vivo analysis and because PpV2383 is similar to L-A, we synthesized a DNA clone of PpV2383 and tested its infectivity in yeast cells. PpV2383 RNA was successfully expressed in yeast, and mass spectrometry confirmed the translation of gag and gag-pol fusion proteins. There was, however, no production of PpV2383 dsRNA, the evidence of viral replication. Neither the presence of endogenous L-A nor the substitution of the 5' and 3' untranslated regions with those from L-A was sufficient to rescue replication of PpV2383. Nevertheless, the proof of transcription and translation from the clone in vivo are steps toward confirming that PpV2383 is a mycovirus. Further development of a surrogate biological system for the study of rust mycoviruses is necessary, and such research may facilitate biological control of rust diseases.

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

    Directory of Open Access Journals (Sweden)

    Lim Kean-Jin

    2009-10-01

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

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

    Science.gov (United States)

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

    2012-07-01

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

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

    Science.gov (United States)

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

    2011-07-01

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

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

    Science.gov (United States)

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

    2008-08-20

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

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

    Science.gov (United States)

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

    2015-10-21

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

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

    Science.gov (United States)

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

    2012-09-10

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

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

    Directory of Open Access Journals (Sweden)

    Sujuan YE

    2008-08-01

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

  20. Constructing and screening a cDNA library. Methods for identification and characterization of novel genes expressed under conditions of environmental stress.

    Science.gov (United States)

    Larade, Kevin; Storey, Kenneth B

    2008-01-01

    Many organisms provide excellent models for studying disease states or for exploring the molecular adaptations that allow cells and organisms to cope with or survive different stresses. The construction of a cDNA library and subsequent screening for genes of interest allows researchers to select for genes that are likely to play key roles in the regulation or response to the condition or stress of interest, those that may not be expressed (or exist) in other systems. Determination of the open reading frame(s) of novel genes, and extensive analysis of the proteins they encode, can open up new avenues of research and promote intelligent design of downstream projects.

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

    Science.gov (United States)

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

    2013-05-24

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

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

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2013-05-01

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

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

    Science.gov (United States)

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

    2013-01-01

    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

  4. Construction and characterization of recombinant Japanese encephalitis virus carrying brainspecific miRNA target sequences

    Directory of Open Access Journals (Sweden)

    Wen-yuan CAO

    2014-08-01

    Full Text Available Objective To construct the recombinant Japanese encephalitis virus ( JEV carrying brain-specific miRNA targeting sequences. Methods The target sequences of brain-specific miR-124 and miR-125 were introduced into the infectious cDNA clone of JEV to generate recombinant plasmids based on reverse genetics technology. The recombinant plasmids were linearized with Xho Ⅰ and served as templates of transcription with SP6 RNA polymerase to generate infectious viral RNA. The RNA transcripts were then transfected into BHK-21 cells, and the supernatant was obtained after incubated at 37℃, 5% CO2 for 3 days. The cytopathic changes of BHK-21 cells inoculated with the supernatant were observed after one passage. The rescued viruses carrying miRNA target sequences were validated by RT-PCR, standard plaque forming test on BHK-21 cells and growth curves analysis. Results Two recombinant viruses carrying miR-124 or miR-125 target sequence were rescued, respectively. The insertion of miRNA target sequences was confirmed by DNA sequencing. The rescued viruses yielded similar plaque morphology and replication efficiency compared with wild type JEV. Conclusion The recombinant JEV containing brain-specific miRNA target sequences can be obtained by reverse genetics technique, which could be used in further studies of miRNA-mediated tissue-specific attenuation mechanism of JEV. DOI: 10.11855/j.issn.0577-7402.2014.06.01

  5. Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus.

    Science.gov (United States)

    Ma, Xueqing; Li, Pinghua; Sun, Pu; Bai, Xingwen; Bao, Huifang; Lu, Zengjun; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

    2015-04-01

    Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids (aa) in most FMDVs examined to date. Specific deletion in the FMDV 3A protein has been associated with the inability of FMDV to grow in primary bovine cells and cause disease in cattle. However, the aa residues playing key roles in these processes are poorly understood. In this study, we constructed epitope-tagged FMDVs containing an 8 aa FLAG epitope, a 9 aa haemagglutinin (HA) epitope, and a 10 aa c-Myc epitope to substitute residues 94-101, 93-101, and 93-102 of 3A protein, respectively, using a recently developed O/SEA/Mya-98 FMDV infectious cDNA clone. Immunofluorescence assay (IFA), Western blot and sequence analysis showed that the epitope-tagged viruses stably maintained and expressed the foreign epitopes even after 10 serial passages in BHK-21 cells. The epitope-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the epitope-tagged viruses exhibited lower growth rates and smaller plaque size phenotypes than those of the parental virus in primary fetal bovine kidney (FBK) cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the decreased ability of FMDV to replicate in primary bovine cells was not associated with the length of 3A, and the genetic determinant thought to play key role in decreased ability to replicate in primary bovine cells could be reduced from 93-102 residues to 8 aa residues at positions 94-101 in 3A protein. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Molecular and Biological Characterization of an Isolate of Cucumber mosaic virus from Glycine soja by Generating its Infectious Full-genome cDNA Clones

    Directory of Open Access Journals (Sweden)

    Mi Sa Vo Phan

    2014-06-01

    Full Text Available Molecular and biological characteristics of an isolate of Cucumber mosaic virus (CMV from Glycine soja (wild soybean, named as CMV-209, was examined in this study. Comparison of nucleotide sequences and phylogenetic analyses of CMV-209 with the other CMV strains revealed that CMV-209 belonged to CMV subgroup I. However, CMV-209 showed some genetic distance from the CMV strains assigned to subgroup IA or subgroup IB. Infectious full-genome cDNA clones of CMV-209 were generated under the control of the Cauliflower mosaic virus 35S promoter. Infectivity of the CMV-209 clones was evaluated in Nicotiana benthamiana and various legume species. Our assays revealed that CMV-209 could systemically infect Glycine soja (wild soybean and Pisum sativum (pea as well as N. benthamiana, but not the other legume species.

  7. Toward the gene catalogue of sea urchin development: the construction and analysis of an unfertilized egg cDNA library highly normalized by oligonucleotide fingerprinting.

    Science.gov (United States)

    Poustka, A J; Herwig, R; Krause, A; Hennig, S; Meier-Ewert, S; Lehrach, H

    1999-07-15

    We describe the use of oligonucleotide fingerprinting for the generation of a normalized cDNA library from unfertilized sea urchin eggs and report the preliminary analysis of this library, which resulted in the establishment of a partial gene catalogue of the sea urchin egg. In an analysis of 21,925 cDNA clones by hybridization with 217 oligonucleotide probes, we were able to identify 6291 clusters corresponding to different transcripts, ranging in size from 1 to 265 clones. This corresponds to an average 3.5-fold normalization of the starting library. The normalized library represents about one-third of all genes expressed in the sea urchin egg. To generate sequence information for the transcripts represented by the clusters, representative clones selected from 711 clusters were sequenced. The construction and preliminary analysis of the normalized library are the first steps in the assembly of an increasingly complete collection of maternal genes expressed in the sea urchin egg, which will provide a number of insights into the early development of this well-characterized model organism. Copyright 1999 Academic Press.

  8. cDNA library preparation.

    Science.gov (United States)

    Kooiker, Maarten; Xue, Gang-Ping

    2014-01-01

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

  9. Rescue of the highly virulent classical swine fever virus strain “Koslov” from cloned cDNA and first insights into genome variations relevant for virulence

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Risager, Peter Christian

    2014-01-01

    Classical swine fever virus (CSFV) strain “Koslov” is highly virulent with a mortality rate of up to 100% in pigs. In this study, we modified non-functional cDNAs generated from the blood of Koslov virus infected pigs bysite-directed mutagenesis, removing non-synonymous mutations step-by-step, th......Classical swine fever virus (CSFV) strain “Koslov” is highly virulent with a mortality rate of up to 100% in pigs. In this study, we modified non-functional cDNAs generated from the blood of Koslov virus infected pigs bysite-directed mutagenesis, removing non-synonymous mutations step......-by-step, thereby producing genomes encoding the consensus amino acid sequence. Viruses rescued from the construct corresponding to the inferred parental form were highly virulent, when tested in pigs, with infected animals displaying pronounced clinical symptoms leading to high mortality. The reconstruction...

  10. [Construction and expression of recombinant adeno-associated virus expressing brain-derived neurotrophic factor].

    Science.gov (United States)

    Li, Huiming; Qiu, Wei; Wang, Feng; Wei, Fang; Chen, Xiafang; Wu, Xiaobing; Huang, Qian

    2008-02-01

    A fusion gene called Ig-BDNF, in which brain-derived neurotrophic factor cDNA fused to the 3' end of signal peptide of Ig coding sequence, was constructed by PCR, digested and subcloned into shuttle plasmid pSNAV to obtain a recombinant plasmid pSNAV-Ig-BDNF. Then the plasmid encoding fusion protein was transfected into 293 cell lines and the stably transfected clones were selected with neomycin. AAV1 containing Ig-BDNF fusion gene vectors were obtained by super-infection by Herpes virus. The resultant adeno-associated virus vectors AAV-Ig-BDNF were confirmed by PCR, Western blotting and a sandwich enzyme-linked immunosorbent assay (ELISA) after infection of 293 cell lines. The results indicated that AAV-Ig-BDNF contained the target gene, and infected cells and produced the fusion protein into the supernatant. The content of BDNF in medium per 5x104 cells over a 24 h incubation period reached 1000 pg/mL. With the help of non-replicative adenovirus during AAV-Ig-BDNF infection, the expression of BDNF increased 7-8 fold, and the enhancement of BDNF gene expression was observed in a concentration-dependent manner. These results suggested that a functional AAV-Ig-BDNF was successfully constructed and it offers basis for further study for gene therapy of neural degeneration diseases.

  11. 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.

    Science.gov (United States)

    Tsetsarkin, Konstantin A; Kenney, Heather; Chen, Rubing; Liu, Guangping; Manukyan, Hasmik; Whitehead, Stephen S; Laassri, Majid; Chumakov, Konstantin; Pletnev, Alexander G

    2016-08-23

    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. The availability of genetic tools and laboratory models determines the progress in understanding mechanisms of virus emergence and pathogenesis. Recent large-scale outbreaks of Zika virus (ZIKV) that were linked to complications during perinatal development and Guillain-Barré syndrome in adults emphasize the urgency for the development of a reverse-genetics system based on an epidemic ZIKV strain. Here, we report a stable infectious cDNA clone for ZIKV isolated during the 2015 epidemic in Brazil, as well as a Vero cell

  12. Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

    Science.gov (United States)

    Wei, Shih-Chung; Chuang, Tsung-Liang; Wang, Da-Shin; Lu, Hui-Hsin; Gu, Frank X.; Sung, Kung-Bin; Lin, Chii-Wann

    2015-02-01

    A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004 ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

  13. [Construction of adeno-associated virus vector containing ANG-1 gene and its expression in pig mesenchymal stem cells].

    Science.gov (United States)

    ZHU, Cheng-chu; CHEN, Shi-lin; LIU, Yu-qing; TANG, Li-jiang; BAO, Wei-guang

    2009-07-01

    To construct recombinant adeno-associated virus (rAAV) vector containing angiopoietin-1 (ANG-1) gene and to express the ANG-1 in targeting cells. ANG-1 cDNA was obtained from human spleen by RT-PCR and was inserted into AAV vectors to form rAAV ANG-1, the virus stocks in high titer were harvested. The rAAVANG-1 and rAAV GFP were transferred into pig mesenchymal stem cells and the expression of ANG-1 was detected by Western blot. The cloned ANG-1 cDNA was 1515bp in length which was in accordance with that reported previously. Titration of rAAVANG-1 stock was 9 X 10(11)v.g/ml. The expression of ANG-1 gene was detected in transfected cells. Forty-eight hours after rAAV GFP was transfected into mesenchymal stem cells, 55% cells expressed GFP. The constructed rAAV ANG-1 vector has successfully transfered and expressed in pig mesenchymal stem cells.

  14. A novel approach to propagate flavivirus infectious cDNA clones in bacteria by introducing tandem repeat sequences upstream of virus genome.

    Science.gov (United States)

    Pu, Szu-Yuan; Wu, Ren-Huang; Tsai, Ming-Han; Yang, Chi-Chen; Chang, Chung-Ming; Yueh, Andrew

    2014-07-01

    Despite tremendous efforts to improve the methodology for constructing flavivirus infectious cDNAs, the manipulation of flavivirus cDNAs remains a difficult task in bacteria. Here, we successfully propagated DNA-launched type 2 dengue virus (DENV2) and Japanese encephalitis virus (JEV) infectious cDNAs by introducing seven repeats of the tetracycline-response element (7×TRE) and a minimal cytomegalovirus (CMVmin) promoter upstream of the viral genome. Insertion of the 7×TRE-CMVmin sequence upstream of the DENV2 or JEV genome decreased the cryptic E. coli promoter (ECP) activity of the viral genome in bacteria, as measured using fusion constructs containing DENV2 or JEV segments and the reporter gene Renilla luciferase in an empty vector. The growth kinetics of recombinant viruses derived from DNA-launched DENV2 and JEV infectious cDNAs were similar to those of parental viruses. Similarly, RNA-launched DENV2 infectious cDNAs were generated by inserting 7×TRE-CMVmin, five repeats of the GAL4 upstream activating sequence, or five repeats of BamHI linkers upstream of the DENV2 genome. All three tandem repeat sequences decreased the ECP activity of the DENV2 genome in bacteria. Notably, 7×TRE-CMVmin stabilized RNA-launched JEV infectious cDNAs and reduced the ECP activity of the JEV genome in bacteria. The growth kinetics of recombinant viruses derived from RNA-launched DENV2 and JEV infectious cDNAs displayed patterns similar to those of the parental viruses. These results support a novel methodology for constructing flavivirus infectious cDNAs, which will facilitate research in virology, viral pathogenesis and vaccine development of flaviviruses and other RNA viruses. © 2014 The Authors.

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Science.gov (United States)

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

    2017-10-01

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

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

    Science.gov (United States)

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

    2009-04-01

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

  18. Assessment the Efficiency of the Constructed Minigenome of Rabies Virus using PV Strain as Helper Virus.

    Science.gov (United States)

    Ajorloo, Mehdi; Bamdad, Taravat; Gholami, Ali Reza; Azadmanesh, Keyhan

    2016-05-01

    Rabies is an acute viral disease that causes encephalomyelitis in mammals and human. The only way to prevent this disease is through vaccination before or after exposure. The aim of this study is to evaluate the efficiency of the Pasteur virus (PV) minigenome, using PV strain. Enhanced Green Fluorescent Protein (EGFP) sequence was placed between the designed necessary elements (Hammerhead, HDV ribozyme, 3' Leader, and 5' Trailer sequences), which resemble the rabies virus PV strain (PV2061) genome and anti-genome. These constructs were placed between T7 polymerase promoter and T7 polymerase terminator sequences. The accuracy of the minigenome was confirmed by the expression of EGFP using the helper virus in T7-BHK cell line. The viral necessary elements of positive and negative sense strands were evaluated for the ability of EGFP expression in the presence of the helper virus. While the positive strand showed background results, no EGFP background was observed in the negative strand application. Establishment of minigenome system does not require advanced biosafety levels. Furthermore, using minigenome system eliminates many potential confounding factors that may be present in coding regions of the genome. Use of the minigenome system is easier and more feasible than the full genome rescue of the virus. This study successfully shows the efficiency of the constructed rabies virus minigenome in expression of inserted gene.

  19. Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses.

    Science.gov (United States)

    Lekcharoensuk, Porntippa; Wiriyarat, Witthawat; Petcharat, Nantawan; Lekcharoensuk, Chalermpol; Auewarakul, Prasert; Richt, Juergen A

    2012-02-14

    Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin-Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 2(9) HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Two viral proteins involved in the proteolytic processing of the cowpea mosaic virus polyproteins.

    NARCIS (Netherlands)

    Vos, P.; Verver, J.; Jaegle, M.; Wellink, J.; Kammen, van A.; Goldbach, R.

    1988-01-01

    A series of specific deletion mutants derived from a full-length cDNA clone of cowpea mosaic virus (CPMV) B RNA was constructed with the aim to study the role of viral proteins in the proteolytic processing of the primary translation products. For the same purpose cDNA clones were constructed having

  1. Simplified methods for the construction of RNA and DNA virus infectious clones.

    Science.gov (United States)

    Nagata, Tatsuya; Inoue-Nagata, Alice Kazuko

    2015-01-01

    Infectious virus clones are one of the most powerful tools in plant pathology, molecular biology, and biotechnology. The construction of infectious clones of RNA and DNA viruses, however, usually requires laborious cloning and subcloning steps. In addition, instability of the RNA virus genome is frequently reported after its introduction into the vector and transference to Escherichia coli. These difficulties hamper the cloning procedures, making it tedious and cumbersome. This chapter describes two protocols for a simple construction of infectious viruses, an RNA virus, the tobamovirus Pepper mild mottle virus, and a DNA virus, a bipartite begomovirus. For this purpose, the strategy of overlap-extension PCR was used for the construction of infectious tobamovirus clone and of rolling circle amplification (RCA) for the construction of a dimeric form of the begomovirus clone.

  2. Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties.

    Science.gov (United States)

    Chambers, T J; Nestorowicz, A; Mason, P W; Rice, C M

    1999-04-01

    A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.

  3. Characterization and conservation of the inner E2 core domain structure of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Construction of a cDNA encoding the entire transacylase (E2b) precursor.

    Science.gov (United States)

    Griffin, T A; Lau, K S; Chuang, D T

    1988-10-05

    A cDNA clone encoding the entire transacylase (E2b) precursor of the bovine branched-chain alpha-keto acid dehydrogenase complex has been constructed from two overlapping incomplete cDNA clones which were isolated from a lambda ZAP library prepared from bovine liver poly(A)+ RNA. Nucleotide sequencing indicates that this bovine E2b cDNA insert (bE2-11) is 2701 base pairs in length with an open reading frame of 1446 base pairs. The bE2-11 cDNA insert encodes a leader peptide of 61 residues and a mature E2b polypeptide of 421 amino acid residues with a calculated monomeric molecular mass of 46,518 daltons. The molecular mass of the native E2b component isolated from bovine liver is 1,110,000 daltons as determined by sedimentation equilibrium. This value establishes the 24-subunit octahedral model for the quaternary structure of bovine E2b. The amino-terminal sequences of two tryptic fragments (A and B) of the E2b protein have been determined. Fragment A comprises residues 175 to 421 of the E2b protein and is the inner E2 core domain which contains the transacylase active site. Fragment B, produced by further tryptic cleavage of fragment, comprises residues 205 to 421, but does not have transacylase activity. Both fragments A and B confer the highly assembled 24-mer structure. The primary structure of the inner E2 core domain of bovine E2b (fragment A) is very similar to those of three other E2 proteins (human E2p, Escherichia coli E2p, and E. coli E2k). These similarities suggest that these E2 proteins are structurally and evolutionarily related.

  4. Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies

    DEFF Research Database (Denmark)

    Gottwein, Judith; Scheel, Troels; Callendret, Benoit

    2010-01-01

    Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic...... analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees...... animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes....

  5. Human parainfluenza virus type 3 (HPIV-3); Construction and rescue of an infectious, recombinant virus expressing the enhanced green fluorescent protein (EGFP).

    Science.gov (United States)

    The ability to rescue an infectious, recombinant, RNA virus from a cDNA clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this protocol, the process of inserting enhanced green fluorescent protein (EGFP) gene into the human parainfluenza vi...

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

    Directory of Open Access Journals (Sweden)

    Burgess J Grant

    2009-06-01

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

  7. Position-Specific Gene Expression Analysis Using a Microgram Dissection Method Combined with On-Bead cDNA Library Construction.

    Science.gov (United States)

    Kajiyama, Tomoharu; Fujii, Akihiko; Arikawa, Kouji; Habu, Toru; Mochizuki, Nobuyoshi; Nagatani, Akira; Kambara, Hideki

    2015-07-01

    Gene expression analysis is a key technology that is used to understand living systems. Multicellular organisms, including plants, are composed of various tissues and cell types, each of which exhibits a unique gene expression pattern. However, because of their rigid cell walls, plant cells are difficult to isolate from the whole plant. Although laser dissection has been used to circumvent this problem, the plant sample needs to be fixed beforehand, which presents several problems. In the present study, we developed an alternative method to conduct highly reliable gene expression profiling. First, we assembled a dissection apparatus that used a narrow, sharpened needle to dissect out a microsample of fresh plant tissue (0.1-0.2 mm on each side) automatically from a target site within a short time frame. Then, we optimized a protocol to synthesize a high-quality cDNA library on magnetic beads using a single microsample. The cDNA library was amplified and subjected to high-throughput sequencing. In this way, a stable and reliable system was developed to conduct gene expression profiling in small regions of a plant. The system was used to analyze the gene expression patterns at successive 50 µm intervals in the shoot apex of a 4-day-old Arabidopsis seedling. Clustering analysis of the data demonstrated that two small, adjacent domains, the shoot apical meristem and the leaf primordia, were clearly distinguishable. This system should be broadly applicable in the investigation of the spatial organization of gene expression in various contexts. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Chimeric Hemagglutinin Constructs Induce Broad Protection against Influenza B Virus Challenge in the Mouse Model.

    Science.gov (United States)

    Ermler, Megan E; Kirkpatrick, Ericka; Sun, Weina; Hai, Rong; Amanat, Fatima; Chromikova, Veronika; Palese, Peter; Krammer, Florian

    2017-06-15

    Seasonal influenza virus epidemics represent a significant public health burden. Approximately 25% of all influenza virus infections are caused by type B viruses, and these infections can be severe, especially in children. Current influenza virus vaccines are an effective prophylaxis against infection but are impacted by rapid antigenic drift, which can lead to mismatches between vaccine strains and circulating strains. Here, we describe a broadly protective vaccine candidate based on chimeric hemagglutinins, consisting of globular head domains from exotic influenza A viruses and stalk domains from influenza B viruses. Sequential vaccination with these constructs in mice leads to the induction of broadly reactive antibodies that bind to the conserved stalk domain of influenza B virus hemagglutinin. Vaccinated mice are protected from lethal challenge with diverse influenza B viruses. Results from serum transfer experiments and antibody-dependent cell-mediated cytotoxicity (ADCC) assays indicate that this protection is antibody mediated and based on Fc effector functions. The present data suggest that chimeric hemagglutinin-based vaccination is a viable strategy to broadly protect against influenza B virus infection.IMPORTANCE While current influenza virus vaccines are effective, they are affected by mismatches between vaccine strains and circulating strains. Furthermore, the antiviral drug oseltamivir is less effective for treating influenza B virus infections than for treating influenza A virus infections. A vaccine that induces broad and long-lasting protection against influenza B viruses is therefore urgently needed. Copyright © 2017 American Society for Microbiology.

  9. Construction and characterization of efficient, stable and safe replication-deficient foamy virus vectors

    NARCIS (Netherlands)

    Bastone, P.; Romen, F.; Liu, W.; Wirtz, R.; Koch, U.; Josephson, N.; Langbein, S.; Löchelt, M.

    2007-01-01

    As serious side effects affected recent virus-mediated gene transfer studies, novel vectors with improved safety profiles are urgently needed. In the present study, replication-deficient retroviral vectors based on feline foamy virus (FFV) were constructed and analyzed. The novel FFV vectors are

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2012-11-01

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

  13. Development of transgenic watermelon resistant to Cucumber mosaic virus and Watermelon mosaic virus by using a single chimeric transgene construct.

    Science.gov (United States)

    Lin, Ching-Yi; Ku, Hsin-Mei; Chiang, Yi-Hua; Ho, Hsiu-Yin; Yu, Tsong-Ann; Jan, Fuh-Jyh

    2012-10-01

    Watermelon, an important fruit crop worldwide, is prone to attack by several viruses that often results in destructive yield loss. To develop a transgenic watermelon resistant to multiple virus infection, a single chimeric transgene comprising a silencer DNA from the partial N gene of Watermelon silver mottle virus (WSMoV) fused to the partial coat protein (CP) gene sequences of Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Watermelon mosaic virus (WMV) was constructed and transformed into watermelon (cv. Feeling) via Agrobacterium-mediated transformation. Single or multiple transgene copies randomly inserted into various locations in the genome were confirmed by Southern blot analysis. Transgenic watermelon R(0) plants were individually challenged with CMV, CGMMV or WMV, or with a mixture of these three viruses for resistance evaluation. Two lines were identified to exhibit resistance to CMV, CGMMV, WMV individually, and a mixed inoculation of the three viruses. The R(1) progeny of the two resistant R(0) lines showed resistance to CMV and WMV, but not to CGMMV. Low level accumulation of transgene transcripts in resistant plants and small interfering (si) RNAs specific to CMV and WMV were readily detected in the resistant R(1) plants by northern blot analysis, indicating that the resistance was established via RNA-mediated post-transcriptional gene silencing (PTGS). Loss of the CGMMV CP-transgene fragment in R1 progeny might be the reason for the failure to resistant CGMMV infection, as shown by the absence of a hybridization signal and no detectable siRNA specific to CGMMV in Southern and northern blot analyses. In summary, this study demonstrated that fusion of different viral CP gene fragments in transgenic watermelon contributed to multiple virus resistance via PTGS. The construct and resistant watermelon lines developed in this study could be used in a watermelon breeding program for resistance to multiple viruses.

  14. A cDNA Clone-Launched Platform for High-Yield Production of Inactivated Zika Vaccine

    Directory of Open Access Journals (Sweden)

    Yujiao Yang

    2017-03-01

    Full Text Available A purified inactivated vaccine (PIV using the Zika virus (ZIKV Puerto Rico strain PRVABC59 showed efficacy in monkeys, and is currently in a phase I clinical trial. High-yield manufacture of this PIV is essential for its development and vaccine access. Here we report an infectious cDNA clone-launched platform to maximize its yield. A single NS1 protein substitution (K265E was identified to increase ZIKV replication on Vero cells (a cell line approved for vaccine production for both Cambodian FSS13025 and Puerto Rico PRVABC59 strains. The NS1 mutation did not affect viral RNA synthesis, but significantly increased virion assembly through an increased interaction between NS1 and NS2A (a known regulator of flavivirus assembly. The NS1 mutant virus retained wild-type virulence in the A129 mouse model, but decreased its competence to infect Aedes aegypti mosquitoes. To further increase virus yield, we constructed an infectious cDNA clone of the clinical trial PIV strain PRVABC59 containing three viral replication-enhancing mutations (NS1 K265E, prM H83R, and NS3 S356F. The mutant cDNA clone produced >25-fold more ZIKV than the wild-type parent on Vero cells. This cDNA clone-launched manufacture platform has the advantages of higher virus yield, shortened manufacture time, and minimized chance of contamination.

  15. A cDNA Clone-Launched Platform for High-Yield Production of Inactivated Zika Vaccine.

    Science.gov (United States)

    Yang, Yujiao; Shan, Chao; Zou, Jing; Muruato, Antonio E; Bruno, Diniz Nunes; de Almeida Medeiros Daniele, Barbosa; Vasconcelos, Pedro F C; Rossi, Shannan L; Weaver, Scott C; Xie, Xuping; Shi, Pei-Yong

    2017-03-01

    A purified inactivated vaccine (PIV) using the Zika virus (ZIKV) Puerto Rico strain PRVABC59 showed efficacy in monkeys, and is currently in a phase I clinical trial. High-yield manufacture of this PIV is essential for its development and vaccine access. Here we report an infectious cDNA clone-launched platform to maximize its yield. A single NS1 protein substitution (K265E) was identified to increase ZIKV replication on Vero cells (a cell line approved for vaccine production) for both Cambodian FSS13025 and Puerto Rico PRVABC59 strains. The NS1 mutation did not affect viral RNA synthesis, but significantly increased virion assembly through an increased interaction between NS1 and NS2A (a known regulator of flavivirus assembly). The NS1 mutant virus retained wild-type virulence in the A129 mouse model, but decreased its competence to infect Aedes aegypti mosquitoes. To further increase virus yield, we constructed an infectious cDNA clone of the clinical trial PIV strain PRVABC59 containing three viral replication-enhancing mutations (NS1 K265E, prM H83R, and NS3 S356F). The mutant cDNA clone produced >25-fold more ZIKV than the wild-type parent on Vero cells. This cDNA clone-launched manufacture platform has the advantages of higher virus yield, shortened manufacture time, and minimized chance of contamination. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2015-05-01

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

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

    Lifescience Database Archive (English)

    Full Text Available List Contact us Dicty_cDB cDNA library information Data detail Data name cDNA library information DOI 10.189...s Data item Description cDNA library name Names of cDNA libraries (AF, AH, CF, CH, FC, FC-IC, FCL, SF, SH, S...(C) 5) sexually fusion-competent KAX3 cells (Gamete phase) (F) cDNA library construction method How to construct cDNA library...dir) 2) Full-length cDNA libraries (oligocapped method)(fl) 3) Gamete-specific subtraction library (sub) cDNA library... construction protocol Link to the webpage describing the protocol for generating cDNA library Size

  18. Recovery of avirulent, thermostable Newcastle disease virus strain NDV4-C from cloned cDNA and stable expression of an inserted foreign gene

    NARCIS (Netherlands)

    Zhang, X.; Liu, H.; Liu, P.; Peeters, B.P.H.; Zhao, C.; Kong, X.

    2013-01-01

    A reverse genetics system for thermostable Newcastle disease virus (NDV) is not currently available. In this study, we developed a reverse genetics system for the avirulent and thermostable NDV4-C strain. Successful recovery of NDV4-C was achieved by using either T7 RNA polymerase or cellular RNA

  19. Expression in E. coli of the cloned cDNA for the major antigen of foot ...

    Indian Academy of Sciences (India)

    DNA produced proteins .which cross-reacted with the antibodies generated against the structural proteins of the virus in an enzyme linked immunosorbent assay, indicating that the. cDNA of the major antigen is expressed in the cloned cell. Keywords. Foot and mouth disease virus; cDNA; cloning; antigen; expression. 1.

  20. Evaluation of the Cross-Protective Efficacy of a Chimeric Porcine Reproductive and Respiratory Syndrome Virus Constructed Based on Two Field Strains.

    Science.gov (United States)

    Shabir, Nadeem; Khatun, Amina; Nazki, Salik; Kim, Bumseok; Choi, Eun-Jin; Sun, Dong; Yoon, Kyoung-Jin; Kim, Won-Il

    2016-08-22

    One of the major hurdles to porcine reproductive and respiratory syndrome (PRRS) vaccinology is the limited or no cross-protection conferred by current vaccines. To overcome this challenge, a PRRS chimeric virus (CV) was constructed using an FL12-based cDNA infectious clone in which open reading frames (ORFs) 3-4 and ORFs 5-6 were replaced with the two Korean field isolates K08-1054 and K07-2273,respectively. This virus was evaluated as a vaccine candidate to provide simultaneous protection against two genetically distinct PRRS virus (PRRSV) strains. Thirty PRRS-negative three-week-old pigs were divided into five groups and vaccinated with CV, K08-1054, K07-2273, VR-2332, or a mock inoculum. At 25 days post-vaccination (dpv), the pigs in each group were divided further into two groups and challenged with either K08-1054 or K07-2273. All of the pigs were observed until 42 dpv and were euthanized for pathological evaluation. Overall, the CV-vaccinated group exhibited higher levels of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin-12 (IL-12) expression and of serum virus-neutralizing antibodies compared with the other groups after vaccination and also demonstrated better protection levels against both viruses compared with the challenge control group. Based on these results, it was concluded that CV might be an effective vaccine model that can confer a broader range of cross-protection to various PRRSV strains.

  1. Structural defect linked to nonrandom mutations in the matrix gene of Biden strain subacute sclerosing panencephalitis virus defined by cDNA cloning and expression of chimeric genes

    Energy Technology Data Exchange (ETDEWEB)

    Ayata, M.; Hirano, A.; Wong, T.C.

    1989-03-01

    Biken strain, a nonproductive measles viruslike agent isolated from a subacute sclerosing panencephalitis (SSPE) patient, contains a posttranscriptional defect affecting matrix (M) protein. A putative M protein was translated in vitro with RNA from Biken strain-infected cells. A similar protein was detected in vivo by an antiserum against a peptide synthesized from the cloned M gene of Edmonston strain measles virus. By using a novel method, full-length cDNAs of the Biken M gene were selectively cloned. The cloned Biken M gene contained an open reading frame which encoded 8 extra carboxy-terminal amino acid residues and 20 amino acid substitutions predicted to affect both the hydrophobicity and secondary structure of the gene product. The cloned gene was expressed in vitro and in vivo into a 37,500 M/sub r/ protein electrophoretically and antigenically distinct from the M protein of Edmonston strain but identical to the M protein in Biken strain-infected cells. Chimeric M proteins synthesized in vitro and in vivo showed that the mutations in the carboxy-proximal region altered the local antigenicity and those in the amino region affected the overall protein conformation. The protein expressed from the Biken M gene was unstable in vivo. Instability was attributed to multiple mutations. These results offer insights into the basis of the defect in Biken strain and pose intriguing questions about the evolutionary origins of SSPE viruses in general.

  2. Construction high-yield candidate influenza vaccine viruses in Vero cells by reassortment.

    Science.gov (United States)

    Yu, Wei; Yang, Fan; Yang, Jinghui; Ma, Lei; Cun, Yina; Song, Shaohui; Liao, Guoyang

    2016-11-01

    Usage of influenza vaccine is the best choice measure for preventing and conclusion of influenza virus infection. Although it has been used of chicken embryo to produce influenza vaccine, following with WHO recommended vaccine strain, there were uncontrollable factors and its deficiencies, specially, during an influenza pandemic in the world. The Vero cells are used for vaccine production of a few strains including influenza virus, because of its homology with human, recommended by WHO. However, as known most of the influenza viruses strains could not culture by Vero cells. It was used two high-yield influenza viruses adapted in Vero cells as donor viruses, such as A/Yunnan/1/2005Va (H3N2) and B/Yunnan/2/2005Va (B), to construct high-yield wild influenza virus in Vero cells under antibody selection pressure. After reassortment and passages, it obtained the new Vaccine strains with A/Tianjin/15/2009Va (H1N1), A/Fujian/196/2009Va (H3N2) and B/Chongqing/1384/2010Va (B), which was not only completely keeping their original antigenic (HA and NA), but also grown well in Vero cells with high-yield. All results of gene analysis and HA, HI shown that this reassortment method could be used to find new direction to product the influenza vaccine. J. Med. Virol. 88:1914-1921, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies.

    Science.gov (United States)

    Krammer, Florian; Pica, Natalie; Hai, Rong; Margine, Irina; Palese, Peter

    2013-06-01

    Current influenza virus vaccine strategies stimulate immune responses toward the globular head domain of the hemagglutinin protein in order to inhibit key steps of the virus life cycle. Because this domain is highly variable across strains, new vaccine formulations are required in most years. Here we demonstrate a novel vaccine strategy that generates immunity to the highly conserved stalk domain by using chimeric hemagglutinin constructs that express unique head and stalk combinations. By repeatedly immunizing mice with constructs that expressed the same stalk but an irrelevant head, we specifically stimulated a stalk-directed response that provided broad-based heterologous and heterosubtypic immunity in mice. Notably, our vaccination scheme provides a universal vaccine approach that protects against challenge with an H5 subtype virus. Furthermore, through in vivo studies using passively transferred antibodies or depletion of CD8(+) T cells, we demonstrated the critical role that humoral mechanisms of immunity play in the protection observed. The present data suggest that a vaccine strategy based on the stalk domain of the hemagglutinin protein could be used in humans to broadly protect against a variety of influenza virus subtypes.

  4. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

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

    1987-01-01

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

  5. Construction and immune efficacy of recombinant pseudorabies virus expressing PrM-E proteins of Japanese encephalitis virus genotype І.

    Science.gov (United States)

    Qian, Ping; Zhi, Xianwei; Wang, Bo; Zhang, Huawei; Chen, Huanchun; Li, Xiangmin

    2015-12-10

    Japanese encephalitis (JE) is an arboviral disease with high case fatality rates and neurologic or psychiatric sequelae among survivors in Asia, western Pacific countries and northern Australia. Japanese encephalitis virus (JEV) is the cause of JE and the emergence of genotype І (GI) JEV has displaced genotype III (GIII) as the dominant strains circulating in some Asian regions. The currently available JE vaccines are safe and effective in preventing this disease, but they are developed based on the GIII JEV strains. The recombinant virus PRV TK(-)/gE(-)/PrM-E(+) which expressed the premembrane (prM) and envelope (E) proteins of JEV SX09S-01 strain (genotype I, GI) was constructed by homologous recombination between the genome of PRV TK(-)/gE(-)/LacZ(+) digested with EcoRI and plasmid pIE-CAG-PrM-E-BGH. Expression of JEV PrM and E proteins was analyzed by Western blot analysis. Immune efficacy of PRV TK(-)/gE(-)/PrM-E(+) was further evaluated in mouse model. A recombinant pseudorabies virus (PRV TK(-)/gE(-)/PrM-E(+)) was successfully constructed. Mice experiments showed that PRV TK(-)/gE(-)/PrM-E(+) could induce a high level of ELISA antibodies against PRV and JEV, as well as high titer of PRV neutralizing antibodies. After challenge with 1 × 10(7) PFU virulent JEV SX09S-01 strain, the time of death was delayed and the survival rate was improved in PRV TK(-)/gE(-)/PrM-E(+) vaccinated mice. PRV TK(-)/gE(-)/PrM-E(+) is a potential vaccine candidate against PRV and JEV GI infection in the future.

  6. Development of a dual recombinant vaccine to protect small ruminants against peste-des-petits-ruminants virus and capripoxvirus infections.

    Science.gov (United States)

    Berhe, G; Minet, C; Le Goff, C; Barrett, T; Ngangnou, A; Grillet, C; Libeau, G; Fleming, M; Black, D N; Diallo, A

    2003-01-01

    A recombinant capripoxvirus vaccine containing a cDNA of the peste-des-petits-ruminants virus (PPRV) fusion protein gene was constructed. A quick and efficient method was used to select a highly purified recombinant virus clone. A trial showed that a dose of this recombinant as low as 0.1 PFU protected goats against challenge with a virulent PPRV strain.

  7. Development of a Dual Recombinant Vaccine To Protect Small Ruminants against Peste-des-Petits-Ruminants Virus and Capripoxvirus Infections

    OpenAIRE

    Berhe, G.; Minet, C.; Le Goff, C.; Barrett, T; Ngangnou, A.; Grillet, C.; Libeau, G.; Fleming, M; Black, D. N.; Diallo, A.

    2003-01-01

    A recombinant capripoxvirus vaccine containing a cDNA of the peste-des-petits-ruminants virus (PPRV) fusion protein gene was constructed. A quick and efficient method was used to select a highly purified recombinant virus clone. A trial showed that a dose of this recombinant as low as 0.1 PFU protected goats against challenge with a virulent PPRV strain.

  8. cDNA cloning and sequencing of ostrich Growth hormone

    Directory of Open Access Journals (Sweden)

    Doosti Abbas

    2012-01-01

    Full Text Available In recent years, industrial breeding of ostrich (Struthio camelus has been widely developed in Iran. Growth hormone (GH is a peptide hormone that stimulates growth and cell reproduction in different animals. The aim of this study was to clone and sequence the ostrich growth hormone gene in E. coli, done for the first time in Iran. The cDNA that encodes ostrich growth hormone was isolated from total mRNA of the pituitary gland and amplified by RT-PCR using GH specific PCR primers. Then GH cDNA was cloned by T/A cloning technique and the construct was transformed into E. coli. Finally, GH cDNA sequence was submitted to the GenBank (Accession number: JN559394. The results of present study showed that GH cDNA was successfully cloned in E. coli. Sequencing confirmed that GH cDNA was cloned and that the length of ostrich GH cDNA was 672 bp; BLAST search showed that the sequence of growth hormone cDNA of the ostrich from Iran has 100% homology with other records existing in GenBank.

  9. Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-)

    Energy Technology Data Exchange (ETDEWEB)

    Hirono, A.; Beutler, E. (Research Institute of Scripps Clinic, La Jolla, CA (USA))

    1988-06-01

    Glucose-6-phosphate dehydrogenase A(-) is a common variant in Blacks that causes sensitivity to drug- and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3{prime} end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C{sup 33} {yields} G, G{sup 202} {yields} A, and A{sup 376} {yields} G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The findings of the same mutation in G6PD A(-) as is found in G6PD A(+) strongly suggests that the G6PD A(-) mutation arose in an individual with G6PD A(+), adding another mutation that causes the in vivo instability of this enzyme protein.

  10. Screening of cDNA libraries on glass slide microarrays.

    Science.gov (United States)

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

    2007-01-01

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

  11. Construction and Immunogenicity Evaluation of Recombinant Influenza A Viruses Containing Chimeric Hemagglutinin Genes Derived from Genetically Divergent Influenza A H1N1 Subtype Viruses.

    Directory of Open Access Journals (Sweden)

    Kara McCormick

    Full Text Available Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling technology to create a panel of chimeric HA genes.Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129 was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8LAIV-129 and a A/swine/Texas/4199-2/98 backbone (TX98-129. When delivered to mice, the PR8LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates.This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines.

  12. Construction and Immunogenicity Evaluation of Recombinant Influenza A Viruses Containing Chimeric Hemagglutinin Genes Derived from Genetically Divergent Influenza A H1N1 Subtype Viruses.

    Science.gov (United States)

    McCormick, Kara; Jiang, Zhiyong; Zhu, Longchao; Lawson, Steven R; Langenhorst, Robert; Ransburgh, Russell; Brunick, Colin; Tracy, Miranda C; Hurtig, Heather R; Mabee, Leah M; Mingo, Mark; Li, Yanhua; Webby, Richard J; Huber, Victor C; Fang, Ying

    2015-01-01

    Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes. Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8LAIV-129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates. This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines.

  13. Cassava cDNA Library Construction: One Tool for Biotechnological Development of the Crop CONSTRUCCIÓN DE UNA LIBRERÍA DE ADNc EN YUCA: UNA HERRAMIENTA PARA EL DESARROLLO BIOTECNOLÓGICO DEL CULTIVO

    Directory of Open Access Journals (Sweden)

    CAROLINA GONZÁLEZ ALMARIO

    el estudio de su función a través de la identificación y la interacción entre proteínas.Cassava is one of the most important crops for global food security and provides food and livelihood for 600 million people in the developing world. It is also good source of starch, with levels between 73.7 y 84.9% of total dry weight in roots (FAO, 2007. Cassava starch can be used in a wide range of industries (textile, cosmetic, nourishing, etc and it has a high potential for the production of biofuel. Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam, is one of the most important diseases that affects cassava. This disease can compromise the starch supply not only for bioetanol production but also affect global food security. The long reproductive cycle, high heterozigosity and tetraploid character of cassava are characteristics that have complicated the genetic breeding for this crop. For these reasons new alternatives based on biotechnology are necessary to accelerate its improvement. In the postgenomic era many experiments rely on the availability of transcript sequences for cloning. As these clones usually originate from cDNA libraries, the quality of these libraries is crucial. In this article we report the construction of the first cassava cDNA library employing the Gateway® system. For this, in vitro grown plants were inoculated with the Xam strain CIO151. The expression library shows a high titer of 1 x 10(7 cfu/ml, with inserts ranging between 600 and 1500 bp. The sequence analyses from 14 random clones confirmed that these are expressed genes and showed similarity with previously cloned genes from species related to cassava. This library is an excellent resource for the identification of novel genes and for functional studies through the identification of their interactions with other proteins.

  14. A cryptic promoter in potato virus X vector interrupted plasmid construction

    Directory of Open Access Journals (Sweden)

    Schultz Ronald D

    2007-03-01

    Full Text Available Abstract Background Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2 VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation. Results A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR. Conclusion It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli can interrupt the downstream work.

  15. Construction and characterisation of a complete reverse genetics system of dengue virus type 3

    Directory of Open Access Journals (Sweden)

    Jefferson Jose da Silva Santos

    2013-12-01

    Full Text Available Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

  16. Protective Effect of Different Anti-Rabies Virus VHH Constructs against Rabies Disease in Mice

    Science.gov (United States)

    Terryn, Sanne; Francart, Aurélie; Lamoral, Sophie; Hultberg, Anna; Rommelaere, Heidi; Wittelsberger, Angela; Callewaert, Filip; Stohr, Thomas; Meerschaert, Kris; Ottevaere, Ingrid; Stortelers, Catelijne; Vanlandschoot, Peter; Kalai, Michael; Van Gucht, Steven

    2014-01-01

    Rabies virus causes lethal brain infection in about 61000 people per year. Each year, tens of thousands of people receive anti-rabies prophylaxis with plasma-derived immunoglobulins and vaccine soon after exposure. Anti-rabies immunoglobulins are however expensive and have limited availability. VHH are the smallest antigen-binding functional fragments of camelid heavy chain antibodies, also called Nanobodies. The therapeutic potential of anti-rabies VHH was examined in a mouse model using intranasal challenge with a lethal dose of rabies virus. Anti-rabies VHH were administered directly into the brain or systemically, by intraperitoneal injection, 24 hours after virus challenge. Anti-rabies VHH were able to significantly prolong survival or even completely rescue mice from disease. The therapeutic effect depended on the dose, affinity and brain and plasma half-life of the VHH construct. Increasing the affinity by combining two VHH with a glycine-serine linker into bivalent or biparatopic constructs, increased the neutralizing potency to the picomolar range. Upon direct intracerebral administration, a dose as low as 33 µg of the biparatopic Rab-E8/H7 was still able to establish an anti-rabies effect. The effect of systemic treatment was significantly improved by increasing the half-life of Rab-E8/H7 through linkage with a third VHH targeted against albumin. Intraperitoneal treatment with 1.5 mg (2505 IU, 1 ml) of anti-albumin Rab-E8/H7 prolonged the median survival time from 9 to 15 days and completely rescued 43% of mice. For comparison, intraperitoneal treatment with the highest available dose of human anti-rabies immunoglobulins (65 mg, 111 IU, 1 ml) only prolonged survival by 2 days, without rescue. Overall, the therapeutic benefit seemed well correlated with the time of brain exposure and the plasma half-life of the used VHH construct. These results, together with the ease-of-production and superior thermal stability, render anti-rabies VHH into valuable

  17. Brief report of the construction of infectious DNA clones of South African genetic variants of grapevine virus A and grapevine virus B.

    Science.gov (United States)

    Goszczynski, D E

    2015-01-01

    Recent research results strongly suggest that certain genetic variants of grapevine virus A (GVA) and grapevine virus B (GVB), two members of the Vitivirus genus of the family Betaflexiviridae, are the cause of Shiraz disease and corky bark disease of grapevines in South Africa, respectively. To investigate this hypothesis, work was undertaken to construct DNA clones of these viruses. Biologically viable and stable DNA clones of genetic variants of GVA and GVB B from South Africa were constructed. The clones share 76.3, 73.2 and 85.2, 77.6 % nt sequence similarity with corresponding clones constructed in Italy and Israel. The results suggest that a derivative of a mini binary vector pCB302 is superior to pCAMBIA1305.1 for the construction of infectious and stable DNA clones of vitiviruses. Successful construction of such DNA clones of GVA and GVB reported in this study is a clear step towards fulfilling Koch's 3rd postulate in investigating the aetiology of Shiraz disease and corky bark disease.

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

    Science.gov (United States)

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

    1984-12-10

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

  19. Construction and characterization of an infectious DNA clone and of mutants of simian immunodeficiency virus isolated from the African green monkey.

    OpenAIRE

    Shibata, R; Miura, T; Hayami, M; Sakai, H.; Ogawa, K; Kiyomasu, T; Ishimoto, A; Adachi, A

    1990-01-01

    We constructed a full-length molecular clone of simian immunodeficiency virus from an African green monkey. Upon transfection, this clone directed the production of virus particles cytopathic and infectious to human CD4+ leukemia cell lines. Mutations were introduced by recombinant DNA techniques into eight open reading frames of simian immunodeficiency virus from the African green monkey thus far identified. The phenotypes of mutant viruses, i.e., infectivity, cytopathogenicity, transactivat...

  20. Construction of stabilized and tagged foot-and-mouth disease virus.

    Science.gov (United States)

    Park, Jeong-Nam; Ko, Mi-Kyeong; Kim, Rae-Hyung; Park, Min-Eun; Lee, Seo-Yong; Yoon, Ji-Eun; Choi, Joo-Hyung; You, Su-Hwa; Park, Jung-Won; Lee, Kwang-Nyeong; Chun, Ji-Eun; Kim, Su-Mi; Tark, Dongseob; Lee, Hyang-Sim; Ko, Young-Joon; Kim, Byounghan; Lee, Myoung-Heon; Park, Jong-Hyeon

    2016-11-01

    Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease that affects cloven-hoofed animals worldwide. Construction and purification of stable antigen for vaccine are necessary but technically difficult and laborious. Here, we have tried to investigate an alternative method by inserting a hexa-histidine tag (6xHIS) in the VP1 C-terminal for easy purification and replacing two amino acids of VP1/VP2 to enhance the stability of the capsid of the FMD virus (FMDV) Asia1/MOG/05. In addition, infectious 6xHIS-tagged stable (S/T) FMDVs were maintained under acidic conditions (pH 6.0) and were readily purified from small-scale cultures using a commercial metal-affinity column. The groups vaccinated with the S/T FMDV antigen showed complete protection comparing to low survival rate in the group vaccinated with non-S/T FMDV against lethal challenge with Asia1 Shamir in mice. Therefore, the present findings indicate that the stabilized and tagged antigen offers an alternative to using the current methods for antigen purification and enhancement of stability and has potential for the development of a new FMD vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Recovery of the Zika virus through an in vitro ligation approach.

    Science.gov (United States)

    Deng, Cheng-Lin; Zhang, Qiu-Yan; Chen, Dong-Dong; Liu, Si-Qing; Qin, Cheng-Feng; Zhang, Bo; Ye, Han-Qing

    2017-07-01

    In this study, an in vitro ligation method was developed to assemble a full-length infectious cDNA clone of the Zika virus (ZIKV). Four contiguous cDNA subclones covering the complete ZIKV genome were constructed with unique BglI restriction sites at the ends of each fragment. The BglI restriction sites only allow in vitro ligation to happen between interconnecting restriction sites from adjacent cDNA fragments, resulting in an intact full-length cDNA of ZIKV. RNA transcripts derived from the full-length cDNA were infectious. The recombinant virus replicated as efficiently as the wild-type virus with similar growth kinetics and plaque morphologies in Vero and C6/36 cells. Both viruses were inhibited by NITD008 treatment. This in vitro ligation method will facilitate manipulation of the viral genome through genetic modifications of four separated subclones of ZIKV for the rapid and rational development of candidate vaccines and viral replication study.

  2. Epitope mapping of Ebola virus dominant and subdominant glycoprotein epitopes facilitates construction of an epitope-based DNA vaccine able to focus the antibody response in mice

    Science.gov (United States)

    2017-04-06

    Epitope mapping of Ebola virus dominant and subdominant glycoprotein epitopes facilitates construction of an epitope-based DNA vaccine able to focus... vaccinated against or infected with EBOV. Using the information obtained along with structural modeling to predict epitope accessibility, we then...constructed two DNA vaccines encoding immunodominant and subdominant epitopes predicted to be accessible on EBOV GP. Although a construct designed to

  3. Linear programming model to construct phylogenetic network for 16S rRNA sequences of photosynthetic organisms and influenza viruses.

    Science.gov (United States)

    Mathur, Rinku; Adlakha, Neeru

    2014-06-01

    Phylogenetic trees give the information about the vertical relationships of ancestors and descendants but phylogenetic networks are used to visualize the horizontal relationships among the different organisms. In order to predict reticulate events there is a need to construct phylogenetic networks. Here, a Linear Programming (LP) model has been developed for the construction of phylogenetic network. The model is validated by using data sets of chloroplast of 16S rRNA sequences of photosynthetic organisms and Influenza A/H5N1 viruses. Results obtained are in agreement with those obtained by earlier researchers.

  4. Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein

    Directory of Open Access Journals (Sweden)

    Zanotto Carlo

    2011-11-01

    Full Text Available Abstract Background Human papilloma virus (HPV-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1 have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species. Methods A new fowlpox virus recombinant encoding HPV-L1 (FPL1 was engineered and evaluated for the correct expression of HPV-L1 in vitro, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays. Results The FPL1 recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector. Conclusion This FPL1 recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.

  5. Construction

    Science.gov (United States)

    2002-01-01

    San Juan, PR Tren Urbano Subway Project, San Juan, PR U.S. Army South, San Juan, PR U.S. Coast Guard Housing Project, San Juan, PR U.S. Coast Guard...construction?, Foresight/CRISP Workshop on Nanotechnology, Royal Society of Arts . Cheltenham, England: 2001, p.5. 56 Concrete Proposals, Economist, July 24

  6. A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9

    Directory of Open Access Journals (Sweden)

    Ming Yuan

    Full Text Available The current method for creation of vaccinia virus (VACV vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (∼90% in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application.

  7. Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV.

    Directory of Open Access Journals (Sweden)

    Benjamin Lamp

    Full Text Available European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV, a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch's postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.

  8. An Analysis of the Effectiveness of a Constructive Induction-Based Virus Detection Prototype

    National Research Council Canada - National Science Library

    Damp, Kevin

    2000-01-01

    Computer viruses remain a tangible threat to systems both within the Department of Defense and throughout the greater international data communications infrastructure on which the DoD increasingly depends...

  9. Horizon scanning for emergence of new viruses: from constructing complex scenarios to online games.

    Science.gov (United States)

    Gale, P; Breed, A C

    2013-10-01

    Horizon scanning techniques can be developed to identify novel routes and sources for the emergence of viruses in the medium to long term. Central to horizon scanning is prediction of the complex scenarios through which viruses could emerge before they occur. One approach involves 'spidergrams' in which complex scenarios are generated by combining factors randomly selected from different categories of events. Spidergrams provide a framework for how different factors could interact, irrespective of the virus, and also enable testing of combinations not previously considered but which would be 'tested' in nature by a virus. The emergence of viruses through new routes is often related to changes, for example, in environmental and social factors, and the Internet will undoubtedly be used to identify long-term trends for consideration. In addition, online games may provide horizon scanners with suggestions for new routes and strategies that could be used by emerging viruses. © 2012 Crown copyright Reproduced with the permission of the Controller of Her Majesty's Stationery Office and Animal Health and Veterinary Laboratories Agency.

  10. Exploring the limits of vector construction based on Citrus tristeza virus.

    Science.gov (United States)

    El-Mohtar, Choaa; Dawson, William O

    2014-01-05

    We examined the limits of manipulation of the Citrus tristeza virus (CTV) genome for expressing foreign genes in plants. We previously created a vector with a foreign gene cassette inserted between the major and minor coat protein genes, which is position 6 from the 3' terminus. Yet, this virus has 10 3'-genes with several other potential locations for expression of foreign genes. Since genes positioned closer to the 3' terminus tend to be expressed in greater amounts, there were opportunities for producing greater amounts of foreign protein. We found that the virus tolerated insertions of an extra gene in most positions within the 3' region of the genome with substantially increased levels of gene product produced throughout citrus trees. CTV was amazingly tolerant to manipulation resulting in a suite of stable transient expression vectors, each with advantages for specific uses and sizes of foreign genes in citrus trees. © 2013 Elsevier Inc. All rights reserved.

  11. Construction of an infectious clone of simian foamy virus of Japanese macaque (SFVjm) and phylogenetic analyses of SFVjm isolates.

    Science.gov (United States)

    Yoshikawa, Rokusuke; Nakagawa, So; Okamoto, Munehiro; Miyazawa, Takayuki

    2014-09-10

    Foamy viruses belong to the genus Spumavirus of the family Retroviridae and have been isolated from many mammalian species. It was reported that simian foamy viruses (SFVs) have co-evolved with host species. In this study, we isolated four strains (WK1, WK2, AR1 and AR2) of SFV (named SFVjm) from Japanese macaques (Macaca fuscata) in main island Honshu of Japan. We constructed an infectious molecular clone of SFVjm strain WK1, termed pJM356. The virus derived from the clone replicated and induced syncytia in human (human embryonic kidney 293T cells), African green monkey (Vero cells) and mouse cell lines (Mus dunni tail fibroblast cells). Phylogenetic analysis also revealed that these four SFVjm strains formed two distinct SFVjm clusters. SFVjm strains WK1 and WK2 and SFV isolated from Taiwanese macaques (Macaca cyclopis) formed one cluster, whereas strains AR1 and AR2 formed the other cluster with SFV isolated from a rhesus macaque (Macaca mulatta). Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Optimization the expression of human papilloma virus E6 and E7 polytopic construct in E. coli expression system

    Directory of Open Access Journals (Sweden)

    Arian Rahimi

    2015-12-01

    Full Text Available Background: Human papilloma virus is a DNA virus from the papillomavirus family that is most prevalent in human cervical cancers and many studies showed the E6 and E7 proteins are present in the majority of cervical cancer cases. Development of universal HPV peptide-based vaccine with more serotypes coverage has considerable value. The aim of the study was to design a multi-epitope universal vaccine for major HPV based on E6 and E7 proteins and optimization the expression of polytopic construct contains E6 and E7 genes from different genotypes of human papilloma virus as a candid vaccine. Methods: In this experimental study that was carried out in Pasteur Institute of Iran, Virology Department from October 2013 to November 2014. In order to design the polytypic construct, we predicted the most probable immunogenic epitopes of E6 and E7 from common high risk HPV16, 18, 31, 45 along with high prevalent type 6 and 11 using bioinformatics methods. The synthetic pET28a expression vector harboring E6 and E7 protein was transformed into Escherichia coli hosts and its expression was analyzed by SDS-PAGE and western blotting. Finally, in order to expression optimization of recombinant protein, cell density, induction time, growth temperature, IPTG (Isopropyl β-D-1-thiogalactopyranoside concentration and cultures media were studied. Results: In the present study the recombinant fusion protein was expressed successfully and the highest expression of target protein was achieved in super broth medium containing 0.1% glucose and 0.2% L-arabinose. In Super broth medium, the optimum condition for recombinant protein expression was occurred at OD600 of 0.8, 0.1mM IPTG, one hour’s incubation time at 37 °C and BL21 (A1 host. Conclusion: The results of this study show that the optimum expression of E6 and E7 proteins from different genotypes of human papilloma virus can be performed. Moreover, by purification of recombinant protein and evaluation of its

  13. Cloning and Expression of cDNA for Rat Heme Oxygenase

    Science.gov (United States)

    Shibahara, Shigeki; Muller, Rita; Taguchi, Hayao; Yoshida, Tadashi

    1985-12-01

    Two cDNA clones for rat heme oxygenase have been isolated from a rat spleen cDNA library in λ gt11 by immunological screening using a specific polyclonal antibody. One of these clones has an insert of 1530 nucleotides that contains the entire protein-coding region. To confirm that the isolated cDNA encodes heme oxygenase, we transfected monkey kidney cells (COS-7) with the cDNA carried in a simian virus 40 vector. The heme oxygenase was highly expressed in endoplasmic reticulum of transfected cells. The nucleotide sequence of the cloned cDNA was determined and the primary structure of heme oxygenase was deduced. Heme oxygenase is composed of 289 amino acids and has one hydrophobic segment at its carboxyl terminus, which is probably important for the insertion of heme oxygenase into endoplasmic reticulum. The cloned cDNA was used to analyze the induction of heme oxygenase in rat liver by treatment with CoCl2 or with hemin. RNA blot analysis showed that both CoCl2 and hemin increased the amount of hybridizable mRNA, suggesting that these substances may act at the transcriptional level to increase the amount of heme oxygenase.

  14. Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

    Science.gov (United States)

    2012-12-12

    and DOBV strain Dobrava (25) were propagated in Vero E6 cells ( Vero C1008; ATCC CRL 1586). Transient-expression experiments were performed with COS...N-specific anti- bodies. PRNT. Plaque-reduction neutralization tests (PRNT) were performed using Vero E6 cells as previously described (15). HTNV...Unexpectedly, vaccination protected hamsters in a lethal disease model of Andes virus (ANDV) in the absence of ANDV cross- neutralizing antibodies. This

  15. [Construction of dengue virus-specific full-length fully human antibody libraries by mammalian display technology].

    Science.gov (United States)

    Wen, Yangming; Lan, Kaijian; Wang, Junjie; Yu, Jingyi; Qu, Yarong; Zhao, Wei; Zhang, Fuchun; Tan, Wanlong; Cao, Hong; Zhou, Chen

    2013-06-01

    To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry. Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)]. Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

  16. Establishment of Self-incompatibility Gene cDNA Microarray to Identify S-genotypes of Pyrus pyrifolia

    Directory of Open Access Journals (Sweden)

    Jiang Nan

    2015-11-01

    Full Text Available Based on the cDNA sequences from hyper variable (HV regions of identified 52 S-alleles in Oriental pear cultivars, S-RNase cDNA probes were designed, and a cDNA microarray for S-RNase detections was established. Each microarray contained 240 sites from 55 cDNA probes, including all specific cDNA sequences from the HV regions of the S-alleles. Using the cDNA of pistils of tested pear cultivars as template and Cy3 fluorescently labeling primers by PCR amplification, microarray hybridization detected the S-genotype of each pear cultivar. The genotypes inferred from the cDNA microarray hybridization signals of pear cultivars such as ‘Lijiang Huangsuanli’, ‘Xiuyu’, ‘Midu Yuli’, ‘Baimianli’, and ‘Deshengxiang’ were similar to the known genotypes of all tested cultivars. The S-RNase cDNA microarrays and the oligonucleotide gene chips were then used to conduct parallel testing of 24 P. pyrifolia cultivars with unknown S-genotypes. In conclusion, the construction of cDNA microarrays has further improved the pear S-RNase detection platform.

  17. [Construction and identification of recombinant avian adeno-associated virus expressing GFP reporter gene].

    Science.gov (United States)

    Wang, An-ping; Sun, Huai-chang; Wang, Jian-ye; Wang, Yong-juan; Yuan, Wei-feng

    2007-07-01

    To generate recombinant avian adeno-associated virus (rAAAV) for gene transfer studies in avian cells, the recombinant plasmid containing the whole genome of AAAV was digested with restriction enzymes to remove the Rep and Cap genes, resulting in AAAV transfer vector pAITR. GFP-expressing cassette was amplified by PCR and inserted into the AAAV transfer vector. The Rep-Cap gene of AAAV amplified by high fidelity PCR was subcloned into eukaryotic expression vector pcDNA3, resulting in an AAAV helper vector pcDNA-ARC. The Rep and Cap genes amplified by high fidelity PCR were subcloned separately into the co-expression vector pVITRO2-mcs, resulting in another AAAV helper vector pVITRO2-ARC. Using calcium phosphate precipitation method, rAAAV-GFP was generated by co-transfecting AAV-293 cells with a cocktail of pAITR-GFP, pcDNA-ARC or pVITRO2-ARC, and adenovirus helper vector pHelper. The three structural proteins VP1, VP2 and VP3 of correct molecular masses were detected by SDS-PAGE and the GFP reporter gene was detected by PCR in purified rAAAV-GFP virions. Chicken embryonic fibroblast (CEF) cells and CEL cell line were transduced with the recombinant virus, the GFP-positive cells were easily observed under fluorescent microscope, expression of which lasted for at least two weeks. These data demonstrate that an efficient helper virus-free packaging system has been established for generating recombinant AAAV particles for gene transfer studies in avian cells and for development of recombinant vaccines against avian diseases.

  18. Infection with strains of Citrus tristeza virus does not exclude superinfection by other strains of the virus.

    Science.gov (United States)

    Folimonova, Svetlana Y; Robertson, Cecile J; Shilts, Turksen; Folimonov, Alexey S; Hilf, Mark E; Garnsey, Stephen M; Dawson, William O

    2010-02-01

    Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were "strains" of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most

  19. Construction of a fusion gene containing hepatitis B virus L gene ...

    African Journals Online (AJOL)

    The results of SDS-PAGE and Western blot showed that the recombinant protein was induced by methanol and stably expressed in P. pastoris, while it has specific reaction with the serum containing anti-HbsAg or anti-Ag85B. However, the successful construction of a recombinant yeast expression vector containing gene ...

  20. Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus.

    Science.gov (United States)

    Clarke, Jihong Liu; Spetz, Carl; Haugslien, Sissel; Xing, Shaochen; Dees, Merete W; Moe, Roar; Blystad, Dag-Ragnar

    2008-06-01

    Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.

  1. Construction of a bivalent DNA vaccine co-expressing S genes of transmissible gastroenteritis virus and porcine epidemic diarrhea virus delivered by attenuated Salmonella typhimurium.

    Science.gov (United States)

    Zhang, Yudi; Zhang, Xiaohui; Liao, Xiaodan; Huang, Xiaobo; Cao, Sanjie; Wen, Xintian; Wen, Yiping; Wu, Rui; Liu, Wumei

    2016-06-01

    Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) can cause severe diarrhea in newborn piglets and led to significant economic losses. The S proteins are the main structural proteins of PEDV and TGEV capable of inducing neutralizing antibodies in vivo. In this study, a DNA vaccine SL7207 (pVAXD-PS1-TS) co-expressing S proteins of TGEV and PEDV delivered by attenuated Salmonella typhimurium was constructed and its immunogenicity in piglets was investigated. Twenty-day-old piglets were orally immunized with SL7207 (pVAXD-PS1-TS) at a dosage of 1.6 × 10(11) CFU per piglet and then booster immunized with 2.0 × 10(11) CFU after 2 weeks. Humoral immune responses, as reflected by virus neutralizing antibodies and specific IgG and sIgA, and cellular immune responses, as reflected by IFN-γ, IL-4, and lymphocyte proliferation, were evaluated. SL7207 (pVAXD-PS1-TS) simultaneously elicited immune responses against TGEV and PEDV after oral immunization. The immune levels started to increase at 2 weeks after immunization and increased to levels statistically significantly different than controls at 4 weeks post-immunization, peaking at 6 weeks and declined at 8 weeks. The humoral, mucosal, and cellular immune responses induced by SL7207 (pAXD-PS1-TS) were significantly higher than those of the PBS and SL7207 (pVAXD) (p < 0.01). In particular, the levels of IFN-γ and IL-4 were higher than those induced by the single-gene vaccine SL7207 (pVAXD-PS1) (p < 0.05). These results demonstrated that SL7207 (pVAXD-PS1-TS) possess the immunological functions of the two S proteins of TGEV and PEDV, indicating that SL7207 (pVAXD-PS1-TS) is a candidate oral vaccine for TGE and PED.

  2. VIRUSES

    Indian Academy of Sciences (India)

    and-mouth disease in livestock was an infectious particle smaller than any bacteria. This was the first clue to the nature of viruses, genetic entities that lie somewhere in the gray area between living and non-living states.

  3. Replication of a chronic hepatitis B virus genotype F1b construct.

    Science.gov (United States)

    Hernández, Sergio; Jiménez, Gustavo; Alarcón, Valentina; Prieto, Cristian; Muñoz, Francisca; Riquelme, Constanza; Venegas, Mauricio; Brahm, Javier; Loyola, Alejandra; Villanueva, Rodrigo A

    2016-03-01

    Genotype F is one of the less-studied genotypes of human hepatitis B virus, although it is widely distributed in regions of Central and South American. Our previous studies have shown that HBV genotype F is prevalent in Chile, and phylogenetic analysis of its full-length sequence amplified from the sera of chronically infected patients identified it as HBV subgenotype F1b. We have previously reported the full-length sequence of a HBV molecular clone obtained from a patient chronically infected with genotype F1b. In this report, we established a system to study HBV replication based on hepatoma cell lines transfected with full-length monomers of the HBV genome. Culture supernatants were analyzed after transfection and found to contain both HBsAg and HBeAg viral antigens. Consistently, fractionated cell extracts revealed the presence of viral replication, with both cytoplasmic and nuclear DNA intermediates. Analysis of HBV-transfected cells by indirect immunofluorescence or immunoelectron microscopy revealed the expression of viral antigens and cytoplasmic viral particles, respectively. To test the functionality of the ongoing viral replication further at the level of chromatinized cccDNA, transfected cells were treated with a histone deacetylase inhibitor, and this resulted in increased viral replication. This correlated with changes posttranslational modifications of histones at viral promoters. Thus, the development of this viral replication system for HBV genotype F will facilitate studies on the regulation of viral replication and the identification of new antiviral drugs.

  4. Cloning a cDNA for the lysosomal alpha-glucosidase

    NARCIS (Netherlands)

    KONINGS, A.; HUPKES, P.; Versteeg, R.; Grosveld, G.; Reuser, A.; Galjaard, H.

    1984-01-01

    Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most

  5. Isolation of an ATP synthase cDNA from Sinonovacula constricta ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... 2Ningbo City College of Vocational Technology, Ningbo, 315100 People's Republic of China. 3National Marine Environmental Monitoring Center. Dalian, 116023, People's ... The SMART cDNA library of S. constricta was constructed by our laboratory. Random sequencing of the library using T3 primer.

  6. Construction of Recombinant HVT Expressing PmpD, and Immunological Evaluation against Chlamydia psittaci and Marek's Disease Virus.

    Directory of Open Access Journals (Sweden)

    Shanshan Liu

    Full Text Available Chlamydia psittaci (C. psittaci is an obligate intracellular zoonotic pathogen that can be transmitted to humans from birds. No efficacious commercial vaccine is available for clearing chlamydial infection due to lack of potential vaccine candidates and effective delivery vehicles. Herpesvirus of turkeys (HVT is an efficacious commercially available vaccine against Marek's Disease virus (MDV. In this study, a recombinant HVT-delivered vaccine against C. psittaci and Marek's disease was developed and examined. The 5'-terminus of pmpD gene (pmpD-N encoding the N-terminal fragment of polymorphic membrane protein D of C. psittaci was inserted into a nonessential region of HVT genome using reverse genetics based on an infectious bacterial artificial chromosome (BAC clone of HVT. The recombinant virus (rHVT-pmpD-N was recovered from primary chicken embryo fibroblast (CEF cells by transfection of modified HVT BAC DNA containing the pmpD-N gene. The rHVT-pmpD-N construct was confirmed to express PmpD-N by immunoblot and immunofluorescence. The rHVT-pmpD-N was stable during 20 passages in vitro. The growth kinetics of rHVT-pmpD-N was comparable to that of parental HVT in vitro and in vivo. One-day-old SPF chickens inoculated subcutaneously with rHVT-pmpD-N displayed increased PmpD-specific antibody levels and a vigorous PmpD-specific lymphocyte proliferation response using HVT vector or CEF cells as control. Furthermore, the percentage of CD4+ cells was significantly elevated in rHVT-pmpD-N-immunized birds as compared to the parental HVT. All chickens vaccinated with rHVT-pmpD-N or parental HVT were protected completely against challenge with a very virulent strain of Marek's Disease virus (MDV RB-1B. Post challenge with C. psittaci CB7 strain, a significant decrease in respiratory distress, lesions and Chlamydia load was found in the rHVT-pmpD-N-vaccinated group compared to the parental HVT. In conclusion, our study suggests that the rHVT-pmpD-N live

  7. Characterization of a cDNA encoding cottonseed catalase.

    Science.gov (United States)

    Ni, W; Turley, R B; Trelease, R N

    1990-06-21

    A 1.7 kb cDNA clone was isolated from our lambda gt11 library constructed from poly(A) RNA of 24-h-old cotyledons. The cDNA encodes a full-length catalase peptide (492 amino acid residues). The calculated molecular mass is 56,800, similar to that determined for purified enzyme (57,000 SDS-PAGE). Among higher plant catalases, this cotton catalase shows the highest amino acid sequence identity (85%) to the subunit of homotetrameric maize CAT 1, a developmental counterpart to the homotetrameric CAT A isoform of cotton seeds. Comparison of sequences from cotton, sweet potato, maize CAT 1, and yeast with bovine catalase revealed that the amino acid residues and regions that are involved in catalytic activity and/or required to maintain basic catalase structure, are highly conserved. The C-terminus region, which has the lowest nucleotide sequence identity between plant and mammalian catalases, does not terminate with a tripeptide, S-K/R/H-L, a putative targeting signal for peroxisomal proteins.

  8. Construction of a doxycycline-dependent simian immunodeficiency virus reveals a nontranscriptional function of tat in viral replication

    NARCIS (Netherlands)

    Das, Atze T.; Klaver, Bep; Harwig, Alex; Vink, Monique; Ooms, Marcel; Centlivre, Mireille; Berkhout, Ben

    2007-01-01

    In the quest for an effective vaccine against human immunodeficiency virus (HIV), live attenuated virus vaccines have proven to be very effective in the experimental model system of simian immunodeficiency virus (SIV) in macaques. However, live attenuated HIV vaccines are considered unsafe for use

  9. Design and generation of recombinant rabies virus vectors

    Science.gov (United States)

    Osakada, Fumitaka; Callaway, Edward M.

    2014-01-01

    Rabies viruses, negative-strand RNA viruses, infect neurons through axon terminals and spread transsynaptically in a retrograde direction between neurons. Rabies viruses whose glycoprotein (G) gene is deleted from the genome cannot spread across synapses. Complementation of G in trans, however, enables transsynaptic spreading of G-deleted rabies viruses to directly-connected, presynaptic neurons. Recombinant rabies viruses can encode genes of interest for labeling cells, controlling gene expression, and monitoring or manipulating neural activity. Cre-dependent or bridge-protein-mediated transduction and single-cell electroporation via EnvA/TVA or EnvB/TVB system allow cell-type-specific or single-cell-specific targeting. These rabies virus-based approaches permit the linking of connectivity to cell morphology and circuit function for particular cell types or single cells. Here we describe methods for construction of rabies viral vectors, recovery of G-deleted rabies viruses from cDNA, amplification of the viruses, pseudotyping them with EnvA or EnvB, and concentration and titration of the viruses. The entire protocol takes 6–8 weeks. PMID:23887178

  10. Efficacy Testing of H56 cDNA Tattoo Immunization against Tuberculosis in a Mouse Model.

    Science.gov (United States)

    Platteel, Anouk C M; Nieuwenhuizen, Natalie E; Domaszewska, Teresa; Schürer, Stefanie; Zedler, Ulrike; Brinkmann, Volker; Sijts, Alice J A M; Kaufmann, Stefan H E

    2017-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), remains a global threat. The only approved vaccine against TB, Mycobacterium bovis bacillus Calmette-Guérin (BCG), provides insufficient protection and, being a live vaccine, can cause disseminated disease in immunocompromised individuals. Previously, we found that intradermal cDNA tattoo immunization with cDNA of tetanus toxoid fragment C domain 1 fused to cDNA of the fusion protein H56, comprising the Mtb antigens Ag85B, ESAT-6, and Rv2660c, induced antigen-specific CD8 + T cell responses in vivo . As cDNA tattoo immunization would be safer than a live vaccine in immunocompromised patients, we tested the protective efficacy of intradermal tattoo immunization against TB with H56 cDNA, as well as with H56_E, a construct optimized for epitope processing in a mouse model. As Mtb antigens can be used in combination with BCG to boost immune responses, we also tested the protective efficacy of heterologous prime-boost, using dermal tattoo immunization with H56_E cDNA to boost BCG immunization in mice. Dermal H56 and H56_E cDNA immunization induced H56-specific CD4 + and CD8 + T cell responses and Ag85B-specific IgG antibodies, but did not reduce bacterial loads, although immunization with H56_E ameliorated lung pathology. Both subcutaneous and intradermal immunization with BCG resulted in broad cellular immune responses, with increased frequencies of CD4 + T effector memory cells, T follicular helper cells, and germinal center B cells, and resulted in reduced bacterial loads and lung pathology. Heterologous vaccination with BCG/H56_E cDNA induced increased H56-specific CD4 + and CD8 + T cell cytokine responses compared to vaccination with BCG alone, and lung pathology was significantly decreased in BCG/H56_E cDNA immunized mice compared to unvaccinated controls. However, bacterial loads were not decreased after heterologous vaccination compared to BCG alone. CD4 + T cells responding to Ag85B- and ESAT-6

  11. Molecular Characterization of Tomato Yellow Leaf Curl Virus in Korea and the Construction of an Infectious Clone

    Directory of Open Access Journals (Sweden)

    Bong Choon Lee

    2015-06-01

    Full Text Available Several tomato production regions in Korea were surveyed for tomato yellow leaf curl disease (TYLCD. Tomato leaf samples showing TYLCD-like symptoms were collected from Tongyeong (To, Geoje (Gi, and Gimhae (Gh cities of the southern part of Korea. Tomato yellow leaf curl virus (TYLCV was detected and the full-length genomes of the isolates were sequenced. The TYLCV isolates found in Korea shared high sequence identity (> 99% with TYLCV-IL [JR:Omu:Ng] (AB110217. Phylogenetic relationship analysis revealed that they formed two groups (with little genetic variability, and the To, Gj, and Gh isolates belonged to the TYLCV-IL group. An infectious clone of TYLCV-To (JQ013089 was constructed and agroinoculated into Nicotiana benthamiana, Nicotiana tabacum var. Xanthi, Petunia hybrida, Capsicum annuum, and Lycopersicon esculentum cv. Hausumomotaro. Agroinfection with a dimeric infectious clone of TYLCV-To induced severe leaf curling and stunting symptoms in these plants, excluding C. annuum. Tomato plants then developed typical yellow leaf curl symptoms.

  12. Digital analysis of cDNA abundance; expression profiling by means of restriction fragment fingerprinting

    Directory of Open Access Journals (Sweden)

    Regenbogen Johannes

    2002-03-01

    Full Text Available Abstract Background Gene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance, that calculates the relative abundance of genes in cDNA libraries. Results DADA is based upon multiple restriction fragment length analysis of pools of clones from cDNA libraries and the identification of gene-specific restriction fingerprints in the resulting complex fragment mixtures. A specific cDNA cloning vector had to be constructed that governed missing or incomplete cDNA inserts which would generate misleading fingerprints in standard cloning vectors. Double stranded cDNA was synthesized using an anchored oligo dT primer, uni-directionally inserted into the DADA vector and cDNA libraries were constructed in E. coli. The cDNA fingerprints were generated in a PCR-free procedure that allows for parallel plasmid preparation, labeling, restriction digest and fragment separation of pools of 96 colonies each. This multiplexing significantly enhanced the throughput in comparison to sequence-based methods (e.g. EST approach. The data of the fragment mixtures were integrated into a relational database system and queried with fingerprints experimentally produced by analyzing single colonies. Due to limited predictability of the position of DNA fragments on the polyacrylamid gels of a given size, fingerprints derived solely from cDNA sequences were not accurate enough to be used for the analysis. We applied DADA to the analysis of gene expression profiles in a model for impaired wound healing (treatment of mice with dexamethasone. Conclusions The method proved to be capable of identifying pharmacologically relevant target genes that had not been identified by other standard methods routinely used to find differentially expressed genes. Due to the above mentioned limited predictability of the fingerprints, the method was yet tested only with

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

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data DOI 10.18908/lsdba.nbdc00838-003 Description of data contents Phred's quality score. P...tion Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality

  14. Construction and characterization of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus capsid proteins of Indian vaccine strain, O/IND/R2/75

    Directory of Open Access Journals (Sweden)

    Ramesh Kumar

    2015-02-01

    Full Text Available Aim: Generation of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus (FMDV capsid protein genes along with full-length 2B, 3B and 3Cpro and its characterization. Materials and Methods: FMD viral RNA isolation, cDNA synthesis, and polymerase chain reaction were performed to synthesize expression cassettes (P1-2AB3BCwt and P1-2AB3BCm followed by cloning in pShuttle-CMV vector. Chemically competent BJ5183-AD-1 cells were transformed with the recombinant pShuttle-CMV to produce recombinant adenoviral plasmids. HEK-293 cells were transfected with the recombinant adenoviral plasmids to generate recombinant adenoviruses (hAd5/P1-2AB3BCwt and hAd5/P1-2AB3BCm. Expression of the target proteins was analyzed by sandwich ELISA and indirect immunofluorescence assay. The recombinant adenoviruses were purified and concentrated by CsCl density gradient ultracentrifugation. Growth kinetics and thermostability of the recombinant adenoviruses were compared with that of non-recombinant replication-defective adenovirus (dAd5. Results: The recombinant adenoviruses containing capsid protein genes of the FMDV O/IND/R2/75 were generated and amplified in HEK-293 cells. The titer of the recombinant adenoviruses was approximately 108, 109.5 and 1011 TCID50/ml in supernatant media, cell lysate and CsCl purified preparation, respectively. Expression of the FMDV capsid protein was detectable in sandwich ELISA and confirmed by immunofluorescence assay. Growth kinetics of the recombinant adenoviruses did not reveal a significant difference when compared with that of dAd5. A decrement of up to 10-fold at 4°C and 21-fold at 37°C was recorded in the virus titers during 60 h incubation period and found to be statistically significant (p<0.01. Conclusion: Recombinant adenoviruses expressing capsid proteins of the FMDV O/IND/R2/75 were constructed and produced in high titers. In vitro expression of the target proteins in the adenovirus vector system was

  15. Interferon-gamma up-regulates a unique set of proteins in human keratinocytes. Molecular cloning and expression of the cDNA encoding the RGD-sequence-containing protein IGUP I-5111

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder

    1993-01-01

    transformation and because peptide sequences were available in the microsequence database. The cDNA was cloned from a fibroblast cDNA library using degenerate oligodeoxyribonucleotides and expressed in AMA cells using the vaccinia virus expression system. Database searches indicated that the predicted protein...

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

    Directory of Open Access Journals (Sweden)

    Poustka Annemarie

    2004-06-01

    Full Text Available Abstract Background cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library. Results We describe a method to construct SMART full-length enriched cDNA libraries with large insert sizes. Sub-libraries were generated from size-fractionated cDNA with an average insert size of up to seven kb. The percentage of full-length clones was calculated for different size ranges from BLAST results of over 12,000 5'ESTs. Conclusions The presented technique is suitable to generate full-length enriched cDNA libraries with large average insert sizes in a straightforward and robust way. The representation of full-coding clones is high also for large cDNAs (70%, 4–10 kb, when high-quality starting mRNA is used.

  17. Modulation of Translation Initiation Efficiency in Classical Swine Fever Virus

    DEFF Research Database (Denmark)

    Friis, Martin Barfred; Rasmussen, Thomas Bruun; Belsham, Graham J.

    Modulation of translation initiation efficiency on classical swine fever virus (CSFV) RNA can be achieved by targeted mutations within the internal ribosome entry site (IRES). In this study, the nucleotides 47 to 427, including the IRES region of the wt CSFV strain Paderborn, were amplified...... and inserted, under T7 promoter control, into mono- and dicistronic plasmids containing the reporter genes rLuc and fLuc. Mutant fragments of the IRES sequence were generated by overlap PCR and inserted into the reporter plasmids. To evaluate IRES functionality, translation of the rLUC was placed under...... viruses were obtained after one cell culture passage from constructs with more than 75 % translation efficiency compared to the wildtype IRES. cDNA was generated from these clones and sequenced to verify the maintenance of the changes in the IRES. These results show that full-length viable mutant viruses...

  18. Infection with Strains of Citrus Tristeza Virus Does Not Exclude Superinfection by Other Strains of the Virus▿

    Science.gov (United States)

    Folimonova, Svetlana Y.; Robertson, Cecile J.; Shilts, Turksen; Folimonov, Alexey S.; Hilf, Mark E.; Garnsey, Stephen M.; Dawson, William O.

    2010-01-01

    Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were “strains” of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most

  19. Verifikasi cDNA T29 sebagai kandidat gen pengkode protein Toxoplasma gondii dengan metode SDS PAGE

    Directory of Open Access Journals (Sweden)

    Ira Djajanegara

    2012-02-01

    Full Text Available The process of cDNA construction from mRNA isolated from Toxoplasma gondii has been done. There were 7 candidates cDNA which one of them is called T29. Since Toxoplasma gondii is the cause of toxoplasmosis infection, cloning the gene encoding protein from this parasite provides an important tool for developing diagnostic kit for detection of toxoplasmosis. Digestion of the cDNA T29 with EcoRI which is the restriction site where the cDNA was inserted yielded a 1.862 bp fragment. The fragment was subcloned into E. coli expression vector pMal-p2x and transformed into E.coli strain TB1. Colonies of TB1 were grown on ampicillin plates and the recombinant plasmid was extracted using the standard procedure. The palsmid was digested using EcoRI and PstI, checked by PCR amplification using malE and M13/pUC primers. The recombinant plasmid was expressed in TB1 and the protein extracted was ran in SDS PAGE to observe the presence of the expressed protein. Based on the data from this experiment, there was no expression result of the expressed cDNA which was confirm by the PCR result. Therefore, it was concluded that cDNA T29 was not carrying the gene coding for protein from parasite Toxoplasma gondii.

  20. Analysis of a tobacco mosaic virus strain capable of overcoming N gene-mediated resistance.

    Science.gov (United States)

    Padgett, H S; Beachy, R N

    1993-05-01

    The genome of Ob, a tobamovirus that overcomes the N gene-mediated hypersensitive response (HR), was cloned as a cDNA, and its nucleotide sequence was determined. The genomic organization of Ob is similar to that of other tobamoviruses, consisting of 6506 nucleotides and containing at least four open reading frames. These open reading frames encode a 126-kD polypeptide with a 183-kD readthrough product, a 30.6-kD movement protein, and an 18-kD coat protein. A bacteriophage T7 promoter sequence was fused to the full-length cDNA clone to obtain infectious RNA transcripts. These transcripts, when inoculated onto tobacco plants, induced disease symptoms indistinguishable from plants inoculated with Ob viral RNA. To determine which viral factor is responsible for the resistance-breaking character of Ob, a recombinant virus was constructed in which the movement protein gene of tobacco mosaic virus was replaced with that of Ob. Cultivar Xanthi NN tobacco plants infected with this virus responded with an HR, indicating that the Ob movement protein alone does not act to overcome the N gene-mediated response. Following mutagenesis of the infectious Ob cDNA clone with hydroxylamine, populations of transcripts from the mutagenized DNA were inoculated onto Xanthi NN tobacco, and a variant that induced the HR was identified. The mutant was analyzed and found to contain a single nucleotide change in the 126-kD gene. Recreating the mutation in the Ob cDNA clone by site-directed mutagenesis resulted in a virus that caused symptoms identical to the chemically induced mutant.

  1. A novel strategy to develop a robust infectious hepatitis C virus cell culture system directly from a clinical isolate.

    Science.gov (United States)

    Lu, Jie; Xiang, Yu; Tao, Wanyin; Li, Qingchao; Wang, Na; Gao, Yongfeng; Xiang, Xiaogang; Xie, Qing; Zhong, Jin

    2014-02-01

    Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases. Progress in the HCV field was greatly enhanced by constructing infectious cDNA clone of JFH-1. Since then, JFH-1-based intra- and intergenotypic recombinants have been developed, and this permitted the study of vaccines and antiviral inhibitors for all genotypes. Recently, highly efficient HCV culture systems have been established by using consensus sequence-based clones. We developed a novel strategy to construct infectious HCV cDNA clone by combining functional screening of sequences directly from a genotype 2a clinical isolate (PR63) and cell culture adaptation. Using JFH-1 cDNA as the starting backbone, we sequentially replaced the JFH-1 fragments with a sequence from the pools of PR63 sequences. Through engineering adaptive mutations that improve HCV infectivity, we finally established a full-length cell culture-derived infectious clone of PR63, named PR63cc, that could efficiently produce virus particles in Huh7-derived cells, with peak titers of 1.6 × 10(5) focus-forming units/ml. The PR63cc could be neutralized by an anti-E2 antibody and inhibited by antiviral agents but appeared more resistant to an NS5A inhibitor than JFH-1. In summary, we developed a new approach to construct an infectious HCV cDNA clone that can produce viruses efficiently in cell culture. This approach could be applied to other viral isolates, with potential implications for individualized treatments of HCV patients.

  2. Cloning and characterization of cDNA encoding xyloglucan ...

    African Journals Online (AJOL)

    We cloned complete cDNA of an XET from pearl millet (Pennisetum glaucum L.) and characterized it using in silico comparative genomics and activity assays. The cloned cDNA was 1266 bp in length, encoding a protein with 291 amino acids having signal peptide targeting it to the cell wall. The protein showed xyloglucan ...

  3. cDNA structure, genomic organization and expression patterns of ...

    African Journals Online (AJOL)

    Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin cDNA cloned from the liver was ...

  4. Virulence in pigs of vPader10 rescued from an infectious cDNA clone of the CSFV strain Paderborn

    DEFF Research Database (Denmark)

    Friis, Martin Barfred; Nielsen, Jens; Uttenthal, Åse

    The BAC clone, pBeloPader10, contains a complete cDNA of the CSFV strain Paderborn. Virus, named vPader10, was rescued from this construct by electroporation of RNA transcripts into porcine PK15 cells. To further study the characteristics of vPader10, we evaluated the virulence of this virus...... in vivo in pigs. An animal experiment was performed where three pigs were inoculated with vPader10 and housed in-contact with two non-inoculated pigs for 5 weeks. Following inoculation with vPader10, two out of three pigs displayed severe clinical signs of CSF from PID 14 that progressed until death...... of the pigs at PID 21 and PID 22, respectively. High fever (>41ºC) was observed in these pigs from PID 14 and remained at a high level until day of death. One of two contact pigs developed similar clinical disease that initiated at PID 21 and progressed until it was euthanised at PID 32 due to severe clinical...

  5. CDNA library from the Latex of Hevea brasiliensis

    Directory of Open Access Journals (Sweden)

    Wilaiwan Chotigeat

    2010-12-01

    Full Text Available Latex from Hevea brasiliensis contains 30-50% (w/w of natural rubber (cis-1,4-polyisoprene, the important rawmaterial for many rubber industries. We have constructed a cDNA library from the latex of H. brasiliensis to investigate theexpressed genes and molecular events in the latex. We analyzed 412 expressed sequence tags (ESTs. More than 90% of theEST clones showed homology to previously described sequences in public databases. Functional classification of the ESTsshowed that the largest category were proteins of unknown function (30.1%, 11.4% of ESTs encoded for rubber synthesisrelatedproteins (RS and 8.5% for defense or stress related proteins (DS. Those with no significant homology to knownsequences (NSH accounted for 8.7%, primary metabolism (PM and gene expression and RNA metabolism were 7.8% and6.6%, respectively. Other categories included, protein synthesis-related proteins (6.6%, chromatin and DNA metabolism(CDM 3.9%, energy metabolism (EM 3.4%, cellular transport (CT 3.2%, cell structure (CS 3.2%, signal transduction (ST2.2%, secondary metabolism (SM 1.7%, protein fate (PF 2.2%, and reproductive proteins (RP 0.7%.

  6. Cloning and expression of a cDNA covering the complete coding region of the P32 subunit of human pre-mRNA splicing factor SF2

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Rasmussen, H H

    1993-01-01

    We have cloned and expressed a cDNA encoding the 32-kDa subunit (P32) of the human pre-mRNA splicing factor, SF2. This cDNA extends beyond the 5'-end of a previously reported cDNA [Krainer et al., Cell 66 (1991) 383-394]. Importantly, our fragment includes an ATG start codon which was absent from...... the previously reported cDNA, where it was suggested that translation might initiate at a CTG codon instead of at an ATG codon. Using the vaccinia virus (Vv) expression system, we demonstrate that translation starts at the conventional ATG start codon and not at the CTG codon. The protein is synthesized as a pro...

  7. Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes.

    Science.gov (United States)

    Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

    2015-07-29

    Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes.

  8. Epitope mapping of Ebola virus dominant and subdominant glycoprotein epitopes facilitates construction of an epitope-based DNA vaccine able to focus the antibody response in mice.

    Science.gov (United States)

    Mitchell, Daniel A J; Dupuy, Lesley C; Sanchez-Lockhart, Mariano; Palacios, Gustavo; Back, Jaap W; Shimanovskaya, Katya; Chaudhury, Sidhartha; Ripoll, Daniel R; Wallqvist, Anders; Schmaljohn, Connie S

    2017-07-12

    We performed epitope mapping studies on the major surface glycoprotein (GP) of Ebola virus (EBOV) using Chemically Linked Peptides on Scaffolds (CLIPS), which form linear and potential conformational epitopes. This method identified monoclonal antibody epitopes and predicted additional epitopes recognized by antibodies in polyclonal sera from animals experimentally vaccinated against or infected with EBOV. Using the information obtained along with structural modeling to predict epitope accessibility, we then constructed two DNA vaccines encoding immunodominant and subdominant epitopes predicted to be accessible on EBOV GP. Although a construct designed to produce a membrane-bound oligopeptide was poorly immunogenic, a construct generating a secreted oligopeptide elicited strong antibody responses in mice. When this construct was administered as a boost to a DNA vaccine expressing the complete EBOV GP gene, the resultant antibody response was focused largely toward the less immunodominant epitopes in the oligopeptide. Taken together, the results of this work suggest a utility for this method for immune focusing of antibody responses elicited by vaccination.

  9. Data of evolutionary structure change: 1CDNA-1J7OA [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1CDNA-1J7OA 1CDN 1J7O A A ---KSPEELKGIFEKYAAKE--GDPNQLSKEELKLLLQTEFPSLLKGGSTLDELFE...1CDN A 1CDNA YAAKE--GDPNQ...1CDN A 1CDNA LFEEL---DKNGD...1CDN A 1CDNA LLQTEFPSLLKGGST...1CDN A 1CDNA DGEVSFEEFQVL

  10. [Construction of an infectious clone of pseudorabies virus strain ZJ genome maintained as a bacterial artificial chromosome].

    Science.gov (United States)

    Yin, Wen-Ling; Yin, Long-Bo; Ye, Wei-Cheng; Sun, Xue-Qiang; Yao, Huo-Chun; Zhang, Miao-Tao; Wang, Yi-Cheng; Zhang, Cun

    2010-07-01

    pHA2 plasmid sequence,with Bacterial Artificial Chromosome(BAC) vector and the GFP expression cassette, was introduced into the UL23(TK) gene of Pseudorabies virus(PRV)strain ZJ by homologous recombination,and the recombinant PRV (rPRV-HA2) was confirmed and isolated by plaque purification. The circular genome of rPRV-HA2 was electroporated into Escherichia coli strain DH10B and then the PRV BAC (pPRV) was recovered. The transfection of pPRV into VeroE6 cells resulted in productive infection. The rescued virus isolated following transfection was indistinguishable from rPRV-HA2 in cytopathic effects (CPE) and replication curve in vitro. The growth kinetics of the viruses indicated that partial deletion of TK gene and BAC vector insertion had no effect on the viral titre and plaque size in vitro. The PRV BAC system will enable quick and reliable manipulation of the viral genome for the functional investigation on the PRV genes and the development of PRV vector in vaccine.

  11. Construction of adeno-associated virus packaging plasmids and cells that directly select for AAV helper functions.

    Science.gov (United States)

    Whiteway, Alistair; Deru, Wale; Prentice, H Grant; Anderson, Robert

    2003-12-01

    Recombinant adeno-associated virus type 2 (rAAV) has promise for use as a gene therapy vector. Potential problems in the production of rAAV stocks are both the limited amount of recombinant virus that is produced by traditional methods and the possibility of wild-type replication competent adeno-associated virus (wtAAV) contamination. The presence of these contaminants is largely dependent upon the helper plasmid used. Whilst wtAAV is not a pathogen, the presence of these contaminants is undesirable as they may affect experiments concerning the biology of rAAV. Additionally as protocols using rAAV with altered tropism are becoming more prevalent, it is important that no recombination be permitted that may cause the creation of a replication competent AAV with modified (targeting) capsids. Many experimental protocols require the generation of large amounts of high titre rAAV stocks. We describe the production of several AAV helper plasmids and cell lines designed to achieve this goal. These plasmids possess split AAV rep and cap genes to eliminate the production of wtAAV and they possess a selection mechanism which is operatively linked to expression from the AAV cap gene. This allows positive selection of those cells expressing the highest level of the structural capsid proteins and therefore those cells which yield the highest amount of rAAV.

  12. The complete nucleotide sequence and genome organization of pea streak virus (genus Carlavirus).

    Science.gov (United States)

    Su, Li; Li, Zhengnan; Bernardy, Mike; Wiersma, Paul A; Cheng, Zhihui; Xiang, Yu

    2015-10-01

    Pea streak virus (PeSV) is a member of the genus Carlavirus in the family Betaflexiviridae. Here, the first complete genome sequence of PeSV was determined by deep sequencing of a cDNA library constructed from dsRNA extracted from a PeSV-infected sample and Rapid Amplification of cDNA Ends (RACE) PCR. The PeSV genome consists of 8041 nucleotides excluding the poly(A) tail and contains six open reading frames (ORFs). The putative peptide encoded by the PeSV ORF6 has an estimated molecular mass of 6.6 kDa and shows no similarity to any known proteins. This differs from typical carlaviruses, whose ORF6 encodes a 12- to 18-kDa cysteine-rich nucleic-acid-binding protein.

  13. Directional random oligonucleotide primed (DROP) global amplification of cDNA: its application to subtractive cDNA cloning.

    OpenAIRE

    Hampson, I N; Hampson, L; Dexter, T M

    1996-01-01

    We describe a method of global PCR amplification of cDNA such that the strand sense is maintained. The products of this process are random primed fragments ranging in size from 100 to 500 bp which facilitates uniform PCR amplification of total cDNA. Directional incorporation of a T7 RNA polymerase initiator/promoter sequence allows efficient synthesis of total sense RNA from this material and the use of a biotinylated primer permits the separation of single-stranded cDNA. Isolation of these p...

  14. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    1994-01-04

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties. GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon.

  15. Unprocessed foot-and-mouth disease virus capsid precursor displays discontinuous epitopes involved in viral neutralization.

    OpenAIRE

    Sáiz, J C; Cairó, J; Medina, M.; Zuidema, D.; Abrams, C; Belsham, G.J.; Domingo, E; Vlak, J.M.

    1994-01-01

    A foot-and-mouth disease virus (FMDV) cDNA cassette containing sequences encoding the capsid precursor P1, peptide 2A and a truncated 2B (abbreviated P1-2A) of type C FMDV, has been modified to generate the authentic amino terminus and the myristoylation signal. This construct has been used to produce a recombinant baculovirus (AcMM53) which, upon infection of Spodoptera frugiperda insect cells, expressed a recombinant P1-2A precursor with a high yield. This polyprotein reacted with neutraliz...

  16. Construction of target-specific virus-like particles for the delivery of algicidal compounds to harmful algae.

    Science.gov (United States)

    Kang, Beom Sik; Eom, Chi-Yong; Kim, Wonduck; Kim, Pyoung Il; Ju, Sun Yi; Ryu, Jaewon; Han, Gui Hwan; Oh, Jeong-Il; Cho, Hoon; Baek, Seung Ho; Kim, Gueeda; Kim, Minju; Hyun, Jaekyung; Jin, EonSeon; Kim, Si Wouk

    2015-04-01

    Harmful algal blooms (HABs) can lead to substantial socio-economic losses and extensive damage to aquatic ecosystems, drinking water sources and human health. Common algicidal techniques, including ozonation, ultrasonic treatment and dispersion of algae-killing chemicals, are unsatisfactory both economically and ecologically. This study therefore presents a novel alternative strategy for the efficient control of deleterious algae via the use of host-specific virus-like particles (VLPs) combined with chemically synthesized algicidal compounds. The capsid protein of HcRNAV34, a single-stranded RNA virus that infects the toxic dinoflagellate, Heterocapsa circularisquama, was expressed in and purified from Escherichia coli and then self-assembled into VLPs in vitro. Next, the algicidal compound, thiazolidinedione 49 (TD49), was encapsidated into HcRNAV34 VLPs for specific delivery to H. circularisquama. Consequently, HcRNAV34 VLPs demonstrated the same host selectivity as naturally occurring HcRNAV34 virions, while TD49-encapsidated VLPs showed a more potent target-specific algicidal effect than TD49 alone. These results indicate that target-specific VLPs for the delivery of cytotoxic compounds to nuisance algae might provide a safe, environmentally friendly approach for the management of HABs in aquatic ecosystems. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  17. Characterization of a cDNA Encoding Lens culinaris Glycolate Oxidase and Developmental Expression of Glycolate Oxidase mRNA in Cotyledons and Leaves

    Science.gov (United States)

    Ludt, Claudia; Kindl, Helmut

    1990-01-01

    mRNA obtained from green leaves of lentil (Lens culinaris) was used to construct a cDNA library in phage λgt11. The cDNA library was screened with antibodies raised against lentil glycolate oxidase and catalase. Clone CL 1 containing the full-length sequence complementary to glycolate oxidase mRNA was characterized and sequenced. In addition, a 800-base pair catalase cDNA clone was sequenced. To prove the correlation of cDNA insert in CL 1 with glycolate oxidase, the cDNA was transcribed in vitro. The mRNA was translated in vitro yielding a 43 kilodalton protein immunoprecipitable with anti-glycolate oxidase serum. Nucleotide sequences of lentil cDNA and spinach cDNA were 86% identical. Lentil glycolate oxidase was characterized by a C-terminal sequence -P-R-A-L-P-R-L. The expression of glycolate oxidase mRNA in cotyledons, leaves and roots was compared with that of catalase. In leaves, the relative amount of glycolate oxidase mRNA increased during the first 2 days of greening, but decreased later, and was hardly detectable during senescence. In cotyledons of germinating seeds, the level of glycolate oxidase mRNA was markedly lower than the catalase mRNA. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:16667816

  18. Citrus psorosis virus coat protein-derived hairpin construct confers stable transgenic resistance in citrus against psorosis A and B syndromes.

    Science.gov (United States)

    De Francesco, A; Costa, N; García, M L

    2017-04-01

    Citrus psorosis virus (CPsV) is the causal agent of psorosis, a serious and widespread citrus disease. Two syndromes of psorosis, PsA and PsB, have been described. PsB is the most aggressive and rampant form. Previously, we obtained Pineapple sweet orange plants transformed with a hairpin construct derived from the CPsV coat protein gene (ihpCP). Some of these plants were resistant to CPsV 90-1-1, a PsA isolate homologous to the transgene. In this study, we found that expression of the ihpCP transgene and siRNA production in lines ihpCP-10 and -15 were stable with time and propagation. In particular, line ihpCP-15 has been resistant for more than 2 years, even after re-inoculation. The ihpCP plants were also resistant against a heterologous CPsV isolate that causes severe PsB syndrome. Line ihpCP-15 manifested complete resistance while line ihpCP-10 was tolerant to the virus, although with variable behaviour, showing delay and attenuation in PsB symptoms. These lines are promising for a biotech product aimed at eradicating psorosis.

  19. Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge.

    Science.gov (United States)

    Montes, Christian; Castro, Álvaro; Barba, Paola; Rubio, Julia; Sánchez, Evelyn; Carvajal, Denisse; Aguirre, Carlos; Tapia, Eduardo; DelÍ Orto, Paola; Decroocq, Veronique; Prieto, Humberto

    2014-10-01

    Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.

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

    Science.gov (United States)

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

    2002-01-01

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

  1. cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome

    Directory of Open Access Journals (Sweden)

    Valenzuela Jesus G

    2007-07-01

    Full Text Available Abstract Background The completion of the Plasmodium falciparum genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions. Results We aimed to obtain cDNA sequences to examine the accuracy of gene prediction in silico. We constructed cDNA libraries from mixed blood stages of P. falciparum parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST, including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons versus 5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6% with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions. Conclusion Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively

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

    Science.gov (United States)

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

    2010-01-01

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

  3. Molecular cloning and functional expression of cDNA encoding a cysteine proteinase inhibitor, cystatin, from Job's tears (Coix lacryma-jobi L. var. Ma-yuen Stapf).

    Science.gov (United States)

    Yoza, Koh-Ichi; Nakamura, Sumiko; Yaguchi, Miki; Haraguchi, Kazutomo; Ohtsubo, Ken-Ichi

    2002-10-01

    A lambdaZAP II cDNA library was constructed from mRNA in immature seeds of the grass Job's tears. A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library. The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues. The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn. The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found. A remarkable characteristic of the peptide sequence of Job's-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice. The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The purified recombinant protein inhibited papain.

  4. Epidemiology and host spectrum of Borna disease virus infections

    National Research Council Canada - National Science Library

    Kinnunen, Paula M; Palva, Airi; Vaheri, Antti; Vapalahti, Olli

    2013-01-01

    Borna disease virus (BDV) has gained lot of interest because of its zoonotic potential, ability to introduce cDNA of its RNA transcripts into host genomes, and ability to cause severe neurobehavioural diseases...

  5. cDNA Microarray Screening in Food Safety

    Science.gov (United States)

    ROY, SASHWATI; SEN, CHANDAN K

    2009-01-01

    The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests. PMID:16466843

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

    Lifescience Database Archive (English)

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

  7. A full-length infectious clone of beet soil-borne virus indicates the dispensability of the RNA-2 for virus survival in planta and symptom expression on Chenopodium quinoa leaves.

    Science.gov (United States)

    Crutzen, François; Mehrvar, Mohsen; Gilmer, David; Bragard, Claude

    2009-12-01

    For a better understanding of the functionality and pathogenicity of beet soil-borne virus (BSBV), full-length cDNA clones have been constructed for the three genomic RNAs. With the aim of assessing their effectiveness and relative contribution to the virus housekeeping functions, transcripts were inoculated on Chenopodium quinoa and Beta macrocarpa leaves using five genome combinations. Both RNAs-1 (putative replicase) and -3 (putative movement proteins) proved to be essential for virus replication in planta and symptom production on C. quinoa, whereas RNA-2 (putative coat protein, CP, and a read-through domain, RT) was not. No symptoms were recorded on B. macrocarpa, but viral RNAs were detected. In both host plants, the 19 kDa CP was detected by Western blotting as well as a 115 kDa protein corresponding to the CP-RT.

  8. Restriction landmark cDNA scanning (RLCS): a novel cDNA display system using two-dimensional gel electrophoresis.

    Science.gov (United States)

    Suzuki, H; Yaoi, T; Kawai, J; Hara, A; Kuwajima, G; Wantanabe, S

    1996-01-01

    We have developed a new method, designated restriction landmark cDNA scanning (RLCS), which displays many cDNA species quantitatively and simultaneously as two-dimensional gel spots. In this method cDNA species of uniform length were prepared for each mRNA species using restriction enzymes. After the restriction enzyme sites were radiolabeled as landmarks, the labeled fragments were subjected to high resolution two-dimensional gel electrophoresis. In analyses of cDNA samples from adult mouse liver and brain (cerebral cortex, cerebellum and brain stem) we detected approximately 500 and >1000 discrete gel spots respectively of various intensities at a time. The spot patterns of the three brain regions were very similar, although not identical, but were quite different from the pattern for the liver. RNA blot hybridization analysis using several cloned spot DNAs as probes showed that differences in intensity of the spots among RLCS profiles correlated well with expression levels of the corresponding mRNA species in the brain regions. Because the spots and their intensities reflect distinct mRNA species and their expression level respectively, the RLCS is a novel cDNA display system which provides a great deal of information and should be useful for systematic documentation of differentially expressed genes. PMID:8628652

  9. An implantable vascularized protein gel construct that supports human fetal hepatoblast survival and infection by hepatitis C virus in mice.

    Directory of Open Access Journals (Sweden)

    Martha J Harding

    2010-04-01

    Full Text Available Widely accessible small animal models suitable for the study of hepatitis C virus (HCV in vivo are lacking, primarily because rodent hepatocytes cannot be productively infected and because human hepatocytes are not easily engrafted in immunodeficient mice.We report here on a novel approach for human hepatocyte engraftment that involves subcutaneous implantation of primary human fetal hepatoblasts (HFH within a vascularized rat collagen type I/human fibronectin (rCI/hFN gel containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC in severe combined immunodeficient X beige (SCID/bg mice. Maturing hepatic epithelial cells in HFH/Bcl-2-HUVEC co-implants displayed endocytotic activity at the basolateral surface, canalicular microvilli and apical tight junctions between adjacent cells assessed by transmission electron microscopy. Some primary HFH, but not Huh-7.5 hepatoma cells, appeared to differentiate towards a cholangiocyte lineage within the gels, based on histological appearance and cytokeratin 7 (CK7 mRNA and protein expression. Levels of human albumin and hepatic nuclear factor 4alpha (HNF4alpha mRNA expression in gel implants and plasma human albumin levels in mice engrafted with HFH and Bcl-2-HUVEC were somewhat enhanced by including murine liver-like basement membrane (mLBM components and/or hepatocyte growth factor (HGF-HUVEC within the gel matrix. Following ex vivo viral adsorption, both HFH/Bcl-2-HUVEC and Huh-7.5/Bcl-2-HUVEC co-implants sustained HCV Jc1 infection for at least 2 weeks in vivo, based on qRT-PCR and immunoelectron microscopic (IEM analyses of gel tissue.The system described here thus provides the basis for a simple and robust small animal model of HFH engraftment that is applicable to the study of HCV infections in vivo.

  10. An implantable vascularized protein gel construct that supports human fetal hepatoblast survival and infection by hepatitis C virus in mice.

    Science.gov (United States)

    Harding, Martha J; Lepus, Christin M; Gibson, Thomas F; Shepherd, Benjamin R; Gerber, Scott A; Graham, Morven; Paturzo, Frank X; Rahner, Christoph; Madri, Joseph A; Bothwell, Alfred L M; Lindenbach, Brett D; Pober, Jordan S

    2010-04-01

    Widely accessible small animal models suitable for the study of hepatitis C virus (HCV) in vivo are lacking, primarily because rodent hepatocytes cannot be productively infected and because human hepatocytes are not easily engrafted in immunodeficient mice. We report here on a novel approach for human hepatocyte engraftment that involves subcutaneous implantation of primary human fetal hepatoblasts (HFH) within a vascularized rat collagen type I/human fibronectin (rCI/hFN) gel containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) in severe combined immunodeficient X beige (SCID/bg) mice. Maturing hepatic epithelial cells in HFH/Bcl-2-HUVEC co-implants displayed endocytotic activity at the basolateral surface, canalicular microvilli and apical tight junctions between adjacent cells assessed by transmission electron microscopy. Some primary HFH, but not Huh-7.5 hepatoma cells, appeared to differentiate towards a cholangiocyte lineage within the gels, based on histological appearance and cytokeratin 7 (CK7) mRNA and protein expression. Levels of human albumin and hepatic nuclear factor 4alpha (HNF4alpha) mRNA expression in gel implants and plasma human albumin levels in mice engrafted with HFH and Bcl-2-HUVEC were somewhat enhanced by including murine liver-like basement membrane (mLBM) components and/or hepatocyte growth factor (HGF)-HUVEC within the gel matrix. Following ex vivo viral adsorption, both HFH/Bcl-2-HUVEC and Huh-7.5/Bcl-2-HUVEC co-implants sustained HCV Jc1 infection for at least 2 weeks in vivo, based on qRT-PCR and immunoelectron microscopic (IEM) analyses of gel tissue. The system described here thus provides the basis for a simple and robust small animal model of HFH engraftment that is applicable to the study of HCV infections in vivo.

  11. cDNA encoding a polypeptide including a hevein sequence

    Science.gov (United States)

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

    1993-02-16

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

  12. Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System

    Science.gov (United States)

    Makvandi, Manoochehr; Teimoori, Ali; Neisi, Niloofar; Samarbafzadeh, Alireza

    2016-01-01

    Background The hepatitis E virus (HEV) accounts for hepatitis E infection with relatively high mortality rate in pregnant women that can lead to fulminant hepatitis. The baculovirus expression system (BES) has the capability to produce high-level recombinant proteins and could be useful for vaccine designing. Objectives The aim of this study was designing a recombinant hepatitis E virus ORF2 and Rotavirus NSP4 (ORF2-NSP4) and to evaluating construction these recombinant proteins in the BES. Methods The truncated ORF2 gene (112-607) and truncated ORF2-NSP4 were subcloned in pFastBac1 plasmid, separately, followed by digestion and confirmed by digestion and sequencing. Then the products were transformed into Escherichia coli DH5α and retransformed in DH10Bac competent cells. Finally the white colonies containing Bacmid DNA subjected to PCR for confirming transformation. Bacmid DNA containing HEV truncated ORF2 and HEV truncated ORF2-NSP4 genes were transfected into SF9 cells using BES. The expressed proteins in the cell lysate were evaluated by SDS-PAGE and determined by the western blot assay. Results The lengths of subcloned genes, truncated ORF2 and truncated ORF2-NSP4 were 1500 and 2000bp, respectively. After retransforming in DH10Bac, the size of PCR products were 300 bp in Bacmid DNA without recombination while it was 4300 and 3800 bp in Bacmid truncated ORF2-NSP4 and Bacmid truncated ORF2 PCR products. The analysis of protein expression by SDS-PAGE and immunoblotting revealed the presence of 56 KDa for truncated ORF2 and 74.5 KDa for truncated ORF2-NSP4 proteins. Conclusions The results of the present study showed that the baculovirus expression system (SF9 cells) was able to express truncated ORF2 and truncated ORF2-NSP4 proteins as a potential candidate vaccine. PMID:28138375

  13. Transformation of Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to the virus.

    Science.gov (United States)

    Soler, Nuria; Plomer, Montserrat; Fagoaga, Carmen; Moreno, Pedro; Navarro, Luis; Flores, Ricardo; Peña, Leandro

    2012-06-01

    Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and/or intercellular level (p20 and p25) to overcome host antiviral defence. Previously, we showed that Mexican lime transformed with an intron-hairpin construct including part of the gene p23 and the adjacent 3' untranslated region displays partial resistance to CTV, with a fraction of the propagations from some transgenic lines remaining uninfected. Here, we transformed Mexican lime with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20 and p23 from CTV strain T36 to silence the expression of these critical genes in CTV-infected cells. Three transgenic lines presented complete resistance to viral infection, with all their propagations remaining symptomless and virus-free after graft inoculation with CTV-T36, either in the nontransgenic rootstock or in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Inoculation with a divergent CTV strain led to partially breaking the resistance, thus showing the role of sequence identity in the underlying mechanism. Our results are a step forward to developing transgenic resistance to CTV and also show that targeting simultaneously by RNA interference (RNAi) the three viral silencing suppressors appears critical for this purpose, although the involvement of concurrent RNAi mechanisms cannot be excluded. © 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  14. Intercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA3.

    OpenAIRE

    French, R; Ahlquist, P

    1987-01-01

    The genome of brome mosaic virus (BMV) is divided among messenger polarity RNA1, RNA2, and RNA3 (3.2, 2.9, and 2.1 kilobases, respectively). cis-Acting sequences required for BMV RNA amplification were investigated with RNA3. By using expressible cDNA clones, deletions were constructed throughout RNA3 and tested in barley protoplasts coinoculated with RNA1 and RNA2. In contrast to requirements for 5'- and 3'-terminal noncoding sequences, either of the two RNA3 coding regions can be deleted in...

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

    Science.gov (United States)

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

    2004-11-01

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

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

    Science.gov (United States)

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

    2003-07-01

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

  17. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain ...

  18. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-02

    Nov 2, 2009 ... RPS20 is a component of the 40S small ribosomal subunit encoded by RPS20 gene, which is conserved between eukaryotes, prokaryotes and archaebacteria. The cDNA and the genomic sequence of RPS20 were cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR ...

  19. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    Science.gov (United States)

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

  20. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences; Volume 26; Issue 3. Cloning, sequencing ... The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ ...

  1. cDNA, genomic sequence cloning and overexpression of ...

    African Journals Online (AJOL)

    Cytochrome c oxidase (COX) is a component of the mitochondria respiratory chain. COX6b1 is one of the COX small subunits encoded by nuclear genes. In currently study, the cDNA and the genomic sequence of COX6b1 were successfully cloned from the Ailuropoda melanoleuca with the RT-PCR technology and ...

  2. Cloning of a cDNA encoding a novel human nuclear phosphoprotein belonging to the WD-40 family

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder

    1994-01-01

    We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79,400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database [Celis et al., Electrophoresis 14 (1993) 1091-1198]. The deduced aa......-134]. The protein contains a nuclear targeting signal (KKKGK), and fractionation of transformed human amnion cells (AMA) in karyoplasts and cytoplasts confirmed that it is predominantly localized in the nucleus. Database searching indicated that IEF SSP 9502 is a putative human homologue of the Saccharomyces...

  3. HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein–Coupled Receptor

    OpenAIRE

    Feng, Yu; Broder, Christopher C.; Kennedy, Paul E.; Berger, Edward A.

    2011-01-01

    A cofactor for HIV-1 (human immunodeficiency virus-type 1) fusion and entry was identified with the use of a novel functional complementary DNA (cDNA) cloning strategy. This protein, designated “fusin,” is a putative G protein–coupled receptor with seven transmembrane segments. Recombinant fusin enabled CD4-expressing nonhuman cell types to support HIV-1 Env-mediated cell fusion and HIV-1 infection. Antibodies to fusin blocked cell fusion and infection with normal CD4-positive human target ce...

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

    Directory of Open Access Journals (Sweden)

    Czosnek Henryk

    2006-04-01

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

  5. Study of the differentially expressed genes in pleomorphic adenoma using cDNA microarrays.

    Science.gov (United States)

    Song, Meng; Xiao, Cuicui; Wang, Tingle; Pei, Qingguo; Wang, Shiwei; Xu, Liqun; Chen, Wantao

    2011-09-01

    Recent studies have determined that gene expression profiling using microarray technology can be used to identify tumor-related molecules. The objective of this study was to screen the differentially expressed genes between pleomorphic adenoma (PA) and the normal tissue adjacent to PA using cDNA microarrays and to further validate the differentially expressed genes by real-time PCR. In this study, we selected five pairs of PA and the surrounding normal salivary gland tissues. The total RNA was isolated from tumor and normal tissues and purified to mRNA. The mRNA was reverse-transcribed to cDNA with the incorporation of fluorescent-labeled dUTP to prepare the hybridization probes. The mixed probes were hybridized to Whole Human Gene Expression Microarrays by Agilent. Tumor-related genes were screened by analyzing the fluorescence intensity. As a result, a total of 447 genes were found to be differentially expressed between PA and normal tissue adjacent to PA. Among them, 185 genes were up-regulated and 262 genes were down-regulated in PA. By constructing a network from the differentially expressed genes, some genes, such as Gli2 and CTNNB1, were identified as being at the core of the network. In addition, differential gene expression was validated for 2 up-regulated genes, Gli2 and LOX, using real-time PCR and the results were consistent with those of the cDNA microarray analysis thus verifying the credibility of the microarray data. Therefore, our microarray data may provide clues for finding novel genes involved in the development of PA, and shed light on finding new targets for diagnosis and therapy of PA. Further characterization of these differentially expressed genes will be useful in understanding the genetic basis for PA.

  6. In Vitro Transcripts of Wild-Type and Fluorescent Protein-Tagged Triticum mosaic virus (Family Potyviridae) are Biologically Active in Wheat.

    Science.gov (United States)

    Tatineni, Satyanarayana; McMechan, Anthony J; Bartels, Melissa; Hein, Gary L; Graybosch, Robert A

    2015-11-01

    Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat. Wheat seedlings inoculated with in vitro transcripts elicited mosaic and mottling symptoms similar to the wild-type virus, and the progeny virus was efficiently transmitted by wheat curl mites, indicating that the cloned virus retained pathogenicity, movement, and wheat curl mite transmission characteristics. A series of TriMV-based expression vectors was constructed by engineering a green fluorescent protein (GFP) or red fluorescent protein (RFP) open reading frame with homologous NIa-Pro cleavage peptides between the P1 and HC-Pro cistrons. We found that GFP-tagged TriMV with seven or nine amino acid cleavage peptides efficiently processed GFP from HC-Pro. TriMV-GFP vectors were stable in wheat for more than 120 days and for six serial passages at 14-day intervals by mechanical inoculation and were transmitted by wheat curl mites similarly to the wild-type virus. Fluorescent protein-tagged TriMV was observed in wheat leaves, stems, and crowns. The availability of fluorescent protein-tagged TriMV will facilitate the examination of virus movement and distribution in cereal hosts and the mechanisms of cross protection and synergistic interactions between TriMV and Wheat streak mosaic virus.

  7. A rat model for hepatitis E virus

    Directory of Open Access Journals (Sweden)

    Yannick Debing

    2016-10-01

    Full Text Available Hepatitis E virus (HEV is one of the prime causes of acute viral hepatitis, and chronic hepatitis E is increasingly recognized as an important problem in the transplant setting. Nevertheless, the fundamental understanding of the biology of HEV replication is limited and there are few therapeutic options. The development of such therapies is partially hindered by the lack of a robust and convenient animal model. We propose the infection of athymic nude rats with the rat HEV strain LA-B350 as such a model. A cDNA clone, pLA-B350, was constructed and the infectivity of its capped RNA transcripts was confirmed in vitro and in vivo. Furthermore, a subgenomic replicon, pLA-B350/luc, was constructed and validated for in vitro antiviral studies. Interestingly, rat HEV proved to be less sensitive to the antiviral activity of α-interferon, ribavirin and mycophenolic acid than genotype 3 HEV (a strain that infects humans. As a proof-of-concept, part of the C-terminal polymerase sequence of pLA-B350/luc was swapped with its genotype 3 HEV counterpart: the resulting chimeric replicon replicated with comparable efficiency as the wild-type construct, confirming that LA-B350 strain is amenable to humanization (replacement of certain sequences or motifs by their counterparts from human HEV strains. Finally, ribavirin effectively inhibited LA-B350 replication in athymic nude rats, confirming the suitability of the rat model for antiviral studies.

  8. Characterization of cis-Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone.

    Science.gov (United States)

    Liu, Zhong-Yu; Yu, Jiu-Yang; Huang, Xing-Yao; Fan, Hang; Li, Xiao-Feng; Deng, Yong-Qiang; Ji, Xue; Cheng, Meng-Li; Ye, Qing; Zhao, Hui; Han, Jian-Feng; An, Xiao-Ping; Jiang, Tao; Zhang, Bo; Tong, Yi-Gang; Qin, Cheng-Feng

    2017-11-01

    Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5'-SLA promoter and 5'-3' cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses.IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable

  9. Isolation and characterization of expression cDNA clones encoding antigens of Onchocerca volvulus infective larvae.

    Science.gov (United States)

    Unnasch, T R; Gallin, M Y; Soboslay, P T; Erttmann, K D; Greene, B M

    1988-01-01

    The isolation of recombinant cDNA clones expressing antigens found in Onchocerca volvulus infective larvae is described. To isolate such clones, an expression cDNA library constructed from adult O. volvulus RNA was screened with antiserum raised against infective larvae. One clone, designated lambda RAL-1 was characterized further. The recombinant antigen produced by lambda RAL-1 stimulates proliferation of peripheral blood mononuclear cells from O. volvulus infected humans. Lambda RAL-1 is derived from a 1450 bases message that encodes a protein with an apparent molecular weight of 42,000 in adult O. volvulus. The inserted DNA of lambda RAL-1 contains an open reading frame of 1008 bp. The amino acid sequence predicted by this open reading frame contains three repeats of the sequence KKPEDWD. The identification of clones such as lambda RAL-1 will provide quantities of purified antigens sufficient to begin to study the immune response to and explore the development of immunity against the infectious form of the parasite. Images PMID:2455736

  10. Automation of cDNA Synthesis and Labelling Improves Reproducibility

    OpenAIRE

    Daniel Klevebring; Marcus Gry; Johan Lindberg; Anna Eidefors; Joakim Lundeberg

    2009-01-01

    Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on c...

  11. The Central Role of the Matrix Protein in Nipah Virus Assembly and Morphogenesis

    Science.gov (United States)

    2007-03-23

    virus (MeV), Sendai virus (SeV), human parainfluenza viruses (hPIV) types 1-4, Simian virus 5 (SV5), Newcastle disease virus (NDV), Mumps virus, and...Malur, and A. K. Banerjee. 2001. Polarity of human parainfluenza virus type 3 infection in polarized human lung epithelial A549 cells: role of...Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV

  12. Classical Swine Fever Virus-Rluc Replicons

    DEFF Research Database (Denmark)

    Risager, Peter Christian; Belsham, Graham J.; Rasmussen, Thomas Bruun

    Classical swine fever virus (CSFV) is the etiologic agent of the severe porcine disease, classical swine fever. Unraveling the molecular determinants of efficient replication is crucial for gaining proper knowledge of the pathogenic traits of this virus. Monitoring the replication competence with...... type CSFV-Paderborn strain, as judged by qRT-PCR, was picked as the candidate and could potentially be useful as a tool for further downstream applications including investigation of CSFV non-structural proteins involvement in viral replication....... cells can be achieved using autonomously replicating genome constructs (replicons) containing a reporter gene that expresses a readily quantifiable enzyme. Here, a newly implemented cloning technique was applied to genome modification of the full-length CSFV cDNA previously inserted into a single......-copy bacterial artificial chromosome (BAC). This technique, the Red/ET counter-selection method, is based upon homologous recombination, thus obviating the need for internal restriction sites. Several CSFV replicons with deletions in regions encoding structural viral proteins considered non-essential for RNA...

  13. Molecular cloning of growth hormone encoding cDNA of Indian ...

    Indian Academy of Sciences (India)

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in ...

  14. cDNA cloning and characterization of a mannose-binding lectin from ...

    Indian Academy of Sciences (India)

    Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a ...

  15. Novel borna virus in psittacine birds with proventricular dilatation disease.

    Science.gov (United States)

    Honkavuori, Kirsi S; Shivaprasad, H L; Williams, Brent L; Quan, Phenix Lan; Hornig, Mady; Street, Craig; Palacios, Gustavo; Hutchison, Stephen K; Franca, Monique; Egholm, Michael; Briese, Thomas; Lipkin, W Ian

    2008-12-01

    Pyrosequencing of cDNA from brains of parrots with proventricular dilatation disease (PDD), an unexplained fatal inflammatory central, autonomic, and peripheral nervous system disease, showed 2 strains of a novel Borna virus. Real-time PCR confirmed virus presence in brain, proventriculus, and adrenal gland of 3 birds with PDD but not in 4 unaffected birds.

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

    Science.gov (United States)

    Colby, S M; Crock, J; Dowdle-Rizzo, B; Lemaux, P G; Croteau, R

    1998-03-03

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, beta-caryophyllene, alpha-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton delta-cadinene synthase (50% identity).

  17. Molecular cloning and characterization of a new cDNA sequence encoding a venom peptide from the centipede Scolopendra subspinipes mutilans.

    Science.gov (United States)

    Liu, Wanhong; Luo, Feng; He, Jing; Cao, Zhijian; Miao, Lixia

    2012-01-01

    Many studies have been performed on venomous peptides derived from animals. However, little of this research has focused on peptides from centipede venoms. Here, a venom gland cDNA library was successfully constructed for the centipede Scolopendra subspinipes mutilans. A new cDNA encoding the precursor of a venom peptide, named SsmTx, was cloned from the venomous gland cDNA library of the centipede S. subspinipes mutilans. The full-length SsmTx cDNA sequence is 465 nt, including a 249 nt ORF, a 45 nt 5' UTR and a 171 nt 3' UTR. There is a signal tail AATAAA 31 nt upstream of the poly (A) tail. The precursor nucleotide sequence of SsmTx encodes a signal peptide of 25 residues and a mature peptide of 57 residues, which is bridged by two pairs of disulfide bonds. SsmTx displays a unique cysteine motif that is completely different from that of other venomous animal toxins. This is the first reported cDNA sequence encoding a venom peptide from the centipede S. subspinipes mutilans.

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

    Directory of Open Access Journals (Sweden)

    Dixon Richard A

    2007-11-01

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

  19. Increased pathogenicity of rabies virus due to modification of a non-coding region.

    Science.gov (United States)

    Virojanapirom, Phatthamon; Yamada, Kentaro; Khawplod, Pakamatz; Nishizono, Akira; Hemachudha, Thiravat

    2016-11-01

    Sub-passaging of QS-05, a street rabies virus (RABV) isolate, in non-neuronal cells resulted in a virus with higher pathogenicity, QS-BHK-P7. Four full-length cDNA plasmids were constructed and the corresponding recombinant viruses were recovered: rQS-05, rQS-BHK-P7 and rQS05-2475G/rQS-BHK-P7-2475A (made by switching of intergenic P-M between these two backbones). rQS-BHK-P7-2475 A virus had eight instead of seven adenosines in its poly(A) sequence. Interestingly, mutant viruses with 6 or 8 adenosines infected more neuroblastoma cells than their parental ones. Mice that were infected intracerebrally and intramuscularly with rQS05-2475G and rQS-BHK-P7 exhibited highest mortality. However, mice infected with rQS-BHK-P7-2475AA had the shortest survival time. This study demonstrates that modifications in the non-coding region may play a role in determining the virulence of RABV.

  20. Monitoring the determinants of efficient viral replication using Classical Swine Fever Virus-reporter replicons

    DEFF Research Database (Denmark)

    Risager, Peter Christian; Everett, Helen; Crooke, Helen

    2012-01-01

    of the CSFV genome within cells can be achieved using autonomously replicating constructs (replicons) containing a reporter gene that expresses a readily quantifiable enzyme. Here, a newly implemented cloning technique was applied to genome modification of the fulllength CSFV cDNA previously inserted...... into a single-copy bacterial artificial chromosome (BAC). This technique, the Red/ET counter-selection method, is based upon homologous recombination, thus obviating the need for internal restriction sites or complex cloning strategies. Several CSFV replicons with deletions in regions encoding virus structural...... proteins considered non-essential for RNA replication were constructed and these deletions were replaced with an in-frame insertion of the Renilla luciferase (Rluc) sequence. RNA transcripts from these replicons should be translated as a single functional open reading frame. Full-genome cDNAs (~10-12,3 kb...

  1. Expression and stability of foreign epitopes introduced into 3A nonstructural protein of foot-and-mouth disease virus.

    Directory of Open Access Journals (Sweden)

    Pinghua Li

    Full Text Available Foot-and-mouth disease virus (FMDV is an aphthovirus that belongs to the Picornaviridae family and causes one of the most important animal diseases worldwide. The capacity of other picornaviruses to express foreign antigens has been extensively reported, however, little is known about FMDV. To explore the potential of FMDV as a viral vector, an 11-amino-acid (aa HSV epitope and an 8 aa FLAG epitope were introduced into the C-terminal different regions of 3A protein of FMDV full-length infectious cDNA clone. Recombinant viruses expressing the HSV or FLAG epitope were successfully rescued after transfection of both modified constructs. Immunofluorescence assay, Western blot and sequence analysis showed that the recombinant viruses stably maintained the foreign epitopes even after 11 serial passages in BHK-21 cells. The 3A-tagged viruses shared similar plaque phenotypes and replication kinetics to those of the parental virus. In addition, mice experimentally infected with the epitope-tagged viruses could induce tag-specific antibodies. Our results demonstrate that FMDV can be used effectively as a viral vector for the delivery of foreign tags.

  2. Research on computer virus database management system

    Science.gov (United States)

    Qi, Guoquan

    2011-12-01

    The growing proliferation of computer viruses becomes the lethal threat and research focus of the security of network information. While new virus is emerging, the number of viruses is growing, virus classification increasing complex. Virus naming because of agencies' capture time differences can not be unified. Although each agency has its own virus database, the communication between each other lacks, or virus information is incomplete, or a small number of sample information. This paper introduces the current construction status of the virus database at home and abroad, analyzes how to standardize and complete description of virus characteristics, and then gives the information integrity, storage security and manageable computer virus database design scheme.

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

    Directory of Open Access Journals (Sweden)

    Heinz Ruth A

    2003-09-01

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

  4. [Construction and identification of non-replication recombinant vaccinia virus co-expressing human papillomavirus type 16 L1/L2/E6/E7 proteins].

    Science.gov (United States)

    Huang, Wei; Tian, Hou-wen; Ren, Jiao; Fan, Jiang-tao; Zhao, Li; Bian, Tao; Lu, Zhen-hua; Ruan, Li

    2005-09-01

    To generate a human papillomavirus (HPV16) prophylactic and therapeutic vaccine candidate for cervical cancer. HPV16 major capsid protein L1 gene/minor capsid protein L2 gene and HPV16 early E6/E7 genes were inserted into a vaccinia virus expression vector. A strain of non-recombinant vaccinia virus containing the sequences was obtained through a homologous recombination and identified. DNA hybridization confirmed that the HPV16L1/L2/E6/E7 genes were integrated into vaccinia virus DNA. Western Blot result showed that full-length L1/L2/E6/E7 proteins were co-expressed in CEF cells infected with the recombinant virus. NTVJE6E7CKL1L2 could be taken as a candidate of prophylactic and therapeutic vaccine for HPV-associated tumors and their precancerous transformations.

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

    African Journals Online (AJOL)

    USER

    2010-08-16

    Aug 16, 2010 ... Abbreviations: LDPCR, Long distance polymerase chain reaction; MCK, muscle creatine kinase; MDM2, murine double minute 2; PCNA, proliferating cell nuclear antigen; PGM, phosphoglycerate mutase; TIGAR, TP53-induced glycolysis and apoptosis regulator; SCO2, synthesis of cytochrome c oxidase 2;.

  6. Construction of a full-length cDNA library and analysis of expressed ...

    African Journals Online (AJOL)

    sunny t

    2015-06-10

    Jun 10, 2015 ... White jute (Corchorus capsularis L.) is recognized as an important industrial raw material fibre crop owing to its elite characters. ... Cluster analysis allowed the identification of 61 unique sequences. These genes were ... textile and paper industry are interested in its potential as. *Corresponding author.

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

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-18

    May 18, 2009 ... secondary metabolism induced by methyl jasmonate. RNA was isolated from leaves of S. miltiorrhiza. ACKNOWLEDGEMENTS. The authors are grateful to the Funds provided by. Shandong Province Key Scientific and Technological. Project (No. 2007GG2NS02051). The authors express their great thanks ...

  8. Sensitive label-free electrochemical analysis of human IgE using an aptasensor with cDNA amplification.

    Science.gov (United States)

    Lee, Cheng-Yu; Wu, Kuan-Ying; Su, Hsiu-Li; Hung, Huan-Yi; Hsieh, You-Zung

    2013-01-15

    In this study, we developed an ultrasensitive label-free aptamer-based electrochemical biosensor, featuring a highly specific anti-human immunoglobulin E (IgE) aptamer as a capture probe, for human IgE detection. Construction of the aptasensor began with the electrodeposition of gold nanoparticles (AuNPs) onto a graphite-based screen-printed electrode (SPE). After immobilizing the thiol-capped anti-human IgE aptamer onto the AuNPs through self-assembly, we treated the electrode with mercaptohexanol (MCH) to ensure that the remaining unoccupied surfaces of the AuNPs would not undergo nonspecific binding. We employed a designed complementary DNA featuring a guanine-rich section in its sequence (cDNA G1) as a detection probe to bind with the unbound anti-human IgE aptamer. We measured the redox current of methylene blue (MB) to determine the concentration of human IgE in the sample. When the aptamer captured human IgE, the binding of cDNA G1 to the aptamer was inhibited. Using cDNA G1 in the assay greatly amplified the redox signal of MB bound to the detection probe. Accordingly, this approach allowed the linear range (coefficient of determination: 0.996) for the analysis of human IgE to extend from 1 to 100,000pM; the limit of detection was 0.16pM. The fabricated aptasensor exhibited good selectivity toward human IgE even when human IgG, thrombin, and human serum albumin were present at 100-fold concentrations. This method should be readily applicable to the detection of other analytes, merely by replacing the anti-human IgE aptamer/cDNA G1 pair with a suitable anti-target molecule aptamer and cDNA. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Sakaki Yoshiyuki

    2008-08-01

    Full Text Available Abstract 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Wallis James G

    2007-07-01

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

  13. Automation of cDNA Synthesis and Labelling Improves Reproducibility

    Directory of Open Access Journals (Sweden)

    Daniel Klevebring

    2009-01-01

    Full Text Available Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

  14. Automation of cDNA synthesis and labelling improves reproducibility.

    Science.gov (United States)

    Klevebring, Daniel; Gry, Marcus; Lindberg, Johan; Eidefors, Anna; Lundeberg, Joakim

    2009-01-01

    Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

  15. A drosophila full-length cDNA resource

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Cellular expression of a functional nodavirus RNA replicon from vaccinia virus vectors.

    Science.gov (United States)

    Ball, L A

    1992-04-01

    RNA replication provides a powerful means for the amplification of RNA, but to date it has been found to occur naturally only among RNA viruses. In an attempt to harness this process for the amplification of heterologous mRNAs, both an RNA replicase and its corresponding RNA templates have been expressed in functional form, using vaccinia virus-bacteriophage T7 RNA polymerase vectors. Plasmids were constructed which contained in 5'-to-3' order (i) a bacteriophage T7 promoter; (ii) a full-length cDNA encoding either the RNA replicase (RNA 1) or the coat protein (RNA 2) of flock house virus (FHV), (iii) a cDNA sequence that encoded the self-cleaving ribozyme of satellite tobacco ringspot virus, and (iv) a T7 transcriptional terminator. Both in vitro and in vivo, circular plasmids of this structure were transcribed by T7 RNA polymerase to produce RNAs with sizes that closely resembled those of the two authentic FHV genomic RNAs, RNA 1 and RNA 2. In baby hamster kidney cells that expressed authentic FHV RNA replicase, the RNA 2 (coat protein) transcripts were accurately replicated. Moreover, the RNA 1 (replicase) transcripts directed the synthesis of an enzyme that could replicate not only authentic virion-derived FHV RNA but also the plasmid-derived transcripts themselves. Under the latter conditions, replicative amplification of the RNA transcripts ensued and resulted in a high rate of synthesis of the encoded proteins. This successful expression from a DNA vector of the complex biological process of RNA replication will greatly facilitate studies of its mechanism and is a major step towards the goal of harnessing RNA replication for mRNA amplification.

  17. In-vitro and in-vivo phenotype of type Asia 1 foot-and-mouth disease viruses utilizing two non-RGD receptor recognition sites

    Science.gov (United States)

    2011-01-01

    Background Foot-and-mouth disease virus (FMDV) uses a highly conserved Arg-Gly-Asp (RGD) triplet for attachment to host cells and this motif is believed to be essential for virus viability. Previous sequence analyses of the 1D-encoding region of an FMDV field isolate (Asia1/JS/CHA/05) and its two derivatives indicated that two viruses, which contained an Arg-Asp-Asp (RDD) or an Arg-Ser-Asp (RSD) triplet instead of the RGD integrin recognition motif, were generated serendipitously upon short-term evolution of field isolate in different biological environments. To examine the influence of single amino acid substitutions in the receptor binding site of the RDD-containing FMD viral genome on virus viability and the ability of non-RGD FMDVs to cause disease in susceptible animals, we constructed an RDD-containing FMDV full-length cDNA clone and derived mutant molecules with RGD or RSD receptor recognition motifs. Following transfection of BSR cells with the full-length genome plasmids, the genetically engineered viruses were examined for their infectious potential in cell culture and susceptible animals. Results Amino acid sequence analysis of the 1D-coding region of different derivatives derived from the Asia1/JS/CHA/05 field isolate revealed that the RDD mutants became dominant or achieved population equilibrium with coexistence of the RGD and RSD subpopulations at an early phase of type Asia1 FMDV quasispecies evolution. Furthermore, the RDD and RSD sequences remained genetically stable for at least 20 passages. Using reverse genetics, the RDD-, RSD-, and RGD-containing FMD viruses were rescued from full-length cDNA clones, and single amino acid substitution in RDD-containing FMD viral genome did not affect virus viability. The genetically engineered viruses replicated stably in BHK-21 cells and had similar growth properties to the parental virus. The RDD parental virus and two non-RGD recombinant viruses were virulent to pigs and bovines that developed typical

  18. Capsid proteins from field strains of foot-and-mouth disease virus confer a pathogenic phenotype in cattle on an attenuated, cell-culture-adapted virus

    DEFF Research Database (Denmark)

    Bøtner, Anette; Kakker, Naresh K.; Barbezange, Cyril

    2011-01-01

    cells than the rescued parental O1K B64 virus. The two chimeric viruses displayed the expected antigenicity in serotype-specific antigen ELISAs. Following inoculation of each virus into cattle, the rescued O1K B64 strain proved to be attenuated whereas, with each chimeric virus, typical clinical signs......Chimeric foot-and-mouth disease viruses (FMDVs) have been generated from plasmids containing full-length FMDV cDNAs and characterized. The parental virus cDNA was derived from the cell-culture-adapted O1Kaufbeuren B64 (O1K B64) strain. Chimeric viruses, containing capsid coding sequences derived...

  19. cDNA - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data ...ontents List of cDNA in locus Data file File name: astra_cdna.zip File URL: ftp://ftp.biosciencedbc.jp/archiv...ing/transcriptional initiation regionss About This Database Database Description Download License Update History of This Database Site Policy | Contact Us cDNA - ASTRA | LSDB Archive ...

  20. Cloning and sequence analysis of H. contortus HC58cDNA gene ...

    African Journals Online (AJOL)

    The complete coding sequence of Hemonchus contortus HC58cDNA was generated by rapid amplification of cDNA ends and polymerase chain reaction using primers based on the 5\\' and 3\\' ends of the parasite mRNA, accession no. AF305964. The HC58cDNA gene was 851bp long, with open reading frame of 717bp, ...

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

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

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

  2. CD40 ligand and tdTomato-armed vaccinia virus for induction of antitumor immune response and tumor imaging.

    Science.gov (United States)

    Parviainen, S; Ahonen, M; Diaconu, I; Hirvinen, M; Karttunen, Å; Vähä-Koskela, M; Hemminki, A; Cerullo, V

    2014-02-01

    Oncolytic vaccinia virus is an attractive platform for immunotherapy. Oncolysis releases tumor antigens and provides co-stimulatory danger signals. However, arming the virus can improve efficacy further. CD40 ligand (CD40L, CD154) can induce apoptosis of tumor cells and it also triggers several immune mechanisms. One of these is a T-helper type 1 (Th1) response that leads to activation of cytotoxic T-cells and reduction of immune suppression. Therefore, we constructed an oncolytic vaccinia virus expressing hCD40L (vvdd-hCD40L-tdTomato), which in addition features a cDNA expressing the tdTomato fluorochrome for detection of virus, potentially important for biosafety evaluation. We show effective expression of functional CD40L both in vitro and in vivo. In a xenograft model of bladder carcinoma sensitive to CD40L treatment, we show that growth of tumors was significantly inhibited by the oncolysis and apoptosis following both intravenous and intratumoral administration. In a CD40-negative model, CD40L expression did not add potency to vaccinia oncolysis. Tumors treated with vvdd-mCD40L-tdtomato showed enhanced efficacy in a syngenic mouse model and induced recruitment of antigen-presenting cells and lymphocytes at the tumor site. In summary, oncolytic vaccinia virus coding for CD40L mediates multiple antitumor effects including oncolysis, apoptosis and induction of Th1 type T-cell responses.

  3. Evaluation of a 3A-truncated foot-and-mouth disease virus in pigs for its potential as a marker vaccine

    Science.gov (United States)

    2014-01-01

    Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals in the world. The disease can be effectively controlled by vaccination of susceptible animals with the conventional inactivated vaccine. However, one major concern of the inactivated FMD virus (FMDV) vaccine is that it does not allow serological discrimination between infected and vaccinated animals, and therefore interferes with serologic surveillance and the epidemiology of disease. A marker vaccine has proven to be of great value in disease eradication and control programs. In this study, we constructed a marker FMDV containing a deletion of residues 93 to 143 in the nonstructural protein 3A using a recently developed FMDV infectious cDNA clone. The marker virus, r-HN/3A93–143, had similar growth kinetics as the wild type virus in culture cell and caused a symptomatic infection in pigs. Pigs immunized with chemically inactivated marker vaccine were fully protected from the wild type virus challenge, and the potency of this marker vaccine was 10 PD50 (50% pig protective dose) per dose, indicating it could be an efficacious vaccine against FMDV. In addition, we developed a blocking ELISA targeted to the deleted epitope that could clearly differentiate animals infected with the marker virus from those infected with the wild type virus. These results indicate that a marker FMDV vaccine can be potentially developed by deleting an immunodominant epitope in NSP 3A. PMID:24885414

  4. Durable Expression of Minicircle DNA-Liposome-Delivered Androgen Receptor cDNA in Mice with Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Tian-You Chang

    2014-01-01

    Full Text Available Background. The most common gene-based cancer therapies involve the suppression of oncogenic molecules and enhancement of the expression of tumor-suppressor genes. Studies in noncancer disease animal models have shown that minicircle (MC DNA vectors are easy to deliver and that the proteins from said MC-carrying DNA vectors are expressed over a long period of time. However, delivery of therapeutic genes via a liposome-mediated, MC DNA complex has never been tested in vascular-rich hepatocellular carcinoma (HCC. Liposome-mediated DNA delivery exhibits high in vivo transfection efficiency and minimal systemic immune response, thereby allowing for repetitive interventions. In this study, we evaluated the efficacy of delivering an MC-liposome vector containing a 3.2 kb androgen receptor (AR; HCC metastasis suppressor cDNA into Hepatitis B Virus- (HBV- induced HCC mouse livers. Results. Protein expression and promoter luciferase assays revealed that liposome-encapsulated MC-AR resulted in abundant functional expression of AR protein (100 kD for up to two weeks. The AR cDNA was also successfully delivered into normal livers and diseased livers, where it was persistently expressed. In both normal livers and livers with tumors, the expression of AR was detectable for up to 60 days. Conclusion. Our results show that an MC/liposome delivery system might improve the efficacy of gene therapy in patients with HCC.

  5. Complete sequence of a cryptic virus from hemp (Cannabis sativa).

    Science.gov (United States)

    Ziegler, Angelika; Matoušek, Jaroslav; Steger, Gerhard; Schubert, Jörg

    2012-02-01

    Hemp (Cannabis sativa) was found to be a useful propagation host for hop latent virus, a carlavirus. However, when virus preparations were analysed by electron microscopy, along with the expected filamentous particles, spherical particles with a diameter of around 34 nm were found. RNA from virus preparations was purified, and cDNA was prepared and cloned. Sequence information was used to search databases, and the greatest similarity was found with Primula malacoides virus 1, a putative new member of the genus Partitivirus. The full sequences of RNA 1 and RNA 2 of this new hemp cryptic virus were obtained.

  6. A preferred region for recombinational patch repair in the 5' untranslated region of primer binding site-impaired murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Mikkelsen, J G; Lund, Anders Henrik; Kristensen, K D

    1996-01-01

    Transduction of primer binding site-impaired Akv murine leukemia virus-based retroviral vectors from the murine packaging cell lines psi-2 and omega E was studied. The efficiency of transduction of the neo marker of all mutated constructs was found to decrease by 5 to 6 orders of magnitude compared...... with that of the wild-type vector. Thirty-two of 60 transduced proviruses analyzed harbored a primer binding site sequence matching a glutamine tRNA primer. Sequence analysis of the regions flanking the glutamine tRNA primer binding site revealed a distinct pattern of nucleotide differences from the Akv-based vector......, suggesting the involvement of a specific endogenous virus-like sequence in patch repair rescue of the primer binding site mutants. The putative recombination partner RNA was found in virions from psi-2 cells as detected by analysis of glutamine tRNA-initiated cDNA and by sequence analysis of regions...

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

    Energy Technology Data Exchange (ETDEWEB)

    Tenchini, M.L.; Bossi, E.; Marchetti, L.; Malcovati, M. (Universita di Milano Via Viotti (Italy)); Lorenzetti, R. (M.M.D.R.I. Via R. Lepetit, Gerenzano (Italy))

    1992-04-01

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

  8. Ebola Virus and Marburg Virus

    Science.gov (United States)

    Ebola virus and Marburg virus Overview Ebola virus and Marburg virus are related viruses that cause hemorrhagic fevers — illnesses marked by severe bleeding (hemorrhage), organ failure and, in many ...

  9. Identification of a novel shrimp protein phosphatase and its association with latency-related ORF427 of white spot syndrome virus.

    Science.gov (United States)

    Lu, Liqun; Kwang, Jimmy

    2004-11-05

    To characterize the role of latency-associated ORF427 of white spot syndrome virus (WSSV), a shrimp cDNA library was constructed to screen interacting proteins of ORF427. Employing the yeast two-hybrid system, a novel shrimp protein phosphatase (named PPs), sharing 93% homology with human protein phosphatase 1, has been identified able to bind ORF427 in yeast. Through co-immunoprecipitation assays, the interaction between PPs and ORF427 was further confirmed both in vitro and in vivo. Interestingly, the novel shrimp protein phosphatase consists of only 199 aa and contains almost all the functional catalytic domains of human protein phosphatase, while it lacks the corresponding C-terminal non-catalytic sequence. Transcription and translation products of the identified cDNA can be detected in both normal and WSSV-infected shrimps; and PPs was found to localize mainly in the lysosome of shrimp cells. To characterize its function, the PPs cDNA was highly expressed in bacteria and the purified protein showed phosphatase activity when tested against pNPP in a standard phosphatase assay. Our results suggest that the identified protein phosphatase, PPs, may represent a novel member of protein phosphatase family and might be involved in the regulation of WSSV's life cycle through interaction with latency-related ORF427 of WSSV.

  10. cDNA table - RPD | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data ...ile URL: ftp://ftp.biosciencedbc.jp/archive/rpd/LATEST/rpd_cdna.zip File size: 15 KB Simple search URL http:...age About This Database Database Description Download License Update History of This Database Site Policy | Contact Us cDNA table - RPD | LSDB Archive ...

  11. Index of /data/medaka-full-length-cdna-db/20110331 [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Index of /data/medaka-full-length-cdna-db/20110331 Name Last modified Size Descript...a_full_length_c..> 26-Aug-2011 15:52 814K medaka_full_length_c..> 26-Aug-2011 15:52 8.7M Index of /data/medaka-full-length-cdna-db/20110331 ...

  12. Cloning and sequencing of complete τ-crystallin cDNA from ...

    Indian Academy of Sciences (India)

    Unknown

    embryonic tissues namely, brain, heart and GAM tissues. In each case, cDNA made by RT-PCR from total RNA isolated from the respective tissue was used as template. 2.6 Sequence analysis and primer design. The completed cDNA sequences were analysed for open reading frame using NCBI-ORF finder and homology.

  13. Cloning of a cDNA encoding the smallest neurofilament protein from the rat

    NARCIS (Netherlands)

    J-P. Julien (Jean-Pierre); K. Ramachadran; F.G. Grosveld (Frank)

    1985-01-01

    textabstractWe have cloned a cDNA coding for the smallest rat neurofilament protein. The cDNA is 861 nucleotides long coding for 287 amino acids from the internal alpha-helical region and the carboxy-terminal tail domain of the neurofilament protein. Comparison of the porcine, mouse and rat

  14. [Generation of Japanese Encephalitis Virus-like Particle Vaccine and Preliminary Evaluation of Its Protective Efficiency].

    Science.gov (United States)

    Zhang, Yanfang; Du, Ruikun; Huang, Shaomei; Zhang, Tao; Liu, Jinliang; Zhu, Bibo; Wang, Hualin; Deng, Fei; Cao, Shengbo

    2016-03-01

    The cDNA fragment of JEV prME gene was cloned into the baculovirus shuttle vector (bacmid) to construct a recombinant baculovirus vector, defined as AcBac-prME. Then the recombinant baculovirus Ac-prME was obtained by transfecting Sf9 cells with AcBac-prME. Western blot analysis and immunofluorescence results indicated that both prM and E proteins were efficiently expressed in Sf9 cells. Electron microscopy suggested that prME was assembled into JEV-VLPs. To further evaluate the potential of JEV-VLPs as vaccine, the mice were immunized with JEV-VLPs and then challenged with lethal JEV. The results of mice survival and pathological changes demonstrated that the JEV-VLPs performed complete protection against JEV-P3 strain and relieved pathological changes in the mice brain significant. This study suggest that JEV-VLPs would be a potential vaccine for Japanese encephalitis virus.

  15. Construction, expression and immunogenicity of a novel anti-hypertension angiotensin II vaccine based on hepatitis A virus-like particle

    OpenAIRE

    Ou, Xia; Guo, Lili; Wu, Jinyuan; Mi, Kai; Yin, Na; Zhang, Guangming; Li, Hongjun; Sun, Maosheng

    2013-01-01

    Hypertension is a serious worldwide public health problem. The aim of this study is to design anti-hypertension angiotensin II (Ang II) vaccine using molecular biology and immunological method. This novel anti-hypertension vaccine, which is a chimeric protein named pHAV–4Ang IIs, presents four successive repeated Ang IIs as the functional epitope on the surface of the hepatitis A virus-like particle(HAVLP). In this study, pHAV–4Ang IIs was expressed using Bac-to-Bac Baculovirus Expression Sys...

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

    Science.gov (United States)

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

    2015-01-01

    The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML) using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM) cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30%) had MYC as the only transgene, and seven mice (70%) had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy. PMID:26606454

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

    Science.gov (United States)

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

    2013-03-01

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

  18. Complete Genome Sequence of Border Disease Virus Genotype 3 Strain Gifhorn

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Höper, Dirk; Beer, Martin

    2014-01-01

    The complete genome sequence of the genotype 3 border disease virus strain Gifhorn has been determined; this strain was originally isolated from pigs. This represents the consensus sequence for the virus used to produce the bacterial artificial chromosome (BAC) cDNA clone pBeloGif3, which yields...

  19. Characterization and simulation of cDNA microarray spots using a novel mathematical model

    Directory of Open Access Journals (Sweden)

    Lee Yong Sung

    2007-12-01

    Full Text Available Abstract Background The quality of cDNA microarray data is crucial for expanding its application to other research areas, such as the study of gene regulatory networks. Despite the fact that a number of algorithms have been suggested to increase the accuracy of microarray gene expression data, it is necessary to obtain reliable microarray images by improving wet-lab experiments. As the first step of a cDNA microarray experiment, spotting cDNA probes is critical to determining the quality of spot images. Results We developed a governing equation of cDNA deposition during evaporation of a drop in the microarray spotting process. The governing equation included four parameters: the surface site density on the support, the extrapolated equilibrium constant for the binding of cDNA molecules with surface sites on glass slides, the macromolecular interaction factor, and the volume constant of a drop of cDNA solution. We simulated cDNA deposition from the single model equation by varying the value of the parameters. The morphology of the resulting cDNA deposit can be classified into three types: a doughnut shape, a peak shape, and a volcano shape. The spot morphology can be changed into a flat shape by varying the experimental conditions while considering the parameters of the governing equation of cDNA deposition. The four parameters were estimated by fitting the governing equation to the real microarray images. With the results of the simulation and the parameter estimation, the phenomenon of the formation of cDNA deposits in each type was investigated. Conclusion This study explains how various spot shapes can exist and suggests which parameters are to be adjusted for obtaining a good spot. This system is able to explore the cDNA microarray spotting process in a predictable, manageable and descriptive manner. We hope it can provide a way to predict the incidents that can occur during a real cDNA microarray experiment, and produce useful data for

  20. A cDNA library of the eutardigrade Hypsibius klebelsbergi Mihelčič, 1959 and analysis of the actin gene

    Directory of Open Access Journals (Sweden)

    Hartmut GREVEN

    2007-09-01

    Full Text Available A cDNA library was constructed from the glacier-dwelling eutardigrade Hypsibius klebelsbergi from more than 2000 individuals collected in the Austrian Central Alps. RNA, DNA and proteins were successively isolated by the Trizol®-method. From the RNA preparation a cDNA library was constructed with the cDNA inserted unidirectionally in the phagemid expression vector TriplEx2. The primary gene library had a titre of 107 pfu ml-1 and the final amplified gene library a titre of 6×109 pfu ml-1. The average insert length was about 1.6 kb. The partial sequence of H. klebelsbergi actin (746 bp showed highest similarity to GenBank data of Drosophila melanogaster actin at the nucleic acid level (84.9% and at the amino acid level (98%. Compared with actin fragments of the eutardigrades Ramazzottius oberhaeuseri (450 bp and Macrobiotus sp. (453 bp the identities were 85% - 81% and 100% - 98% with respect to the nucleic/amino acids. Identity with actin fragments (359 bp of Hypsibius dujardini from GenBank was 96% - 100%.

  1. In vitro transcripts of wild-type and fluorescent protein-tagged triticum mosaic virus (family potyviridae) are biologically active in wheat

    Science.gov (United States)

    Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat, and the progeny virus was efficiently transmitted by wheat curl m...

  2. Identification of Eukaryotic Open Reading Frames in Metagenomic cDNA Libraries Made from Environmental Samples†

    Science.gov (United States)

    Grant, Susan; Grant, William D.; Cowan, Don A.; Jones, Brian E.; Ma, Yanhe; Ventosa, Antonio; Heaphy, Shaun

    2006-01-01

    Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at −20°C. Protocols for library construction were established on total RNA extracted from Acanthamoeba polyphaga trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes. PMID:16391035

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

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

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

  4. [Construction and application of a lentiviral vector of single-chain variable fragment antibody against human hepatocyte growth factor receptor].

    Science.gov (United States)

    Chen, Yonghua; Guo, Jia; Yin, Yanxin; Jiang, Ming; Zhu, Hongsheng; Zhang, Guodong; Li, Bingyu

    2014-09-01

    To construct a lentiviral expression vector carrying the single-chain variable fragment (scFv) antibody against human hepatocyte growth factor receptor (HGFR), express it in transfected HEK293 cells, and observe its biological function of specific binding to antigen. The variable regions of the heavy chain (VH) and light chain (VL) genes were amplified directly from the cDNA of hybridoma cell line 8E8 secreting mouse anti-human HGFR antibody and assembled using the splice overlap extension-PCR (SOE-PCR). The constructed HGFR-scFv gene with the signal peptide SP-VH-linker-VL was ligated into the cloning vector pCR-Blunt. After cut off from pCR-Blunt using enzyme digestion, HGFR-scFv gene was subcloned into the lentiviral transfer vector pRRL-CMV, which was identified by the restriction enzyme digestion and sequencing. The lentiviral expression vector pRRL HGFR-scFv was then tansfected together with the packaging plasmids into HEK293T cells to obtain virus particles, and green fluorescent protein (GFP) expression was detected under a fluorescent microscope. Then the virus particles were used to infect HEK293 cells. The scFv expression was detected by RT-PCR and its biological affinity as antibody was measured by ELISA. The lentiviral expression vector pRRL HGFR-scFv was constructed correctly. After HEK293T cells were transfected with the pRRL HGFR-scFv plasmid, the GFP was visible. After HEK293 cells were infected with virus particles, the scFv antibody expressed could bind to HGFR specifically. The lentiviral expression vector of HGFR-scFv was constructed successfully, which would help to study the important role of HGFR in following experiments.

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

    Science.gov (United States)

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

    2009-01-01

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

  6. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon [College of Medicine, Univ. of Korea, Seoul (Korea, Republic of)

    2003-07-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  7. Magnetic purification of biotinylated cDNA removes false priming and ensures strand-specificity of RT-PCR for enteroviral RNAs.

    Science.gov (United States)

    Boncristiani, Humberto F; Rossi, Reginaldo D; Criado, Miriã Ferreira; Furtado, Felipe Magalhães; Arruda, Eurico

    2009-10-01

    The detection of replicative intermediate RNAs as markers of active replication of RNA viruses is an essential tool to investigate pathogenesis in acute viral infections, as well as in their long-term sequelae. In this regard, strand-specific PCR has been used widely to distinguish (-) and (+) enteroviral RNAs in pathogenesis studies of diseases such as dilated cardiomyopathy. It has been generally assumed that oligonucleotide-primed reverse transcription of a given RNA generates only the corresponding specific cDNA, thus assuring the specificity of a PCR product amplified from it. Nevertheless, such assumed strand-specificity is a fallacy, because falsely primed cDNAs can be produced by RNA reverse transcription in the absence of exogenously added primers, (cDNA(primer)(-)), and such falsely primed cDNAs are amplifiable by PCR in the same way as the correctly primed cDNAs. Using as a prototype the coxsackievirus B5 (CVB5), a (+) strand RNA virus, it was shown that cDNA(primer)(-) renders the differential detection of viral (-) and (+) RNAs by conventional PCR virtually impossible, due to gross non-specificity. Using in vitro transcribed CVB5 RNAs (+) and (-), it was shown that cDNA(primer)(-) could be removed effectively by magnetic physical separation of correctly primed biotinylated cDNA. Such strategy enabled truly strand-specific detection of RNA (-) and (+), not only for CVB5, but also for other non-polio enteroviruses. These findings indicate that previous conclusions supporting a role for the persistence of actively replicating enterovirus in the pathogenesis of chronic myocarditis should be regarded with strong skepticism and purification of correctly primed cDNA should be used for strand-specific PCR of viral RNA in order to obtain reliable information on this important subject.

  8. Unprocessed foot-and-mouth disease virus capsid precursor displays discontinuous epitopes involved in viral neutralization.

    Science.gov (United States)

    Sáiz, J C; Cairó, J; Medina, M; Zuidema, D; Abrams, C; Belsham, G J; Domingo, E; Vlak, J M

    1994-07-01

    A foot-and-mouth disease virus (FMDV) cDNA cassette containing sequences encoding the capsid precursor P1, peptide 2A and a truncated 2B (abbreviated P1-2A) of type C FMDV, has been modified to generate the authentic amino terminus and the myristoylation signal. This construct has been used to produce a recombinant baculovirus (AcMM53) which, upon infection of Spodoptera frugiperda insect cells, expressed a recombinant P1-2A precursor with a high yield. This polyprotein reacted with neutralizing monoclonal antibodies (MAbs) that bind to continuous epitopes of the major antigenic site A (also termed site 1) of capsid protein VP1. Unexpectedly, it also reacted with neutralizing MAbs which define complex, discontinuous epitopes previously identified on FMDV particles. The reactivity of MAbs with P1-2A was quantitatively similar to their reactivity with intact virus and, in both cases, the reactivity with MAbs that recognized discontinuous epitopes was lost upon heat denaturation of the antigen. The finding that a capsid precursor may fold in such a way as to maintain discontinuous epitopes involved in virus neutralization present on the virion surface opens the possibility of using unprocessed capsid precursors as novel antiviral immunogens.

  9. A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice.

    Science.gov (United States)

    Yun, Sang-Im; Song, Byung-Hak; Kim, Jin-Kyoung; Yun, Gil-Nam; Lee, Eun-Young; Li, Long; Kuhn, Richard J; Rossmann, Michael G; Morrey, John D; Lee, Young-Min

    2014-07-01

    Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, 1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

  10. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones and quality score...s Data detail Data name 5'-end sequences of budding yeast full-length cDNA clones and quality score...or-capping method, the sequence quality score generated by the Phred software, and links to SGD, dbEST and U...es. FASTA format. Quality Phred's quality score About This Database Database Desc...g yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive ...

  11. Factors influencing cDNA microarray hybridization on silylated glass slides

    NARCIS (Netherlands)

    Franssen-Hal, van N.L.W.; Vorst, O.F.J.; Kramer, E.H.M.; Hall, R.D.; Keijer, J.

    2002-01-01

    cDNA microarray technology is becoming the technique of choice for studying gene expression and gene expression patterns. Although experimental protocols are available, only limited methodological information on microarray manufacture, hybridization, and signal interpretation has been published. The

  12. cDNA cloning and mRNA expression of heat shock protein 70 gene ...

    African Journals Online (AJOL)

    In this study, the full-length heat shock protein 70 of Tegillarca granosa was cloned from cDNA library by rapid amplification of cDNA end (RACE). The open reading frame (ORF) of heat shock protein 70 was 1968 bp, and it encoded a protein of 655 amino acids with a predicted molecular weight of 71.48 kDa and an ...

  13. Cloning of rat aorta lysyl oxidase cDNA: Complete codons and predicted amino acid sequence

    Energy Technology Data Exchange (ETDEWEB)

    Trackman, P.C.; Pratt, A.M.; Wolanski, A.; Tang, Shiowshih; Offner, G.D.; Troxler, R.F.; Kagan, H.M. (Boston Univ. School of Medicine, MA (USA))

    1990-05-22

    Lysyl oxidase cDNA clones were identified by their reactivity with anti-bovine lysyl oxidase in a neonatal rat aorta cDNA {lambda}gt11 expression library. A 500-bp cDNA sequence encoding four of six peptides derived from proteolytic digests of bovine aorta lysyl oxidase was found from the overlapping cDNA sequences of two positive clones. The library was rescreened with a radiolabeled cDNA probe made from one of these clones, thus identifying an additional 13 positive clones. Sequencing of the largest two of these overlapping clones resulted in 2,672 bp of cDNA sequence containing partial 5{prime}- and 3{prime}-untranslated sequences of 286 and 1,159 nucleotides, respectively, and a complete open reading frame of 1,227 bp encoding a polypeptide of 409 amino acids (46 kDa), consistent with the 48 {plus minus} 3 kDa cell-free translation product of rat smooth muscle cell RNA that was immunoprecipitated by anti-bovine lysyl oxidase. The rat aorta cDNA-derived amino acid sequence contains the sequence of each of the six peptides isolated and sequenced from the 32-kDa bovine aorta enzyme, including the C-terminal peptide with sequence identity of 96%. Southern blotting of rat genomic DNA with lysyl oxidase cDNA probes indicated that the lysyl oxidase gene is located at a single locus and does not appear to be a member of a multigene family. A potential stem-loop structure was found in the 3{prime}-untranslated region of the cDNA. The deduced amino acid sequence contains a putative signal peptide, in addition to sequences that are similar to those of other known copper proteins.

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

    Science.gov (United States)

    Venkatesh, Balakrishnan; Hettwer, Ursula; Koopmann, Birger; Karlovsky, Petr

    2005-01-01

    Background Gene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Commonly used protocols for gel-based transcript profiling are cDNA differential display (DDRT-PCR) and cDNA-AFLP. Both methods have been used merely as qualitative gene discovery tools so far. Results We developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer and detector signals are converted into virtual gel images suitable for semi-automatic analysis. Data processing consists of four steps: (i) cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, (ii) intensity of bands is determined by densitometry, (iii) densitometric values are normalized, and (iv) intensity ratio is calculated for each pair of corresponding bands. Transcription profiles are represented by sets of intensity ratios (control vs. treatment) for cDNA fragments defined by primer combination and DNA mobility. We demonstrated the procedure by analyzing DDRT-PCR data on the effect of secondary metabolites of oilseed rape Brassica napus on the transcriptome of the pathogenic fungus Leptosphaeria maculans. Conclusion We developed a data processing procedure for the quantitative analysis of amplified cDNA fragments separated by electrophoresis. The system utilizes common software and provides an open-end alternative to DNA microarray analysis of the transcriptome. It is expected to work equally well with DDRT-PCR and cDNA-AFLP data and be useful particularly in reseach on organisms for which microarray analysis is not available or economical. PMID:15807902

  15. Modulation of Translation Initiation Efficiency in Classical Swine Fever Virus

    DEFF Research Database (Denmark)

    Friis, Martin Barfred; Rasmussen, Thomas Bruun; Belsham, Graham

    2012-01-01

    Modulation of translation initiation efficiency on classical swine fever virus (CSFV) RNA can be achieved by targeted mutations within the internal ribosome entry site (IRES). In this study, cDNAs corresponding to the wild type (wt) or mutant forms of the IRES of CSFV strain Paderborn were...... in vitro and electroporated into porcine PK15 cells. Rescued mutant viruses were obtained from RNAs that contained mutations within domain IIIf which retained more than 75% of wt translation efficiency. Sequencing of cDNA generated from these rescued viruses verified the maintenance of the introduced...... changes within the IRES. The growth characteristics of each rescued mutant virus were compared to that of the wt virus. It was shown that viable mutant viruses with reduced translation initiation efficiency can be designed and generated and that viruses containing mutations within domain IIIf of the IRES...

  16. Constructed Wetlands

    Science.gov (United States)

    these systems can improve water quality, engineers and scientists construct systems that replicate the functions of natural wetlands. Constructed wetlands are treatment systems that use natural processes

  17. Nucleotide sequence of Phaseolus vulgaris L. alcohol dehydrogenase encoding cDNA and three-dimensional structure prediction of the deduced protein.

    Science.gov (United States)

    Amelia, Kassim; Khor, Chin Yin; Shah, Farida Habib; Bhore, Subhash J

    2015-01-01

    Common beans (Phaseolus vulgaris L.) are widely consumed as a source of proteins and natural products. However, its yield needs to be increased. In line with the agenda of Phaseomics (an international consortium), work of expressed sequence tags (ESTs) generation from bean pods was initiated. Altogether, 5972 ESTs have been isolated. Alcohol dehydrogenase (AD) encoding gene cDNA was a noticeable transcript among the generated ESTs. This AD is an important enzyme; therefore, to understand more about it this study was undertaken. The objective of this study was to elucidate P. vulgaris L. AD (PvAD) gene cDNA sequence and to predict the three-dimensional (3D) structure of deduced protein. positive and negative strands of the PvAD cDNA clone were sequenced using M13 forward and M13 reverse primers to elucidate the nucleotide sequence. Deduced PvAD cDNA and protein sequence was analyzed for their basic features using online bioinformatics tools. Sequence comparison was carried out using bl2seq program, and tree-view program was used to construct a phylogenetic tree. The secondary structures and 3D structure of PvAD protein were predicted by using the PHYRE automatic fold recognition server. The sequencing results analysis showed that PvAD cDNA is 1294 bp in length. It's open reading frame encodes for a protein that contains 371 amino acids. Deduced protein sequence analysis showed the presence of putative substrate binding, catalytic Zn binding, and NAD binding sites. Results indicate that the predicted 3D structure of PvAD protein is analogous to the experimentally determined crystal structure of s-nitrosoglutathione reductase from an Arabidopsis species. The 1294 bp long PvAD cDNA encodes for 371 amino acid long protein that contains conserved domains required for biological functions of AD. The predicted deduced PvAD protein's 3D structure reflects the analogy with the crystal structure of Arabidopsis thaliana s-nitrosoglutathione reductase. Further study is required

  18. The construction and characterization of the bi-directional promoter between pp38 gene and 1.8-kb mRNA transcripts of Marek's disease viruses

    Directory of Open Access Journals (Sweden)

    Ding Jiabo

    2009-11-01

    Full Text Available Abstract Background Marek's disease virus (MDV has a bi-directional promoter between pp38 gene and 1.8-kb mRNA transcripts. By sequencing for the promoters from 8 different strains (CVI988, 814, GA, JM, Md5, G2, RB1B and 648A, it is found, comparing with the other 7 MDV strains, CVI988 has a 5-bp (from -628 to -632 deletion in this region, which caused a Sp1 site destroyed. In order to analysis the activity of the promoter, the complete bi-directional promoters from GA and CVI988 were, respectively, cloned into pCAT-Basic vector in both directions for the recombinants pPGA(pp38-CAT, pPGA(1.8 kb-CAT, pPCVI(pp38-CAT and pPCVI(1.8 kb-CAT. The complete promoter of GA was divided into two single-direction promoters from the replication of MDV genomic DNA, and cloned into pCAT-Basic for pdPGA(pp38-CAT and pdPGA(1.8 kb-CAT as well. The above 6 recombinants were then transfected into chicken embryo fibroblasts (CEFs infected with MDV, and the activity of chloramphenicol acetyltransferase (CAT was measured from the lysed CEFs 48 h post transfection. Results The results showed the activity of the divided promoters was decreased on both directions. In 1.8-kb mRNA direction, it is nearly down to 2.4% (19/781 of the whole promoter, while it keeps 65% (34/52 activity in pp38 direction. The deletion of Sp1 site in CVI988 causes the 20% activity decreased, and has little influence in pp38 direction. Conclusion The present study confirmed their result, and the promoter for the 1.8-kb mRNA transcripts is a much stronger promoter than that in the orientation for pp38.

  19. Construction and immunogenicity analysis of Lactobacillus plantarum expressing a porcine epidemic diarrhea virus S gene fused to a DC-targeting peptide.

    Science.gov (United States)

    Huang, Ke-Yan; Yang, Gui-Lian; Jin, Yu-Bei; Liu, Jing; Chen, Hong-Liang; Wang, Peng-Bo; Jiang, Yan-Long; Shi, Chun-Wei; Huang, Hai-Bin; Wang, Jian-Zhong; Wang, Guan; Kang, Yuan-Huan; Yang, Wen-Tao; Wang, Chun-Feng

    2017-12-27

    Porcine epidemic diarrhea virus (PEDV) is one of the most important causative pathogens of swine diarrhea, which is widely prevalent throughout the world and is responsible for significant economic losses in the commercial pig industry, both domestic and abroad. The spike (S) protein in the PEDV capsid structure can carry the major B lymphocyte epitope, which induces production of neutralizing antibodies and provides immunoprotective effects. Moreover, the conserved region encoded by the S gene can be considered a target for establishing a new diagnostic method and is a new candidate for vaccine design. In this study, use of anchorin pgsA' allowed the fusion protein of S-DCpep to express on the surface of recombinant Lactobacillus plantarum (NC8-pSIP409-pgsA'-S-DCpep) NC8 strain. Mice were immunized by lavage administration of the recombinant NC8-pSIP409-pgsA'-S-DCpep, which was observed to induce DC activation and high production of sIgA and IgG antibodies in experimental animals, while also eliciting production of significantly more IgA + B220 + B cells. More importantly, secretion of cytokines IFN-γ, IL-4 and IL-17 in mice that were vaccinated with NC8-pSIP409-pgsA'-S-DCpep was remarkably increased. The results of our study suggest that NC8-pSIP409-pgsA'-S-DCpep potently triggers cellular and humoral immune responses. The obtained experimental results can provide a theoretical basis that lays the foundation for production of a novel oral vaccine against PED. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. The full-ORF clone resource of the German cDNA Consortium

    Directory of Open Access Journals (Sweden)

    Michel Guenter

    2007-10-01

    Full Text Available Abstract Background With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. Results Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen. A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. Conclusion The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available

  1. cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

    Directory of Open Access Journals (Sweden)

    F.H.R. Fagundes

    2010-03-01

    Full Text Available To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR, we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49, from the venom of Crotalus durissus collilineatus (Cdc PLA2. The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC. The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.

  2. Cloning and expression of cDNA encoding a cysteine protease inhibitor from clamworm and its possible use in managing Anoplophora glabripennis Motschulsky (Coleoptera: Cerambycidae).

    Science.gov (United States)

    Li, Shengnan; Guo, Daosen; Zhao, Boguang; Ye, Jianling; Tian, Jie; Ren, Wenqing; Ju, Yunwei; Cui, Peng; Li, Ronggui

    2010-08-01

    A cDNA encoding cysteine protease inhibitor (CPI) was isolated from the cDNA library of clamworm Perinereis aibuhitensis Grube. The deduced amino acid sequence analysis showed that the protein had 51%, 48%, and 48% identity with Zgc:153129 from Danio rerio, cystatin B from Theromyzon tessulatum and ChainA, stefin B tetramer from Homo sapiens, respectively. The gene was cloned into the intracellular expression vector pET-15b and expressed in Escherichia coli. The recombinant CPI (PA-CPI) was purified by affinity chromatography on Ni-charged resin and ion-exchange chromatography on DEAE-Sepharose FF. The relative molecular mass of PA-CPI was 16 KDa deduced by SDS-PAGE. Activity analysis showed that the recombinant protein could inhibit the proteolytic activity of papain. A constitutive and secretive expression vector was also constructed, and the cDNA encoding CPI was subcloned into the vector for extracellular expression. Western blotting analysis results showed that the PA-CPI was secreted into the medium. Bioassay demonstrated that E. coli DH5alpha harboring pUC18ompAcat-CPI showed a significant difference in mortality to the Asian longhorned beetle Anoplophora glabripennis compared with untransformed E. coli DH5alpha and control.

  3. Identification of Potential Vaccine and Drug Target Candidates by Expressed Sequence Tag Analysis and Immunoscreening of Onchocerca volvulus Larval cDNA Libraries

    Science.gov (United States)

    Lizotte-Waniewski, Michelle; Tawe, Wilson; Guiliano, David B.; Lu, Wenhong; Liu, Jing; Williams, Steven A.; Lustigman, Sara

    2000-01-01

    The search for appropriate vaccine candidates and drug targets against onchocerciasis has so far been confronted with several limitations due to the unavailability of biological material, appropriate molecular resources, and knowledge of the parasite biology. To identify targets for vaccine or chemotherapy development we have undertaken two approaches. First, cDNA expression libraries were constructed from life cycle stages that are critical for establishment of Onchocerca volvulus infection, the third-stage larvae (L3) and the molting L3. A gene discovery effort was then initiated by random expressed sequence tag analysis of 5,506 cDNA clones. Cluster analyses showed that many of the transcripts were up-regulated and/or stage specific in either one or both of the cDNA libraries when compared to the microfilariae, L2, and both adult stages of the parasite. Homology searches against the GenBank database facilitated the identification of several genes of interest, such as proteinases, proteinase inhibitors, antioxidant or detoxification enzymes, and neurotransmitter receptors, as well as structural and housekeeping genes. Other O. volvulus genes showed homology only to predicted genes from the free-living nematode Caenorhabditis elegans or were entirely novel. Some of the novel proteins contain potential secretory leaders. Secondly, by immunoscreening the molting L3 cDNA library with a pool of human sera from putatively immune individuals, we identified six novel immunogenic proteins that otherwise would not have been identified as potential vaccinogens using the gene discovery effort. This study lays a solid foundation for a better understanding of the biology of O. volvulus as well as for the identification of novel targets for filaricidal agents and/or vaccines against onchocerciasis based on immunological and rational hypothesis-driven research. PMID:10816503

  4. Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Y. Cui

    2008-05-01

    Full Text Available Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1 allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+, expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG, 267-277 (NYHAVNIVGYG and 284-303 (YWIVRNSWDTTWGDSGYGYF. Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%, an extended strand (17.13%, a ß turn (5.61%, and a random coil (43.30%. A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

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

    Directory of Open Access Journals (Sweden)

    Alamar Santiago

    2009-09-01

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

  6. Construction management

    CERN Document Server

    Pellicer, Eugenio; Teixeira, José C; Moura, Helder P; Catalá, Joaquín

    2014-01-01

    The management of construction projects is a wide ranging and challenging discipline in an increasingly international industry, facing continual challenges and demands for improvements in safety, in quality and cost control, and in the avoidance of contractual disputes. Construction Management grew out of a Leonardo da Vinci project to develop a series of Common Learning Outcomes for European Managers in Construction. Financed by the European Union, the project aimed to develop a library of basic materials for developing construction management skills for use in a pan-European context. Focused exclusively on the management of the construction phase of a building project from the contractor's point of view, Construction Management covers the complete range of topics of which mastery is required by the construction management professional for the effective delivery of new construction projects. With the continued internationalisation of the construction industry, Construction Management will be required rea...

  7. cDNA2Genome: A tool for mapping and annotating cDNAs

    Directory of Open Access Journals (Sweden)

    Suhai Sandor

    2003-09-01

    Full Text Available Abstract Background In the last years several high-throughput cDNA sequencing projects have been funded worldwide with the aim of identifying and characterizing the structure of complete novel human transcripts. However some of these cDNAs are error prone due to frameshifts and stop codon errors caused by low sequence quality, or to cloning of truncated inserts, among other reasons. Therefore, accurate CDS prediction from these sequences first require the identification of potentially problematic cDNAs in order to speed up the posterior annotation process. Results cDNA2Genome is an application for the automatic high-throughput mapping and characterization of cDNAs. It utilizes current annotation data and the most up to date databases, especially in the case of ESTs and mRNAs in conjunction with a vast number of approaches to gene prediction in order to perform a comprehensive assessment of the cDNA exon-intron structure. The final result of cDNA2Genome is an XML file containing all relevant information obtained in the process. This XML output can easily be used for further analysis such us program pipelines, or the integration of results into databases. The web interface to cDNA2Genome also presents this data in HTML, where the annotation is additionally shown in a graphical form. cDNA2Genome has been implemented under the W3H task framework which allows the combination of bioinformatics tools in tailor-made analysis task flows as well as the sequential or parallel computation of many sequences for large-scale analysis. Conclusions cDNA2Genome represents a new versatile and easily extensible approach to the automated mapping and annotation of human cDNAs. The underlying approach allows sequential or parallel computation of sequences for high-throughput analysis of cDNAs.

  8. [Cloning and tissue expression pattern analysis of the human citrate synthase cDNA].

    Science.gov (United States)

    Liu, Q; Yu, L; Han, X F; Fu, Q; Zhang, J X; Tang, H; Zhao, S Y

    2000-09-01

    Tricarboxylic acid (TCA) cycle is an important way to generate ATP, which is widely distributed in the cells of animal, plant or microorganism. It catalyses the catabolism of sugar as well as protein and fat. Citrate synthase plays a key role in regulating TCA cycle and is responsible for catalysing the synthesis of citrate from oxaloacetate and acetyl CoA. Screening of genomic informatics was performed by using pig citrate synthase cDNA as a probe and a contig which is 1636 bp long and has highly homologous to the pig citrate synthase cDNA was obtained from selected ESTs with the ASSEMBLY program. According to the sequence of this contig, a pair of primers was designed and used to amplify cDNA libraries. A 1492 bp cDNA containing an open reading frame encoding 466 amino acids was cloned from human testis and skeletal muscle cDNA libraries. The deduced amino acid sequence of the cDNA showed 95%, 92% and 60.9% identity to pig, chicken and yeast citrate synthase respectively. Because the deduced amino acids sequence contains a highly conserved motif of citrate synthase from three different species, it is believed that this cDNA may be a transcript of human citrate synthase gene. Northern analysis showed that the human citrate synthase was expressed at high level in heart and muscle, at middle level in brain, kidney and pancreas tissues, not detectable in thymus and small intestine tissues, and at low level in other nine tested human tissues.

  9. ECHO virus

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/001340.htm ECHO virus To use the sharing features on this page, please enable JavaScript. Enteric cytopathic human orphan (ECHO) viruses are a group of viruses that can lead ...

  10. An introduction to computer viruses

    Energy Technology Data Exchange (ETDEWEB)

    Brown, D.R.

    1992-03-01

    This report on computer viruses is based upon a thesis written for the Master of Science degree in Computer Science from the University of Tennessee in December 1989 by David R. Brown. This thesis is entitled An Analysis of Computer Virus Construction, Proliferation, and Control and is available through the University of Tennessee Library. This paper contains an overview of the computer virus arena that can help the reader to evaluate the threat that computer viruses pose. The extent of this threat can only be determined by evaluating many different factors. These factors include the relative ease with which a computer virus can be written, the motivation involved in writing a computer virus, the damage and overhead incurred by infected systems, and the legal implications of computer viruses, among others. Based upon the research, the development of a computer virus seems to require more persistence than technical expertise. This is a frightening proclamation to the computing community. The education of computer professionals to the dangers that viruses pose to the welfare of the computing industry as a whole is stressed as a means of inhibiting the current proliferation of computer virus programs. Recommendations are made to assist computer users in preventing infection by computer viruses. These recommendations support solid general computer security practices as a means of combating computer viruses.

  11. Construction and analysis of subtractive hybridization library of ...

    African Journals Online (AJOL)

    Eightweek- old C57BL/6 and A/J mouse were infected with S. suis serotype 2, a 200-μL volume of a bacterial suspension (1 × 108 CFU/mL) was administrated by intraperitoneal injection to the mice, and cDNA subtraction libraries were constructed by suppression subtraction hybridization (SSH). Genes involved in immune ...

  12. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

    OpenAIRE

    Bertagnoli, Stéphane; Gelfi, Jacqueline; Le Gall, Ghislaine; Boilletot, Eric; Vautherot, Jean-François; Rasschaert, Denis; Laurent, Sylvie; Petit, Frédérique; Boucraut-Baralon, Corine; Milon, Alain

    1996-01-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma vir...

  13. Identifikasi Molekuler Tobacco mosaic virus pada Anggrek di Sleman, Yogyakarta

    Directory of Open Access Journals (Sweden)

    Soesamto Somowiyarjo

    2016-05-01

    Full Text Available Tobamovirus is a group of virus with a wide host range, including orchid plant which considered as an economically important plant. This research aimed to identify Tobamovirus infecting orchids. Virus isolates were collected from orchid nursery in Sleman, Yogyakarta. Plant extract from orchid showing necrotic flex symptom was inoculated to indicator plants Chenopodium amaranticolor. Chlorotic local lesion symptoms occurred within 3 days after inoculation. RNA total from symptomatic C. amaranticolor was extracted by using a commercial kit. cDNA was synthesized using oligo d(T primer. Amplification of cDNA using partial movement protein specific primers TMV-1F and TMV-2R was successfully amplified the amplicon with size ± 422 bp. The nucleotide sequences of this amplicon  showed highest DNA homology (98% with Tobacco mosaic virus Yongren-2 isolat from China.

  14. Functional annotation of 19,841 Populus nigra full-length enriched cDNA clones

    Science.gov (United States)

    Nanjo, Tokihiko; Sakurai, Tetsuya; Totoki, Yasushi; Toyoda, Atsushi; Nishiguchi, Mitsuru; Kado, Tomoyuki; Igasaki, Tomohiro; Futamura, Norihiro; Seki, Motoaki; Sakaki, Yoshiyuki; Shinozaki, Kazuo; Shinohara, Kenji

    2007-01-01

    Background Populus is one of favorable model plants because of its small genome. Structural genomics of Populus has reached a breakpoint as nucleotides of the entire genome have been determined. Reaching the post genome era, functional genomics of Populus is getting more important for well-comprehended plant science. Development of bioresorce serving functional genomics is making rapid progress. Huge efforts have achieved deposits of expressed sequence tags (ESTs) in various plant species consequently accelerating functional analysis of genes. ESTs from full-length cDNA clones are especially powerful for accurate molecular annotation. We promoted collection and annotation of the ESTs from Populus full-length enriched cDNA clones as part of functional genomics of tree species. Results We have been collecting the full-length enriched cDNA of the female poplar (Populus nigra var. italica) for years. By sequencing P. nigra full-length (PnFL) cDNA libraries, we generated about 116,000 5'-end or 3'-end ESTs corresponding to 19,841 nonredundant PnFL clones. Population of PnFL cDNA clones represents 44% of the predicted genes in the Populus genome. Conclusion Our resource of P. nigra full-length enriched clones is expected to provide valuable tools to gain further insight into genome annotation and functional genomics in Populus. PMID:18053163

  15. Functional annotation of 19,841 Populus nigra full-length enriched cDNA clones

    Directory of Open Access Journals (Sweden)

    Seki Motoaki

    2007-12-01

    Full Text Available Abstract Background Populus is one of favorable model plants because of its small genome. Structural genomics of Populus has reached a breakpoint as nucleotides of the entire genome have been determined. Reaching the post genome era, functional genomics of Populus is getting more important for well-comprehended plant science. Development of bioresorce serving functional genomics is making rapid progress. Huge efforts have achieved deposits of expressed sequence tags (ESTs in various plant species consequently accelerating functional analysis of genes. ESTs from full-length cDNA clones are especially powerful for accurate molecular annotation. We promoted collection and annotation of the ESTs from Populus full-length enriched cDNA clones as part of functional genomics of tree species. Results We have been collecting the full-length enriched cDNA of the female poplar (Populus nigra var. italica for years. By sequencing P. nigra full-length (PnFL cDNA libraries, we generated about 116,000 5'-end or 3'-end ESTs corresponding to 19,841 nonredundant PnFL clones. Population of PnFL cDNA clones represents 44% of the predicted genes in the Populus genome. Conclusion Our resource of P. nigra full-length enriched clones is expected to provide valuable tools to gain further insight into genome annotation and functional genomics in Populus.

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

    Science.gov (United States)

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

    2003-04-20

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

  17. Characterization and phylogenetic analysis of lectin gene cDNA isolated from sea cucumber ( Apostichopus japonicus) body wall

    Science.gov (United States)

    Xue, Zhuang; Li, Hui; Liu, Yang; Zhou, Wei; Sun, Jing; Wang, Xiuli

    2017-12-01

    As a `living fossil' of species origin and `rich treasure' of food and nutrition development, sea cucumber has received a lot of attentions from researchers. The cDNA library construction and EST sequencing of blood had been conducted previously in our lab. The bioinformatic analysis provided a gene fragment which is highly homologous with the genes of lectin family, named AjL ( Apostichopus japonicus lectin). To characterize and determine the phylogeny of AjL genes in early evolution, we isolated a full-length cDNA of lectin gene from the body wall of A. japonicus. The open reading frame of this gene contained 489 bp and encoded a 163 amino acids secretory protein being homologous to lectins of mammals and aquatic organisms. The deduced protein included a lectin-like domain. SDS-PAGE analysis showed that AjL migrated as a specific band (about 36.09 kDa under reducing), and agglutinated against rabbit red blood cells. AjL was similar to chain A of CEL-IV in space structure. We predicted that AjL may play the same role of CEL-IV. Our results suggested that more than one lectin gene functioned in sea cucumber and most of other species, which was fused by uncertain sequences during the evolution and encoded different proteins with diverse functions. Our findings provided the insights into the function and characteristics of lectin genes invertebrates. The results will also be helpful for the identification and structural, functional, and evolutionary analyses of lectin genes.

  18. Bitis gabonica (Gaboon viper) snake venom gland: toward a catalog for the full- length transcripts (cDNA) and proteins

    Science.gov (United States)

    Francischetti, Ivo M. B.; My-Pham, Van; Harrison, Jim; Garfield, Mark K.; Ribeiro, José M. C.

    2010-01-01

    The venom gland of the snake Bitis gabonica (Gaboon viper) was used for the first time to construct a unidirectional cDNA phage library followed by high-throughput sequencing and bioinformatic analysis. Hundreds of cDNAs were obtained and clustered into contigs. We found mostly novel full-length cDNA coding for metalloproteases (P-II and P-III classes), Lys49-phospholipase A2, serine proteases with essential mutations in the active site, Kunitz protease inhibitors, several C-type lectins, bradykinin-potentiating peptide, vascular endothelial growth factor, nucleotidases and nucleases, nerve growth factor, and L-amino acid oxidases. Two new members of the recently described short coding region family of disintegrin, displaying RGD and MLD motifs are reported. In addition, we have identified for the first time a cytokine-like molecule and a multi-Kunitz protease inhibitor in snake venoms. The CLUSTAL alignment and the unrooted cladograms for selected families of B. gabonica venom proteins are also presented. A significant number of sequences were devoid of database matches, suggesting that their biologic function remains to be identified. This paper also reports the N-terminus of the 15 most abundant venom proteins and the sequences matching their corresponding transcripts. The electronic version of this manuscript, available on request, contains spreadsheets with hyperlinks to FASTA-formatted files for each contig and the best match to the GenBank and Conserved Domain Databases, in addition to CLUSTAL alignments of each contig. We have thus generated a comprehensive catalog of the B. gabonica venom gland, containing for each secreted protein: i) the predicted molecular weight, ii) the predicted isoelectric point, iii) the accession number, and iv) the putative function. The role of these molecules is discussed in the context of the envenomation caused by the Gaboon viper. PMID:15276202

  19. Conserved elements within the genome of foot-and mouth disease virus; their influence on virus replication

    DEFF Research Database (Denmark)

    Kjær, Jonas; Poulsen, Line D.; Vinther, Jeppe

    mutations, to disrupt the 3Dpol RNA secondary structure, were generated in a FMDV replicon containing Gaussia luciferase. 2) Sequence changes encoding selected modifications of the 2A peptide (as described by Donnelly et al., 2001) were introduced into a full-length FMDV cDNA and in a FMDV replicon c......DNA containing Gaussia luciferase. RNA transcripts were generated in vitro from the plasmids, and introduced into BHK cells by electroporation. The replication efficiency was assessed by measurement of luciferase activity or by rescue of mutant viruses. The rescued viruses derived from the 2A mutant cDNAs were...... able to undergo replication, although at a lower rate than for the wt FMDV replicon. One mutant which previously (Donnelly et al., 2001) was found not to undergo “cleavage” was still replication competent. Analysis of rescued viruses by sequencing of the third passage revealed that the 2A mutants...

  20. Detection and analysis of the Chameleon WiFi access point virus

    National Research Council Canada - National Science Library

    Milliken, Jonny; Selis, Valerio; Marshall, Alan

    2013-01-01

    This paper analyses and proposes a novel detection strategy for the 'Chameleon’ WiFi AP-AP virus. Previous research has considered virus construction, likely virus behaviour and propagation methods...

  1. Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

    Energy Technology Data Exchange (ETDEWEB)

    Du, Yijun [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan (China); Pattnaik, Asit K. [School of Veterinary Medicine and Biomedical Sciences and the Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583-0900 (United States); Song, Cheng [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Yoo, Dongwan, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Li, Gang, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing (China)

    2012-03-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 ({omega} - 2, where {omega} is the GPI moiety at E160), P159 ({omega} - 1), and M162 ({omega} + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.

  2. The reverse genetics applied to fish RNA viruses

    Directory of Open Access Journals (Sweden)

    Biacchesi Stéphane

    2011-01-01

    Full Text Available Abstract Aquaculture has expanded rapidly to become a major economic and food-producing sector worldwide these last 30 years. In parallel, viral diseases have emerged and rapidly spread from farm to farm causing enormous economic losses. The most problematic viruses encountered in the field are mainly, but not exclusively, RNA viruses belonging to the Novirhabdovirus, Aquabirnavirus, Alphavirus and Betanodavirus genera. The recent establishment of reverse genetics systems to recover infectious fish RNA viruses entirely from cDNA has made possible to genetically manipulate the viral genome. These systems have provided powerful tools to study all aspects of the virus biology and virus-host interactions but also gave the opportunity to use these viruses as live vaccines or as gene vectors. This review provides an overview on the recent breakthroughs achieved by using these reverse genetics systems in terms of viral protein function, virulence and host-specificity factor, vaccine development and vector design.

  3. Zika Virus Encoding Non-Glycosylated Envelope Protein is Attenuated and Defective in Neuroinvasion.

    Science.gov (United States)

    Annamalai, Arun S; Pattnaik, Aryamav; Sahoo, Bikash R; Muthukrishnan, Ezhumalai; Natarajan, Sathish Kumar; Steffen, David; Vu, Hiep L X; Delhon, Gustavo; Osorio, Fernando A; Petro, Thomas M; Xiang, Shi-Hua; Pattnaik, Asit K

    2017-09-20

    Zika virus (ZIKV), a mosquito-transmitted flavivirus, responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, re-emerged in the last decade causing serious human diseases including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently outstanding. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic, being absent in many of the African isolates while present in all isolates from the recent outbreaks. In the present study, we interrogated the role of this sequence motif and glycosylation of the E protein in pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or from which N-linked glycosylation site is mutated by single amino acid substitution, are highly attenuated and non-lethal. The mutant viruses replicate poorly in the brain of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE Recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic

  4. Usability Constructs

    DEFF Research Database (Denmark)

    Hertzum, Morten; Clemmesen, Torkil; Hornbæk, Kasper Anders Søren

    2007-01-01

    Whereas research on usability predominantly employs universal definitions of the aspects that comprise usability, people experience their use of information systems through personal constructs. Based on 48 repertory-grid interviews, this study investigates how such personal constructs are affecte...

  5. Accelerated construction

    Science.gov (United States)

    2004-01-01

    Accelerated Construction Technology Transfer (ACTT) is a strategic process that uses various innovative techniques, strategies, and technologies to minimize actual construction time, while enhancing quality and safety on today's large, complex multip...

  6. Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA

    Directory of Open Access Journals (Sweden)

    Karlovsky Petr

    2008-10-01

    Full Text Available Abstract Background cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively. Results With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation was written in Perl. cDNA-AFLP protocols described in the literatur and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and Arabidopsis thaliana. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set. Conclusion Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each

  7. Gene therapy for bladder pain with gene gun particle encoding pro-opiomelanocortin cDNA.

    Science.gov (United States)

    Chuang, Yao-Chi; Chou, A-K; Wu, P-C; Chiang, Po-Hui; Yu, T-J; Yang, L-C; Yoshimura, Naoki; Chancellor, Michael B

    2003-11-01

    Interstitial cystitis is a bladder hypersensitivity disease associated with bladder pain that has been a major challenge to understand and treat. We hypothesized that targeted and localized expression of endogenous opioid peptide in the bladder could be useful for the treatment of bladder pain. Pro-opiomelanocortin (POMC) is one of such precursor molecules. In this study we developed a gene gun method for the transfer of POMC cDNA in vivo and investigated its therapeutic effect on acetic acid induced bladder hyperactivity in rats. Human POMC cDNA was cloned into a modified pCMV plasmid and delivered into the bladder wall of adult female rats by direct injection or the gene gun. Three days after gene therapy continuous cystometrograms were performed using urethane anesthesia by filling the bladder (0.08 ml per minute) with saline, followed by 0.3% acetic acid. Bladder immunohistochemical testing was used to detect endorphin after POMC cDNA transfer. The intercontraction interval was decreased after intravesical instillation of acetic acid (73.1% or 68.1% decrease) in 2 control groups treated with saline or the gene gun without POMC cDNA, respectively. However, rats that received POMC cDNA via the gene gun showed a significantly decreased response (intercontraction interval 35% decreased) to acetic acid instillation, whereas this antinociceptive effect was not detected in the plasmid POMC cDNA direct injection group. This effect induced by POMC gene gun treatment was reversed by intramuscular naloxone (1 mg/kg), an opioid antagonist. Increased endorphin immunoreactivity with anti-endorphin antibodies was observed in the bladder of gene gun treated animals. The POMC gene can be transferred in the bladder using the gene gun and increased bladder expression of endorphin can suppress nociceptive responses induced by bladder irritation. Thus, POMC gene gun delivery may be useful for the treatment of interstitial cystitis and other types of visceral pain.

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

    Science.gov (United States)

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

    2017-08-01

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

  9. Database Description - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project Database Description General information of database D...ases Organism Taxonomy Name: Saccharomyces cerevisiae Taxonomy ID: 4932 Database description 5'-end sequence...nuine 5'-end, mapping the 5'-end sequence to the genome will lead to accurate identification of the transcript... title: A large-scale full-length cDNA analysis to explore the budding yeast transcriptome. Author name(s): ...rvices Not available URL of Web services - Need for user registration - About This Database Database Descript

  10. Development and Evaluation of an Automated Annotation Pipeline and cDNA Annotation System

    OpenAIRE

    Kasukawa, Takeya; Furuno, Masaaki; Nikaido, Itoshi; Bono, Hidemasa; Hume, David A.; Bult, Carol; Hill, David P; Baldarelli, Richard; Gough, Julian; Kanapin, Alexander; Matsuda, Hideo; Schriml, Lynn M.; Hayashizaki, Yoshihide; Okazaki, Yasushi; Quackenbush, John

    2003-01-01

    Manual curation has long been held to be the “gold standard” for functional annotation of DNA sequence. Our experience with the annotation of more than 20,000 full-length cDNA sequences revealed problems with this approach, including inaccurate and inconsistent assignment of gene names, as well as many good assignments that were difficult to reproduce using only computational methods. For the FANTOM2 annotation of more than 60,000 cDNA clones, we developed a number of methods and tools ...

  11. Download - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available English ]; } else { document.getElementById(lang).innerHTML= '[ Japanese | English ]'; } } window.onload = ...List Contact us Budding yeast cDNA sequencing project Download First of all, please read the license of this... database. Data names and data descriptions are about the downloadable data in this page. They might not cor... # Data name File Simple search and download 1 README README_e.html - 2 5'-end se...quences of budding yeast full-length cDNA clones and quality scores yeast_seq_qual.zip (59.9MB) Simple search and dow

  12. Isolation and characterization of a Paracentrotus lividus cDNA encoding a stress-inducible chaperonin

    Science.gov (United States)

    Gianguzza, Fabrizio; Antonietta Ragusa, Maria; Roccheri, Maria Carmela; Liegro, Italia Di; Rinaldi, Anna Maria

    2000-01-01

    Chaperonins are ubiquitous proteins that facilitate protein folding in an adenosine triphosphate–dependent manner. Here we report the isolation of a sea urchin cDNA (Plhsp60) coding for mitochondrial chaperonin (Cpn60), whose basal expression is further enhanced by heat shock. The described cDNA corresponds to a full-length mRNA encoding a protein of 582 amino acids, the first 32 of which constitute a putative mitochondrial targeting leader sequence. Comparative analysis has demonstrated that this protein is highly conserved in evolution. PMID:11147969

  13. Role of CD151, A tetraspanin, in porcine reproductive and respiratory syndrome virus infection

    Directory of Open Access Journals (Sweden)

    Kapil Sanjay

    2007-06-01

    Full Text Available Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV is a RNA virus causing respiratory and reproductive diseases in swine. The susceptibility for PRRSV varies between the different breeds of swine. In cell culture, PRRSV virus can be propagated in primary porcine alveolar macrophages and some African green monkey kidney cell lines, such as MARC-145 cells. Previous studies have shown that 3' untranslated region (UTR RNAs of the arteriviruses play an important role in the replication of the virus through interactions with cellular proteins. To better understand the differences in the replication capability of PRRSV in different cell lines, we sought to identify the host cellular proteins interacting with PRRSV 3' UTR RNA. We constructed a cDNA library of MARC-145 cell line in lambda ZAP Express vector and screened the library with the positive sense 3' UTR RNA of PRRSV. Results We found that CD151, a host cellular protein, interacting with PRRSV 3' UTR RNA. The specificity of the interaction between CD151 and PRRSV 3' UTR RNA was examined by gel shift assay as well as North-Western hybridization. The transfection of CD151 expression clone into BHK-21 rendered these cells susceptible to PRRSV infection, and the transfection of siRNA against CD151 into MARC-145 significantly reduced the level of PRRSV infection. Also, anti-CD151 antibody treatment to MARC-145 completely blocked PRRSV infection. Conclusion Based on our results, we suggest that CD151 should cooperate in PRRSV infection in vitro in MARC-145 and BHK-21 cells.

  14. Secretion of rat serum albumin by COS cells transfected with a spliced cDNA gene. A system to study protein sorting.

    Science.gov (United States)

    McCracken, A A; Fishman, N F

    1986-01-15

    Certain structural features of secreted proteins may function as "sorting signals" to direct the various steps required in the secretory pathway. In order to identify and study the function of these signals we have cloned a complete cDNA gene encoding rat serum albumin (RSA) and expressed this gene in COS-1 cells via an SV40-plasmid shuttle vector. The gene was constructed by splicing together a segment of genomic DNA and three cDNA fragments excised from recombinant plasmids. DNA endonuclease digestion and ligation at restriction sites common to overlapping regions of these four RSA DNA fragments assured the maintenance of the translation reading frame during the construction of this gene. COS-1 cells transfected with the recombinant vector containing the full-length RSA gene (pSV2rsa) synthesize and secrete RSA immunoreactive material into the culture medium. This mammalian expression system provides a means to study the signals and processes involved in intracellular transport of secreted proteins.

  15. Development of pGEMINI, a Plant Gateway Destination Vector Allowing the Simultaneous Integration of Two cDNA via a Single LR-Clonase Reaction.

    Science.gov (United States)

    Exposito-Rodriguez, Marino; Laissue, Philippe P; López-Calcagno, Patricia E; Mullineaux, Philip M; Raines, Christine A; Simkin, Andrew J

    2017-11-12

    Gateway technology has been used to facilitate the generation of a large number of constructs for the modification of plants for research purposes. However, many of the currently available vectors only allow the integration of a single cDNA of interest into an expression clone. The ability to over-express multiple genes in combination is essential for the study of plant development where several transcripts have a role to play in one or more metabolic processes. The tools to carry out such studies are limited, and in many cases rely on the incorporation of cDNA into expression systems via conventional cloning, which can be both time consuming and laborious. To our knowledge, this study reports on the first development of a vector allowing the simultaneous integration of two independent cDNAs via a single LR-clonase reaction. This vector " pGEMINI " represents a powerful molecular tool offering the ability to study the role of multi-cDNA constructs on plant development, and opens up the process of gene stacking and the study of gene combinations through transient or stable transformation procedures.

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

    Directory of Open Access Journals (Sweden)

    Tuomas Rönnberg

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

  17. Pilot study of an EST approach of the coccolithophorid Emiliania huxleyi during a virus infection.

    Science.gov (United States)

    Kegel, Jessica; Allen, Michael J; Metfies, Katja; Wilson, William H; Wolf-Gladrow, Dieter; Valentin, Klaus

    2007-12-30

    Blooms of the coccolithophorid Emiliania huxleyi can be infected by viruses, which can lead to bloom-termination. This pilot study used an expressed sequence tag (EST) approach to get a first view of gene-expression changes that occur during viral infection of E. huxleyi. cDNA libraries were constructed from uninfected cultures and 6, 12, and 24 h after infection with E. huxleyi-specific virus 86 (EhV-86). From each library 60-90 ESTs were randomly selected and annotated manually with PhyloGena. Viral genes were identified using BLAST-Search of the known viral genome. The data of this study show, that 6 h after viral infection the algal transcriptome changed significantly although few viral transcripts were present. At this point, changes mainly concerned transcripts related to photosynthesis and protein metabolism. However, after 24 h viral transcripts were most abundant. Viral transcripts found at this stage of viral infection encode proteins involved in protein degradation, nucleic acid degradation, transcription and replication.

  18. Principles of Virus Structural Organization

    Science.gov (United States)

    Prasad, B.V. Venkataram; Schmid, Michael F

    2013-01-01

    Viruses, the molecular nanomachines infecting hosts ranging from prokaryotes to eukaryotes, come in different sizes, shapes and symmetries. Questions such as what principles govern their structural organization, what factors guide their assembly, how these viruses integrate multifarious functions into one unique structure have enamored researchers for years. In the last five decades, following Caspar and Klug's elegant conceptualization of how viruses are constructed, high resolution structural studies using X-ray crystallography and more recently cryo-EM techniques have provided a wealth of information on structures of variety of viruses. These studies have significantly furthered our understanding of the principles that underlie structural organization in viruses. Such an understanding has practical impact in providing a rational basis for the design and development of antiviral strategies. In this chapter, we review principles underlying capsid formation in a variety of viruses, emphasizing the recent developments along with some historical perspective. PMID:22297509

  19. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

    Science.gov (United States)

    Bertagnoli, S; Gelfi, J; Le Gall, G; Boilletot, E; Vautherot, J F; Rasschaert, D; Laurent, S; Petit, F; Boucraut-Baralon, C; Milon, A

    1996-08-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    % identity and 87% similarity at the amino acid level. Sequence comparisons between mouse and human tetranectin and some C-type lectins confirmed a complete conservation in the position of six cysteines as well as numerous other amino acid residues, indicating an essential structure for potential function...... regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed...

  1. Primary structure of a lipoxygenase from barley grain as deduced from its cDNA sequence

    NARCIS (Netherlands)

    Mechelen, J.R. van; Smits, M.; Douma, A.C.; Rouster, J.; Cameron-Mills, V.; Heidekamp, F.; Valk, B.E.

    1995-01-01

    A full length cDNA sequence for a barley grain lipoxygenase was obtained. It includes a 5' untranslated region of 69 nucleotides, an open reading frame of 2586 nucleotides encoding a protein of 862 amino acid residues and a 3' untranslated region of 142 nucleotides. The molecular mass of the encoded

  2. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    enoh

    2012-03-13

    Mar 13, 2012 ... Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The. cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown- ...

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

    Directory of Open Access Journals (Sweden)

    Maria Cartolano

    Full Text Available 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.

  4. Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

    NARCIS (Netherlands)

    Platteel, Anouk C M|info:eu-repo/dai/nl/375805613; Marit de Groot, A; Andersen, Peter; Ovaa, Huib; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M|info:eu-repo/dai/nl/115553843

    2016-01-01

    Most vaccines are based on protective humoral responses while for intracellular pathogens CD8(+) T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8(+) T cell priming, hampering vaccine efficacy. The multistage cDNA

  5. GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN CDNA MICROARRAY ANALYSES

    Science.gov (United States)

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSESB.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt11Department of Reproductiv...

  6. CLONING AND CHARACTERIZATION OF CDNA ENCODING GIARDIA LAMBLIA d-GIARDIN

    Science.gov (United States)

    A cDNA coding for d-giardin was cloned from Giardia lamblia trophozoites in order to localize the protein and study its function in mediating surface attachment. Recombinant d-giardin antigen was produced in Escherichia coli as a poly-histidine fusion protein and was purified by affinity chromatogr...

  7. cDNA cloning and characterization of a mannose-binding lectin from ...

    Indian Academy of Sciences (India)

    Unknown

    pension method while hot-shock method was adopted in. E. coli transformation in terms of the protocol of Sam- brook et al (1989). The PCR positive clone was sequenced using T7/SP6 primers (Sangon). 2.4 5′ RACE. The 5′ RACE System for RACE (GIBCO BRL, Life. Technologies, USA) was used for 5′ cDNA cloning.

  8. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown-PCR technology ...

  9. cDNA sequence and tissue expression analysis of glucokinase from ...

    African Journals Online (AJOL)

    Tissue distribution of GK mRNA in brain, mesenteric adipose tissue, spleen, white muscle and liver of grass carp was analyzed by SYBR real-time fluorescence quantitative RT-PCR method using β-actin as an internal control for cDNA normalization. The result shows that the expression level of GK mRNA in liver was ...

  10. Molecular cloning and characterization of ATX1 cDNA from the mole cricket, Gryllotalpa orientalis.

    Science.gov (United States)

    Kim, Iksoo; Lee, Kwang Sik; Hwang, Jae Sam; Ahn, Mi Young; Yun, Eun Young; Li, Jian Hong; Sohn, Hung Dae; Jin, Byung Rae

    2006-04-01

    To search for an insect homologue of antioxidant protein 1 (ATX1), a mole cricket, Gryllotalpa orientalis, cDNA library was screened and a cDNA clone, which encodes a 73 amino acid polypeptide with a predicted molecular mass of 8.0 kDa and pI of 5.68, was isolated. The G. orientalis ATX1 (GoATX1) cDNA features both a MTCXXC copper-binding site in the N-terminus and a KTGK lysine-rich region in the C-terminus. The deduced amino acid sequence of the GoATX1 cDNA showed 63% identity to Drosophila melanogaster ATX1 and 55% to Ixodes pacificus ATX1. Northern blot analysis revealed the presence of GoATX1 transcripts in midgut, fat body, and epidermis. When H2O2 was injected into the body cavity of G. orientalis adult, GoATX1 mRNA expression was up-regulated in the fat body tissue. Fat body expression level of GoATX1 mRNA in the fat body was increased following exposure to low (4 degrees C) and high (37 degrees C) temperatures, suggesting that GoATX1 plays a protective role against oxidative stress caused by temperature shock. This is the first report about a functional role of insect ATX1 in antioxidant defense. Copyright 2006 Wiley-Liss, Inc.

  11. Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

    DEFF Research Database (Denmark)

    Cowell, G M; Kønigshøfer, E; Danielsen, E M

    1988-01-01

    The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250...

  12. Observation of intermittency in gene expression on cDNA microarrays

    CERN Document Server

    Peterson, L E

    2002-01-01

    We used scaled factorial moments to search for intermittency in the log expression ratios (LERs) for thousands of genes spotted on cDNA microarrays (gene chips). Results indicate varying levels of intermittency in gene expression. The observation of intermittency in the data analyzed provides a complimentary handle on moderately expressed genes, generally not tackled by conventional techniques.

  13. Cloning and sequencing of complete τ-crystallin cDNA from ...

    Indian Academy of Sciences (India)

    The study thus provides the first report on cDNA sequence of -crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution ...

  14. KLONING cDNA HORMON PERTUMBUHAN DARI IKAN GURAME (Osphronemus gouramy

    Directory of Open Access Journals (Sweden)

    Estu Nugroho

    2016-11-01

    Full Text Available Penelitian mengenai kloning cDNA pengkode hormon pertumbuhan ikan gurame telah dilakukan. Tujuan dari penelitian ini adalah untuk memperoleh sekuens DNA komplemen hormon pertumbuhan sebagai langkah awal dalam rangka pengembangan teknologi rekayasa genetik ikan gurame. Empat buah kelenjar hifopisa ikan gurame digunakan sebagai bahan bakunya dan dilakukan proses ekstraksi RNA total dari kelenjar hipofisa, dilanjutkan dengan sintesis cDNA, amplifikasi PCR, purifikasi fragmen DNA dari gel, ligasi produk PCR dengan vektor kloning, transformasi dan inkubasi bakteri, seleksi koloni bakteri putih, isolasi plasmid, dan sekuensing. Hasil sekuensing menunjukkan bahwa panjang produk amplifikasi PCR adalah 843 bp yang menyandikan 204 asam amino residu dan mengandung sekuens-sekuens yang konserf untuk gen hormon pertumbuhan (GH. Analisis homologi menunjukkan kesamaan sekuens hasil isolasi antara 52,4%--97,6% dengan gen GH ikan lainnya, dengan persentase homologi tertinggi adalah dengan ikan sepat. Dengan demikian dapat disimpulkan bahwa sekuens hasil isolasi merupakan sekuens gen GH. Dari hasil analisis sekuens terlihat bahwa gen GH ikan gurame secara evolusi adalah konserf. Research on cDNA cloning encoded the gouramy growth hormone was conducted. The aim of the research was to get complementary DNA, cDNA, sequences of growth hormone as an initial step to develop genetic engineering of gouramy fish. Four pituitary glands of the gouramy were taken and then processed with total RNA extraction, and continued with cDNA synthesis, PCR amplification, DNA fragment purification from the gel, PCR product legation with cloning vector, transformation and incubation of bacteria, white colony bacteria selection, plasmid isolation and sequencing analysis. Sequencing result showed that the amplified PCR product length had 834 bp, encoding 204 amino acid residue and contained conserve sequence for GH (growth hormone gen. Homolog analysis showed sequence similarity of

  15. Construction Management.

    Science.gov (United States)

    Abbott, James F.

    This article provides a detailed discussion of a team approach to building that involves a construction manager, an architect, and a contractor. Bidding methods are outlined; the major components in construction management -- value engineering and fast track scheduling -- and the use of performance specifications are discussed. The construction…

  16. Superstring construction

    CERN Document Server

    1989-01-01

    The book includes a selection of papers on the construction of superstring theories, mainly written during the years 1984-1987. It covers ten-dimensional supersymmetric and non-supersymmetric strings, four-dimensional heterotic strings and four-dimensional type-II strings. An introduction to more recent developments in conformal field theory in relation to string construction is provided.

  17. ' CONSTRUCTION INDUSTRY

    African Journals Online (AJOL)

    CONSTRUCTION INDUSTRY. N. Aniekwu. Civil Engineering Department,. University of Benin,. Benin City. Nigeria. ABSTRACT. This paper deals with safety in the Nigerian construction industry. Three aspects of safety were identified as paramount in the study and include accident causations, accident prevention and the.

  18. Chikungunya virus

    Science.gov (United States)

    Chikungunya virus infection; Chikungunya ... Where Chikungunya is Found Before 2013, the virus was found in Africa, Asia, Europe, and the Indian and Pacific oceans. In late 2013, outbreaks occurred for the first time in the ...

  19. Zika Virus

    Science.gov (United States)

    ... through blood transfusions. There have been outbreaks of Zika virus in the United States, Africa, Southeast Asia, the ... not travel to areas where there is a Zika virus outbreak. If you do decide to travel, first ...

  20. Chikungunya Virus

    Science.gov (United States)

    ... Gaines, PhD, MPH, MA, CHES Differentiating Chikungunya From Dengue: A Clinical Challenge For Travelers CDC Travelers' Health Chikungunya Virus Home Prevention Transmission Symptoms & Treatment Geographic Distribution Chikungunya virus in the United States ...

  1. Zika Virus

    Science.gov (United States)

    ... Funding CDC Activities For Healthcare Providers Clinical Evaluation & Disease Sexual Transmission HIV Infection & Zika Virus Testing for Zika Test Specimens – At Time of Birth Diagnostic Tests Understanding Zika Virus Test Results ...

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

    Directory of Open Access Journals (Sweden)

    Sebastian Boltaña

    2017-02-01

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

  3. Taxol biosynthesis: Molecular cloning of a benzoyl- CoA:taxane 2α-O-benzoyltransferase cDNA from Taxus and functional expression in Escherichia coli

    Science.gov (United States)

    Walker, Kevin; Croteau, Rodney

    2000-01-01

    A cDNA clone encoding a taxane 2α-O-benzoyltransferase has been isolated from Taxus cuspidata. The recombinant enzyme catalyzes the conversion of 2-debenzoyl-7,13-diacetylbaccatin III, a semisynthetic substrate, to 7,13-diacetylbaccatin III, and thus appears to function in a late-stage acylation step of the Taxol biosynthetic pathway. By employing a homology-based PCR cloning strategy for generating acyltransferase oligodeoxynucleotide probes, several gene fragments were amplified and used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-induced Taxus cells, from which several full-length acyltransferases were obtained and individually expressed in Escherichia coli. The functionally expressed benzoyltransferase was confirmed by radio-HPLC, 1H-NMR, and combined HPLC-MS verification of the product, 7,13-diacetylbaccatin III, derived from 2-debenzoyl-7,13-diacetylbaccatin III and benzoyl-CoA as cosubstrates in the corresponding cell-free extract. The full-length cDNA has an open reading frame of 1,320 base pairs and encodes a protein of 440 residues with a molecular weight of 50,089. The recombinant benzoyltransferase has a pH optimum of 8.0, Km values of 0.64 mM and 0.30 mM for the taxoid substrate and benzoyl-CoA, respectively, and is apparently regiospecific for acylation of the 2α-hydroxyl group of the functionalized taxane nucleus. This enzyme may be used to improve the production yields of Taxol and for the semisynthesis of drug analogs bearing modified aroyl groups at the C2 position. PMID:11095755

  4. The yellow fever 17D vaccine virus: molecular basis of viral attenuation and its use as an expression vector

    Directory of Open Access Journals (Sweden)

    Galler R.

    1997-01-01

    Full Text Available The yellow fever (YF virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed

  5. Generation of EST and cDNA Microarray Resources for the Study of Bovine Immunobiology*

    Directory of Open Access Journals (Sweden)

    Coussens PM

    2003-03-01

    Full Text Available Recent developments in expressed sequence tag (EST and cDNA microarray technology have had a dramatic impact on the ability of scientists to study the responses of thousands of genes to external stimuli, such as infection, nutrient flux, and stress. To date however, these studies have largely been limited to human and rodent systems. Despite the tremendous potential benefit of EST and cDNA microarray technology to studies of complex problems in domestic animal species, a lack of integrated resources has precluded application of these technologies to domestic species. To address this problem, the Center for Animal Functional Genomics (CAFG at Michigan State University has developed a normalized bovine total leukocyte (BOTL cDNA library, generated EST clones from this library, and printed cDNA microarrays suitable for studying bovine immunobiology. Our data revealed that the normalization procedure successfully reduced highly abundant cDNA species while enhancing the relative percentage of clones representing rare transcripts. To date, a total of 932 EST sequences have been generated from this library (BOTL and the sequence information plus BLAST results made available through a web-accessible database http://gowhite.ans.msu.edu. Cluster analysis of the data indicates that a total of 842 unique cDNAs are present in this collection, reflecting a low redundancy rate of 9.7%. For creation of first generation cDNA microarrays, inserts from 720 unique clones in this library were amplified and microarrays were produced by spotting each insert or amplicon 3 times on glass slides in a 48-patch arrangement with 64 total spots (including blanks and positive controls per patch. To test our BOTL microarray, we compared gene expression patterns of concanavalin A stimulated and unstimulated peripheral blood mononuclear cells (PBMCs. In total, hybridization signals on over 90 amplicons showed upregulation (>3× in response to Con A stimulation, relative to

  6. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

    Energy Technology Data Exchange (ETDEWEB)

    Dalton, J.P.; Tom, T.D.; Strand, M.

    1987-06-01

    Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in lambdagt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein.

  7. Helicase domain encoded by Cucumber mosaic virus RNA1 determines systemic infection of Cmr1 in pepper.

    Directory of Open Access Journals (Sweden)

    Won-Hee Kang

    Full Text Available The Cmr1 gene in peppers confers resistance to Cucumber mosaic virus isolate-P0 (CMV-P0. Cmr1 restricts the systemic spread of CMV strain-Fny (CMV-Fny, whereas this gene cannot block the spread of CMV isolate-P1 (CMV-P1 to the upper leaves, resulting in systemic infection. To identify the virulence determinant of CMV-P1, six reassortant viruses and six chimeric viruses derived from CMV-Fny and CMV-P1 cDNA clones were used. Our results demonstrate that the C-terminus of the helicase domain encoded by CMV-P1 RNA1 determines susceptibility to systemic infection, and that the helicase domain contains six different amino acid substitutions between CMV-Fny and CMV-P1(. To identify the key amino acids of the helicase domain determining systemic infection with CMV-P1, we then constructed amino acid substitution mutants. Of the mutants tested, amino acid residues at positions 865, 896, 957, and 980 in the 1a protein sequence of CMV-P1 affected the systemic infection. Virus localization studies with GFP-tagged CMV clones and in situ localization of virus RNA revealed that these four amino acid residues together form the movement determinant for CMV-P1 movement from the epidermal cell layer to mesophyll cell layers. Quantitative real-time PCR revealed that CMV-P1 and a chimeric virus with four amino acid residues of CMV-P1 accumulated more genomic RNA in inoculated leaves than did CMV-Fny, indicating that those four amino acids are also involved in virus replication. These results demonstrate that the C-terminal region of the helicase domain is responsible for systemic infection by controlling virus replication and cell-to-cell movement. Whereas four amino acids are responsible for acquiring virulence in CMV-Fny, six amino acid (positions at 865, 896, 901, 957, 980 and 993 substitutions in CMV-P1 were required for complete loss of virulence in 'Bukang'.

  8. Genetic determinism and evolutionary reconstruction of a host jump in a plant virus

    DEFF Research Database (Denmark)

    Vassilakos, Nikon; Simon, Vincent; Tzima, Aliki

    2016-01-01

    analysis, and 3) locus-by-locus analysis of molecular variance (AMOVA) allowed reconstructing the Potato virus Y (PVY; genus Potyvirus, family Potyviridae) jump to pepper (Capsicum annuum), probably from other solanaceous plants. Synthetic chimeras between infectious cDNA clones of two PVY isolates...

  9. Complementation and recombination between alfalfa mosaic virus RNA3 mutants in tobacco plants

    NARCIS (Netherlands)

    van der Kuyl, A. C.; Neeleman, L.; Bol, J. F.

    1991-01-01

    Deletions were made in an infectious cDNA clone of alfalfa mosaic virus (AIMV) RNA3 and the replication of RNA transcripts of these cDNAs was studied in tobacco plants transformed with AIMV replicase genes (P12 plants). Previously, we found that deletions in the P3 gene did not affect accumulation

  10. Envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus

    NARCIS (Netherlands)

    Wissink, E.H.J.; Kroese, M.V.; Wijk, van H.A.; Rijsewijk, F.A.M.; Meulenberg, J.J.; Rottier, P.J.M.

    2005-01-01

    Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2a, E, GP3, and GP4. Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA

  11. Modelling Constructs

    DEFF Research Database (Denmark)

    Kindler, Ekkart

    2009-01-01

    , these notations have been extended in order to increase expressiveness and to be more competitive. This resulted in an increasing number of notations and formalisms for modelling business processes and in an increase of the different modelling constructs provided by modelling notations, which makes it difficult...... to compare modelling notations and to make transformations between them. One of the reasons is that, in each notation, the new concepts are introduced in a different way by extending the already existing constructs. In this chapter, we go the opposite direction: We show that it is possible to add most...... constructs and how they can be combined with more advanced constructs. Second, it will help combining different modelling notations with each other. Note that, though we introduce a notation for blocks in this chapter, we are not so much interested in promoting this notation here. The notation should just...

  12. Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    Soumen Naskar

    2012-01-01

    Full Text Available In the present study, water buffalo MHC (Bubu-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

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

    Science.gov (United States)

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

    1990-09-25

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

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

    DEFF Research Database (Denmark)

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

    1991-01-01

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

  15. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    Directory of Open Access Journals (Sweden)

    Juana M Sánchez-Puig

    Full Text Available Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

  16. Isolation of the putative cDNA encoding cholesterol side chain cleavage cytochrome P450 (CYP11A) of the southern stingray (Dasyatis americana).

    Science.gov (United States)

    Nunez, S; Trant, J M

    1997-03-10

    Cholesterol side chain cleavage cytochrome P450 (P450scc; CYP11A) catalyzes the first step in the production of steroid hormones. By utilizing degenerate oligonucleotide primers in a reverse transcriptase-coupled polymerase chain reaction (RT-PCR), a specific 252 bp fragment of the putative P450scc was amplified from RNA of interrenal tissue (the adrenal cortex homolog) from the southern stingray (Dasyatis americana), blacktip shark (Carcharhinus limbatus), and the spiny dogfish shark (Squalus acanthias). The amino-acid sequences predicted by these PCR products were 73-90% identical to each other. Using the homologous PCR-generated probe, five positive clones were isolated from a cDNA library constructed from interrenal mRNA of the southern stingray. The longest clone (4619 bp) contained the 3'-untranslated region, including four putative polyadenylation signals. Northern blot analysis of stingray interrenal RNA revealed a single transcript of 4.2 kb in length. The incomplete amino-acid sequence predicted by the open reading frame of the cDNA (514 residues in length) is 48% homologous to the trout form and 39-40% homologous to mammalian forms. Even though the stingray P450scc contains an amino terminus longer than the other forms of P450scc, no translation initiation signal (ATG) was evident within the open reading frame. This report presents the first sequence of cytochrome P450scc from this evolutionary unique taxon of vertebrates.

  17. Molecular cloning and characterization of a tomato cDNA encoding a systemically wound-inducible bZIP DNA-binding protein

    Science.gov (United States)

    Stankovic, B.; Vian, A.; Henry-Vian, C.; Davies, E.

    2000-01-01

    Localized wounding of one leaf in intact tomato (Lycopersicon esculentum Mill.) plants triggers rapid systemic transcriptional responses that might be involved in defense. To better understand the mechanism(s) of intercellular signal transmission in wounded tomatoes, and to identify the array of genes systemically up-regulated by wounding, a subtractive cDNA library for wounded tomato leaves was constructed. A novel cDNA clone (designated LebZIP1) encoding a DNA-binding protein was isolated and identified. This clone appears to be encoded by a single gene, and belongs to the family of basic leucine zipper domain (bZIP) transcription factors shown to be up-regulated by cold and dark treatments. Analysis of the mRNA levels suggests that the transcript for LebZIP1 is both organ-specific and up-regulated by wounding. In wounded wild-type tomatoes, the LebZIP1 mRNA levels in distant tissue were maximally up-regulated within only 5 min following localized wounding. Exogenous abscisic acid (ABA) prevented the rapid wound-induced increase in LebZIP1 mRNA levels, while the basal levels of LebZIP1 transcripts were higher in the ABA mutants notabilis (not), sitiens (sit), and flacca (flc), and wound-induced increases were greater in the ABA-deficient mutants. Together, these results suggest that ABA acts to curtail the wound-induced synthesis of LebZIP1 mRNA.

  18. cDNA cloning and expression analysis of a mannose-binding lectin ...

    Indian Academy of Sciences (India)

    Pinellia pedatisecta agglutinin (PPA) is a very basic protein that accumulates in the tuber of P. pedatisecta. PPA is a hetero-tetramer protein of 40 kDa, composed of two polypeptide chains A (about 12 kDa) and two polypeptides chains B (about 12 kDa). The full-length cDNA of PPA was cloned from P. pedatisecta using ...

  19. Escaping introns in COI through cDNA barcoding of mushrooms: Pleurotus as a test case.

    Science.gov (United States)

    Avin, Farhat A; Subha, Bhassu; Tan, Yee-Shin; Braukmann, Thomas W A; Vikineswary, Sabaratnam; Hebert, Paul D N

    2017-09-01

    DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648-bp segment near the 5' terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus, the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5' region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus. Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.

  20. dCAS: a desktop application for cDNA sequence annotation

    OpenAIRE

    Guo, Yongjian; Ribeiro, Jose M.C.; Anderson, Jennifer M; Bour, Stephan

    2009-01-01

    Motivation: Understanding gene regulation and expression is the key to the advancement of biology. EST sequence assembly and analysis provide unique benefits in this regard. We have developed a standalone application, dCAS (Desktop cDNA Annotation System), which performs automated EST cleaning, clustering, assembly and annotation on a desktop computer. Compared with other available tools, dCAS provides a more convenient and user-friendly solution to biologists for extracting biological meanin...

  1. Reuse of cDNA Microarrays Hybridized With cRNA by Stripping With RNase H

    Science.gov (United States)

    2008-11-01

    improved stripping procedure for in situ microarrays. J. Biotechnol. 128:1- 13. 5. Hausen, P. and H. Stein. 1970. Ribonuclease H: an enzyme degrading...DATE 01 NOV 2008 2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Reuse of cDNA microarrays hybridized with cRNA by stripping with...studies. This technique may be appli- cable to other types of oligonucleotide or DNA microarrays hybridized with sense or antisense RNA

  2. Detection of differentially expressed genes in healing mouse corneas, using cDNA microarrays.

    Science.gov (United States)

    Cao, Zhiyi; Wu, Helen K; Bruce, Amenda; Wollenberg, Kurt; Panjwani, Noorjahan

    2002-09-01

    To identify differentially expressed genes in healing mouse corneas by using cDNA microarrays. Transepithelial excimer laser ablations were performed on mouse corneas, and the wounds were allowed to heal partially in vivo for 18 to 22 hours. Total RNA was isolated from both normal and healing corneas and was used for synthesis of cDNA probes. 33P-labeled exponential cDNA probes were hybridized to mouse cDNA nylon arrays. Of the 1176 genes on the nylon arrays, the expression of 37 was upregulated and that of 27 was downregulated more than fivefold in the healing corneas compared with the normal, uninjured corneas. Interleukin (IL)-1beta, laminin-5, and thrombospondin-1, which have been shown to be upregulated in healing corneas, were all found to be induced in the corneas in response to excimer laser treatment. Many genes were identified for the first time to be differentially regulated during corneal wound healing. Among the upregulated genes were intercellular adhesion molecule (ICAM)-1, macrophage inflammatory proteins, suppressors of cytokine signaling proteins (SOCS), IL-10 receptor, and galectin-7. Among the downregulated genes were connexin-31, a gap junction protein; ZO1 and occludin, tight junction proteins; and Smad2, a key component in the TGFbeta signaling pathway. Microarray data were validated on a limited number of genes by semiquantitative RT-PCR and Western blot analyses. Gene array technology was used to identify for the first time many genes that are differentially regulated during corneal wound healing. These differentially expressed genes have not previously been investigated in the context of wound healing and represent novel factors for further study of the mechanism of wound healing.

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

    Science.gov (United States)

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

    2015-01-01

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

  4. A proteomic approach to identify phosphoproteins encoded by cDNA libraries.

    Science.gov (United States)

    Shi, Xudong; Belton, Robert J; Burkin, Heather R; Vieira, Ana P; Miller, David J

    2004-06-15

    We report a method for large-scale rapid analysis of phosphoproteins in tissues or cells by combining immobilized metal affinity chromatography (IMAC) with phage display cDNA library screening. We expressed a testis cDNA library as fusion proteins on phage and, using IMAC, enriched for sequences encoding phosphoproteins. Selected clones were polymerase chain reaction amplified and sequenced. The majority of the clones sequenced (80%) encoded known proteins previously identified as phosphoproteins. Immunoblotting with phosphotyrosine antibodies confirmed that some of the selected sequences encoded tyrosine phosphorylated proteins when expressed on phage. An advantage of this method is the rapid identification of phosphoproteins encoded by a cDNA library, which can identify proteins that are potentially phosphorylated in vivo. When this method is combined with limited enzymatic digestion and tandem mass spectrometric techniques, the specific phosphorylation site in a protein can be identified. This technique can be used in proteomics studies to effectively detect phosphorylated proteins and avoid time-consuming and expensive peptide sequencing.

  5. Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

    Directory of Open Access Journals (Sweden)

    Zhongwei Wu

    2010-01-01

    Full Text Available In this paper we describe an isothermal rolling-circle amplification (RCA protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP.The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  6. Cloning and sequencing of cDNA and genomic DNA encoding PDM phosphatase of Fusarium moniliforme.

    Science.gov (United States)

    Yoshida, Hiroshi; Iizuka, Mari; Narita, Takao; Norioka, Naoko; Norioka, Shigemi

    2006-12-01

    PDM phosphatase was purified approximately 500-fold through six steps from the extract of dried powder of the culture filtrate of Fusarium moniliforme. The purified preparation appeared homogeneous on SDS-PAGE although the protein band was broad. Amino acid sequence information was collected on tryptic peptides from this preparation. cDNA cloning was carried out based on the information. A full-length cDNA was obtained and sequenced. The sequence had an open reading frame of 651 amino acid residues with a molecular mass of 69,988 Da. Cloning and sequencing of the genomic DNA corresponding to the cDNA was also conducted. The deduced amino acid sequence could account for many but not all of the tryptic peptides, suggesting presence of contaminant protein(s). SDS-PAGE analysis after chemical deglycosylation showed two proteins with molecular masses of 58 and 68 kDa. This implied that the 58 kDa protein had been copurified with PDM phosphatase. Homology search showed that PDM phosphatase belongs to the purple acid phosphatase family, which is widely distributed in the biosphere. Sequence data of fungal purple acid phosphatases were collected from the database. Processing of the data revealed presence of two types, whose evolutionary relationships were discussed.

  7. Nucleotide sequence and expression of a cDNA encoding rabbit liver cytosolic serine hydroxymethyltransferase.

    Science.gov (United States)

    Byrne, P C; Sanders, P G; Snell, K

    1992-08-15

    A rabbit liver cDNA library in phage lambda gt10 was screened using a portion of the coding sequences for rabbit cytosolic serine hydroxymethyltransferase (amino acids 244-420) that had been amplified by PCR, with total rabbit liver RNA as a template. A clone of 2.3 kb (pUS1203) was isolated and the nucleotide sequence showed that it contained an open reading frame of 1452 bp, which coded for serine hydroxymethyltransferase and was flanked by 155 bp at the 5' end and 653 bp at the 3' end. The full-length cDNA was cloned into an expression vector and transfected into COS-1 cells. Serine hydroxymethyltransferase activity was increased by 33% in the transfected cells and a new protein band of the appropriate size was seen by SDS/PAGE analysis of proteins extracted from transfected cells. The protein sequence for rabbit cytosolic serine hydroxymethyltransferase derived from the cDNA nucleotide sequence was compared with three other derived or known prokaryotic and eukaryotic sequences. An overall sequence similarity of 34% was noted between all four sequences, whereas the similarity between the rabbit cytosolic and mitochondrial isoforms was 62%.

  8. Cloning and endogenous expression of a Eucalyptus grandis UDP-glucose dehydrogenase cDNA

    Directory of Open Access Journals (Sweden)

    Mônica T. Veneziano Labate

    2010-01-01

    Full Text Available UDP-glucose dehydrogenase (UGDH catalyzes the oxidation of UDP-glucose (UDP-Glc to UDP-glucuronate (UDP-GlcA, a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis.

  9. Ohanin, a novel protein from king cobra venom: its cDNA and genomic organization.

    Science.gov (United States)

    Pung, Yuh Fen; Kumar, Sanjeed Vijaya; Rajagopalan, Nandhakishore; Fry, Bryan G; Kumar, Prakash P; Kini, R Manjunatha

    2006-04-26

    Ohanin, from king cobra venom, is a novel protein which induces hypolocomotion and hyperalgesia in mice [Pung, Y.F., Wong, P.T.H., Kumar, P.P., Hodgson W.C., Kini, R.M., 2005. Ohanin, a novel protein from king cobra venom induces hypolocomotion and hyperalgesia in mice. J. Biol. Chem. 280, 13137-13147.]. It is weakly similar to PRY-SPRY domains (B30.2-like domain). Here we report the complete cDNA and genomic organization of ohanin. Interestingly, cDNA sequence does not show significant sequence similarity to any known sequences, including those of B30.2-like domain-containing proteins. Its full-length cDNA sequence of 1558 bp encodes for prepro-ohanin with a propeptide segment at the C-terminal. Ohanin is the first member of a new subfamily of proteins containing B30.2-like domain with short N-terminal segment. We named this subfamily as vespryns. There are two mRNA subtypes differing in their 5'-untranslated regions. Southern hybridization study shows that ohanin is encoded by a single gene. Its genomic sequence is 7086 bp with five exons and four introns, and the two types of mRNAs are generated by alternative splicing of exon 2. Our results indicate that ohanin and vespryns may have evolved from the same ancestral gene as B30.2 domain.

  10. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

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

    Science.gov (United States)

    Rebrikov, Denis V

    2008-07-01

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

  12. Intercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA3.

    Science.gov (United States)

    French, R; Ahlquist, P

    1987-05-01

    The genome of brome mosaic virus (BMV) is divided among messenger polarity RNA1, RNA2, and RNA3 (3.2, 2.9, and 2.1 kilobases, respectively). cis-Acting sequences required for BMV RNA amplification were investigated with RNA3. By using expressible cDNA clones, deletions were constructed throughout RNA3 and tested in barley protoplasts coinoculated with RNA1 and RNA2. In contrast to requirements for 5'- and 3'-terminal noncoding sequences, either of the two RNA3 coding regions can be deleted individually and both can be simultaneously inactivated by N-terminal frameshift mutations without significantly interfering with amplification of RNA3 or production of its subgenomic mRNA. However, simultaneous major deletions in both coding regions greatly attenuate RNA3 accumulation. RNA3 levels can be largely restored by insertion of a heterologous, nonviral sequence in such mutants, suggesting that RNA3 requires physical separation of its terminal domains or a minimum overall size for normal replication or stability. Unexpectedly, deletions in a 150-base segment of the intercistronic noncoding region drastically reduce RNA3 accumulation. This segment contains a sequence element homologous to sequences found near the 5' ends of BMV RNA1 and RNA2 and in analogous positions in the three genomic RNAs of the related cucumber mosaic virus, suggesting a possible role in plus-strand synthesis.

  13. Construction safety

    CERN Document Server

    Li, Rita Yi Man

    2013-01-01

    A close-to-ideal blend of suburb and city, speedy construction of towers of Babylon, the sparkling proportion of glass and steel buildings’ facade at night showcase the wisdom of humans. They also witness the footsteps, sweats and tears of architects and engineers. Unfortunately, these signatures of human civilizations are swathed in towering figures of construction accidents. Fretting about these on sites, different countries adopt different measures on sites. This book firstly sketches the construction accidents on sites, followed by a review on safety measures in some of the developing countries such as Bermuda, Egypt, Kuwait and China; as well as developed countries, for example, the United States, France and Singapore. It also highlights the enormous compensation costs with the courts’ experiences in the United Kingdom and Hong Kong.

  14. A Stochastic Dynamic Model of Computer Viruses

    Directory of Open Access Journals (Sweden)

    Chunming Zhang

    2012-01-01

    Full Text Available A stochastic computer virus spread model is proposed and its dynamic behavior is fully investigated. Specifically, we prove the existence and uniqueness of positive solutions, and the stability of the virus-free equilibrium and viral equilibrium by constructing Lyapunov functions and applying Ito's formula. Some numerical simulations are finally given to illustrate our main results.

  15. High Genetic Stability of Dengue Virus Propagated in MRC-5 Cells as Compared to the Virus Propagated in Vero Cells

    OpenAIRE

    Chia-Chyi Liu; Shiang-Chi Lee; Michael Butler; Suh-Chin Wu

    2008-01-01

    This work investigated the replication kinetics of the four dengue virus serotypes (DEN-1 to DEN-4), including dengue virus type 4 (DEN-4) recovered from an infectious cDNA clone, in Vero cells and in MRC-5 cells grown on Cytodex 1 microcarriers. DEN-1 strain Hawaii, DEN-2 strain NGC, DEN-3 strain H-87, and DEN-4 strain H-241 , and DEN-4 strain 814669 derived from cloned DNA, were used to infect Vero cells and MRC-5 cells grown in serum-free or serum-containing microcarrier cultures. Serum-fr...

  16. Hunting the subset-specific genes of neuroblastoma: expression profiling and differential screening of the full-length-enriched oligo-capping cDNA libraries.

    Science.gov (United States)

    Ohira, M; Shishikura, T; Kawamoto, T; Inuzuka, H; Morohashi, A; Takayasu, H; Kageyama, H; Takada, N; Takahashi, M; Sakiyama, S; Suzuki, Y; Sugano, S; Kuma, H; Nozawa, I; Nakagawara, A

    2000-12-01

    Neuroblastoma (NBL) has a distinct nature in different prognostic subgroups. To understand the molecular mechanism of NBL's genesis and biology as well as that of the neural crest development, we constructed full-length-enriched cDNA libraries by an oligo-capping method from two different subsets of primary NBL, one with favorable biology and the other with MYCN amplification. Sequencing analysis of these libraries revealed that the expression profile was markedly different between both subsets. To identify the genes differentially expressed between the subsets, semi-quantitative RT-PCR analyses are proceeding. So far, 54 transcripts have been found to be expressed at high levels in favorable NBLs, and significantly at low levels in unfavorable NBLs. Copyright 2000 Wiley-Liss, Inc.

  17. Construction work

    CERN Multimedia

    2004-01-01

    Construction work on building 179 will start on the 16th February 2004 and continue until November 2004. The road between buildings 179 and 158 will temporarily become a one way street from Route Democrite towards building 7. The parking places between buildings 179 and 7 will become obsolete. The ISOLDE collaboration would like to apologize for any inconveniences.

  18. Stability of RNA silencing-based traits after virus infection

    DEFF Research Database (Denmark)

    Jørgensen, Bodil; Albrechtsen, Merete

    2007-01-01

    engineered virus resistance based on either a simple sense or an inverted repeat construct. We decided to use genetically engineered virus resistance in potato as a model system for further studies of the effect of virus infection on genetically engineered traits. We present for the first time a comparison...

  19. CHLORELLA VIRUSES

    Science.gov (United States)

    Yamada, Takashi; Onimatsu, Hideki; Van Etten, James L.

    2007-01-01

    Chlorella viruses or chloroviruses are large, icosahedral, plaque‐forming, double‐stranded‐DNA—containing viruses that replicate in certain strains of the unicellular green alga Chlorella. DNA sequence analysis of the 330‐kbp genome of Paramecium bursaria chlorella virus 1 (PBCV‐1), the prototype of this virus family (Phycodnaviridae), predict ∼366 protein‐encoding genes and 11 tRNA genes. The predicted gene products of ∼50% of these genes resemble proteins of known function, including many that are completely unexpected for a virus. In addition, the chlorella viruses have several features and encode many gene products that distinguish them from most viruses. These products include: (1) multiple DNA methyltransferases and DNA site‐specific endonucleases, (2) the enzymes required to glycosylate their proteins and synthesize polysaccharides such as hyaluronan and chitin, (3) a virus‐encoded K+ channel (called Kcv) located in the internal membrane of the virions, (4) a SET domain containing protein (referred to as vSET) that dimethylates Lys27 in histone 3, and (5) PBCV‐1 has three types of introns; a self‐splicing intron, a spliceosomal processed intron, and a small tRNA intron. Accumulating evidence indicates that the chlorella viruses have a very long evolutionary history. This review mainly deals with research on the virion structure, genome rearrangements, gene expression, cell wall degradation, polysaccharide synthesis, and evolution of PBCV‐1 as well as other related viruses. PMID:16877063

  20. Virus Crystallography

    Science.gov (United States)

    Fry, Elizabeth; Logan, Derek; Stuart, David

    Crystallography provides a means of visualizing intact virus particles as well as their isolated constituent proteins and enzymes (1-3) at near-atomic resolution, and is thus an extraordinarily powerful tool in the pursuit of a fuller understanding of the functioning of these simple biological systems. We have already expanded our knowledge of virus evolution, assembly, antigenic variation, and host-cell interactions; further studies will no doubt reveal much more. Although the rewards are enormous, an intact virus structure determination is not a trivial undertaking and entails a significant scaling up in terms of time and resources through all stages of data collection and processing compared to a traditional protein crystallographic structure determination. It is the methodology required for such studies that will be the focus of this chapter. The computational requirements were satisfied in the late 1970s, and when combined with the introduction of phase improvement techniques utilizing the virus symmetry (4,5), the application of crystallography to these massive macromolecular assemblies became feasible. This led to the determination of the first virus structure (the small RNA plant virus, tomato bushy stunt virus), by Harrison and coworkers in 1978 (6). The structures of two other plant viruses followed rapidly (7,8). In the 1980s, a major focus of attention was a family of animal RNA viruses; the Picornaviridae.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sugantham Priyanka Annabel

    2010-10-01

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

  3. p lambda Zd39: a new type of cDNA expression vector for low background, high efficiency directional cloning.

    OpenAIRE

    Murphy, A J; Schimke, R T

    1991-01-01

    We have developed a new type of bacteriophage lambda vector which provides a strong biological selection against non-recombinants that is independent of the sequences immediately surrounding the cloning site. This system, which we call 'selective substitution', is ideally suited for cDNA expression vectors where it is necessary to flank the cDNA insert with sequence elements (promoters etc.) required to produce a biologically active mRNA in vivo. Selective substitution is a general method, wh...

  4. Isolation and sequence analysis of a chalcone synthase cDNA of Matthiola incana R. Br. (Brassicaceae).

    Science.gov (United States)

    Epping, B; Kittel, M; Ruhnau, B; Hemleben, V

    1990-06-01

    A cDNA clone (pcM12) of the chalcone synthase (CHS) of Matthiola incana R. Br. (Brassicaceae) was isolated from a cDNA library, sequenced and analysed. It comprises the complete coding sequence for the CHS and 5' and 3' untranslated regions. The deduced amino acid sequence shows that the Matthiola incana CHS consists of 394 amino acid residues. Comparison with CHS amino acid sequences of other plants indicates more than 82% homology.

  5. Generation of an infectious clone of VR-2332, a highly virulent North American type isolate of porcine reproductive and respiratory syndrome virus

    DEFF Research Database (Denmark)

    Nielsen, H.S.; Liu, G.; Nielsen, Jens

    2003-01-01

    -2332 strain. However, the cloned virus was clearly distinguishable from the parental VR-2332 strain by an engineered marker, a BstZ171 restriction site. The full-length cDNA clone had 11 nucleotide changes, 2 of which affected coding, compared to the parental VR-2332 strain. Additionally...... virus were consistently slightly lower. In experimentally infected 5.5-week-old pigs, the cloned virus produced blue discoloration of the ears, a classical clinical symptom of PRRSV. Also, the seroconversion kinetics of pigs infected with the cloned virus and VR-2332 were very similar. Hence, virus...

  6. Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a P-Glycoprotein cDNA.

    Directory of Open Access Journals (Sweden)

    Kristen M Pluchino

    Full Text Available The efflux transporter P-glycoprotein (P-gp is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.

  7. Enrichment of measles virus-like RNA in the nucleocapsid fraction isolated from subacute sclerosing panencephalitis brains

    Energy Technology Data Exchange (ETDEWEB)

    Bedows, E.; Payne, F.E. (Michigan Univ., Ann Arbor (USA). School of Public Health); Kohne, D.E. (Center for Neurologic Study, San Diego, CA, USA); Tourtellotte, W.W. (Neurology Service, V.A. Wadsworth Hospital Center, Los Angeles, CA, USA)

    1982-02-01

    A procedure has been developed which facilitates the detection of measles virus RNA sequences in human brains. The procedure involves isolating subviral components (nucleocapsids) from brain tissues prior to RNA purification, followed by hybridization of these RNAs to cDNA synthesized from measles virus 50 S RNA template. Using these techniques we were able to obtain an RNA fraction which was manyfold enriched in measles virus-specific RNA, relative to unfractionated subacute sclerosing panencephalitis (SSPE) brain RNAs. 70-100% of the measles virus-specific RNA present in these SSPE brain samples were recovered in this enriched fraction.

  8. Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration

    Directory of Open Access Journals (Sweden)

    Bryant Susan V

    2009-01-01

    Full Text Available Abstract Background Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR and denervated (DL forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. Results Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa. Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST contigs from the Ambystoma EST database more than doubled (3935 to 9411 the number of non-redundant human-A. mexicanum orthologous sequences. Conclusion Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

  9. Nucleic acid hybridization techniques for the detection of bluetongue virus

    Energy Technology Data Exchange (ETDEWEB)

    Schoepp, R.J.

    1989-01-01

    Virus isolation, antigen detection, and in situ hybridization were compared in their abilities to detect in cell culture, the five serotypes of bluetongue virus (BTV) occurring in the United States, serotypes 2, 10, 11, 13, and 17. For isolation, virus was propagated in baby hamster kidney (BHK-21) cell culture. For antigen detection, two techniques, indirect fluorescent-antibody (IFA) and enzyme immunocytoassay (EICA) were used. For in situ hybridization, a complementary DNA (cDNA) of the L3 RNA genome segment of BTV, serotype 17 (BTV-17) labeled with {sup 35}S was used as a group-specific probe. Virus isolation was the most sensitive technique, often detecting input virus and then detecting virus throughout the course of the study. IFA and EICA were of similar sensitivity and detected BTV antigen shortly after detection of virus by isolation. A direct-blot hybridization technique using a {sup 32}P-labeled, strand-specific RNA transcript probe was developed, optimized, and used to detect BTV in pools of infected Culicoides variipennis midges. The technique was able to detect as few as one infected Culicoides midge in a pool of 100 and as little as 3.5 log{sub 10} TCID{sub 50} per ml of virus. A sandwich hybridization technique was developed and used to detect BTV in pools of infected Culicoides variipennis midges. The sandwich hybridization technique used a single-stranded DNA catcher sequence bound to a solid support and a {sup 32}P-labeled, single-stranded RNA detector sequence. Sandwich hybridization was compared to direct blot hybridization using a strand-specific RNA transcript probe or a cDNA probe. Sandwich hybridization was able to detect as few as one infected Culicoides midge in a pool of 50; however, the technique was approximately tenfold less sensitive than direct blot hybridization.

  10. Reconstructing the highly virulent Classical Swine Fever Virus strain Koslov

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Nielsen, Jens

    DNA by site directed mutagenesis, removing non-synonymous mutations step by step. In vitro testing of modified constructs did indeed lead to fully functional viruses with similar growth kinetics as the wild-type strain. Moreover, viruses rescued from the construct had the ancestral amino acid sequence and...

  11. Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus

    Directory of Open Access Journals (Sweden)

    De Iaco Alberto

    2011-12-01

    Full Text Available Abstract Background HIV-1 infects non-dividing cells. This implies that the virus traverses the nuclear pore before it integrates into chromosomal DNA. Recent studies demonstrated that TNPO3 is required for full infectivity of HIV-1. The fact that TNPO3 is a karyopherin suggests that it acts by directly promoting nuclear entry of HIV-1. Some studies support this hypothesis, while others have failed to do so. Additionally, some studies suggest that TNPO3 acts via HIV-1 Integrase (IN, and others indicate that it acts via capsid (CA. Results To shed light on the mechanism by which TNPO3 contributes to HIV-1 infection we engineered a panel of twenty-seven single-cycle HIV-1 vectors each bearing a different CA mutation and characterized them for the ability to transduce cells in which TNPO3 had been knocked down (KD. Fourteen CA mutants were relatively TNPO3-independent, as compared to wild-type (WT HIV-1. Two mutants were more TNPO3-dependent than the WT, and eleven mutants were actually inhibited by TNPO3. The efficiency of the synthesis of viral cDNA, 2-LTR circles, and proviral DNA was then assessed for WT HIV-1 and three select CA mutants. Controls included rescue of TNPO3 KD with non-targetable coding sequence, RT- and IN- mutant viruses, and pharmacologic inhibitors of RT and IN. TNPO3 KD blocked transduction and establishment of proviral DNA by wild-type HIV-1 with no significant effect on the level of 2-LTR circles. PCR results were confirmed by achieving TNPO3 KD using two different methodologies (lentiviral vector and siRNA oligonucleotide transfection; by challenging three different cell types; by using two different challenge viruses, each necessitating different sets of PCR primers; and by pseudotyping virus with VSV G or using HIV-1 Env. Conclusion TNPO3 promotes HIV-1 infectivity at a step in the virus life cycle that is detectable after the preintegration complex arrives in the nucleus and CA is the viral determinant for TNPO3

  12. Hepatitis C Virus(Topics in Transfusion Medicine 1990 : Autologous Transfusion and Post-Transfusion Hepatitis)

    OpenAIRE

    渡辺, 勇四郎; 宮村, 達男; Watanabe, Yushiro; Miyamura, Tatsuo

    1990-01-01

    The agent that causes blood-borne non-A, non-B hepatitis has been identified by M.Houghton and his colleagues. A cDNA of this virus has been cloned and assay system of HCV-antibody has been established. By using this assay system, we have been accumulating new epidemiological and clinical findings. We also isolated HCVcDNA clones from the serum of a Japanese healthy HCV carrier. Nucleotide sequences of these cDNA clones showed high homology with the Houghton's clones. Amino acid homology in t...

  13. Generation and Analysis of Full-length cDNA Sequences from Elephant Shark (Callorhinchus milii)

    KAUST Repository

    Kodzius, Rimantas

    2009-03-17

    Cartilaginous fishes are the oldest living group of jawed vertebrates and therefore is an important group for understanding the evolution of vertebrate genomes including the human genome. Our laboratory has proposed elephant shark (C. milii) as a model cartilaginous fish genome because of its relatively small genome size (910 Mb). The whole genome of C. milii is being sequenced (first cartilaginous fish genome to be sequenced completely). To characterize the transcriptome of C. milii and to assist in annotating exon-intron boundaries, transcriptional start sites and alternatively spliced transcripts, we are generating full-length cDNA sequences from C. milii.

  14. Cloning and sequencing of Indian Water buffalo (Bubalus bubalis) interleukin-3 cDNA

    KAUST Repository

    Sugumar, Thennarasu

    2011-12-12

    Full-length cDNA (435 bp) of the interleukin-3(IL-3) gene of the Indian water buffalo was amplified by reverse transcriptase-polymerase chain reaction and sequenced. This sequence had 96% nucleotide identity and 92% amino acid identity with bovine IL-3. There are 10 amino acid substitutions in buffalo compared with that of bovine. The amino acid sequence of buffalo IL-3 also showed very high identity with that of other ruminants, indicating functional cross-reactivity. Structural homology modelling of buffalo IL-3 protein with human IL-3 showed the presence of five helical structures.

  15. Nucleotide sequence and expression of a cDNA encoding rabbit liver cytosolic serine hydroxymethyltransferase.

    OpenAIRE

    Byrne, P C; Sanders, P. G.; Snell, K

    1992-01-01

    A rabbit liver cDNA library in phage lambda gt10 was screened using a portion of the coding sequences for rabbit cytosolic serine hydroxymethyltransferase (amino acids 244-420) that had been amplified by PCR, with total rabbit liver RNA as a template. A clone of 2.3 kb (pUS1203) was isolated and the nucleotide sequence showed that it contained an open reading frame of 1452 bp, which coded for serine hydroxymethyltransferase and was flanked by 155 bp at the 5' end and 653 bp at the 3' end. The...

  16. Detection of reverse transcriptase termination sites using cDNA ligation and massive parallel sequencing

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz J; Boyd, Mette; Sandelin, Albin

    2013-01-01

    Detection of reverse transcriptase termination sites is important in many different applications, such as structural probing of RNAs, rapid amplification of cDNA 5' ends (5' RACE), cap analysis of gene expression, and detection of RNA modifications and protein-RNA cross-links. The throughput...... amplification, Illumina adapters and index sequences are introduced, thereby allowing amplicons to be pooled and sequenced on the standard Illumina platform for genomic DNA sequencing. Moreover, we demonstrate how to map sequencing reads and perform analysis of the sequencing data with freely available tools...

  17. Sequence and expression of a microspore cDNA clone with homology to a ribosomal protein

    Directory of Open Access Journals (Sweden)

    Michael P. Turcich

    2014-01-01

    Full Text Available A cDNA library was made to RNA from corn anthers containing developing pollen at the uninucleate microspore stage. A randomly selected clone from this library which contained an insert (531 bp was isolated and sequenced. An open reading frame of 330 bp was located. Computer alignments of the putative amino acid sequence with sequences from GenBank and the SwissProt protein databases indicated homology to L12, an acidic ribosomal protein. RNA blot analysis showed highest levels of this mRNA in mature pollen. The significance of this observation in light of the known biochemistry of ribosome synthesis in developing pollen is discussed.

  18. [Cloning and analyzing of the cDNA sequence of CHS-A gene of Narcissus].

    Science.gov (United States)

    Huang, Yin Yi; Shen, Ming Shan; Chen, Liang; Li, Peng; Chen, Mu Zhuan

    2002-09-01

    Chalcone synthase (CHS) is a key enzyme in the biosynthesis of all classes of flavonoids. The production of flower pigment is specifically regulated by the activity of CHS. We cloned the cDNA sequence of CHS-A gene from Narcissus by PCR and analyzed the coding sequence of gene. The result demonstrated that the sequence of the coding region was 1167bp, encoding a protein of 389 amino acid which was more than 80% homology with CHS of the other 8 plants, such as Nicotine abacus and Solana tuberosum.

  19. Intra-genomic GC heterogeneity in sauropsids: evolutionary insights from cDNA mapping and GC3 profiling in snake

    Science.gov (United States)

    2012-01-01

    Background Extant sauropsids (reptiles and birds) are divided into two major lineages, the lineage of Testudines (turtles) and Archosauria (crocodilians and birds) and the lineage of Lepidosauria (tuatara, lizards, worm lizards and snakes). Karyotypes of these sauropsidan groups generally consist of macrochromosomes and microchromosomes. In chicken, microchromosomes exhibit a higher GC-content than macrochromosomes. To examine the pattern of intra-genomic GC heterogeneity in lepidosaurian genomes, we constructed a cytogenetic map of the Japanese four-striped rat snake (Elaphe quadrivirgata) with 183 cDNA clones by fluorescence in situ hybridization, and examined the correlation between the GC-content of exonic third codon positions (GC3) of the genes and the size of chromosomes on which the genes were localized. Results Although GC3 distribution of snake genes was relatively homogeneous compared with those of the other amniotes, microchromosomal genes showed significantly higher GC3 than macrochromosomal genes as in chicken. Our snake cytogenetic map also identified several conserved segments between the snake macrochromosomes and the chicken microchromosomes. Cross-species comparisons revealed that GC3 of most snake orthologs in such macrochromosomal segments were GC-poor (GC3 < 50%) whereas those of chicken orthologs in microchromosomes were relatively GC-rich (GC3 ≥ 50%). Conclusion Our results suggest that the chromosome size-dependent GC heterogeneity had already occurred before the lepidosaur-archosaur split, 275 million years ago. This character was probably present in the common ancestor of lepidosaurs and but lost in the lineage leading to Anolis during the diversification of lepidosaurs. We also identified several genes whose GC-content might have been influenced by the size of the chromosomes on which they were harbored over the course of sauropsid evolution. PMID:23140509

  20. Intra-genomic GC heterogeneity in sauropsids: evolutionary insights from cDNA mapping and GC3 profiling in snake

    Directory of Open Access Journals (Sweden)

    Matsubara Kazumi

    2012-11-01

    Full Text Available Abstract Background Extant sauropsids (reptiles and birds are divided into two major lineages, the lineage of Testudines (turtles and Archosauria (crocodilians and birds and the lineage of Lepidosauria (tuatara, lizards, worm lizards and snakes. Karyotypes of these sauropsidan groups generally consist of macrochromosomes and microchromosomes. In chicken, microchromosomes exhibit a higher GC-content than macrochromosomes. To examine the pattern of intra-genomic GC heterogeneity in lepidosaurian genomes, we constructed a cytogenetic map of the Japanese four-striped rat snake (Elaphe quadrivirgata with 183 cDNA clones by fluorescence in situ hybridization, and examined the correlation between the GC-content of exonic third codon positions (GC3 of the genes and the size of chromosomes on which the genes were localized. Results Although GC3 distribution of snake genes was relatively homogeneous compared with those of the other amniotes, microchromosomal genes showed significantly higher GC3 than macrochromosomal genes as in chicken. Our snake cytogenetic map also identified several conserved segments between the snake macrochromosomes and the chicken microchromosomes. Cross-species comparisons revealed that GC3 of most snake orthologs in such macrochromosomal segments were GC-poor (GC3 3 ≥ 50%. Conclusion Our results suggest that the chromosome size-dependent GC heterogeneity had already occurred before the lepidosaur-archosaur split, 275 million years ago. This character was probably present in the common ancestor of lepidosaurs and but lost in the lineage leading to Anolis during the diversification of lepidosaurs. We also identified several genes whose GC-content might have been influenced by the size of the chromosomes on which they were harbored over the course of sauropsid evolution.

  1. cDNA cloning and characterization of UDP-glucose: anthocyanidin 3-O-glucosyltransferase in Freesia hybrida.

    Science.gov (United States)

    Sui, Xin; Gao, Xiang; Ao, Man; Wang, Qinmei; Yang, Dan; Wang, Meng; Fu, Yang; Wang, Li

    2011-07-01

    The enzyme that catalyzes the formation of the first stable anthocyanin in the biosynthesis of natural compounds is UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UF3GT). A cDNA clone (Fh3GT1) encoding UF3GT was isolated from Freesia hybrida. Phylogenetic tree analysis indicated that Fh3GT1 was a novel member of glycosyltransferase, which was classified into monocot subgroups. Semi-quantitative RT-PCR analysis detected transcripts of Fh3GT1 in different organs of F. hybrida and in petals of Freesia cultivars of different colors, and the expression level reached the maximum at the fully opened stage of petals. Characterization of the enzymatic assays indicated that Fh3GT1 had a role in anthocyanin glycoside biosyntheses in vitro. To elucidate the function of Fh3GT1, RNA interference vector (pART-Fh3GT1i) was constructed, and introduced into Petunia grandiflora by Agrobacterium-mediated transformation. Integration of the Fh3GT1 in petunia genome was confirmed by PCR and Southern blotting. SqRT-PCR revealed that the endogenous Ph3GT1 mRNA expression levels decreased in transgenic lines compared with the wild-type. The content of total anthocyanin pigments also decreased with the reduction of mRNA transcript levels, and the transgenic petunia plants had significant changes on their flower colors. In summary, this work identified a UF3GT gene from Freesia hybrida and demonstrated a method to modify plant flower color by redirecting the anthocyanin biosynthesis.

  2. CHANDIPURA VIRUS

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. CHANDIPURA VIRUS. First isolated from a village called Chandipura near Nagpur in 1965 in India. Belongs to rhabdoviridae family. Used as a Model System to study RNA virus multiplication in the infected cell at molecular level. Notes:

  3. Chimeric Bovine Respiratory Syncytial Virus with Attachment and Fusion Glycoproteins Replaced by Bovine Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase and Fusion Proteins

    Science.gov (United States)

    Stope, Matthias B.; Karger, Axel; Schmidt, Ulrike; Buchholz, Ursula J.

    2001-01-01

    Chimeric bovine respiratory syncytial viruses (BRSV) expressing glycoproteins of bovine parainfluenza virus type 3 (BPIV-3) instead of BRSV glycoproteins were generated from cDNA. In the BRSV antigenome cDNA, the open reading frames of the major BRSV glycoproteins, attachment protein G and fusion protein F, were replaced individually or together by those of the BPIV-3 hemagglutinin-neuraminidase (HN) and/or fusion (F) glycoproteins. Recombinant virus could not be recovered from cDNA when the BRSV F open reading frame was replaced by the BPIV-3 F open reading frame. However, cDNA recovery of the chimeric virus rBRSV-HNF, with both glycoproteins replaced simultaneously, and of the chimeric virus rBRSV-HN, with the BRSV G protein replaced by BPIV-3 HN, was successful. The replication rates of both chimeras were similar to that of standard rBRSV. Moreover, rBRSV-HNF was neutralized by antibodies specific for BPIV-3, but not by antibodies specific to BRSV, demonstrating that the BRSV glycoproteins can be functionally replaced by BPIV-3 glycoproteins. In contrast, rBRSV-HN was neutralized by BRSV-specific antisera, but not by BPIV-3 specific sera, showing that infection of rBRSV-HN is mediated by BRSV F. Hemadsorption of cells infected with rBRSV-HNF and rBRSV-HN proved that BPIV-3 HN protein expressed by rBRSV is functional. Colocalization of the BPIV-3 glycoproteins with BRSV M protein was demonstrated by confocal laser scan microscopy. Moreover, protein analysis revealed that the BPIV-3 glycoproteins were present in chimeric virions. Taken together, these data indicate that the heterologous glycoproteins were not only expressed but were incorporated into the envelope of recombinant BRSV. Thus, the envelope glycoproteins derived from a member of the Respirovirus genus can together functionally replace their homologs in a Pneumovirus background. PMID:11533200

  4. Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

    Directory of Open Access Journals (Sweden)

    Farzaneh Shafaghat

    2015-03-01

    Full Text Available Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs. We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems.

  5. Constructing Catalonia

    Directory of Open Access Journals (Sweden)

    Bill Philips

    2009-07-01

    Full Text Available Catalonia, in common with other nations, has long been concerned with the question of identity and difference. Its problematic relationship with Spain has led to an emphasis on differentiating itself from its larger neighbour (if we are to accept, as most Spaniards do not, that Catalonia is not Spain, a situation complicated by the loss of the Spanish colonies of Cuba and The Philippines in 1898, and the Spanish Civil War and subsequent dictatorship from 1936 to 1976. Beginning in the late nineteenth century, the construction of a Catalan identity followed a similar route to that taken by other European nations such as England, Ireland and, indeed, Spain, including an emphasis on rural values, activities and the countryside, and the conversion of specifically local traditions into national past times. It is only in the last ten years or so that this model of Catalan identity has been recognised for what it is – a model constructed and encouraged for and by specific nationalist political interests. Ironically, Catalonia’s identity abroad has also been constructed and manipulated for political purposes, but from quite a different perspective. Orwell’s /Homage to Catalonia/ (1938 narrates an extremely blinkered version of the Spanish Civil War which has achieved iconic status as a result of cold war politics. Subsequent portrayals of the Spanish Civil War – Valentine Cunningham’s /The Penguin Book of Spanish Civil War Verse/ (ed., Penguin, 1980, or Ken Loach’s 1995 film /Land and Freedom/ base their arguments unquestioningly on /Homage to Catalonia/, perpetuating a view of the nation’s recent history that is both reductive and inaccurate

  6. Constructing dialogues

    DEFF Research Database (Denmark)

    Bundgaard, Charlotte

    2017-01-01

    The concept of montage is proposed as a driver for establishing heterogeneous, open and dynamic new-industrial architecture. Drawing parallels between the intense industrialization that occurred in Denmark in the 1960s and current scenarios of IT-based production methods, montage is unfolded both...... as an aesthetic strategy and a technical procedure, taking inspiration from both visual arts and architecture. Montage is presented as a means of creating active, architectural interactions - material as well as in terms of meaning - not only in the realm of industrialization but also in the intersection between...... industrial production methods and on-site, craft-based fields of construction....

  7. Constructing History

    DEFF Research Database (Denmark)

    Jørgensen, Michael Riber

    2010-01-01

    The Icelandic sagas can be read and interpreted in many ways. This article examines the sagas both as literary expressions of a longstanding oral tradition and as part of a collective and cultural memory. The focus in the first part is on people and places in the sagas as ‘realms of memory’: things...... that help construct a common past and a common identity. The second part of the article explores the role of the sagas in medieval Iceland as ‘key myths’ that explain the origin and uniqueness of a society, and as moral and legal role models legitimizing the current social order....

  8. Characterizations of sea urchin fibrillar collagen and its cDNA clone.

    Science.gov (United States)

    Tomita, M; Kinoshita, T; Izumi, S; Tomino, S; Yoshizato, K

    1994-03-01

    Collagens were isolated from the adult test of the sea urchin species, Hemicentrotus pulcherrimus and Strongylocentrotus purpuratus, and their molecular properties were compared with those of Asthenosoma ijimai collagen. Collagens from H. pulcherrimus and S. purpuratus comprised two major alpha-chains (alpha 120 and alpha 90) and a minor chain (alpha 140), while collagen from A. ijimai contained four alpha-chains (alpha 1, alpha 2, alpha 3 and alpha 4). Based on their molecular and immunological properties, the alpha 90 chain of H. pulcherrimus and S. purpuratus, and the alpha 2 and alpha 4 chains of A. ijimai are grouped together, while the alpha 120 and alpha 140 chains of H. pulcherrimus and S. purpuratus, and the alpha 1 and alpha 3 chains of A. ijimai are classified into another group. It is likely that collagen molecules of sea urchins are heterotrimers composed of these two types of alpha-chains. A cDNA of collagen was cloned from the cDNA library prepared from mRNA of H. pulcherrimus test and denoted as Hpcol1. This clone contained sequences for uninterrupted triple helical domain (378 amino acids), carboxyl telopeptide (28 amino acids) and carboxyl propeptide (225 amino acids). This structure is characteristic for fibril-forming collagens and was shown to encode alpha 120 and alpha 140 chains of H. pulcherrimus collagen. Hpcol1-mRNA was expressed in embryos as early as the prism stage.

  9. Purification, characterisation and cDNA cloning of an antimicrobial peptide from Macadamia integrifolia.

    Science.gov (United States)

    Marcus, J P; Goulter, K C; Green, J L; Harrison, S J; Manners, J M

    1997-03-15

    An antimicrobial peptide with no significant amino acid sequence similarity to previously described peptides has been isolated from the nut kernels of Macadcamia integrifolia. The peptide, termed MiAMP1, is highly basic with an estimated pI of 10.1, a mass of 8.1 kDa and contains 76 amino acids including 6 cysteine residues. A cDNA clone containing the entire coding region corresponding to the peptide was obtained. The deduced amino acid sequence of the cDNA indicated a 26-amino-acid signal peptide at the N-terminus of the preprotein. Purified MiAMP1 inhibited the growth of a variety of fungal, oomycete and gram-positive bacterial phytopathogens in vitro. Some pathogens exhibited close to 100% inhibition in less than 1 microM peptide (5 microg/ml). Antimicrobial activity was diminished against most, but not all, microbes in the presence of calcium and potassium chloride salts (1 mM and 50 mM, respectively). MiAMP1 was active against bakers yeast, was inactive against Escherichia coli and was non-toxic to plant and mammalian cells. Analysis of genomic DNA indicated that MiAMP1 was encoded on a single copy gene containing no introns. The MiAMP1 gene may prove useful in genetic manipulations to increase disease resistance in transgenic plants.

  10. Development of a cDNA array for chicken gene expression analysis

    Directory of Open Access Journals (Sweden)

    Burt David

    2005-02-01

    Full Text Available Abstract Background The application of microarray technology to functional genomic analysis in the chicken has been limited by the lack of arrays containing large numbers of genes. Results We have produced cDNA arrays using chicken EST collections generated by BBSRC, University of Delaware and the Fred Hutchinson Cancer Research Center. From a total of 363,838 chicken ESTs representing 24 different adult or embryonic tissues, a set of 11,447 non-redundant ESTs were selected and added to an existing collection of clones (4,162 from immune tissues and a chicken bursal cell line (DT40. Quality control analysis indicates there are 13,007 useable features on the array, including 160 control spots. The array provides broad coverage of mRNAs expressed in many tissues; in addition, clones with expression unique to various tissues can be detected. Conclusions A chicken multi-tissue cDNA microarray with 13,007 features is now available to academic researchers from genomics@fhcrc.org. Sequence information for all features on the array is in GenBank, and clones can be readily obtained. Targeted users include researchers in comparative and developmental biology, immunology, vaccine and agricultural technology. These arrays will be an important resource for the entire research community using the chicken as a model.

  11. Cloning and sequence analysis of a partial cDNA for chicken cartilage proteoglycan core protein.

    Science.gov (United States)

    Sai, S; Tanaka, T; Kosher, R A; Tanzer, M L

    1986-01-01

    A chicken embryo sternal cartilage cDNA library, created in the plasmid expression vector pUC9, was screened for sequences coding for immunologically detectable core protein of the large, major proteoglycan of cartilage. A 1229-base-pair cDNA clone was isolated that contained only one extended open reading frame, which had sequences coding for a polypeptide of 379 amino acid residues. These deduced sequences corresponded to those anticipated from current models of proteoglycan structure; a deduced sequence encompassing 21 amino acids was almost identical to a known sequence of bovine nasal cartilage proteoglycan. Significant homology was found between the deduced amino acid sequence of the proteoglycan and two regions of a chicken hepatic lectin. Immunoprecipitation of the products of cell-free translation yielded a component of about 340 kDa, and transfer blot hybridization of sternal cartilage RNA showed a single mRNA of about 8.1 kilobases. Hybridizable mRNA sequences were readily detectable by dot-blot analyses of the cytoplasm of cartilaginous tissues of the chicken embryo, whereas similar analyses of prechondrogenic limb mesenchymal cells did not demonstrate such hybridizable mRNA signals. Images PMID:3460082

  12. Characterization of an Atlantic cod (Gadus morhua embryonic stem cell cDNA library

    Directory of Open Access Journals (Sweden)

    Holen Elisabeth

    2009-05-01

    Full Text Available Abstract Background The Atlantic cod is an ecologically and economically important North Atlantic fish species and also an emerging aquaculture species. To study gene expression in Atlantic cod embryonic stem (ES cells, our goal was to generate and analyze expressed sequence tags (ESTs from an ES cell cDNA library of mRNA consisting of approximately 3,900 ESTs. Results We sequenced 3,935 EST clones using a directional cDNA library made from pooled ES cells harvested at the blastula stage. Quality filtering of these ESTs allowed identification of 2,719 high-quality sequences with an average length of 442 bp containing 368 contigs and 1,276 singletons (1,644 unique sequences. BLASTX searches produced 889 significant (E-value -3 hits, of which 698 (42.5% were annotated with Gene Ontology terms (E-value -6. The number of unknown unique sequences was 946 (57.5%. All the high-quality EST sequences have been deposited in GenBank (GenBank: 2,719 sequences in UniGene library dbEST id: 22,021. Gene discovery and annotations are presented and discussed. Conclusion This set of ESTs represents one of the first attempts to describe mRNA in ES cells from a marine cold-water fish species, and provides a basis for gene expression studies of Atlantic cod ES cells.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-08-15

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Hoskins, Roger A.; Stapleton, Mark; George, Reed A.; Yu, Charles; Wan, Kenneth H.; Carlson, Joseph W.; Celniker, Susan E.

    2005-04-22

    The production of comprehensive cDNA clone collections is an important goal of the human and model organism genome projects. cDNA sequences are used to determine the structures of transcripts, including splice junctions, polyadenylation sites, and 5' and 3' untranslated regions (UTRs). cDNA collections are also valuable resources for functional studies of genes and proteins. Expressed Sequence Tag (EST)sequencing is the method of choice for recovering cDNAs representing a majority of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a library at random, so it realizes diminishing returns as the project progresses. To drive cDNA collections toward completion new methods are needed to recover cDNAs representing specific genes and alternative transcripts, including transcripts with low expression levels. We describe a simple and effective inverse-PCR-based method for screening plasmid libraries to recover intact cDNAs for specific transcripts. We tested the method by screening libraries used in our Drosophila EST projects for 153 transcription factor genes that were not yet represented by full-length cDNAs. We recovered target-specific clones for 104 of the genes: 46 exactly match, 30 improve and 28partially match current gene annotations. Successful application of the screening method depends on cDNA library complexity and quality of the gene models. The approach should be effective for improving cDNA collections for other model organisms and the human. It also provides a simple and rapid method for isolating cDNAs of interest in any system for which plasmid cDNA libraries and complete or partial gene sequences are available.

  16. Partial antiviral activities detection of chicken Mx jointing with neuraminidase gene (NA against Newcastle disease virus.

    Directory of Open Access Journals (Sweden)

    Yani Zhang

    Full Text Available As an attempt to increase the resistance to Newcastle Disease Virus (NDV and so further reduction of its risk on the poultry industry. This work aimed to build the eukaryotic gene co-expression plasmid of neuraminidase (NA gene and myxo-virus resistance (Mx and detect the gene expression in transfected mouse fibroblasts (NIH-3T3 cells, it is most important to investigate the influence of the recombinant plasmid on the chicken embryonic fibroblasts (CEF cells. cDNA fragment of NA and mutant Mx gene were derived from pcDNA3.0-NA and pcDNA3.0-Mx plasmid via PCR, respectively, then NA and Mx cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-Mx-NA. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequencing, and it was transfected into the mouse fibroblasts (NIH-3T3 cells. The expression of genes in pVITRO2-Mx-NA were measured by RT-PCR and indirect immunofluorescence assay (IFA. The recombinant plasmid was transfected into CEF cells then RT-PCR and the micro-cell inhibition tests were used to test the antiviral activity for NDV. Our results showed that co-expression vector pVITRO2-Mx-NA was constructed successfully; the expression of Mx and NA could be detected in both NIH-3T3 and CEF cells. The recombinant proteins of Mx and NA protect CEF cells from NDV infection until after 72 h of incubation but the individually mutagenic Mx protein or NA protein protects CEF cells from NDV infection till 48 h post-infection, and co-transfection group decreased significantly NDV infection compared with single-gene transfection group (P<0. 05, indicating that Mx-NA jointing contributed to delaying the infection of NDV in single-cell level and the co-transfection of the jointed genes was more powerful than single one due to their synergistic effects.

  17. Computer viruses

    Science.gov (United States)

    Denning, Peter J.

    1988-01-01

    The worm, Trojan horse, bacterium, and virus are destructive programs that attack information stored in a computer's memory. Virus programs, which propagate by incorporating copies of themselves into other programs, are a growing menace in the late-1980s world of unprotected, networked workstations and personal computers. Limited immunity is offered by memory protection hardware, digitally authenticated object programs,and antibody programs that kill specific viruses. Additional immunity can be gained from the practice of digital hygiene, primarily the refusal to use software from untrusted sources. Full immunity requires attention in a social dimension, the accountability of programmers.

  18. Construction and Characterization of a Recombinant Invertebrate Iridovirus

    NARCIS (Netherlands)

    Ozgen, A.; Muratoglu, H.; Demirbag, Z.; Vlak, J.M.; Oers, van M.M.; Nalcacioglu, R.

    2014-01-01

    Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral

  19. cDNA cloning of a snake venom metalloproteinase from the eastern diamondback rattlesnake (Crotalus adamanteus), and the expression of its disintegrin domain with anti-platelet effects.

    Science.gov (United States)

    Suntravat, Montamas; Jia, Ying; Lucena, Sara E; Sánchez, Elda E; Pérez, John C

    2013-03-15

    A 5' truncated snake venom metalloproteinase was identified from a cDNA library constructed from venom glands of an eastern diamondback rattlesnake (Crotalus adamanteus). The 5'-rapid amplification of cDNA ends (RACE) was used to obtain the 1865 bp full-length cDNA sequence of a snake venom metalloproteinase (CamVMPII). CamVMPII encodes an open reading frame of 488 amino acids, which includes a signal peptide, a pro-domain, a metalloproteinase domain, a spacer, and an RGD-disintegrin domain. The predicted amino acid sequence of CamVMPII showed a 91%, 90%, 83%, and 82% sequence homology to the P-II class enzymes of C. adamanteus metalloproteinase 2, Crotalus atrox CaVMP-II, Gloydius halys agkistin, and Protobothrops jerdonii jerdonitin, respectively. Disintegrins are potent inhibitors of both platelet aggregation and integrin-dependent cell adhesion. Therefore, the disintegrin domain (Cam-dis) of CamVMPII was amplified by PCR, cloned into a pET-43.1a vector, and expressed in Escherichia coli BL21. Affinity purified recombinantly modified Cam-dis (r-Cam-dis) with a yield of 8.5 mg/L culture medium was cleaved from the fusion tags by enterokinase cleavage. r-Cam-dis was further purified by two-step chromatography consisting of HiTrap™ Benzamidine FF column, followed by Talon Metal affinity column with a final yield of 1 mg/L culture. r-Cam-dis was able to inhibit all three processes of platelet thrombus formation including platelet adhesion with an estimated IC(50) of 1 nM, collagen- and ADP-induced platelet aggregation with the estimated IC(50)s of 18 and 6 nM, respectively, and platelet function on clot retraction. It is a potent anti-platelet inhibitor, which should be further investigated for drug discovery to treat stroke patients or patients with thrombotic disorders. Published by Elsevier Ltd.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-04-01

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

  1. Evaluation of the protective immunogencity of the N, P, M, NV and G proteins of infectious hematopoietic necrosis virus in rainbow trout Oncorhynchus mykiss using DNA vaccines

    Science.gov (United States)

    Corbeil, S.; LaPatra, S.E.; Anderson, E.D.; Jones, J.; Vincent, B.; Hsu, Ya Li; Kurath, G.

    1999-01-01

    The protective immunogenicity of the nucleoprotein (N), phosphoprotein (P), matrix protein (M), non-virion protein (NV) and glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV) was assessed in rainbow trout using DNA vaccine technology. DNA vaccines were produced by amplifying and cloning the viral genes in the plasmid pCDNA 3.1. The protective immunity elicited by each vaccine was evaluated through survival of immunized fry after challenge with live virus. Neutralizing antibody titers were also determined in vaccinated rainbow troutOncorhynchus mykiss fry (mean weight 2 g) and 150 g sockeye salmon Oncorhynchus nerka. The serum from the 150 g fish was also used in passive immunization studies with naïve fry. Our results showed that neither the internal structural proteins (N, P and M) nor the NV protein of IHNV induced protective immunity in fry or neutralizing antibodies in fry and 150 g fish when expressed by a DNA vaccine construct. The G protein, however, did confer significant protection in fry up to 80 d post-immunization and induced protective neutralizing antibodies. We are currently investigating the role of different arms of the fish immune system that contribute to the high level of protection against IHNV seen in vaccinated fish.

  2. eEF1G interaction with foot-and-mouth disease virus nonstructural protein 2B: Identification by yeast two-hybrid system.

    Science.gov (United States)

    Zhang, Zhongwang; Pan, Li; Ding, Yaozhong; Lv, Jianliang; Zhou, Peng; Fang, Yuzhen; Liu, Xinsheng; Zhang, Yongguang; Wang, Yonglu

    2017-11-01

    Foot-and-mouth disease virus (FMDV) is a picornavirus that causes an economically significant disease in cattle and swine. Replication of FMDV is dependent on both viral proteins and cellular factors. Nonstructural protein 2B of FMDV plays multiple roles during viral infection and replication. We investigated the roles of 2B in virus-host interactions by constructing a cDNA library obtained from FMDV-infected swine tissues, and used a split-ubiquitin-based yeast two-hybrid system to identify host proteins that interacted with 2B. We found that 2B interacted with amino acids 208-437 in the C-terminal region of the eEF1G subunit of eukaryotic elongation factor 1, which is essential for protein synthesis. The 2B-eEF1G interaction was confirmed by co-immunoprecipitation of 2B and eEF1G in HEK293T cells. Collectively, our results suggest that eEF1G interacts with the 2B protein of FMDV. The identified 2B interaction partner may help to elucidate the mechanisms of FMDV infection and replication. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study

    Science.gov (United States)

    Cao, Wuxiong; Epstein, Charles; Liu, Hong; DeLoughery, Craig; Ge, Nanxiang; Lin, Jieyi; Diao, Rong; Cao, Hui; Long, Fan; Zhang, Xin; Chen, Yangde; Wright, Paul S; Busch, Steve; Wenck, Michelle; Wong, Karen; Saltzman, Alan G; Tang, Zhihua; Liu, Li; Zilberstein, Asher

    2004-01-01

    Background Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip® technology. In this "case study" we explored the transcriptional pattern changes during the in vitro differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology. Results The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results. Conclusions This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays

  4. Extraterrestrial Viruses?

    OpenAIRE

    Jurado Hernández, Daniel José

    2017-01-01

    Fundamentals of Life - Origin and Fundamentals of Living Things. Evaluation rubric to evaluate the debate and presentation about the point of view regarding the possibility of viruses from the outer space.

  5. ISOLASI cDNA SUCROSE TRANSPORTER (SUT DARI BATANG TANAMAN TEBU (Saccharum officinarum L.

    Directory of Open Access Journals (Sweden)

    - Slameto

    2010-09-01

    Full Text Available Sucrose Transporter (SUT is kind of protein transporter that control in sucrose translocation. Sucrose Transporter is intermediate in translocation of sucrose from apoplasmic to simplasmic. SUT facilitates sucrose transportation from vascular tissues to parenchyma cells toward in node sugarcane stem. This research was purposed to isolate cDNA SUT from sugarcane stem, and cloned in Escherichia coli strain DH5α. Total RNA of sugarcane stem was isolated by single step method, then add with oligo dT in order to obtain the first strand of SUT cDNA then used as template for PCR. The primer used for PCR is 5’ –ggg ctg att gtg gcc atg tc- ‘3 (SUT-F and 5’ –tgc cct ttg tct ccg gaa cc- ‘3 (SUT-R. PCR was programmed as follow denaturation at 94°C for 2 minutes and 30 second, annealing at 54°C for 30 s, extension at 72°C 2 min and 7 min, and storage at 4°C for unlimited, It was for 30 cycles. Complementary DNA SUT from PCR ligalized to pTOPO bunt-end, then it cloned in to E. coli strain DH5α. The cloning resulted then be sequenced in order to observe the homologues with other nucleotides sequences of some plant using BLASTn program in GENE BANK NCBI and the level of homology determined by Genetyx program. The concentrated of total RNA isolated was 5,024 μg/μl, with purity of 1,85. Complementary DNA SUT fragment from PCR with size 2037 bp appropriated to the both of primer was used. Complementary DNA SUT fragment showed by analyzed some of restriction enzyme e.g. EcoRI, PstI and BamHI. Homologues of this cDNA SUT fragment was 100% to SoSUT 2A of sugarcane stem and 84% to OsSUT of rice plant (Casu et al ., 2003.

  6. Evaluation of normalization methods for cDNA microarray data by k-NN classification

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wei; Xing, Eric P; Myers, Connie; Mian, Saira; Bissell, Mina J

    2004-12-17

    Non-biological factors give rise to unwanted variations in cDNA microarray data. There are many normalization methods designed to remove such variations. However, to date there have been few published systematic evaluations of these techniques for removing variations arising from dye biases in the context of downstream, higher-order analytical tasks such as classification. Ten location normalization methods that adjust spatial- and/or intensity-dependent dye biases, and three scale methods that adjust scale differences were applied, individually and in combination, to five distinct, published, cancer biology-related cDNA microarray data sets. Leave-one-out cross-validation (LOOCV) classification error was employed as the quantitative end-point for assessing the effectiveness of a normalization method. In particular, a known classifier, k-nearest neighbor (k-NN), was estimated from data normalized using a given technique, and the LOOCV error rate of the ensuing model was computed. We found that k-NN classifiers are sensitive to dye biases in the data. Using NONRM and GMEDIAN as baseline methods, our results show that single-bias-removal techniques which remove either spatial-dependent dye bias (referred later as spatial effect) or intensity-dependent dye bias (referred later as intensity effect) moderately reduce LOOCV classification errors; whereas double-bias-removal techniques which remove both spatial- and intensity effect reduce LOOCV classification errors even further. Of the 41 different strategies examined, three two-step processes, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, all of which removed intensity effect globally and spatial effect locally, appear to reduce LOOCV classification errors most consistently and effectively across all data sets. We also found that the investigated scale normalization methods do not reduce LOOCV classification error. Using LOOCV error of k-NNs as the evaluation criterion, three double

  7. Zika Virus

    OpenAIRE

    Musso, Didier; Gubler, Duane J.

    2016-01-01

    Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) in the genus Flavivirus and the family Flaviviridae. ZIKV was first isolated from a nonhuman primate in 1947 and from mosquitoes in 1948 in Africa, and ZIKV infections in humans were sporadic for half a century before emerging in the Pacific and the Americas. ZIKV is usually transmitted by the bite of infected mosquitoes. The clinical presentation of Zika fever is nonspecific and can be misdiagnosed as other infectious diseases, especi...

  8. Molecular cloning and sequence analysis of growth hormone cDNA of Neotropical freshwater fish Pacu (Piaractus mesopotamicus

    Directory of Open Access Journals (Sweden)

    Janeth Silva Pinheiro

    2008-01-01

    Full Text Available RT-PCR was used for amplifying Piaractus mesopotamicus growth hormone (GH cDNA obtained from mRNA extracted from pituitary cells. The amplified fragment was cloned and the complete cDNA sequence was determined. The cloned cDNA encompassed a sequence of 543 nucleotides that encoded a polypeptide of 178 amino acids corresponding to mature P. mesopotamicus GH. Comparison with other GH sequences showed a gap of 10 amino acids localized in the N terminus of the putative polypeptide of P. mesopotamicus. This same gap was also observed in other members of the family. Neighbor-joining tree analysis with GH sequences from fishes belonging to different taxonomic groups placed the P. mesopotamicus GH within the Otophysi group. To our knowledge, this is the first GH sequence of a Neotropical characiform fish deposited in GenBank.

  9. Sample processing and cDNA preparation for microbial metatranscriptomics in complex soil communities.

    Science.gov (United States)

    Carvalhais, Lilia C; Schenk, Peer M

    2013-01-01

    Soil presents one of the most complex environments for microbial communities as it provides many microhabitats that allow coexistence of thousands of species with important ecosystem functions. These include biomass and nutrient cycling, mineralization, and detoxification. Culture-independent DNA-based methods, such as metagenomics, have revealed operational taxonomic units that suggest a high diversity of microbial species and associated functions in soil. An emerging but technically challenging area to profile the functions of microorganisms and their activities is mRNA-based metatranscriptomics. Here, we describe issues and important considerations of soil sample processing and cDNA preparation for metatranscriptomics from bacteria and archaea and provide a set of methods that can be used in the required experimental steps. © 2013 Elsevier Inc. All rights reserved.

  10. Genes expression by using cDNA Microarray in Whallak-tang

    Directory of Open Access Journals (Sweden)

    Cheol-kyung Sin

    2008-12-01

    Full Text Available Objective : This study was undertaken to determine the effect of Whallak-tang on expression of CD/cytokine Genes. Methods : The expression of CD/Cytokine Genes were examined by cDNA microarray using the human mast cell line(HMC-l. Results : The expression of ATP5F1, FLJ20671, unknown, KIAA0342, OAS2, unknown genes were increased in 200~300% range. The expression of unknown, MDS006, IFITM1, MRPL3, ZNF207, FTH1, FBP1, NRGN, NR1H2, KIAA0747 genes were decreased in 0~33% range. Conclusion : These results would provide important basic data on the possibility of the clinical treatment of Whallaktang in musculoskeletal disease.

  11. Gene expression profile analysis in human hepatocellular carcinoma by cDNA microarray.

    Science.gov (United States)

    Chung, Eun Jung; Sung, Young Kwan; Farooq, Mohammad; Kim, Younghee; Im, Sanguk; Tak, Won Young; Hwang, Yoon Jin; Kim, Yang Il; Han, Hyung Soo; Kim, Jung-Chul; Kim, Moon Kyu

    2002-12-31

    We performed gene expression profiling of normal and hepatocellular carcinoma (HCC) liver tissues using a high-density microarray that contained 3,063 human cDNA. The results of a microarray hybridization experiment from eight different HCC tissues were analyzed and classified by the Cluster program. Among these differentially-expressed genes, the galectin-3, serine/threonine kinase SGK, translation factor eIF-4A, -4B, -3, fibroblast growth factor receptor, and ribosomal protein L35A were up-regulated; the mRNAs of Nip3, decorin, and the insulin-like growth factor binding protein-3 were down-regulated in HCC. The differential expression of these genes was further confirmed by an RT-PCR analysis. In addition, our data suggest that the gene expression profile of HCC varies according to the histological types.

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

    Science.gov (United States)

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

    2009-09-01

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

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

    DEFF Research Database (Denmark)

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

    1985-01-01

    obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication......DNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence...

  14. Antisense oligonucleotide inhibition of hepatitis C virus genotype 4 replication in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Omran Moataza H

    2006-06-01

    Full Text Available Abstract Background Hepatitis C (HCV viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy. It is estimated that over 15% of Egyptians are infected by the virus and thus finding a cure for this disease is of utmost importance. Current therapies for hepatitis C virus (HCV genotype 4 with interferon/ribavirin have not been successful and thus the development of alternative therapy for this genotype is disparately needed. Results Although previous studies utilizing viral subgenomic or full cDNA fragments linked to reporter genes transfected into adhered cells or in a cell free system showed promise, demonstration of efficient viral replication was lacking. Thus, we utilized HepG2 cells infected with native HCV RNA genomes in a replication competent system and used antisense phosphorothioate Oligonucleotides (S-ODN against stem loop IIId and the AUG translation start site of the viral polyprotein precursor to monitor viral replication. We were able to show complete arrest of intracellular replication of HCV-4 at 1 uM S-ODN, thus providing a proof of concept for the potential antiviral activity of S-ODN on native genomic replication of HCV genotype 4. Conclusion We have successfully demonstrated that by using two S-ODNs [(S-ODN1 (nt 326–348 and S-ODN-2 (nt 264–282], we were able to completely inhibit viral replication in culture, thus confirming earlier reports on subgenomic constructs and suggesting a potential therapeutic value in HCV type 4.

  15. Optimizing a custom tiling microarray for low input detection and identification of unamplified virus targets.

    Science.gov (United States)

    Yu, Christine; Wales, Samantha Q; Mammel, Mark K; Hida, Kaoru; Kulka, Michael

    2016-08-01

    Viruses are major pathogens causing foodborne illnesses and are often present at low levels in foods, thus requiring sensitive techniques for their detection in contaminated foods. The lack of efficient culture methods for many foodborne viruses and the potential for multi-species viral contamination have driven investigation toward non-amplification based methods for virus detection and identification. A custom DNA microarray (FDA_EVIR) was assessed for its sensitivity in the detection and identification of low-input virus targets, human hepatitis A virus, norovirus, and coxsackievirus, individually and in combination. Modifications to sample processing were made to accommodate low input levels of unamplified virus targets, which included addition of carrier cDNA, RNase treatment, and optimization of DNase I-mediated target fragmentation. Amplification-free detection and identification of foodborne viruses were achieved in the range of 250-500 copies of virus RNA. Alternative data analysis methods were employed to distinguish the genotypes of the viruses particularly at lower levels of target input and the single probe-based analysis approach made it possible to identify a minority species in a multi-virus complex. The oligonucleotide array is shown to be a promising platform to detect foodborne viruses at low levels close to what are anticipated in food or environmental samples. Published by Elsevier B.V.

  16. Characterisation of lettuce virus X, a new potexvirus infecting lettuce in Iran.

    Science.gov (United States)

    Dizadji, A; Koohi-Habibi, M; Izadpanah, K; Dietrich, C; Mossahebi, G H; Winter, S

    2008-01-01

    A virus with flexuous rod-shaped particle morphology was found in samples from lettuce during a survey of viruses infecting lettuce in Tehran province in Iran. This virus was subjected to a complete analysis of its biological and molecular features. The entire nucleotide sequence of the virus was determined, revealing a polyadenylated ssRNA genome consisting of 7,212 nucleotides [without poly (A) tail] and possessing an organization typical for potexviruses. Comparative genome analysis showed that the lettuce virus is closely related to Alstroemeria virus X, narcissus mosaic virus and asparagus virus 3. Based on particle morphology, physico-chemical properties and the complete genome sequence, this virus is a member of a new species in the genus Potexvirus, for which the name lettuce virus X (LeVX) is proposed. Biological assays using an infectious cDNA clone and a wild-type isolate of LeVX revealed that the virus, despite reaching high concentrations in all lettuce cultivars tested, does not cause symptoms in lettuce.

  17. Collagenolytic serine protease PC and trypsin PC from king crab Paralithodes camtschaticus: cDNA cloning and primary structure of the enzymes

    Directory of Open Access Journals (Sweden)

    Rebrikov Denis V

    2004-01-01

    Full Text Available Abstract Background In this paper, we describe cDNA cloning of a new anionic trypsin and a collagenolytic serine protease from king crab Paralithodes camtschaticus and the elucidation of their primary structures. Constructing the phylogenetic tree of these enzymes was undertaken in order to prove the evolutionary relationship between them. Results The mature trypsin PC and collagenolytic protease PC contain 237 (Mcalc 24.8 kDa and 226 amino acid residues (Mcalc 23.5 kDa, respectively. Alignments of their amino acid sequences revealed a high degree of the trypsin PC identity to the trypsin from Penaeus vannamei (approximately 70% and of the collagenolytic protease PC identity to the collagenase from fiddler crab Uca pugilator (76%. The phylogenetic tree of these enzymes was constructed. Conclusions Primary structures of the two mature enzymes from P. camtschaticus were obtained and compared with those of other proteolytic proteins, including some enzymes from brachyurans. A phylogenetic analysis was also carried out. These comparisons revealed that brachyurins are closely related to their vertebrate and bacterial congeners, occupy an intermediate position between them, and their study significantly contributes to the understanding of the evolution and function of serine proteases.

  18. Cloning of fusion protein gene of Newcastle disease virus into a baculovirus derived bacmid shuttle vector, in order to express it in insect cell line

    Directory of Open Access Journals (Sweden)

    Hashemzadeh MS

    2015-05-01

    Full Text Available Abstract Background: Newcastle disease virus (NDV is one of the major pathogens in poultry and vaccination is intended to control the disease, as an effective solution, yet. Fusion protein (F on surface of NDV, has a fundamental role in virus pathogenicity and can induce protective immunity, alone. With this background, here our aim was to construct a baculovirus derived recombinant bacmid shuttle vector (encoding F-protein in order to express it in insect cell line. Materials and Methods: In this experimental study, at first complete F gene from avirulent strain La Sota of NDV was amplified by RT-PCR to produce F cDNA. The amplicon was cloned into T/A cloning vector and afterwards into pFastBac Dual donor plasmid. After the verification of cloning process by two methods, PCR and enzymatic digestion analysis, the accuracy of F gene sequence was confirmed by sequencing. Finally, F-containing recombinant bacmid was subsequently generated in DH10Bac cell and the construct production was confirmed by a special PCR panel, using F specific primers and M13 universal primers. Results: Analysis of confirmatory tests showed that the recombinant bacmid, expressing of F-protein gene in correct sequence and framework, has been constructed successfully. Conclusion: The product of this F-containing recombinant bacmid, in addition to its independent application in the induction of protective immunity, can be used with the other individual recombinant baculoviruses, expressing HN and NP genes to produce NDV-VLPs in insect cell line.

  19. [The effects of culture supernatant of human GRC-1 cells transfected with PF4 cDNA on the VEGF expression and the growth of ECV304 cells].

    Science.gov (United States)

    Chang, Zheng; Liu, Fan; Han, Hua; Sun, Qiang

    2004-05-01

    To construct the eukaryotic expression vector pcDNA3-PF4-SS, and to detect the effects of the culture supernatant of transfected GRC-1 cells on the VEGF expression in transfected GRC-1 cells and the growth of ECV304 cells. The eukaryotic expression vector pcDNA3-PF4-SS was constructed and identified with Bgl II/BamH I digestion. The pcDNA3-PF4-SS was transfected stably into GRC-1 cells with lipofectamine mediation. The VEGF expression in transfected GRC-1 cells was detected by immunohistochemical staining, and the effect of the culture supernatant of transfected GRC-1 cells on ECV304 cells was detected by MTT colorimetry. Restrictive enzyme (Bgl II/BamH I)digestion analysis showed that the recombinant expression vector pcDNA3-PF4-SS had been constructed successfully. RT-PCR detection proved that hPF4 cDNA had been transfected into GRC-1 cells. The result of immunohistochemical staining showed that the VEGF expression could be seen in the cytoplasm and on cytomembrane of GRC-1 cells transfected with pcDNA3-PF4-SS, but the expression obviously weakened as comparison with that before transfection. Cell counting and MTT colorimetry manifested that the culture supernatant of transfected GRC-1 cells could inhibit markedly growth of ECV304 cells. The eukaryotic expression vector pcDNA3-PF4-SS has been constructed successfully, and stably transfected into the GRC-1 cells. The culture supernatant of transfected GRC-1 cells has obviously inhibitory effect on the growth of ECV304 cells and the VEGF expression in the GRC-1 cells, which lays some foundation for exploring the mechanism for anti-tumor growth and developing tumor vaccine for kidney neoplasms.

  20. Newcastle Disease Virus (PDQ)

    Science.gov (United States)

    ... to Ask about Your Treatment Research Newcastle Disease Virus (PDQ®)–Patient Version Overview Go to Health Professional ... Question 8 ). Questions and Answers About Newcastle Disease Virus What is Newcastle disease virus? Newcastle disease virus ( ...

  1. Powassan (POW) Virus Basics

    Science.gov (United States)

    ... Professionals Related Topics For International Travelers Powassan (POW) Virus Basics Download this fact sheet formatted for print: ... POW) Virus Fact Sheet (PDF) What is Powassan virus? Powassan (POW) virus is a flavivirus that is ...

  2. Molecular cloning and expression analysis of 12-oxophytodienoate reductase cDNA by wounding in Solanum tuberosum

    OpenAIRE

    Díaz, Mauricio; Polanco, Victor; Ramírez, Ingrid; Peña-Cortés, Hugo

    2012-01-01

    Jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) are signal molecules involved in the stress and defense responses in plants. A full-length cDNA clon of OPR3 encoding 12-oxophytodienoate reductase 3, key enzyme involved in the biosynthesis of JA from linolenic acid was obtained from a Solanum tuberosum cDNA library. Sequence analysis showed that OPR3 encoded a polypeptide of 400 amino acids with a predicted molecular mass of 43.9 kDa and pI of 7.72. The deduced amino acid sequence of OPR...

  3. Isolation of human cDNA clones of myb-related genes, A-myb and B-myb.

    OpenAIRE

    Nomura, N; Takahashi, M; Matsui, M; Ishii, S.; Date, T; Sasamoto, S; Ishizaki, R

    1988-01-01

    cDNA clones of the myb-related genes A-myb and B-myb were obtained by screening human cDNA libraries. The predicted open reading frame of B-myb could encode a protein of 700 amino acid residues. Although the C-terminal end has not been cloned yet, an almost entire coding region of A-myb, which is 745 amino acid long, was determined. The A-myb and B-myb proteins are highly homologous with the myb protein in three regions. Domain I, which is 161 amino acid long, is well conserved in the myb gen...

  4. Limits in virus filtration capability? Impact of virus quality and spike level on virus removal with xenotropic murine leukemia virus.

    Science.gov (United States)

    Roush, David J; Myrold, Adam; Burnham, Michael S; And, Joseph V; Hughes, Joseph V

    2015-01-01

    aggregate (5 vol/vol %). Significant differences were seen for the process performance over a number of lots of the less-pure Ultra 1 virus preparations. Experiments utilizing a developmental lot of the chromatographic purified XMuLV (Ultra 2 Development lot) that had elevated levels of host cell residuals (vs. the final Ultra 2 preparations) suggest that these contaminant residuals can impact virus filter fouling, even if the virus prep is essentially monodisperse. Process studies utilizing an Ultra 2 virus with substantially less host cell residuals and highly monodispersed virus particles demonstrated superior performance and an LRV in excess of 7.7 log10 . A model was constructed demonstrating the linear dependence of filtration flux versus filter loading which can be used to predict the V75 for a range of virus spike levels conditions using this highly purified virus. Fine tuning the virus spike level with this model can ultimately maximize the LRV for the virus filter step, essentially adding the LRV equivalent of another process step (i.e. protein A or CEX chromatography). © 2014 American Institute of Chemical Engineers.

  5. Computer Viruses. Technology Update.

    Science.gov (United States)

    Ponder, Tim, Comp.; Ropog, Marty, Comp.; Keating, Joseph, Comp.

    This document provides general information on computer viruses, how to help protect a computer network from them, measures to take if a computer becomes infected. Highlights include the origins of computer viruses; virus contraction; a description of some common virus types (File Virus, Boot Sector/Partition Table Viruses, Trojan Horses, and…

  6. Viruses Avian influenza, bovine herpes, bovine viral diarrhea virus ...

    Indian Academy of Sciences (India)

    ... human cytomegalovirus, herpes simplex virus, human immunodeficiency virus I, influenza, lymphocytic choriomeningitis virus, measles, papilloma, rabies, respiratory syncitial virus, simian immunodeficiency virus, simian virus 40. Bacteria Borrelia burgdorferi (Lyme disease), Moraxella bovis, Mycobacterium tuberculosis, ...

  7. Computer viruses

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, F.B.

    1986-01-01

    This thesis investigates a recently discovered vulnerability in computer systems which opens the possibility that a single individual with an average user's knowledge could cause widespread damage to information residing in computer networks. This vulnerability is due to a transitive integrity corrupting mechanism called a computer virus which causes corrupted information to spread from program to program. Experiments have shown that a virus can spread at an alarmingly rapid rate from user to user, from system to system, and from network to network, even when the best-availability security techniques are properly used. Formal definitions of self-replication, evolution, viruses, and protection mechanisms are used to prove that any system that allows sharing, general functionality, and transitivity of information flow cannot completely prevent viral attack. Computational aspects of viruses are examined, and several undecidable problems are shown. It is demonstrated that a virus may evolve so as to generate any computable sequence. Protection mechanisms are explored, and the design of computer networks that prevent both illicit modification and dissemination of information are given. Administration and protection of information networks based on partial orderings are examined, and probably correct automated administrative assistance is introduced.

  8. Identification of Plum pox virus pathogenicity determinants in herbaceous and woody hosts.

    Science.gov (United States)

    Salvador, B; Delgadillo, M O; Sáenz, P; García, J A; Simón-Mateo, C

    2008-01-01

    Plum pox virus (PPV) is a member of the genus Potyvirus that is able to infect a large variety of plant species, including trees of the genus Prunus, its natural host. When some PPV isolates are propagated for an extended time in herbaceous plants, their ability to infect trees is reduced. The molecular basis of this change in host infectivity is poorly understood. We report the construction of hybrid viruses from cDNA clones of two D-strain isolates of PPV, PPV-D and PPV-R, which differ in their host range. PPV-D can infect GF305 peach seedlings efficiently, however, it is unable to infect Nicotiana clevelandii plants. Conversely, PPV-R infects N. clevelandii, but not GF305 peach seedlings. The analyses of the hybrid viruses showed that, although determinants of PPV pathogenicity are extensively spread throughout the PPV genome, the 3' terminal region of the PPV-R genome, including the 3' noncoding region and the coding regions for the coat protein (CP), NIb, and part of NIa protein, is sufficient to confer infectivity of N. clevelandii in a PPV-D background. Our data demonstrate a high concentration of amino acid substitutions in the CP and a host-specific effect of a deletion at the N terminus of this protein in PPV pathogenicity in peach and N. clevelandii infectivity experiments. These results suggest that relevant host specificity determinants are located in the N-terminal region of the CP. The analyses of the PPV-R and PPV-D chimeras also showed that key host-specific pathogenicity determinants lie in the 5' terminal third of the PPV genome, a region that spans proteins P1, HCPro, and P3. The selection of mutations in only a few specific residues in proteins P1, P3, and 6K1 after partial adaptation of a chimeric virus (BD-GFP) to N. clevelandii further suggests a relevant role for these proteins in host adaptation.

  9. Multiple isoforms of immune-related genes from hemocytes and eyestalk cDNA libraries of swimming crab Portunus trituberculatus.

    Science.gov (United States)

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

    2011-07-01

    Expressed sequence tags (ESTs) analysis has been shown to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. Two full-length enriched cDNA libraries were constructed from hemocytes and eyestalk of Portunus trituberculatus, respectively, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. A total of 99 unigenes including 64 unigenes (6.00% of 1066 unigenes) in hemocytes library and 35 unigens (6.86% of 510 unigenes) in eyestalk library are identified to be immune genes. These genes are categorized into six classes, viz. antimicrobial peptides, redox proteins, melanization related proteins, chaperone proteins, clottable proteins and other immune factors. The content and category of immune genes in eyestalk library indicate eyestalk might have unrecognized role in crab immunity. Five immune genes containing multiple protein isoforms are identified and characterized, including anti-lipopolysaccharide factor (PtALF1-7), crustin (PtCrustin1-3), thioredoxin (PtTrx1-2), clip domain serine proteinase (PtcSP1-5) and kazal-type proteinase inhibitor (PtKPI1-4). Sequence alignment and phylogenetic analysis reveal PtALF1-7 contain two conserved cysteine residues and might be encoded by multiple genomic loci. PtCrustin1-3 share the consensus cysteine motif and are considered as Type I crustins. PtTrx1 possesses the critical structural cysteine residue C⁷³ of Trx-1, while PtTrx2 has the N-terminal mitochondrial translocation signal of Trx-2. Sequence analysis shows PtcSP1-5 contain one clip domain and one partial SP catalytic triad domain. PtKPI1-4 present one typical Kazal domain consisting of six conserved cysteine residues. Some protein isoforms are tissue-specific, which might suggest they have different origins and perform diverse functions. Except PtALF1-3 and PtCrustin1, the other isoformes in this study are firstly identified from P. trituberculatus

  10. Hendra virus.

    Science.gov (United States)

    Middleton, Deborah

    2014-12-01

    Hendra virus infection of horses occurred sporadically between 1994 and 2010 as a result of spill-over from the viral reservoir in Australian mainland flying-foxes, and occasional onward transmission to people also followed from exposure to affected horses. An unprecedented number of outbreaks were recorded in 2011 leading to heightened community concern. Release of an inactivated subunit vaccine for horses against Hendra virus represents the first commercially available product that is focused on mitigating the impact of a Biosafety Level 4 pathogen. Through preventing the development of acute Hendra virus disease in horses, vaccine use is also expected to reduce the risk of transmission of infection to people. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

  11. Marburg virus.

    Science.gov (United States)

    Dowdle, W R

    1976-01-01

    Marburg virus disease, which produced 20 per cent mortality when it first occured during 1967 in Germany and Yugoslavia, recently appeared again in South Africa. The source of the first outbreak was monkeys shipped from Africa; the origin of the second episode is unclear. Because distribution of the virus in nature is unknown, its threat to man cannot be readily determined. Differential laboratory diagnoses of hemorrhagic fevers should be encouraged in order to learn more about the epidemiology of these diseases and to better assess the risks which their etiologic agents may pose for attending medical personnel.

  12. Cloning and functional characterization of a testicular TSH receptor cDNA from the African catfish (Clarias gariepinus)

    NARCIS (Netherlands)

    Vischer, H F; Bogerd, J.

    A cDNA encoding a putative thyroid-stimulating hormone receptor (cfTSH-R) was cloned from the testis of the African catfish (Clarias gariepinus). The cfTSH-R showed the highest amino acid sequence identity with the TSH-Rs of other fish species. In addition, an insertion of approximately 50 amino

  13. Recombinant bovine dihydrofolate reductase produced by mutagenesis and nested PCR of murine dihydrofolate reductase cDNA.

    Science.gov (United States)

    Cody, Vivian; Mao, Qilong; Queener, Sherry F

    2008-11-01

    Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quantities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.

  14. Isolation of a cDNA encoding a putative cellulase in the red claw crayfish Cherax quadricarinatus.

    Science.gov (United States)

    Byrne, K A; Lehnert, S A; Johnson, S E; Moore, S S

    1999-11-01

    Amino acid sequences of cellulases have been determined in insects, nematodes, plants, slime moulds and bacteria but not in crustaceans. However, cellulase activity has been demonstrated in the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. In order to obtain information on the nature of this cellulase, a C. quadricarinatus hepatopancreas cDNA library was screened with a PCR product generated using degenerate oligonucleotide primers derived from conserved regions of known cellulases. Two identical 1.56kb cDNAs with sequence similarities to known cellulases, particularly the termite endoglucanases, were identified and sequenced. The clones contain the complete cDNA open reading frame for an endo-1, 4-beta-glucanase of 469 amino acids termed Cherax quadricarinatus endoglucanase (CqEG). The endogenous origin of the gene was confirmed by PCR amplification and sequencing of a 1012bp PCR product from genomic DNA. This fragment contains four exon sequences identical to the cDNA and is interrupted by three introns of 371, 102, 194bp respectively, with one intron exhibiting typical eukaryotic splice sites. The isolation of an endo-1,4-beta-glucanase encoding cDNA from the crayfish C. quadricarinatus provides the first endogenous cellulase sequence in a crustacean species.

  15. Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns

    Science.gov (United States)

    Luis F. Larrondo; Marcela Avila; Loreto Salas; Dan Cullen; Rafael Vicuna

    2003-01-01

    Analysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described lcs-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active...

  16. Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, E; Spiering, M; Chow, KS; Mulder, PPMFA; Subroto, T; Beintema, JJ

    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus

  17. Update History of This Database - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data ...List Contact us Budding yeast cDNA sequencing project Update History of This Database Date Update contents 2...cription Download License Update History of This Database Site Policy | Contact Us Update Histor

  18. Expressed sequence tags of randomly selected cDNA clones from Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza.

    Science.gov (United States)

    Tagu, D; Martin, F

    1995-01-01

    Random sequencing of cDNA clones from Eucalyptus globulus-Pisolithus tinctorius ectomycorrhizal tissues was carried out to generate expressed sequence tags (ESTs). Database comparisons revealed that 42% of the cDNAs corresponded to previously sequenced genes. These ESTs represent efficient molecular markers to analyze changes in gene expression during the formation of the ectomycorrhizal symbiosis.

  19. Cloning of the human carnitine-acylcarnitine carrier cDNA and identification of the molecular defect in a patient

    NARCIS (Netherlands)

    Huizing, M.; Iacobazzi, V.; IJlst, L.; Savelkoul, P.; Ruitenbeek, W.; van den Heuvel, L.; Indiveri, C.; Smeitink, J.; Trijbels, F.; Wanders, R.; Palmieri, F.

    1997-01-01

    The carnitine-acylcarnitine carrier (CAC) catalyzes the translocation of long-chain fatty acids across the inner mitochondrial membrane. We cloned and sequenced the human CAC cDNA, which has an open reading frame of 903 nucleotides. Northern blot studies revealed different expression levels of CAC

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

    NARCIS (Netherlands)

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

    2002-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Stear Michael

    2003-03-01

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

  2. Cloning and sequence analysis of an Ophiophagus hannah cDNA encoding a precursor of two natriuretic peptide domains.

    Science.gov (United States)

    Lei, Weiwei; Zhang, Yong; Yu, Guoyu; Jiang, Ping; He, Yingying; Lee, Wenhui; Zhang, Yun

    2011-04-01

    The king cobra (Ophiophagus hannah) is the largest venomous snake. Despite the components are mainly neurotoxins, the venom contains several proteins affecting blood system. Natriuretic peptide (NP), one of the important components of snake venoms, could cause local vasodilatation and a promoted capillary permeability facilitating a rapid diffusion of other toxins into the prey tissues. Due to the low abundance, it is hard to purify the snake venom NPs. The cDNA cloning of the NPs become a useful approach. In this study, a 957 bp natriuretic peptide-encoding cDNA clone was isolated from an O. hannah venom gland cDNA library. The open-reading frame of the cDNA encodes a 210-amino acid residues precursor protein named Oh-NP. Oh-NP has a typical signal peptide sequence of 26 amino acid residues. Surprisingly, Oh-NP has two typical NP domains which consist of the typical sequence of 17-residue loop of CFGXXDRIGC, so it is an unusual NP precursor. These two NP domains share high amino acid sequence identity. In addition, there are two homologous peptides of unknown function within the Oh-NP precursor. To our knowledge, Oh-NP is the first protein precursor containing two NP domains. It might belong to another subclass of snake venom NPs. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    1995-08-01

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

  4. cDNA cloning and expression analysis of a mannose-binding lectin ...

    Indian Academy of Sciences (India)

    PRAKASH

    Many plants contain carbohydrate-binding proteins that are commonly known as lectins, ... virus (HIV) and chemotherapy of acquired immunodeficiency syndrome (AIDS; Van Damme et ... Email, kxtangl@yahoo.com). Pinellia pedatisecta agglutinin (PPA) is a very basic protein that accumulates in the tuber of P. pedatisecta.

  5. Identification of a novel Getah virus by Virus-Discovery-cDNA random amplified polymorphic DNA (RAPD

    Directory of Open Access Journals (Sweden)

    Hu Tingsong

    2012-12-01

    Full Text Available Abstract Background The identification of new virus strains is important for the study of infectious disease, but current (or existing molecular biology methods are limited since the target sequence must be known to design genome-specific PCR primers. Thus, we developed a new method for the discovery of unknown viruses based on the cDNA - random amplified polymorphic DNA (cDNA-RAPD technique. Getah virus, belonging to the family Togaviridae in the genus Alphavirus, is a mosquito-borne enveloped RNA virus that was identified using the Virus-Discovery-cDNA RAPD (VIDISCR method. Results A novel Getah virus was identified by VIDISCR from suckling mice exposed to mosquitoes (Aedes albopictus collected in Yunnan Province, China. The non-structural protein gene, nsP3, the structural protein gene, the capsid protein gene, and the 3'-untranslated region (UTR of the novel Getah virus isolate were cloned and sequenced. Nucleotide sequence identities of each gene were determined to be 97.1–99.3%, 94.9–99.4%, and 93.6–99.9%, respectively, when compared with the genomes of 10 other representative strains of Getah virus. Conclusions The VIDISCR method was able to identify known virus isolates and a novel isolate of Getah virus from infected mice. Phylogenetic analysis indicated that the YN08 isolate was more closely related to the Hebei HB0234 strain than the YN0540 strain, and more genetically distinct from the MM2021 Malaysia primitive strain.

  6. Cloning of NAD-SDH cDNA from plum fruit and its expression and characterization.

    Science.gov (United States)

    Guo, Zhi-Xiong; Pan, Teng-Fei; Li, Kai-Tuo; Zhong, Feng-Lin; Lin, Lin; Pan, Dong-Ming; Lu, Liu-Xin

    2012-08-01

    A full-length cDNA consisting of 1444 bp for NAD dependent sorbitol dehydrogenase (NAD-SDH) was cloned from fruit of plum (Prunus salicina var. cordata cv. Younai) by means of RT-PCR and RACE. The cDNA containing an open reading frame (ORF) of 1101 bp encoded a polypeptide of 367 amino acid residues. The maltose binding protein fusion SDH (MBP-SDH) was expressed and partially purified from Escherichia coli cells, and biochemical properties of MBP-SDH and SDH cleaved from the fusion protein by factor Xa were characterized. The MBP-SDH had the specific affinity for NAD and was able to oxidize sorbitol, xylitol, l-ribitol and mannitol but not ethyl alcohol, arabitol and other polyols. The optimum pH for the oxidation of sorbitol and the reduction of fructose was 9.0 and 7.0, respectively; the maximum reaction rate occurred when temperature increased up to 50 °C in the presence of sorbitol. The MBP-SDH with a subunit of 80 kDa appears to be a hexamer. Its molecular weight was 478.6 kDa estimated by gel filtration and 493.2 kDa estimated using native linear gradient PAGE. The K(m) values for sorbitol, NAD, fructose and NADH were 95.86 mM, 0.31 mM, 1.04 M and 0.038 mM, respectively. However, when MBP was cleaved from the fusion enzyme, the SDH exists as a homotetramer with the native molecular weight of 164.8 kDa estimated by gel filtration. The K(m) values were 111.8 mM, 0.35 mM, 1.25 M and 0.048 mM for sorbitol, NAD, fructose and NADH, respectively. The MBP-SDH and the SDH were similar with respect to their kinetic characteristics despite their difference in quaternary structures. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-30

    Centipedes are one of the oldest venomous arthropods using toxin as their weapon to capture prey. But little attention was focused on them and only few centipede toxins were demonstrated with activity on ion channels. Therefore, more deep works are needed to understand the diversity of centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins were more complicated than any other animal toxins and even exhibited large differences in homologues. Meanwhile, a large number of variants generated by alternative cleavage sites were detected by mass spectra. Odd number of cystein (3, 5, 7) found in the mature peptides were seldom seen in peptide toxins. In additional, two novel cysteine frameworks (C-C-C-CCC, C-C-C-C-CC-CC) were identified from 16 different cysteine frameworks from centipede peptides. Only 29 precursors have clear targets, while others may provide a potential diversity function for centipede. These findings highlight the extensive diversity of centipede toxins and provide powerful tools to understand the capture and defense weapon of centipede. Peptide toxins from venomous animal have attracted increasing attentions due to their extraordinary chemical and pharmacological diversity. Centipedes are one of the most used Chinese traditional medicines, but little was known about the active components. The venom of Scolopendra subspinipes mutilans L. Koch is first deeply analyzed in this work and most of peptides were never discovered before. Interestingly, the number and arrangement of cysteine showed a larger different to known peptide toxins such spider or scorpion toxins. Moreover, only 29 peptides from this centipede venom were identified with known function. It suggested that our work not only important to

  8. Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine.

    Directory of Open Access Journals (Sweden)

    Utut Widyastuti Suharsono

    2008-11-01

    Full Text Available Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE.

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

    Energy Technology Data Exchange (ETDEWEB)

    Oyake, M.; Onodera, O.; Ikeuchi, T. [Niigata Univ. (Japan)] [and others

    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.

  10. All-fish gene constructs for growth hormone gene transfer in fish.

    Science.gov (United States)

    Cavari, B; Hong, Y; Funkenstein, B; Moav, B; Schartl, M

    1993-07-01

    In order to develop all-fish expression vectors for microinjection into fertilized fish eggs, we have prepared the following constructs: rainbow trout metallothionein a/b and the gilthead seabream growth hormone cDNA (ptMTa-gbsGHcDNA, ptMTb-gsbGHcDNA), carp β-actin gilthead seabream GH cDNA (pcAβ-gsbGHcDNA). The inducible metallothionein promoters a and b were cloned from rainbow trout, and the constitutive promoter β-actin was isolated from carp.The metallothionein promoters were cloned by using the PCR technique. The tMTa contains 430 bp, while the tMTb contains 260 bp (Hong et al. 1992). These two promoters were introduced to pGEM-3Z containing the GH cDNA of Sparus aurata to form ptMTa-gsbGH and ptMTb-gsbGH, respectively. The carp cytoplasmic β-actin gene was chosen as a source for isolating strong constitutive regulatory sequences. One of these regulatory sequences in pUC118 was ligated to GH cDNA of S. aurata to form the pcAβ-gsbGHcDNA.Expression of the constructs containing the metallothionein promoters was tested in fish cell culture and was found to be induced effectively by zinc. The ptMTa gsb-GH cDNA construct was microinjected into fertilized carp eggs, and integration in the genome of carp was detected in the DNA isolated from fins at the age of two months.

  11. Nucleotide sequence of a cDNA coding for the barley seed protein CMa: an inhibitor of insect α-amylase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Johansson, A.

    1992-01-01

    The primary structure of the insect alpha-amylase inhibitor CMa of barley seeds was deduced from a full-length cDNA clone pc43F6. Analysis of RNA from barley endosperm shows high levels 15 and 20 days after flowering. The cDNA predicts an amino acid sequence of 119 residues preceded by a signal...

  12. HUMAN PAPILLOMA VIRUS — ONCOGENIC VIRUS

    Directory of Open Access Journals (Sweden)

    A.N. Mayansky

    2010-01-01

    Full Text Available The lecture is devoted to oncogenic viruses, particularly human papilloma virus. Papilloma viral infection is found in all parts of the globe and highly contagious. In addition to exhaustive current data on classification, specifics of papilloma viruses composition and epidemiology, the author describes in great detail the malignization mechanisms of papilloma viruses pockets. Also, issues of diagnostics and specific prevention and treatment of diseases caused by this virus are illustrated. Key words: oncogenic viruses, papilloma viruses, prevention, vaccination. (Pediatric Pharmacology. – 2010; 7(4:48-55

  13. Prevalence of Hepatitis C virus Genotype 3a in patients with Hodgkin and Non-Hodgkin Lymphoma.

    Science.gov (United States)

    Radmehr, Hashem; Makvandi, Manoochehr; Samarbafzadeh, Alireza; Teimoori, Ali; Neisi, Niloofar; Rasti, Mojtaba; Abasifar, Sara; Soltani, Hasan; Abbasi, Samaneh; Kiani, Hadis; Mehravaran, Hamide; Azaran, Azarakhsh; Shahani, Toran

    2016-12-01

    Hepatitis C virus (HCV) is a major public health problem worldwide. Replication and persistence of HCV genome have been described in the liver tissue as well as B cells lymphocyte. Several investigations have reported that long-term persistence of HCV in B cells may result in Hodgkin and Non-Hodgkin lymphoma. This study was aimed to determine frequency of HCV RNA in histological tissues obtained from patients suffered from Hodgkin and Non-Hodgkin lymphoma. 52 formalin-fixed paraffin-embedded tissue blocks including 23 (44.3%) Hodgkin and 29 (55.7%) Non-Hodgkin samples were collected and five micrometer sections were prepared. RNA was extracted and cDNA was synthesized. Two consecutive Nested RT-PCR assays were carried out for detection of HCV 5' UTR and core gene. RT-PCR products were sequenced and aligned to construct HCV phylogenic tree to evaluate the homology of sequences in comparison to the reference sequences retrieved from Genbank. Overall, 6 Non-Hodgkin (20.6%) and 3 Hodgkin lymphoma (13.04%) samples showed positive PCR results for both 5' UTR and HCV core RNA via nested PCR (PHodgkin and Non-Hodgkin lymphoma. To improve treatment regimens, screening of HCV RNA in patients suffered from Hodgkin or Non-Hodgkin lymphoma is recommended which can be done through highly sensitive molecular means before and after immunosuppression status.

  14. Protective efficacy and immunogenicity of an adenoviral vector vaccine encoding the codon-optimized F protein of respiratory syncytial virus.

    Science.gov (United States)

    Kohlmann, Rebekka; Schwannecke, Sarah; Tippler, Bettina; Ternette, Nicola; Temchura, Vladimir V; Tenbusch, Matthias; Uberla, Klaus; Grunwald, Thomas

    2009-12-01

    Adenoviral vectors (AdV) have received considerable attention for vaccine development because of their high immunogenicity and efficacy. In previous studies, it was shown that DNA immunization of mice with codon-optimized expression plasmids encoding the fusion protein of respiratory syncytial virus (RSV F) resulted in enhanced protection against RSV challenge compared to immunization with plasmids carrying the wild-type cDNA sequence of RSV F. In this study, we constructed AdV carrying the codon-optimized full-length RSV F gene (AdV-F) or the soluble form of the RSV F gene (AdV-Fsol). BALB/c mice were immunized twice with AdV-F or AdV-Fsol and challenged with RSV intranasally. Substantial levels of antibody to RSV F were induced by both AdV vaccines, with peak neutralizing-antibody titers of 1:900. Consistently, the viral loads in lung homogenates and bronchoalveolar lavage fluids were significantly reduced by a factor of more than 60,000. The protection against viral challenge could be measured even 8 months after the booster immunization. AdV-F and AdV-Fsol induced similar levels of immunogenicity and protective efficacy. Therefore, these results encourage further development of AdV vaccines against RSV infection in humans.

  15. Serine Protease Variants Encoded by Echis ocellatus Venom Gland cDNA: Cloning and Sequencing Analysis

    Directory of Open Access Journals (Sweden)

    S. S. Hasson

    2010-01-01

    Full Text Available Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent.

  16. Gene expression of panaxydol-treated human melanoma cells using radioactive cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Joong Youn; Yu, Su Jin; Soh, Jeong Won; Kim, Meyoung Kon [College of Medicine, Korea Univ., Seoul (Korea, Republic of)

    2001-07-01

    Polyacetylenic alcohols derived from Panax ginseng have been studied to be an anticancer reagent previously. One of the Panax ginseng polyacetylenic alcohols, i.e., panaxydol, has been studied to possess an antiproliferative effect on human melanoma cell line (SK-MEL-1). In ths study, radioactive cDNA microarrays enabled an efficient approach to analyze the pattern of gene expression (3.194 genes in a total) simultaneously. The bioinformatics selection of human cDNAs, which is specifically designed for immunology, apoptosis and signal transduction, were arrayed on nylon membranes. Using with {sup 33}P labeled probes, this method provided highly sensitive gene expression profiles of our interest including apoptosis, cell proliferation, cell cycle, and signal transduction. Gene expression profiles were also classified into several categories in accordance with the duration of panaxydol treatment. Consequently, the gene profiles of our interest were significantly up (199 genes, > 2.0 of Z-ratio) or down-(196 genes, < 2.0 of Z-ratio) regulated in panaxydol-treated human melanoma cells.

  17. Monitoring gene expression of potato under salinity using cDNA microarrays.

    Science.gov (United States)

    Legay, Sylvain; Lamoureux, Didier; Hausman, Jean-François; Hoffmann, Lucien; Evers, Danièle

    2009-12-01

    The molecular response to salt exposure was studied in the leaves of a Solanum tuberosum clone using cDNA microarray. Differentially expressed genes were classified according to their known or predicted function and their expression ratio as compared to the control. The major changes upon a 150 mM NaCl exposure in potato leaves occurred in the photosystem apparatus and Calvin cycle: many transcripts coding for proteins belonging to photosystems I and II and chlorophyll synthesis were repressed. On the other hand, we observed the induction of various kinds of transcription factors implicated in osmotic stress response via ABA-dependent or ABA-independent pathways but also in plant defense pathways. This revealed a crosstalk between abiotic and biotic stress responses during salt exposure, which activated several adaptation mechanisms including heat shock proteins, late embryogenesis abundant, dehydrins and PR proteins. Gene expression changes related to carbohydrate and amino acid metabolism were also observed, pointing at putative modifications at the metabolic level.

  18. Cloning and Sequencing of Protein Kinase cDNA from Harbor Seal (Phoca vitulina Lymphocytes

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2004-01-01

    Full Text Available Protein kinases (PKs play critical roles in signal transduction and activation of lymphocytes. The identification of PK genes provides a tool for understanding mechanisms of immunotoxic xenobiotics. As part of a larger study investigating persistent organic pollutants in the harbor seal and their possible immunomodulatory actions, we sequenced harbor seal cDNA fragments encoding PKs. The procedure, using degenerate primers based on conserved motifs of human protein tyrosine kinases (PTKs, successfully amplified nine phocid PK gene fragments with high homology to human and rodent orthologs. We identified eight PTKs and one dual (serine/threonine and tyrosine kinase. Among these were several PKs important in early signaling events through the B- and T-cell receptors (FYN, LYN, ITK and SYK and a MAP kinase involved in downstream signal transduction. V-FGR, RET and DDR2 were also expressed. Sequential activation of protein kinases ultimately induces gene transcription leading to the proliferation and differentiation of lymphocytes critical to adaptive immunity. PKs are potential targets of bioactive xenobiotics, including persistent organic pollutants of the marine environment; characterization of these molecules in the harbor seal provides a foundation for further research illuminating mechanisms of action of contaminants speculated to contribute to large-scale die-offs of marine mammals via immunosuppression.

  19. Cloning, Characterization, and Functional Expression of Phospholipase Dα cDNA from Banana (Musa acuminate L.

    Directory of Open Access Journals (Sweden)

    Li Li

    2017-01-01

    Full Text Available Phospholipase D (PLD plays a key role in adaptive responses of postharvest fruits. A cDNA clone of banana (Musa acuminate L. PLDα (MaPLDα was obtained by RT-PCR in this study. The MaPLDα gene contains a complete open reading frame (ORF encoding a 92-kDa protein composed of 832 amino acid residues and possesses a characteristic C2 domain and two catalytic H×K×××D (abbr. HKD motifs. The two HKD motifs are separated by 341 amino acid residues in the primary structure. Relatively higher PLD activity and expression of MaPLDα mRNA were detected in developing tissues compared to senescent or mature tissues in individual leaves, flower, stem, and fruit organs, respectively. The expression profile of PLDα mRNA in postharvest banana fruits at different temperatures was determined, and the MaPLDα mRNA reached the highest expression peak on day 5 at 25°C and on day 7 at 12°C. The results provide useful information for maintaining postharvest quality and extending the storage life of banana fruit.

  20. Characterization of porcine ENO3: genomic and cDNA structure, polymorphism and expression

    Directory of Open Access Journals (Sweden)

    Xiong Yuanzhu

    2008-09-01

    Full Text Available Abstract In this study, a full-length cDNA of the porcine ENO3 gene encoding a 434 amino acid protein was isolated. It contains 12 exons over approximately 5.4 kb. Differential splicing in the 5'-untranslated sequence generates two forms of mRNA that differ from each other in the presence or absence of a 142-nucleotide fragment. Expression analysis showed that transcript 1 of ENO3 is highly expressed in liver and lung, while transcript 2 is highly expressed in skeletal muscle and heart. We provide the first evidence that in skeletal muscle expression of ENO3 is different between Yorkshire and Meishan pig breeds. Furthermore, real-time polymerase chain reaction revealed that, in Yorkshire pigs, skeletal muscle expression of transcript 1 is identical at postnatal day-1 and at other stages while that of transcript 2 is higher. Moreover, expression of transcript 1 is lower in skeletal muscle and all other tissue samples than that of transcript 2, with the exception of liver and kidney. Statistical analysis showed the existence of a polymorphism in the ENO3 gene between Chinese indigenous and introduced commercial western pig breeds and that it is associated with fat percentage, average backfat thickness, meat marbling and intramuscular fat in two different populations.