WorldWideScience

Sample records for characterized clone resource

  1. Cloning and characterization of new bioluminescent proteins

    Science.gov (United States)

    Szent-Gyorgyi, Christopher; Ballou, Byron T.; Dagnal, Erich; Bryan, Bruce

    1999-07-01

    Over the past two years Prolume has undertaken a comprehensive program to clone luciferases and associated 'green fluorescent proteins' (GFPs) from marine animals that use coelenterazine as the luciferin. To data we have cloned several bioluminescent proteins, including two novel copepod luciferases and two anthozoan GFPs. These four proteins have sequences that differ greatly form previously cloned analogous proteins; the sequence diversity apparently is due to independent evolutionary origins and unusual evolutionary constraints. Thus coelenterazine-based bioluminescent systems may also manifest a variety of useful properties. We discuss form this taxonomic perspective the initial biochemical and spectral characterization of our cloned proteins. Emphasis is placed on the anthozoan luciferase-GFP systems, whose efficient resonance energy transfer has elicited much current interest.

  2. Distribution and uses of legume DNA clone resources

    International Nuclear Information System (INIS)

    Since 1990, my lab has developed and distributed various DNA clone resources for the legumes. In the first several years, the focus was on members of the tropical genus, Vigna, including the widely cultivated species, mungbean (V. radiata) and cowpea (V. unguiculata). Both of these grain legumes play key roles in agriculture in developing countries of Asia (mungbean) and Africa (cowpea). Moreover, because there is substantial genome conservation among legumes, these genetic resources have also been utilized by a wide range of researchers in other crop species. In 1997, my lab began to focus on the development and distribution of a new generation of DNA clone resources; Bacterial Artificial Chromosomes (BAC). A library of these clones was constructed in soybean (Glycine max) the most important legume species worldwide in terms of economic value. Again, the library has become a valuable resource for the legume research community and has been widely used in studies of legume genomics. (author)

  3. Recombinant laccase: I. Enzyme cloning and characterization.

    Science.gov (United States)

    Nicolini, Claudio; Bruzzese, Debora; Cambria, Maria Teresa; Bragazzi, Nicola Luigi; Pechkova, Eugenia

    2013-03-01

    We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white-rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system. The resulting expression plasmid, pET-28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl-β-d-thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II. PMID:22991171

  4. Characterizing and Evaluating the Impact of Software Interface Clones

    Directory of Open Access Journals (Sweden)

    Hani Abdeen

    2013-02-01

    Full Text Available Software Interfaces are meant to describe contracts governing interactions between logic modules.Interfaces, if well designed, significantly reduce software complexity and ease maintainability. However,as software evolves, the organization and the quality of software interfaces gradually deteriorate. As aconsequence, this often leads to increased development cost, lower code quality and reduced reusability.Code clones are one of the most known bad smells in source code. This design defect may occur ininterfaces by duplicating method/API declarations in several interfaces. Such interfaces are similar fromthe point of view of public services/APIs they specify, thus they indicate a bad organization of applicationservices. In this paper, we characterize the interface clone design defect and illustrate it via examplestaken from real-world open source software applications. We conduct an empirical study covering ninereal-world open source software applications to quantify the presence of interface clones and evaluatetheir impact on interface design quality. The results of the empirical study show that interface clones arewidely present in software interfaces. They also show that the presence of interface clones may cause adegradation of interface cohesion and indicate a considerable presence of code clones at implementationslevel.

  5. Characterizing seamless ligation cloning extract for synthetic biological applications.

    Science.gov (United States)

    Messerschmidt, Katrin; Hochrein, Lena; Dehm, Daniel; Schulz, Karina; Mueller-Roeber, Bernd

    2016-09-15

    Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular and synthetic biology projects. PMID:27311554

  6. Construction characterization and application of Flavivirus infectious clones

    NARCIS (Netherlands)

    Jiang, Xiaohong

    2013-01-01

    The topics in this thesis revolve around a group of plus-strand RNA viruses that belong to the Flavivirus genus, a general introduction of which is presented in Chapter 1. The experimental chapters in this thesis mainly focus on the construction and characterization of flavivirus infectious clones a

  7. Molecular cloning and functional characterization of avian interleukin-19

    Science.gov (United States)

    The present study describes the cloning and functional characterization of avian interleukin (IL)-19, a cytokine that, in mammals, alters the balance of Th1 and Th2 cells in favor of the Th2 phenotype. The full-length avian IL-19 gene, located on chromosome 26, was amplified from LPS-stimulated chi...

  8. Molecular Cloning, Overexpression and Characterization of Human Interleukin 1α

    OpenAIRE

    Rajalingam, Dakshinamurthy; Kacer, Doreen; Prudovsky, Igor; Kumar, Thallapuranam Krishnaswamy Suresh

    2007-01-01

    Interleukin-1 alpha (IL-1α) regulates a wide range of important cellular processes. In this study for the first time we report the cloning, expression, biophysical and biological characterization of the human interleukin-1α. Human IL-1α has been expressed in Escherichia coli in high yields (~ 4 mg per liter of the bacterial culture). The protein was purified to homogeneity (~ 98% purity) using affinity chromatography and size exclusion chromatography. Results of the steady state fluorescence ...

  9. Cloning

    Science.gov (United States)

    ... copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. NIH: National Human Genome Research Institute

  10. Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

    Science.gov (United States)

    Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

    1997-02-01

    Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

  11. Cloning

    Science.gov (United States)

    ... Care Online Health Resources For Health Professionals Competency & Curricular Resources Genetics 101 Genomic Medicine and Health Care ... as bacteria , produce genetically identical offspring through a process called asexual reproduction. In asexual reproduction, a new ...

  12. Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana

    Science.gov (United States)

    Wang, Zhonghua; Yeats, Trevor; Han, Hong; Jetter, Reinhard

    2010-01-01

    The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C30H50O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761–779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores. PMID:20610397

  13. Cloning, characterization and targeting of the mouse HEXA gene

    Energy Technology Data Exchange (ETDEWEB)

    Wakamatsu, N.; Trasler, J.M.; Gravel, R.A. [McGill Univ., Quebec (Canada)] [and others

    1994-09-01

    The HEXA gene, encoding the {alpha} subunit of {beta}-hexosaminidase A, is essential for the metabolism of ganglioside G{sub M2}, and defects in this gene cause Tay-Sachs disease in humans. To elucidate the role of the gene in the nervous system of the mouse and to establish a mouse model of Tay-Sachs disease, we have cloned and characterized the HEXA gene and targeted a disruption of the gene in mouse ES cells. The mouse HEXA gene spans {approximately}26 kb and consists of 14 exons, similar to the human gene. A heterogeneous transcription initiation site was identified 21-42 bp 5{prime} of the initiator ATG, with two of the sites fitting the consensus CTCA (A = start) as seen for some weak initiator systems. Promoter analysis showed that the first 150 bp 5{prime} of the ATG contained 85% of promoter activity observed in constructs containing up to 1050 bp of 5{prime} sequence. The active region contained a sequence matching that of the adenovirus major late promoter upstream element factor. A survey of mouse tissues showed that the highest mRNA levels were in (max to min): testis (5.5 x brain cortex), adrenal, epididymis, heart, brain, lung, kidney, and liver (0.3 x brain cortex). A 12 kb BstI/SalI fragment containing nine exons was disrupted with the insertion of the bacterial neo{sup r} gene in exon 11 and was targeted into 129/Sv ES cells by homologous recombination. Nine of 153 G418 resistant clones were correctly targeted as confirmed by Southern blotting. The heterozygous ES cells were microinjected into mouse blastocysts and implanted into pseudo-pregnant mice. Nine male chimeric mice, showing that 40-95% chimerism for the 129/Sv agouti coat color marker, are being bred in an effort to generate germline transmission of the disrupted HEXA gene.

  14. [Cloning and functional characterization of phytoene desaturase in Andrographis paniculata].

    Science.gov (United States)

    Shen, Qin-qin; Li, Li-xia; Zhan, Peng-lin; Wang, Qiang

    2015-10-01

    A full-length cDNA of phytoene desaturase (PDS) gene from Andrographis paniculata was obtained through RACE-PCR. The cDNA sequence consists of 2 224 bp with an intact ORF of 1 752 bp (GeneBank: KP982892), encoding a ploypeptide of 584 amino acids. Homology analysis showed that the deduced protein has extensive sequence similarities to PDS from other plants, and contains a conserved NAD ( H) -binding domain of plant dehydrase cofactor binding-domain in N-terminal. Phylogenetic analysis demonstrated that ApPDS was more related to PDS of Sesamum indicum and Pogostemon cablin. The semi-quantitative RT-PCR analysis revealed that ApPDS expressed in whole aboveground tissues with the highest expression in leaves. Virus induced gene silencing (VIGS) was performed to characterize the functional of ApPDS in planta. Significant photobleaching was not observed in infiltrated leaves, while the PDS gene has been down-regulated significantly at the yellowish area. To the best of our knowledge, this represents the first report of PDS gene cloning and functional characterization from A. paniculata, which lays the foundation for further investigation of new genes, especially that correlative to andrographolide biosynthetic pathway. PMID:26975098

  15. Construction and characterization of two Citrus BAC libraries and identification of clones containing the phytoene synthase gene.

    Science.gov (United States)

    Baig, M N R; Yu, An; Guo, Wenwu; Deng, Xiuxin

    2009-05-01

    Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck 'Cara Cara' navel orange and Citrus reticulata (L.) Blanco 'Egan No. 1' Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 x 109 bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 x 109 bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in genomics research in the two important species C. sinensis and C. reticulata. Resources, high-density filters, individual clones, and whole libraries are available for public distribution and are accessible at the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

  16. Cloning and characterization of a murine SIL gene

    Energy Technology Data Exchange (ETDEWEB)

    Collazo-Garcia, N.; Scherer, P.; Aplan, P.D. [Roswell Park Cancer Institute, Buffalo, NY (United States)

    1995-12-10

    The human SIL gene is disrupted by a site-specific interstitial deletion in 25% of children with T-cell acute lymphoblastic leukemia. Since transcriptionally active genes are prone to recombination events, the recurrent nature of this lesion suggests that the SIL gene product is transcriptionally active in the cell type that undergoes this interstitial deletion and that the SIL gene product may play a role in normal lymphoid development. To facilitate studies of SIL gene function, we have cloned and characterized a murine SIL gene. The predicted murine SIL protein is 75% identical to the human gene, with good homology throughout the open reading frame. An in vitro translated SIL cDNA generated a protein slightly larger than the predicted 139-kDa protein. Although a prior report detected SIL mRNA expression exclusively in hematopoietic tissues, a sensitive RT-PCR assay demonstrated SIL expression to be ubiquitous, detectable in all tissues examined. Since the RT-PCR assay suggested that SIL mRNA expression was higher in rapidly proliferating tissues, we assayed SIL mRNA expression using a murine erythroleukemia model of terminal differentiation and found it to be dramatically decreased in conjunction with terminal differentiation. These studies demonstrate that the human SIL gene product is quite well conserved in rodents and suggest that the SIL gene product may play a role in cell proliferation. 26 refs., 6 figs.

  17. Cloning and characterization of metallothionein gene in ayu Plecoglossus altivelis

    Energy Technology Data Exchange (ETDEWEB)

    Lin, C.-H.; John, Joseph Abraham Christopher; Ou, L.-W.; Chen, J.-C.; Lin, C.-H.; Chang, C.-Y

    2004-02-10

    Metallothionein (MT) has been used widely as a potential molecular marker to detect the deleterious effects of heavy metals in aquatic ecosystem. Here we exposed ayu, Plecoglossus altivelis, to zinc (Zn) and tested the distribution as well as the induction of MT in various tissues such as liver, kidney, intestine and stomach. MT induction was significant in liver tissue, followed by kidney and intestine, whereas no induction was detected in stomach. The gene encoding ayu MT was successfully cloned and characterized. Complete nucleotide sequencing and analysis of the 4.5 kb DNA fragment containing the ayu MT gene revealed that the gene has three exons interrupted by two introns, a 5'-flanking region of about 2.5 kb and about 1.6 kb of 3'-flanking region. In grouper heart and kidney cells, the 2.5 kb promoter containing eight metal responsive elements (MREs), two hepatic nuclear factor 5 responsive elements (HNF5REs) and one cAMP responsive element (CRE) had the highest reporter activity.

  18. Cloning and characterization of a cDNA clone encoding calreticulin from Haemaphysalis qinghaiensis (Acari: Ixodidae).

    Science.gov (United States)

    Gao, Jinliang; Luo, Jianxun; Fan, Ruiquan; Fingerle, Volker; Guan, Guiquan; Liu, Zhijie; Li, Youquan; Zhao, Haiping; Ma, Miling; Liu, Junlong; Liu, Aihong; Ren, Qiaoyun; Dang, Zhisheng; Sugimoto, Chihiro; Yin, Hong

    2008-03-01

    The application of anti-tick vaccine has been shown to be the most promising alternative strategy compared to the current use of acaricides that suffer from a number of serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. Previously, we have cloned 21 positive clones (named from Hq02 to Hq22) by immunoscreening complimentary DNA (cDNA) libraries of Haemaphysalis qinghaiensis; however, some of those clones did not contain open reading frames (ORF). In this study, we amplified the entire sequence of Hq07 by using rapid amplification of the cDNA ends. Hq07 contains an ORF of 1,233 bp that encodes for 410 amino acid residues with a coding capacity of 47 kDa. Search of the cloned sequences against GenBank revealed that Hq07 is a calreticulin (CRT)-similar clone and designated HqCRT. Expression analysis by reverse transcription-polymerase chain reaction showed that this gene is ubiquitously expressed at different developmental stages and in different tissues of H. qinghaiensis. The gene was expressed as glutathione S-transferase-fused proteins in a prokaryotic system. Western blot analysis revealed that native HqCRT was secreted into their hosts by ticks during blood sucking. Vaccination of sheep with rHqCRT conferred protective immunity against ticks, resulting in 54.3% mortality in adult ticks, compared to the 38.7% death rate in the control group. These results demonstrated that rHqCRT might be a useful vaccine candidate antigen for biological control of H. qinghaiensis.

  19. Construction and characterization of human chromosome 2-specific cosmid, fosmid, and PAC clone libraries

    Energy Technology Data Exchange (ETDEWEB)

    Gingrich, J.C.; Boehrer, D.M.; Garnes, J.A. [Lawrence Livermore National Lab., CA (United States)] [and others

    1996-02-15

    This article discusses the construction and characterization of three human chromosome 2-specific clone libraries. A chromosome 2-specific PAC library was also constructed from a hybrid cell line. The chromosome 2 coverage of each of the three libraries was further determined by PCR screening clone pools with 82 chromosome 2-specific STSs. 47 refs., 3 figs., 1 tab.

  20. Cloning and characterization of a pathogen-induced chitinase in Brassica napus

    DEFF Research Database (Denmark)

    Rasmussen, U.; Bojsen, K.; Collinge, D.B.

    1992-01-01

    A chitinase cDNA clone from rapeseed (Brassica napus L. ssp. oleifera) was isolated. The cDNA clone, ChB4, represents a previously purified and characterized basic chitinase isozyme. The longest open reading frame in ChB4 encodes a polypeptide of 268 amino acids. This polypeptide consists of a 24...

  1. Characterization of glyphosate resistance in cloned Amaranthus palmeri plants

    Science.gov (United States)

    Glyphosate resistant Palmer amaranth from Georgia (GA) possesses multiple copies of the target site, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of this herbicide. Cloned plants of glyphosate-resistant Palmer amaranth biotypes from Mississippi (MS) were compared with GA populations using le...

  2. Molecular cloning and characterization of duck interleukin-17

    Science.gov (United States)

    Interleukin-17 (IL-17) belonging to the Th17 family is a proinflammatory cytokine produced by activated T cells. A 1034-bp cDNA encoding duck IL-17 (duIL-17) was cloned from ConA-activated splenic lymphocytes of ducks. The encoded protein, predicted to consisted of 169 amino acids, displayed a molec...

  3. Molecular cloning and characterization of multidomain xylanase from manure library

    Science.gov (United States)

    The gene (manf-x10) encoding xylanase from an environmental genomic DNA library was cloned and expressed in Escherichia coli. The encoded enzyme was predicted to be 467 amino acids with a molecular mass of 50.3 kD. The recombinant ManF-X10 was purified by HisTrap affinity column and showed activit...

  4. Characterization and multivariate classification of grapes and wines of two Cabernet Sauvignon clones

    Directory of Open Access Journals (Sweden)

    Vívian Maria Burin

    2011-05-01

    Full Text Available The objective of this work was to assess and characterize two clones, 169 and 685, of Cabernet Sauvignon grapes and to evaluate the wine produced from these grapes. The experiment was carried out in São Joaquim, SC, Brazil, during the 2009 harvest season. During grape ripening, the evolution of physical-chemical properties, phenolic compounds, organic acids, and anthocyanins was evaluated. During grape harvest, yield components were determined for each clone. Individual and total phenolics, individual and total anthocyanins, and antioxidant activity were evaluated for wine. The clones were also assessed regarding the duration of their phenological cycle. During ripening, the evolution of phenolic compounds and of physical-chemical parameters was similar for both clones; however, during harvest, significant differences were observed regarding yield, number of bunches per plant and berries per bunch, leaf area, and organic acid, polyphenol, and anthocyanin content. The wines produced from these clones showed significant differences regarding chemical composition. The clones showed similar phenological cycle and responses to bioclimatic parameters. Principal component analysis shows that clone 685 is strongly correlated with color characteristics, mainly monomeric anthocyanins, while clone 169 is correlated with individual phenolic compounds.

  5. A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization

    OpenAIRE

    Valéria Pereira Ferrer; Thiago Lopes de Mari; Luiza Helena Gremski; Dilza Trevisan Silva; Rafael Bertoni da Silveira; Waldemiro Gremski; Olga Meiri Chaim; Andrea Senff-Ribeiro; Helena Bonciani Nader; Silvio Sanches Veiga

    2013-01-01

    Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide...

  6. Cloning, characterization and expression analysis of porcine microRNAs

    Directory of Open Access Journals (Sweden)

    Desilva Udaya

    2009-02-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are small ~22-nt regulatory RNAs that can silence target genes, by blocking their protein production or degrading the mRNAs. Pig is an important animal in the agriculture industry because of its utility in the meat production. Besides, pig has tremendous biomedical importance as a model organism because of its closer proximity to humans than the mouse model. Several hundreds of miRNAs have been identified from mammals, humans, mice and rats, but little is known about the miRNA component in the pig genome. Here, we adopted an experimental approach to identify conserved and unique miRNAs and characterize their expression patterns in diverse tissues of pig. Results By sequencing a small RNA library generated using pooled RNA from the pig heart, liver and thymus; we identified a total of 120 conserved miRNA homologs in pig. Expression analysis of conserved miRNAs in 14 different tissue types revealed heart-specific expression of miR-499 and miR-208 and liver-specific expression of miR-122. Additionally, miR-1 and miR-133 in the heart, miR-181a and miR-142-3p in the thymus, miR-194 in the liver, and miR-143 in the stomach showed the highest levels of expression. miR-22, miR-26b, miR-29c and miR-30c showed ubiquitous expression in diverse tissues. The expression patterns of pig-specific miRNAs also varied among the tissues examined. Conclusion Identification of 120 miRNAs and determination of the spatial expression patterns of a sub-set of these in the pig is a valuable resource for molecular biologists, breeders, and biomedical investigators interested in post-transcriptional gene regulation in pig and in related mammals, including humans.

  7. Entamoeba Clone-Recognition Experiments: Morphometrics, Aggregative Behavior, and Cell-Signaling Characterization.

    Science.gov (United States)

    Espinosa, Avelina; Paz-Y-Miño-C, Guillermo; Hackey, Meagan; Rutherford, Scott

    2016-05-01

    Studies on clone- and kin-discrimination in protists have proliferated during the past decade. We report clone-recognition experiments in seven Entamoeba lineages (E. invadens IP-1, E. invadens VK-1:NS, E. terrapinae, E. moshkovskii Laredo, E. moshkovskii Snake, E. histolytica HM-1:IMSS and E. dispar). First, we characterized morphometrically each clone (length, width, and cell-surface area) and documented how they differed statistically from one another (as per single-variable or canonical-discriminant analyses). Second, we demonstrated that amebas themselves could discriminate self (clone) from different (themselves vs. other clones). In mix-cell-line cultures between closely-related (E. invadens IP-1 vs. E. invadens VK-1:NS) or distant-phylogenetic clones (E. terrapinae vs. E. moshkovskii Laredo), amebas consistently aggregated with same-clone members. Third, we identified six putative cell-signals secreted by the amebas (RasGap/Ankyrin, coronin-WD40, actin, protein kinases, heat shock 70, and ubiquitin) and which known functions in Entamoeba spp. included: cell proliferation, cell adhesion, cell movement, and stress-induced encystation. To our knowledge, this is the first multi-clone characterization of Entamoeba spp. morphometrics, aggregative behavior, and cell-signaling secretion in the context of clone-recognition. Protists allow us to study cell-cell recognition from ecological and evolutionary perspectives. Modern protistan lineages can be central to studies about the origins and evolution of multicellularity. PMID:26990199

  8. Entamoeba Clone-Recognition Experiments: Morphometrics, Aggregative Behavior, and Cell-Signaling Characterization.

    Science.gov (United States)

    Espinosa, Avelina; Paz-Y-Miño-C, Guillermo; Hackey, Meagan; Rutherford, Scott

    2016-05-01

    Studies on clone- and kin-discrimination in protists have proliferated during the past decade. We report clone-recognition experiments in seven Entamoeba lineages (E. invadens IP-1, E. invadens VK-1:NS, E. terrapinae, E. moshkovskii Laredo, E. moshkovskii Snake, E. histolytica HM-1:IMSS and E. dispar). First, we characterized morphometrically each clone (length, width, and cell-surface area) and documented how they differed statistically from one another (as per single-variable or canonical-discriminant analyses). Second, we demonstrated that amebas themselves could discriminate self (clone) from different (themselves vs. other clones). In mix-cell-line cultures between closely-related (E. invadens IP-1 vs. E. invadens VK-1:NS) or distant-phylogenetic clones (E. terrapinae vs. E. moshkovskii Laredo), amebas consistently aggregated with same-clone members. Third, we identified six putative cell-signals secreted by the amebas (RasGap/Ankyrin, coronin-WD40, actin, protein kinases, heat shock 70, and ubiquitin) and which known functions in Entamoeba spp. included: cell proliferation, cell adhesion, cell movement, and stress-induced encystation. To our knowledge, this is the first multi-clone characterization of Entamoeba spp. morphometrics, aggregative behavior, and cell-signaling secretion in the context of clone-recognition. Protists allow us to study cell-cell recognition from ecological and evolutionary perspectives. Modern protistan lineages can be central to studies about the origins and evolution of multicellularity.

  9. Molecular cloning and enzymatic characterization of sheep CYP2J

    OpenAIRE

    Messina, Andrea; Nencioni, Simona; Gervasi, Pier Giovanni; Gotlinger, K. H.; Schwartzman, Michael Linado; Longo, Vincenzo

    2010-01-01

    Abstract 1. Cytochrome P450 (CYP) 2Js have been studied in various mammals, but not in sheep, as an animal model used to test veterinary drug metabolism. 2. Sheep CYP2J was cloned from liver messenger RNA (mRNA) by RACE. The cDNA, after modification at its N- and C-terminals, was expressed in Escherichia coli and the sheep CYP2J protein, purified by chromatography, was 80% homologous to human and monkey CYP2J2. 3. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed tha...

  10. Cloning and characterization of a ribitol dehydrogenase from Zymomonas mobilis

    DEFF Research Database (Denmark)

    Moon, Hee-Jung; Tiwari, Manish; Jeya, Marimuthu;

    2010-01-01

    Ribitol dehydrogenase (RDH) catalyzes the conversion of ribitol to D-ribulose. A novel RDH gene was cloned from Zymomonas mobilis subsp. mobilis ZM4 and overexpressed in Escherichia coli BL21(DE3). DNA sequence analysis revealed an open reading frame of 795 bp, capable of encoding a polypeptide...... of 266 amino acid residues with a calculated molecular mass of 28,426 Da. The gene was overexpressed in E. coli BL21(DE3) and the protein was purified as an active soluble form using glutathione S-transferase affinity chromatography. The molecular mass of the purified enzyme was estimated...

  11. Cloning and Characterization of Gene Promoters from Bacillus pumilus

    Institute of Scientific and Technical Information of China (English)

    Pan Jiao(潘皎); Zhang Yizheng

    2004-01-01

    DNA fragments obtained from Sau3AI partially digested total DNA of Bacillus pumilus UN31-C-42 are first inserted into BamHI site of pSUPV4, a promoter-probe vector. The recombinant DNA molecules are transformed into Escherichia coli cells and eight-three Kanr clones (named pSUBp1- pSUBp83) are obtained. The inserted fragments in pSUBp53, pSUBp57, pSUBp21, which showed high level of kanamycin - resistance, are sequenced and analyzed, respectively. These fragments contain some conserved sequences of prokaryotic gene promoters, such as TATAAT and TTGACA box. The promoter fragment Bp53 could efficiently promote the alkaline protease gene of B.pumilus expression not only in E.coli but also in B.subtilis cells.

  12. Cloning and characterization of temperature-related gene TRS1.

    Science.gov (United States)

    Han, X-B; Zhou, X-C; Hu, Z-Y; Zhang, Z-H; Liu, Y-X

    2002-01-01

    To investigate the mechanism of spermatogenesis arrest derived from heat treatment and to screen temperature-related genes involved in spermatogenesis, the authors analyzed the differences in gene expression between cryptorchid and scrotal testes in rats, and cloned a full-length cDNA named TRS1. In situ hybridization showed that TRS1 mRNA was mainly expressed in spermatocyte and round spermatids in testis. The expression level decreased in cryptorchid testis, suggesting that the lower scrotal temperature is a key factor in keeping the normal expression of TRS1. At the N-terminal of TRS1, there was a plecstrin homology (PH) domain signature. This PH domain has high similarity to that in PEPP2, a homosapien protein, which has a characteristic of binding phosphatidylinositol 3-phosphate via its PH domain in vitro. These findings suggest that TRS1 may be important in spermatogenesis and give clues for further research on the function of TRS1. PMID:12137588

  13. Molecular Cloning, Expression and Characterization of Ribokinase of Leishmania major

    Institute of Scientific and Technical Information of China (English)

    Patrick. O.J. OGBUNUDE; Nadia LAMOUR; Michael P. BARRETT

    2007-01-01

    Ribokinase (EC 2.1.7.15) from Leishmania major was cloned, sequenced and overexpressed in Escherichia coli. The gene expressed an active enzyme that had comparable activity to the same enzyme studied in E. coli. It specifically phosphorylated D-ribose. Under defined conditions, the Km for the substrates D-ribose and ATP were 0.3±0.04 mM and 0.2±0.02 mM, respectively. The turnover numbers of the enzyme for the substrates were 10.8 s-1 and 10.2 s-1, respectively. The enzyme product ribose 5-phosphate inhibited the phosphorylation of D-ribose with an apparent Ki of 0.4 mM, which is close to the Km (0.3 mM) of D-ribose, suggesting that it might play a role in regulating flux through the enzyme.

  14. Cloning and functional characterization of SAD genes in potato.

    Directory of Open Access Journals (Sweden)

    Fei Li

    Full Text Available Stearoyl-acyl carrier protein desaturase (SAD, locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8 against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.

  15. Cloning and functional characterization of SAD genes in potato.

    Science.gov (United States)

    Li, Fei; Bian, Chun Song; Xu, Jian Fei; Pang, Wan Fu; Liu, Jie; Duan, Shao Guang; Lei, Zun-Guo; Jiwan, Palta; Jin, Li-Ping

    2015-01-01

    Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato. PMID:25825911

  16. Characterization of a Forest Soil Metagenome Clone That Confers Indirubin and Indigo Production on Escherichia coli

    OpenAIRE

    Lim, He Kyoung; Chung, Eu Jin; Kim, Jin-Cheol; Choi, Gyung Ja; Jang, Kyoung Soo; Chung, Young Ryun; Cho, Kwang Yun; Lee, Seon-Woo

    2005-01-01

    A microbial community analysis of forest soil from Jindong Valley, Korea, revealed that the most abundant rRNA genes were related to Acidobacteria, a major taxon with few cultured representatives. To access the microbial genetic resources of this forest soil, metagenomic libraries were constructed in fosmids, with an average DNA insert size of more than 35 kb. We constructed 80,500 clones from Yuseong and 33,200 clones from Jindong Valley forest soils. The double-agar-layer method allowed us ...

  17. Cloning, expression and characterization of phycoerythrin gene from Ceramium boydenn.

    Science.gov (United States)

    Zhang, Xiaowen; Zhao, Fangqing; Qin, Song; Yan, Binlun

    2006-04-01

    Phycobiliproteins function as a major light harvesting protein-pigment complex in the cyanobacteria and the eukaryotic algae. Phycoerythrin (PE) is a kind of phycobiliproteins, widely located in all rhodophytes, some species of cyanobacteria and cryptophytes, and different ecotypes of Prochlorococcus populations. PeBA encoding beta and alpha subunits of PE from Ceramium boydenn was cloned and sequenced in this research. A peBA specific PCR primer was synthesized, based on the peBA gene conserved sequences. The beta subunit encoding gene (peB) contained an open reading frame of 534 bp, while the alpha subunit (peA) was 495 bp. Recombinant expression plasmid pET-peAB was constructed and expressed in Escherichia coli BL21. The molecular weight of expressive product of peB and peA was about 23.3 and 18.2 KD, respectively. Results of codon usage analysis show that G + C content is heterogeneous among different groups of PE and spacers have dramatically lower G + C contents than coding regions. Also there is a high variance in G + C content among sequences at the third position sites. It is also found in this paper that several sequence regions, which might reflect functional or structural requirements of the PE organization, and several residues known for their functional importance are conserved in almost all the sequences.

  18. Cloning and characterization of nanos gene in silkworm Bombyx mori.

    Science.gov (United States)

    Zhao, Guoli; Chen, Keping; Yao, Qin; Wang, Weihua

    2008-02-01

    Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5'-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mori and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5. 38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee (Apis mellifera). This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells. PMID:18407054

  19. Cloning and partial characterization of Entamoeba histolytica PTPases

    International Nuclear Information System (INIS)

    Reversible protein tyrosine phosphorylation is an essential signal transduction mechanism that regulates cell growth, differentiation, mobility, metabolism, and survival. Two genes coding for protein tyrosine phophatases, designed EhPTPA and EhPTPB, were cloned from Entamoeba histolytica. EhPTPA and EhPTPB proteins showed amino acid sequence identity of 37%, both EhPTPases showed similarity with Dictyostelium discoideum and vertebrate trasmembranal PTPases. mRNA levels of EhPTPA gene are up-regulated in trophozoites recovered after 96 h of liver abscess development in the hamster model. EhPTPA protein expressed as a glutathione S-transferase fusion protein (GST::EhPTPA) showed enzymatic activity with p-nitrophenylphosphate as a substrate and was inhibited by PTPase inhibitors vanadate and molybdate. GST::EhPTPA protein selectively dephosphorylates a 130 kDa phosphotyrosine-containing protein in trophozoite cell lysates. EhPTPA gene codifies for a 43 kDa native protein. Up-regulation of EhPTPA expression suggests that EhPTPA may play an important role in the adaptive response of trophozoites during amoebic liver abscess development

  20. Cloning and characterization of DELLA genes in Artemisia annua.

    Science.gov (United States)

    Shen, Q; Cui, J; Fu, X Q; Yan, T X; Tang, K X

    2015-01-01

    Gibberellins (GA) are some of the most important phytohormones involved in plant development. DELLA proteins are negative regulators of GA signaling in many plants. In this study, the full-length cDNA sequences of three DELLA genes were cloned from Artemisia annua. Phylogenetic analysis revealed that AaDELLA1 and AaDELLA2 were located in the same cluster, but AaDELLA3 was not. Subcellular localization analysis suggested that AaDELLAs can be targeted to the nucleus and/or cytoplasm. Real-time PCR indicated that all three AaDELLA genes exhibited the highest expression in seeds. Expression of all AaDELLA genes was enhanced by exogenous MeJA treatment but inhibited by GA3 treatment. Yeast two-hybrid assay showed that AaDELLAs could interact with basic helix-loop-helix transcription factor AaMYC2, suggesting that GA and JA signaling may be involved in cross-talk via DELLA and MYC2 interaction in A. annua. PMID:26345940

  1. Cloning and characterization of human DNA repair genes

    International Nuclear Information System (INIS)

    The isolation of two addition human genes that give efficient restoration of the repair defects in other CHO mutant lines is reported. The gene designated ERCC2 (Excision Repair Complementing Chinese hamster) corrects mutant UV5 from complementation group 1. They recently cloned this gene by first constructing a secondary transformant in which the human gene was shown to have become physically linked to the bacterial gpt dominant-marker gene by cotransfer in calcium phosphate precipitates in the primary transfection. Transformants expressing both genes were recovered by selecting for resistance to both UV radiation and mycophenolic acid. Using similar methods, the human gene that corrects CHO mutant EM9 was isolated in cosmids and named XRCC1 (X-ray Repair Complementing Chinese hamster). In this case, transformants were recovered by selecting for resistance to CldUrd, which kills EM9 very efficiently. In both genomic and cosmid transformants, the XRCC1 gene restored resistance to the normal range. DNA repair was studied using the kinetics of strand-break rejoining, which was measured after exposure to 137Cs γ-rays

  2. Cloning and characterization of nanos gene in silkworm Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    Guoli Zhao; Keping Chen; Qin Yao; Weihua Wang

    2008-01-01

    Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Droso phila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5'-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mori and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5. 38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee (Apis mellifera). This gene has been registered in the GenBank under the accession number EF647589. One encoding se quence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.

  3. Lignin Characterization of Triploid Clones of Populus tomentosa Carr.

    Institute of Scientific and Technical Information of China (English)

    Jin Xiao-juan; Pu Jun-wen; Xie Yi-min; Takeshi Furuno; Liu Xin-yu

    2005-01-01

    In order to understand the structural characteristics of lignin in triploid clones ofPopulus tomentosa and its changes in the processes of pulping and bleaching, milled wood lignin (MWL), lignin carbohydrate complex (LCC) and the residual lignin from kraft pulp (KP) and sulfite pulp (SP) were isolated and analyzed by Fourier transform infrared (FTIR) spectrum and 13C nuclear magnetic resonance (NMR). The most diagnostic peaks were assigned and the differences were discussed. The spectral patterns reveal that triploid P. tomentosa shows the specific features of hardwood from temperate areas, but in the spectrum of FTIR, the strength ratio of A1270 cm-1 to A1226 cm-1 is 0.88, higher than the average of hardwood from temperate areas, which will make the lignin delignification more difficult during pulping and bleaching. The LCC from triploid P. tomentosa is mainly composed of xyloglucan and glucuronic acid, and other glucides have much lower ratio. In LCC FTIR, there are three peaks at 1 427, 1 329 and 1046 cm--1, indicating that both semi-cellulose and cellulose could exist in LCC, and that there might be relationships between cellulose and lignin. Compared with the residual lignin from KP and SP, the condensed structure in KP is more than that in SP.

  4. CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

    Science.gov (United States)

    CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

  5. Cloning and characterization of type III iodothyronine deiodinase from the fish Oreochromis niloticus

    NARCIS (Netherlands)

    J.P. Sanders; S. van der Geyten; E. Kaptein (Ellen); V.M. Darras (Veerle); E.R. Kuhn; J.L. Leonard; T.J. Visser (Ton)

    1999-01-01

    textabstractType III iodothyronine deiodinase (D3) catalyzes the inner ring deiodination (IRD) of T4 and T3 to the inactive metabolites rT3 and 3,3'-diiodothyronine (3,3'-T2), respectively. Here we describe the cloning and characterization of complementary DNA (cDNA) co

  6. Molecular cloning, characterization and developmental expression of porcine β-synuclein

    DEFF Research Database (Denmark)

    Larsen, Knud; Frandsen, Pernille Munk; Madsen, Lone Bruhn;

    2010-01-01

    The synuclein family includes three known proteins: alpha-synuclein, beta-synuclein and gamma-synuclein. beta-Synuclein inhibits the aggregation of alpha-synuclein, a protein involved in Parkinson's disease. We have cloned and characterized the cDNA sequence for porcine beta-synuclein (SNCB) from...

  7. Molecular Cloning and Characterization of a Broad Substrate Terpenoid Oxidoreductase from Artemisia annua

    NARCIS (Netherlands)

    Ryden, Anna-Margareta; Ruyter-Spira, Carolien; Litjens, Ralph; Takahashi, Shunji; Quax, Wim; Osada, Hiroyuki; Bouwmeester, Harro; Kayser, Oliver

    2010-01-01

    From Artemisia annua L., a new oxidoreductase (Red 1) was cloned, sequenced and functionally characterized. Through bioinformatics, heterologous protein expression and enzyme substrate conversion assays, the elucidation of the enzymatic capacities of Red1 was achieved. Red1 acts on monoterpenoids, a

  8. Molecular cloning and characterization of a broad substrate terpenoid oxidoreductase from Artemisia annua.

    NARCIS (Netherlands)

    Ryden, A.M.; Ruyter-Spira, C.P.; Litjens, R.; Takahashi, S.; Quax, W.J.; Osada, H.; Bouwmeester, H.J.; Kayser, O.

    2010-01-01

    From Artemisia annua L., a new oxidoreductase (Red 1) was cloned, sequenced and functionally characterized. Through bioinformatics, heterologous protein expression, and enzyme substrate conversion assays, the elucidation of the enzymatic capacities of Red1 was achieved. Red1 acts on monoterpenoids,

  9. Establishment and characterization of porcine cytolytic cell lines and clones

    NARCIS (Netherlands)

    Bruin, de M.C.M.; Rooij, van E.M.A.; Voermans, J.L.M.; Visser, de Y.E.; Bianchi, A.T.J.; Kimman, T.G.

    1997-01-01

    Although non-major-histocompatibility-complex-restricted cytolytic cells appear to significantly influence antiviral immunity in pigs, the phenotype and functional characteristics of these cells are not well defined. To allow a detailed analysis of these subsets, we established and characterized cel

  10. Characterization and molecular cloning in Escherichia coli of a plasmid from the mollicute Spiroplasma citri.

    OpenAIRE

    Mouches, C; Barroso, G.; Bové, J M

    1983-01-01

    Two plasmids, pMH1 with 7 kilobase pairs and pM41 with 8 kilobase pairs, were purified from the plant pathogen Spiroplasma citri and characterized by restriction mapping. Upon in vitro DNA recombination with plasmid pBR328 as a vector, we have cloned pMH1 in Escherichia coli. A radioactive probe obtained upon nick translation of the recombinant plasmid was used to further characterize and compare pMH1 and pM41.

  11. Rapid Characterization of Garlic Clones with Locus-Specific DNA Markers

    OpenAIRE

    İPEK, Meryem; İPEK, AHMET; Simon, Philipp W

    2008-01-01

    Maintenance of redundant garlic (Allium sativum L.) accessions is expensive due to the necessity of yearly regenerating garlic accessions in germplasm centers. Therefore, rapid characterization of garlic accessions is important for avoiding duplicated genotypes. For this purpose we developed several locus-specific polymerase chain reaction (PCR)-based DNA markers, and tested them for the characterization of garlic clones that were previously analyzed using amplified fragment length polymorphi...

  12. Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones

    Science.gov (United States)

    Deepe, George S., Jr.; Smith, James G.; Denman, David; Bullock, Ward E.; Sonnenfeld, Gerald

    1986-01-01

    Several Histoplasma capsulatum-reactive murine cloned T-cell lines (TCLs) were isolated from spleens of C57BL/6 mice immunized with viable H. capsulatum yeast cells, using the methodology of Kimoto and Fathman (1980). These T-cells were characterized phenotypically as Thy-1.2(+) Lyt-1(+) L3T4(+) Lyt-2(-), that is, as the helper/inducer phenotype. The cloned T cells proliferate in response to histoplasmin and, in some cases, to heterologous fungal anigens. Upon injection of mice with the antigen, the T-cells mediate local delayed-type hypersensitivity responses and, after stimulation, release regulatory lymphokines.

  13. Molecular cloning and biological characterization of the human excision repair gene ERCC-3.

    OpenAIRE

    Weeda, G; van Ham, R C; Masurel, R; Westerveld, A; Odijk, H; Wit, J.; Bootsma, D; van der Eb, A J; Hoeijmakers, J. H.

    1990-01-01

    In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent m...

  14. Cloning, Expression and Characterization of PprI gene in Escherichia coli

    OpenAIRE

    Amir Mirzaie; Jalil Fallah Mehrabadi; Noor Amirmozafari; Taher Nejadsattari

    2014-01-01

    Background and Objectives: PprI is one of newly gene identified in Deinococcus radiodurans and plays a critical role in DNA repair and protection against ultraviolet radiation stress. The aim of this study was to clone, express and characterize PprI gene in Escherichia coli.Materials and Methods: The PprI gene of D. radiodurans was constructed in pGEM-B1 vector and the cloned gene was subcloned into pET21a expression vector. The pET21a-PprI was expressed in E. coli (Origami strain). Expressio...

  15. Recent Advances in Cloning and Characterization of Disease Resistance Genes in Rice

    Institute of Scientific and Technical Information of China (English)

    Liang-Ying Dai; Xiong-Lun Liu; Ying-Hui Xiao; Guo-Liang Wang

    2007-01-01

    Rice diseases caused by fungi, bacteria and viruses are one of the major constraints for sustainable rice (Oryza sativa L.) production worldwide. The use of resistant cultivars is considered the most economical and effective method to control rice diseases. In the last decade, a dozen resistance genes against the fungal pathogen Magnaporthe grisea and the bacterial pathogen Xanthomonas oryzae pv. oryzae have been cloned. Approximately half of them encode nuclear binding site (NBS) and leucine rich repeat (LRR)-containing proteins, the most common type of cloned plant resistance genes. Interestingly, four of them encode novel proteins which have not been identified in other plant species, suggesting that unique mechanisms might be involved in rice defense responses. This review summarizes the recent advances in cloning and characterization of disease resistance genes in rice and presents future perspectives for in-depth molecular analysis of the function and evolution of rice resistance genes and their interaction with avirulence genes in pathogens.

  16. Cloning, expression, purification and characterization of tryptophan hydroxylase variants

    DEFF Research Database (Denmark)

    Boesen, Jane

    such as depression and obsessive-compulsive disorder (OCD). Characterization of TPH and elucidation of the enzymes regulation and catalytic mechanism is therefore vital to our understanding of the serotonin balance. This study concerns variants of both human TPH isoform 1 (hTPH1) and human TPH isoform 2 (h PH2...... the variant. The GST-rhTPH1 variant could be purified using affinity chromatography followed by gel filtration with high purity and a yield of 40 mg/l culture. The purified GST-rhTPH1 exists as a dimer in solution due to dimerization of GST. The GST could be cleaved successfully from the fusion protein using...

  17. Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the Metagenome

    Directory of Open Access Journals (Sweden)

    Liu Yu

    2008-12-01

    Full Text Available Abstract Background Pyrethroids and pyrethrins are widely used insecticides. Extensive applications not only result in pest resistance to these insecticides, but also may lead to environmental issues and human exposure. Numerous studies have shown that very high exposure to pyrethroids might cause potential problems to man and aquatic organisms. Therefore, it is important to develop a rapid and efficient disposal process to eliminate or minimize contamination of surface water, groundwater and agricultural products by pyrethroid insecticides. Bioremediation is considered to be a reliable and cost-effective technique for pesticides abatement and a major factor determining the fate of pyrethroid pesticides in the environment, and suitable esterase is expected to be useful for potential application for detoxification of pyrethroid residues. Soil is a complex environment considered as one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches provide a powerful tool for accessing novel valuable genetic resources (novel enzymes and developing various biotechnological applications. Results The pyrethroid pesticides residues on foods and the environmental contamination are a public safety concern. Pretreatment with pyrethroid-hydrolyzing esterase has the potential to alleviate the conditions. To this end, a pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, sequence analysis of the DNA responsible for the pye3 gene revealed an open reading frame of 819 bp encoding for a protein of 272 amino acid residues. Extensive multiple sequence alignments of the deduced amino acid of Pye3 with the most homologous carboxylesterases revealed moderate identity (45–49%. The recombinant Pye3 was heterologously expressed in E. coli BL21(DE3

  18. The role of 5'-adenylylsulfate reductase in the sulfur assimilation pathway of soybean: molecular cloning, kinetic characterization, and gene expression

    Science.gov (United States)

    Soybean seeds are a major source of protein, but contain low levels of sulfur-containing amino acids. With the objective of studying the sulfur assimilation pathway of soybean, a full-length cDNA clone for 5’-adenylylsulfate reductase (APS reductase) was isolated and characterized. The cDNA clone ...

  19. Molecular cloning and characterization of a calmodulin-dependent phosphodiesterase enriched in olfactory sensory neurons.

    OpenAIRE

    C. Yan; Zhao, A Z; Bentley, J K; Loughney, K; Ferguson, K; Beavo, J. A.

    1995-01-01

    The sensing of an odorant by an animal must be a rapid but transient process, requiring an instant response and also a speedy termination of the signal. Previous biochemical and electrophysiological studies suggest that one or more phosphodiesterases (PDEs) may play an essential role in the rapid termination of the odorant-induced cAMP signal. Here we report the molecular cloning, expression, and characterization of a cDNA from rat olfactory epithelium that encodes a member of the calmodulin-...

  20. Molecular Cloning and Characterization of OsCDase, a Ceramidase Enzyme from Rice

    OpenAIRE

    Pata, Mickael O.; Wu, Bill X.; Bielawski, Jacek; Xiong, Tou Cheu; Hannun, Yusuf A.; Ng, Carl K. -Y.

    2008-01-01

    Sphingolipids are a structurally diverse group of molecules based on long-chain sphingoid bases found in animal, fungal and plant cells. In contrast to the situation in animals and yeast, we know much less about the spectrum of sphingolipid species in plants and the roles they play in mediating cellular processes. Here, we report the cloning and characterization of a plant ceramidase from rice (Oryza sativa spp. Japonica cv. Nipponbare). Sequence analysis suggests that the rice ceramidase (Os...

  1. Gene cloning, expression, and characterization of the Bacillus amyloliquefaciens PS35 lipase

    OpenAIRE

    Palanisamy Kanmani; Kuppamuthu Kumaresan; Jeyaseelan Aravind

    2015-01-01

    Abstract Lipases are enzymes of immense industrial relevance, and, therefore, are being intensely investigated. In an attempt to characterize lipases at molecular level from novel sources, a lipase gene from Bacillus amyloliquefaciens PS35 was cloned, heterologously expressed in Escherichia coli DH5α cells and sequenced. It showed up to 98% homology with other lipase sequences in the NCBI database. The recombinant enzyme was then purified from E. coli culture, resulting in a 19.41-fold purifi...

  2. Construction and characterization of an infectious molecular clone of Koala retrovirus.

    Science.gov (United States)

    Shojima, Takayuki; Hoshino, Shigeki; Abe, Masumi; Yasuda, Jiro; Shogen, Hiroko; Kobayashi, Takeshi; Miyazawa, Takayuki

    2013-05-01

    Koala retrovirus (KoRV) is a gammaretrovirus that is currently endogenizing into koalas. Studies on KoRV infection have been hampered by the lack of a replication-competent molecular clone. In this study, we constructed an infectious molecular clone, termed plasmid pKoRV522, of a KoRV isolate (strain Aki) from a koala reared in a Japanese zoo. The virus KoRV522, derived from pKoRV522, grew efficiently in human embryonic kidney (HEK293T) cells, attaining 10(6) focus-forming units/ml. Several mutations in the Gag (L domain) and Env regions reported to be involved in reduction in viral infection/production in vitro are found in pKoRV522, yet KoRV522 replicated well, suggesting that any effects of these mutations are limited. Indeed, a reporter virus pseudotyped with pKoRV522 Env was found to infect human, feline, and mink cell lines efficiently. Analyses of KoRV L-domain mutants showed that an additional PPXY sequence, PPPY, in Gag plays a critical role in KoRV budding. Altogether, our results demonstrate the construction and characterization of the first infectious molecular clone of KoRV. The infectious clone reported here will be useful for elucidating the mechanism of endogenization of the virus in koalas and screening for antiretroviral drugs for KoRV-infected koalas.

  3. A five-fold pig bacterial artificial chromosome library:a resource for positional cloning and physical mapping

    Institute of Scientific and Technical Information of China (English)

    LIU Wei; LI Ning; ZHANG Ying; LIU Zhaoliang; GUO Li; WANG Xiaobo; FEI Jing; FENG Jidong; ZHAO Rui; HU Xiaoxiang

    2006-01-01

    A pig BAC library was constructed with genomic DNA from a male Erhualian pig. After partial digestion with Hind Ⅲ or BamH I the fragments obtained were cloned into the pBeloBAC11 vector. The library consists of 184320 clones which stored in 480pieces 384-well plates (20 plates per superpool). A two-step 4-dimension PCR screening system was established to screen the positive clones. An average insert size of 128 kb was estimated from 105 randomly isolated clones, which indicates that the library is more than five times of genomic coverage. For the demonstration of the probability to pick out any unique genes or DNA markers from the library, 10single-copy genes were screened out and the positive clones were yielded between 1 and 8 with an average of 3.6. Positive superpools were obtained for 32 microsatellite markers selected from different regions of pig genome. The number of positive superpools for each marker varies from 1 to 9 with an average of 4.78. This BAC library provides an additional resource for pig physical mapping and gene identification.

  4. Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli

    OpenAIRE

    Dorismey Vieira Tokano; Marisa Emiko Kawaichi; Emerson José Venâncio; Marilda Carlos Vidotto

    2008-01-01

    The aim of this work was to isolate, clone and characterize the iron uptake gene iutA from avian pathogenic E. coli (APEC). The iutA gene was isolated from the strain APEC 9, serotype O2:H9, which was cloned in the expression vector pET101/D-TOPO. The gene of 2.2 Kb was sequenced (AY602767, which showed high similarity to the iutA gene from three plasmids, two from APEC, pAPEC-02-ColV (AY545598.4) and pTJ100 (AY553855.1), and one from a human invasive E. coli strain, the pColV K30. The recomb...

  5. Molecular Cloning and Characterization of the Actin-depolymerizing Factor Gene in Gossypium barbadense

    Institute of Scientific and Technical Information of China (English)

    MA Zhi-ying; CHI Ji-na; WANG Xing fen; ZHOU Hong-mei; ZHANG Gui-yin

    2008-01-01

    @@ Sea Island cotton (Gossypium barbadense L.) has been highly valued in Verticillium wilt resistance and many fiber qualities including fiber length,strength,and fineness.To identify whether it had some special genes in fiber development in comparison with the upland cotton (G.hirsutum L.),an actin-depolymerizing factor (ADF) gene was cloned and characterized in this research.A 420 bp open reading frame of the cloned gene,termed GbADF1,encoded a protein of 139 amino acids,which included39.57% nonpolar amino acids,17.27% acidic amino acids,15.83% basic amino acids,and 31.92% hydrophobic amino aids.

  6. cDNA Cloning, Prokaryotic and Eukaryotic Expression and Characterization of Porcine Leukemia Inhibitory Factor

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Molecular cloning of the porcine leukemia inhibitor factor(pLIF) has not been reported. A full-length cDNA encoding pLIF was cloned, expressed and characterized. The full-length porcine LIF cDNA encodes a 202 amino acid protein that has an 84% sequence identity to mouse LIF and 86% sequence identity to human LIF. The deduced amino acid sequence of a pLIF protein contains six conserved consensus N-linked glycosylation sites and six cysteine groups to form potential disulfide bonds. The pLIF was expressed in E coli, as a mature form, and in CHO cells as a secreted form. Both the forms of the recombinant pLIFs can maintain murine embryonic stem cells in an undifferentiated state in a culture. The recombinant pLIFs will be useful in establishing a long-term culture of stable pluripotent porcine embryonic stem cells for further manipulation.

  7. Characterization of a TK6-Bcl-xL gly-159-ala Human Lymphoblast Clone

    Energy Technology Data Exchange (ETDEWEB)

    Chyall, L.: Gauny, S.; Kronenberg, A.

    2006-01-01

    TK6 cells are a well-characterized human B-lymphoblast cell line derived from WIL-2 cells. A derivative of the TK6 cell line that was stably transfected to express a mutated form of the anti-apoptotic protein Bcl-xL (TK6-Bcl-xL gly-159- ala clone #38) is compared with the parent cell line. Four parameters were evaluated for each cell line: growth under normal conditions, plating efficiency, and frequency of spontaneous mutation to 6‑thioguanine resistance (hypoxanthine phosphoribosyl transferase locus) or trifluorothymidine resistance (thymidine kinase locus). We conclude that the mutated Bcl-xL protein did not affect growth under normal conditions, plating efficiency or spontaneous mutation frequencies at the thymidine kinase (TK) locus. Results at the hypoxanthine phosphoribosyl transferase (HPRT) locus were inconclusive. A mutant fraction for TK6‑Bcl-xL gly-159-ala clone #38 cells exposed to 150cGy of 160kVp x-rays was also calculated. Exposure to x-irradiation increased the mutant fraction of TK6‑Bcl-xL gly-159-ala clone #38 cells.

  8. Cloning and characterization of the beer foaming gene CFG1 from Saccharomyces pastorianus.

    Science.gov (United States)

    Blasco, Lucía; Veiga-Crespo, Patricia; Sánchez-Pérez, Angeles; Villa, Tomás G

    2012-10-31

    Foam production is an essential characteristic of beer, generated mainly from the proteins present in the malt and, to a minor extent, from the mannoproteins in brewer's yeast cell walls. Here, we describe the isolation and characterization of the novel fermentation gene CFG1 (Carlsbergensis foaming gene) from Saccharomyces pastorianus. CFG1 encodes the cell wall protein Cfg1p, a 105 kDa protein highly homologous to Saccharomyces cerevisiae cell wall mannoproteins, particularly those involved in foam formation, such as Awa1p and Fpg1p. Further characterization of Cfg1p revealed that this novel protein is responsible for beer foam stabilization. This report represents the first time that a brewing yeast foaming gene has been cloned and its action fully characterized. PMID:23039128

  9. Characterization of a circulating PRRSV strain by means of random PCR cloning and full genome sequencing

    Directory of Open Access Journals (Sweden)

    Nauwynck Hans J

    2011-04-01

    Full Text Available Abstract PRRS is a pig disease of major economic importance that causes respiratory and reproductive problems in pigs. Over the last years it has become clear that PRRSV heterogeneity is increasing. Consequently, this has a potential impact on diagnosis and strategies to counter this disease. The use of sequence-independent PCR techniques for the detection and characterization of PRRSV could be useful to bypass problems associated with the heterogeneity of this virus. A random PCR cloning approach was tested for the characterization of PRRSV strain 07V063 of unknown genetic background that circulated on a Belgian farm. By using this approach, 7305 bp of sequence data were obtained, distributed randomly across the genome. Using RT-PCR with strain-specific primers, the full length sequence (15014 nt was obtained. Phylogenetic relationships using ORF5 and ORF1a (NSP2 sequences showed that 07V063 was classified in type 1 subtype 1 and that 07V063 was genetically different from prototype Lelystad Virus (LV. 07V063 showed 87-93% aa identity with LV ORFs coding for structural proteins. Most variation (compared to LV was noticed in Nsp2 (81% identity with a deletion of 28 aa. This deletion was different from other known deletions in this ORF. In conclusion, it is shown that this random PCR cloning approach can be used for the characterization of new PRRSV strains of unknown genetic background.

  10. Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum

    Directory of Open Access Journals (Sweden)

    Safar Farajnia

    2011-01-01

    Full Text Available Parasite of the genus Leishmania is reliant on the salvage pathway for recycling of ribonucleotides. A class I nuclease enzyme also known as P4 nuclease is involved in salvage of purines in cutaneous Leishmania species but the relevant enzymes have not been characterized in Leishmania infantum (L. infantum. The aim of this study was to clone and characterize the gene encoding class I nuclease in L. infantum. DNA extracted from L. infantum was used for amplification of P4 nuclease gene (Li-P4 by PCR. The product was cloned, sequenced, and expressed in E. coli for further characterization. Analysis of the sequence of Li-P4 revealed that the gene consists of an ORF of 951 bp. Sequence similarity analysis indicated that Li-P4 has a high homology to relevant enzymes of other kintoplastids with the highest homology (88% to p1/s1 class I nuclease from L. donovani. Western blotting of antirecombinant Li-P4 with promastigote and amastigote stages of L. infantum showed that this nuclease is present in both stages of parasite with higher expression in amastigote stage. The highly conserved nature of this essential enzyme in Leishmania parasites suggests it as a promising drug target for leishmaniasis.

  11. Cloning and characterization of type III iodothyronine deiodinase from the fish Oreochromis niloticus

    OpenAIRE

    Sanders, J.P.; Van Der Geyten, S; Kaptein, Ellen; Darras, Veerle; Kuhn, E.R.; Leonard, J L; Visser, Ton

    1999-01-01

    textabstractType III iodothyronine deiodinase (D3) catalyzes the inner ring deiodination (IRD) of T4 and T3 to the inactive metabolites rT3 and 3,3'-diiodothyronine (3,3'-T2), respectively. Here we describe the cloning and characterization of complementary DNA (cDNA) coding for D3 in fish (Oreochromis niloticus, tilapia). This cDNA contains 1478 nucleotides and codes for a protein of 267 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 131....

  12. Cloning and molecular characterization of Cu,Zn superoxide dismutase from Actinobacillus pleuropneumoniae.

    OpenAIRE

    Langford, P R; Loynds, B M; Kroll, J S

    1996-01-01

    Copper-zinc superoxide dismutases (Cu,Zn SODs), until recently considered very unusual in bacteria, are now being found in a wide range of gram-negative bacterial species. Here we report the cloning and characterization of sodC, encoding Cu,Zn SOD in Actinobacillus pleuropneumoniae, a major pathogen of pigs and the causative organism of porcine pleuropneumonia. sodC was shown to lie on a monocistronic operon, at the chromosomal locus between the genes asd (encoding aspartate semialdehyde dehy...

  13. Molecular cloning and biological characterization of the human excision repair gene ERCC-3

    International Nuclear Information System (INIS)

    In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion

  14. Cloning, expression, and characterization of pollen allergens from Humulus scandens (Lour) Merr and Ambrosia artemisiifolia L

    Institute of Scientific and Technical Information of China (English)

    Ai-lin TAO; Shao-heng HE

    2005-01-01

    Aim: To clone the pollen allergen genes in Humulus scandens (Lour) Merr (LüCao in Chinese) and short ragweed (Ambrosia artemisiifolia L) for recombinant allergen production and immunotherapy. Methods: The allergen genes were selectively amplified in the weed pollen cDNA pool by using a special PCR profile, with the primers designed by a modeling procedure. Following truncated gene cloning and confirmation of the pollen source, unknown 3'cDNA ends were identified by using the 3'-RACE method. The gene function conferred by the full-length coding region was evaluated by a homologue search in the GenBank database. Recombinant proteins expressed in Escherichia coli pET-44 RosettaBlue cells were subsequently characterized by N-terminal end sequencing, IgE binding, and crossreactivity. Results: Three full-length cDNAs were obtained in each weed. Multiple alignment analysis revealed that the deduced amino acid sequences were 83% identical to each other and 56%-90% identical to panallergen profilins from other species. Five recombinant proteins were abundantly expressed in nonfusion forms and were confirmed by using the N-terminal end sequence identity.Sera from patients who were allergic to A artemisiifolia reacted not only with rAmb a 8(D03) derived from A artemisiifolia, but also with recombinant protein rHum s 1(LCM9) derived from H scandens, which confirmed the allergenicity and cross-reactivity of the recombinant proteins from the 2 sources. Comparison of the degenerate primers used for truncated gene cloning with the full-length cDNA demonstrated that alternative nucleotide degeneracy occurred. Conclusion: This study demonstrates a useful method for cloning homologous allergen genes across different species, particularly for little-studied species. The recombinant allergens obtained might be useful for the immunotherapeutic treatment of H scandens and/or A artemisiifolia pollen allergies.

  15. Cloning, expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus

    OpenAIRE

    Lakshmi, Hanumanthu Prasanna; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Vasu, Dudipeta; Swarupa, Vimjam; Kumar, Pasupuleti Santhosh; Narasu, Mangamoori Lakshmi; Krishna Sarma, Potukuchi Venkata Gurunadha

    2013-01-01

    Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was...

  16. Molecular cloning and characterization of Hymenolepis diminuta alpha-tubulin gene.

    Science.gov (United States)

    Mohajer-Maghari, Behrokh; Amini-Bavil-Olyaee, Samad; Webb, Rodney A; Coe, Imogen R

    2007-02-01

    To isolate a full-length alpha-tubulin cDNA from an eucestode, Hymenolepis diminuta, a lambda phage cDNA library was constructed. The alpha-tubulin gene was cloned, sequenced and characterized. The H. diminuta alpha-tubulin consisted of 450 amino acids. This protein contained putative sites for all posttranslational modifications as detyrosination/tyrosination at the carboxyl-terminal of protien, phosphorylation at residues R79 and K336, glycylation/glutamylation at residue G445 and acetylation at residue K40. Comparisons of H. diminuta alpha-tubulin with all full-length alpha-tubulin proteins revealed that H. diminuta alpha-tubulin possesses 10 distinctive residues, which are not found in any other alpha-tubulins. Phylogenetic analysis showed that H. diminuta alpha-tubulin has grouped in a separated branch adjacent eucestode and trematodes branch with 92% bootstrap value (1000 replicates). In conclusion, this is the first report of H. diminuta cDNA library construction, cloning and characterization of H. diminuta alpha-tubulin gene.

  17. Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in gastrointestinal system

    Institute of Scientific and Technical Information of China (English)

    Shun-Ying Jin; Yan-Li Liu; Li-Na Xu; Yong Jiang; Ying Wang; Bao-Xue Yang; Hong Yang; Tong-Hui Ma

    2006-01-01

    AIM: To clone and characterize the porcine aquaporins (AQPs) in the gastrointestinal system.METHODS: A PCR-based cloning strategy and RACE were used to clone full-length AQP coding sequence from reversely transcribed pig liver cDNA. Stopped-flow light scattering and a YFP-based fluorescence method were used to measure the osmotic water permeability of erythrocytes and the stably transfected CHO cells.RT-PCR, Northern blot, and immunohistochemistry were used to determine the gastrointestinal expression and localization of cloned AQPs. Protein expression in transfected cells and red blood cells was analyzed by Western blot.RESULTS: An 813 bp cDNA encoding a 271 amino acid porcine aquaporin (designated pAQP1) was cloned from liver mRNA (pAQP1 has a 93% identity with human AQP1 and contains two NPA motifs conserved in AQP family, one consensus sequence for N-linked glycosylation, and one mercury-sensitive site at cysteine 191). RT-PCR analysis revealed extensive expression of pAQP1 mRNA in porcine digestive glands and gut.Northern blot showed a single 3.0 kb transcript in selected digestive organs, pAQP1 protein was localized at central lacteals of the small intestine, microvessles of salivary glands, as well as epithelium of intrahepatic bile ducts by immunoperoxydase. High osmotic water permeability that is inhibitable by HgCl2 was detected in porcine erythrocytes and CHO cells stably transfected with pAQP1 cDNA. Tmmunoblot analysis of porcine erythrocytes and pAQP-transfected CHO cells revealed an unglycosylated 28 ku band and larger glycosylated proteins.CONCLUSION: pAQP1 is the first porcine aquaporin that can be molecularly identified so far. The broad distribution of pAQP1 in epithelium and endothelium of porcine digestive organs may suggest an important role of channel-mediated water transport in fluid secretion/absorption as well as in digestive function and pathophysiology of the gastrointestinal system.

  18. Characterization of T cell clones from chagasic patients: predominance of CD8 surface phenotype in clones from patients with pathology

    Directory of Open Access Journals (Sweden)

    Washington R. Cuna

    1995-08-01

    Full Text Available Human Chagas' disease, caused by the protozoan Trypanosoma cruzi, is associated with pathological processes whose mechanisms are not known. To address this question, T cell lines were developed from chronic chagasic patients peripheral blood mononuclear cells (PBMC and cloned. These T cell clones (TCC were analyzed phenotypically with monoclonal antibodies by the use of a fluorescence microscope. The surface phenotype of the TCC from the asymptomatic patient were predominantly CD4 positive (86%. On the contrary, the surface phenotype CD8 was predominant in the TCC from the patients suffering from cardiomegaly with right bundle branch block (83%, bradycardia with megacolon (75 % and bradycardia (75%. Future studies will be developed in order to identify the antigens eliciting these T cell subpopulations.

  19. Molecular cloning, bioinformatics analysis and functional characterization of HWTX-XI toxin superfamily from the spider Ornithoctonus huwena.

    Science.gov (United States)

    Jiang, Liping; Deng, Meichun; Duan, Zhigui; Tang, Xing; Liang, Songping

    2014-04-01

    Spider venom contains a very valuable repertoire of natural resources to discover novel components for molecular diversity analyses and therapeutic applications. In this study, HWTX-XI toxins from the spider venom glands of Ornithoctonus huwena which are Kunitz-type toxins (KTTs) and were directly cloned, analyzed and functionally characterized. To date, the HWTX-XI superfamily consists of 38 members deduced from 121 high-quality expressed sequence tags, which is the largest spider KTT superfamily with significant molecular diversity mainly resulted from cDNA tandem repeats as well as focal hypermutation. Among them, HW11c40 and HW11c50 may be intermediate variants between native Kunitz toxins and sub-Kunitz toxins based on evolutionary analyses. In order to elucidate their biological activities, recombinant HW11c4, HW11c24, HW11c27 and HW11c39 were successfully expressed, further purified and functionally characterized. Both HW11c4 and HW11c27 display inhibitory activities against trypsin, chymotrypsin and kallikrein. Moreover, HW11c4 is also an inhibitor relatively specific for Kv1.1 channels. HW11c24 and HW11c39 are found to be inactive on chymotrysin, trypsin, kallikrein, thrombin and ion channels. These findings provide molecular evidence for toxin diversification of the HWTX-XI superfamily and useful molecular templates of serine protease inhibitors and ion channel blockers for the development of potentially clinical applications.

  20. Wind resource characterization in the Arabian Peninsula

    KAUST Repository

    Yip, Chak Man Andrew

    2015-12-28

    Wind energy is expected to contribute to alleviating the rise in energy demand in the Middle East that is driven by population growth and industrial development. However, variability and intermittency in the wind resource present significant challenges to grid integration of wind energy systems. These issues are rarely addressed in the literature of wind resource assessment in the Middle East due to sparse meteorological observations with varying record lengths. In this study, the wind field with consistent space–time resolution for over three decades at three hub heights (50m, 80m, 140m) over the whole Arabian Peninsula is constructed using the Modern Era Retrospective-Analysis for Research and Applications (MERRA) dataset. The wind resource is assessed at a higher spatial resolution with metrics of temporal variations in the wind than in prior studies. Previously unrecognized locations of interest with high wind abundance and low variability and intermittency have been identified in this study and confirmed by recent on-site observations. In particular, the western mountains of Saudi Arabia experience more abundant wind resource than most Red Sea coastal areas. The wind resource is more variable in coastal areas along the Arabian Gulf than their Red Sea counterparts at a similar latitude. Persistent wind is found along the coast of the Arabian Gulf.

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

    Science.gov (United States)

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

    2014-01-01

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

  2. Cloning and Characterization of a Putative CTR1 Gene from Wheat

    Institute of Scientific and Technical Information of China (English)

    BI Cai-li; WEN Xiao-jie; ZHANG Xue-yong; LIU Xu

    2010-01-01

    CTR1 is a key negative regulator in ethylene signal transduction.A salt-induced CTR1 like gene(TaCTR1)was cloned from wheat,its expression under abiotie stresses,subcellular localization and the effect of overexpression of TaCTR1 on salt tolerance in tobacco was studied.A putative CTR1 gene was cloned and characterized from wheat via rapid amplification of cDNA ends(RACE)and RT-PCR.TaCTR1 expression under stresses was analyzed using semi-quantitative RT-PCR and the effect of overexpression of TaCTR1 on salt tolerance was conducted in tobacco.The full-length cDNA of TaCTR1is 2635 bp which codes for a polypeptide of 759 amino acids.There is a conserved serine/threonine protein kinase domain at the carboxyl terminus containing an ATP-binding site.Southern blot analysis revealed that TaCTR1 consisted of a gene family in wheat.The amino acid homologies of CTR1 among different organisms share higher similarities.Expression analysis revealed that TaCTR1 was induced by NaCl and drought stress but inhibited by ABA treatment.Transient expression of TaCTR1-GFP in the onion epidermal cells indicated that TaCTR1 was probably targeted to the plasma membrane.Overexpression of TaCTR1 decreased salt tolerance in transgenic tobacco(Nicotiana tabacum L.)plants compared with the control.To our knowledge,TaCTR1 is the first CTR1 gene cloned in wheat and may be involved in various abiotic stresses.Overexpression of TaCTR1 decreased the salt tolerance in tobacco suggested that TaCTR1 may act as a negative regulator of salt stress in plants.

  3. Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel

    Energy Technology Data Exchange (ETDEWEB)

    Leng, Q.; Mercier, R.W.; Yao, W.; Berkowitz, G.A.

    1999-11-01

    Cyclic nucleotide-gated (cng) non-selective cation channels have been cloned from a number of animal systems. These channels are characterized by direct gating upon cAMO or cGMO binding to the intracellular portion of the channel protein, which leads to an increase in channel conductance. Animal cng channels are involved in signal transduction systems; they translate stimulus-induced changes in cytosolic cyclic nucleotide into altered cell membrane potential and/or cation flux as part of a signal cascade pathway. Putative plant homologs of animal cng channels have been identified. However, functional characterization (i.e., demonstration of cyclic-nucleotide-dependent ion currents) of a plant cng channel has not yet been accomplished. The authors report the cloning and first functional characterization of a plant member of this family of ion channels. The Arabidopsis cDNA AtCNGC2 encodes a polypeptide with deduced homology to the {alpha}-subunit of animal channels, and facilitates cyclic nucleotide-dependent cation currents upon expression in a number of heterologous systems. AtCNGC2 expression in a yeast mutant lacking a low-affinity K{sup +} uptake system complements growth inhibition only when lipophilic nucleotides are present in the culture medium. Voltage clamp analysis indicates that Xenopus lawvis oocytes injected with AtCNGC2 cRNA demonstrate cyclic-nucleotide-dependent, inward-rectifying K{sup +} currents. Human embryonic kidney cells (HEK293) transfected with AtCNGC2 cDNA demonstrate increased permeability to Ca{sup 2+} only in the presence of lipophilic cyclic nucleotides. The evidence presented here supports the functional classification of AtCNGC2 as a cyclic-nucleotide-gated cation channel, and presents the first direct evidence identifying a plant member of this ion channel family.

  4. Molecular cloning and functional characterization of a rainbow trout liver Oatp

    International Nuclear Information System (INIS)

    Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrain fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologues OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a Km value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a Km value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications. - Highlights: • A new Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. • rtOatp1d1 is predominantly expressed in the liver. • rtOatp1d1 displays multi-specific transport of endogenous and xenobiotic substrates. • rtOatp1d1 is a homologue of the OATP1A1, OATP1B1 and OATP1B3. • rtOatp1d1 is a microcystin (MC) transporter

  5. Molecular cloning and functional characterization of a rainbow trout liver Oatp

    Energy Technology Data Exchange (ETDEWEB)

    Steiner, Konstanze, E-mail: konstanze.steiner@uni-konstanz.de [University of Konstanz, Human- and Environmental Toxicology, 78464 Konstanz (Germany); Hagenbuch, Bruno, E-mail: bhagenbuch@kumc.edu [Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City 66160, KS (United States); Dietrich, Daniel R., E-mail: daniel.dietrich@uni-konstanz.de [University of Konstanz, Human- and Environmental Toxicology, 78464 Konstanz (Germany)

    2014-11-01

    Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrain fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologues OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a K{sub m} value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a K{sub m} value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications. - Highlights: • A new Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. • rtOatp1d1 is predominantly expressed in the liver. • rtOatp1d1 displays multi-specific transport of endogenous and xenobiotic substrates. • rtOatp1d1 is a homologue of the OATP1A1, OATP1B1 and OATP1B3. • rtOatp1d1 is a microcystin (MC) transporter.

  6. Cloning and molecular characterization of Omp31 gene from Brucella melitensis Rev 1 strain

    Directory of Open Access Journals (Sweden)

    Yousefi, S.

    2016-07-01

    Full Text Available Brucellosis, caused by the genus Brucella bacterium, is a well-known infection among domestic animals. Considering the serious economic and medical consequences of this infection, various preventive efforts have been made through using recombinant vaccines, based on outer membrane protein (OMP antigens of Brucella species. The objective of the present study was to clone, analyze the sequence, and predict the epitopes of Omp31 gene as a major B. melitensis antigen. The full-length open reading frame (ORF for this gene was amplified by specific primers and cloned into the pTZ57R/T vector. The gene sequence of B. melitensis Rev 1 strain was submitted to NCBI database. The results of phylogenetic analysis showed that Omp31 is almost similar in different Brucella species. Online prediction software programs were also used to predict B- and Tcell epitopes, secondary and tertiary structures, antigenicity, and enzymatic degradation sites. The bioinformatic tools in the current study were confirmed by the results of three different experimental epitope prediction studies. Bioinformatic analysis identified one T-cell and three B-cell epitopes for Omp31 antigen. Finally, based on the antigenicity and proteosome recognition sites, common B- and T-cell epitopes were predicted for Omp31 (amino acids 191-204. Bioinformatic analysis showed that these regions had proper epitope characterization and could be useful for recombinant vaccine development.

  7. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  8. The cloning and expression characterization of the centrosome protein genes family (centrin genes) in rat testis

    Institute of Scientific and Technical Information of China (English)

    SUN; Xiaodong(孙晓冬); GE; Yehua(葛晔华); MA; Jing(马静); YU; Zuoren(俞作仁); LI; Sai(李赛); WANG; Yongchao(王永潮); XUE; Shepu(薛社普); HAN; Daishu(韩代书)

    2002-01-01

    Centrins are members of the centrosome protein family, which is highly conserved during revolution. The homologous genes of centrin in many organisms had been cloned, but the sequences of the rat centrin genes were not reported yet in GenBank. We cloned the cDNA fragments of centrin-1, -2 and -3 from the rat testis by RT-PCR, and analyzed the homology of the deduced amino acid sequences. The expression characterization of centrin genes in rat spermatogenesis was carried out by semi-quantitative RT-PCR. The results show that the homology of the corresponding centrin proteins in human, mouse and rat is high. The expression of centrin-1 is testis-specific, spermatogenic cell-specific and developmental stage-related. Centrin-1 begins to be transcribed when the meiosis occurs, and its mRNA level reaches the peak in round spermatids. Centrin-2 and centrin-3 are highly expressed in spermatogonia and their mRNA level decreases markedly when meiosis occurs. These results suggest that centrin-1 may play roles in meiosis and spermiogenesis, and centrin-2 and centrin-3 may be related to mitosis.

  9. Cloning and characterization of an immunoglobulin A Fc receptor from cattle.

    Science.gov (United States)

    Morton, H Craig; Pleass, Richard J; Storset, Anne K; Dissen, Erik; Williams, John L; Brandtzaeg, Per; Woof, Jenny M

    2004-02-01

    Here, we describe the cloning, sequencing and characterization of an immunoglobulin A (IgA) Fc receptor from cattle (bFcalphaR). By screening a translated EST database with the protein sequence of the human IgA Fc receptor (CD89) we identified a putative bovine homologue. Subsequent polymerase chain reaction (PCR) amplification confirmed that the identified full-length cDNA was expressed in bovine cells. COS-1 cells transfected with a plasmid containing the cloned cDNA bound to beads coated with either bovine or human IgA, but not to beads coated with bovine IgG2 or human IgG. The bFcalphaR cDNA is 873 nucleotides long and is predicted to encode a 269 amino-acid transmembrane glycoprotein composed of two immunoglobulin-like extracellular domains, a transmembrane region and a short cytoplasmic tail devoid of known signalling motifs. Genetically, bFcalphaR is more closely related to CD89, bFcgamma2R, NKp46, and the KIR and LILR gene families than to other FcRs. Moreover, the bFcalphaR gene maps to the bovine leucocyte receptor complex on chromosome 18. Identification of the bFcalphaR will aid in the understanding of IgA-FcalphaR interactions, and may facilitate the isolation of FcalphaR from other species. PMID:15027906

  10. Cloning, expression and characterization of a feruloyl esterase C from Penicillium chrysogenum

    Institute of Scientific and Technical Information of China (English)

    LV Shan-shan; LI Gui-Iong; YANG Shao-Iong

    2016-01-01

    Objective: To clone feruloyl esterase gene C from Penicillium chrysogenum and characterize the general properties of the enzyme. Methods:The feruloyl esterase C gene was amplified by PCR based on the Penicillium chrysogenum feruloyl esterase C gene sequence and cloned into the expression vector pPIC9K, resulting the recombinant plasmid pPIC9K-PcfaeC. The recombiant plasmid was linerized and transformed into P. pastoris by electroporation. The transformants was screened based on the transparent zone technology. The screened transformants was then induced by methanol. the enzymatic properties of the protein were then measured. Results:SDS-PAGE analysis showed that the molecular mass of the enzyme was about 30 kD. The length of the gene was 762 bp. It comprised one open reading framwork(ORF) and annotated to encode 249 amino acid. The optimal temperature and pH was found to be 40℃and 6, respectively. Moreover, the recombinant enzyme was stable at 40-50℃and pH 5-7. Conclusion:The enzyme successfully expressed in P. pastoris could laid theoretical foundation in food, fodder and paper making industry.

  11. Molecular cloning and characterization of a threonine/serine protein kinase lvakt from Litopenaeus vannamei

    Science.gov (United States)

    Ruan, Lingwei; Liu, Rongdiao; Xu, Xun; Shi, Hong

    2014-07-01

    The phosphatidylinositol 3-kinase (PI3K)-AKT pathway is involved in various cellular functions, including anti-apoptosis, protein synthesis, glucose metabolism and cell cycling. However, the role of the PI3K-AKT pathway in crustaceans remains unclear. In the present study, we cloned and characterized the AKT gene lvakt from Litopenaeus vannamei. The 511-residue LVAKT was highly conserved; contained a PH domain, a catalytic domain and a hydrophobic domain; and was highly expressed in the heart and gills of L. vannamei. We found, using Real-Time Quantitative PCR (Q-PCR) analysis, that lvakt was up-regulated during early white spot syndrome virus (WSSV) infection. Moreover, the PI3K-specific inhibitor, LY294002, reduced viral gene transcription, implying that the PI3K-AKT pathway might be hijacked by WSSV. Our results therefore suggest that LVAKT may play an important role in the shrimp immune response against WSSV.

  12. Cloning and characterization of ftsZ and pyrF from the archaeon Thermoplasma acidophilum.

    Science.gov (United States)

    Yaoi, T; Laksanalamai, P; Jiemjit, A; Kagawa, H K; Alton, T; Trent, J D

    2000-09-01

    To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif. PMID:10973825

  13. Cloning and characterization of PhPI9 involved in floral development from Phalaenopsis Orchid

    Institute of Scientific and Technical Information of China (English)

    GUO Bin; DAI Wei; CHEN Donghong; WEI Xing; MING Feng

    2007-01-01

    In the attempt to discover new genes involved in the floral development in monoeotyledonousin species,we have cloned and characterized the homologous PISTALLATA-like (PI-like) gone from Phalaenopsis hybrid cultivar named PhPI9 (Phalaenopsis PI STILLATA # 9).The eDNA of PhPI9 has a fragment of 834 bp and has 60% identity with the PISTILATA from Arabidopsis.The deduced amino acid sequence of PhPI9 had the typical PI-motif.It also formed a subelade with other monoeot PI-type genes in phylogenetie analysis.Southern analysis showed that PhPI9 was present in the Phalaenopsis orchid genome as a single copy.Furthermore,it was expressed only in the lip of the Phalaenopsis flower and no expression was detected in vegetative organs.Thus,as a B-function MADS-box gone,PhP19 specifies floral organ identity in orchids.

  14. First characterization of infectious cDNA clones of Olive mild mosaic virus

    OpenAIRE

    Cardoso, Joana M.S.; Félix, M. Rosário; Clara, M.Ivone; Oliveira, Solange

    2012-01-01

    Full-length cDNA clones of an Olive mild mosaic virus (OMMV) isolate were constructed in order to find infectious cDNA clones. The sequencing of three individual full-length clones revealed some differences between them. In vitro transcription of these clones was performed and the effect of spontaneous mutations in the biological behaviour of the in vitro transcripts was evaluated by symptomatology, RNA accumulation and virus replication in inoculated plants. In vitro synthesized RNA from one...

  15. Cloning, and Molecular Characterization of Polymorphic Iranian Isolate Theileria annulata Surface Protein (Tasp

    Directory of Open Access Journals (Sweden)

    E Ebrahimzadeh

    2012-06-01

    Full Text Available Background: Because of the strong immunologic responses of surface protein TaSp in Theileria annu­lata infected host, we tried to characterize this protein in a T. annulata isolate from Iran.Methods: The RNA prepared from T. annulata infected cells was used to produce SMART-DS-cDNA. The Double strand cDNA was then amplified with primers derived from TaSp mRNA se­quences. The PCR product was cloned in pTZ57R/T vector, sequenced and registered under acces­sion no. JQ003240 in GenBank.Results: The sequence analysis showed 90%-94% nucleotide sequence identity and 68%-94% amino acid homology to the corresponding sequences of TaSp gene by T. annulata, T. sp. china I, T. sp. china and T. lestoquardi and three T. annulata reported from Iran respectively. Interestingly, the sequence analysis also showed small nucleotide sequence region near the 5` end in which the presented TaSp protein differed very strongly from the other known TaSp sequences. For the preparation of the recombi­nant protein, the cDNA was cloned in pQE-32 vector, the recombinant protein was pre­pared and assayed by Theileria infected bovine serum.Conclusion: The polymorphism in TaSp gene could be detected in intra- as well as inter species. The different characterized TaSp proteins had a common identic region, which may be helpful for develop­ment of broad band vaccine based on the recombinant proteins. The polymorphism in this gene, make this protein also interesting for the diagnostic purposes.

  16. Molecular cloning and partial characterization of a plant VAP33 homologue with a major sperm protein domain

    NARCIS (Netherlands)

    Laurent, F.; Labesse, G.; Wit, de P.

    2000-01-01

    In a search for proteins interacting with the resistance protein Cf9 from tomato, a new cDNA was cloned and characterized. Protein sequence database searches suggested that the 120 residue-N terminal domain of the encoded protein (named VAP27) is highly similar to the VAP33 protein family from anima

  17. Cloning and Characterization of an Endoglucanase Gene from Actinomyces sp. Korean Native Goat 40.

    Science.gov (United States)

    Kim, Sung Chan; Kang, Seung Ha; Choi, Eun Young; Hong, Yeon Hee; Bok, Jin Duck; Kim, Jae Yeong; Lee, Sang Suk; Choi, Yun Jaie; Choi, In Soon; Cho, Kwang Keun

    2016-01-01

    A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-β-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) DH5α. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli DH5α harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine-Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was 55°C, but it retained over 90% of maximum activity in a broad temperature range (40°C to 60°C). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, Km and Vmax of rEG1 were 0.39% CMC and 143 U/mg, respectively. PMID:26732336

  18. A novel hyaluronidase from brown spider (Loxosceles intermedia venom (Dietrich's Hyaluronidase: from cloning to functional characterization.

    Directory of Open Access Journals (Sweden)

    Valéria Pereira Ferrer

    Full Text Available Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (∼45 kDa in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA and chondroitin sulfate (CS, while dermatan sulfate (DS and heparan sulfate (HS were not affected. Zymograph experiments resulted in ∼45 kDa lytic zones in hyaluronic acid (HA and chondroitin sulfate (CS substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1. These data support the hypothesis that hyaluronidase is a "spreading factor". Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.

  19. Cloning and functional characterization of the rabbit C-C chemokine receptor 2

    Directory of Open Access Journals (Sweden)

    Hamdouchi Chafiq

    2005-07-01

    Full Text Available Abstract Background CC-family chemokine receptor 2 (CCR2 is implicated in the trafficking of blood-borne monocytes to sites of inflammation and is implicated in the pathogenesis of several inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and atherosclerosis. The major challenge in the development of small molecule chemokine receptor antagonists is the lack of cross-species activity to the receptor in the preclinical species. Rabbit models have been widely used to study the role of various inflammatory molecules in the development of inflammatory processes. Therefore, in this study, we report the cloning and characterization of rabbit CCR2. Data regarding the activity of the CCR2 antagonist will provide valuable tools to perform toxicology and efficacy studies in the rabbit model. Results Sequence alignment indicated that rabbit CCR2 shares 80 % identity to human CCR2b. Tissue distribution indicated that rabbit CCR2 is abundantly expressed in spleen and lung. Recombinant rabbit CCR2 expressed as stable transfectants in U-937 cells binds radiolabeled 125I-mouse JE (murine MCP-1 with a calculated Kd of 0.1 nM. In competition binding assays, binding of radiolabeled mouse JE to rabbit CCR2 is differentially competed by human MCP-1, -2, -3 and -4, but not by RANTES, MIP-1α or MIP-1β. U-937/rabbit CCR2 stable transfectants undergo chemotaxis in response to both human MCP-1 and mouse JE with potencies comparable to those reported for human CCR2b. Finally, TAK-779, a dual CCR2/CCR5 antagonist effectively inhibits the binding of 125I-mouse JE (IC50 = 2.3 nM to rabbit CCR2 and effectively blocks CCR2-mediated chemotaxis. Conclusion In this study, we report the cloning of rabbit CCR2 and demonstrate that this receptor is a functional chemotactic receptor for MCP-1.

  20. Virulent clones of Klebsiella pneumoniae: identification and evolutionary scenario based on genomic and phenotypic characterization.

    Directory of Open Access Journals (Sweden)

    Sylvain Brisse

    Full Text Available Klebsiella pneumoniae is found in the environment and as a harmless commensal, but is also a frequent nosocomial pathogen (causing urinary, respiratory and blood infections and the agent of specific human infections including Friedländer's pneumonia, rhinoscleroma and the emerging disease pyogenic liver abscess (PLA. The identification and precise definition of virulent clones, i.e. groups of strains with a single ancestor that are associated with particular infections, is critical to understand the evolution of pathogenicity from commensalism and for a better control of infections. We analyzed 235 K. pneumoniae isolates of diverse environmental and clinical origins by multilocus sequence typing, virulence gene content, biochemical and capsular profiling and virulence to mice. Phylogenetic analysis of housekeeping genes clearly defined clones that differ sharply by their clinical source and biological features. First, two clones comprising isolates of capsular type K1, clone CC23(K1 and clone CC82(K1, were strongly associated with PLA and respiratory infection, respectively. Second, only one of the two major disclosed K2 clones was highly virulent to mice. Third, strains associated with the human infections ozena and rhinoscleroma each corresponded to one monomorphic clone. Therefore, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis should be regarded as virulent clones derived from K. pneumoniae. The lack of strict association of virulent capsular types with clones was explained by horizontal transfer of the cps operon, responsible for the synthesis of the capsular polysaccharide. Finally, the reduction of metabolic versatility observed in clones Rhinoscleromatis, Ozaenae and CC82(K1 indicates an evolutionary process of specialization to a pathogenic lifestyle. In contrast, clone CC23(K1 remains metabolically versatile, suggesting recent acquisition of invasive potential. In conclusion, our results reveal the existence of

  1. Gene cloning, expression, and characterization of the Bacillus amyloliquefaciens PS35 lipase

    Directory of Open Access Journals (Sweden)

    Palanisamy Kanmani

    2015-01-01

    Full Text Available AbstractLipases are enzymes of immense industrial relevance, and, therefore, are being intensely investigated. In an attempt to characterize lipases at molecular level from novel sources, a lipase gene from Bacillus amyloliquefaciens PS35 was cloned, heterologously expressed in Escherichia coli DH5α cells and sequenced. It showed up to 98% homology with other lipase sequences in the NCBI database. The recombinant enzyme was then purified from E. coli culture, resulting in a 19.41-fold purification with 9.7% yield. It displayed a preference for long-chain para-nitrophenyl esters, a characteristic that is typical of true lipases. Its optimum pH and temperature were determined to be 8.0 and 40 °C, respectively. The half-lives were 2.0, 1.0 and 0.5 h at 50 °C, 60 °C and 70 °C, respectively. The metal ions K+and Fe3+ enhanced the enzyme activity. The enzyme displayed substantial residual activity in the presence of various tested chemical modifiers, and interestingly, the organic solvents, such as n-hexane and toluene, also favored the enzyme activity. Thus, this study involves characterization of B. amyloliquefaciens lipase at molecular level. The key outcomes are novelty of the bacterial source and purification of the enzyme with desirable properties for industrial applications.

  2. A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

    Directory of Open Access Journals (Sweden)

    Yue-Zhong Li

    2011-10-01

    Full Text Available Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG and catalytic triad residues (Ser114, Asp250 and His284. Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP esters of short or medium chain fatty acids (≤C10, and the maximal activity was on pNP acetate.The r-LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the Km values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening.

  3. Molecular cloning and characterization of a GH11 endoxylanase from Chaetomium globosum, and its use in enzymatic pretreatment of biomass

    DEFF Research Database (Denmark)

    Singh, Raushan Kumar; Tiwari, Manish Kumar; Kim, Dongwook;

    2013-01-01

    An endo-1,4-β-xylanase gene, xylcg, was cloned from Chaetomium globosum and successfully expressed in Escherichia coli. The complete gene of 675 bp was amplified, cloned into the pET 28(a) vector, and expressed. The optimal conditions for the highest activity of the purified recombinant XylCg were......Cg belongs to the GH11 family. Rice straw pretreated with XylCg showed 30 % higher conversion yield than the rice straw pretreated with a commercial xylanase. Although xylanases have been characterized from fungal and bacterial sources, C. globosum XylCg is distinguished from other xylanases by its high...

  4. Caracterização agronômica da produção de rizomas de clones de taro Characterization of yield factors in taro clones

    Directory of Open Access Journals (Sweden)

    Francisco Hevilásio F. Pereira

    2003-03-01

    Full Text Available Objetivou-se caracterizar agronomicamente 36 acessos (clones de taro pertencentes ao Banco de Germoplasma de Hortaliças da UFV quanto às características relativas à produção de rizomas. O experimento foi conduzido na horta de pesquisas da UFV, de 19/09/2000 a 13/07/2001. Foi utilizado o delineamento de blocos casualizados, com 36 tratamentos (clones e cinco repetições. A parcela foi composta de quatro fileiras, com quatro metros de comprimento, com as plantas espaçadas de 1,0 x 0,5 m. As características avaliadas foram peso médio de rizomas comerciáveis; número de rizomas comerciáveis por planta; produtividade de rizomas comerciáveis e total; razão de formato (diâmetro longitudinal/diâmetro transversal de rizomas comerciáveis; produtividade de rizomas em classes (com base no diâmetro transversal filho grande (>47 mm, filho médio (40-47 mm, filho pequeno (33-40 mm e refugo (Clones of taro, from the Germoplasm Bank of the Universidade Federal de Viçosa, Brazil, were agronomically characterized. The experiment was carried out from 19/09/2000 to 13/07/2001, in a randomized complete block design, with 36 treatments (clones and five replicates. The experimental unit was comprised of four rows spaced 1.0 m apart, with four meters in length. The distance between plants in the row was 0.5 m. We evaluated the average commercial rhizome weight; number of commercial rhizomes/plant; total and commercial rhizome yield; format ratio (longitudinal/transversal diameter of the commercial production, and rhizomes classification according to the transversal diameter (large offspring >47 mm; medium offspring = 40-47 mm; small offspring = 33-40 mm; and discard < 33 mm. Clones BGH 5916, BGH 6137, and BGH 6298 presented good rhizome shape (ratio between 1.1 and 1.7 and higher values of mean corm fresh commercial weight, commercial and total yields. These clones did not differ from BGH 5925 (Japanese, presenting significantly higher values than BGH

  5. Cloning and characterization of gene-resistant analogs (RGAs) involved in rust (Puccinia psidii) resistance in Eucalyptus grandis

    Institute of Scientific and Technical Information of China (English)

    Marcelo Luiz Laia; Acelino Couto Alfenas; Sergio Hermnio Brommonschenkel; Shinitiro Oda; Eduardo Jose de Melo; Inae Marie de Arau jo Silva; Janana Fernandes Goncalves; Ariadne Marques

    2015-01-01

    Disease-resistant genes play an important role in defending against a variety of pathogens and insect pests in plants. Most of the disease-resistant genes encode pro-teins with conserved leucine rich repeat and nucleotide binding site domains. In this study, we cloned and char-acterized gene-resistant analogs (RGAs) from Eucalyptus grandis using degenerate PCR, with primers specifically targeting these two domains. The amplified fragments were cloned into the pGEM-T vector and transformed into Escherichia coli. Among the 90 clones obtained, 13 were sequenced and compared with each other and with previ-ously identified gene-resistant diseases. A BLASTX search in GenBank revealed high similarities among the con-served domains of these cloned genes with RGA genes. Some clones, however, showed no significant similarity with DNA sequences in GenBank. Southern blotting ana-lysis identified several polymorphic RFLP loci between distinct genotypes. However, none of them co-segregated with the Puccinia psidii Winter resistance gene 1 (Ppr1) in a population study.

  6. Characterization of an Unusual Cytoplasmic Chimera Detected in Bolting Garlic (Allium sativum L.) Clones

    Science.gov (United States)

    Production of a visible flower stalk or bolting has been used as a major trait to categorize garlic clones. Analysis of mitochondrial genome variation with PCR revealed differences between bolting and non-bolting clones of garlic. Screening 333 garlic accessions from diverse geographic origins rev...

  7. Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

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    Sakuko Ueshima

    2010-01-01

    Full Text Available The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3 were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD+-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienylserine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO2.

  8. Molecular Cloning, Expression Analysis, and Functional Characterization of the H(+)-Pyrophosphatase from Jatropha curcas.

    Science.gov (United States)

    Yang, Yumei; Luo, Zhu; Zhang, Mengru; Liu, Chang; Gong, Ming; Zou, Zhurong

    2016-04-01

    H(+)-pyrophosphatase (H(+)-PPase) is a primary pyrophosphate (PPi)-energized proton pump to generate electrochemical H(+) gradient for ATP production and substance translocations across membranes. It plays an important role in stress adaptation that was intensively substantiated by numerous transgenic plants overexpressing H(+)-PPases yet devoid of any correlated studies pointing to the elite energy plant, Jatropha curcas. Herein, we cloned the full length of J. curcas H(+)-PPase (JcVP1) complementary DNA (cDNA) by reverse transcription PCR, based on the assembled sequence of its ESTs highly matched to Hevea brasiliensis H(+)-PPase. This gene encodes a polypeptide of 765 amino acids that was predicted as a K(+)-dependent H(+)-PPase evolutionarily closest to those of other Euphorbiaceae plants. Many cis-regulatory elements relevant to environmental stresses, molecular signals, or tissue-specificity were identified by promoter prediction within the 1.5-kb region upstream of JcVP1 coding sequence. Meanwhile, the responses of JcVP1 expression to several common abiotic stresses (salt, drought, heat, cold) were characterized with a considerable accordance with the inherent stress tolerance of J. curcas. Moreover, we found that the heterologous expression of JcVP1 could significantly improve the salt tolerance in both recombinant Escherichia coli and Saccharomyces cerevisiae, and this effect could be further fortified in yeast by N-terminal addition of a vacuole-targeting signal peptide from the H(+)-PPase of Trypanosoma cruzi. PMID:26643082

  9. Cloning and Characterization of Glyceraldehyde-3-phosphate Dehydrogenase Encoding Gene in Gracilaria/Gracilariopsis lemaneiformis

    Institute of Scientific and Technical Information of China (English)

    REN Xueying; SUI Zhenghong; ZHANG Xuecheng

    2006-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. A cytosolic GAPDH encoding gene (gpd) of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5'-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues (CL to LF), thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

  10. Molecular cloning and characterization of orange-spotted grouper (Epinephelus coioides) CXC chemokine ligand 12.

    Science.gov (United States)

    Wu, Chen-Shiou; Wang, Ting-Yu; Liu, Chin-Feng; Lin, Hao-Ping; Chen, Young-Mao; Chen, Tzong-Yueh

    2015-12-01

    Chemokines are a family of soluble peptides that can recruit a wide range of immune cells to sites of infection and disease. The CXCL12 is a chemokine that binds to its cognate receptor CXCR4 and thus involved in multiple physiological and pathophysiological processes. In this study, we cloned and characterized CXCL12 from Epinephelus coioides (osgCXCL12). We found that the open reading frame of osgCXCL12 consists of 98 amino acid residues with the small cytokine C-X-C domain located between residues 29 and 87. Higher expression levels for osgCXCL12 were detected at the kitting stage, compared with the prolarva and larva shape stages. The expression patterns revealed that osgCXCL12 may play a key role in early grouper development. We detected mRNA transcripts for osgCXCL12 in healthy tissues and found the highest osgCXCL12 expression in the head kidney. Furthermore, a time-course analysis revealed significantly increased osgCXCL12 and osgCXCR4 expression levels after the nervous necrosis virus (NNV) challenge. In addition, expression of osgCXCL12 was affected by injection with microbial mimics [LPS and poly(I:C)]. These results suggest that osgCXCL12 is associated with inflammatory and developmental processes in the grouper.

  11. Cloning, expression and characterization of 3-hydroxyisobutyrate dehydrogenase from Pseudomonas denitrificans ATCC 13867.

    Directory of Open Access Journals (Sweden)

    Shengfang Zhou

    Full Text Available The gene encoding an NAD(+-dependent, 3-hydroxyisobutyrate dehydrogenase (3HIBDH-IV from Pseudomonas denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL 21 (DE3 and characterized to understand its physiological relevance in the degradation of 3-hydroxypropionic acid (3-HP. The deduced amino acid sequence showed high similarity to other 3-hydroxyisobutyrate dehydrogenase isozymes (3HIBDHs of P. denitrificans ATCC 13867. A comparison of 3HIBDH-IV with its relevant enzymes along with molecular docking studies suggested that Lys171, Asn175 and Gly123 are important for its catalytic function on 3-hydroxyacids. The recombinant 3HIBDH-IV was purified to homogeneity utilizing a Ni-NTA-HP resin column in high yield. 3HIBDH-IV was very specific to (S-3-hydroxyisobutyrate, but also catalyzed the oxidation of 3-HP to malonate semialdehyde. The specific activity and half-saturation constant (K m for 3-HP at 30°C and pH 9.0 were determined to be 17 U/mg protein and 1.0 mM, respectively. Heavy metals, such as Ag(+ and Hg(2+, completely inhibited the 3HIBDH-IV activity, whereas dithiothreitol, 2-mercaptoethanol and ethylenediaminetetraacetic acid increased its activity 1.5-1.8-fold. This paper reports the characteristics of 3HIBDH-IV as well as its probable role in 3-HP degradation.

  12. Glucoamylase starch-binding domain of Aspergillus niger B1: molecular cloning and functional characterization.

    Science.gov (United States)

    Paldi, Tzur; Levy, Ilan; Shoseyov, Oded

    2003-01-01

    Carbohydrate-binding modules (CBMs) are protein domains located within a carbohydrate-active enzyme, with a discrete fold that can be separated from the catalytic domain. Starch-binding domains (SBDs) are CBMs that are usually found at the C-terminus in many amylolytic enzymes. The SBD from Aspergillus niger B1 (CMI CC 324262) was cloned and expressed in Escherichia coli as an independent domain and the recombinant protein was purified on starch. The A. niger B1 SBD was found to be similar to SBD from A. kawachii, A. niger var. awamori and A. shirusami (95-96% identity) and was classified as a member of the CBM family 20. Characterization of SBD binding to starch indicated that it is essentially irreversible and that its affinity to cationic or anionic starch, as well as to potato or corn starch, does not differ significantly. These observations indicate that the fundamental binding area on these starches is essentially the same. Natural and chemically modified starches are among the most useful biopolymers employed in the industry. Our study demonstrates that SBD binds effectively to both anionic and cationic starch. PMID:12646045

  13. Cloning and characterization of a novel cysteine protease gene (HbCP1) from Hevea brasiliensis

    Indian Academy of Sciences (India)

    Shi-Qing Peng; Jia-Hong Zhu; Hui-Liang Li; Wei-Min Tian

    2008-12-01

    The full-length cDNA encoding a cysteine protease, designated HbCP1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCP1 contained a 1371 bp open reading frame encoding 457 amino acids. The deduced HbCP1 protein, which showed high identity to cysteine proteases of other plant species, was predicted to possess a putative repeat in toxin (RTX) domain at the N-terminal and a granulin (GRAN) domain at the C-terminal. Southern blot analysis indicated that the HbCP1 gene is present as a single copy in the rubber tree. Transcription pattern analysis revealed that HbCP1 had high transcription in laticifer, and low transcription in bark and leaf. The transcription of HbCP1 in latex was induced by ethylene and tapping. Cloning of the HbCP1 gene will enable us to further understand the molecular characterization of cysteine protease and its possible function in the rubber tree.

  14. Cloning and characterization of indolepyruvate decarboxylase from Methylobacterium extorquens AM1.

    Science.gov (United States)

    Fedorov, D N; Doronina, N V; Trotsenko, Yu A

    2010-12-01

    For the first time for methylotrophic bacteria an enzyme of phytohormone indole-3-acetic acid (IAA) biosynthesis, indole-3-pyruvate decarboxylase (EC 4.1.1.74), has been found. An open reading frame (ORF) was identified in the genome of facultative methylotroph Methylobacterium extorquens AM1 using BLAST. This ORF encodes thiamine diphosphate-dependent 2-keto acid decarboxylase and has similarity with indole-3-pyruvate decarboxylases, which are key enzymes of IAA biosynthesis. The ORF of the gene, named ipdC, was cloned into overexpression vector pET-22b(+). Recombinant enzyme IpdC was purified from Escherichia coli BL21(DE3) and characterized. The enzyme showed the highest k(cat) value for benzoylformate, albeit the indolepyruvate was decarboxylated with the highest catalytic efficiency (k(cat)/K(m)). The molecular mass of the holoenzyme determined using gel-permeation chromatography corresponds to a 245-kDa homotetramer. An ipdC-knockout mutant of M. extorquens grown in the presence of tryptophan had decreased IAA level (46% of wild type strain). Complementation of the mutation resulted in 6.3-fold increase of IAA concentration in the culture medium compared to that of the mutant strain. Thus involvement of IpdC in IAA biosynthesis in M. extorquens was shown. PMID:21314613

  15. Cloning, expression, and characterization of thermostable manganese superoxide dismutase from Thermoascus aurantiacus var. levisporus.

    Science.gov (United States)

    Song, Ning-Ning; Zheng, Yan; E, Shi-Jin; Li, Duo-Chuan

    2009-02-01

    A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 microg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN(3), but not by KCN or H(2)O(2). These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55 degrees C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60 degrees C and the half-life at 80 degrees C was approximately 40 min. PMID:19229500

  16. Cloning, expression and characterization of a mucin-binding GAPDH from Lactobacillus acidophilus.

    Science.gov (United States)

    Patel, Dhaval K; Shah, Kunal R; Pappachan, Anju; Gupta, Sarita; Singh, Desh Deepak

    2016-10-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis. It is also referred to as a moonlighting protein as it has many diverse functions like regulation of apoptosis, iron homeostasis, cell-matrix interactions, adherence to human colon etc. apart from its principal role in glycolysis. Lactobacilli are lactic acid bacteria which colonize the human gut and confer various health benefits to humans. In the present study, we have cloned, expressed and purified the GAPDH from Lactobacillus acidophilus to get a recombinant product (r-LaGAPDH) and characterized it. Size exclusion chromatography shows that r-LaGAPDH exists as a tetramer in solution and have a mucin binding and hemagglutination activity indicating carbohydrate like binding adhesion mechanism. Fluorescence spectroscopy studies showed an interaction of r-LaGAPDH with mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine with a Kd of 3.6±0.7×10(-3)M, 4.34±0.09×10(-3)M, 4±0.87×10(-3)M and 3.7±0.28×10(-3)M respectively. We hope that this preliminary data will generate more interest in further elucidation of the roles of GAPDH in the adhesion processes of the bacteria. PMID:27180300

  17. Molecular Cloning, Expression Analysis, and Functional Characterization of the H(+)-Pyrophosphatase from Jatropha curcas.

    Science.gov (United States)

    Yang, Yumei; Luo, Zhu; Zhang, Mengru; Liu, Chang; Gong, Ming; Zou, Zhurong

    2016-04-01

    H(+)-pyrophosphatase (H(+)-PPase) is a primary pyrophosphate (PPi)-energized proton pump to generate electrochemical H(+) gradient for ATP production and substance translocations across membranes. It plays an important role in stress adaptation that was intensively substantiated by numerous transgenic plants overexpressing H(+)-PPases yet devoid of any correlated studies pointing to the elite energy plant, Jatropha curcas. Herein, we cloned the full length of J. curcas H(+)-PPase (JcVP1) complementary DNA (cDNA) by reverse transcription PCR, based on the assembled sequence of its ESTs highly matched to Hevea brasiliensis H(+)-PPase. This gene encodes a polypeptide of 765 amino acids that was predicted as a K(+)-dependent H(+)-PPase evolutionarily closest to those of other Euphorbiaceae plants. Many cis-regulatory elements relevant to environmental stresses, molecular signals, or tissue-specificity were identified by promoter prediction within the 1.5-kb region upstream of JcVP1 coding sequence. Meanwhile, the responses of JcVP1 expression to several common abiotic stresses (salt, drought, heat, cold) were characterized with a considerable accordance with the inherent stress tolerance of J. curcas. Moreover, we found that the heterologous expression of JcVP1 could significantly improve the salt tolerance in both recombinant Escherichia coli and Saccharomyces cerevisiae, and this effect could be further fortified in yeast by N-terminal addition of a vacuole-targeting signal peptide from the H(+)-PPase of Trypanosoma cruzi.

  18. Cloning and characterization of the nicotianamine synthase gene in Eruca vesicaria subsp sativa.

    Science.gov (United States)

    Huang, B L; Cheng, C; Zhang, G Y; Su, J J; Zhi, Y; Xu, S S; Cai, D T; Zhang, X K; Huang, B Q

    2015-12-22

    Nicotianamine (NA) is a ubiquitous metabolite in plants that bind heavy metals, is crucial for metal homeostasis, and is also an important metal chelator that facilitates long-distance metal transport and sequestration. NA synthesis is catalyzed by the enzyme nicotianamine synthase (NAS). Eruca vesicaria subsp sativa is highly tolerant to Ni, Pb, and Zn. In this study, a gene encoding EvNAS was cloned and characterized in E. vesicaria subsp sativa. The full-length EvNAS cDNA sequence contained a 111-bp 5'-untranslated region (UTR), a 155-bp 3'-UTR, and a 966-bp open reading frame encoding 322-amino acid residues. The EvNAS genomic sequence contained no introns, which is similar to previously reported NAS genes. The deduced translation of EvNAS contained a well-conserved NAS domain (1-279 amino acids) and an LIKI-CGEAEG box identical to some Brassica NAS and to the LIRL-box in most plant NAS, which is essential for DNA binding. Phylogenetic analysis indicated that EvNAS was most closely related to Brassica rapa NAS3 within the Cruciferae, followed by Thlaspi NAS1, Camelina NAS3, and Arabidopsis NAS3. A reverse transcription-polymerase chain reaction indicated that EvNAS expression was greatest in the leaves, followed by the flower buds and hypocotyls. EvNAS was moderately expressed in the roots.

  19. Cloning and characterization of cotton heteromeric acetyl-CoA carboxylase genes

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Heteromeric acetyl-coanzyme A(CoA)carboxylese(ACCase)catalyzes the formation of malonyl-CoA from acetyl-CoA.It plays an essential role in fatty acid synthesis in prokaryotes and most of plants.The heteromeric ACCase is composed of four subunits:biotin carboxylase (BC),biotin carboxyl carrier protein (BCCP),and α-and β-subunits of carboxyltransferese(α-andβ-CT).In this study,we cloned five novel genes encoding the subunits of heteromeric ACCese(one BC,BCCP and β-CT,and two α-CTs) from cotton (Gossypium hirsutum cv.zhongmian 35)by RACE-PCR.The deduced amino acid sequence of these cDNAs shares high similarity with other reported heteromeric ACCese subunits.The phylogenetic analysis indicated that the different subunits of heteromeric ACCase were grouped in a similar pattern.Southern blot analysis revealed the milti-copy patterns of these heteromeric ACCase genes in cotton genome.Semi-quantitative RT-PCR demonstrated that heteromeric ACCese genes were constitutively expressed in all of the cotton tissues,but the transcripts accumulated at a relatively low level in roots.To our knowledge,this is the first report about characterization of the heteromeric ACCase genes in cotton.

  20. Cloning and characterization of giant panda (Ailuropoda melanoleuca) IL-18 binding protein.

    Science.gov (United States)

    Yan, Yue; Deng, Jiabo; Niu, Lili; Wang, Qiang; Yu, Jianqiu; Shao, Huanhuan; Cao, Qinghua; Zhang, Yizheng; Tan, Xuemei

    2016-06-01

    The giant panda (Ailuropoda melanoleuca) is an endangered species. Interleukin-18 (IL-18) plays an important role in the innate and adaptive immune responses by inducing IFN-γ. IL-18 has been implicated in the pathogenesis of various diseases. IL-18 binding protein (IL-18BP) is an intrinsic inhibitor of IL-18 that possesses higher affinity to IL-18. In this study, we cloned and characterized IL-18BP in giant panda (AmIL-18BP) from the spleen. The amino acid sequence of giant panda IL-18BP ORF shared about 65% identities with other species. To evaluate the effects of AmIL-18BP on the immune responses, we expressed the recombinant AmIL-18BP in Escherichia coli BL21 (DE3).The fusing protein PET-AmIL-18BP was purified by nickel affinity column chromatography. The biological function of purified PET-AmIL-18BP was determined on mice splenocyte by qRT-PCR. The results showed that AmIL-18BP was functional and could significantly reduce IFN-γ production in murine splenocytes. These results will facilitate the study of protecting giant panda on etiology and immunology. PMID:27234556

  1. Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725

    Directory of Open Access Journals (Sweden)

    Yanyan Chen

    2014-04-01

    Full Text Available We cloned the gene ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3. After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA. The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA, and released monogalacturonic acid as its sole product. The Vmax and Km of CbPelA were 384.6 U·mg−1 and 0.31 mg·mL−1, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%–34% and 85% methylated pectin, respectively. The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry.

  2. Cloning, expression, and characterization of a novel diketoreductase from Acinetobacter baylyi

    Institute of Scientific and Technical Information of China (English)

    Xuri Wu; Nan Liu; Yunmian He; Yijun Chen

    2009-01-01

    Reductions of carbonyl groups catalyzed by oxidoreduc-tases are involved in all biological processes and are often a class of important biocatalyst. In this article, we report a novel enzyme designated as diketoreductase (DKR) that was able to reduce two carbonyl groups in a diketo ester to corresponding dihydroxy ester with excel-lent stereoselectivity. The DKR was cloned from Acinetobacter baylyi by reverse genetic method, heteroge-neously expressed in Escherichia coli, and purified to homogeneity by two chromatographic steps. This novel enzyme exhibited dual cofactor specificity, with a prefer-ence of NADH over NADPH. The dihydroxy ester product catalyzed by the DKR was only 3R,5S-stereoi-somer with both diastereomeric excess and enantiomeric excess values more than 99.5%. In addition, some bio-chemical properties of the enzyme, such as the optimal pH and temperature, were also characterized. Furthermore, sequence analysis indicated that this new enzyme was homologous to bacterial 3-hydroxyacyl coenzyme-A dehydrogenase. More importantly, based on the unique catalytic activity and excellent stereoselec-tivity, the DKR could be utilized in the synthesis of valuable chiral drug intermediates, such as Lipitor .

  3. Cloning and characterization of ADP-glucose pyrophosphorylase small subunit gene in Cyperus esculentus (yellow nutsedge).

    Science.gov (United States)

    Cheng, C; Hu, J; Zhi, Y; Su, J J; Zhang, X K; Huang, B Q

    2015-01-01

    ADP-glucose pyrophosphorylase (ADPGlcPPase) controls the first committed step of starch synthesis by catalyzing the biosynthesis of ADP-glucose from glucose-phosphate and ATP. It is a tetrameric protein consisting of two small and two large subunits. The small subunits have a catalytic function, while the large subunits regulate the enzyme activity. Cyperus esculentus (yellow nutsedge) is a perennial C4 plant grown from rhizomes and tubers. Previous studies on yellow nutsedge have mostly focused on the morphology and cultivation of tubers, their application in food, and biochemical analyses of the tubers. In this study, the gene encoding the ADPGlcPPase small subunit (CeAGPS) in yellow nutsedge was cloned and characterized. The full-length CeAGPS cDNA sequence contained an 81-bp 5'-untranslated region (UTR), a 188-bp 3'-UTR, and a 1539-bp open reading frame encoding 512-amino acid residues. The genomic sequence of CeAGPS comprises a nine exon-eight intron structure similar to the previously reported cotton and Arabidopsis thaliana AGPS genes. The deduced translation product of the CeAGPS gene contained a well-conserved catalytic domain and regulatory elements typical of plant AGPS. Reverse transcriptase polymerase chain reaction amplification of the target gene in various plant parts using gene-specific primers indicated that the expression of CeAGPS was most abundant in the tuber, and relatively lower in nutsedge roots. PMID:26782478

  4. Cloning and characterization of a glucosyltransferase from Crocus sativus stigmas involved in flavonoid glucosylation

    Directory of Open Access Journals (Sweden)

    Ahrazem Oussama

    2009-08-01

    Full Text Available Abstract Background Flavonol glucosides constitute the second group of secondary metabolites that accumulate in Crocus sativus stigmas. To date there are no reports of functionally characterized flavonoid glucosyltransferases in C. sativus, despite the importance of these compounds as antioxidant agents. Moreover, their bitter taste makes them excellent candidates for consideration as potential organoleptic agents of saffron spice, the dry stigmas of C. sativus. Results Using degenerate primers designed to match the plant secondary product glucosyltransferase (PSPG box we cloned a full length cDNA encoding CsGT45 from C. sativus stigmas. This protein showed homology with flavonoid glucosyltransferases. In vitro reactions showed that CsGT45 catalyses the transfer of glucose from UDP_glucose to kaempferol and quercetin. Kaempferol is the unique flavonol present in C. sativus stigmas and the levels of its glucosides changed during stigma development, and these changes, are correlated with the expression levels of CsGT45 during these developmental stages. Conclusion Findings presented here suggest that CsGT45 is an active enzyme that plays a role in the formation of flavonoid glucosides in C. sativus.

  5. Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579.

    Science.gov (United States)

    Abfalter, Carmen M; Schönauer, Esther; Ponnuraj, Karthe; Huemer, Markus; Gadermaier, Gabriele; Regl, Christof; Briza, Peter; Ferreira, Fatima; Huber, Christian G; Brandstetter, Hans; Posselt, Gernot; Wessler, Silja

    2016-01-01

    Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications. PMID:27588686

  6. Cloning and characterization of two xyloglucanases from Paenibacillus sp. strain KM21.

    Science.gov (United States)

    Yaoi, Katsuro; Nakai, Tomonori; Kameda, Yoshiro; Hiyoshi, Ayako; Mitsuishi, Yasushi

    2005-12-01

    Two xyloglucan-specific endo-beta-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley beta-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74. PMID:16332739

  7. Cloning and characterization of mouse cullin4B/E3 ubiquitin ligase

    Indian Academy of Sciences (India)

    Rachana Tripathi; K Seetharama; Satya Keerthi Kota; Usha K Srinivas

    2005-06-01

    Heat induced differentiation of mouse embryonal carcinoma cells PCC4 has been reported earlier. We have further characterized the phenotype of the differentiated cells and by DD-RT-PCR identified several partial cDNAs that are differentially expressed during differentiation. Nucleotide homology search revealed that the genes corresponding to some of the up-regulated partial cDNAs are indeed part of differentiation pathway. 5′ extension of an EST that has homology to one of the partial cDNAs led to the identification of mouse cullin4B. Cullin4B is coded by a separate gene and has a unique and longer amino-terminal end with a putative nuclear localization signal sequence (NLS). We have cloned, expressed and raised antibodies against the amino and carboxy-terminal halves of cullin4B. Immuno staining of differentiated PCC4 cells with N-terminal Cul4B antibody showed enhanced expression of Cul4B and its translocation into the nucleus upon differentiation. Transient transfection of a chimeric gene encoding the N-terminal part of Cul4B fused to green fluorescent protein into PCC4 cells revealed that the protein was localized in the nucleus confirming the functional significance of the putative NLS. Since cullins are involved in recognition of specific proteins for degradation, based on the evidence presented here, we hypothesize that cullin4B is probably involved in differentiation specific degradation/modification of nuclear proteins.

  8. Cloning, sequencing, and characterization of the Azospirillum brasilense fhuE gene.

    Science.gov (United States)

    Cui, Yanhua; Tu, Ran; Guan, Yue; Ma, Luyan; Chen, Sanfeng

    2006-03-01

    The fhuE gene of Escherichia coli encodes the FhuE protein, which is a receptor protein in the coprogen-mediated siderophore iron-transport system. A fhuE gene homologue from Azospirillum brasilense, a nitrogen-fixing soil bacterium that lives in association with the roots of cereal grasses, was cloned, sequenced, and characterized. The A. brasilense fhuE encodes a protein of 802 amino acids with a predicted molecular weight of approximately 87 kDa. The deduced amino-acid sequence showed a high level of homology to the sequences of all the known fhuE gene products. The fhuE mutant was sensitive to iron starvation and defective in coprogen-mediated iron uptake. The mutant failed to express one membrane protein of approximately 78 kDa that was induced by iron starvation in the wild type. Complementation studies showed that the A. brasilense fhuE gene, when present on a low-copy number plasmid, could restore the functions of the mutant. Mutation in fhuE gene did not affect nitrogen fixation.

  9. A Metagenomic Advance for the Cloning and Characterization of a Cellulase from Red Rice Crop Residues.

    Science.gov (United States)

    Meneses, Carlos; Silva, Bruna; Medeiros, Betsy; Serrato, Rodrigo; Johnston-Monje, David

    2016-01-01

    Many naturally-occurring cellulolytic microorganisms are not readily cultivable, demanding a culture-independent approach in order to study their cellulolytic genes. Metagenomics involves the isolation of DNA from environmental sources and can be used to identify enzymes with biotechnological potential from uncultured microbes. In this study, a gene encoding an endoglucanase was cloned from red rice crop residues using a metagenomic strategy. The amino acid identity between this gene and its closest published counterparts is lower than 70%. The endoglucanase was named EglaRR01 and was biochemically characterized. This recombinant protein showed activity on carboxymethylcellulose, indicating that EglaRR01 is an endoactive lytic enzyme. The enzymatic activity was optimal at a pH of 6.8 and at a temperature of 30 °C. Ethanol production from this recombinant enzyme was also analyzed on EglaRR01 crop residues, and resulted in conversion of cellulose from red rice into simple sugars which were further fermented by Saccharomyces cerevisiae to produce ethanol after seven days. Ethanol yield in this study was approximately 8 g/L. The gene found herein shows strong potential for use in ethanol production from cellulosic biomass (second generation ethanol). PMID:27347917

  10. Molecular cloning, characterization, and expression studies of water buffalo (Bubalus bubalis) somatotropin.

    Science.gov (United States)

    Sadaf, S; Khan, M A; Wilson, D B; Akhtar, M W

    2007-02-01

    Cloning, high-level expression, and characterization of the somatotropin (ST) gene of an indigenous Nili-Ravi breed of water buffalo Bubalus bubalis (BbST) are described. Coding, non-coding, and promoter regions of BbST were amplified and sequenced. Sequence analysis revealed several silent and two interesting point mutations on comparison with STs of other vertebrate species. One interesting variation in the BbST sequence was the replacement of a conserved glutamine residue by arginine. A plasmid was also constructed for the production of BbST in Escherichia coli BL21 (RIPL) CodonPlus, under the control of IPTG-inducible T7-lac promoter. High-level expression could be obtained by synthesizing a codon-optimized ST gene and expressing it in the form of inclusion bodies. The inclusion bodies represented over 20% of the E. coli cellular proteins. The biologically active conformation of purified BbST was confirmed by its efficient growth promoting activity in Nb2 cell proliferation assay. The expression system and purification strategy employed promise to be a useful approach to produce BbST for further use in structure-function studies and livestock industry. PMID:17367293

  11. Cloning, expression, purification and characterization of the stress kinase YeaG from Escherichia coli.

    Science.gov (United States)

    Tagourti, Jihen; Landoulsi, Ahmed; Richarme, Gilbert

    2008-05-01

    We cloned, overexpressed and purified the Escherichia coli yeaG gene product, whose amino acid sequence displays homology to prokaryotic serine protein kinases. The gene coding for YeaG was generated by amplifying the yeaG gene from E. coli by polymerase chain reaction. It was inserted into the expression plasmid pET-21a, under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21(DE3) E. coli strain transformed with the YeaG-expression vector pET-21a-yeaG accumulates large amounts of a soluble protein with a molecular mass of 76kDa in SDS-PAGE, which matches the expected YeaG molecular weight of 74.5kDa. YeaG, although soluble, has a marked tendency to aggregate in the absence of detergents, so that it was purified in the presence of 0.1% Triton X-100, by ion exchange chromatography and hydroxyapatite chromatography. The purified protein is monomeric and displays autokinase and casein kinase activities which are optimal in the presence of 10mM Mn(2+). The purification of the active protein kinase YeaG described in this study should allow us to characterize its biochemical target(s) in E. coli extracts. PMID:18276156

  12. Cloning and characterizing of the murine IRF-3 gene promoter region.

    Science.gov (United States)

    Xu, Hua-Guo; Liu, Lifei; Gao, Shan; Jin, Rui; Ren, Wei; Zhou, Guo-Ping

    2016-08-01

    The interferon regulatory factor 3 (IRF-3) plays essential roles in inflammation and immune response. Here, we cloned the nucleotide sequence of the 5'-flanking region of the murine IRF-3 gene (mIRF-3) and characterized the molecular mechanisms controlling the mIRF-3 transcriptional activity in NIH3T3 cells. Analyses of a series of 5' deletion constructs demonstrated that a 301 bp region (-255/+46) of the mIRF-3 gene is sufficient for full promoter activity. This region contains IK1, Egr2, Cmyb, E2F1 and YY1 putative transcription factor binding sites. Mutation of Egr2 or YY1 site led to 52-68 % decrease of the mIRF-3 promoter activity, and double Egr2 and YY1 mutation reduced the promoter activity to 20 % of the wild-type promoter activity. Furthermore, knockingdown of endogenous Egr2 or YY1 by a siRNA strategy markedly inhibited the mIRF-3 promoter activity. Chromatin immunoprecipitation assays showed that Egr2 and YY1 interact with the mIRF-3 promoter in vivo. These results suggested that the basal promoter activity of the mIRF-3 gene is regulated by transcription factors Egr2 and YY1 in NIH3T3 cells. PMID:26740329

  13. RESOLVE Projects: Lunar Water Resource Demonstration and Regolith Volatile Characterization

    Science.gov (United States)

    2008-01-01

    To sustain affordable human and robotic space exploration, the ability to live off the land at the exploration site will be essential. NASA calls this ability in situ resource utilization (ISRU) and is focusing on finding ways to sustain missions first on the Moon and then on Mars. The ISRU project aims to develop capabilities to technology readiness level 6 for the Robotic Lunar Exploration Program and early human missions returning to the Moon. NASA is concentrating on three primary areas of ISRU: (1) excavating, handling, and moving lunar regolith, (2) extracting oxygen from lunar regolith, and (3) finding, characterizing, extracting, separating, and storing volatile lunar resources, especially in the permanently shadowed polar craters. To meet the challenges related to technology development for these three primary focus areas, the Regolith and Environment Science and Oxygen and Lunar Volatile Extraction (RESOLVE) project was initiated in February 2005, through funding by the Exploration Systems Mission Directorate. RESOLVE's objectives are to develop requirements and conceptual designs and to perform breadboard concept verification testing of each experiment module. The final goal is to deliver a flight prototype unit that has been tested in a relevant lunar polar environment. Here we report progress toward the third primary area creating ways to find, characterize, extract, separate, and store volatile lunar resources. The tasks include studying thermal, chemical, and electrical ways to collect such volatile resources as hydrogen, water, nitrogen, methane, and ammonia. We approached this effort through two subtasks: lunar water resource demonstration (LWRD) and regolith volatile characterization (RVC).

  14. Cloning and functional characterization of the 5' regulatory region of ovine Hormone Sensitive Lipase (HSL) gene.

    Science.gov (United States)

    Lampidonis, Antonis D; Stravopodis, Dimitrios J; Voutsinas, Gerassimos E; Messini-Nikolaki, Niki; Stefos, George C; Margaritis, Lukas H; Argyrokastritis, Alexandros; Bizelis, Iosif; Rogdakis, Emmanuel

    2008-12-31

    Hormone Sensitive Lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signaling cascade reactions. HSL constitutes the critical enzyme in the modulation of lipid stores and the only component being subjected to hormonal control in terms of the recently identified Adipose Triglyceride Lipase (ATGL). In order to acquire detailed knowledge with regard to the mechanisms regulating ovine HSL (ovHSL) gene transcription activity, we initially isolated and cloned the 5' proximal and distal promoter regions through a genome walking approach, with the utilization of the already characterized ovHSL cDNAs. As evinced by BLAST analysis and a multiple alignment procedure, the isolated genomic fragment of 2.744 kb appeared to contain the already specified 5'-untranslated region (5'-UTR), which was interrupted by a relatively large intron of 1.448 kb. Regarding the upstream remaining part of 1.224 kb, it was demonstrated to represent a TATA-less promoter area, harboring several cis-regulatory elements that could be putatively recognized by relatively more general transcription factors, mainly including Stimulating protein 1 (Sp1), CCAAT-box Binding Factors (CBFs), Activator Protein 2 (AP2) and Glucocorticoid Receptor (GR), as well as other cis-acting regions denominated as Insulin Response Element (IRE), Glucose Response Element (GRE), Fat Specific Element (FSE) and cAMP Response Element (CRE), which could likely function in a nourishment (i.e. glucose)-/hormone-dependent fashion. When different genomic fragments were directionally (5' to 3') cloned into a suitable reporter vector upstream of a promoter-less luciferase gene and

  15. Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches.

    Directory of Open Access Journals (Sweden)

    Ákos Tóth

    Full Text Available Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T. Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5, their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM of type 2 with a 23-25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don't show homology to each other, but all of them are built up from 3-6 times repeated tetrapeptide motifs (PTDP-Tc, TEEP-Tf, DPGT-Th. All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively while their temperature optima span within the range of 70-75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9-1.7mM of KM and 80-120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial

  16. Hydrokinetic Resource Characterization on the Tanana River Near Nenana, Alaska

    Science.gov (United States)

    Toniolo, H.; Duvoy, P.; Schmid, J.; Johnson, J.

    2012-12-01

    The field of hydrokinetics, in general, is developing rapidly due to high fossil fuel costs and the desire to use renewable energy sources to reduce greenhouse gases. Alaska, in particular, has tidal and in-stream hydrokinetic resources. This presentation focuses on resource characterization in rivers; specifically, the Tanana River near Nenana, Alaska. We present a comprehensive approach to characterize the existing resource and the conditions for installing hydrokinetic devices. The methodology includes: a) extensive field measurements, b) numerical modeling, and c) turbulence analysis. Field work efforts involve bathymetric surveys, velocity measurements, and sediment sampling. Modeling encompasses an existing 2D-dimensional hydrodynamic model, and the calculation of power density along the river reach. Turbulence analysis provides insights on channel stability and energy partition. As results of this combined research approach, preliminary sediment-rating curves were developed, distribution of available power density was calculated and possible sites for turbine deployment were defined.

  17. Characterization of Porcine Endogenous Retrovirus Clones from the NIH Miniature Pig BAC Library

    Directory of Open Access Journals (Sweden)

    Seong-Lan Yu

    2012-01-01

    Full Text Available Pigs have been considered as donors for xenotransplantation in the replacement of human organs and tissues. However, porcine endogenous retroviruses (PERVs might transmit new infectious disease to humans during xenotransplantation. To investigate PERV integration sites, 45 PERV-positive BAC clones, including 12 PERV-A, 16 PERV-B, and 17 PERV-C clones, were identified from the NIH miniature pig BAC library. The analysis of 12 selected full-length sequences of PERVs, including the long terminal repeat (LTR region, identified the expected of open reading frame length, an indicative of active PERV, in all five PERV-C clones and one of the four PERV-B clones. Premature stop codons were observed in only three PERV-A clones. Also, eleven PERV integration sites were mapped using a 5000-rad IMpRH panel. The map locations of PERV-C clones have not been reported before, thus they are novel PERV clones identified in this study. The results could provide basic information for the elimination of site-specific PERVs in selection of pigs for xenotransplantation.

  18. The characterization of cDNA clones coding for wheat storage proteins.

    OpenAIRE

    Bartels, D.; Thompson, R D

    1983-01-01

    Poly(A)+ RNA isolated from the developing wheat endosperm var. Chinese Spring, has been used as template for the construction of a cDNA library. Within the library, clones have been identified by in vitro translation of hybrid-selected mRNA which encode alpha/beta gliadin related sequences and gamma-gliadin related sequences. The DNA sequence of one such clone has been determined and it shows homology with that of a clone encoding a barley storage protein, B-hordein. The sequence includes a t...

  19. Histological and immunohistochemical characterization of joint inflammation and flare-up reactions induced by cloned MT4+, Lyt-2-T cells

    NARCIS (Netherlands)

    I. Klasen (Ina); R.M.T. Ladestein (R. M T); A.A. Grandia (A.); Th.H. van der Kwast (Theo); R. Benner (Robbert)

    1990-01-01

    markdownabstractAbstract This report describes the histological and immunohistochemical characterization of joint inflammations and flare-up reactions in mice induced by cloned MT4+, Lyt-2− T cells. The T-cell clone used was specific for the antigen methylated bonne serum albumin (mBSA) and was in

  20. Cloning and characterization of the S fimbrial adhesin II complex of an Escherichia coli O18:K1 meningitis isolate.

    OpenAIRE

    Hacker, J; Kestler, H; Hoschützky, H; Jann, K; Lottspeich, F; Korhonen, T K

    1993-01-01

    S fimbrial adhesins (Sfa), which are able to recognize sialic acid-containing receptors on eukaryotic cells, are produced by Escherichia coli strains causing urinary tract infections or newborn meningitis. We recently described the cloning and molecular characterization of a determinant, termed sfaI, from the chromosome of an E. coli urinary tract infection strain. Here we present data concerning a S fimbria-specific gene cluster, designated sfaII, of an E. coli newborn meningitis strain. Lik...

  1. Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones.

    OpenAIRE

    Deepe, G S; Smith, J G; Sonnenfeld, G; Denman, D.; Bullock, W E

    1986-01-01

    Experimental studies have suggested that antigen-specific T lymphocytes are important mediators of resistance to infection with the pathogenic fungus Histoplasma capsulation. To gain a better understanding of the role of T lymphocytes, we developed murine T-cell lines and clones that recognized Histoplasma antigens. These T cells were of the helper/inducer phenotype (Thy-1.2+ Lyt-1+ L3T4+ Lyt-2-) and exerted multiple immunological functions. T-cell lines and 12 clones proliferated vigorously ...

  2. Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum

    Directory of Open Access Journals (Sweden)

    Tzeng Kuo-Ching

    2011-05-01

    Full Text Available Abstract Background Most isolates of Pectobacterium carotovorum subsp. carotovorum (Pcc produce bacteriocins. In this study, we have determined that Pcc strain F-rif-18 has a chromosomal gene encoding the low-molecular-weight bacteriocin, Carocin S2, and that this bacteriocin inhibits the growth of a closely related strain. Carocin S2 is inducible by ultraviolet radiation but not by mutagenic agents such as mitomycin C. Results A carocin S2-defective mutant, TF1-2, was obtained by Tn5 insertional mutagenesis using F-rif-18. A 5706-bp DNA fragment was detected by Southern blotting, selected from a genomic DNA library, and cloned to the vector, pMS2KI. Two adjacent complete open reading frames within pMS2KI were sequenced, characterized, and identified as caroS2K and caroS2I, which respectively encode the killing protein and immunity protein. Notably, carocin S2 could be expressed not only in the mutant TF1-2 but also in Escherichia coli DH5α after entry of the plasmid pMS2KI. Furthermore, the C-terminal domain of CaroS2K was homologous to the nuclease domains of colicin D and klebicin D. Moreover, SDS-PAGE analysis showed that the relative mass of CaroS2K was 85 kDa and that of CaroS2I was 10 kDa. Conclusion This study shown that another nuclease type of bacteriocin was found in Pectobacterium carotovorum. This new type of bacteriocin, Carocin S2, has the ribonuclease activity of CaroS2K and the immunity protein activity of CaroS2I.

  3. Cloning, expression and characterization of β-xylosidase from Aspergillus niger ASKU28.

    Science.gov (United States)

    Choengpanya, Khuanjarat; Arthornthurasuk, Siriphan; Wattana-amorn, Pakorn; Huang, Wan-Ting; Plengmuankhae, Wandee; Li, Yaw-Kuen; Kongsaeree, Prachumporn T

    2015-11-01

    β-Xylosidases catalyze the breakdown of β-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 β-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the α-mating factor signal sequence (α-MF) and a poly-histidine tag. The expression level was increased to 5.7 g/l in a fermenter system as a result of optimization of only five codons near the 5' end of the α-MF sequence. The recombinant AnBX was purified to homogeneity through a single-step Phenyl Sepharose chromatography. The enzyme exhibited an optimal activity at 70°C and at pH 4.0-4.5, and a very high kinetic efficiency toward a xyloside substrate. AnBX demonstrated an exo-type activity with retention of the β-configuration, and a synergistic action with xylanase in hydrolysis of beechwood xylan. This study provides comprehensive data on characterization of a glycoside hydrolase family 3 β-xylosidase that have not been determined in any prior investigations. Our results suggested that AnBX may be useful for degradation of lignocellulosic biomass in bioethanol production, pulp bleaching process and beverage industry. PMID:26166179

  4. cDNA cloning and characterization of two trehalases from Spodoptera litura (Lepidoptera; Noctuidade).

    Science.gov (United States)

    Zou, Q; Wei, P; Xu, Q; Zheng, H Z; Tang, B; Wang, S G

    2013-01-01

    The oriental leafworm moth, Spodoptera litura, is a major agricultural pest in southeast Asia and nearby Pacific regions. Two distinct trehalases have been identified in insects: soluble trehalase (Treh1) and membrane-bound trehalase (Treh2), although there is currently no information on these genes in S. litura. To characterize the distribution and function of treh, cDNAs of Treh proteins were cloned from S. litura. SpoliTreh1 cDNA has an open reading frame of 1758 nucleotides, which encodes a protein of 585 amino acids, with a predicted mass of approximately 67.07 kDa and an isoelectric point of 4.86. SpoliTreh2 cDNA has an open reading frame of 2325 nucleotides, encoding a protein of 645 amino acids, a mass of approximately 73.62 kDa, and an isoelectric point of 5.90. Northern blotting analysis revealed that SpoliTreh1 transcripts are in the midgut, fat body, tracheae, and epidermis, but not in the brain and Malpighian tubules of S. litura larvae, whereas SpoliTreh2 transcripts were found in all 6 tissues. SpoliTreh1 transcripts were highly expressed in the fat body of the pre-pupal stage, and SpoliTreh2 transcripts were highly expressed in the fat body of 3-day-old larvae of the 6th instar and during the 1st 6 days of the pupal stage, except the 2nd day. Both SpoliTreh1 and SpoliTreh2 were highly expressed in third-instar larvae. PMID:23613237

  5. Chemokine receptor CXCR3 in turbot (Scophthalmus maximus): cloning, characterization and its responses to lipopolysaccharide.

    Science.gov (United States)

    Chen, Yadong; Zhou, Shuhong; Jiang, Zhiqiang; Wang, Xiuli; Liu, Yang

    2016-04-01

    Chemokine (C-X-C motif) receptor 3, a member of the G protein-coupled receptors superfamily, regulates the responses of many immune responses. In this experiment, we cloned and characterized the cDNA of CXCR3 in Scophthalmus maximus (turbot). A 5'-UTR of 216-bp, a 259-bp 3'-UTR with a poly (A) tail and a 1089-bp CDS encoding 362 amino acids form the cDNA of CXCR3, which is 1564-bp long. Phylogenetic analyses indicated that turbot CXCR3 shared a high similarity with other CXCR3s and shared more similarity with CXCR5 than the other subfamilies of chemokines. The CXCR3 protein in turbot showed the highest similarity with the CXCR3b from rainbow trout (44.5%), which indicated that this CXCR3 gene/protein may be a CXCR3b isoform. Quantitative real-time PCR analysis showed that CXCR3 transcripts were constitutively expressed in all the tissues of the non-injected turbot used in this study, with the highest expression occurring in blood. Several immune-related tissues of fish, such as the spleen, head kidney, liver and blood, tissues, which were abundant of lymphocyte, were investigated in this study. CXCR3 gene was expressed at the highest level in blood than the other tested tissues. The injection experiment suggested that the CXCR3 expression level after LPS injection was significantly up-regulated in all immune-related tissues in turbot. These results improve our understanding of the functions of CXCR3 in the turbot immune response. PMID:26585996

  6. Cloning and characterization of nif structural and regulatory genes in the purple sulfur bacterium, Halorhodospira halophila.

    Science.gov (United States)

    Tsuihiji, Hisayoshi; Yamazaki, Yoichi; Kamikubo, Hironari; Imamoto, Yasushi; Kataoka, Mikio

    2006-03-01

    Halorhodospira halophila is a halophilic photosynthetic bacterium classified as a purple sulfur bacterium. We found that H. halophila generates hydrogen gas during photoautotrophic growth as a byproduct of a nitrogenase reaction. In order to consider the applied possibilities of this photobiological hydrogen generation, we cloned and characterized the structural and regulatory genes encoding the nitrogenase, nifH, nifD and nifA, from H. halophila. This is the first description of the nif genes for a purple sulfur bacterium. The amino-acid sequences of NifH and NifD indicated that these proteins are an Fe protein and a part of a MoFe protein, respectively. The important residues are conserved completely. The sequence upstream from the nifH region and sequence similarities of nifH and nifD with those of the other organisms suggest that the regulatory system might be a NifL-NifA system; however, H. halophila lacks nifL. The amino-acid sequence of H. halophila NifA is closer to that of the NifA of the NifL-NifA system than to that of NifA without NifL. H. halophila NifA does not conserve either the residue that interacts with NifL or the important residues involved in NifL-independent regulation. These results suggest the existence of yet another regulatory system, and that the development of functional systems and their molecular counterparts are not necessarily correlated throughout evolution. All of these Nif proteins of H. halophila possess an excess of acidic residues, which acts as a salt-resistant mechanism.

  7. Molecular cloning and characterization of SoxB2 gene from Zhikong scallop Chlamys farreri

    Science.gov (United States)

    He, Yan; Bao, Zhenmin; Guo, Huihui; Zhang, Yueyue; Zhang, Lingling; Wang, Shi; Hu, Jingjie; Hu, Xiaoli

    2013-11-01

    The Sox proteins play critical roles during the development of animals, including sex determination and central nervous system development. In this study, the SoxB2 gene was cloned from a mollusk, the Zhikong scallop ( Chlamys farreri), and characterized with respect to phylogeny and tissue distribution. The full-length cDNA and genomic DNA sequences of C. farreri SoxB2 ( Cf SoxB2) were obtained by rapid amplification of cDNA ends and genome walking, respectively, using a partial cDNA fragment from the highly conserved DNA-binding domain, i.e., the High Mobility Group (HMG) box. The full-length cDNA sequence of Cf SoxB2 was 2 048 bp and encoded 268 amino acids protein. The genomic sequence was 5 551 bp in length with only one exon. Several conserved elements, such as the TATA-box, GC-box, CAAT-box, GATA-box, and Sox/sry-sex/testis-determining and related HMG box factors, were found in the promoter region. Furthermore, real-time quantitative reverse transcription PCR assays were carried out to assess the mRNA expression of Cf SoxB 2 in different tissues. SoxB2 was highly expressed in the mantle, moderately in the digestive gland and gill, and weakly expressed in the gonad, kidney and adductor muscle. In male and female gonads at different developmental stages of reproduction, the expression levels of Cf SoxB2 were similar. Considering the specific expression and roles of SoxB 2 in other animals, in particular vertebrates, and the fact that there are many pallial nerves in the mantle, cerebral ganglia in the digestive gland and gill nerves in gill, we propose a possible essential role in nervous tissue function for Sox B 2 in C. farreri.

  8. Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

    Directory of Open Access Journals (Sweden)

    F.C. Cançado

    2008-11-01

    Full Text Available cDNA coding for two digestive lysozymes (MdL1 and MdL2 of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8 at low ionic strength (μ = 0.02, which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (μ = 0.2. However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9 is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI indicate that MdL2 (pI = 6.7 is less positively charged than MdL1 (pI = 7.7 at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

  9. Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca.

    Science.gov (United States)

    Irwin, Diana C; Cheng, Mark; Xiang, Bosong; Rose, Jocelyn K C; Wilson, David B

    2003-07-01

    Thermobifida fusca xyloglucan-specific endo-beta-1,4-glucanase (Xeg)74 and the Xeg74 catalytic domain (CD) were cloned, expressed in Escherichia coli, purified and characterized. This enzyme has a glycohydrolase family-74 CD that is a specific xyloglucanase followed by a family-2 carbohydrate binding module at the C terminus. The Michaelis constant (Km) and maximal rate (Vmax) values for hydrolysis of tamarind seed xyloglucan (tamXG) are 2.4 micro m and 966 micro mol xyloglucan oligosaccharides (XGOs) min-1. micro mol protein-1. More than 75% of the activity was retained after a 16-h incubation at temperatures up to 60 degrees C. The enzyme was most active at pH 6.0-9.4. NMR analysis showed that its catalytic mechanism is inverting. The oligosaccharide products from hydrolysis of tamXG were determined by MS analysis. Cel9B, an active carboxymethylcellulose (CMC)ase from T. fusca, was also found to have activity on xyloglucan (XG) at 49 micro mol.min-1. micro mol protein-1, but it could not hydrolyze XG units containing galactose. An XG/cellulose composite was prepared by growing Gluconacetobacterxylinus on glucose with tamXG in the medium. Although a mixture of purified cellulases was unable to degrade this material, the composite material was fully hydrolyzed when Xeg74 was added. T. fusca was not able to grow on tamXG, but Xeg74 was found in the culture supernatant at the same level as was found in cultures grown on Solka Floc. The function of this enzyme appears to be to break down the XG surrounding cellulose fibrils found in biomass so that T. fusca can utilize the cellulose as a carbon source.

  10. Molecular cloning and characterization of an S-adenosylmethionine synthetase gene from Chorispora bungeana.

    Science.gov (United States)

    Ding, Chenchen; Chen, Tao; Yang, Yu; Liu, Sha; Yan, Kan; Yue, Xiule; Zhang, Hua; Xiang, Yun; An, Lizhe; Chen, Shuyan

    2015-11-10

    S-adenosylmethionine synthetase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) which is a molecule essential for polyamines and ethylene biosynthesis, methylation modifications of protein, DNA and lipids. SAMS also plays an important role in abiotic stress response. Chorispora bungeana (C. bungeana) is an alpine subnival plant species which possesses strong tolerance to cold stress. Here, we cloned and characterized an S-adenosylmethionine synthetase gene, CbSAMS (C. bungeana S-adenosylmethionine synthetase), from C. bungeana, which encodes a protein of 393 amino acids containing a methionine binding motif GHPDK, an ATP binding motif GAGDQG and a phosphate binding motif GGGAFSGDK. Furthermore, an NES (nuclear export signal) peptide was identified through bioinformatics analysis. To explore the CbSAMS gene expression regulation, we isolated the promoter region of CbSAMS gene 1919bp upstream the ATG start codon, CbSAMSp, and analyzed its cis-acting elements by bioinformatics method. It was revealed that a transcription start site located at 320 bp upstream the ATG start codon and cis-acting elements related to light, ABA, auxin, ethylene, MeJA, low temperature and drought had been found in the CbSAMSp sequence. The gene expression pattern of CbSAMS was then analyzed by TR-qPCR and GUS assay method. The result showed that CbSAMS is expressed in all examined tissues including callus, roots, petioles, leaves, and flowers with a significant higher expression level in roots and flowers. Furthermore, the expression level of CbSAMS was induced by low temperature, ethylene and NaCl. Subcellular localization revealed that CbSAMS was located in the cytoplasm and nucleus but has a significant higher level in the nucleus. These results indicated a potential role of CbSAMS in abiotic stresses and plant growth in C. bungeana. PMID:26205258

  11. Molecular cloning and characterization of enhanced disease susceptibility 1 (EDS1) from Gossypium barbadense.

    Science.gov (United States)

    Su, Xiaofeng; Qi, Xiliang; Cheng, Hongmei

    2014-06-01

    Arabidopsis enhanced disease susceptibility 1 (EDS1) plays an important role in plant defense against biotrophic and necrotrophic pathogens. The necrotrophic pathogen Verticillium dahliae infection of Gossypium barbadense could lead to Verticillium wilt which seriously reduces the cotton production. Here, we cloned and characterized a G. barbadense homolog of EDS1, designated as GbEDS1. The full-length cDNA of the GbEDS1 gene was obtained by the technique of rapid-amplification of cDNA ends. The open reading frame of the GbEDS1 gene was 1,647 bp long and encoded a protein of 548 amino acids residues. Comparison of the cDNA and genomic DNA sequence of GbEDS1 indicated that this gene contained a single intron and two exons. Like other EDS1s, GbEDS1 contained a conserved N-terminal lipase domain and an EDS1-specific KNEDT motif. Subcellular localization assay revealed that GbEDS1-green fluorescence protein fusion protein was localized in both cytosol and nucleus. Interestingly, the transcript levels of GbEDS1 were dramatically increased in response to pathogen V. dahliae infection. To investigate the role of GbEDS1 in plant resistance against V. dahliae, a conserved fragment derived from GbEDS1 was used to knockdown the endogenous EDS1 in Nicotiana benthamiana by heterologous virus-induced gene silencing. Our data showed that silencing of NbEDS1 resulted in increased susceptibility to V. dahliae infection in N. benthamiana, suggesting a possible involvement of the novelly isolated GbEDS1 in the regulation of plant defense against V. dahliae.

  12. Cloning and characterization of the rice low phytic acid 1 gene

    International Nuclear Information System (INIS)

    The rice low phytic acid 1 (lpa1) mutant exhibits a 45% reduction in seed phytic acid with a molar-equivalent increase in inorganic phosphorus; however, it does not appear to differ significantly in productivity from its wild-type progenitor. Using a positional cloning strategy, we have identified a single candidate gene at the rice Lpa1 locus. Sequence analysis of the candidate gene from the original rice lpa1 mutant and a second recently identified lpa1 mutant revealed two independent mutations (a single base pair substitution and a single base pair deletion) that confirmed the identification of this candidate as the rice low phytic acid 1 gene, OsLpa1. The OsLpa1 gene has three expressed splice variants. The location and nature of the two mutations suggests that these lesions should only affect the translation of the predicted protein derived from the longest transcript. The proteins encoded by OsLpa1 do not have homology to any of the inositol phosphate metabolism genes characterized in plants to date, although there is homology to 2-phosphoglycerate kinase, an enzyme found in hyperthermophilic methanogens that catalyzes the formation of 2,3-bisphosphoglycerate from 2-phosphoglycerate. It has previously been shown that 2,3-bisphosphoglycerate is a competitive inhibitor of inositol polyphosphate 5-phosphatases. These phosphatases are known to breakdown inositol polyphosphate intermediates, suggesting a possible indirect role for OsLpa1 in phytic acid biosynthesis and accumulation. Functional analysis of OsLpa1 is underway and our progress will be reported. (author)

  13. Thermostable lipases from extremely radioresistant bacterium Deinococcus radiodurans: cloning, expression, and biochemical characterization.

    Science.gov (United States)

    Shao, Hua; Xu, Li; Yan, Yunjun

    2014-09-01

    A search for lipases was conducted in the radiophile of Deinococcus radiodurans R1. Four putative lipase genes, encoding DR0334, DR2078, DR1485, and DR2522, were cloned and expressed in Escherichia coli. The recombinant enzymes were subsequently purified and characterized. The results showed DR0334 and DR2078 had the ability to hydrolyze long-chain length p-nitrophenyl esters (C12-C16), while DR1485 and DR2522 hydrolyzed short- and medium-chain length p-nitrophenyl esters (C2-C10). DR0334, DR1485, DR2078, and DR2522 showed optimum pH at 8.5, and optimum temperature at 40, 50, 60, and 60 °C, respectively. DR0334 almost lost its whole activity after 60 min pretreatment at 60 °C, while DR1485, DR2078, and DR2522 retained more than 70% of their original activities after 6 h incubation at 80 °C. The activities of DR2078, DR1485, and DR2522 were enhanced by Mg(2+) , Ba(2+) , and Mn(2+) , but strongly inhibited by EDTA. Nevertheless, DR2078, DR1485, and DR2522 showed moderate stability in organic solvents and detergents. Phylogenetic analysis revealed that DR0334 and DR2078, respectively belong to family IV and family IX, while each of DR1485 and DR2522 forms a new separate branch. The unique properties of DR2078, DR1485, and DR2522, thermostability and organic solvent tolerance, make them useful in industrial applications.

  14. Molecular cloning and characterization of taurocyamine kinase from Clonorchis sinensis: a candidate chemotherapeutic target.

    Directory of Open Access Journals (Sweden)

    Jing-Ying Xiao

    2013-11-01

    Full Text Available BACKGROUND: Adult Clonorchis sinensis lives in the bile duct and causes endemic clonorchiasis in East Asian countries. Phosphagen kinases (PK constitute a highly conserved family of enzymes, which play a role in ATP buffering in cells, and are potential targets for chemotherapeutic agents, since variants of PK are found only in invertebrate animals, including helminthic parasites. This work is conducted to characterize a PK from C. sinensis and to address further investigation for future drug development. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] A cDNA clone encoding a putative polypeptide of 717 amino acids was retrieved from a C. sinensis transcriptome. This polypeptide was homologous to taurocyamine kinase (TK of the invertebrate animals and consisted of two contiguous domains. C. sinensis TK (CsTK gene was reported and found consist of 13 exons intercalated with 12 introns. This suggested an evolutionary pathway originating from an arginine kinase gene group, and distinguished annelid TK from the general CK phylogenetic group. CsTK was found not to have a homologous counterpart in sequences analysis of its mammalian hosts from public databases. Individual domains of CsTK, as well as the whole two-domain enzyme, showed enzymatic activity and specificity toward taurocyamine substrate. Of the CsTK residues, R58, I60 and Y84 of domain 1, and H60, I63 and Y87 of domain 2 were found to participate in binding taurocyamine. CsTK expression was distributed in locomotive and reproductive organs of adult C. sinensis. Developmentally, CsTK was stably expressed in both the adult and metacercariae stages. Recombinant CsTK protein was found to have low sensitivity and specificity toward C. sinensis and platyhelminth-infected human sera on ELISA. CONCLUSION: CsTK is a promising anti-C. sinensis drug target since the enzyme is found only in the C. sinensis and has a substrate specificity for taurocyamine, which is different from its mammalian counterpart

  15. Molecular cloning, characterization and expression of cathepsin D from grass carp (Ctenopharyngodon idella).

    Science.gov (United States)

    Dong, Zhong-dian; Zhang, Jiao; Ji, Xiang-shan; Zhou, Fen-na; Fu, Yong; Chen, Weiyun; Zeng, Yong-qing; Li, Tong-ming; Wang, Hui

    2012-11-01

    Cathepsin D is a lysosomal aspartic proteinase which participates in various degradation functions within the cell. In this current study, we cloned and characterized the complete cDNA of grass carp cathepsin D through 5'- and 3'-RACE. The cathepsin D contained a 56 bp 5' terminal untranslated region (5'-UTR), a 1197 bp open reading frame encoding 398 amino acids, and a 394 bp 3'-UTR. Grass carp cathepsin D shared high similarity with those from other species, and showed the highest amino acid identity of 91% to Danio rerio. Unlike many other organisms, the grass carp cathepsin D contains only one N-glycosylation site closest to the N-terminal. Real-time quantitative RT-PCR demonstrated that Cathepsin D expressed in all twelve tissues (bladder, brain, liver, heart, gill, muscle, fin, eye, intestines, spleen, gonad and head kidney). The relative expression levels of Cathepsin D in gonad and liver were 26.58 and 24.95 times as much as those in fin, respectively. The expression level of Cathepsin D in muscle approximately 16-fold higher, in intestines and spleen were 12-fold higher. The cathepsin D expression showed an upward trend during embryonic development. After challenged with Aeromonas hydrophil, the expression of grass carp cathepsin D gene showed significant changes in the four test tissues (liver, head kidney, spleen and intestines). The fact that the bacterial infection can obviously improve the cathepsin D expression in immune-related organs, may suggest that cathepsin D plays an important role in the innate immune response of grass carp.

  16. Molecular cloning and characterization of four caspases members in Apostichopus japonicus.

    Science.gov (United States)

    Shao, Yina; Li, Chenghua; Zhang, Weiwei; Duan, Xuemei; Li, Ye; Jin, Chunhua; Xiong, Jinbo; Qiu, Qiongfen

    2016-08-01

    The caspase family representing aspartate-specific cysteine proteases have been demonstrated to possess key roles in apoptosis and immune response. We previously demonstrated that LPS challenged Apostichopus japonicus coelomocyte could significantly induced apoptosis in vitro. However, apoptosis related molecules were scarcely investigated in this economic species. In the present work, we cloned and characterized four members caspase family from A. japonicus (designated as Ajcaspase-2, Ajcaspase-3, Ajcaspase-6, and Ajcaspase-8, respectively) by RACE. Multiple sequence alignment and structural analysis revealed that all Ajcaspases contained the conservative CASC domain at C terminal, in which some unique features for each Ajcaspase made them different from each other. These specific domains together with phylogenetic analysis supported that all these four identified proteins belonged to novel members of apoptotic signaling pathway in sea cucumber. Tissue distribution analysis revealed that four Ajcaspase genes were constitutively expressed in all examined tissues. The expression of Ajcaspase-2 was tightly correlated with that of Ajcaspase-8 in each detected tissues. Ajcaspase-3 and Ajcaspase-6 transcripts were both highly expressed in immune tissue of coelomocytes. Furthermore, the Vibrio splendidus challenged sea cucumber coelomocytes could significantly up-regulate the mRNA expressions of four genes. The expression levels of Ajcaspase-2 and Ajcaspase-8 were relative earlier than those of Ajcaspase-6 and Ajcaspase-3, respectively, which could be inferred that Ajcapase-2 might directly modulate Ajcaspase-6, and Ajcaspase-8 initiate the expression of Ajcaspase-3. The induce expressions differed among each Ajcaspase depending upon their roles such as initiator or effector caspase. All our results demonstrated that four Ajcaspases present diversified functions in apoptotic cascade signaling pathway of sea cucumber under immune response. PMID:27245866

  17. Molecular cloning and characterization of multiple isoforms of the snowdrop (Galanthus nivalis L.) lectin.

    Science.gov (United States)

    Van Damme, E J; De Clercq, N; Claessens, F; Hemschoote, K; Peeters, B; Peumans, W J

    1991-12-01

    Screening of a copy-DNA (cDNA) library constructed from RNA isolated from young developing ovaries of snowdrop (Galanthus nivalis) resulted in the isolation of five lectin clones which clearly differed from each other with regard to their nucleotide sequence and deduced amino-acid sequence. Sequence comparison between the coding regions of different lectin cDNAs revealed the highest homology between lectin clones LECGNA 3 and LECGNA 5, showing 96.4% and 93.6% similarity at the nucleotide level and at the deduced amino-acid level, respectively, whereas lectin clones LECGNA 1 and LECGNA 3 showed the lowest homology of 81.6% and 68.6% for the nucleotide sequence and the amino-acid sequence, respectively. Only very few lectin cDNA clones containing a polyadenylated tail could be isolated. Moreover all these cDNA clones were derived from isolectin 3 and showed some variability within the length of the 3' untranslated region. The major transcription initiation site was located 30 bases upstream from the AUG codon as could be deduced from primer-extension analysis. Taking into account the small 5' untranslated region of the lectin clones, the size of the lectin mRNA, which is approx. 780 nucleotides as determined by Northern blot analysis, is in good agreement with the length of the cDNA clones isolated. Besides the ovary tissue, both the leaf and the flower tissue were also shown to express the lectin mRNA in a flowering snowdrop plant.

  18. Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli

    Directory of Open Access Journals (Sweden)

    Dorismey Vieira Tokano

    2008-06-01

    Full Text Available The aim of this work was to isolate, clone and characterize the iron uptake gene iutA from avian pathogenic E. coli (APEC. The iutA gene was isolated from the strain APEC 9, serotype O2:H9, which was cloned in the expression vector pET101/D-TOPO. The gene of 2.2 Kb was sequenced (AY602767, which showed high similarity to the iutA gene from three plasmids, two from APEC, pAPEC-02-ColV (AY545598.4 and pTJ100 (AY553855.1, and one from a human invasive E. coli strain, the pColV K30. The recombinant protein IutA was over expressed in E. coli BL21(DE-3 and was solubilized with urea and purified by Ni-NTA column. This method produced a relatively high yield of r-IutA of approximately 74kDa, which was used to produce the antibody anti-IutA. This anti-IutA reacted with the protein r-IutA and native IutA of APEC 9, as demonstrated by Western blot, showing that the r-IutA conserved epitopes and its antigenicity was preserved. The anti-IutA IgY was able to inhibit the IutA biological activity, inhibiting the sensitivity to cloacin DF13 of APEC9. However, it did not inhibit the growth of APEC9 in M9 and did not protect the chickens inoculated with the APEC, suggesting that the APEC possessed another iron acquisition mechanism distinct of aerobactin.A proteína de membrane externa IutA (iron uptake transport é o receptor para aerobactina férrica, um fator de virulência encontrado mais frequentemente entre as amostras de E. coli pathogênicas para aves (APEC do que entre os isolados fecais de aves saudáveis. O gene iutA da amostra APEC 9, sorotipo O2:H9, foi amplificado e clonado no vetor pET101/D-TOPO. O gene iutA 2.2 Kb foi sequenciado (AY602767 e mostrou alta similaridade para gene iutA de três plasmidios, dois da APEC, pAPEC-02-ColV (AY545598.4 e pTJ100 (AY553855.1, e um da amostra E. coli invasiva humana, pColV K30. A proteína IutA recombinante (r-IutA foi produzida em Escherichia coli BL21(DE-3, solubilizada com uréia e purificada em coluna de n

  19. Cloning, characterization, and tissue expression pattern of mouse Nma/BAMBI during odontogenesis.

    Science.gov (United States)

    Knight, C; Simmons, D; Gu, T T; Gluhak-Heinrich, J; Pavlin, D; Zeichner-David, M; MacDougall, M

    2001-10-01

    Degenerate oligonucleotides to consensus serine kinase functional domains previously identified a novel, partial rabbit tooth cDNA (Zeichner-David et al., 1992) that was used in this study to identify a full-length mouse clone. A 1390-base-pair cDNA clone was isolated encoding a putative 260-amino-acid open reading frame containing a hydrophobic 25-amino-acid potential transmembrane domain. This clone shares some homology with the TGF-beta type I receptor family, but lacks the intracellular kinase domain. DNA database analysis revealed that this clone has 86% identity to a newly isolated human gene termed non-metastatic gene A and 80% identity to a Xenopus cDNA clone termed BMP and activin membrane bound inhibitor. Here we report the mouse Nma/BAMBI cDNA sequence, the tissue expression pattern, and confirmed expression in dental cell lines. This study demonstrates that Nma/BAMBI is a highly conserved protein across species and is expressed at high levels during odontogenesis. PMID:11706948

  20. Caracterização de clones de mandioca utilizando marcadores microssatélites Characterization of cassava clones using microsatellite markers

    OpenAIRE

    Marcus Vanner Carvalho de Oliveira; Danielle Pereira Baliza; Genaina Aparecida de Souza; Samuel Pereira de Carvalho; Luiz Henrique Bambine Assis

    2012-01-01

    As características dos clones utilizados no cultivo da mandioca variam de acordo com a aptidão comercial da cultura, ou seja, para o processo industrial e consumo humano "in natura". O presente trabalho objetivou identificar clones novos de mandioca obtidos por policruzamento na Universidade Federal de Lavras (UFLA) em 1998 com relação a essas aptidões. Para tanto, procurou-se identificar clones com padrões moleculares semelhantes aos padrões apresentados pelos clones comerciais. A metodologi...

  1. Cloning and characterization of a human c-myc promoter-binding protein.

    OpenAIRE

    Ray, R; Miller, D M

    1991-01-01

    A human cDNA clone encoding a c-myc promoter-binding protein was detected by screening a HeLa cell lambda phage expression cDNA library. The library was screened by using an XhoI-NaeI human c-myc P2 promoter fragment as a probe. The recombinant phage encoded a fusion protein, myc-binding protein 1 (MBP-1), which had an apparent molecular size of 40 kDa. A corresponding protein with a molecular size of 35 kDa was present in a HeLa cell extract. Sequence analysis of the cloned gene reveals an o...

  2. The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-Xylosidase I.

    Science.gov (United States)

    Graciano, Luciana; Corrêa, Juliana Moço; Gandra, Rinaldo Ferreira; Seixas, Flavio Augusto Vicente; Kadowaki, Marina Kimiko; Sampaio, Silvio César; Silva, José Luis da Conceição; Osaku, Clarice Aoki; Simão, Rita de Cássia Garcia

    2012-09-01

    The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved β-Xylosidase and α-L-Arabinofuranosidase (β-Xyl I-α-L-Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant β-Xyl I-α-L-Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant β-Xyl I-α-L-Ara exhibited a specific β-Xylosidase I activity of 1.25 U mg(-1) to oNPX and a specific α-L-Arabinofuranosidase activity of 0.47 U mg(-1) to pNPA. The predominant activity of the recombinant enzyme was its β-Xylosidase I activity, and the enzymatic characterization was focused on it. The β-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45 °C, and a high degree of stability was verified over 240 min at this temperature. Moreover, β-Xylosidase activity was inhibited in the presence of the metals Zn(2+) and Cu(2+), and the enzyme exhibited K(M) and V(Max) values of 2.89 ± 0.13 mM and 1.4 ± 0.04 μM min(-1) to oNPX, respectively. The modeled structure of β-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form. PMID:22806729

  3. Cloning and characterization of a novel hepatitis B virus core binding protein C12

    Institute of Scientific and Technical Information of China (English)

    Yin-Ying Lu; Jun Cheng; Yong-Ping Yang; Yan Liu; Lin Wang; Ke Li; Ling-Xia Zhang

    2005-01-01

    AIM: To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS: HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade)containing X-α-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating.pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein.RESULTS: C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray,of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function

  4. Molecular cloning, characterization and expression analysis of melanotransferrin from the sea cucumber Apostichopus japonicus.

    Science.gov (United States)

    Qiu, Xuemei; Li, Dong; Cui, Jun; Liu, Yang; Wang, Xiuli

    2014-06-01

    Melanotransferrin (MTf), a member of the transferrin families, plays an important role in immune response. But the research about MTf in sea cucumber is limited till now. In this study, the Melanotransferrin (Aj-MTf) gene was firstly cloned and characterized from the sea cucumber Apostichoupus japonicus by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The full-length cDNA of Aj-MTf is 2,840 bp in length and contains a 2,184 bp open reading frame that encodes a polypeptide of 727 amino acids. An iron-responsive element-like structure is located at the 5'-UTR of Aj-MTf cDNA. Sequence analysis shows that the Aj-MTf contains two conserved domains, and the binding-iron (III) sites, including eight amino acid residues (D81,Y109,Y215,H283,D425,Y454,Y565 and H634) and three N-linked glycosylation sites (N121V122S123,N173A174S175 and N673S674T675). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses suggested that the Aj-MTf expressions in the coelomic fluid, body cavity wall and respiratory trees were significantly changed from 4 to 24 h post lipopolysaccharide (LPS) injection. The mRNA levels of Aj-MTf in coelomic fluid was significantly up-regulated at 12 and 24 h in treatment group, and Aj-MTf shared a similar expression pattern with C-type lectin in coelomic fluid, while both genes appears to gradually increase after 4 h of LPS injection. These results indicate that the Aj-MTf plays a pivotal role in immune responses to the LPS challenge in sea cucumber, and provide new information that it is complementary to the sea cucumber immune genes and initiate new researches concerning the genetic basis of the holothurian immune response. PMID:24535270

  5. The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-Xylosidase I.

    Science.gov (United States)

    Graciano, Luciana; Corrêa, Juliana Moço; Gandra, Rinaldo Ferreira; Seixas, Flavio Augusto Vicente; Kadowaki, Marina Kimiko; Sampaio, Silvio César; Silva, José Luis da Conceição; Osaku, Clarice Aoki; Simão, Rita de Cássia Garcia

    2012-09-01

    The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved β-Xylosidase and α-L-Arabinofuranosidase (β-Xyl I-α-L-Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant β-Xyl I-α-L-Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant β-Xyl I-α-L-Ara exhibited a specific β-Xylosidase I activity of 1.25 U mg(-1) to oNPX and a specific α-L-Arabinofuranosidase activity of 0.47 U mg(-1) to pNPA. The predominant activity of the recombinant enzyme was its β-Xylosidase I activity, and the enzymatic characterization was focused on it. The β-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45 °C, and a high degree of stability was verified over 240 min at this temperature. Moreover, β-Xylosidase activity was inhibited in the presence of the metals Zn(2+) and Cu(2+), and the enzyme exhibited K(M) and V(Max) values of 2.89 ± 0.13 mM and 1.4 ± 0.04 μM min(-1) to oNPX, respectively. The modeled structure of β-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form.

  6. Molecular Cloning and Characterization of an Allene Oxide Cyclase Gene Associated with Fiber Strength in Cotton

    Institute of Scientific and Technical Information of China (English)

    WANG Li-man; ZHU You-min; TONG Xiang-chao; HU Wen-jing; CAI Cai-ping; GUO Wang-zhen

    2014-01-01

    Allene oxide cyclase (AOC) is one of the most important enzymes in the biosynthetic pathway of the plant hormone jasmonic acid (JA). AOC catalyzes the conversion of allene oxide into 12-oxo-phytodienoic acid (OPDA), a precursor of JA. Using 28K cotton genome array hybridization, an expressed sequence tag (EST;GenBank accession no. ES792958) was investigated that exhibited signiifcant expression differences between lintless-fuzzless XinWX and linted-fuzzless XinFLM isogenic lines during ifber initiation stages. The EST was used to search the Gossypium EST database (http://www.ncbi.nlm.nih.gov/) for corresponding cDNA sequences encoding full-length open reading frames (ORFs). Identiifed ORFs were conifrmed using transcriptional and genomic data. As a result, a novel gene encoding AOC in cotton (Gossypium hirsutum AOC;GenBank accession no. KF383427) was cloned and characterized. The 741-bp GhAOC gene comprises three exons and two introns and encodes a polypeptide of 246 amino acids. Two homologous copies were identiifed in the tetraploid cotton species G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124, and one copy in the diploid cotton species G. herbaceum and G. raimondii. qRT-PCR showed that the GhAOC transcript was abundant in cotton ifber tissues from 8 to 23 days post anthesis (DPA), and the expression proifles were similar in the two cultivated tetraploid cotton species G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124, with a higher level of transcription in the former. One copy of GhAOC in tetraploid cotton was localized to chromosome 24 (Chr. D8) using the subgenome-speciifc single nucleotide polymorphism (SNP) marker analysis, which co-localized GhAOC to within 10 cM of a ifber strength quantitative trait locus (QTL) reported previously. GhAOC was highly correlated with ifber quality and strength (P=0.014) in an association analysis, suggesting a possible role in cotton ifber development, especially in secondary cell wall thickening.

  7. Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila

    Directory of Open Access Journals (Sweden)

    Anthi Karnaouri

    2013-02-01

    Full Text Available The β-glucosidase gene bgl3a from Myceliophthora thermophila, member of the fungal glycosyl hydrolase (GH family 3, was cloned and expressed in Pichia pastoris. The mature β-glucosidase gene, which results after the excision of one intron and the secreting signal peptide, was placed under the control of the strong alcohol oxidase promoter (AOX1 in the plasmid pPICZαC. The recombinant enzyme (90 kDa was purified and characterized in order to evaluate its biotechnological potential. Recombinant P. pastoris efficiently secreted β-glucosidase into the medium and produced high level of enzymatic activity (41 U/ml after 192 h of growth, under methanol induction. MtBgl3a was able to hydrolyze low molecular weight substrates and polysaccharides containing β-glucosidic residues. The Km was found to be 0.39 mM on p-β-NPG and 2.64 mM on cellobiose. Optimal pH and temperature for the p-β-NPG hydrolysis were 5.0 and 70 °C. The β-glucosidase exhibits a half life of 143 min at 60 °C. Kinetic parameters of inhibition were determined for D-glucose, D-xylose and D-gluconic acid, indicating tolerance of the enzyme for these sugars and oxidized products. The recombinant enzyme was stimulated by short chain alcohols and has been shown to efficiently synthesize methyl-D-glucoside in the presence of methanol due to its transglycosylation activity. The stability of MtBgl3a in ethanol was prominent, and it retained most of its original activity after we exposed it to 50% ethanol for 6 h. The high catalytic performance, good thermal stability and tolerance to elevated concentrations of ethanol, D-xylose and D-glucose qualify this enzyme for use in the hydrolysis of lignocellulosic biomass for biofuel production, as part of an efficient complete multi-enzyme cocktail.

  8. Cloning, expression, and characterization of recombinant nitric oxide synthase-like protein from Bacillus anthracis

    International Nuclear Information System (INIS)

    Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substrate and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with L-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of L-arginine, N ω-hydroxy-L-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS-like protein

  9. Identification, cloning and characterization of sis7 and sis10 sugar-insensitive mutants of Arabidopsis

    Directory of Open Access Journals (Sweden)

    Biddle Kelly D

    2008-10-01

    Full Text Available Abstract Background The levels of soluble sugars, such as glucose and sucrose, help regulate many plant metabolic, physiological and developmental processes. Genetic screens are helping identify some of the loci involved in plant sugar response and reveal extensive cross-talk between sugar and phytohormone response pathways. Results A forward genetic screen was performed to identify mutants with increased resistance to the inhibitory effects of high levels of exogenous sugars on early Arabidopsis seedling development. The positional cloning and characterization of two of these sugar insensitive (sis mutants, both of which are also involved in abscisic acid (ABA biosynthesis or response, are reported. Plants carrying mutations in SIS7/NCED3/STO1 or SIS10/ABI3 are resistant to the inhibitory effects of high levels of exogenous Glc and Suc. Quantitative RT-PCR analyses indicate transcriptional upregulation of ABA biosynthesis genes by high concentrations of Glc in wild-type germinating seeds. Gene expression profiling revealed that a significant number of genes that are expressed at lower levels in germinating sis7-1/nced3-4/sto1-4 seeds than in wild-type seeds are implicated in auxin biosynthesis or transport, suggesting cross-talk between ABA and auxin response pathways. The degree of sugar insensitivity of different sis10/abi3 mutant seedlings shows a strong positive correlation with their level of ABA insensitivity during seed germination. Conclusion Mutations in the SIS7/NCED3/STO1 gene, which is primarily required for ABA biosynthesis under drought conditions, confer a sugar-insensitive phenotype, indicating that a constitutive role in ABA biosynthesis is not necessary to confer sugar insensitivity. Findings presented here clearly demonstrate that mutations in ABI3 can confer a sugar-insensitive phenotype and help explain previous, mixed reports on this topic by showing that ABA and sugar insensitivity exhibit a strong positive correlation in

  10. Molecular cloning, tissue distribution, and pharmacological characterization of melanocortin-4 receptor in spotted scat, Scatophagus argus.

    Science.gov (United States)

    Li, Jian-Tao; Yang, Zhao; Chen, Hua-Pu; Zhu, Chun-Hua; Deng, Si-Ping; Li, Guang-Li; Tao, Ya-Xiong

    2016-05-01

    Melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure in mammals. The functions of the MC4R in fish have not been investigated extensively. We herein reported on the cloning, tissue distribution, and pharmacological characterization of spotted scat (Scatophagus argus) MC4R (SAMC4R). It consisted of a 984bp open reading frame predicted to encode a protein of 327 amino acids. Sequence analysis revealed that SAMC4R was highly homologous (>80%) at amino acid levels to several teleost MC4Rs. Phylogenetic analyses showed that SAMC4R was closely related to piscine MC4R. Using RT-PCR, we showed that in addition to brain, pituitary, and gonads, mc4r mRNA was also widely expressed in peripheral tissues of spotted scat in sexually divergent pattern. With human MC4R (hMC4R) as a control, several agonists including α-melanocyte stimulating hormone (α-MSH), [Nle(4), D-Phe(7)]-α-MSH (NDP-MSH), adrenocorticotropic hormone (ACTH) and THIQ (N-[(3R)-1,2,3,4-tetrahydroisoquinolinium3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine), were used to investigate the binding and signaling properties of SAMC4R. The results showed that SAMC4R bound NDP-MSH with the highest affinity followed by ACTH (1-24) and α-MSH. Similar ranking was also found for hMC4R, although SAMC4R had two to five-fold higher affinities for these ligands. THIQ did not displace NDP-MSH from SAMC4R, different from hMC4R. α-MSH, NDP-MSH, and ACTH (1-24) were identified as potent agonists to stimulate cAMP generation followed by THIQ in SAMC4R. The availability of SAMC4R and its pharmacological characteristics will facilitate the investigation of its function in regulating diverse physiological processes in spotted scat. PMID:27080551

  11. Cloning and Characterization of a Flavonoid 3′-Hydroxylase Gene from Tea Plant (Camellia sinensis

    Directory of Open Access Journals (Sweden)

    Tian-Shan Zhou

    2016-02-01

    Full Text Available Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21 is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3′H, designated as CsF3′H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309. Bioinformatic analysis revealed that CsF3′H was highly homologous with the characterized F3′Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3′H-specific conserved motifs were discovered in the protein sequence of CsF3′H. Enzymatic analysis of the heterologously expressed CsF3′H in yeast demonstrated that tea F3′H catalyzed the 3′-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 μM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min−1, respectively. Transcription level of CsF3′H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3′,4′-flavan-3-ols, 3′,4′,5′-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3′H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3′H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3′H in the biosynthesis of 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols in tea leaves.

  12. Human granulocyte colony stimulating factor (hG-CSF: cloning, overexpression, purification and characterization

    Directory of Open Access Journals (Sweden)

    Vanz Ana LS

    2008-04-01

    Full Text Available Abstract Background Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Results Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3 host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4% to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. Conclusion The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost

  13. Transfer of the cloned Salmonella SPI-1 type III secretion system and characterization of its expression mechanisms in Gram negative bacteria in comparison with cloned SPI-2.

    Science.gov (United States)

    Cangelosi, Chris; Hannagan, Susan; Santiago, Clayton P; Wilson, James W

    2015-11-01

    Cloned type III secretion systems have much potential to be used for bacterial engineering purposes involving protein secretion and substrate translocation directly into eukaryotic cells. We have previously cloned the SPI-1 and SPI-2 type III systems from the Salmonella enterica serovar Typhimurium genome using plasmid R995 which can conveniently capture large genomic segments for transfer between bacterial strains. However, though expressed and functional in Salmonella strains, cloned SPI-1 was previously observed to have a serious expression defect in other Gram negative bacteria including Escherichia coli. Here we show that cloned SPI-1 expression and secretion can be detected in the secretion preps from E. coli and Citrobacter indicating the first observation of non-Salmonella SPI-1 expression. We describe a compatible plasmid system to introduce engineered SPI-1 substrates into cloned SPI-1 strains. However, a SPI-1 translocation defect is still observed in E. coli, and we show that this is likely due to a defect in SipB expression/secretion in this species. In addition, we also examined the requirement for the hilA and ssrAB regulators in the expression of cloned SPI-1 and SPI-2, respectively. We found a strict requirement for hilA for full cloned SPI-1 expression and secretion. However, though we found that ssrAB is required for full cloned SPI-2 expression in a range of media across different bacteria, it is not required for cloned SPI-2 expression in MgM8 inducing media in S. Typhimurium. This suggests that under SPI-2 inducing conditions in S. Typhimurium, other factors can substitute for loss of ssrAB in cloned SPI-2 expression. The results provide key foundational information for the future use of these cloned systems in bacteria.

  14. Resources

    Science.gov (United States)

    ... palate - resources Colon cancer - resources Cystic fibrosis - resources Depression - resources Diabetes - resources Digestive disease - resources Drug abuse - resources Eating disorders - resources Elder care - resources Epilepsy - resources Family troubles - ...

  15. Measles virus-specific murine T cell clones: characterization of fine specificity function.

    NARCIS (Netherlands)

    P. de Vries (Petra); J.P.M. Versteeg-van Oosten (José); I.K.G. Visser (Ilona); R.S. van Binnendijk (Rob); S.A. Langeveld (Sacha); A.D.M.E. Osterhaus (Ab); F.G.C.M. Uytdehaag (Fons)

    1989-01-01

    textabstractMeasles virus (MV)-specific murine helper T cell clones (Thy-1.2+, CD4+, CD8-) were generated from mice immunized with MV-infected mouse brain homogenate by limiting dilution and in vitro stimulation of spleen cells with UV-inactivated MV Ag. The protein specificity of 7 out of 37 stable

  16. Isolation, cloning, and characterization of the 2S albumin: A new allergen from hazelnut

    NARCIS (Netherlands)

    C. Garino; L. Zuidmeer; J. Marsh; A. Lovegrove; M. Morati; S. Versteeg; P. Schilte; P. Shewry; M. Arlorio; R. van Ree

    2010-01-01

    Scope: 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. Methods: 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP-

  17. Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01

    NARCIS (Netherlands)

    Nieuwenhuizen, W.F.; Leeuwen, S. van; Jack, R.W.; Egmond, M.R.; Götz, F.

    2003-01-01

    Ceramidase (CDase) hydrolyzes the amide bond in ceramides to yield free fatty acid and sphingosine. From a 3-L Pseudomonas aeruginosa PA01 culture, 70 μg of extracellular alkaline, Ca2+-dependent CDase, was purified to homogeneity, the N-terminal sequence was determined, and the CDase gene was clone

  18. Cloning, expression and characterization of a lipase encoding gene from human oral metagenome.

    Science.gov (United States)

    Preeti, Arivaradarajan; Hemalatha, Devaraj; Rajendhran, Jeyaprakash; Mullany, Peter; Gunasekaran, Paramasamy

    2014-09-01

    The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6-7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes. PMID:24891735

  19. Cloning and characterization of a thermostable xylitol dehydrogenase from Rhizobium etli CFN42

    DEFF Research Database (Denmark)

    Tiwari, Manish Kumar; Moon, Hee-Jung; Jeya, Marimuthu;

    2010-01-01

    An NAD(+)-dependent xylitol dehydrogenase from Rhizobium etli CFN42 (ReXDH) was cloned and overexpressed in Escherichia coli. The DNA sequence analysis revealed an open reading frame of 1,044 bp, capable of encoding a polypeptide of 347 amino acid residues with a calculated molecular mass of 35...

  20. Thorough Characterization of Brazilian New Generation of Eucalypt Clones and Grass for Pulp Production

    Directory of Open Access Journals (Sweden)

    Fernando José Borges Gomes

    2015-01-01

    Full Text Available Eucalypt wood is becoming the most important raw material for the pulp industries in South America. However, due to the high wood cost in comparison to other raw material sources, nonwoody materials are also being investigated aiming at pulp production. In this way, this paper aimed at the evaluation of eighteen eucalypt clones obtained from the Brazilian Genolyptus project, regarding their potential characteristics for pulp production. Aiming at the same goal, two species of elephant grass were also evaluated as alternative raw material sources. Through the analyses of the anatomic and chemical characteristics, five eucalypt clones and one elephant grass species were indicated for pulp production and biorefinery application. The results of this study indicate the high technological quality of Eucalyptus clones evaluated and indicate that they can be used for biorefinery applications since they have the suitable characteristics. In general, the eucalypt clones are less moist and denser and contain fewer minerals and extraneous materials than the elephant grass species, which make them more attractive for utilization in deconstruction studies aiming at production of bioproducts.

  1. CLONING AND CHARACTERIZATION OF A NUCLEAR, SITE-SPECIFIC SSDNA BINDING-PROTEIN

    NARCIS (Netherlands)

    SMIDT, MP; RUSSCHEN, B; SNIPPE, L; WIJNHOLDS, J; AB, G

    1995-01-01

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of

  2. 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...

  3. Characterization of nonprimate hepacivirus and construction of a functional molecular clone

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Kapoor, Amit; Nishiuchi, Eiko;

    2015-01-01

    consensus cDNA clone, replication was not observed in primary equine fetal liver cultures or after electroporation of selectable replicons. However, intrahepatic RNA inoculation of a horse initiated infection, yielding high RNA titers in the serum and liver. Delayed seroconversion, slightly elevated...

  4. 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

    -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...

  5. Cloning,Characterization,and Gene Annotation of Cellulose Synthase Genes from Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    BALASUBRAMANI G; AMUDHA J; KATEGERI I S; KHADI B M

    2008-01-01

    @@ The mechanistic basis of cellulose biosynthesis in plants has gained ground during last decade or so.The isolation of plant eDNA clones encoding cotton homologs of the bacterial cellulose synthase catalytic subunit was a significant achievement,which promises the elucidation of cellulose biosynthesis.

  6. Cloning, sequence analysis, and characterization of the genes involved in isoprimeverose metabolism in Lactobacillus pentosus

    NARCIS (Netherlands)

    Chaillou, S.; Lokman, B.C.; Leer, R.J.; Posthuma, C.; Postma, P.W.; Pouwels, P.H.

    1998-01-01

    Two genes, xylP and xylQ, from the xylose regulon of Lactobacillus pentosus were cloned and sequenced. Together with the repressor gene of the regulon, xylR, the xylPQ genes form an operon which is inducible by xylose and which is transcribed from a promoter located 145 bp upstream of xylP. A putati

  7. Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos

    Directory of Open Access Journals (Sweden)

    Wang Ya-Ping

    2008-03-01

    Full Text Available Abstract Background Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development. Results A novel gene, K31, was identified as an up-regulated gene in fish cross-subfamily cloned embryos using SSH approach and RACE method. K31 complete cDNA sequence is 1106 base pairs (bp in length, with a 342 bp open reading frame (ORF encoding a putative protein of 113 amino acids (aa. Comparative analysis revealed no homologous known gene in zebrafish and other species database. K31 protein contains a putative transmembrane helix and five putative phosphorylation sites but without a signal peptide. Expression pattern analysis by real time RT-PCR and whole-mount in situ hybridization (WISH shows that it has the characteristics of constitutively expressed gene. Sub-cellular localization assay shows that K31 protein can not penetrate the nuclei. Interestingly, over-expression of K31 gene can cause lethality in the epithelioma papulosum cyprinid (EPC cells in cell culture, which gave hint to the inefficient reprogramming events occurred in cloned embryos. Conclusion Taken together, our findings indicated that K31 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos and over-expression of K31 gene can cause lethality of cultured fish cells. To our knowledge, this is the first report on the determination of novel genes involved in nucleo-cytoplasmic interaction of fish cross-subfamily cloned embryos.

  8. Cloning, Sequencing and Characterization of 3-Hydroxybuty- rate Dehydrogenase Encoding Gene (bdh A) in Bradyrhizobium japonicum USDA110 Strain

    Institute of Scientific and Technical Information of China (English)

    DAI Mei-xue; WU Bo; BAI Xue-liang; ZHANG Cheng-gang; MA Qing-sheng; Charles Trevor C

    2002-01-01

    The current study describes the molecular characterization of a clone which can restore the ability of bdhA mutant strains NGRPA2 and Rm11107 to utilize 3-hydroxybutyrate as a sole carbon source (Hbu+). This clone was screened out by complementation experiment from Bradyrhizobium japonicum USDA110 genomic library, and the presence of bdhA gene in the clone was verified by Bdh assay and Southern blot analysis. Furthermore, the entire sequence of bdhA gene was sequenced and the sequence was deposited in GenBank database under the accession number AY077581. bdhA gene comprises 789 base pairs and encodes Bdh with 262 amino acid of MW 27.59 kDa. Interposon ΩKm was inserted into the bdh A ORF at EcoR I site and the bdhA mutant was constructed in B .japonicum by homologous recombination. Plant assay result did not show obvious effects of mutation of bdhA gene on nodulation and nitrogen-fixation.

  9. Preliminary functional characterization, cloning and primary sequence of Fastuosain, a cysteine peptidase isolated from fruits of Bromelia fastuosa.

    Science.gov (United States)

    Cabral, Hamilton; Leopoldino, Andréia M; Tajara, Eloiza H; Greene, Lewis J; Faça, Vitor M; Mateus, Rogério P; Ceron, Carlos R; de Souza Judice, Wagner A; Julianod, Luiz; Bonilla-Rodriguez, Gustavo O

    2006-01-01

    The present work reports the characterization of Fastuosain, a novel cysteine protease of 25kDa, purified from the unripe fruits of Bromelia fastuosa, a wild South American Bromeliaceae. Proteolytic activity, measured using casein and synthetic substrates, was dependent on the presence of thiol reagents, having maximum activity at pH 7.0. The present work reports cDNA cloning of Fastuosain; cDNA was amplified by PCR using specific primers. The product was 1096pb long. Mature fastuosain has 217 residues, and with the proregion has a total length of 324 residues. Its primary sequence showed high homology with ananain(74%), stem bromelain (66%) and papain (44%).

  10. Cloning and characterization of high mobility group box protein 1 (HMGB1) of Wuchereria bancrofti and Brugia malayi

    OpenAIRE

    Thirugnanam, Sivasakthivel; Munirathinam, Gnanasekar; Veerapathran, Anandharaman; Dakshinamoorthy, Gajalakshmi; Reddy, Maryada V.; RAMASWAMY, KALYANASUNDARAM

    2012-01-01

    A human homologue of high mobility group box 1 (HMGB1) protein was cloned and characterized from the human filarial parasites Wuchereria bancrofti and Brugia malayi. Sequence analysis showed that W. bancrofti HMGB1 (WbHMGB1) and B. malayi HMGB1 (BmHMGB1) proteins share 99 % sequence identity. Filarial HMGB1 showed typical architectural sequence characteristics of HMGB family of proteins and consisted of only a single HMG box domain that had significant sequence similarity to the pro-inflammat...

  11. Comment on: Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in the gastrointestinal system

    Institute of Scientific and Technical Information of China (English)

    Ali Mobasheri

    2006-01-01

    @@ TO THE EDITOR Sir, I read with great interest the recently published article in the World Journal of Gastroenterology by Jin and co-workers[1] on the cloning and characterization of porcine aquaporin 1 water channel from the pig liver and studies on its expression in the porcine gastrointestinal system. The authors should be congratulated for making this important and valuable contribution to the field of aquaporin biology and porcine gastrointestinal physiology.However, there are a number of unresolved issues and controversies concerning the expression of aquaporins (especially aquaporin 1) in the gastrointestinal system that are worthy of additional comment and discussion by Jin and co-workers.

  12. Cloning and characterization of the rec2 gene of Ustilago maydis

    International Nuclear Information System (INIS)

    The authors are exploring the molecular basis for genetic recombination using the corn smut fungus Ustilago maydis, from which the first two eucaryotic DNA repair and recombination mutants, rec1 and rec2, were described. Cells mutant at the rec2 locus are unable to repair lethal damage to their DNA from UV and X irradiation or from chemical alkylating agents such as N-methyl-nitrosoguanidine. Rec2 mutants retain only a residual level of DNA-damage inducible mitotic recombination, and are unable to complete meiosis. Using an autonomously replicating plasmid vector for Ustilago, they established the first nonintegrating plasmid library of the Ustilago genome. The rec2 locus was cloned by complementation of the rec2 mutation in vivo. One clone was found to restore all of the deficient activities. Although this rec2 complementing clone is present on a multicopy plasmid, the authors observed that it fully restored but did not further increase the fifty-fold inducibility of heteroallelic recombination at the nitrate reductase and inositol loci of rec2 or wild type cells. Northern blot analysis using the rec2 complementing clone revealed three UV inducible transcripts, one of which is absent in a rec2 mutant strain. This transcript organization resembles that of the yeast rad10 and the human ERCC-1 genes (MCB 9:1794), but sequence obtained to date from rec2 does not show homology with these genes. They have also observed that the rec2 mutation may alter the level of homologous integration of transformed DNA markers. Integration of a Leu1 complementing plasmid by Scott Fotheringham of the lab has shown that while much of plasmid integration in wild type Ustilago is nonhomologous, integration in at least some rec2 strains is entirely homologous. They are using the cloned rec2 gene to confirm that rec2 is indeed involved in altering the level of homologous integration in Ustilago, and if so, they plan to clone a mammalian analogue of rec2

  13. Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    1985-01-01

    The gene, prs, encoding phosphoribosylpyrophosphate (PRPP) synthetase of Escherichia coli was isolated from a library of E. coli genes cloned in the bacteriophage λD69 vector. A strain with a temperature-lethal defect in PRPP synthetase, (prs-2), was used as the host and cloning was performed...... by lysogenic complementation. The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI. The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment. Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent...... deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene. Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity. The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells...

  14. Cloning, Expression, and Characterization of Thermotolerant Manganese Superoxide Dismutase from Bacillus sp. MHS47

    OpenAIRE

    Supatra Areekit; Paisarn Khawsak; Arda Pakpitchareon; Kajeenart Potivejkul; Gaysorn Chansiri; Kosum Chansiri; Pornpimon Kanjanavas

    2011-01-01

    A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83...

  15. Cloning, overexpression, and characterization of a bacterial Ca2+-dependent phospholipase D

    OpenAIRE

    Yang, Hongying; Roberts, Mary F.

    2002-01-01

    Phospholipase D (PLD), an important enzyme involved in signal transduction in mammals, is also secreted by many microorganisms. A highly conserved HKD motif has been identified in most PLD homologs in the PLD superfamily. However, the Ca2+-dependent PLD from Streptomyces chromofuscus exhibits little homology to other PLDs. We have cloned (using DNA isolated from the ATCC type strain), overexpressed in Escherichia coli (two expression systems, pET-23a(+) and pTYB11), and purified the S. chromo...

  16. Molecular Cloning and Characterization of G Alpha Proteins from the Western Tarnished Plant Bug, Lygus hesperus

    OpenAIRE

    J Joe Hull; Meixian Wang

    2014-01-01

    The Gα subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-based cloning methods and transcriptome data mining to identity Gα subunits in the western tarnished plant bug (Lygus hesperus Knight). Among the nine sequences identified were single variants of the Gαi, Gαo...

  17. Cloning and characterization of the beta-amylase gene from Bacillus polymyxa.

    OpenAIRE

    Friedberg, F; Rhodes, C.

    1986-01-01

    The gene for beta-amylase was isolated from Bacillus polymyxa by molecular cloning in B. subtilis. B. subtilis cells containing this gene express and secrete an amylase which resembles the B. polymyxa beta-amylase and barley beta-amylase in terms of the products it generates during carbohydrate hydrolysis. Starch hydrolysis with this beta-amylase produces maltose, not glucose, whereas maltotriose and cycloheptaose are resistant to the action of this beta-amylase. The enzyme has a molecular we...

  18. The ABCG2 efflux transporter from rabbit placenta: Cloning and functional characterization.

    Science.gov (United States)

    Halwachs, Sandra; Kneuer, Carsten; Gohlsch, Katrin; Müller, Marian; Ritz, Vera; Honscha, Walther

    2016-02-01

    In human placenta, the ATP-binding cassette efflux transporter ABCG2 is highly expressed in syncytiotrophoblast cells and mediates cellular excretion of various drugs and toxins. Hence, physiological ABCG2 activity substantially contributes to the fetoprotective placenta barrier function during gestation. Developmental toxicity studies are often performed in rabbit. However, despite its toxicological relevance, there is no data so far on functional ABCG2 expression in this species. Therefore, we cloned ABCG2 from placenta tissues of chinchilla rabbit. Sequencing showed 84-86% amino acid sequence identity to the orthologues from man, rat and mouse. We transduced the rabbit ABCG2 clone (rbABCG2) in MDCKII cells and stable rbABCG2 gene and protein expression was shown by RT-PCR and Western blot analysis. The rbABCG2 efflux activity was demonstrated with the Hoechst H33342 assay using the specific ABCG2 inhibitor Ko143. We further tested the effect of established human ABCG2 (hABCG2) drug substrates including the antibiotic danofloxacin or the histamine H2-receptor antagonist cimetidine on H33342 accumulation in MDCKII-rbABCG2 or -hABCG2 cells. Human therapeutic plasma concentrations of all tested drugs caused a comparable competitive inhibition of H33342 excretion in both ABCG2 clones. Altogether, we first showed functional expression of the ABCG2 efflux transporter in rabbit placenta. Moreover, our data suggest a similar drug substrate spectrum of the rabbit and the human ABCG2 efflux transporter. PMID:26907376

  19. Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

    Institute of Scientific and Technical Information of China (English)

    JIAN HONG YAO; XIU YUN ZHAO; ZHI HUA LIAO; JUAN LIN; ZHONG HAI CHEN; FEI CHEN; JUN SONG; XIAO FEN SUN; KE XUAN TANG

    2003-01-01

    The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.

  20. Molecular Cloning and Characterization of Enolase from Oilseed Rape (Brassica napus)

    Institute of Scientific and Technical Information of China (English)

    ZHAOJing-Ya; ZUOKai-Jing; QINJie; TANGKe-Xuan

    2004-01-01

    An enolase-encoding cDNA clone in oilseed rape (Brassica napus L.) was isolated. This gene (accession number: AY307449) had a total length of 1 624 bp with an open reading frame of 1 335 bp, and encoded a predicted polypeptide of 444 amino acids with a molecular weight of 47.38 kD. The deduced amino acid sequence shared identity with a number of enolases ranging from Bacillus subtilis to human beings and had much higher identity with other plant enolases than with enolases from Bacillus, yeast and human beings. Comparison of its primary structure with those of other enolases revealed the presence of an insertion of five amino acids in enolase of B. napus. Southern blotting analysis of genomic DNA indicated that enolase was likely to be a low-copy gene in the oilseed rape genome. Expression of the cloned enolase gene increased under salt stress, but decreased in response to low temperature. Our studies suggested that the cloned gene was a new member of plant enolase gene family, which contributed to the energy supply in stress-treated tissues.

  1. Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L.

    Institute of Scientific and Technical Information of China (English)

    WANG Sheng; ZHAO Guo-hong; JIA Yin-hua; DU Xiong-ming

    2009-01-01

    The aim of this study was to clone CAP (adenylyl cyclase-associated protein) gene from Gossypium arboreum L. and develop a platform for expressing and purifying CAP protein, which is a base for the construction and function researches of CAP. In this work, a CAP homolog from cotton (DPL971) ovule was identified and cloned. And the cDNA sequence consisted of an open reading frame of 1416 nucleotides encoding a protein of 471 amino acid residues with a calculated molecular weight of 50.6 kDa. To gain insight on the CAP role in cotton fiber development, the cloned CAP cDNA was expressed. A significant higher yield pure protein was obtained with the chromatographic method. Further experiments showed that the purified protein can bind with the actin in vitro indicating that the recombinant cotton CAP is functional. The procedure described here produced high yield pure protein through one chromatographic step, suitable for further structure-function studies.

  2. CLONING AND CHARACTERIZATION OF HUMAN UBIQUITIN BINDING ENZYME 2 cDNA

    Institute of Scientific and Technical Information of China (English)

    李光涛; 吕鸿雁; 周严; 金鉴; 蒋科艺; 彭小忠; 袁建刚; 强伯勤

    2002-01-01

    Objective.To clone and identify the gene encoding human ubiquitin binding enzyme 2 and study its expression pattern. Methods.According to the sequence of human EST,which is highly homologous to the mouse ubiquitin binding/conjugating enzyme (E2),primers were synthesized to screen the human fetal brain cDNA library.The gene was analyzed by bioinformatics technique and its expression pattern was studied by using multiple tissue Northern blot. Results.Two cDNA clones encoding human ubiquitin conjugating enzyme have been isolated and identified.Both containing the ubiquitin conjugating domain,the 2 cDNA clones are 88% identical in amino acid sequences and splicing isoforms to each other only with an exon excised to form the short sequence.They belong to a highly conserved and widely expressed E2 enzyme family.Northern blot shows that they are expressed exclusively in adult human heart,placenta,and pancreas but no transcripts can be detected in brain,lung,liver,skeletal muscle or kidney.Conclusions.The gene encoding human ubiquitin binding enzyme is expressed under temporal control.As a key enzyme in the degradation of proteins,ubiquitin conjugating enzymes play a central role in the expression regulation on the level of post translation.

  3. Establishment and characterization of a chimeric infectious cDNA clone of classical swine fever virus.

    Science.gov (United States)

    Zhao, T S; Xia, Y H

    2016-06-01

    Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. There are two important CSFV strains in China, Shimen and hog cholera lapinized virus (HCLV). Shimen strain is highly virulent while HCLV, also referred to as C-strain, is a live attenuated vaccine strain considered to be one of the most effective and safest live vaccines. In this study, a chimeric infectious cDNA clone of CSFV named pT7SM-c was engineered by replacing the E(rns) genomic region of an infectious clone of CSFV Shimen strain, pT7SM, with the same region obtained from HCLV. RNA transcripts of pT7SM-c containing an engineered EcoRI site that served as a genetic marker were directly infectious in PK15 cells. The rescued virus vT7SM-c showed similar growth kinetics and cytopathic effect with the parental virus vT7SM in the cells. The chimeric infectious cDNA clone can be used as a practical tool for further studying of the virulence, protein function and pathogenesis of CSFV through genetic manipulation. PMID:27265471

  4. Characterization of wind power resource and its intermittency

    Science.gov (United States)

    Gunturu, U. B.; Schlosser, C. A.

    2011-12-01

    Wind resource in the continental and offshore United States has been calculated and characterized using metrics that describe - apart from abundance - its availability, persistence and intermittency. The Modern Era Retrospective-Analysis for Research and Applications (MERRA) boundary layer flux data has been used to construct wind power density profiles at 50, 80, 100 and 120 m turbine hub heights. The wind power density estimates at 50 m are qualitatively similar to those in the US wind atlas developed by the National Renewable Energy Laboratory (NREL), but quantitatively a class less in some regions, but are within the limits of uncertainty. We also show that for long tailed distributions like those of the wind power density, the mean is an overestimation and median is a more robust metric for summary representation of wind power resource.Generally speaking, the largest and most available wind power density resources are found in off-shore regions of the Atlantic and Pacific coastline, and the largest on-shore resource potential lies in the central United States. However, the intermittency and widespread synchronicity of on-shore wind power density are substantial, and highlights areas where considerable back-up generation technologies will be required. Generation-duration curves are also presented for the independent systems operator (ISO) zones of the U.S. to highlight the regions with the largest capacity factor (MISO, ERCOT, and SWPP) as well as the periods and extent to which all ISOs contain no wind power and the potential benefits of aggregation on wind power intermittency in each region. The impact of raising the wind turbine hub height on metrics of abundance, persistence, variability and intermittency is analyzed. There is a general increase in availability and abundance of wind resource but there is also an increase in intermittency with respect to a 'usable wind power' crossing level in low resource regions. A similar perspective of wind resource for

  5. Cloning and functional characterization of Δ6 fatty acid desaturase (FADS2) in Eurasian perch (Perca fluviatilis).

    Science.gov (United States)

    Geay, F; Tinti, E; Mellery, J; Michaux, C; Larondelle, Y; Perpète, E; Kestemont, P

    2016-01-01

    The Eurasian perch (Perca fluviatilis) is a freshwater carnivorous species of high interest to diversify inland aquaculture. However, little is known about its ability to bioconvert polyunsaturated fatty acids (PUFAs) from plant oils into long chain polyunsaturated fatty acids (LC-PUFAs). In this study, special attention has been given to the fatty acid desaturase 2 (FADS2) which is commonly described to be a rate-limiting enzyme of the LC-PUFA biosynthesis. This work reports on the cloning, tissue expression and functional characterization of the Eurasian perch fads2, but also on the cloning of two alternative splicing transcripts named fads2-AS1 and fads2-AS2. The fads2 cDNA cloned is composed of an open reading frame (ORF) of 1338 nucleotides (nt) and encodes a protein of 445 amino acids. This deduced amino acid sequence displays the typical structure of microsomal FADS2 including two transmembrane domains and an N-terminal cytochrome b5 domain with the "HPGG" motif. Quantitative real-time PCR assay of fads2, fads2-AS1 and fads2-AS2 expressions revealed that the fads2 transcript was mainly expressed in the liver and intestine and exhibited a typical gene expression pattern of freshwater species while fads2-AS1 and fads2-AS2 genes were highly expressed in the brain, followed by the liver and intestine. Functional characterization of Eurasian perch FADS2 in transgenic yeast showed a fully functional Δ6 desaturation activity toward C18 PUFA substrates, without residual Δ5 and Δ8 desaturase activities.

  6. Cloning, Structural Characterization, and Phylogenetic Analysis of Flower MADS-Box Genes from Crocus (Crocus sativus L.

    Directory of Open Access Journals (Sweden)

    Athanasios S. Tsaftaris

    2007-01-01

    Full Text Available Crocus (Crocus sativus L. is a crop species cultivated for its flowers and, more specifically, for its red stigmas. The flower of crocus is bisexual and sterile, since crocus is a triploid species. Its perianth consists of six petaloid tepals: three tepals in whorl 1 (outer tepals and three tepals in whorl 2 (inner tepals. The androecium consists of three distinct stamens and the gynoecium consists of a single compound pistil with three carpels, a single three-branched style, and an inferior ovary. The dry form of the stigmas constitutes the commercial saffron used as a food additive, in the coloring industry, and in medicine. In order to uncover and understand the molecular mechanisms controlling flower development in cultivated crocus and its relative wild progenitor species, and characterize a number of crocus flower mutants, we have cloned and characterized different, full-length, cDNA sequences encoding MADS-box transcription factor proteins involved in flower formation.

  7. Functional Identification and Characterization of Genes Cloned from Halophyte Seashore Paspalum Conferring Salinity and Cadmium Tolerance

    Directory of Open Access Journals (Sweden)

    Yu eChen

    2016-02-01

    Full Text Available Salinity-affected and heavy metal-contaminated soils limit the growth of glycophytic plants. Identifying genes responsible for superior tolerance to salinity and heavy metals in halophytes has great potential for use in developing salinity- and Cd-tolerant glycophytes. The objective of this study was to identify salinity- and Cd-tolerance related genes in seashore paspalum (Paspalum vaginatum, a halophytic perennial grass species, using the yeast cDNA expression library screening method. Based on the Gateway-compatible vector system, a high quality entry library was constructed, which contained 9.9×106 clones with an average inserted fragments length of 1.48 kb representing a 100% full-length rate. The yeast expression libraries were screened in a salinity-sensitive and a Cd-sensitive yeast mutant. The screening yielded 32 salinity-tolerant clones harboring 18 salinity-tolerance genes and 20 Cd-tolerant clones, including 5 Cd-tolerance genes. qPCR analysis confirmed that most of the 18 salinity-tolerance and 5 Cd-tolerance genes were up-regulated at the transcript level in response to salinity or Cd stress in seashore paspalum. Functional analysis indicated that salinity-tolerance genes from seashore paspalum could be mainly involved in photosynthetic metabolism, antioxidant systems, protein modification, iron transport, vesicle traffic, and phospholipid biosynthesis. Cd-tolerance genes from seashore paspalum could be associated with regulating pathways involved in phytochelatin synthesis, HSFA4-relsted stress protection, CYP450 complex and sugar metabolism. The 18 salinity-tolerance genes and 5 Cd-tolerance genes could be potentially used as candidate genes for genetic modification of glycophytic grass species to improve salinity and Cd tolerance and for further analysis of molecular mechanisms regulating salinity and Cd tolerance.

  8. Functional Identification and Characterization of Genes Cloned from Halophyte Seashore Paspalum Conferring Salinity and Cadmium Tolerance

    Science.gov (United States)

    Chen, Yu; Chen, Chuanming; Tan, Zhiqun; Liu, Jun; Zhuang, Lili; Yang, Zhimin; Huang, Bingru

    2016-01-01

    Salinity-affected and heavy metal-contaminated soils limit the growth of glycophytic plants. Identifying genes responsible for superior tolerance to salinity and heavy metals in halophytes has great potential for use in developing salinity- and Cd-tolerant glycophytes. The objective of this study was to identify salinity- and Cd-tolerance related genes in seashore paspalum (Paspalum vaginatum), a halophytic perennial grass species, using yeast cDNA expression library screening method. Based on the Gateway-compatible vector system, a high-quality entry library was constructed, which contained 9.9 × 106 clones with an average inserted fragment length of 1.48 kb representing a 100% full-length rate. The yeast expression libraries were screened in a salinity-sensitive and a Cd-sensitive yeast mutant. The screening yielded 32 salinity-tolerant clones harboring 18 salinity-tolerance genes and 20 Cd-tolerant clones, including five Cd-tolerance genes. qPCR analysis confirmed that most of the 18 salinity-tolerance and five Cd-tolerance genes were up-regulated at the transcript level in response to salinity or Cd stress in seashore paspalum. Functional analysis indicated that salinity-tolerance genes from seashore paspalum could be involved mainly in photosynthetic metabolism, antioxidant systems, protein modification, iron transport, vesicle traffic, and phospholipid biosynthesis. Cd-tolerance genes could be associated with regulating pathways that are involved in phytochelatin synthesis, HSFA4-related stress protection, CYP450 complex, and sugar metabolism. The 18 salinity-tolerance genes and five Cd-tolerance genes could be potentially used as candidate genes for genetic modification of glycophytic grass species to improve salinity and Cd tolerance and for further analysis of molecular mechanisms regulating salinity and Cd tolerance. PMID:26904068

  9. Characterization of a novel MICA allele, MICA*012:05, by cloning and sequencing.

    Science.gov (United States)

    Wang, W Y; Tian, W; Wang, F; Zhu, F M; Li, L X

    2016-08-01

    A new MICA allelic variant, MICA*012:05, has been identified in a Chinese Mongolian population. Following polymerase chain reaction-sequence-based typing (PCR-SBT), this new allele was further confirmed by cloning and sequencing. MICA*012:05 was linked to an HLA-A*24-C*01-B*55:02-DRB1*09 haplotype. MICA*012:05 differs from MICA*012:01 by a single synonymous C to T substitution at nucleotide position 269 in exon 3.

  10. Cloning and characterization of a tryptophanase gene from Enterobacter aerogenes SM-18

    OpenAIRE

    Kawasaki, K.; Yokota, A; Oita, S; Kobayashi, C.; Yoshikawa, S; KAWAMOTO, S.; Takao, S.; Tomita, F.

    1993-01-01

    A tryptophanase gene from Enterobacter aerogenes SM-18 was cloned and sequenced. The structural gene for tryptophanase, tnaA, consisted of 1389 bp encoding 462 amino acid residues, and its nucleotide sequence and deduced amino acid sequence showed significant homology to those of tnaA from Escherichia coli K12. A short open reading frame consisting of 31 amino acid residues was found upstream of tnaA, and it showed some similarity to the E. coli tnaC gene known to be a cis-acting regulatory e...

  11. Cloning, characterization, and regulation of nifF from Rhodobacter capsulatus.

    OpenAIRE

    G. de Gennaro; Hübner, P.; Sandmeier, U; Yakunin, A. F.; Hallenbeck, P C

    1996-01-01

    The Rhodobacter capsulatus nifF gene and upstream sequence were cloned by using a probe based on the N-terminal sequence of NifF. nifF was found to not be contained in the previously described nif regions I, II, and III. Comparison of the deduced amino acid sequence showed that it is highly similar to NifF from Azotobacter vinelandii and NifF from Klebsiella pneumoniae. Analysis of translational fusions demonstrated that the regulation of transcription was the same as previously reported at t...

  12. Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins

    OpenAIRE

    Jönsson, Klas; Guo, Betty P.; Monstein, Hans-Jürg; Mekalanos, John J.; Kronvall, Göran

    2004-01-01

    Helicobacter pylori binds a number of host cell proteins, including the plasma protein plasminogen, which is the proenzyme of the serine protease plasmin. Two H. pylori plasminogen-binding proteins have been described; however, no genes were identified. Here we report the use of a phage display library to clone two genes from the H. pylori CCUG 17874 genome that mediate binding to plasminogen. DNA sequence analysis of one of these genes revealed 96.6% homology with H. pylori 26695 HP0508. A s...

  13. Mutant radiation-resistance alleles from the Escherichia coli Gamr444 mutant: Cloning and preliminary characterization

    International Nuclear Information System (INIS)

    Mutant alleles Gamr, which are able to increase the resistance to radiation of Escherichia coli wild-type cells, were cloned from the hyperradioresistant mutant Gamr444 on plasmid mini-Mu-vector MudII4042. The influence of recombinant plasmids on the sensitivity of wild-type and mutant (recA and htpR) cells to γ-irradiation was studied. It was shown that the enhanced resistance of the Gamr444 strain to radiation was caused by mutations of two different classes, dominant and recessive. The cloned recessive mutation gamr12 increases resistance to radiation only after homogenotization, that is, radiation-induced transfer from the plasmid to the chromosome, and it imposes constitutive expression of the heat-shock promoter htpG. Dominant mutant gamr alleles are active in the trans-position. A mutation-insertion into a chromosomal gene impaired by one of the dominant mutations, gamr18, was constructed. The insertion causes drastic cell radiosensitization on the recBC sbcB background and probably disturbs the RecF pathway of recombination and repair. Dominant plasmids of the second type lead to the RecA-independent inhibition of DNA postirradiation degradation. The radioprotective action of recessive and dominant gamr mutations is additive

  14. Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase from Geobacillus sp. CHB1

    Science.gov (United States)

    2016-01-01

    Catalases are widely used in many scientific areas. A catalase gene (Kat) from Geobacillus sp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed in Escherichia coli (E. coli), which was the first time to clone and express this type of catalase of genus Geobacillus strains as far as we know. This Kat gene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studied Bacillus sp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble in E. coli and made up 30% of the total E. coli protein. Fermentation broth of the recombinant E. coli showed a high catalase activity level up to 35,831 U/mL which was only lower than recombinant Bacillus sp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg and Km of 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications. PMID:27579320

  15. Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase from Geobacillus sp. CHB1

    Directory of Open Access Journals (Sweden)

    Xianbo Jia

    2016-01-01

    Full Text Available Catalases are widely used in many scientific areas. A catalase gene (Kat from Geobacillus sp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed in Escherichia coli (E. coli, which was the first time to clone and express this type of catalase of genus Geobacillus strains as far as we know. This Kat gene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studied Bacillus sp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble in E. coli and made up 30% of the total E. coli protein. Fermentation broth of the recombinant E. coli showed a high catalase activity level up to 35,831 U/mL which was only lower than recombinant Bacillus sp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg and Km of 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications.

  16. Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

    Indian Academy of Sciences (India)

    Hari S. Misra; Nivedita P. Khairnar; Manjula Mathur; N. Vijayalakshmi; Remesh S. Hire; T. K. Dongre; S. K. Mahajan

    2002-04-01

    A sporulating culture of Bacillus thuringiensis subsp. kenyae strain HD549 is toxic to larvae of lepidopteran insect species such as Spodoptera litura, Helicoverpa armigera and Phthorimaea operculella, and a dipteran insect, Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed in E. coli. The recombinant protein produced 92% mortality in first-instar larvae of Spodoptera litura and 86% inhibition of adult emergence in Phthorimaea operculella, but showed very low toxicity against Helicoverpa armigera, and lower mortality against third-instar larvae of dipteran insects Culex fatigans, Anopheles stephensi and Aedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene from Bacillus thuringiensis var. kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.

  17. Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae.

    Science.gov (United States)

    Misra, Hari S; Khairnar, Nivedita P; Mathur, Manjula; Vijayalakshmi, N; Hire, Ramesh S; Dongre, T K; Mahajan, S K

    2002-04-01

    A sporulating culture of Bacillus thuringiensis subsp. kenyae strain HD549 is toxic to larvae of lepidopteran insect species such as Spodoptera litura, Helicoverpa armigera and Phthorimaea operculella, and a dipteran insect, Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed in E. coli. The recombinant protein produced 92% mortality in first-instar larvae of Spodoptera litura and 86% inhibition of adult emergence in Phthorimaea operculella, but showed very low toxicity against Helicoverpa armigera, and lower mortality against third-instar larvae of dipteran insects Culex fatigans, Anopheles stephensi and Aedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene from Bacillus thuringiensis var. kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block. PMID:12357073

  18. Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

    Science.gov (United States)

    Coskun, Cagil; Purali, Nuhan

    2016-06-01

    Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue. PMID:27032955

  19. Homologous cloning, characterization and expression of a new halophyte phytochelatin synthase gene in Suaeda salsa

    Science.gov (United States)

    Cong, Ming; Zhao, Jianmin; Lü, Jiasen; Ren, Zhiming; Wu, Huifeng

    2016-09-01

    The halophyte Suaeda salsa can grow in heavy metal-polluted areas along intertidal zones having high salinity. Since phytochelatins can eff ectively chelate heavy metals, it was hypothesized that S. salsa possessed a phytochelatin synthase (PCS) gene. In the present study, the cDNA of PCS was obtained from S. salsa (designated as SsPCS) using homologous cloning and the rapid amplification of cDNA ends (RACE). A sequence analysis revealed that SsPCS consisted of 1 916 bp nucleotides, encoding a polypeptide of 492 amino acids with one phytochelatin domain and one phytochelatin C domain. A similarity analysis suggested that SsPCS shared up to a 58.6% identity with other PCS proteins and clustered with PCS proteins from eudicots. There was a new kind of metal ion sensor motif in its C-terminal domain. The SsPCS transcript was more highly expressed in elongated and fibered roots and stems ( P<0.05) than in leaves. Lead and mercury exposure significantly enhanced the mRNA expression of SsPCS ( P<0.05). To the best of our knowledge, SsPCS is the second PCS gene cloned from a halophyte, and it might contain a diff erent metal sensing capability than the first PCS from Thellungiella halophila. This study provided a new view of halophyte PCS genes in heavy metal tolerance.

  20. Identification, characterization and cloning of Entamoeba histolytica adhesins: Their potential use in diagnosis

    International Nuclear Information System (INIS)

    Entamoeba histolytica adhesins were detected by the generation of monoclonal antibodies (MAbs) inhibitors of adhesion. In other experiments trophozoites were radiolabeled with 125I or with (35S)-methionine and incubated with red blood cells (RBS's) or epithelial MDCK cells. Labeled amebic proteins of 210, 160, 112, 90, 70, 50 and 24 kDa were detected adhered on RBCs. Iodinated MDCK proteins formed ligand-receptor complexes with amebic proteins of 112 kDa, 90 and 48 to 50 kDa, when incubated with amebic proteins separated by gel electrophoresis. When RBC's interacted with adherence-deficient mutants, used in this work as highly negative genetic controls, proteins of 112 and 90 kDa appeared diminished on RBS surface, in comparison with wild type strain. The 112 kDa adhesin was not detected in a monoxenic nonpathogenic E. histolytica strain, isolated from an asymptomatic carrier. An E. histolytica DNA library was constructed in lambda gt-11, and recombinant clones were selected using polyclonal antibodies against the 112 kDa adhesin. In spite of the fact that DNA of the parasite was cloned, production of the hybrid protein in Escherichia coli was relatively efficient. Correlation of the presence of adhesins with the virulence of E. histolytica trophozoites encourage us to propose these proteins and their antibodies as good candidates in the design of reliable and inexpensive diagnosis and vaccine methods. (author). 23 refs, 7 figs, 2 tabs

  1. Cloning and characterization of novel isoforms of the human kallikrein 6 gene

    International Nuclear Information System (INIS)

    Human kallikrein 6 (protease M/zyme/neurosin) was originally identified based on its aberrant expression in tumor cells and is considered a biomarker for ovarian cancer. Here, we describe the identification, cloning, and tissue expression of three novel transcript variants of the KLK6 gene that encode for wild-type kallikrein 6. Contrary to the classical form, transcript variants contain one untranslated exon, exploit intronic sequences, and are likely products of alternative promoters. In addition, we cloned splice variants 2 and 3 produced by splicing out exons 3 and 4, respectively. Given the potential diagnostic applications of kallikrein 6 at both the mRNA and protein levels, we developed a duplex RT-PCR, in order to differentially detect and quantitate mRNA species corresponding to splice variants. We show that in normal mammary epithelial cells and mammary tumor cell lines that overexpress the KLK6 gene, splice variants account for approximately 10-20% of all mRNA species

  2. Cloning and characterization of a tuberous root-specific promoter from cassava (Manihot esculenta Crantz).

    Science.gov (United States)

    Koehorst-van Putten, Herma J J; Wolters, Anne-Marie A; Pereira-Bertram, Isolde M; van den Berg, Hans H J; van der Krol, Alexander R; Visser, Richard G F

    2012-12-01

    In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava.

  3. Characterization and cDNA cloning of aminopeptidase A from the venom of Gloydius blomhoffi brevicaudus.

    Science.gov (United States)

    Ogawa, Yuko; Murayama, Nobuhiro; Fujita, Yoshiaki; Yanoshita, Ryohei

    2007-06-15

    The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)(18)-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile. PMID:17383704

  4. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

    Energy Technology Data Exchange (ETDEWEB)

    Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

    1985-02-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, /sup 32/P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

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

    Science.gov (United States)

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

    2014-02-01

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

  6. Physical Characterization of human centromeric regions using transformation-associated recombination cloning technology

    Energy Technology Data Exchange (ETDEWEB)

    Vladimir Larionov, Ph D

    2007-06-05

    A special interest in the organization of human centromeric DNA was stimulated a few years ago when two independent groups succeeded in reconstituting a functional human centromere, using constructs carrying centromere-specific alphoid DNA arrays. This work demonstrated the importance of DNA components in mammalian centromeres and opened a way for studying the structural requirements for de novo kinetochore formation and for construction of human artificial chromosomes (HACs) with therapeutic potential. To elucidate the structural requirements for formation of HACs with a functional kinetochore, we developed a new method for cloning of large DNA fragments for human centromeric regions that can be used as a substrate for HAC formation. This method exploits in vivo recombination in yeast (TAR cloning). In addition, a new strategy for the construction of alphoid DNA arrays was developed in our lab. The strategy involves the construction of uniform or hybrid synthetic alphoid DNA arrays by the RCA-TAR technique. This technique comprises two steps: rolling circle amplification of an alphoid DNA dimer and subsequent assembling of the amplified fragments by in vivo homologous recombination in yeast (Figure 1). Using this system, we constructed a set of different synthetic alphoid DNA arrays with a predetermined sequence varying in size from 30 to 140 kb and demonstrated that some of the arrays are competent in HAC formation. Because any nucleotide can be changed in a dimer before its amplification, this new technique is optimal for identifying the structural requirements for de novo kinetochore formation in HACs. Moreover, the technique makes possible to introduce into alphoid DNA arrays recognition sites for DNA-binding proteins. We have made the following progress on the studying of human centromeric regions using transformation-associated recombination cloning technology: i) minimal size of alphoid DNA array required for de novo kinetochore formation was estimated; ii

  7. Molecular cloning and characterization of the light-harvesting chlorophyll a/b gene from the pigeon pea (Cajanus cajan).

    Science.gov (United States)

    Qiao, Guang; Wen, Xiao-Peng; Zhang, Ting

    2015-12-01

    Light-harvesting chlorophyll a/b-binding proteins (LHCB) have been implicated in the stress response. In this study, a gene encoding LHCB in the pigeon pea was cloned and characterized. Based on the sequence of a previously obtained 327 bp Est, a full-length 793 bp cDNA was cloned using the rapid amplification of cDNA ends (RACE) method. It was designated CcLHCB1 and encoded a 262 amino acid protein. The calculated molecular weight of the CcLHCB1 protein was 27.89 kDa, and the theoretical isoelectric point was 5.29. Homology search and sequence multi-alignment demonstrated that the CcLHCB1 protein sequence shared a high identity with LHCB from other plants. Bioinformatics analysis revealed that CcLHCB1 was a hydrophobic protein with three transmembrane domains. By fluorescent quantitative real-time polymerase chain reaction (PCR), CcLHCB1 mRNA transcripts were detectable in different tissues (leaf, stem, and root), with the highest level found in the leaf. The expression of CcLHCB1 mRNA in the leaves was up-regulated by drought stimulation and AM inoculation. Our results provide the basis for a better understanding of the molecular organization of LCHB and might be useful for understanding the interaction between plants and microbes in the future. PMID:26329890

  8. Cloning, Characterization and Heterologous Expression of the Indolocarbazole Biosynthetic Gene Cluster from Marine-Derived Streptomyces sanyensis FMA

    Directory of Open Access Journals (Sweden)

    Wenli Li

    2013-02-01

    Full Text Available The indolocarbazole (ICZ alkaloids have attracted much attention due to their unique structures and potential therapeutic applications. A series of ICZs were recently isolated and identified from a marine-derived actinomycete strain, Streptomyces sanyensis FMA. To elucidate the biosynthetic machinery associated with ICZs production in S. sanyensis FMA, PCR using degenerate primers was carried out to clone the FAD-dependent monooxygenase gene fragment for ICZ ring formation, which was used as a probe to isolate the 34.6-kb DNA region containing the spc gene cluster. Sequence analysis revealed genes for ICZ ring formation (spcO, D, P, C, sugar unit formation (spcA, B, E, K, J, I, glycosylation (spcN, G, methylation (spcMA, MB, as well as regulation (spcR. Their involvement in ICZ biosynthesis was confirmed by gene inactivation and heterologous expression in Streptomyces coelicolor M1152. This work represents the first cloning and characterization of an ICZ gene cluster isolated from a marine-derived actinomycete strain and would be helpful for thoroughly understanding the biosynthetic mechanism of ICZ glycosides.

  9. Cloning and structural and expressional characterization of BcpLH gene preferentially expressed in folding leaf of Chinese cabbage

    Institute of Scientific and Technical Information of China (English)

    余旭红; 彭洁松; 冯献忠; 杨素欣; 郑志仁; 唐向荣; 沈瑞娟; 刘平林; 何玉科

    2000-01-01

    Vegetative growth of Chinese cabbage undergoes the four successive stages which are characterized with the definite types of juvenile, rosette, folding and head leaves. From shoot tips of Chinese cabbage at early folding stage, we constructed a cDNA library and screened the differentially expressed cDNA clones using the cDNAs derived from developing folding leaves and rosette leaves as probes. One complete length of cDNA clone is designated as BcpLH. Computer alignment matched BcpLH to the domains of double-stranded RNA binding (DBRM) and the homologous regions were recognized between BcpLH and human and mouse double-stranded RNA-binding protein TRBP. PCR expression analysis shows that during vegetative growth BcpLH gene was expressed preferentially in folding leaves at folding stage. Transcripts of BcpLH gene were increased when plants were sprayed with IAA. It is deduced that BcpLH gene may be related to initiation of folding leaf and leafy head and induced by auxin in the aspect of transcriptional ex

  10. Molecular cloning and characterization of the light-harvesting chlorophyll a/b gene from the pigeon pea (Cajanus cajan).

    Science.gov (United States)

    Qiao, Guang; Wen, Xiao-Peng; Zhang, Ting

    2015-12-01

    Light-harvesting chlorophyll a/b-binding proteins (LHCB) have been implicated in the stress response. In this study, a gene encoding LHCB in the pigeon pea was cloned and characterized. Based on the sequence of a previously obtained 327 bp Est, a full-length 793 bp cDNA was cloned using the rapid amplification of cDNA ends (RACE) method. It was designated CcLHCB1 and encoded a 262 amino acid protein. The calculated molecular weight of the CcLHCB1 protein was 27.89 kDa, and the theoretical isoelectric point was 5.29. Homology search and sequence multi-alignment demonstrated that the CcLHCB1 protein sequence shared a high identity with LHCB from other plants. Bioinformatics analysis revealed that CcLHCB1 was a hydrophobic protein with three transmembrane domains. By fluorescent quantitative real-time polymerase chain reaction (PCR), CcLHCB1 mRNA transcripts were detectable in different tissues (leaf, stem, and root), with the highest level found in the leaf. The expression of CcLHCB1 mRNA in the leaves was up-regulated by drought stimulation and AM inoculation. Our results provide the basis for a better understanding of the molecular organization of LCHB and might be useful for understanding the interaction between plants and microbes in the future.

  11. Cloning and structural and expressional characterization of BcpLH gene preferentially expressed in folding leaf of Chinese cabbage

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Vegetative growth of Chinese cabbage undergoes the four successive stages which are characterized with the definite types of juvenile,rosette,folding and head leaves.From shoot tips of Chinese cabbage at early folding stage,we constructed a cDNA library and screened the differentially expressed cDNA clones using the cDNAs derived from developing folding leaves and rosette leaves as probes.One complete length of cDNA clone is designated as BcpLH.Computer alignment matched BcpLH to the domains of double-stranded RNA binding (DBRM) and the homologous regions were recognized between BcpLH and human and mouse double-stranded RNA-binding protein TRBP.PCR expression analysis shows that during vegetative growth BcpLH gene was expressed preferentially in folding leaves at folding stage.Transcripts of BcpLH gene were increased when plants were sprayed with IAA.It is deduced that BcpLH gene may be related to initiation of folding leaf and leafy head and induced by auxin in the aspect of transcriptional expression.

  12. Establishment and characterization of Epstein-Barr virus gp350-expressing transfected human lymphoid (Raji) cell clones.

    Science.gov (United States)

    Khyatti, M; Patel, P C; Menezes, J

    1991-06-19

    Gp350, a late Epstein-Barr-virus (EBV) glycoprotein expressed on both the envelope of viral particles and EBV-producing cells, is also the candidate for the development of an anti-EBV subunit vaccine. This glycoprotein is thought to play an important role in anti-EBV immunity. However, studies on the role of this viral antigen in cellular cytotoxicity and other immune functions have been hampered by the lack of a suitable model expressing gp350. We describe here a study in which we successfully transfected a gp350-negative cell line resistant to natural-killer(NK)-cell activity (i.e., Raji) with a recombinant plasmid (pZIP-MA) containing the EBV-gp350 and the neomycin resistance gene. Three clones with a stable and strong expression of gp350 on their surface membrane, as demonstrated using a gp350-specific (i.e., 2LI0) monoclonal antibody (MAb) were isolated, characterized and used as targets in an antibody-dependent cellular cytotoxicity (ADCC) assay. However, gp350 expression on 2 of the 3 isolated clones was not recognized by an anti-gp350 MAb (72AI) which is specific to a unique gp350 epitope with a dual function (i.e., involved in both EBV binding to its target cell receptors and in inducing virus-neutralizing antibody). We have also found that gp350 expression on our 3 selected clones does not affect EBV-receptor (CR2) density. Our model of gp350-expressing, NK-cell-activity-resistant targets revealed very useful in determining that gp350 serves as a target antigen for EBV-specific ADCC. These gp350-expressing cell clones appear to represent a valuable tool for diagnostic purposes (i.e., for detecting and titrating gp350 antibodies in patients with EBV-associated diseases). Our approach should also prove useful for studying the expression of other cell-surface-associated viral and tumor antigens and their role in specific cellular immunity and immunosurveillance. PMID:1646179

  13. Cloning and characterization of a new actin gene from Oryza sativa L.

    Institute of Scientific and Technical Information of China (English)

    LIANG Weihong; TANG Chaorong; WU Naihu

    2004-01-01

    Using Rho family member osRACD as bait, a new member of actin gene family -Act was isolated from Oryza sativa by yeast two-hybrid system. The full-length cDNA was cloned with 5' RACE technology, which contains an open reading frame of 1134 bp with a predicted protein of 377 amino acids. Sequence alignment revealed 96% to 81.8% identities with some known actin proteins in plants. The method of bioinformatics was used to analyze the protein modification sites, structure and evolution of the gene. Southern blot analysis showed that Act is a single-copy gene in the genome. The result of RT-PCR showed it is ubiquitously expressed in root, shoot, callus and panicle in a temporal fashion. The relationship between Rho family and actin family in evolution and function was also studied.

  14. Cloning and Characterization of an Abalone (Haliotis discus hannai) Actin Gene

    Institute of Scientific and Technical Information of China (English)

    MA Hongming; XU Wei; MAI Kangsen; LIUFU Zhiguo; CHEN Hong

    2004-01-01

    An actin encoding gene was cloned by using RT-PCR, 3' RACE and 5' RACE from abalone Haliotis discus hannai. The full length of the gene is 1532 base pairs, which contains a long 3' untranslated region of 307 base pairs and 79 base pairs of 5' untranslated sequence. The open reading frame encodes 376 amino acid residues. Sequence comparison with those of human and other mollusks showed high conservation among species at amino acid level. The identities was 96%, 97% and 96% respectively compared with Aplysia californica, Biomphalaria glabrata and Homo sapience β-actin. It is also indicated that this actin is more similar to the human cytoplasmic actin(β-actin)than to human muscle actin.

  15. Cloning, expression and mo-lecular characterization of promoter elements from Ba-cillus pumilus

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Promoter elements from random chromosomal DNA of a rice epiphytic Bacillus pumilus were cloned into promoter probe shuttle vector ECE7 and sequenced. The results showed that these elements were all new DNA sequences. Six strong promoter elements were obtained by determination of CAT enzyme activity in both E. coli and B. pumilus. Transcription start sites of the cat mRNA were located by primer extension using total RNA. Comparison of the promoter sequences indicated that three of them contain -10 and -35 regions like B. pumilus s43 consensus sequence and another one is similar to B. pumilus s29. The other two have no typical consensus sequences of known sigma factors so far.

  16. Molecular Cloning and Characterization of cDNA Encoding Fibrinolytic Enzyme-3 from Earthworm Eisenia foetida

    Institute of Scientific and Technical Information of China (English)

    Guo-Qing DONG; Xiao-Ling YUAN; Ya-Jun SHAN; Zhen-Hu ZHAO; Jia-Pei CHEN; Yu-Wen CONG

    2004-01-01

    The earthworm fibrinolytic enzyme-3 (EFE-3, GenBank accession No: AY438622), from the earthworm Eiseniafoetida, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the EFE-3 was cloned by RT-PCR. The eDNA contained an open reading frame of 741 nucleotides, which encoded a deduced protein of 247 amino acid residues, including signal sequences. EFE-3 showed a high degree of homology to earthworm (Lumbricus rebullus) proteases F-III-1, F-III-2, and bovine trypsin. The recombinant EFE-3 was expressed in E. coli as inclusion bodies, and the gene encoding the native form of EFE-3 was expressed in COS-7 cells in the medium. Both the refolding product of inclusion bodies and the secreted protease could dissolve the artificial fibrin plate.

  17. Cloning, Characterization and Anti-Inflammatory Properties of Bothrops jararaca Snake Antithrombin.

    Science.gov (United States)

    Morais-Zani, Karen de; Grego, Kathleen F; Torquato, Ricardo J S; Silva, Caroline S; Tanaka, Aparecida S; Tanaka-Azevedo, Anita M

    2015-01-01

    Antithrombin inhibits blood coagulation through the interaction with serine proteases in both intrinsic and extrinsic pathways. In addition, antithrombin also shows anti-inflammatory properties, which are independent of its effects on coagulation. This work shows for the first time the cloning and sequencing of antithrombin from a snake species. This predicted protein is composed by 430 amino acids and presents about 64.5% sequence identity to human antithrombin. Biacore experiments revealed that the binding affinity of Bothrops jararaca snake antithrombin to heparin was ~30 times higher than that of human antithrombin. Furthermore, Bothrops jararaca antithrombin is more effective in preventing acute inflammation induced by carrageenan when compared to human antithrombin. Hence, the results showed herein suggest that Bothrops jararaca antithrombin can play a key role in the control of acute inflammation and that this molecule might be used as a pharmacological tool and as a prototype for drug development. PMID:25687119

  18. Cloning, characterization, and regulation of nifF from Rhodobacter capsulatus.

    Science.gov (United States)

    Gennaro, G; Hübner, P; Sandmeier, U; Yakunin, A F; Hallenbeck, P C

    1996-07-01

    The Rhodobacter capsulatus nifF gene and upstream sequence were cloned by using a probe based on the N-terminal sequence of NifF. nifF was found to not be contained in the previously described nif regions I, II, and III. Comparison of the deduced amino acid sequence showed that it is highly similar to NifF from Azotobacter vinelandii and NifF from Klebsiella pneumoniae. Analysis of translational fusions demonstrated that the regulation of transcription was the same as previously reported at the protein level. Insertional mutagen esis showed that NifF contributes significantly to nitrogenase activity under normal nitrogen-fixing conditions and that it is absolutely required for nitrogen fixation under iron limitation. PMID:8682802

  19. Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from bufo melanostictus.

    Science.gov (United States)

    Liu, Wa; Ji, Senlin; Zhang, A-Mei; Han, Qinqin; Feng, Yue; Song, Yuzhu

    2013-01-01

    Cystatins are efficient inhibitors of papain-like cysteine proteinases, and they serve various important physiological functions. In this study, a novel cystatin, Cystatin-X, was cloned from a cDNA library of the skin of Bufo melanostictus. The single nonglycosylated polypeptide chain of Cystatin-X consisted of 102 amino acid residues, including seven cysteines. Evolutionary analysis indicated that Cystatin-X can be grouped with family 1 cystatins. It contains cystatin-conserved motifs known to interact with the active site of cysteine proteinases. Recombinant Cystatin-X expressed and purified from Escherichia coli exhibited obvious inhibitory activity against cathepsin B. rCystatin-X at a concentration of 8 µM inhibited nearly 80% of cathepsin B activity within 15 s, and about 90% of cathepsin B activity within 15 min. The Cystatin-X identified in this study can play an important role in host immunity and in the medical effect of B. melanostictus.

  20. Gene cloning, expression, purification and characterization of lipoprotein- associated phospholipase A2 in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Fu-junZHANG; Yi-pingWANG

    2005-01-01

    AIM To express and purify Lipoprotein -associated phospholipase A2 (Lp-PLA2), and to establish a screening model for Lp-PLA2 inhibitors through the recombinant Lp-PLA2. METHODS The full-length gene of Lp-PLA2 was cloned from the differentiated THP-1 cells by RT-PCR and PCR. The Lp-PLA2 gene was subcloned into the Pichia expression vector pPIC9 and introduced a sequence encoding a C-terminal stretch of six histidine residues at the same time. The recombinant plasmid was transformed into Pichia pastoris GS115 by spheroplasting and the gene was then integrated into the GS115 genome. Lp- PLA2 was expressed in the yeast strain GS115 by inducing with 0.5% methanol.

  1. Cloning and characterization of a glucose 6-phosphate/phosphate translocator from Oryza sativa

    Institute of Scientific and Technical Information of China (English)

    姜华武; 佃蔚敏; 刘非燕; 吴平

    2003-01-01

    Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy, and glucose 6-phosphate is the preferred hexose phosphate taken up by nongreen plastids. A cDNA clone encoding glucose 6-phosphate/phosphate translocator (GPT) was isolated from a cDNA library of immature seeds of rice and named as OsGPT. The cDNA has one uninterrupted open reading frame encoding a 42 kDa polypeptide possessing transit poptide consisting of 70 amino acid residues. The OsGPT gene maps on chromosome 8 of rice and is linked to the quantitative trait locus for 1000-grain weight. The expression of OsGPT is mainly re-stricted to heteretrephic tissues. These results suggest that glucose 6-phosphate imported via GPT can be used for starch biosynthesis in rice nongreen plastids

  2. Cloning and characterization of a glucose 6-phosphate/phosphate translocator from Oryza sativa

    Institute of Scientific and Technical Information of China (English)

    姜华武; 佃蔚敏; 刘非燕; 吴平

    2003-01-01

    Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy, and glucose 6-phosphate is the preferred hexose phosphate taken up by nongreen plastids. A cDNA clone encoding glucose 6-phosphate/phosphate translocator (GPT) was isolated from a cDNA library of immature seeds of rice and named as OsGPT. The cDNA has one uninterrupted open reading frame encoding a 42 kDa polypeptide possessing transit peptide consisting of 70 amino acid residues. The OsGPT gene maps on chromosome 8 of rice and is linked to the quantitative trait locus for 1000-grain weight. The expression of OsGPT is mainly restricted to heterotrophic tissues. These results suggest that glucose 6-phosphate imported via GPT can be used for starch biosynthesis in rice nongreen plastids.

  3. Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone

    Directory of Open Access Journals (Sweden)

    Karlsson Johnny

    2005-08-01

    Full Text Available Abstract Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal.

  4. Wood Quality and Growth Characterization across Intra- and Inter-Specific Hybrid Aspen Clones

    Directory of Open Access Journals (Sweden)

    Shawn D. Mansfield

    2013-09-01

    Full Text Available Trembling aspen (Populus tremuloides Michx. is one of the most abundant poplar species in North America; it is native, displays substantial breadth in distribution inhabiting several geographical and climatic ecoregions, is notable for its rapid growth, and is ecologically and economically important. As the demand for raw material continues to increase rapidly, there is a pressing need to improve both tree quality and growth rates via breeding efforts. Hybridization is considered one of the most promising options to simultaneously accelerate these tree characteristics, as it takes advantage of heterosis. Two aspen species showing particular promise for hybridization with trembling aspen are European aspen (P. tremula and Chinese aspen (P. davidiana because their native climates are similar to that of P. tremuloides and are also very easy to hybridize. In 2003, aspen clones were planted in Athabasca, Alberta from the following species crosses: open pollinated (OP P. tremuloides (NN, OP P. davidiana (CC, P. tremula × P. tremula (EE, P. tremula × P. tremuloides (EN, and P. tremuloides × P. davidiana (CN. In November 2010, growth measurements and core samples were taken from seven-year field grown clones. Comparisons of the mean growth and cell wall traits were made between crosses using generalized linear model least squares means tests for stem volume, fiber length, fiber width, coarseness, wood density, microfibril angle, total cell wall carbohydrate and lignin content, and lignin composition. The results clearly indicated that the inter-specific crosses offer a means to breed for more desirable wood characteristics than the intra-specific Populus spp. crosses.

  5. Molecular cloning and pharmacological characterization of giant panda (Ailuropoda melanoleuca) melanocortin-4 receptor.

    Science.gov (United States)

    Wang, Zhi-Qiang; Wang, Wei; Shi, Lin; Chai, Ji-Tian; Zhang, Xin-Jun; Tao, Ya-Xiong

    2016-04-01

    The melanocortin-4 receptor (MC4R) is critical in regulating mammalian food intake and energy expenditure. Giant panda (Ailuropoda melanoleuca), famous as the living fossil, is an endangered species endemic to China. We are interested in exploring the functions of the giant panda MC4R (amMC4R) in regulating energy homeostasis and report herein the molecular cloning and pharmacology of the amMC4R. Sequence analysis revealed that amMC4R was highly homologous (>88%) at nucleotide and amino acid sequences to several mammalian MC4Rs. Western blot revealed that the expression construct myc-amMC4R in pcDNA3.1 was successfully constructed and expressed in HEK293T cells. With human MC4R (hMC4R) as a control, pharmacological characteristics of amMC4R were analyzed with binding and signaling assays. Four agonists, including [Nle(4), D-Phe(7)]-α-melanocyte stimulating hormone (NDP-MSH), α- and β-MSH, and a small molecule agonist, THIQ, were used in binding and signaling assays. We showed that amMC4R bound NDP-MSH with the highest affinity followed by THIQ, α-MSH, and β-MSH, with the same ranking order as hMC4R. Treatment of HEK293T cells expressing amMC4R with different concentrations of agonists resulted in dose-dependent increase of intracellular cAMP levels, with similar EC50s for the four agonists. The results suggested that the cloned amMC4R encoded a functional MC4R. The availability of amMC4R and its binding and signaling properties will facilitate the investigation of amMC4R in regulating food intake and energy homeostasis. PMID:26896843

  6. Gene cloning, expression and characterization of avian cathelicidin orthologs, Cc-CATHs, from Coturnix coturnix.

    Science.gov (United States)

    Feng, Feifei; Chen, Chen; Zhu, Wenjuan; He, Weiyu; Guang, Huijuan; Li, Zheng; Wang, Duo; Liu, Jingze; Chen, Ming; Wang, Yipeng; Yu, Haining

    2011-05-01

    Cathelicidins comprise a family of antimicrobial peptides sharing a highly conserved cathelin domain, which play a central role in the early innate host defense against infection. In the present study, we report three novel avian cathelicidin orthologs cloned from a constructed spleen cDNA library of Coturnix coturnix, using a nested-PCR-based cloning strategy. Three coding sequences containing ORFs of 447, 465 and 456 bp encode three mature antimicrobial peptides (named Cc-CATH1, 2 and 3) of 26, 32 and 29 amino acid residues, respectively. Phylogenetic analysis indicated that precursors of Cc-CATHs are significantly conserved with known avian cathelicidins. Synthetic Cc-CATH2 and 3 displayed broad and potent antimicrobial activity against most of the 41 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, with minimum inhibitory concentration values in the range 0.3-2.5 μm for most strains with or without the presence of 100 mm NaCl. Cc-CATH2 and 3 showed considerable reduction of cytotoxic activity compared to other avian cathelicidins, with average IC(50) values of 20.18 and 17.16 μm, respectively. They also exerted a negligible hemolytic activity against human erythrocytes, lysing only 3.6% of erythrocytes at a dose up to 100 μg·mL(-1) . As expected, the recombinant Cc-CATH2 (rCc-CATH2) also showed potent bactericidal activity. All these features of Cc-CATHs encourage further studies aiming to estimate their therapeutic potential as drug leads, as well as coping with current widespread antibiotic resistance, especially the new prevalent and dangerous 'superbug' that is resistant to almost all antibiotics. PMID:21375690

  7. Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Z.; Chen, S.; Wilson, D.B.

    1999-11-01

    An Mn{sup 2+} and Cd{sup 2+} uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd{sup 2+} sensitivity as well as energy-dependent Cd{sup 2+} uptake activity. Both transcription and translation of mntA were induced by Mn{sup 2+} starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd{sup 2+} uptake systems have been identified in L. plantarum: one is a high-affinity Mn{sup 2+} and Cd{sup 2+} uptake system that is expressed in Mn{sup 2+}-starved cells, and the other is a nonsaturable Cd{sup 2+} uptake system that is expressed in Cd{sup 2+}-sufficient cells. MntA was not detected in an Mn{sup 2+}-dependent mutant of L. plantarum which had lost high-affinity Mn{sup 2+} and Cd{sup 2+} uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn{sup 2+} and Cd{sup 2+} transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn{sup 2+}- and Cd{sup 2+}-binding sites of MntA are discussed. A second clone containing a low-affinity Cd{sup 2+} transport system was also isolated.

  8. Characterization of nonprimate hepacivirus and construction of a functional molecular clone

    Science.gov (United States)

    Scheel, Troels K. H.; Kapoor, Amit; Nishiuchi, Eiko; Brock, Kenny V.; Yu, Yingpu; Andrus, Linda; Gu, Meigang; Renshaw, Randall W.; Dubovi, Edward J.; McDonough, Sean P.; Van de Walle, Gerlinde R.; Lipkin, W. Ian; Divers, Thomas J.; Tennant, Bud C.; Rice, Charles M.

    2015-01-01

    Nonprimate hepacivirus (NPHV) is the closest known relative of hepatitis C virus (HCV) and its study could enrich our understanding of HCV evolution, immunity, and pathogenesis. High seropositivity is found in horses worldwide with ∼3% viremic. NPHV natural history and molecular virology remain largely unexplored, however. Here, we show that NPHV, like HCV, can cause persistent infection for over a decade, with high titers and negative strand RNA in the liver. NPHV is a near-universal contaminant of commercial horse sera for cell culture. The complete NPHV 3′-UTR was determined and consists of interspersed homopolymer tracts and an HCV-like 3′-terminal poly(U)-X-tail. NPHV translation is stimulated by miR-122 and the 3′-UTR and, similar to HCV, the NPHV NS3-4A protease can cleave mitochondrial antiviral-signaling protein to inactivate the retinoic acid-inducible gene I pathway. Using an NPHV consensus cDNA clone, replication was not observed in primary equine fetal liver cultures or after electroporation of selectable replicons. However, intrahepatic RNA inoculation of a horse initiated infection, yielding high RNA titers in the serum and liver. Delayed seroconversion, slightly elevated circulating liver enzymes and mild hepatitis was observed, followed by viral clearance. This establishes the molecular components of a functional NPHV genome. Thus, NPHV appears to resemble HCV not only in genome structure but also in its ability to establish chronic infection with delayed seroconversion and hepatitis. This NPHV infectious clone and resulting acute phase sera will facilitate more detailed studies on the natural history, pathogenesis, and immunity of this novel hepacivirus in its natural host. PMID:25646476

  9. Cloning, functional characterization and catalytic mechanism of a bergaptol O-methyltransferase from Peucedanum praeruptorum Dunn

    Directory of Open Access Journals (Sweden)

    Yucheng eZhao

    2016-05-01

    Full Text Available Coumarins are main active components of Peucedanum praeruptorum Dunn. Among them, methoxylated coumarin compound, such as bergapten, xanthotoxin and isopimpinellin, has high officinal value and plays an important role in medicinal field. However, major issues associated with the biosynthesis mechanism of coumarins remain unsolved and no corresponding enzyme has been cloned from P. praeruptorum. In this study, a local BLASTN program was conducted to find the candidate genes from P. praeruptorum transcriptome database using the nucleotide sequence of Ammi majus bergaptol O-methyltransferase (AmBMT, GenBank accession No: AY443006 as a template. As a result, a 1335 bp full-length of cDNA sequence which contains an open reading frame of 1080 bp encoding a BMT polypeptide of 359 amino acids was obtained. The recombinant protein was functionally expressed in Escherichia coli and displayed an observed activity to bergaptol. In vitro experiments show that the protein has narrow substrate specificity for bergaptol. Expression profile indicated that the cloned gene had a higher expression level in roots and can be induced by methyl jasmonate (MeJA. Subcellular localization analysis showed that the BMT protein was located in cytoplasm in planta. Homology modeling and docking based site-directed mutagenesis have been employed to investigate the amino acid residues in BMT required for substrate binding and catalysis. Conservative amino acid substitutions at residue H264 affected BMT catalysis, whereas substitutions at residues F171, M175, D226 and L312 affected substrate binding. The systemic study summarized here will enlarge our knowledge on OMTs and provide useful information in investigating the coumarins biosynthesis mechanism in P. praeruptorum.

  10. Cloning and characterization of BKV(MM) DNA and its use for detection of BKV DNA in human urine

    International Nuclear Information System (INIS)

    The two fragments produced by restriction of BKV(MM) DNA with the endonucleases Pst I and Eco RI have been cloned separately into the vector pBR322 and amplified in E. coli HB101. Eight recombinant plasmids were characterized by gel electrophoresis of Pst I/Eco RI double digestions or Hind III digestions of the DNA and by hybridization of Southern gel blots to a nick-translated BKV(MM) DNA probe. Four of the recombinant plasmids contained the large Pst I/Eco RI BKV(MM) DNA fragment and four contained the small fragment. Two of these recombinant plasmids were then used to make a probe for the identification of BK DNA in a urine specimen from a patient known to be exreting particles with the morphological features of papovavirus

  11. Cloning,expression and characterization of L-cysteine desulfhydrase gene from Pseudomonas sp.TS1138

    Institute of Scientific and Technical Information of China (English)

    YU Yangsheng; BAI Gang; LIU Chunqin; LI Yang; JIN Yongjie; YANG Wenbo

    2007-01-01

    L-cysteine desulthydrase (CD) plays an important role in L-cysteine decomposition.To identify the CD gene in Pseudomonas sp.TS 1138 and investigate its effect on the L-cysteine biosynthetic pathway,the CD gene was cloned from Pseudomonas sp.TS 1138 by polymerase chain reaction (PCR) method.The nucleotide sequence of CD gene was determined to be 1,215 bp,and its homology with other sequences encoding CD was analyzed.Then the CD gene was subcloned into pET-21a(+) vector and expressed in Escherichia coli (E.coli) by isopropyl-β-D-thiogalactopyranoside (IPTG) inducement.The recombinant CD was purified by Ni-NTA His-Bind resin,and its activity was identified by the CD activity staining.The enzymatic properties of the recombinant CD were characterized and its critical role involved in the L-cysteine biosynthetic pathway was also discussed.

  12. Molecular Phylogenetics and Functional Evolution of Major RNA Recognition Domains of Recently Cloned and Characterized Autoimmune RNA-Binding Particle

    Institute of Scientific and Technical Information of China (English)

    Erhan Süleymano(g)lu

    2003-01-01

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) are spliceosomal macromolecular assemblages and thus actively participate in pre-mRNA metabolism. They are composed of evolutionarily conserved and tandemly repeated motifs, where both RNA-binding and protein-protein recognition occur to achieve cellular activities. By yet unknown mechanisms, these ribonucleoprotein (RNP) particles are targeted by autoantibodies and hence play significant role in a variety of human systemic autoimmune diseases. This feature makes them important prognostic markers in terms of molecular epidemiology and pathogenesis of autoimmunity.Since RNP domain is one of the most conserved and widespread scaffolds, evolutiona lyses of these RNA-binding domains can provide further clues on disease-specific epitope formation. The study presented herein represents a sequence comparison of RNA-recognition regions of recently cloned and characterized human hnRNP A3 with those of other relevant hnRNP A/B-type proteins.Their implications in human autoimmunity are particularly emphasized.

  13. Cloning and Characterization of Adinbitor,a Novel Disintegrin from the Snake Venom of Agkistrodon halys brevicaudus stejneger

    Institute of Scientific and Technical Information of China (English)

    Ji-Hong WANG; Yu WU; Feng REN; Li LU; Bao-Chang ZHAO

    2004-01-01

    Adinbitor was cloned from Agkistrodon halys brevicaudus stejneger and characterized as a novel disintegrin.In this study,total RNA was extracted from venom gland and used in RT-PCR to generate a cDNA which is 219 bp long.The sequence encoded a polypeptide composed of 73 amino acids,including 12 cysteines,an RGD motif,and the signature motif of disintergrin.Recombinant Adinbitor (rAdinbitor) was expressed in E.coli and purified by using the His@ Bind affinity chromatography.The IC50 for inhibiting human platelet aggregation and bFGF-induced proliferation of ECV304 cells was 6 μM and 0.89 μM respectively.Furthermore,Adinbitor significantly inhibited angiogenesis both in vivo and in vitro.Taken together,these results suggested that Adinbitor had typical functions of disintegrins.

  14. DNA cloning, characterization, and inhibition studies of an α-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

    Science.gov (United States)

    Del Prete, Sonia; Isik, Semra; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; Scozzafava, Andrea; Supuran, Claudiu T; Capasso, Clemente

    2012-12-13

    We have cloned, purified, and characterized an α-carbonic anhydrase (CA, EC 4.2.1.1) from the human pathogenic bacterium Vibrio cholerae, VchCA. The new enzyme has significant catalytic activity, and an inhibition study with sulfonamides and sulfamates led to the detection of a large number of low nanomolar inhibitors, among which are methazolamide, acetazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide, and indisulam (KI values in the range 0.69-8.1 nM). As bicarbonate is a virulence factor of this bacterium and since ethoxzolamide was shown to inhibit the in vivo virulence, we propose that VchCA may be a target for antibiotic development, exploiting a mechanism of action rarely considered until now. PMID:23181552

  15. Why Clone?

    Science.gov (United States)

    ... How might cloning be used in medicine? Cloning animal models of disease Much of what researchers learn ... the first place, such as habitat destruction and hunting. But cloning may be one more tool that ...

  16. Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

    Directory of Open Access Journals (Sweden)

    Gong Xia

    2012-10-01

    Full Text Available Abstract Background Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow. Results Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum. Conclusion The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.

  17. Cloning, expression and pharmacological characterization of rabbit adenosine A1 and A3 receptors.

    Science.gov (United States)

    Hill, R J; Oleynek, J J; Hoth, C F; Kiron, M A; Weng, W; Wester, R T; Tracey, W R; Knight, D R; Buchholz, R A; Kennedy, S P

    1997-01-01

    The role of adenosine A1 and A3 receptors in mediating cardioprotection has been studied predominantly in rabbits, yet the pharmacological characteristics of rabbit adenosine A1 and A3 receptor subtypes are unknown. Thus, the rabbit adenosine A3 receptor was cloned and expressed, and its pharmacology was compared with that of cloned adenosine A1 receptors. Stable transfection of rabbit A1 or A3 cDNAs in Chinese hamster ovary-K1 cells resulted in high levels of expression of each of the receptors, as demonstrated by high-affinity binding of the A1/A3 adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA). For both receptors, binding of 125I-ABA was inhibited by the GTP analog 5'-guanylimidodiphosphate, and forskolin-stimulated cyclic AMP accumulation was inhibited by the adenosine receptor agonist (R)-phenylisopropyladenosine. The rank orders of potency of adenosine receptor agonists for inhibition of 125I-ABA binding were as follows: rabbit A1, N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N-ethylcarboxamidoadenosine > or = I-ABA > or = N6-2-(4-aminophenyl) ethyladenosine > > N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > N6-(4-amino-3-benzyl)adenosine; rabbit A3, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > or = I-ABA > > N-ethylcarboxamidoadenosine > N6-2-(4-aminophenyl) ethyladenosine = N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N6-(4-amino-3-benzyl)adenosine. The adenosine receptor antagonist rank orders were as follow: rabbit A1, 8-cyclopentyl-1,3-dipropylxanthine > 1,3- dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amine congener > > 8-(p-sulfophenyl)theophylline; rabbit A3, xanthine amine congener > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = 8-cyclopentyl-1,3-dipropylxanthine > > 8-(p-sulfophenyl)theophylline. These observations confirm the identity of the expressed proteins as A1 and A3 receptors. The results will facilitate further in-depth studies of the roles of A1 and A3 receptors in

  18. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein.

    OpenAIRE

    Rozenblatt, S; Eizenberg, O; Englund, G.; Bellini, W J

    1985-01-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, 32P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-...

  19. Characterization and molecular cloning of a serine hydroxymethyltransferase 1 (OsSHM1) in rice

    Institute of Scientific and Technical Information of China (English)

    Dekai Wang; Heqin Liu; Sujuan Li; Guowei Zhai; Jianfeng Shao; Yuezhi Tao

    2015-01-01

    Serine hydroxymethyltransferase (SHMT) is impor-tant for one carbon metabolism and photorespiration in higher plants for its participation in plant growth and development, and resistance to biotic and abiotic stresses. A rice serine hydroxymethyltransferase gene, OsSHM1, an ortholog of Arabidopsis SHM1, was isolated using map-based cloning. The osshm1 mutant had chlorotic lesions and a considerably smaller, lethal phenotype under natural ambient CO2 concentrations, but could be restored to wild type with normal growth under elevated CO2 levels (0.5% CO2), showing a typical photo-respiratory phenotype. The data from antioxidant enzymes activity measurement suggested that osshm1 was subjected to significant oxidative stress. Also, OsSHM1 was expressed in al organs tested (root, culm, leaf, and young panicle) but predominantly in leaves. OsSHM1 protein is localized to the mitochondria. Our study suggested that molecular function of the OsSHM1 gene is conserved in rice and Arabidopsis.

  20. Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6.

    Science.gov (United States)

    Zhu, Yanbing; Zheng, Wenguang; Ni, Hui; Liu, Han; Xiao, Anfeng; Cai, Huinong

    2015-10-01

    A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249-amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p-nitrophenyl butyrate. When p-nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H-257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X-100, sodium deoxycholate, urea, and guanidine hydrochloride.

  1. Molecular cloning and characterization of a malic enzyme gene from the oleaginous yeast Lipomyces starkeyi.

    Science.gov (United States)

    Tang, Wei; Zhang, Sufang; Tan, Haidong; Zhao, Zongbao K

    2010-06-01

    The malic enzyme-encoding cDNA (GQ372891) from the oleaginous yeast Lipomyces starkeyi AS 2.1560 was isolated, which has an 1719-bp open reading frame flanked by a 290-bp 5' untranslated sequence and a 92-bp 3' untranslated sequence. The proposed gene, LsME1, encoded a protein with 572 amino acid residues. The protein presented 58% sequence identity with the malic enzymes from Yarrowia lipolytica CLIB122 and Aspergillus fumigatus Af293. The LsME1 gene was cloned into the vector pMAL-p4x to express a fusion protein (MBP-LsME1) in Escherichia coli TB1. The fusion protein was purified and then cleaved by Factor Xa to give the recombinant LsME1. This purified enzyme took either NAD(+) or NADP(+) as the coenzyme but preferred NAD(+). The K (m) values for malic acid, NAD(+) and NADP(+) were 0.85 +/- 0.05 mM, 0.34 +/- 0.08 mM, and 7.4 +/- 0.32 mM, respectively, at pH 7.3.

  2. Cloning and Characterization of Glutamate Receptors in Californian Sea Lions (Zalophus californianus

    Directory of Open Access Journals (Sweden)

    Santokh Gill

    2010-05-01

    Full Text Available Domoic acid produced by marine algae has been shown to cause acute and chronic neurologic sequelae in Californian sea lions following acute or low-dose exposure. Histological findings in affected animals included a degenerative cardiomyopathy that was hypothesized to be caused by over-excitation of the glutamate receptors (GluRs speculated to be present in the sea lion heart. Thus tissues from five sea lions without lesions associated with domoic acid toxicity and one animal with domoic acid-induced chronic neurologic sequelae and degenerative cardiomyopathy were examined for the presence of GluRs. Immunohistochemistry localized mGluR 2/3, mGluR 5, GluR 2/3 and NMDAR 1 in structures of the conducting system and blood vessels. NMDAR 1 and GluR 2/3 were the most widespread as immunoreactivity was observed within sea lion conducting system structures. PCR analysis, cloning and subsequent sequencing of the seal lion GluRs showed only 80% homology to those from rats, but more than 95% homologous to those from dogs. The cellular distribution and expression of subtypes of GluRs in the sea lion hearts suggests that exposure to domoic acid may induce cardiac damage and functional disturbances.

  3. Molecular cloning and characterization of a new peptide deformylase from human pathogenic bacterium Helicobacter pylori

    International Nuclear Information System (INIS)

    Helicobacter pylori is a gram-negative pathogenic bacterium, which is associated with peptic ulcer disease and gastric cancer. It is urgent to discover novel drug targets for appropriate antimicrobial agents against this human pathogen. In bacteria, peptide deformylase (PDF) catalyzes the removal of a formyl group from the N-termini of nascent polypeptides. Due to its essentiality and absence in mammalian cells, PDF has been considered as an attractive target for the discovery of novel antibiotics. In this work, a new PDF gene (def) from H. pylori strain SS1 was cloned, expressed, and purified in Escherichia coli system. Sequence alignment shows that H. pylori PDF (HpPDF) shares about 40% identity to E. coli PDF (EcPDF). The enzymatic properties of HpPDF demonstrate its relatively high activity toward formyl-Met-Ala-Ser, with Kcat of 3.4 s-1, Km of 1.7 mM, and Kcat/Km of 2000 M-1 s-1. HpPDF enzyme appears to be fully active at pH between 8.0 and 9.0, and temperature 50 deg. C. The enzyme activity of Co2+-containing HpPDF is apparently higher than that of Zn2+-containing HpPDF. This present work thereby supplies a potential platform that facilitates the discovery of novel HpPDF inhibitors and further of possible antimicrobial agents against H. pylori

  4. Cloning, Purification, and Characterization of Recombinant Human Extracellular Superoxide Dismutase in SF9 Insect Cells.

    Science.gov (United States)

    Shrestha, Pravesh; Yun, Ji-Hye; Kim, Woo Taek; Kim, Tae-Yoon; Lee, Weontae

    2016-03-01

    A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1-240) and truncated (residues 19-240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity. PMID:26912083

  5. Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana.

    Science.gov (United States)

    Villand, P; Olsen, O A; Kleczkowski, L A

    1993-12-01

    PCR amplification of cDNA prepared from poly(A)+ RNA from aerial parts of Arabidopsis thaliana, using degenerate nucleotide primers based on conserved regions between the large and small subunits of ADP-glucose pyrophosphorylase (AGP), yielded four different cDNAs of ca. 550 nucleotides each. Based on derived amino acid sequences, the identities between the clones varied from 49 to 69%. Sequence comparison to previously published cDNAs for AGP from various species and tissues has revealed that three of the amplified cDNAs (ApL1, ApL2 and ApL3) correspond to the large subunit of AGP, and one cDNA (ApS) encodes the small subunit of AGP. Both ApL1 and ApS were subsequently found to be present in a cDNA library made from Arabidopsis leaves. All four PCR products are encoded by single genes, as found by genomic Southern analysis. PMID:8292792

  6. Cloning, expression and characterization of a nucleoside diphosphate kinase (NDPK) gene from tobacco

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nucleoside diphosphate kinase (NDPK) is a housekeeping enzyme that maintains the intracellular levels of all (d)NTPs used in biosynthesis except ATP. Here we report that a full-length cDNA encoding nucleoside diphosphate kinase (NDPK) was cloned using yeast two-hybrid approach. A tobacco NDPK gene was obtained and designated as NtNDPK1 . NtNDPK1 is 704 bp in length and encodes a putative 16.2 kD protein of 148 amino acids. Phylogenic analysis showed that NtNDPK1 is highly homologous to other plant NDPK genes and identified as type Ⅰ (NDPK1). RNA-gel blot analysis showed that there was no significant difference of NtNDPK1 expression in roots, stems, leaves and buds. And expression of NtNDPK1 was induced by ABA and PEG and repressed by NaCl, but not significantly affected by Paraquat, wounding and low temperature (4℃) treatments, indicating that NtNDPK1 may play a certain role in response to abiotic stress. In vitro phosphorylation assay demonstrated that NtNDPK1 had autophosphorylation activity.

  7. Homologous cloning, characterization and expression of a new halophyte phytochelatin synthase gene in Suaeda salsa

    Science.gov (United States)

    Cong, Ming; Zhao, Jianmin; Lü, Jiasen; Ren, Zhiming; Wu, Huifeng

    2016-01-01

    The halophyte Suaeda salsa can grow in heavy metal-polluted areas along intertidal zones having high salinity. Since phytochelatins can eff ectively chelate heavy metals, it was hypothesized that S. salsa possessed a phytochelatin synthase (PCS) gene. In the present study, the cDNA of PCS was obtained from S. salsa (designated as SsPCS) using homologous cloning and the rapid amplification of cDNA ends (RACE). A sequence analysis revealed that SsPCS consisted of 1 916 bp nucleotides, encoding a polypeptide of 492 amino acids with one phytochelatin domain and one phytochelatin C domain. A similarity analysis suggested that SsPCS shared up to a 58.6% identity with other PCS proteins and clustered with PCS proteins from eudicots. There was a new kind of metal ion sensor motif in its C-terminal domain. The SsPCS transcript was more highly expressed in elongated and fibered roots and stems (P mercury exposure significantly enhanced the mRNA expression of SsPCS (P metal sensing capability than the first PCS from Thellungiella halophila. This study provided a new view of halophyte PCS genes in heavy metal tolerance.

  8. Molecular cloning and characterization of a galectin-1 homolog in orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Chen, Xiuli; Wei, Jingguang; Xu, Meng; Yang, Min; Li, Pingfei; Wei, Shina; Huang, Youhua; Qin, Qiwei

    2016-07-01

    As a member of animal lectin family, galectin has the functions of pathogen recognition, anti-bacteria and anti-virus. In the present study, a galectin-1 homolog (EcGel-1) from grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcGel-1 is 504 bp, including a 408 bp open reading frame (ORF) which encodes 135 amino acids with a molecular mass of 15.19 kDa. Quantitative real-time PCR analysis indicated that EcGel-1 was constitutively expressed in all analyzed tissues of healthy grouper. The expression of EcGel-1 in the spleen of grouper was differentially up-regulated challenged with Singapore grouper iridovirus (SGIV), poly (I:C), and LPS. EcGel-1 was abundantly distributed in the cytoplasm in GS cells. Recombinant EcGel-1(rEcGel-1) protein can make chicken erythrocyte aggregation, and combine with gram negative bacteria and gram positive bacteria in the presence of 2-Mercaptoethanol (β-ME). Taken together, the results showed that EcGel-1 may be an important molecule involved in pathogen recognition and pathogen elimination in the innate immunity of grouper.

  9. Cloning, characterization, and expression analysis of a thioredoxin from orange-spotted grouper (Epinephelus coioides).

    Science.gov (United States)

    Wei, Jingguang; Guo, Minglan; Ji, Huasong; Yan, Yang; Ouyang, Zhengliang; Huang, Xiaohong; Hang, Youhua; Qin, Qiwei

    2012-09-01

    Thioredoxins (TRXs) are a family of small, highly conserved proteins that are essential for the maintenance of cellular homeostasis. In this study, a thioredoxin gene was cloned from orange-spotted grouper, Epinephelus coioides (designated as Ec-TRX). The full-length cDNA of Ec-TRX was comprised of 767bp with a 327bp open reading frame that encodes a putative protein of 108 amino acids. Quantitative real-time PCR analysis revealed that the Ec-TRX mRNA was distributed abundantly in grouper, E. coioides skin and liver, and the expression in liver was up-regulated after viral challenge with Singapore grouper iridovirus (SGIV). Recombinant Ec-TRX (rEc-TRX) was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-Ec-TRX serum preparation. The rEc-TRX fusion protein was demonstrated to possess the expected redox activity in enzymatic analysis, and scavenge free radicals and protect supercoiled DNA from oxidative damage induced by a metal-ion catalyzed oxidation reaction. Subcellular localization revealed that Ec-TRX was distributed in both cytoplasm and nucleus. Overexpression of Ec-TRX in grouper spleen (GS) cells could promote the growth of GS cells and inhibit the replication of SGIV. These results suggest that Ec-TRX could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to viral challenge.

  10. Molecular cloning, characterization and functional analysis of QRFP in orange-spotted grouper (Epinephelus coioides).

    Science.gov (United States)

    Shu, Hu; Chen, Huapu; Liu, Yun; Yang, Lidong; Yang, Yuqing; Zhang, Haifa

    2014-10-01

    The peptide QRFP plays an important role in the regulation of vertebrate feeding behavior. In this study, we cloned the full length cDNA of a QRFP precursor in a teleost fish, the orange-spotted grouper (Epinephelus coioides). Sequence analysis has shown that the functional regions of QRFP in other vertebrates (QRFP-25 and QRFP-7) are conserved in orange-spotted grouper. RT-PCR demonstrated that the pre-processed mRNA of QRFP is widely expressed in orange-spotted grouper. Three days of food deprivation did not change the hypothalamic pre-processed QRFP expression. However, QRFP expression significantly increased when the fish were reefed after three days of fasting. Intraperitoneal injection of QRFP-25 peptide to orange-spotted grouper suppressed expression of orexin, but elevated expression of pro-opiomelanocortin (POMC) in the hypothalamus. We also investigated the effects of QRFP-25 on the expression of reproductive genes. The peptide suppressed the expression of seabream-type gonadotropin-releasing hormones (sbGnRH), luteinizing hormone beta subunit (LHβ) and follicle-stimulating hormone beta subunit (FSHβ) in vivo, as well as inhibited the expression of LHβ and FSHβ in pituitary cells in primary culture. Our results indicate that QRFP may play an inhibitory role in the regulation of feeding behavior and reproduction in orange-spotted grouper.

  11. Molecular cloning and functional characterization of spexin in orange-spotted grouper (Epinephelus coioides).

    Science.gov (United States)

    Li, Shuisheng; Liu, Qiongyu; Xiao, Ling; Chen, Huapu; Li, Guangli; Zhang, Yong; Lin, Haoran

    2016-01-01

    Spexin is a newly discovered neuropeptide in vertebrates. Comprehensive comparative studies are required to unveil its biological functions. In order to ascertain the neuroendocrine function of spexin in orange-spotted grouper, its full-length cDNA and genomic DNA sequences were cloned and analyzed. Sequence analyses showed that the spexin gene structure is composed of six exons and five introns, and the amino acids of mature peptide (spexin-14) in grouper are identical to that of other fish. Tissue expression analysis found that grouper spexin is highly expressed in the brain, liver and ovary. Real time-PCR analysis demonstrated that the hypothalamic expression of spexin declined gradually during the ovarian development, and was up-regulated by food deprivation. Intraperitoneal administration of spexin-14 peptides to grouper significantly elevated the mRNA levels of proopiomelanocortin (pomc) and suppressed the orexin expression in the hypothalamus, but could not change the hypothalamic expression of gonadotropin releasing hormone 1 (gnrh1). Both in vivo and in vitro administration of spexin could not significantly influence the expression of follicle-stimulating hormone β (fshβ) and luteinizing hormone β (lhβ) in the pituitary with the exception of an inhibition of gh expression. Our data suggested that the spexin has a significant role in the regulation of energy metabolism and food intake in orange-spotted grouper.

  12. Molecular cloning and functional characterization of porcine E74-like factor 4 (ELF4).

    Science.gov (United States)

    Shi, Yanling; Wang, Dang; Zhu, Xinyu; Wu, Qiong; Chen, Huanchun; Xiao, Shaobo; Fang, Liurong

    2016-12-01

    E74-like factor 4 (ELF4) is a novel transcription factor that initiates transcription of type I interferon (IFN) genes to control diverse pathogens. Here, porcine ELF4 (poELF4) was cloned and its role in type I IFN signaling was investigated in different porcine cell lines. Full-length cDNA of poELF4 encodes 663 amino acid residues and ectopic expression of poELF4 significantly induced IFN-β production. Interestingly, difference from the human ELF4 (huELF4), poELF4 mutants lacking the serine/threonine rich domain, which has been demonstrated to be responsible for the phosphorylation of huELF4, were still capable of activating IFN-β promoter. Using pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) as the models of DNA virus and RNA virus, respectively, we found that the replication of both PRV and PRRSV was reduced with poELF4 overexpression and enhanced with poELF4 knockdown. Taken together, these results suggested that poELF4 is an important antiviral host restriction factor. PMID:27426928

  13. Molecular cloning, characterization, and expression profiles of androgen receptors in spotted scat (Scatophagus argus).

    Science.gov (United States)

    Chen, H P; Deng, S P; Dai, M L; Zhu, C H; Li, G L

    2016-01-01

    Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARβ common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding. PMID:27173207

  14. Immunological characterizations of a cloned 13.1-kilodalton protein from pathogenic Naegleria fowleri.

    Science.gov (United States)

    Cho, Myoung-Soo; Jung, Suk-Yul; Park, Sun; Kim, Kyongmin Hwang; Kim, Hyung-Il; Sohn, Seonghyang; Kim, Han-Jip; Im, Kyung-Il; Shin, Ho-Joon

    2003-09-01

    We previously cloned an antigenic gene (named nfa1) from a cDNA library of Naegleria fowleri by immunoscreening. The nfa1 gene had a coding nucleotide sequence consisting of 357 bases and produced a recombinant 13.1-kDa protein (Nfa1). In this study, to get more information regarding the recombinant Nfa1 protein (rNfa1), we produced an anti-Nfa1 polyclonal antibody from mice immunized with rNfa1 and used a peroxidase staining method to carry out immunocytochemistry experiments. In addition, we observed the effect of the presence of an anti-Nfa1 antibody on the in vitro cytotoxicity of N. fowleri against Chinese hamster ovary (CHO) cells. Trophozoites of N. fowleri in cultivation reacted strongly with a peroxidase-labeled anti-Nfa1 antibody. In inflammatory and necrotic regions of brain tissue infected with N. fowleri, labeled trophozoites that were stained brown were also observed. When examined using a transmission electron microscope, the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. When examined using a light microscope, CHO cells grown in cocultures with N. fowleri trophozoites (group I) for 48 h showed morphologically severe destruction but CHO cells grown in cocultures with N. fowleri trophozoites and an anti-Nfa1 polyclonal antibody (group II) showed less destruction. The results of a lactate dehydrogenase release assay showed that group I CHO cells exhibited 81% cytotoxicity and group II CHO cells exhibited 13.8% cytotoxicity.

  15. Cloning and characterization of a gibberellin-induced RNase expressed in barley aleurone cells

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, S.W.; Rogers, J.C. (Washington State Univ., Pullman, WA (United States). Inst. of Biological Chemistry)

    1999-04-01

    The authors cloned a cDNA for a gibberellin-induced ribonuclease (RNase) expressed in barley (Hordeum vulgare) aleurone and the gene for a second barley RNase expressed in leaf tissue. The protein encoded by the cDNA is unique among RNases described to date in that it contains a novel 23-amino acid insert between the C2 and C3 conserved sequences. Expression of the recombinant protein in tobacco (Ncotiana tabacum) suspension-cultured protoplasts gave an active RNase of the expected size, confirming the enzymatic activity of the protein. Analyses of hormone regulation of re-expression of mRNA for the aleurone RNase revealed that, like the pattern for [alpha]-amylase, mRNA levels increased in the presence of gibberellic acid, and its antagonist abscisic acid prevented this effect. Quantitative studies at early times demonstrated that cycloheximide treatment of aleurone layers increased mRNA levels 4-fold, whereas a combination of gibberellin plus cycloheximide treatment was required to increase [alpha]-amylase mRNA levels to the same extent. These results are consistent with loss of repression as an initial effect of gibberellic acid on transcription of those genes, although the regulatory pathways for the two genes may differ.

  16. Molecular cloning and structural characterization of Ecdysis Triggering Hormone from Choristoneura fumiferana.

    Science.gov (United States)

    P, Bhagath Kumar; K, Kasi Viswanath; S, Tuleshwori Devi; R, Sampath Kumar; Doucet, Daniel; Retnakaran, Arthur; Krell, Peter J; Feng, Qili; Ampasala, Dinakara Rao

    2016-07-01

    At the end of each stadium, insects undergo a precisely orchestrated process known as ecdysis which results in the replacement of the old cuticle with a new one. This physiological event is necessary to accommodate growth in arthropods since they have a rigid chitinous exoskeleton. Ecdysis is initiated by the direct action of Ecdysis Triggering Hormones on the central nervous system. Choristoneura fumiferana is a major defoliator of coniferous forests in Eastern North America. It is assumed that, studies on the ecdysis behavior of this pest might lead to the development of novel pest management strategies. Hence in this study, the cDNA of CfETH was cloned. The open reading frame of the cDNA sequence was found to encode three putative peptides viz., Pre-Ecdysis Triggering Hormone (PETH), Ecdysis Triggering Hormone (ETH), and Ecdysis Triggering Hormone Associated Peptide (ETH-AP). The CfETH transcript was detected in the epidermal tissue of larval and pupal stages, but not in eggs and adults. In order to explore the structural conformation of ETH, ab initio modelling and Molecular Dynamics (MD) Simulations were performed. Further, a library of insecticides was generated and virtual screening was performed to identify the compounds displaying high binding capacity to ETH. PMID:27012894

  17. Molecular cloning and characterization of lymphocyte cell kinase from humphead snapper (Lutjanus sanguineus).

    Science.gov (United States)

    Huang, Y; Cai, J; Wang, B; Tang, J-F; Jian, J-C; Wu, Z-H; Gan, Z; Lu, Y-S

    2016-07-01

    Lymphocyte cell kinase (LCK) belongs to the Src family of tyrosine kinases, which involves in the proliferation control of lymphocytes. In this study, we cloned the LCK gene of humphead snapper (Lutjanus sanguineus) (designed as LsLCK). Sequence analysis showed that the full-length cDNA of LsLCK was 2279 bp, contained a 1506-bp open reading frame (ORF), encoding a polypeptide of 501 amino acids. The deduced amino acid possessed the typical structural features of known LCK proteins, including four Src homology (SH) domains arranged as the SH1 domain followed by a regulatory C-terminal tail (COOH-domain), SH2 and SH3 adapter domains and SH4 domain which required for membrane attachment and CD4/CD8 binding. Fluorescent quantitative real-time PCR analysis indicated that LsLCK transcripts were expressed mainly in thymus, spleen and head kidney in healthy fish. Moreover, the mRNA expressions in these tissues were significantly up-regulated after challenge with Vibrio harveyi. The results of immunohistochemistry showed that LsLCK protein localized distinctly in cytoplasm of cell in thymus, spleen and head kidney. Taken together, these findings indicated that LsLCK may play an important role in the immune response of humphead snapper against bacterial infection.

  18. Direct detection, cloning and characterization of a glucoside hydrolase from forest soil.

    Science.gov (United States)

    Hua, Mei; Zhao, Shubo; Zhang, Lili; Liu, Dongbo; Xia, Hongmei; Li, Fan; Chen, Shan

    2015-06-01

    A glucoside hydrolase gene, egl01, was cloned from the soil DNA of Changbai Mountain forest by homologous PCR amplification. The deduced sequence of 517 amino acids included a catalytic domain of glycoside hydrolase family 5 and was homologous to a putative cellulase from Bacillus licheniformis. The recombinant enzyme, Egl01, was maximally active at pH 5 and 50 °C and it was stable at pH 3-9, 4-50 °C, and also stable in the presence of metal ions, organic solvents, surfactants and salt. Its activity was above 120 % in 2-3 M NaCl/KCl and over 70 % was retained in 1-4 M NaCl/KCl for 6d. Egl01 hydrolyzed carboxymethyl cellulose, beechwood xylan, crop stalk, laminarin, filter paper, and avicel but not pNPG, indicating its broad substrate specificity. These properties make this recombinant enzyme a promising candidate for industrial applications. PMID:25700816

  19. Characterization of grass carp (Ctenopharyngodon idella) IL-17D: molecular cloning, functional implication and signal transduction.

    Science.gov (United States)

    Du, Linyong; Qin, Lei; Wang, Xinyan; Zhang, Anying; Wei, He; Zhou, Hong

    2014-02-01

    Although the roles of IL-17 family members during inflammation have been extensively studied in mammals, their knowledge in lower vertebrates is limited. In particular, the biological activities of fish IL-17 and their functional roles are largely unknown. In this study, we cloned grass carp IL-17D (gcIL-17D) and found that its putative protein possessed the conserved features of IL-17 family members. Tissue distribution analysis showed that gcIL-17D was preferentially expressed in the mucosal tissues, including skin, gill and intestine. Subsequently, the involvement of gcIL-17D in inflammatory response was demonstrated by examining the expression profiles of gcIL-17D in head kidney and head kidney leukocytes following in vivo bacterial infection and in vitro LPS treatment, respectively. Furthermore, recombinant gcIL-17D (rgcIL-17D) was prepared in grass carp kidney cells and was able to promote the gene expression of some pro-inflammatory cytokines (IL-1β, TNF-α and CXCL-8) in grass carp primary head kidney cells, revealing gcIL-17D can function as a pro-inflammatory cytokine. Moreover, rgcIL-17D appeared to activate NF-κB signaling by modulating the phosphorylation of IκBα and up-regulated CXCL-8 mRNA expression possibly through NF-κB pathway. Our data shed new light on the functional role of teleost IL-17D in inflammatory response. PMID:24120974

  20. Molecular cloning and characterization of a galectin-1 homolog in orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Chen, Xiuli; Wei, Jingguang; Xu, Meng; Yang, Min; Li, Pingfei; Wei, Shina; Huang, Youhua; Qin, Qiwei

    2016-07-01

    As a member of animal lectin family, galectin has the functions of pathogen recognition, anti-bacteria and anti-virus. In the present study, a galectin-1 homolog (EcGel-1) from grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcGel-1 is 504 bp, including a 408 bp open reading frame (ORF) which encodes 135 amino acids with a molecular mass of 15.19 kDa. Quantitative real-time PCR analysis indicated that EcGel-1 was constitutively expressed in all analyzed tissues of healthy grouper. The expression of EcGel-1 in the spleen of grouper was differentially up-regulated challenged with Singapore grouper iridovirus (SGIV), poly (I:C), and LPS. EcGel-1 was abundantly distributed in the cytoplasm in GS cells. Recombinant EcGel-1(rEcGel-1) protein can make chicken erythrocyte aggregation, and combine with gram negative bacteria and gram positive bacteria in the presence of 2-Mercaptoethanol (β-ME). Taken together, the results showed that EcGel-1 may be an important molecule involved in pathogen recognition and pathogen elimination in the innate immunity of grouper. PMID:27109200

  1. Zebrafish Cx35: cloning and characterization of a gap junction gene highly expressed in the retina.

    Science.gov (United States)

    McLachlan, Elizabeth; White, Thomas W; Ugonabo, Chioma; Olson, Carl; Nagy, James I; Valdimarsson, Gunnar

    2003-09-15

    The vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304-amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT-PCR, at 2 days post-fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos.

  2. Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

    Institute of Scientific and Technical Information of China (English)

    FAN Weimin; HU Yilin; LI Changzhong; XIE Liping; ZHANG Rongqing

    2008-01-01

    Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sar-coplasmic/endoplasmic reticulum.A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pead oyster.This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata,namely PCRT.PCRT encodes a deduced 414-amino acid protein,which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL.The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species.Semi-quantitative RT-PCR indicates that PCRT is ubiquitously ex-pressed in all tissues tested with the highest expression in the hemolymph and the mantle.In situ hybridiza-tion analysis of PCRT in the mantle showed strong signals in the inner fold,the inner side of middle fold,and the inner side of outer fold of the mantle epithelium.All these results suggest PCRT might be involved in Ca2+ transport and storage during oyster biomineralization.

  3. Cloning and Preliminary Characterization of Three Receptor-like Kinase Genes in Soybean

    Institute of Scientific and Technical Information of China (English)

    Yuan-Yuan Ma; Li-Wen Zhang; Peng-Li Li; Rui Gan; Xiao-Ping Li; Ren Zhang; Yong Wang; Ning-Ning Wang

    2006-01-01

    Leaf senescence that occurs in the last stage of leaf development is a genetically programmed process. It is very significant to isolate the upstream components in the senescence signaling pathway and to elucidate the molecular mechanisms that control the initiation and progression of leaf senescence. In this study, full-length cDNAs of three receptor-like protein kinase genes, designated rlpk1, rlpk2 and rlpk3,were cloned from artificially-induced senescent soybean (Glycine max L.) primary leaves (GenBank accession AY687390, AY687391, AF338813). The deduced amino acid sequences indicated that they belonged to a receptor-like kinase family. Each of rlpk1 and rlpk2 encodes a leucine-rich repeat (LRR) receptor-like protein kinase. They both comprise a typical signal peptide, several LRR motifs, a single-pass transmembrane domain, and a cytoplasmic protein kinase domain. No typical extracellular domain of RLPK3 was predicted. Organ-specific expression pattern analysis by reverse-transcription polymerase chain reaction (RT-PCR) revealed higher expression levels of the three genes in cotyledons, roots and flowers. Phylogenetic analysis indicated that RLPK1 and RLPK2 belonged to an independent branch, whereas RLPK3 shared common nodes with several known RLKs responding to abiotic and biotic stresses. The evident alternations of expression profiles of rlpk1 and rlpk2 induced by the artificial senescence-inducing treatment implied involvements of these two RLKs in regulating soybean leaf senescence.

  4. Molecular cloning and characterization of a novel splicing variant of PIASx

    Institute of Scientific and Technical Information of China (English)

    Ying ZHENG; Zuo-min ZHOU; Lan-lan YIN; Jian-ming LI; Jia-hao SHA

    2004-01-01

    AIM: To investigate molecular mechanism of testis development and spermatogenesis. METHODS: A human testis cDNA microarray was hybridized with probes from human adult testis, embryo testis and human sperm, and the differential expressed clones were sequenced and analyzed. Expression of PIAS-NY gene was analyzed by RTPCR. RESULT: A new isoform of PIAS family, named PIAS-NY, was isolated from human testis cDNA liabrary.It was strongly expressed in adult testis and weakly expressed in both embryo testis and human sperm. Analysis of the open reading frame of PIAS-NY indicated that PIAS-NY was a polypeptide of 405 amino acid residues, and the sequence from the 15th amino acid to the end of PIAS-NY protein was the same as the N-terminal amino acids of PIASx-o and PIASx-β protein. PIAS-NY protein contained two conserved putative LXXLL signature motifs and a zinc binding motif. Tissue distribution analysis revealed that PIAS-NY was predominantly expressed in testis,weakly in the pancreas, and almost imperceptibly in the other organs. CONCLUSION: PIAS-NY may play important role in testis development and/or spermatogenesis.

  5. Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos

    Directory of Open Access Journals (Sweden)

    Jianrong Wang

    2014-12-01

    Full Text Available Poria cocos (P. cocos has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%. The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP from geranyl diphosphate (GPP and isopentenyl diphosphate (IPP. Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

  6. Cloning, expression, and characterization of a new phytase from the phytopathogenic bacterium Pectobacterium wasabiae DSMZ 18074.

    Science.gov (United States)

    Shao, Na; Huang, Huoqing; Meng, Kun; Luo, Huiying; Wang, Yaru; Yang, Peilong; Yao, Bin

    2008-07-01

    The soft rot bacterium Pectobacterium wasabiae is an economically important pathogen of many crops. A new phytase gene, appA, was cloned from P. wasabiae by degenerate PCR and TAIL-PCR. The open reading frame of appA consisted of 1,302 bp encoding 433 amino acid residues, including 27 residues of a putative signal peptide. The mature protein had a molecular mass of 45 kDa and a theoretical pI of 5.5. The amino acid sequence contained the conserved active site residues RHGXRXP and HDTN of typical histidine acid phosphatases, and showed the highest identity of 48.5% to PhyM from Pseudomonas syringae. The gene fragment encoding the mature phytase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant phytase had a specific activity of 1,072+/-47 U/mg for phytate substrate. The optimum pH and temperature for the purified phytase were pH 5.0 and 50 degrees C, respectively. The Km value was 0.17 mM, with a Vmax of 1,714 micromol/min/mg. This is the first report of the identification and isolation of phytase from Pectobacterium. PMID:18667849

  7. Molecular cloning, chromosomal mapping, and characterization of the mouse UDP-galactose: Ceramide galactosyltransferase gene

    Energy Technology Data Exchange (ETDEWEB)

    Coetzee, T.; Fujita, N.; Marcus, J. [Univ. of North Carolina, Chapel Hill, NC (United States)] [and others

    1996-07-01

    UDP-galactose:ceramide galactosyltransferase (CGT) (EC 2.11.62) catalyzes the final step in the synthesis of galactocerebroside, a glycosphingolipid characteristically abundant in myelin. In this report, we describe the isolation of genomic clones spanning the mouse CGT gene. The mouse CGT gene consists of six exons that span a minimum of 70 kb of DNA and that encode a 541 amino acid translation product with extensive sequence similarity to the rat CGT enzyme and to UDP-glucuronosyltransferases (UGT). The 5{prime}-untranslated region of the mouse CGT gene is encoded by a separate exon located approximately 25 kb upstream of the first protein-encoding exon. Furthermore, the genomic organization of the five encoding region exons of the mouse CGT gene resembles that of the human UGT1 and rat UGT2B1 genes. Finally, analysis of somatic cell hybrids by PCR and fluorescence in situ hybridization to metaphase chromosomes has localized the mouse CGT gene to chromosome 3, bands E3-F1. 26 refs., 5 figs., 1 tab.

  8. Cloning and characterization of an up-regulated GA 20-oxidase gene in hybrid maize

    Institute of Scientific and Technical Information of China (English)

    Jinkun Du; Yingyin Yao; Zhongfu Ni; Qixin Sun

    2009-01-01

    Previous studies revealed that GA content and metabolism are positively correlated with a faster shoot growth rate of hybrid, and recently, genes participating in both GA biosynthesis and GA response pathways were also found to be differentially expressed between wheat hybrid and its parental inbreds. In this study, an up-regulated GA 20-oxidase gene in a maize hybrid, designated ZmGA20, was cloned. ZmGA20 contains an open reading frame (ORF) encoding 391 amino acid residues. BLASTX searches in GenBank revealed that the ZmGA20 is homologous to the sequences of GA20ox proteins from different species, and analysis also indicated that ZmGA20 had typical features of GA 20-oxidase proteins, including a "LPWKET" sequence. Semi-quantitative RT-PCR analysis showed that ZmGA20 was expressed in different tissues and/or organs. The expression level of ZmGA20 in the hybrid was higher than that in two parents (in roots, leaves, stems and embryo, and ears). The abundance of ZmGA20 transcript was equal to that of the highly expressed parents, which provided molecular evidence for the observed GA content heterosis in maize hybrids.

  9. Molecular Cloning and Characterization of G Alpha Proteins from the Western Tarnished Plant Bug, Lygus hesperus

    Directory of Open Access Journals (Sweden)

    J. Joe Hull

    2014-12-01

    Full Text Available The Gα subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-based cloning methods and transcriptome data mining to identity Gα subunits in the western tarnished plant bug (Lygus hesperus Knight. Among the nine sequences identified were single variants of the Gαi, Gαo, Gαs, and Gα12 subfamilies and five alternative splice variants of the Gαq subfamily. Sequence alignment and phylogenetic analyses of the putative L. hesperus Gα subunits support initial classifications and are consistent with established evolutionary relationships. End-point PCR-based profiling of the transcripts indicated head specific expression for LhGαq4, and largely ubiquitous expression, albeit at varying levels, for the other LhGα transcripts. All subfamilies were amplified from L. hesperus chemosensory tissues, suggesting potential roles in olfaction and/or gustation. Immunohistochemical staining of cultured insect cells transiently expressing recombinant His-tagged LhGαi, LhGαs, and LhGαq1 revealed plasma membrane targeting, suggesting the respective sequences encode functional G protein subunits.

  10. Gene cloning and characterization of a novel esterase from activated sludge metagenome

    Directory of Open Access Journals (Sweden)

    Liu Zhi-Pei

    2009-12-01

    Full Text Available Abstract A metagenomic library was prepared using pCC2FOS vector containing about 3.0 Gbp of community DNA from the microbial assemblage of activated sludge. Screening of a part of the un-amplified library resulted in the finding of 1 unique lipolytic clone capable of hydrolyzing tributyrin, in which an esterase gene was identified. This esterase/lipase gene consists of 834 bp and encodes a polypeptide (designated EstAS of 277 amino acid residuals with a molecular mass of 31 kDa. Sequence analysis indicated that it showed 33% and 31% amino acid identity to esterase/lipase from Gemmata obscuriglobus UQM 2246 (ZP_02733109 and Yarrowia lipolytica CLIB122 (XP_504639, respectively; and several conserved regions were identified, including the putative active site, HSMGG, a catalytic triad (Ser92, His125 and Asp216 and a LHYFRG conserved motif. The EstAS was overexpressed, purified and shown to hydrolyse p-nitrophenyl (NP esters of fatty acids with short chain lengths (≤ C8. This EstAS had optimal temperature and pH at 35°C and 9.0, respectively, by hydrolysis of p-NP hexanoate. It also exhibited the same level of stability over wide temperature and pH ranges and in the presence of metal ions or detergents. The high level of stability of esterase EstAS with its unique substrate specificities make itself highly useful for biotechnological applications.

  11. Molecular cloning, expression and characterization of acylpeptide hydrolase in the silkworm, Bombyx mori.

    Science.gov (United States)

    Fu, Ping; Sun, Wei; Zhang, Ze

    2016-04-10

    Acylpeptide hydrolase (APH) can catalyze the release of the N-terminal amino acid from acetylated peptides. There were many documented examples of this enzyme in various prokaryotic and eukaryotic organisms. However, knowledge about APH in insects still remains unknown. In this study, we cloned and sequenced a putative silkworm Bombyx mori APH (BmAPH) gene. The BmAPH gene encodes a protein of 710 amino acids with a predicted molecular mass of 78.5kDa. The putative BmAPH and mammal APHs share about 36% amino acid sequence identity, yet key catalytic residues are conserved (Ser566, Asp654, and His686). Expression and purification of the recombinant BmAPH in Escherichia coli showed that it has acylpeptide hydrolase activity toward the traditional substrate, Ac-Ala-pNA. Furthermore, organophosphorus (OP) insecticides, chlorpyrifos, phoxim, and malathion, significantly inhibited the activity of the APH both in vitro and in vivo. In addition, BmAPH was expressed in all tested tissues and developmental stages of the silkworm. Finally, immunohistochemistry analysis showed that BmAPH protein was localized in the basement membranes. These results suggested that BmAPH may be involved in enhancing silkworm tolerance to the OP insecticides. In a word, our results provide evidence for understanding of the biological function of APH in insects. PMID:26778207

  12. Cloning and characterization of the glutamate dehydrogenase gene in Bacillus licheniformis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The gdhA genes of IRC-3 GDH-strain and IRC-8 GDH+ strain were cloned,and they both successfully complemented the nutritional lesion of an E.coli glutamate auxotroph,Q100 GDH-.However,the gdhA gene from the mutant IRC-8 GDH+ strain failed to complement the glutamate deficiency of the wild type strain IRC-3.The gdhA genes of the wild type and mutant origin were sequenced separately.No nucleotide difference was detected between them.Further investigations indicated that the gdhA genes were actively expressed in both the wild type and the mutant.Additionally,no GDH inhibitor was found in the wild type strain IRC-3.It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of the gdhA expression.Examination of the deduced amino acid sequence of Bacillus licheniformis GDH revealed the presence of the motifs characteristic of the familyⅠ-type hexameric protein,while the GDH of Bacillus subtilis belongs to family II.

  13. Cloning and characterization of the glutamate dehydrogenase gene in Bacillus licheniformis

    Institute of Scientific and Technical Information of China (English)

    朱冰; 俞冠翘; 朱家璧; 沈善炯

    2000-01-01

    The gdhA genes of IRC-3 GDH strain and IRC-8 GDH+ strain were cloned, and they both successfully complemented the nutritional lesion of an E. coli glutamate auxotroph, Q100 GDH". However, the gdhA gene from the mutant IRC-8 GDH+ strain failed to complement the glutamate deficiency of the wild type strain IRC-3. The gdhA genes of the wild type and mutant origin were sequenced separately. No nucleotide difference was detected between them. Further investigations indicated that the gdhA genes were actively expressed in both the wild type and the mutant. Additionally, no GDH inhibitor was found in the wild type strain IRC-3. It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of the gdhA expression. Examination of the deduced amino acid sequence of Bacillus licheniformis GDH revealed the presence of the motifs characteristic of the family I -type hexameric protein, while the GDH of Bacillus subtilis belongs to family II.

  14. Cloning and characterization of an Eimeria acervulina sporozoite gene homologous to aspartyl proteinases.

    Science.gov (United States)

    Laurent, F; Bourdieu, C; Kaga, M; Chilmonczyk, S; Zgrzebski, G; Yvoré, P; Péry, P

    1993-12-01

    A lambda ZapII cDNA library was constructed using mRNA from Eimeria acervulina sporulated oocysts and screened with monoclonal antibodies raised against Eimeria tenella sporulated oocytes. Monoclonal antibody N3C8B12 identified a clone (6S2) potentially encoding an aspartyl proteinase since significant homology with cathepsin D, pepsin and renin proteinases was revealed by sequence comparisons. The 1500-bp cDNA fragment containing the coccidial gene was subcloned into pGEX-FA expression vector, leading to the production of an 80-kDa fusion protein (FA6S2) which was used to immunize rabbits. The anti-FA6S2 rabbit sera revealed a single 43-kDa protein present in Eimeria acervulina, Eimeria tenella, Eimeria maxima and Eimeria falciformis sporulated oocyst antigens. Indirect immunofluorescence and electron microscopy with mAb N3C8B12 localized the putative aspartyl proteinase in the refractile bodies of Eimeria tenella sporozoites.

  15. Cloning of the Bacillus thuringiensis serovar sotto chitinase (Schi gene and characterization of its protein

    Directory of Open Access Journals (Sweden)

    Wan-Fang Zhong

    2005-12-01

    Full Text Available Chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. We used touchdown PCR to clone the chitinase (Schi gene from Bacillus thuringiensis serovar sotto (Bt sotto chromosomal DNA. Our DNA sequencing analysis revealed that the Bt sotto Schi gene consists of an open reading frame (ORF of 2067 nucleotides with codes for the chitinase precursor. We also found that the putative promoter consensus sequences (the -35 and -10 regions of the Bt soto Schi gene are identical to those of the chiA71 gene from Bt Pakistani, the chiA74 gene from Bt kenyae and the ichi gene from Bt israelensis. The Schi chitinase precursor is 688 amino acids long with an estimated molecular mass of 75.75 kDa and a theoretical isoelectric point of 5.74, and contains four domains, which are, in sequence, a signal peptide, an N-terminal catalytic domain, a fibronectin type III like domain and a C-terminal chitin-binding domain. Sequence comparison and the evolutionary relationship of the Bt sotto Schi chitinase to other chitinase and chitinase-like proteins are also discussed.

  16. Molecular cloning and characterization of violaxanthin de-epoxidase (CsVDE in cucumber.

    Directory of Open Access Journals (Sweden)

    Xin Li

    Full Text Available Violaxanthin de-epoxidase (VDE plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V to intermediate product antheraxanthin (A and final product zeaxanthin (Z under high light stress. We have cloned a violaxanthin de-epoxidase gene (CsVDE from cucumber. The amino acid sequence of CsVDE has high homology with VDEs in other plants. RT-PCR analysis and histochemical staining show that CsVDE is expressed in all green tissues in cucumber and Arabidopsis. Using GFP fusion protein and immunogold labeling methods, we show that CsVDE is mainly localized in chloroplasts in cucumber. Under high light stress, relative expression of CsVDE and the de-epoxidation ratio (A+Z/(V+A+Z is increased rapidly, and abundance of the gold particles was also increased. Furthermore, CsVDE is quickly induced by cold and drought stress, reaching maximum levels at the 2(nd hour and the 9(th day, respectively. The ratio of (A+Z/(V+A+Z and non-photochemical quenching (NPQ is reduced in transgenic Arabidopsis down-regulated by the antisense fragment of CsVDE, compared to wild type (WT Arabidopsis under high light stress. This indicates decreased functionality of the xanthophyll cycle and increased sensitivity to photoinhibition of photosystem II (PSII in transgenic Arabidopsis under high light stress.

  17. Cloning and characterization of the goadsporin biosynthetic gene cluster from Streptomyces sp. TP-A0584.

    Science.gov (United States)

    Onaka, Hiroyasu; Nakaho, Mizuho; Hayashi, Keiko; Igarashi, Yasuhiro; Furumai, Tamotsu

    2005-12-01

    The biosynthetic gene cluster of goadsporin, a polypeptide antibiotic containing thiazole and oxazole rings, was cloned from Streptomyces sp. TP-A0584. The cluster contains a structural gene, godA, and nine god (goadsporin) genes involved in post-translational modification, immunity and transcriptional regulation. Although the gene organization is similar to typical bacteriocin biosynthetic gene clusters, each goadsporin biosynthetic gene shows low homology to these genes. Goadsporin biosynthesis is initiated by the translation of godA, and the subsequent cyclization, dehydration and acetylation are probably catalysed by godD, godE, godF, godG and godH gene products. godI shows high similarity to the 54 kDa subunit of the signal recognition particle and plays an important role in goadsporin immunity. Furthermore, four goadsporin analogues were produced by site-directed mutagenesis of godA, suggesting that this biosynthesis machinery is used for the heterocyclization of peptides. PMID:16339937

  18. Cloning and characterization of the Cu/Zn superoxide dismutase of Trichinella pseudospiralis.

    Science.gov (United States)

    Wu, W K; Mak, C H; Ko, R C

    2006-03-01

    Copper/zinc (Cu/Zn) superoxide dismutase (SOD) activity was identified for the first time in both crude somatic extracts (CE) and excretory/secretory (E/S) products of Trichinella pseudospiralis. It was the dominant SOD in infective-stage larvae. Native polyacrylamide gel electrophoresis of CE and E/S products yielded a prominent band, which was cyanide-sensitive and was partly inhibited by hydrogen peroxide in SOD assay. Cytosolic Cu/Zn SOD was cloned. The 471-bp full-length cDNA sequence contained an open reading frame of 157 amino acids. The gene contained three introns. Quantitative reverse transcription-polymerase chain reaction indicated that the expression of cytosolic Cu/Zn SOD was substantially higher in infective-stage larvae than in adult worms. Cluster analysis showed that the sequence of the Cu/Zn SOD of T. pseudospiralis, an adenophorean nematode, is related to those of Brugia pahangi, Acanthocheilonema viteae, Onchocerca volvulus, and Haemonchus contortus (all belonging to the sercenentean group). PMID:16341881

  19. Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species.

    Science.gov (United States)

    Arnórsdottir, Jóhanna; Smáradóttir, Rúna B; Magnússon, Olafur Th; Thorbjarnardóttir, Sigrídur H; Eggertsson, Gudmundur; Kristjánsson, Magnús M

    2002-11-01

    The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli. The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa. The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence. Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity. The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases. With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same. Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio-proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues. The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index. These factors may contribute to the adaptation of these proteinases to different temperature conditions.

  20. Bacterial phytoene synthase: molecular cloning, expression, and characterization of Erwinia herbicola phytoene synthase.

    Science.gov (United States)

    Iwata-Reuyl, Dirk; Math, Shivanand K; Desai, Shrivallabh B; Poulter, C Dale

    2003-03-25

    Phytoene synthase (PSase) catalyzes the condensation of two molecules of geranylgeranyl diphosphate (GGPP) to give prephytoene diphosphate (PPPP) and the subsequent rearrangement of the cyclopropylcarbinyl intermediate to phytoene. These reactions constitute the first pathway specific step in carotenoid biosynthesis. The crtB gene encoding phytoene synthase was isolated from a plasmid containing the carotenoid gene cluster in Erwinia herbicola and cloned into an Escherichia coli expression system. Upon induction, recombinant phytoene synthase constituted 5-10% of total soluble protein. To facilitate purification of the recombinant enzyme, the structural gene for PSase was modified by site-directed mutagenesis to incorporate a C-terminal Glu-Glu-Phe (EEF) tripepetide to allow purification by immunoaffinity chromatography on an immobilized monoclonal anti-alpha-tubulin antibody YL1/2 column. Purified recombinant PSase-EEF gave a band at 34.5 kDa upon SDS-PAGE. Recombinant PSase-EEF was then purified to >90% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The enzyme required Mn(2+) for activity, had a pH optimum of 8.2, and was strongly stimulated by detergent. The concentration of GGPP needed for half-maximal activity was approximately 35 microM, and a significant inhibition of activity was seen at GGPP concentrations above 100 microM. The sole product of the reaction was 15,15'-Z-phytoene. PMID:12641468

  1. Molecular Cloning and Characterization of Fruit Softening Related Gene Mannanase from Banana Fruit

    Institute of Scientific and Technical Information of China (English)

    ZHUANG Jun-ping; SU Jing; CHEN Wei-xin

    2006-01-01

    A 1 250 bp cDNA fragment encoding β-mannanase, named MaMAN, was cloned from banana (Musa spp cv. Baxi) fruit using degenerate primers designed with reference to the conserved nucleic acid sequences of known β-mannanase genes by RT-PCR. Sequence analysis showed that MaMAN cDNA encompassed a 1 085 bp open-reading frame (ORF), encoding a predicted polypeptide of 395 amino acids. Alignment of the deduced amino acid sequence of MaMAN and other putative β-mannanases showed that MaMAN has an identity of 86, 70, 69, 54, and 57%, respectively, to β-mannanases from tomato, lettuce, arabidopsis, carrot and oryza sativa. The catalytic residues: Asn203, Glu204, Glu318 and the active site residues: Arg86, His277, Tyr279, and Trp360, which were strictly conserved in the glycoside hydrolase family 5 to which all 3-mannanases belonged, were found in MaMAN. Semi-quantitative RT-PCR revealed that the level of MaMAN transcript in the pulp increased during banana fruit ripening, suggesting that MaMAN was likely to be involved highly in banana fruit softening.

  2. Cloning and characterization of a FLORICAULA/LEAFY ortholog, PFL, in polygamous papaya

    Institute of Scientific and Technical Information of China (English)

    Qingyi YU; Paul H. MOORE; Henrik H. ALBERT; Adrienne H.K. ROADER; Ray MING

    2005-01-01

    The homologous genes FLORICAULA (FLO) in Antirrhinum and LEAFY (LFY) in Arabidopsis are known to regulate the initiation of flowering in these two distantly related plant species. These genes are necessary also for the expression of downstream genes that control floral organ identity. We used Arabidopsis LFY cDNA as a probe to clone and sequence a papaya ortholog of LFY, PFL. It encodes a protein that shares 61% identity with the Arabidopsis LFY gene and 71% identity with the LFYhomologs of the two woody tree species: California sycamore (Platanus racemosa) and black cottonwood (Populus trichocarpa). Despite the high sequence similarity within two conserved regions, the N-terminal proline-rich motif in papaya PFL differs from other members in the family. This difference may not affect the gene function of papaya PFL, since an equally divergent but a functional LFY ortholog NEEDLY of Pinus radiata has been reported. Genomic and BAC Southern analyses indicated that there is only one copy of PFL in the papaya genome. In situ hybridization experiments demonstrated that PFL is expressed at a relatively low level in leaf primordia,but it is expressed at a high level in the floral meristem. Quantitative PCR analyses revealed that PFL was expressed in flower buds of all three sex types - male, female, and hermaphrodite with marginal difference between hermaphrodite and unisexual flowers. These data suggest that PFL may play a similar role as LFY in flower development and has limited effect on sex differentiation in papaya.

  3. Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    L. Avilán

    1997-12-01

    Full Text Available We cloned the streptokinase (STK gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

  4. Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

    Directory of Open Access Journals (Sweden)

    Bin Yang

    Full Text Available BACKGROUND: There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. METHODOLOGY/PRINCIPAL FINDINGS: We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. CONCLUSIONS/SIGNIFICANCE: Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

  5. Cloning and characterization of two putative seven-transmembrane receptor genes from cotton

    Institute of Scientific and Technical Information of China (English)

    Peng Gao; Piming Zhao; Juan Wang; Haiyun Wang; Guiling Wang; Guixian Xia

    2008-01-01

    Using rapid amplification of cDNA ends (RACE)-PCR,two full-length cDNAs encoding putative seven-transmembrane receptors (designated Gh7TMpR1 and Gh7TMpR2) were cloned from cotton plants.Southern blot and an ApaLl restriction site polymorphism analyses revealed that GhTTMpR1 was derived from the ancestral A diploid genome,while Gh7TMpR2 was from the D subgenome.Northern blot hybridization indicated that both Gh7TMpR1 and Gh7TMpR2 were expressed preferentially in the elongation phase of fiber development.Majority of the Gh7TMpR1 proteins were located within the membrane structure and displayed a punctuate pattern of distribution.Overexpression of Gh7TMpR1 in fission yeast disrupted the polar growth and caused the formation of rounded cells.These results suggest that GhT7MpRI may play a critical role in cotton fiber development,perhaps as a signaling receptor that is involved in controlling fiber elongation.

  6. Cloning and characterization of a sucrose isomerase from Erwinia rhapontici NX-5 for isomaltulose hyperproduction.

    Science.gov (United States)

    Li, Sha; Cai, Heng; Qing, Yujia; Ren, Ben; Xu, Hong; Zhu, Hongyang; Yao, Jun

    2011-01-01

    The sucrose isomerase (SIase) gene from an efficient strain of Erwinia rhapontici NX-5 for isomaltulose hyperproduction was cloned and overexpressed in Escherichia coli. Protein sequence alignment revealed that SIase was a member of the glycoside hydrolase 13 family. The molecular mass of the purified recombinant protein was estimated at 66 kDa by SDS-PAGE. The SIase had an optimal pH and temperature of 5.0 and 30 °C, respectively, with a K (m) of 257 mmol/l and V (max) of 48.09 μmol/l/s for sucrose. To the best of our knowledge, the recombinant SIase has the most acidic optimum pH for isomaltulose synthesis. When the recombinant E. coli (pET22b- palI) cells were used for isomaltulose synthesis, almost complete conversion of sucrose (550 g/l solution) to isomaltulose was achieved in 1.5 h with high isomaltulose yields (87%). The immobilized E. coli cells remained stable for more than 30 days in a "batch"-type enzyme reactor. This indicated that the recombinant SIase could continuously and efficiently produce isomaltulose.

  7. Cloning and characterization of a phosphate transporter gene in Dunaliella salina.

    Science.gov (United States)

    Li, Shao-He; Xia, Bing-Bing; Zhang, Chi; Cao, Jiao; Bai, Lin-Han

    2012-08-01

    The full-length cDNA of a Na(+) -dependent Pi transport gene (DsSPT1) in Dunaliella salina was cloned by 3' and 5' Rapid Amplification of cDNA Ends (RACE), with an open reading frame (ORF) encoding 716 predicted amino acids, which exhibited 60.5% identity to that of Na(+) -dependent Pi transport 1 (DvSPT1) from Dunaliella viridis. Hydrophobicity and secondary structure prediction revealed 11 conserved transmembrane domains similar to those found in DvSPT1 from D. viridis and PHO89 from Saccharomyces cerevisiae. The result of real-time quantitative PCR showed that expression level of DsSPT1 was enhanced at first and reached its peak at 90 min after salt stress; however, D. salina cells rapidly absorbed extracellular inorganic phosphorus which was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) during the first 5 min under salt stress. It suggested that D. salina on the absorption of inorganic phosphorus was regulated at DsSPTI posttranslational level. PMID:22052620

  8. Molecular cloning and characterization of a trehalose-6-phosphate synthase/phosphatase from Dunaliella viridis.

    Science.gov (United States)

    Zhang, Nan; Wang, Fei; Meng, Xiangzong; Luo, Saifan; Li, Qiyun; Dong, Hongyun; Xu, Zhengkai; Song, Rentao

    2011-04-01

    Dunaliella is a group of green algae with exceptional stress tolerance capability, and is considered as an important model organism for stress tolerance study. Here we cloned a TPS (trehalose-6-phosphate synthase) gene from Dunaliella viridis and designated it as DvTPS (D. viridis trehalose-6-phosphate synthase/phosphatase).The DvTPS cDNA contained an ORF of 2793 bp encoding 930 aa. DvTPS had both TPS and TPP domain and belonged to the Group II TPS/TPP fusion gene family. Southern blots showed it has a single copy in the genome. Genome sequence analysis revealed that it has 18 exons and 17 introns. DvTPS had a constitutive high expression level under various NaCl culture conditions, however, could be induced by salt shock. Promoter analysis indicated there were ten STREs (stress response element) in its promoter region, giving a possible explanation of its inducible expression pattern upon salt shock. Yeast functional complementation analysis showed that DvTPS had neither TPS nor TPP activity. However, DvTPS could improve the salt tolerance of yeast salt sensitive mutant G19. Our results indicated that despite DvTPS showed significant similarity with TPS/TPP, its real biological function is still remained to be revealed. PMID:20878239

  9. Molecular Cloning and Characterization of G Alpha Proteins from the Western Tarnished Plant Bug, Lygus hesperus.

    Science.gov (United States)

    Hull, J Joe; Wang, Meixian

    2014-01-01

    The Gα subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-based cloning methods and transcriptome data mining to identity Gα subunits in the western tarnished plant bug (Lygus hesperus Knight). Among the nine sequences identified were single variants of the Gαi, Gαo, Gαs, and Gα12 subfamilies and five alternative splice variants of the Gαq subfamily. Sequence alignment and phylogenetic analyses of the putative L. hesperus Gα subunits support initial classifications and are consistent with established evolutionary relationships. End-point PCR-based profiling of the transcripts indicated head specific expression for LhGαq4, and largely ubiquitous expression, albeit at varying levels, for the other LhGα transcripts. All subfamilies were amplified from L. hesperus chemosensory tissues, suggesting potential roles in olfaction and/or gustation. Immunohistochemical staining of cultured insect cells transiently expressing recombinant His-tagged LhGαi, LhGαs, and LhGαq1 revealed plasma membrane targeting, suggesting the respective sequences encode functional G protein subunits. PMID:26463065

  10. Cloning and Characterization of Porcine TSARG7 Gene and Analysis of Its Tissue-Specific Expression

    Institute of Scientific and Technical Information of China (English)

    LI Mei-li; LI Gui-qiang; FANG Wei; WANG Wei; SONG Xiao-guang; LI Er-lin; JIA Chao; XU Yin-xue

    2009-01-01

    TSARG7 is a novel member of the acyltransferase family since its sequence possesses the highly conserved phosphate acyltransferase (PIsC) domain existing in all acyltransferase-like proteins. The porcine TSARG7 had been identified by cloning in silico but had not been confirmed experimentally. The full-length mRNA of porcine TSARG7 gene was sequenced and two splice variants were discovered. The full-length cDNA of TSARG7 variant 1 was 2 513 bp and variant 2 was 2 634 bp. The putative porcine TSARG7 proteins, which were located in the cytoplasm, encoded 458 and 456 amino acids, respectively. Real-time PCR analysis showed that TSARG7 gene was expressed in various tissues, but at different levels. The expression levels of this gene were higher in the skeletal muscle, heart, and testis than that in other tissues, suggesting that the TSARG7 gene played a role in procine skeletal muscle, heart, and testis functions.

  11. Purification, characterization, and cDNA cloning of opine dehydrogenases from the polychaete rockworm Marphysa sanguinea.

    Science.gov (United States)

    Endo, Noriyuki; Kan-no, Nobuhiro; Nagahisa, Eizoh

    2007-06-01

    Alanopine dehydrogenase (AlDH) and three isoforms of strombine/alanopine dehydrogenase (St/AlDH) were purified from muscle tissue of the polychaete rockworm Marphysa sanguinea. The four enzymes, which can be distinguished by the isoelectric point, are monomeric 42 kDa proteins, possess similar pH-activity profiles, and display specificity for pyruvate and NAD(H). The three isoforms of St/AlDH show equivalent Km and Vmax for glycine and L-alanine and for D-strombine and meso-alanopine. Free amino acid levels in the muscle and D-strombine accumulation in vivo during muscle activity suggest that St/AlDHs function physiologically as StDH. AlDH shows specificity for L-alanine and meso-alanopine, but not for glycine or D-strombine. The amino acid sequences of AlDH and one of the St/AlDH isoforms were determined by a combination of amino acid sequence analysis and cDNA cloning. St/AlDH cDNA consisted of 1586 bp nucleotides that encode a 399-residue protein (43,346.70 Da), and AlDH cDNA consisted of 1587 bp nucleotides that encode a 399-residue protein (43,886.68 Da). The two amino acid sequences deduced from the cDNA displayed 67% amino acid identity, with greatest similarity to that of tauropine dehydrogenase from the polychaete Arabella iricolor. PMID:17350870

  12. Characterization, cDNA cloning and expression pattern of relaxin gene during embryogenesis of Danio rerio.

    Science.gov (United States)

    Fiengo, Marcella; Donizetti, Aldo; del Gaudio, Rosanna; Minucci, Sergio; Aniello, Francesco

    2012-06-01

    We report the identification, the cDNA cloning, the temporal and spatial expression pattern analysis of the rln gene in the zebrafish Danio rerio. The deduced Rln B and A domains show different evolutionary conservation. Rln B domain shows higher similarity when compared to zebrafish and human RLN3 B domain than human RLN1 and RLN2 B domain. Differently, the zebrafish Rln A domain shows relatively low amino acid sequence similarity when compared with the same sequences. The rln gene is transcribed both during embryogenesis and in adult organism, where higher transcript level has been particularly evidenced in the brain. Moreover, we provide the first description of rln spatial expression pattern during embryonic development. In particular, we show restricted transcript localization starting at the pharyngula stage in olfactory placode, branchial arch region, and in a cell cluster near to otic vesicle. In larval stage, new transcription territories have been detected in both neural and non-neural regions. In particular, in the brain, rln expression has been revealed in telencephalic region around anterior commissure, in the preoptic area, and in restricted rombencephalic cell clusters. Expression of rln gene in extra-neural territories has been detected in the pancreatic and thyroid gland regions. Danio rerio rln expression pattern analysis reveals shared features with the mammalian RLN gene, particularly in the brain, where it might have a role in the neurophysiological processes. In addition, expression in the thyroid and pancreas region suggests a function as a paracrine and endocrine hormone.

  13. Vacuolar invertases in sweet potato: molecular cloning, characterization, and analysis of gene expression.

    Science.gov (United States)

    Wang, Li-Ting; Wang, Ai-Yu; Hsieh, Chang-Wen; Chen, Chih-Yu; Sung, Hsien-Yi

    2005-05-01

    Two cDNAs (Ib beta fruct2 and Ib beta fruct3) encoding vacuolar invertases were cloned from sweet potato leaves, expressed in Pichia pastoris, and the recombinant proteins were purified by ammonium sulfate fractionation and chromatography on Ni-NTA agarose. The deduced amino acid sequences encoded by the cDNAs contained characteristic conserved elements of vacuolar invertases, including the sequence R[G/A/P]xxxGVS[E/D/M]K[S/T/A/R], located in the prepeptide region, Wxxx[M/I/V]LxWQ, located around the starting site of the mature protein, and an intact beta-fructosidase motif. The pH optimum, the substrate specificity, and the apparent K(m) values for sucrose exhibited by the recombinant proteins were similar to those of vacuolar invertases purified from sweet potato leaves and cell suspensions, thus confirming that the proteins encoded by Ib beta fruct2 and Ib beta fruct3 are vacuolar invertases. Moreover, northern analysis revealed that the expression of the two genes was differentially regulated. With the exception of mature leaves and sprouting storage roots, Ib beta fruct2 mRNA is widely expressed among the tissues of the sweet potato and is more abundant in young sink tissues. By contrast, Ib beta fruct3 mRNA was only detected in shoots and in young and mature leaves. It appears, therefore, that these two vacuolar invertases play different physiological roles during the development of the sweet potato plant.

  14. Molecular cloning and characterization of a cotton phosphoen01pyruVate carboxylase gene

    Institute of Scientific and Technical Information of China (English)

    Zhixin Qiao; Jin-Yuan Liu

    2008-01-01

    Phosphoenolpyruvate carboxylase(PEPC)plays diverse physiological functions during plant development.In this study,a new phosphoenolpyruvate carboxylase gene GhPEPC2 is isolated from cotton(Gossypium hirsutum CV.zhongmian 35)by RACE-PCR.The cloned eDNA of GhPEPC2 is 3364 bp in length,and has an open reading frame of 2913 bp,encoding for 971 putative amino acids with a calculated molecular mass of 110.6 kD and pI of 5.56.The deduced amino acid sequence Of GhPEPC2 shares high similarity with other reported plant PEPCs.Southern blot analysis indicates that the cotton PEPC exists as a small gene family and the GhPEPC2 might have two copies in the cotton genome.The semi-quantitative RT-PCR reveals that GhPEPC2 constitutively expresses in all the tissues of cotton and accumulated highly in roots.flowers and embryos but relatively low in stems and fibers.In addition.the recombinant GhPEPC2 has been purified by expressing it in Escherichia coli and the catalytic properties of it were also investigated.The results showed that GhPEPC2 is a typical C3 PEPC with a higher Km(83.6 μM)and lower Vmax(8.0 μmol min-1mg-1)compared with the C3 PEPCs previously reported.

  15. Cloning, expression, purification, and characterization of the catalytic domain of sika deer MMP-13.

    Science.gov (United States)

    Zhang, Xueliang; Wang, Jiawen; Liu, Meichen; Wang, Siming; Zhang, Hui; Zhao, Yu

    2016-11-01

    Matrix metalloproteinase 13 is one of three mammalian collagenases that are capable of initiating the degradation of interstitial collagens during wound healing. Herein, we report for the first time the molecular cloning of the catalytic domain (CD) of sika deer MMP-13, followed by protein expression in Escherichia coli and purification by affinity chromatography. The final yield was approximately 90.4 mg per liter of growth culture with a purity of 91.6%. The mass recovery during the purification and renaturation were 70.2% and 81.5%, respectively. Using gelatin zymography and a degradation assay, we found that the refolded sika deer MMP-13 (CD) could digest gelatin. The optimal pH and temperature for the enzyme bioactivity was 8.0 and 37 °C, respectively. The Km value for the enzyme-catalyzed digestion of gelatin was 136+/-8 μg/mL, and the Vmax was 4.12 × 10(3) U/μg. sdMMP13 (CD) was able to completely degrade collagen II and gelatin, and partially degrade fibronectin. The sdMMP-13 (CD) activity was significantly inhibited by several chemicals including 1, 10-phenanthroline, EDTA, Fe(2+), Cu(2+), and Mn(2+). PMID:27338011

  16. Production, characterization, gene cloning, and nematocidal activity of the extracellular protease from Stenotrophomonas maltophilia N4.

    Science.gov (United States)

    Jankiewicz, Urszula; Larkowska, Ewa; Swiontek Brzezinska, Maria

    2016-06-01

    A rhizosphere strain of the bacterium Stenotrophomonas maltophilia N4 secretes the serine protease PN4, whose molecular mass is approximately 42 kDa. The optimal temperature for the enzyme activity of the 11-fold purified protein was 50°C and the optimal pH was 10.5. The activity of the enzyme was strongly inhibited by specific serine protease inhibitors, which allowed for its classification as an alkaline serine protease family. Ca(2+) ions stimulated the activity of the protease PN4, while Mg(2+) ions stabilized its activity, and Zn(2+) and Cd(2+) ions strongly inhibited its activity. The enzyme has broad substrate specificity. For example, it is able to hydrolyse casein, keratin, albumin, haemoglobin, and gelatin, as well as the insoluble modified substrates azure keratin and azocoll. The gene that encodes the 1740 bp precursor form of the enzyme (accession number: LC031815) was cloned. We then deduced that its amino acid sequence includes the region of the conserved domain of the S8 family of peptidases as well as the catalytic triad Asp/His/Ser. The bacterial culture fluid as well as the purified protease PN4 demonstrated biocidal activity with regard to the nematodes Caenorhabditis elegans and Panagrellus spp. PMID:26896861

  17. Reference Inflow Characterization for River Resource Reference Model (RM2)

    Energy Technology Data Exchange (ETDEWEB)

    Neary, Vincent S [ORNL

    2011-12-01

    Sandia National Laboratory (SNL) is leading an effort to develop reference models for marine and hydrokinetic technologies and wave and current energy resources. This effort will allow the refinement of technology design tools, accurate estimates of a baseline levelized cost of energy (LCoE), and the identification of the main cost drivers that need to be addressed to achieve a competitive LCoE. As part of this effort, Oak Ridge National Laboratory was charged with examining and reporting reference river inflow characteristics for reference model 2 (RM2). Published turbulent flow data from large rivers, a water supply canal and laboratory flumes, are reviewed to determine the range of velocities, turbulence intensities and turbulent stresses acting on hydrokinetic technologies, and also to evaluate the validity of classical models that describe the depth variation of the time-mean velocity and turbulent normal Reynolds stresses. The classical models are found to generally perform well in describing river inflow characteristics. A potential challenge in river inflow characterization, however, is the high variability of depth and flow over the design life of a hydrokinetic device. This variation can have significant effects on the inflow mean velocity and turbulence intensity experienced by stationary and bottom mounted hydrokinetic energy conversion devices, which requires further investigation, but are expected to have minimal effects on surface mounted devices like the vertical axis turbine device designed for RM2. A simple methodology for obtaining an approximate inflow characterization for surface deployed devices is developed using the relation umax=(7/6)V where V is the bulk velocity and umax is assumed to be the near-surface velocity. The application of this expression is recommended for deriving the local inflow velocity acting on the energy extraction planes of the RM2 vertical axis rotors, where V=Q/A can be calculated given a USGS gage flow time

  18. Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis.

    Science.gov (United States)

    Guo, Xi-Zhi J; Dash, Pradyot; Calverley, Matthew; Tomchuck, Suzanne; Dallas, Mari H; Thomas, Paul G

    2016-01-01

    Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αβTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αβ and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα(-)β(-)) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics. PMID:26858965

  19. Cloning and characterization of the gene product of the form II ribulose-1,5-bisphosphate carboxylase gene of Rhodopseudomonas sphaeroides.

    OpenAIRE

    Muller, E D; Chory, J; Kaplan, S

    1985-01-01

    We report the cloning and characterization of the gene product of the gene for the form II ribulose bisphosphate carboxylase from Rhodopseudomonas sphaeroides. We present evidence that the form II enzyme is encoded by a single gene in R. sphaeroides; however, this gene does hybridize to a second chromosomal locus.

  20. Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

    Science.gov (United States)

    This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

  1. Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus.

    Science.gov (United States)

    Iqbal, Irfana; Aftab, Muhammad Nauman; Afzal, Mohammed; Ur-Rehman, Asad; Aftab, Saima; Zafar, Asma; Ud-Din, Zia; Khuharo, Ateeque Rahman; Iqbal, Jawad; Ul-Haq, Ikram

    2015-02-01

    Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg(-1) , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg(-1) . The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.

  2. Cloning, expression and characterization of potential immunogenic recombinant hemagglutinin-neuraminidase protein of Porcine rubulavirus.

    Science.gov (United States)

    Cuevas-Romero, Julieta Sandra; Rivera-Benítez, José Francisco; Hernández-Baumgarten, Eliseo; Hernández-Jaúregui, Pablo; Vega, Marco; Blomström, Anne-Lie; Berg, Mikael; Baule, Claudia

    2016-12-01

    Blue eye disease caused by Porcine rubulavirus (PorPV) is an endemic viral infection of swine causing neurological and respiratory disease in piglets, and reproductive failure in sows and boars. The hemagglutinin-neuraminidase (HN) glycoprotein of PorPV is the most abundant component in the viral envelope and the main target of the immune response in infected animals. In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by testing against serum samples from pigs experimentally infected with PorPV. The immunogenicity of the rHN-PorPV protein was tested by inoculation of BALB/c mice with AbISCO-100(®) as adjuvant. Analysis of the humoral immune responses in mice showed an increased level of specific antibodies 14 days after the first immunization, compared to the control group (P < 0.0005). The results show the ability of the rHN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the rHN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease.

  3. Molecular cloning, characterization, and bioactivity analysis of interleukin 18 in giant panda (Ailuropoda melanoleuca).

    Science.gov (United States)

    Yan, Y; Wang, Q; Niu, L L; Deng, J B; Yu, J Q; Zhang J X Wang, Y Z; Yin, M M; Tan, X M

    2014-01-01

    Interleukin 18 (IL-18), as a member of IL-1 superfamily, is an important pleiotropic cytokine that modulates Th1 immune responses. In this report, we cloned and identified a homolog of IL-18 in giant panda (Ailuropoda melanoleuca) (designated as AmIL-18) from peripheral blood mononuclear cells stimulated with lipopolysaccharide. The open readin g frame of AmIL-18 cDNA is 579 bp encoding a deduced protein of 192 amino acids. AmIL-18 gDNA fragments contained 5 exons and 4 introns. The amino acid sequence of AmIL-18 shared 23.9 to 87.0% identity with other species. To evaluate the effects of AmIL-18 on the immune response, we expressed the recombinant AmIL-18 in Escherichia coli BL21 (DE3). The fusion protein PET-AmIL-18 was purified by nickel affinity column chromatography and verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. The biological function of purified PET-AmIL-18 was determined on mouse splenocytes by quantitative real-time polymerase chain reaction. INF-γ and other cytokines were increased when stimulated by PET-AmIL-18, particularly when combined with recombinant human interleukin 12, while a Th2-type cytokine, interleukin-4, was strikingly suppressed. These results will provide information for the potential use of recombinant proteins to manipulate the immune response in giant pandas and facilitate the study to protect this treasured species. PMID:25501180

  4. Cloning, Expression, and Characterization of Thermotolerant Manganese Superoxide Dismutase from Bacillus sp. MHS47

    Directory of Open Access Journals (Sweden)

    Supatra Areekit

    2011-01-01

    Full Text Available A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47 was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322, B. anthracis (accession no. NP_846724, B. cereus (accession no. ZP_04187911, B. weihenstephanensis (accession no. YP_001646918, and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169 show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7–8.5, with an optimum pH of 7.5, and at temperatures in the range 30–45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.

  5. Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus.

    Directory of Open Access Journals (Sweden)

    Shiwanthi L Ranasinghe

    2015-12-01

    Full Text Available The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE, a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic.

  6. Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus.

    Science.gov (United States)

    Ranasinghe, Shiwanthi L; Fischer, Katja; Zhang, Wenbao; Gobert, Geoffrey N; McManus, Donald P

    2015-12-01

    The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic. PMID:26645974

  7. Cloning, expression, and physicochemical characterization of a new diheme cytochrome c from Shewanella baltica OS155.

    Science.gov (United States)

    Di Rocco, Giulia; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Borsari, Marco; Ferrari, Erika; Monari, Stefano; Sola, Marco

    2011-03-01

    The 16-kDa diheme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in Escherichia coli and investigated through UV-vis, magnetic circular dichroism, and (1)H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides diheme cytochrome c (Rs-DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three α-helices and a compact overall folding. The C-terminal domain is less helical than the corresponding domain of Rs-DHC. The two heme groups are bridged by Tyr26 in correspondence with the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid-base equilibria with pK (a) values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and a pH-linked conformational change of the protein, respectively. Reduction potentials of -0.144 and -0.257 V (vs. the standard hydrogen electrode), were determined for the C- and N-terminal heme groups, respectively. An approach based on the extended Debye-Hückel equation was applied for the first time to a two-centered metalloprotein and was found to reproduce successfully the ionic strength dependence of E°'. PMID:21161306

  8. Cloning, expression and characterization of potential immunogenic recombinant hemagglutinin-neuraminidase protein of Porcine rubulavirus.

    Science.gov (United States)

    Cuevas-Romero, Julieta Sandra; Rivera-Benítez, José Francisco; Hernández-Baumgarten, Eliseo; Hernández-Jaúregui, Pablo; Vega, Marco; Blomström, Anne-Lie; Berg, Mikael; Baule, Claudia

    2016-12-01

    Blue eye disease caused by Porcine rubulavirus (PorPV) is an endemic viral infection of swine causing neurological and respiratory disease in piglets, and reproductive failure in sows and boars. The hemagglutinin-neuraminidase (HN) glycoprotein of PorPV is the most abundant component in the viral envelope and the main target of the immune response in infected animals. In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by testing against serum samples from pigs experimentally infected with PorPV. The immunogenicity of the rHN-PorPV protein was tested by inoculation of BALB/c mice with AbISCO-100(®) as adjuvant. Analysis of the humoral immune responses in mice showed an increased level of specific antibodies 14 days after the first immunization, compared to the control group (P < 0.0005). The results show the ability of the rHN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the rHN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease. PMID:27496728

  9. Cloning, Characterization, and Expression Profile of Estrogen Receptor in Common Chinese Cuttlefish, Sepiella japonica.

    Science.gov (United States)

    Lü, Zhen-Ming; Liu, Wan; Liu, Li-Qin; Wang, Tian-Ming; Shi, Hui-Lai; Ping, Hong-Ling; Chi, Chang-Feng; Yang, Jing-Wen; Wu, Chang-Wen

    2016-03-01

    Sex steroid hormones are widely detected in molluscs and play important roles in sex determination, gonadal tissue maturation, and gametogenesis. Nevertheless, the signaling pathways of sex steroids in cephalopod have not yet been clearly elucidated. In the present study, a full-length sequence encoding the estrogen receptor (ER) was isolated from common Chinese cuttlefish, Sepiella japonica. The sjER cDNA clone was found to contain 1,788 nucleotides including a 1,470 bp open reading frame encoding 489 amino acid (aa) residues. The deduced ER protein consisted of six nuclear receptor characteristic domains. Based on a phylogenetic analysis, the ER DNA-binding domain and ligand-binding domain are highly conserved compared to other mollusc ERs. Highest aa identities were found for sjER with common octopus (Octopus vulgaris) ER (89%) and pacific oyster (Crassostrea gigas) ER (61%). Tissue expression analysis confirmed that sjER was widely distributed among tissues and predominantly expressed in the brain, liver, gonad (testis and ovary), and other accessory sexual gland (nidamental gland). The ER expression was temporally upregulated in the brain, liver, and ovary during the early sexual maturation period in S. japonica, which is coincident with the fluctuation of ovary estradiol content. These suggest that sjER may be involved in regulating the reproductive cycle of S. japonica. A fusion protein transient transfections assay showed that sjER was mainly located in the nucleus, suggesting a possible orthodox working mechanism of S. japonica ER in the nucleus through a ligand-dependent activation of specific gene transcription. PMID:27076436

  10. Cloning, Purification and Characterization of a Highly Thermostable Amylase Gene of Thermotoga petrophila into Escherichia coli.

    Science.gov (United States)

    Zafar, Asma; Aftab, Muhammad Nauman; ud Din, Zia; Aftab, Saima; Iqbal, Irfana; ul Haq, Ikram

    2016-02-01

    A putative α-amylase gene of Thermotoga petrophila was cloned and expressed in Escherichia coli BL21 (DE3) using pET-21a (+), as an expression vector. The growth conditions were optimized for maximal expression of the α-amylase using various parameters, such as pH, temperature, time of induction and addition of an inducer. The optimum temperature and pH for the maximum expression of α-amylase were 22 °C and 7.0 pH units, respectively. Purification of the recombinant enzyme was carried out by heat treatment method, followed by ion exchange chromatography with 34.6-fold purification having specific activity of 126.31 U mg(-1) and a recovery of 56.25%. Molecular weight of the purified α-amylase, 70 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 100 °C temperature and at pH of 7.0. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA indicating that the α-amylase was a metalloenzyme. However, addition of 1% Tween 20, Tween 80 and β-mercaptoethanol constrained the enzyme activity to 87, 96 and 89%, respectively. No considerable effect of organic solvents (ethanol, methanol, isopropanol, acetone and n-butanol) was observed on enzyme activity. With soluble starch as a substrate, the enzyme activity under optimized conditions was 73.8 U mg(-1). The α-amylase enzyme was active to hydrolyse starch forming maltose. PMID:26526464

  11. Molecular cloning, characterization and expression analysis of a catalase gene inPaphia textile

    Institute of Scientific and Technical Information of China (English)

    WU Xiangwei; LI Jiakai; TAN Jing; LIU Xiande

    2016-01-01

    Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase cDNA ofPaphia textile (PtCAT) was cloned using RT-PCR and rapid amplification of cDNA ends (RACE).PtCAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′-UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 kD and an estimated isoelectric point of 8.2. Sequence alignment indicated that PtCAT contained a highly conserved catalytic signature motif (61FNRERIPERVVHAKGAG77), a proximal heme-ligand signature sequence (352RLFSYSDP359), and three catalytic amino acid residues (H72, N145, and Y356). PtCAT also contains two putative N-glycosylation sites (34NKT36 and437NFT439) and a peroxisome-targeting signal (511AQL513). Furthermore, PtCAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences.PtCAT mRNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns ofPtCAT mRNA: first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that PtCAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors.

  12. [Gene cloning, expression and characterization of two cold-adapted lipases from Penicillium sp. XMZ-9].

    Science.gov (United States)

    Zheng, Xiaomei; Wu, Ningfeng; Fan, Yunliu

    2012-04-01

    Cold-adapted lipases are attractive biocatalysts that can be used at low temperatures as additives in food products, laundry detergents, and the organic synthesis of chiral intermediates. Cold-adapted lipases are normally found in microorganisms that survive at low temperatures. A fungi strain XMZ-9 exhibiting lipolytic activity was isolated from the soil of glaciers in Xinjiang by the screening plates using 1% tributyrin as the substrate and Victoria blue as an indicator. Based on morphological characteristics and phylogenetic comparisons of its 18S rDNA, the strain was identified as Penicillium sp. The partial nucleotide sequences of these two lipase related genes, LipA and LipB, were obtained by touchdown PCR using the degenerate primers designed according to the conservative domains of lipase. The full-length sequences of two genes were obtained by genome walking. The gene lipA contained 1 014 nucleotides, without any intron, comprising one open reading frame encoding a polypeptide of 337 amino acids. The gene lipB comprised two introns (61 bp and 49 bp) and a coding region sequence of 1 122 bp encoding a polypeptide of 373 amino acids, cDNA sequences of both lipA and lipB were cloned and expressed in Escherichia coli BL21 (DE3). The recombinant LipA was mostly expressed as inclusion bodies, and recovered lipase activity at low temperature after in vitro refolded by dilution. Differently, the recombinant LipB was expressed in the soluble form and then purified by Ni-NTA affinity chromatography Column. It showed high lipase activity at low temperature. These results indicated that they were cold-adapted enzymes. This study paves the way for the further research of these cold-adapted lipases for application in the industry. PMID:22803398

  13. Cloning, expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues.

    Science.gov (United States)

    Gao, Zhifang; Maurousset, Laurence; Lemoine, Remi; Yoo, Sang-Dong; van Nocker, Steven; Loescher, Wayne

    2003-04-01

    The acyclic polyol sorbitol is a primary photosynthetic product and the principal photosynthetic transport substance in many economically important members of the family Rosaceace (e.g. almond [Prunus dulcis (P. Mill.) D.A. Webber], apple [Malus pumila P. Mill.], cherry [Prunus spp.], peach [Prunus persica L. Batsch], and pear [Pyrus communis]). To understand key steps in long-distance transport and particularly partitioning and accumulation of sorbitol in sink tissues, we have cloned two sorbitol transporter genes (PcSOT1 and PcSOT2) from sour cherry (Prunus cerasus) fruit tissues that accumulate large quantities of sorbitol. Sorbitol uptake activities and other characteristics were measured by heterologous expression of PcSOT1 and PcSOT2 in yeast (Saccharomyces cerevisiae). Both genes encode proton-dependent, sorbitol-specific transporters with similar affinities (K(m) sorbitol of 0.81 mM for PcSOT1 and 0.64 mM for PcSOT2). Analyses of gene expression of these transporters, however, suggest different roles during leaf and fruit development. PcSOT1 is expressed throughout fruit development, but especially when growth and sorbitol accumulation rates are highest. In leaves, PcSOT1 expression is highest in young, expanding tissues, but substantially less in mature leaves. In contrast, PcSOT2 is mainly expressed only early in fruit development and not in leaves. Compositional analyses suggest that transport mediated by PcSOT1 and PcSOT2 plays a major role in sorbitol and dry matter accumulation in sour cherry fruits. Presence of these transporters and the high fruit sorbitol concentrations suggest that there is an apoplastic step during phloem unloading and accumulation in these sink tissues. Expression of PcSOT1 in young leaves before completion of the transition from sink to source is further evidence for a role in determining sink activity. PMID:12692316

  14. Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus.

    Science.gov (United States)

    Ranasinghe, Shiwanthi L; Fischer, Katja; Zhang, Wenbao; Gobert, Geoffrey N; McManus, Donald P

    2015-12-01

    The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic.

  15. Cloning, characterization and functional expression of Taenia solium 17 beta-hydroxysteroid dehydrogenase.

    Science.gov (United States)

    Aceves-Ramos, A; de la Torre, P; Hinojosa, L; Ponce, A; García-Villegas, R; Laclette, J P; Bobes, R J; Romano, M C

    2014-07-01

    The 17β-hydroxysteroid dehydrogenases (17β-HSD) are key enzymes involved in the formation (reduction) and inactivation (oxidation) of sex steroids. Several types have been found in vertebrates including fish, as well as in invertebrates like Caenorhabditis elegans, Ciona intestinalis and Haliotis diversicolor supertexta. To date limited information is available about this enzyme in parasites. We showed previously that Taenia solium cysticerci are able to synthesize sex steroid hormones in vitro when precursors are provided in the culture medium. Here, we identified a T. solium 17β-HSD through in silico blast searches in the T. solium genome database. This coding sequence was amplified by RT-PCR and cloned into the pcDNA 3.1(+) expression vector. The full length cDNA contains 957bp, corresponding to an open reading frame coding for 319 aa. The highest identity (84%) at the protein level was found with the Echinococcus multilocularis 17β-HSD although significant similarities were also found with other invertebrate and vertebrate 17β-HSD sequences. The T. solium Tsol-17βHSD belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily. HEK293T cells transiently transfected with Tsol17β-HSD induced expression of Tsol17β-HSD that transformed 3H-androstenedione into testosterone. In contrast, 3H-estrone was not significantly transformed into estradiol. In conclusion, T. solium cysticerci express a 17β-HSD that catalyzes the androgen reduction. The enzyme belongs to the short chain dehydrogenases/reductase family and shares motifs and activity with the type 3 enzyme of some other species.

  16. Molecular cloning and characterization of growth factor receptor bound-protein in Clonorchis sinensis.

    Directory of Open Access Journals (Sweden)

    Xuelian Bai

    Full Text Available BACKGROUND: Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2 is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2 from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. METHODOLOGY/PRINCIPAL FINDINGS: A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. CONCLUSION: Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths.

  17. Cloning and expressional characterization of soybean GmLls1 gene

    Institute of Scientific and Technical Information of China (English)

    ZHAO Ru; LI Pengli; CHENG Zhou; MA Yuanyuan; LU Weifeng; LI Xiaoping; LU Baorong; ZHANG Liwen; WANG Yong; WANG Ningning

    2006-01-01

    Maize Lls1 (lethal leaf-spot 1) gene and its Arabidopsis orthologue AtAcd1 have been suggested to encode pheide a oxygenase (PaO), a key enzyme catalyzing chlorophyll breakdown. To further elucidate the molecular mechanism that regulates chlorophyll catabolism during soybean leaf senescence, a soybean Lls1 homolog was cloned and designated as GmLls1 (GenBank Accession No. DQ154009). Database searches using the deduced protein sequence revealed that it was highly ho mologous to Lls1 genes or Lis1 orthologues in Arabidopsis, maize, cowpea and tomato. Structural analysis of the predicted GmLLS1 protein revealed typical Rieske [2Fe-2S] and mononuclear iron-binding domains as well as the C-terminus CxxC motif that were conserved in and featured PaO homologues. RT-PCR results showed that the transcription of GmLls1 was up-regulated in all the three tested senescence systems: the natural leaf senescence process, the dark-induced primary leaf senescence and senescing cotyledons. We have previously described the involvement of an LRR receptor-like kinase (RLK), RLPK2 in regulation of soybean leaf senescence. Here we report that the expression of GmLls1 gene was dramatically down-regulated by the RNAi-mediated suppression of rlpk2 and, as expected, greatly up regulated by the CaMV 35S promoter derived overexpression of this RLK gene.These results suggested that the expression of GmLls1 was controlled by the RLPK2-mediated senescence signaling pathway. The observation that the detached rlpk2-RNAi transgenic leaves exhibited light-dependent necrotic lesions, which featured the Ils1 mutation, further supported this hypothesis.

  18. Molecular cloning, characterization and expression profiles of thioredoxin 1 and thioredoxin 2 genes in Mytilus galloprovincialis

    Science.gov (United States)

    Wang, Qing; Ning, Xuanxuan; Pei, Dong; Zhao, Jianmin; You, Liping; Wang, Chunyan; Wu, Huifeng

    2013-05-01

    Thioredoxin (Trx) proteins are involved in many biological processes especially the regulation of cellular redox homeostasis. In this study, two Trx cDNAs were cloned from the mussel Mytilus galloprovincialis using rapid amplifi cation of cDNA ends-polymerase chain reaction (RACE-PCR). The two cDNAs were named MgTrx1 and MgTrx2, respectively. The open reading frames of MgTrx1 and MgTrx2 were 318 and 507 base pairs (bp) and they encoded proteins of 105 and 168 amino acids with estimated molecular masses of 11.45 and 18.93 kDa, respectively. Sequence analysis revealed that both proteins possessed the conserved active site dithiol motif Cys-Gly-Pro-Cys. In addition, MgTrx2 also possessed a putative mitochondrial targeting signal suggesting that it is located in the mitochondria. Quantitative real-time polymerase chain reaction (qPCR) revealed that both MgTrx1 and MgTrx2 were constitutively expressed in all tissues examined. The MgTrx1 transcript was most abundant in hemocytes and gills, whereas the MgTrx2 transcript was most abundant in gonad, hepatopancreas, gill and hemocytes. Following Vibrio anguillarum challenge, the expression of MgTrx1 was up-regulated and reached its peak, at a value 10-fold the initial value, at 24 h. Subsequently, expression returned back to the original level. In contrast, the expression level of MgTrx2 was down-regulated following bacterial stimulation, with one fi fth of the control level evident at 12 h post challenge. These results suggest that MgTrx1 and MgTrx2 may play important roles in the response of M. galloprovincialis to bacterial challenge.

  19. Molecular cloning, characterization and expression profiles of thioredoxin 1 and thioredoxin 2 genes in Mytilus galloprovincialis

    Institute of Scientific and Technical Information of China (English)

    WANG Qing; NING Xuanxuan; PEI Dong; ZHAO Jianmin; YOU Liping; WANG Chunyan; WU Huifeng

    2013-01-01

    Thioredoxin (Trx) proteins are involved in many biological processes especially the regulation of cellular redox homeostasis.In this study,two Trx cDNAs were cloned from the mussel Mytilus galloprovincialis using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR).The two cDNAs were named MgTrx1 and MgTrx2,respectively.The open reading frames of MgTrx1 and MgTrx2 were 318 and 507 base pairs (bp) and they encoded proteins of 105 and 168 amino acids with estimated molecular masses of 11.45 and 18.93 kDa,respectively.Sequence analysis revealed that both proteins possessed the conserved active site dithiol motif Cys-Gly-Pro-Cys.In addition,MgTrx2 also possessed a putative mitochondrial targeting signal suggesting that it is located in the mitochondria.Quantitative real-time polymerase chain reaction (qPCR) revealed that both MgTrx1 and MgTrx2 were constitutively expressed in all tissues examined.The MgTrxl transcript was most abundant in hemocytes and gills,whereas the MgTrx2 transcript was most abundant in gonad,hepatopancreas,gill and hemocytes.Following Vibrio anguillarum challenge,the expression of MgTrxl was up-regulated and reached its peak,at a value 10-fold the initial value,at 24 h.Subsequently,expression returned back to the original level.In contrast,the expression level of MgTrx2 was down-regulated following bacterial stimulation,with one fifth of the control level evident at 12 h post challenge.These results suggest that MgTrxl and MgTrx2 may play important roles in the response of M.galloprovincialis to bacterial challenge.

  20. Cloning, expression and characterization of human tissue-specific DNA polymerase λ2

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    DNA polymerase (POL) λ plays an important role during DNA repair and DNA nonhomologous recom-bination processes. A novel POL λ variant was cloned from a human liver cDNA library and named POL λ2 (GenBank Accession No. AY302442). POL λ2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL λ2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL λ2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL λ and POL λ2 in normal liver tissues and hepato-cellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL λ. This abnormality might be involved in hepato-carcinogenesis. The recombinant POL λ2 with His-tag was expressed as a soluble active protein in E. coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chro-matography in an FPLC system. The analysis using isotope α-32P-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.

  1. Cloning, expression and characterization of human tissue-specific DNA polymerase λ2

    Institute of Scientific and Technical Information of China (English)

    GU Fu; LI YuYang; L(U) Hong; YOU Chun; LIU JianPing; CHEN Ao; YU Yao; WANG Xiang; WAN DaFang; GU JianRen; YUAN HanYing

    2007-01-01

    DNA polymerase (POL) λ plays an important role during DNA repair and DNA nonhomologous recombination processes. A novel POL λ variant was cloned from a human liver cDNA library and named POL λ2 (GenBank Accession No. AY302442). POL λ2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL λ2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL λ2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL λ and POL λ2 in normal liver tissues and hepatocellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL λ. This abnormality might be involved in hepatocarcinogenesis. The recombinant POL λ2 with His-tag was expressed as a soluble active protein in E.coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chromatography in an FPLC system. The analysis using isotope α-32p-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.

  2. Molecular cloning and characterization of three beta-defensins from canine testes.

    Science.gov (United States)

    Sang, Yongming; Ortega, M Teresa; Blecha, Frank; Prakash, Om; Melgarejo, Tonatiuh

    2005-05-01

    Mammalian beta-defensins are small cationic peptides possessing broad antimicrobial and physiological activities. Because dogs are particularly resilient to sexually transmitted diseases, it has been proposed that their antimicrobial peptide repertoire might provide insight into novel antimicrobial therapeutics and treatment regimens. To investigate this proposal, we cloned the full-length cDNA of three canine beta-defensin isoforms (cBD-1, -2, and -3) from canine testicular tissues. Their predicted peptides share identical N-terminal 65-amino-acid residues, including the beta-defensin consensus six-cysteine motif. The two longer isoforms, cBD-2 and -3, possess 4 and 34 additional amino acids, respectively, at the C terminus. To evaluate the antimicrobial activity of cBD, a 34-amino-acid peptide derived from the shared mature peptide region was synthesized. Canine beta-defensin displayed broad antimicrobial activity against gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus; MICs of 6 and 100 mug/ml, respectively), gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, and Neisseria gonorrhoeae; MICs of 20 to 50, 20, and 50 mug/ml, respectively), and yeast (Candida albicans; MIC of 5 to 50 mug/ml) and lower activity against Ureaplasma urealyticum and U. canigenitalium (MIC of 200 mug/ml). Antimicrobial potency was significantly reduced at salt concentrations higher than 140 mM. All three canine beta-defensins were highly expressed in testis. In situ hybridization indicated that cBD-1 was expressed primarily in Sertoli cells within the seminiferous tubules. In contrast, cBD-2 was located primarily within Leydig cells. The longest isoform, cBD-3, was detected in Sertoli cells and to a lesser extent in the interstitium. The tissue-specific expression and broad antimicrobial activity suggest that canine beta-defensins play an important role in host defense and other physiological functions of the male reproductive system. PMID:15845463

  3. Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79.

    Science.gov (United States)

    Khakhum, Nittaya; Yordpratum, Umaporn; Boonmee, Atcha; Tattawasart, Unchalee; Rodrigues, Jorge L M; Sermswan, Rasana W

    2016-12-01

    The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn(2+). When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5-9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases. PMID:27637947

  4. Cloning, characterization and expression of the D2 dopamine receptor from the tilapia pituitary.

    Science.gov (United States)

    Levavi-Sivan, Berta; Aizen, Joseph; Avitan, Ayelet

    2005-05-31

    A full-length cDNA encoding a dopamine receptor (DA-R) was obtained from the pituitary of tilapia (ta). This cDNA encodes a protein of 469 amino acids that exhibits the typical arrangement of GPCR. The taDA-R shows high similarity to the DA-Rs of mullet and fugu, and over 70% similarity to Xenopus, mouse and turkey D2 DA-Rs. Northern blot analysis revealed transcript for a single transcript in the pituitary, of approximately 3 kb. In a Southern analysis, the tilapia probe recognized specific bands in the genomic DNA of both mullet and catfish, suggesting high similarity between the corresponding genes. Phylogenetic analysis clearly aligned the taDA-D2-R with all vertebrate D2-like receptor sequences cloned to date, and it was therefore designated taDA-D2-R. taDA-D2-R was transiently expressed in COS-7 cells together with the reporter construct CRE-luciferase. Addition of the specific D2 dopamine agonists quinpirole or bromocriptine, in the presence of forskolin, led to a dose-dependent decrease in forskolin-induced cAMP levels. Both agonists yielded the same maximal inhibition (around 40%). However, the potency of taDA-D2-R for bromocriptine was higher than for quinpirole. As established for mammalian D2-like receptors, stimulation of the taDA-D2-R with quinpirole triggers pertussis-toxin-sensitive Gi/o-mediated, but not Gs-mediated signaling. In contrast to mammals, PCR analysis gave no evidence of alternative splicing in taDA-D2-R. Pharmacological and genetic manipulation of the taDA-D2-R should enable us to better define its physiological role and to further explore the usefulness of fish as a model system for understanding dopaminergic function in higher organisms. PMID:15876479

  5. Cloning and characterization of two novel chloroplastic glycerol-3-phosphate dehydrogenases from Dunaliella viridis.

    Science.gov (United States)

    He, Yunxia; Meng, Xiangzong; Fan, Qianlan; Sun, Xiaoliang; Xu, Zhengkai; Song, Rentao

    2009-09-01

    Dunaliella, a unicellular green alga, has the unusual ability to survive dramatic osmotic stress by accumulating high concentrations of intracellular glycerol as a compatible solute. The chloroplastic glycerol-3-phosphate dehydrogenase (GPDH) has been considered to be the key enzyme that produces glycerol for osmoregulation in Dunaliella. In this study, we cloned the two most prominent GPDH cDNAs (DvGPDH1 and DvGPDH2) from Dunaliella viridis, which encode two polypeptides of 695 and 701 amino acids, respectively. Unlike higher plant GPDHs, both proteins contained extra phosphoserine phosphatase (SerB) domains at their N-termini in addition to C-terminal GPDH domains. Such bi-domain GPDHs represent a novel type of GPDH and are found exclusively in the chlorophyte lineage. Transient expression of EGFP fusion proteins in tobacco leaf cells demonstrated that both DvGPDH1 and DvGPDH2 are localized in the chloroplast. Overexpression of DvGPDH1 or DvGPDH2 could complement a yeast GPDH mutant (gpd1Delta), but not a yeast SerB mutant (ser2Delta). In vitro assays with purified DvGPDH1 and DvGPDH2 also showed apparent GPDH activity for both, but no SerB activity was detected. Surprisingly, unlike chloroplastic GPDHs from plants, DvGPDH1 and DvGPDH2 could utilize both NADH and NADPH as coenzymes and exhibited significantly higher GPDH activities when NADH was used as the coenzyme. Q-PCR analysis revealed that both genes exhibited transient transcriptional induction of gene expression upon hypersalinity shock, followed by a negative feedback of gene expression. These results shed light on the regulation of glycerol synthesis during salt stress in Dunaliella. PMID:19551475

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

    Science.gov (United States)

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

    2014-01-01

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

  7. Cloning and Characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Stephanie O. Palmer

    2013-01-01

    Full Text Available We have cloned genes encoding elongation factors EF-Tu and EF-Ts from Pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. Sequence analysis indicated that P. aeruginosa EF-Tu and EF-Ts are 84% and 55% identical to E. coli counterparts, respectively. P. aeruginosa EF-Tu was active when assayed in GDP exchange assays. Kinetic parameters for the interaction of EF-Tu with GDP in the absence of EF-Ts were observed to be = 33 μM, = 0.003 s−1, and the specificity constant was  s−1 μM−1. In the presence of EF-Ts, these values were shifted to = 2 μM, = 0.005 s−1, and the specificity constant was  s−1 μM−1. The equilibrium dissociation constants governing the binding of EF-Tu to GDP ( were 30–75 nM and to GTP ( were 125–200 nM. EF-Ts stimulated the exchange of GDP by EF-Tu 10-fold. P. aeruginosa EF-Tu was active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in poly(U-dependent binding of Phe-tRNAPhe at the A-site of P. aeruginosa ribosomes. P. aeruginosa EF-Tu was active in poly(U-programmed polyphenylalanine protein synthesis system composed of all P. aeruginosa components.

  8. Cloning, Characterization and Primary Function Study of a Novel Gene, Cymgl, Related to Family 2 Cystatins

    Institute of Scientific and Technical Information of China (English)

    Yang XIANG; Dong-Song NIE; Jian WANG; Xiao-Jun TAN; Yun DENG; Shu-Wei LUO; Guang-Xiu LU

    2005-01-01

    Cystatins are cysteine proteinase inhibitors. We found two expression sequence tags (ESTs),CA463109 and AV042522, from a mouse testis library using Digital differential display (DDD). By electrical hybridization, a novel gene, Cymgl (GenBank accession No. AY600990), which has a full length of 0.78 kb,and contains four exons and three introns, was cloned from a mouse testis cDNA library. The gene is located in the 2G3 area of chromosome 2. The full cDNA encompasses the entire open reading frame, encoding 141amino acid residues. The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the CRES subfamily, which are related to the family 2 cystatins and are expressed specifically in the male reproductive tract. CYMG1 has a 44% (48/108) identity with mouse CRES and 30% (42/140)identity with mouse cystatin C. Northern blot analysis showed that the Cymgl is specifically expressed in adult mouse testes. Cell location studies showed that the GFP-tagged CYMG1 protein was localized in the cytoplasm of HeLa cells. Immunohistochemistry revealed that the CYMG1 protein was expressed in mouse testes spermatogonium, spermatocytes, round spermatids, elongating spermatids and spermatozoa. RT-PCR results also showed that Cymgl was expressed in mouse testes and spermatogonium. The Cymgl expression level varied in different developmental stages: it was low 1 week postpartum, steadily increased 2 to 5 weeks postpartum, and was highest 7 weeks postpartum. The expression level at 5 weeks postpartum was maintained during 13 to 57 weeks postpartum. The Cymgl expression level in the testes over different developmental stages correlates with the mouse spermatogenesis and sexual maturation process. All these indicate that Cymgl might play an important role in mouse spermatogenesis and sexual maturation.

  9. cDNA Cloning, Expression and Characterization of an Allergenic 60s Ribosomal Protein of Almond (Prunus dulcis

    Directory of Open Access Journals (Sweden)

    Abolhassani Mohsen

    2009-06-01

    Full Text Available Tree nuts, including almond (prunus dulcis are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

  10. Cloning and functional characterization of two abiotic stress-responsive Jerusalem artichoke (Helianthus tuberosus) fructan 1-exohydrolases (1-FEHs).

    Science.gov (United States)

    Xu, Huanhuan; Liang, Mingxiang; Xu, Li; Li, Hui; Zhang, Xi; Kang, Jian; Zhao, Qingxin; Zhao, Haiyan

    2015-01-01

    Two fructan hydrolases were previously reported to exist in Jerusalem artichoke (Helianthus tuberosus) and one native fructan-β-fructosidase (1-FEH) was purified to homogeneity by SDS-PAGE, but no corresponding cDNA was cloned. Here, we cloned two full-length 1-FEH cDNA sequences from Jerusalem artichoke, named Ht1-FEH I and Ht1-FEH II, which showed high levels of identity with chicory 1-FEH I and 1-FEH II. Functional characterization of the corresponding recombinant proteins in Pichia pastoris X-33 demonstrated that both Ht1-FEHs had high levels of hydrolase activity towards β(2,1)-linked fructans, but low or no activity towards β(2,6)-linked levan and sucrose. Like other plant FEHs, the activities of the recombinant Ht1-FEHs were greatly inhibited by sucrose. Real-time quantitative PCR analysis showed that Ht1-FEH I transcripts accumulated to high levels in the developing leaves and stems of artichoke, whereas the expression levels of Ht1-FEH II increased in tubers during tuber sprouting, which implies that the two Ht1-FEHs play different roles. The levels of both Ht1-FEH I and II transcript were significantly increased in the stems of NaCl-treated plants. NaCl treatment also induced transcription of both Ht1-FEHs in the tubers, while PEG treatments slightly inhibited the expression of Ht1-FEH II in tubers. Analysis of sugar-metabolizing enzyme activities and carbohydrate concentration via HPLC showed that the enzyme activities of 1-FEHs were increased but the fructose content was decreased under NaCl and PEG treatments. Given that FEH hydrolyzes fructan to yield Fru, we discuss possible explanations for the inconsistency between 1-FEH activity and fructan dynamics in artichokes subjected to abiotic stress. PMID:25522837

  11. Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification, Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker.

    Science.gov (United States)

    Kawashima, Satoshi; Ikehata, Hiroki; Tada, Chihiro; Ogino, Tomohiro; Kakizaki, Hiromi; Ikeda, Mana; Fukushima, Hideto; Matsumiya, Masahiro

    2016-01-20

    Fish express two different chitinases, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), in the stomach. AFCase-1 and AFCase-2 have different degradation patterns, as fish efficiently degrade chitin ingested as food. For a comparison with the enzymatic properties and the primary structures of chitinase isozymes obtained previously from the stomach of demersal fish, in this study, we purified chitinase isozymes from the stomach of Japanese sardine Sardinops melanostictus, a surface fish that feeds on plankton, characterized the properties of these isozymes, and cloned the cDNAs encoding chitinases. We also predicted 3D structure models using the primary structures of S. melanostictus stomach chitinases. Two chitinase isozymes, SmeChiA (45 kDa) and SmeChiB (56 kDa), were purified from the stomach of S. melanostictus. Moreover, two cDNAs, SmeChi-1 encoding SmeChiA, and SmeChi-2 encoding SmeChiB were cloned. The linker regions of the deduced amino acid sequences of SmeChi-1 and SmeChi-2 (SmeChi-1 and SmeChi-2) are the longest among the fish stomach chitinases. In the cleavage pattern groups toward short substrates and the phylogenetic tree analysis, SmeChi-1 and SmeChi-2 were classified into AFCase-1 and AFCase-2, respectively. SmeChi-1 and SmeChi-2 had catalytic domains that consisted of a TIM-barrel (β/α)₈-fold structure and a deep substrate-binding cleft. This is the first study showing the 3D structure models of fish stomach chitinases.

  12. SABATH Methyltransferases from White Spruce (Picea glauca [Moench] Voss): Gene Cloning, Functional Characterization and Structural Analysis

    Science.gov (United States)

    Known members of the plant SABATH family of methyltransferases have important biological functions by methylating hormones, signaling molecules and other metabolites. While all previously characterized SABATH genes were isolated from angiosperms, in this article, we report on the isolation and funct...

  13. Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus microplus (Acari: Ixodidae

    Directory of Open Access Journals (Sweden)

    Flores-Fernández José Miguel

    2014-01-01

    Full Text Available The cattle tick Rhipicephalus (Boophilus microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus.

  14. Molecular cloning and characterization of a glycine-like receptor gene from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

    Science.gov (United States)

    Flores-Fernández, José Miguel; Gutiérrez-Ortega, Abel; Padilla-Camberos, Eduardo; Rosario-Cruz, Rodrigo; Hernández-Gutiérrez, Rodolfo; Martínez-Velázquez, Moisés

    2014-01-01

    The cattle tick Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus. PMID:25174962

  15. Identification, cDNA Cloning, and Characterization of Luteinizing Hormone Beta Subunit (lhb) Gene in Catla catla.

    Science.gov (United States)

    Rather, Mohd Ashraf; Bhat, Irfan Ahmad; Sharma, Rupam

    2016-01-01

    Reproductive hormones play a significant role in the gonadal development and gametogenesis process of animals. In the present study luteinizing hormone beta, (lhb) subunit gene was cloned and characterized from the brain of Catla catla. The lhb full-length of cDNA sequence is 629 bp which consists of 43bp 5'-UTR (untranslated region) 447bp, ORF(open reading frame) and 139 bp of 3'-UTR respectively. The coding region of lhb gene encoded a peptide of 148 amino acids. The coding sequence of lhb gene consist of a single N-linked glycosylation site (NET) and 12 cysteine knot residues. Phylogenetic analysis of C. catla Lhβ deduced amino acid sequence showed high similarity with Carassius auratus followed by Gobiocypris rarus. 3D structure Lhβ protein comprises of five β-sheets and six coils/loops. The qPCR results revealed lhb mRNA is mainly expressed in the pituitary, ovary while moderate expression was observed in brain and testis. To best our knowledge, this is the first report on the identification, molecular characterization and structural information regarding luteinizing hormone in Indian major carp. PMID:26980432

  16. Acyl-ACP thioesterases from Camelina sativa: cloning, enzymatic characterization and implication in seed oil fatty acid composition.

    Science.gov (United States)

    Rodríguez-Rodríguez, Manuel Fernando; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2014-11-01

    Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo fatty acid biosynthesis in the plastids of higher plants by hydrolyzing the thioester bond between ACP and the fatty acid synthesized. Free fatty acids are then esterified with coenzyme A prior to being incorporated into the glycerolipids synthesized through the eukaryotic pathway. Acyl-ACP thioesterases belong to the TE14 family of thioester-active enzymes and can be classified as FatAs and FatBs, which differ in their amino acid sequence and substrate specificity. Here, the FatA and FatB thioesterases from Camelina sativa seeds, a crop of interest in plant biotechnology, were cloned, sequenced and characterized. The mature proteins encoded by these genes were characterized biochemically after they were heterologously expressed in Escherichia coli and purified. C. sativa contained three different alleles of both the FatA and FatB genes. These genes were expressed most strongly in expanding tissues in which lipids are very actively synthesized, such as developing seed endosperm. The CsFatA enzyme displayed high catalytic efficiency on oleoyl-ACP and CsFatB acted efficiently on palmitoyl-ACP. The contribution of these two enzymes to the synthesis of C. sativa oil was discussed in the light of these results.

  17. Acyl-ACP thioesterases from Camelina sativa: cloning, enzymatic characterization and implication in seed oil fatty acid composition.

    Science.gov (United States)

    Rodríguez-Rodríguez, Manuel Fernando; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2014-11-01

    Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo fatty acid biosynthesis in the plastids of higher plants by hydrolyzing the thioester bond between ACP and the fatty acid synthesized. Free fatty acids are then esterified with coenzyme A prior to being incorporated into the glycerolipids synthesized through the eukaryotic pathway. Acyl-ACP thioesterases belong to the TE14 family of thioester-active enzymes and can be classified as FatAs and FatBs, which differ in their amino acid sequence and substrate specificity. Here, the FatA and FatB thioesterases from Camelina sativa seeds, a crop of interest in plant biotechnology, were cloned, sequenced and characterized. The mature proteins encoded by these genes were characterized biochemically after they were heterologously expressed in Escherichia coli and purified. C. sativa contained three different alleles of both the FatA and FatB genes. These genes were expressed most strongly in expanding tissues in which lipids are very actively synthesized, such as developing seed endosperm. The CsFatA enzyme displayed high catalytic efficiency on oleoyl-ACP and CsFatB acted efficiently on palmitoyl-ACP. The contribution of these two enzymes to the synthesis of C. sativa oil was discussed in the light of these results. PMID:25212866

  18. Cloning and characterization of Arabidopsis thaliana AtNAP57--a homologue of yeast pseudouridine synthase Cbf5p.

    Science.gov (United States)

    Maceluch, J; Kmieciak, M; Szweykowska-Kulińska, Z; Jarmołowski, A

    2001-01-01

    Rat Nap57 and its yeast homologue Cbf5p are pseudouridine synthases involved in rRNA biogenesis, localized in the nucleolus. These proteins, together with H/ACA class of snoRNAs compose snoRNP particles, in which snoRNA guides the synthase to direct site-specific pseudouridylation of rRNA. In this paper we present an Arabidopsis thaliana protein that is highly homologous to Cbf5p (72% identity and 85% homology) and NAP57 (67% identity and 81% homology). Moreover, the plant protein has conserved structural motifs that are characteristic features of pseudouridine synthases of the TruB class. We have named the cloned and characterized protein AtNAP57 (Arabidopsis thaliana homologue of NAP57). AtNAP57 is a 565 amino-acid protein and its calculated molecular mass is 63 kDa. The protein is encoded by a single copy gene located on chromosome 3 of the A. thaliana genome. Interestingly, the AtNAP57 gene does not contain any introns. Mutations in the human DKC1 gene encoding dyskerin (human homologue of yeast Cbf5p and rat NAP57) cause dyskeratosis congenita a rare inherited bone marrow failure syndrome characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia.

  19. Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

    Science.gov (United States)

    Payá-Milans, Miriam; Venegas-Calerón, Mónica; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2015-03-01

    The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants. PMID:25618244

  20. Genomic characterization of the aquaculture resource Mytilus galloprovincialis

    Directory of Open Access Journals (Sweden)

    Maria Murgarella

    2014-06-01

    Full Text Available In the autonomous community of Galicia (NW Spain, M. galloprovincialis is a natural resource of high economic importance to the economy of this region. More than 250,000 MTs of mussels are produced per year, representing 40% of the European production of this bivalve and 80% of the marine aquacultural production of the country. Besides its aquaculture interest, study of the biology M. galloprovincialis may also help to understand better fundamental aspects of molluscan biology. On the other hand, some fascinating features of mediterranean mussels are unique to this species such as its extraordinary resistance to diseases. In fact, in comparison with other bivalves, no records of massive mortality of this organism caused by pathogens has been reported so far. Although some of the molecular components of the innate immune system, responsible of this resistance, have been previously identified and characterized, the genetic mechanisms underlying their diversity are poorly understood. In order to understand better this organism at genome level and search for the genetic basis of its disease resistance, we sequenced its genome at high depth using Illumina technology. After assembling the resulting reads and assisted with transcriptomic data, we annotated its genome sequence. First, we analysed the content of repetitive elements, finding those that are common among molluscs as well as identifying new repeat families unique to M. galloprovincialis. Second, we predicted and annotated its gene repertoire, defining the total number of genes and their density in its genome. Finally, the resulting in silico predicted genes were functionally annotated. Altogether, this information will allow the identification of genes of interest, such as those participating in the innate immunity and will allow us to delve into the possibility of whether their variability has a genetic basis or relies on post-transcriptional events. In addition to this annotation, some

  1. Cloning and molecular characterization of the murine macrophage "68-kDa" protein kinase C substrate and its regulation by bacterial lipopolysaccharide.

    OpenAIRE

    Seykora, J T; Ravetch, J V; Aderem, A

    1991-01-01

    We have isolated and characterized a cDNA clone encoding the murine macrophage 68-kDa protein kinase C substrate, which is homologous to the 80- to 87-kDa protein identified by the acronym MARCKS (myristoylated alanine-rich C kinase substrate). The murine MARCKS cDNA clone encodes an acidic protein of 309 amino acids with a calculated molecular weight of 29,661. Transfection of the murine MARCKS gene into TK-L fibroblasts produced a myristoylated protein kinase C substrate that migrated on SD...

  2. Cloning, periplasmic expression, purification and structural characterization of human ribosomal protein L10

    International Nuclear Information System (INIS)

    The ribosomal protein L10 (RP L10) is a strong candidate to be included in the class of tumor suppressor proteins. This protein, also denominated as QM, is known to participate in the binding of ribosomal subunits 60S and 40S and the translation of mRNAs. It has a molecular weight that varies between 24 and 26 kDa and an isoelectric point of (pI) 10.5. The sequence of the protein QM is highly conserved in mammals, plants, invertebrates, insects and yeast which indicates its critical functions in a cell. As a tumor suppressor, RP L10 has been studied in strains of Wilm's tumor (WT-1) and tumor cells in the stomach, where was observed a decrease in the amount of its mRNA. More recently, the RP L10 was found in low amounts in the early stages of prostate adenoma and showed some mutation in ovarian cancer, what indicates its role as a suppressor protein in the development of these diseases. It has also been described that this protein interacts with c-Jun and c-Yes inhibiting growth factors and consequently, cell division. This work has an important role on the establishment of soluble expression of QM to give base information for further studies on expression that aim to evaluate the specific regions where it acts binding the 60S and 40S ribosomal subunits and translation, as well as its binding to proto-oncogenes. The cDNA for QM protein was amplified by PCR and cloned into periplasmic expression vector p3SN8. The QM protein was expressed in E. coli BL21 (DE3) in the region of cytoplasm and periplasm, the best condition was obtained from the expression of the recombinant plasmid QM p1813QM at 25 degree C or 30 degree C, the soluble protein was obtained with small amounts of contaminants. The assays of secondary structure showed that the QM protein is predominantly alpha-helix, but when it loses the folding, this condition changes and the protein is replaced by β- sheet feature. (author)

  3. Molecular Cloning and Characterization of Human Homeobox Gene Nkx3.1 Promoter

    Institute of Scientific and Technical Information of China (English)

    An-LiJIANG; Jian-YeZHANG; CharlesYOUNG; Xiao-YanHU; Yong-MeiWANG; Zhi-FangLIU; Mei-LanHAO

    2004-01-01

    Nkx3.1 is a prostate-specific homeobox gene related strongly to prostate development and prostate cancer. To study its regulation of transcription, 1.06 kb 5′ flanking region of Nkx3.1 gene and its 5′ deletion mutants (861,617,417 and 238 bp) were obtained by PCR and cloned into pGL3-basic, a promoter-less luciferase reporter vector, to examine their promoter activities driving the reporter gene transcription, pRL-TK, a Renilla luciferase reporter vector was used as internal control, and pGL3-control and pGL3-basic were used as positive and negative control respectively. The promoter activities were determined by dual-luciferase reporter assay 48h after pGL3 constructs were cotransfected with pRL-TK into prostate cancer cell LNCaP. The results showed that dual-luciferase reporter assay (M/M2) of pGL3-1.06kb cotransfection with pRL-TK was 2.7, which was about 1.5-fold higher than that of pGL3-control cotransfection with pRL-TK and 50-fold higher than that of pGL3-basic cotransfection with pRL-TK. The results also showed that the relative activities (M1/M2) were 0.71, 0.84, 0.44 and 2.07 respectively for pGL3-861bp, pGL3-617bp, pGL3-417bp, pGL3-238bp, the last one still had 80% promoter activity compared with pGL3-1.06kb, which showed that deletion from 1.06kb to 238 bp had small effects on promoter activity. The conclusion was that the 238bp fragment containing a TATA box and two CAAT boxes had strong promoter activity. However, the deletion from 1.06kb to 861bp reduced activity 3.8-fold while the deletion from 417bp to 238bp enhanced activity 4.7-fold, which indicated that these deleted sequences might contain some important positive or negative regulatory elements. It will be important to identify the elements within the Nkx3.1 promoter that contribute to regulation of the gene transcription in the future studies.

  4. Molecular Cloning and Characterization of Human Homeobox Gene Nkx3.1 Promoter

    Institute of Scientific and Technical Information of China (English)

    An-Li JIANG; Jian-Ye ZHANG; Charles YOUNG; Xiao-Yan HU; Yong-Mei WANG; Zhi-Fang LIU; Mei-Lan HAO

    2004-01-01

    Nkx3.1 is a prostate-specific homeobox gene related strongly to prostate development andprostate cancer. To study its regulation of transcription, 1.06 kb 5 ′ flanking region of Nkx3.1 gene and its5 ′deletion mutants (861,617, 417 and 238 bp) were obtained by PCR and cloned into pGL3-basic, apromoter-less luciferase reporter vector, to examine their promoter activities driving the reporter genetranscription, pRL-TK, a Renilla luciferase reporter vector was used as internal control, and pGL3-controland pGL3-basic were used as positive and negative control respectively. The promoter activities were deter-mined by dual-luciferase reporter assay 48 h after pGL3 constructs were cotransfected with pRL-TK intoprostate cancer cell LNCaP. The results showed that dual-luciferase reporter assay (M1/M2) of pGL3-1.06 kbcotransfection with pRL-TK was 2.7, which was about 1.5-fold higher than that of pGL3-control cotrans-fection with pRL-TK and 50-fold higher than that of pGL3-basic cotransfection with pRL-TK. The resultsalso showed that the relative activities (M1/M2) were 0.71, 0.84, 0.44 and 2.07 respectively for pGL3-861 bp,pGL3-617 bp, pGL3-417 bp, pGL3-238 bp, the last one still had 80% promoter activity compared with pGL3-1.06 kb, which showed that deletion from 1.06 kb to 238 bp had small effects on promoter activity. Theconclusion was that the 238 bp fragment containing a TATA box and two CAAT boxes had strong promoteractivity. However, the deletion from 1.06 kb to 861 bp reduced activity 3.8-fold while the deletion from 417bp to 238 bp enhanced activity 4.7-fold, which indicated that these deleted sequences might contain someimportant positive or negative regulatory elements. It will be important to identify the elements within theNkx3.1 promoter that contribute to regulation of the gene transcription in the future studies.

  5. Cloning, characterization and expression of $OsFMO_{(t)}$ in rice encoding a flavin monooxygenase

    Indian Academy of Sciences (India)

    Jicai Yi; Lanna Liu; Youpei Cao; Jiazuo Li; Mantong Mei

    2013-12-01

    Flavin monooxygenases (FMO) play a key role in tryptophan (Trp)-dependent indole-acetic acid (IAA) biosynthesis in plants and regulate plant growth and development. In this study, the full-length genomic DNA and cDNA of $OsFMO_{(t)}$, a FMO gene that was originally identified from a rolled-leaf mutant in rice, was isolated and cloned from wild type of the rolled-leaf mutant. $OsFMO_{(t)}$ was found to have four exons and three introns, and encode a protein with 422 amino acid residues that contains two basic conserved motifs, with a ‘G×××G’ characteristic structure. OsFMO(t) showed high amino acid sequence identity with FMO proteins from other plants, in particular with YUCCA from Arabidopsis, FLOOZY from Petunia, and OsYUCCA1 from rice. Our phylogenetic analysis showed that OsFMO$_{\\text{(t)}}$ and the homologous FMO proteins belong to the same clade in the evolutionary tree. Overexpression of $OsFMO_{(t)}$ in transformed rice calli produced IAA-excessive phenotypes that showed browning and lethal effects when exogenous auxins such as naphthylacetic acid (NAA) were added to the medium. These results suggested that the OsFMO$_{\\text{(t)}}$ protein is involved in IAA biosynthesis in rice and its overexpression could lead to the malformation of calli. Spatio-temporal expression analysis using RT-PCR and histochemical analysis for GUS activity revealed that expression of $OsFMO_{(t)}$ was totally absent in the rolled-leaf mutant. However, in the wild type variety, this gene was expressed at different levels temporally and spatially, with the highest expression observed in tissues with fast growth and cell division such as shoot apexes, tender leaves and root tips. Our results demonstrated that IAA biosynthesis regulated by $OsFMO_{(t)}$ is likely localized and might play an essential role in shaping local IAA concentrations which, in turn, is critical for regulating normal growth and development in rice.

  6. A New Farnesyl Diphosphate Synthase Gene from Taxus media Rehder: Cloning, Characterization and Functional Complementation

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hua Liao; Min Chen; Yi-Fu Gong; Zhu-Gang Li; Kai-Jing Zuo; Peng Wang; Feng Tan; Xiao-Fen Sun; Ke-Xuan Tang

    2006-01-01

    Farnesyl diphosphate synthase (FPS; EC 2.5.1.10) catalyzes the production of 15-carbon farnesyl diphosphate which is a branch-point intermediate for many terpenoids. This reaction is considered to be a ratelimiting step in terpenoid biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl diphosphate synthase from a gymnosperm plant species, Taxus media Rehder,designated as TmFPS1. The full-length cDNA of TmFPS1 (GenBank accession number: AY461811) was 1 464bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptide with a calculated molecular weight of 40.3 kDa and a theoretical pl of 5.07. Bioinformatic analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetic analysis showed that farnesyl diphosphate synthases can be divided into two groups: one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homologybased structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature is the arrangement of 13 core helices around a large central cavity in which the catalytic reaction takes place. Our bioinformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPS gene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, including the needles, stems and roots of T. media. Subsequently, functional complementation with TmFPS1 in a FPS-deficient mutant yeast demonstrated that TmFPS1 did encode farnesyl diphosphate synthase, which rescued the yeast mutant.This study will be helpful in future investigations aiming at understanding the detailed role of FPS in terpenoid biosynthesis flux control at the molecular genetic level.

  7. MOLECULAR CLONING AND CHARACTERIZATION OF NOVEL THERMOSTABLE LIPASE FROM SHEWANELLA PUTREFACIENS AND USING ENZYMATIC BIODIESEL PRODUCTION

    Directory of Open Access Journals (Sweden)

    Fahri Akbas

    2015-02-01

    Full Text Available A novel thermostable lipase from Shewanella putrefaciens was identified, expressed in Escherichia coli, characterized and used in biodiesel production. Enzyme characterization was carried out by enzyme assay, SDS-PAGE and other biochemical reactions. The recombinant lipase was found to have a molecular mass of 29 kDa and exhibited lipase activity when Tween 80 was used as the substrate. The purified enzyme showed maximum activity at pH 5.0 and at 80°C. The recombinant lipase was used for the transesterification of canola oil and waste oil. The enzyme retains 50% of its activity at 90°C for 30 minutes. It is also able to retain 20% of its activity even at 100 °C for 20 minutes. These properties of the obtained new recombinant thermostable lipase make it promising as a biocatalyst for industrial processes.

  8. Echinococcus granulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein.

    Science.gov (United States)

    Cortez-Herrera, E; Yamamoto, R R; Rodrigues, J J; Farias, S E; Ferreira, H B; Zaha, A

    2001-04-01

    We report the isolation and characterization of an Echinococcus granulosus gene that codes for a protein with actin filament fragmenting and nucleating activities (EgAFFP). The genomic region corresponding to the EgAFFP gene presents a coding sequence of 1110 bp that is interrupted by eight introns. The EgAFFP deduced amino acid sequence is about 40% homologous to those of several members of the gelsolin family, such as Physarum polycephalum fragmin, Dictyostelium discoideum severin, and Lumbricus terrestris actin modulator. As do other proteins of the same family, EgAFFP presents three repeated domains, each one characterized by internal conserved amino acid motifs. Assays with fluorescence-labeled actin showed that the full-length recombinant EgAFFP effectively binds actin monomers in both a calcium-dependent and calcium-independent manner and also presents actin nucleating and severing activities.

  9. Cloning and characterization of a second member of the mouse mdr gene family.

    OpenAIRE

    Gros, P.; Raymond, M; Bell, J.; Housman, D

    1988-01-01

    The mammalian mdr gene family comprises a small number of closely related genes. Previously, we have shown that one member, mdr1, has the capacity to convey multidrug resistance to drug-sensitive recipient cells in a gene transfer protocol. However, the functional characteristics of other members of this gene family have not been examined. In this report, we characterize a second member of the mdr gene family which we designated mdr2. We determined the nucleotide sequence corresponding to the...

  10. Cloning and molecular characterization of two invertebrate-type lysozymes from Anopheles gambiae

    OpenAIRE

    Paskewitz, S M; Li, B.; Kajla, M. K.

    2008-01-01

    We sequenced and characterized two novel invertebrate-type lysozymes from the mosquito Anopheles gambiae. Alignment and phylogenetic analysis of these and a number of related insect proteins identified through bioinformatics strategies showed a high degree of conservation of this protein family throughout the Class Insecta. Expression profiles were examined for the two mosquito genes through semiquantitative and real-time PCR analysis. Lys i-1 transcripts were found in adult females in the fa...

  11. Identification and characterization of CTX-M-15 producing Klebsiella pneumoniae clone ST101 in a Hungarian university teaching hospital.

    Science.gov (United States)

    Melegh, Szilvia; Schneider, György; Horváth, Marianna; Jakab, Ferenc; Emődy, Levente; Tigyi, Zoltán

    2015-09-01

    We investigated the molecular epidemiology of extended spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae isolates derived from the teaching hospitals of University of Pécs, Pécs, Hungary in the time period 2004-2008. Molecular typing, antimicrobial susceptibility testing, detection of common β-lactamase genes (bla(CTX-M), bla(TEM) and bla(SHV)) and virulence associated traits (hypermucoviscosity, magA, k2a, rmpA, siderophores, type 1 and 3 fimbria, biofilm formation, serum resistance) were performed for 102 isolates. The results showed the presence of three major ciprofloxacin resistant CTX-M-15 producing clones (ST15 n = 69, ST101 n = 10, and ST147 n = 9), of which ST15 was predominant and universally widespread. Considering distribution in time and place, ST101 and ST147 were detected at fewer inpatient units and within a narrower time frame, as compared to ST15. Beside major clones, eleven minor clones were identified, and were shown to harbour the following β-lactamase genes: six clones carried bla(CTX-M), four clones harboured bla(SHV-5) and one clone possessed both bla(CTX-M) and ESBL type bla(SHV). Among the SHV-5 producing K. pneumoniae clones a novel sequence type was found, namely ST1193, which harboured a unique infB allele. Different virulence factor content and peculiar antimicrobial susceptibility profile were characteristic for each clone. In contrast to major clone isolates, which showed high level resistance to ciprofloxacin, minor clone isolates displayed significantly lower MIC values for ciprofloxacin suggesting a role for fluoroquinolones in the dissemination of the major K. pneumoniae clones. This is the first description of the CTX-M-15 producing K. pneumoniae clone ST101 in Hungary. PMID:26551567

  12. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    Energy Technology Data Exchange (ETDEWEB)

    Bhaskar,; Kumari, Neeti [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India); Goyal, Neena, E-mail: neenacdri@yahoo.com [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests

  13. Genetic characterization and molecular cloning of the tripeptide permease (tpp) genes of Salmonella typhimurium.

    OpenAIRE

    Gibson, M M; Price, M; Higgins, C F

    1984-01-01

    Of the three bacterial peptide transport systems only one, the oligopeptide permease, has been characterized in any detail. We have now isolated Salmonella typhimurium mutants deficient in a second transport system, the tripeptide permease (Tpp), using the toxic peptide alafosfalin. Alafosfalin resistance mutations map at three loci, the gene encoding peptidase A (pepA) and two transport-defective loci, tppA and tppB. Locus tppA has been mapped to 74 min on the S. typhimurium chromosome, cotr...

  14. Molecular cloning and characterization of the human RNase κ, an ortholog of Cc RNase

    OpenAIRE

    Economopoulou, Marie-angela I.; Fragoulis, Emmanouel G.; Sideris, Diamantis C.

    2007-01-01

    A novel protein family, designated hereafter as RNase κ (kappa) family, has been recently introduced with the characterization of the specific Cc RNase, isolated from the insect Ceratitis capitata. The human ortholog of this family consists of 98 amino acids and shares > 98% identity with its mammalian counterparts. This RNase is encoded by a single-copy gene found to be expressed in a wide spectrum of normal and cancer tissues. The cDNA of the human ribonuclease has been isolated and subclon...

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

    Science.gov (United States)

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

    2012-12-01

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

  16. Cloning, Characterization and Expression Pattern Analysis of a Cytosolic Copper/Zinc Superoxide Dismutase (SaCSD1) in a Highly Salt Tolerant Mangrove (Sonneratia alba)

    OpenAIRE

    Enze Yang; Shanze Yi; Fang Bai; Dewei Niu; Junjie Zhong; Qiuhong Wu; Shufang Chen; Renchao Zhou; Feng Wang

    2015-01-01

    Mangroves are critical marine resources for their remarkable ability to tolerate seawater. Antioxidant enzymes play an especially significant role in eliminating reactive oxygen species and conferring abiotic stress tolerance. In this study, a cytosolic copper/zinc superoxide dismutase (SaCSD1) cDNA of Sonneratia alba, a mangrove species with high salt tolerance, was successfully cloned and then expressed in Escherichia coli Rosetta-gami (designated as SaCSD1). SaCSD1 comprised a complete ope...

  17. Cloning and characterization of GST fusion tag stabilized large subunit of Escherichia coli acetohydroxyacid synthase I.

    Science.gov (United States)

    Li, Heng; Liu, Nan; Wang, Wen-Ting; Wang, Ji-Yu; Gao, Wen-Yun

    2016-01-01

    There are three acetohydroxyacid synthase (AHAS, EC 4.1.3.18) isozymes (I, II, and III) in the enterobacteria Escherichia coli among which AHAS I is the most active. Its large subunit (LSU) possesses full catalytic machinery, but is unstable in the absence of the small subunit (SSU). To get applicable LSU of AHAS I, we prepared and characterized in this study the polypeptide as a His-tagged (His-LSU) and a glutathione S-transferase (GST)-tagged (GST-LSU) fusion protein, respectively. The results showed that the His-LSU is unstable, whereas the GST-LSU displays excellent stability. This phenomenon suggests that the GST polypeptide fusion tag could stabilize the target protein when compared with histidine tag. It is the first time that the stabilizing effect of the GST tag was observed. Further characterization of the GST-LSU protein indicated that it possesses the basic functions of AHAS I with a specific activity of 20.8 μmol min(-1) mg(-1) and a Km value for pyruvate of 0.95 mM. These observations imply that introduction of the GST fusion tag to LSU of AHAS I does not affect the function of the protein. The possible reasons that the GST fusion tag could make the LSU stable are initially discussed.

  18. Molecular cloning and characterization of the allatotropin precursor and receptor in the desert locust, Schistocerca gregaria.

    Science.gov (United States)

    Lismont, Els; Vleugels, Rut; Marchal, Elisabeth; Badisco, Liesbeth; Van Wielendaele, Pieter; Lenaerts, Cynthia; Zels, Sven; Tobe, Stephen S; Vanden Broeck, Jozef; Verlinden, Heleen

    2015-01-01

    Allatotropins (ATs) are pleiotropic neuropeptides initially isolated from the tobacco hornworm, Manduca sexta. In 2008, the first receptor for AT-like peptides (ATR) was characterized in Bombyx mori. Since then, ATRs have also been characterized in M. sexta, Tribolium castaneum, Aedes aegypti and Bombus terrestris. These receptors show sequence similarity to vertebrate orexin (ORX) receptors. When generating an EST-database of the desert locust (Schistocerca gregaria) central nervous system, we found cDNA sequences encoding the Schgr-AT precursor and a fragment of its putative receptor. This receptor cDNA has now been completed and functionally expressed in mammalian cell lines. Activation of this receptor, designated as Schgr-ATR, by Schgr-AT caused an increase in intracellular calcium ions, as well as cyclic AMP (cAMP), with an EC50 value in the nanomolar range. In addition, the transcript distribution of both the Schgr-AT precursor and Schgr-ATR was investigated by means of quantitative real-time PCR. Moreover, we found more evidence for the myotropic and allatostimulatory actions of Schgr-AT in the desert locust. These data are discussed and situated in a broader context by comparison with literature data on AT and ATR in insects. PMID:25814925

  19. Molecular cloning and characterization of the allatotropin precursor and receptor in the desert locust, Schistocerca gregaria.

    Directory of Open Access Journals (Sweden)

    Els eLismont

    2015-03-01

    Full Text Available Allatotropins (ATs are pleiotropic neuropeptides initially isolated from the tobacco hornworm, Manduca sexta. In 2008, the first receptor for AT-like peptides (ATR was characterized in Bombyx mori. Since then, ATRs have also been characterized in M. sexta, Tribolium castaneum, Aedes aegypti and Bombus terrestris. These receptors show sequence similarity to vertebrate orexin (ORX receptors. When generating an EST-database of the desert locust (Schistocerca gregaria central nervous system, we found cDNA sequences encoding the Schgr-AT precursor and a fragment of its putative receptor. This receptor cDNA has now been completed and functionally expressed in mammalian cell lines. Activation of this receptor, designated as Schgr-ATR, by Schgr-AT caused an increase in intracellular calcium ions, as well as cyclic AMP (cAMP, with an EC50 value in the nanomolar range. In addition, the transcript distribution of both the Schgr-AT precursor and Schgr-ATR was investigated by means of quantitative real-time PCR. Moreover, we found more evidence for the myotropic and allatostimulatory actions of Schgr-AT in the desert locust. These data are discussed and situated in a broader context by comparison with literature data on AT and ATR in insects.

  20. Cloning and functional characterization of the intersex homologous gene in the pest lepidopteron Maruca vitrata.

    Science.gov (United States)

    Cavaliere, Daniela; Di Cara, Francesca; Polito, Lino C; Digilio, Filomena Anna

    2009-01-01

    The intersex (ix) gene works in concert with doublesex (dsx) at the bottom of the sex-determination hierarchy to control somatic sexual differentiation in Drosophila melanogaster females. Here we report the isolation and characterization of the Drosophila intersex (ix) homologue in the pest lepidopteron Maruca vitrata (Mvix). The Mvix gene exhibits major complexity with respect to the Drosophila homolog. It is expressed in males and females and its pre-mRNA is subject to differential splicing events which affect both the protein coding and the non-coding regions. Moreover, Northern blot experiments revealed the presence of a female-specific transcript in pupae RNA, which appears to be the first described sex specific transcript of ix homologs characterized to date. The expression of Mvix cDNA in D.melanogaster transgenic flies indicates that the MvIX product, which shares a relatively high degree of homology with the D.melanogaster IX protein, is able to partially rescues the Drosophila mutant phenotype.

  1. Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis).

    Science.gov (United States)

    Kwon, Moonhyuk; Cochrane, Stephen A; Vederas, John C; Ro, Dae-Kyun

    2014-12-20

    Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (-)-drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (-)-drimenol synthesis suggests that drimenol synthase is likely to use a protonation-initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (-)-drimenol, from which useful drimane-type terpenes can be synthesized. PMID:25447532

  2. Cloning and characterization of the Pichia pastoris MET2 gene as a selectable marker.

    Science.gov (United States)

    Thor, Der; Xiong, See; Orazem, Claire C; Kwan, An-Chun; Cregg, James M; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2005-07-01

    We describe the isolation and characterization of a new biosynthetic gene, MET2, from the methylotrophic yeast Pichia pastoris. The predicted product of PpMET2 is significantly similar to its Saccharomyces cerevisiae counterpart, ScMET2, which encodes homoserine-O-transacetylase. The ScMET2 was able to complement the P. pastoris met2 strain; however, the converse was not true. Expression vectors based on PpMET2 for the intracellular and secreted production of foreign proteins and corresponding auxotrophic strains were constructed and tested for use in heterologous expression. The expression vectors and corresponding strains provide greater flexibility when using P. pastoris for recombinant protein expression. PMID:15996626

  3. Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus

    DEFF Research Database (Denmark)

    Zhang, Ye-Wang; Tiwari, Manish Kumar; Gao, Hui;

    2012-01-01

    .6 and temperature 65 °C, and k(cat)/K(m) of 3.77×10(7) s(-1) M(-1), the highest ever reported. Heat inactivation studies revealed that LrNox had high thermostability, with a half-life of 120 min at 80 °C. Molecular dynamics simulation studies shed light on the factors contributing to the high activity of Lr......Nox. Although the properties of Nox from several microorganisms have been reported, this is the first report on the characterization of a recombinant H(2)O-forming Nox with high activity and thermostability. The characteristics of the LrNox enzyme could prove to be of interest in industrial applications...

  4. An ice nucleation protein from Fusarium acuminatum: cloning, expression, biochemical characterization and computational modeling.

    Science.gov (United States)

    Lagzian, Milad; Latifi, Ali Mohammad; Bassami, Mohammad Reza; Mirzaei, Morteza

    2014-10-01

    Ice nucleation proteins (INP) are a major cause of frost damage in plants and crops. Here, an INP gene from Fusarium acuminatum was optimized, synthesized, expressed in E.coli and subsequently purified and characterized. The protein belongs to the second class of ice nucleation proteins with an optimum pH 5.5, relative activity and stability between pH 5 and 9.5 and up to 45 °C. The protein was fully active and stable in the presence of dimethyl sulfoxide (DMSO), dioxane, acetone and ethyl acetate. Moreover, it retained over 50 % of its original activity in the presence of polyvinyl alcohol. The 3D structure model of the INP-F indicated the protein had three distinct domains as exist in other ice nucleation proteins with some variations. Considering these promising results, INP-F could be a novel candidate for industrial applications. PMID:24930104

  5. Molecular cloning, characterization and regulation of two different NADH-glutamate synthase cDNAs in bean nodules

    Science.gov (United States)

    NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary ammonia assimilation in bean (Phaseolus vulgaris L.) nodules. Two different types of cDNA clones of PvNADH-GOGAT were isolated from two independent nodule cDNA libraries. The full-length cDNA clones of PvNADH-GOGA...

  6. Cloning, characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea.

    Science.gov (United States)

    De Luca, Viviana; Vullo, Daniela; Del Prete, Sonia; Carginale, Vincenzo; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-02-15

    We have cloned, purified and characterized the γ-carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Antarctic bacterium Colwellia psychrerythraea, which is an obligate psychrophile. The enzyme shows a significant catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with the following kinetic parameters: kcat of 6.0×10(5)s(-1) and a kcat/Km of 4.7×10(6)M(-1)×s(-1). This activity was inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a KI of 502nM. A range of anions was also investigated for their inhibitory action against the new enzyme CpsCA. Perchlorate, tetrafluoroborate, fluoride and bromide were not inhibitory, whereas cyanate, thiocyanate, cyanide, hydrogensulfide, carbonate and bicarbonate showed KIs in the range of 1.4-4.4mM. Diethyldithiocarbamate was a better inhibitor (KI of 0.58mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with KIs ranging between 8 and 38μM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here. PMID:26778292

  7. Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas

    Science.gov (United States)

    Li, Xing-Xia; Yu, Wen-Chao; Cai, Zhong-Qiang; He, Cheng; Wei, Na

    2016-01-01

    The shell of the pearl oyster (Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization. PMID:27703977

  8. Molecular Cloning, Expression Analysis, and Preliminarily Functional Characterization of the Gene Encoding Protein Disulfide Isomerase from Jatropha curcas.

    Science.gov (United States)

    Wang, Haibo; Zou, Zhurong; Gong, Ming

    2015-05-01

    Reactive oxygen species (ROS) in plants, arising from various environmental stresses, impair the thiol-contained proteins that are susceptible to irregular oxidative formation of disulfide bonds, which might be alleviated by a relatively specific modifier called protein disulfide isomerase (PDI). From our previous data of the transcriptome and digital gene expression of cold-hardened Jatropha curcas, a PDI gene was proposed to be cold-relevant. In this study, its full-length cDNA (JcPDI) was cloned, with the size of 1649 bp containing the entire open reading frame (ORF) of 1515 bp. This ORF encodes a polypeptide of 504 amino acids with theoretical molecular weight of 56.6 kDa and pI value of 4.85. One N-terminal signal peptide (-MASKGSIWSCMFLFSLI VAISAGEG-) and the C-terminal anchoring sequence motif (-KDEL-) specific to the endoplasmic reticulum, as well as two thioredoxin domains (-CGHC-), are also found by predictions. Through semi-quantitative RT-PCR, the expression of JcPDI was characterized to be tissue-differential strongly in leaves and roots, but weakly in stems, and of cold-induced alternations. Furthermore, JcPDI overexpression in yeast could notably enhance the cold resistance of host cells. Conclusively, these results explicitly suggested a considerable association of JcPDI to cold response and a putative application potential for its correlated genetic engineering. PMID:25825250

  9. Cloning and functional characterization of Chondrichthyes, cloudy catshark, Scyliorhinus torazame and whale shark, Rhincodon typus estrogen receptors.

    Science.gov (United States)

    Katsu, Yoshinao; Kohno, Satomi; Narita, Haruka; Urushitani, Hiroshi; Yamane, Koudai; Hara, Akihiko; Clauss, Tonya M; Walsh, Michael T; Miyagawa, Shinichi; Guillette, Louis J; Iguchi, Taisen

    2010-09-15

    Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor-ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark (Scyliorhinus torazame) and whale shark (Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17beta-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor-ligand interactions and estrogen disrupting mechanisms in Elasmobranchii.

  10. PhAP protease from Pseudoalteromonas haloplanktis TAC125: Gene cloning, recombinant production in E. coli and enzyme characterization

    Science.gov (United States)

    de Pascale, D.; Giuliani, M.; De Santi, C.; Bergamasco, N.; Amoresano, A.; Carpentieri, A.; Parrilli, E.; Tutino, M. L.

    2010-08-01

    Cold-adapted proteases have been found to be the dominant activity throughout the cold marine environment, indicating their importance in bacterial acquisition of nitrogen-rich complex organic compounds. However, few extracellular proteases from marine organisms have been characterized so far, and the mechanisms that enable their activity in situ are still largely unknown. Aside from their ecological importance and use as model enzyme for structure/function investigations, cold-active proteolytic enzymes offer great potential for biotechnological applications. Our studies on cold adapted proteases were performed on exo-enzyme produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. By applying a proteomic approach, we identified several proteolytic activities from its culture supernatant. PhAP protease was selected for further investigations. The encoding gene was cloned and the protein was recombinantly produced in E. coli cells. The homogeneous product was biochemically characterised and it turned out that the enzyme is a Zn-dependent aminopeptidase, with an activity dependence from assay temperature typical of psychrophilic enzymes.

  11. Cloning and characterization of the gene encoding the PepF endopeptidase from the aquatic bacterium Caulobacter crescentus

    Directory of Open Access Journals (Sweden)

    Braz Vânia S.

    2002-01-01

    Full Text Available The metallopeptidases have a very important role in bacteria, being involved in several processes that rely on protein turnover, such as nutrition, degradation of signal peptides, protein localization and virulence. We have cloned and characterized the gene of the metalloendopeptidase PepF from the aquatic bacterium Caulobacter crescentus. The gene upstream of pepF (orf1 encodes a conserved hypothetical protein found in Mycobacterium and Streptomyces. pepF is co-transcribed with the gene downstream (orf3, which encodes a protein that belongs to the ABC1 protein kinase family, suggesting that these two proteins may share a common function in the cell. The C. crescentus PepF protein possesses the conserved HEXGH motif present in zinc binding domains of PepF homologs. Disruption of the pepF gene by insertion of a vector sequence did not produced any growth defect, but the mutant strain possesses only 30% of the specific activity of endopeptidases present in the wild type strain. Deletions and point mutations in the regulatory region showed that there are two putative promoter regions, and the operon expression is independent of the transcription regulator CtrA. The results indicate that PepF is not essential for either growth or development of this bacterium using peptides as the sole carbon source, suggesting that other peptidases can be sharing this function.

  12. Purification, characterization and cloning of tensilin, the collagen-fibril binding and tissue-stiffening factor from Cucumaria frondosa dermis.

    Science.gov (United States)

    Tipper, Jennifer P; Lyons-Levy, Gillian; Atkinson, Mark A L; Trotter, John A

    2002-12-01

    The inner dermis of the sea cucumber, Cucumaria frondosa, is a mutable collagenous tissue characterized by rapid and reversible changes in its mechanical properties regulated by one or more protein effectors that are released from neurosecretory cells. One such effector, tensilin, is a collagen-fibril binding protein, named for its ability to induce dermis stiffening. Tensilin was purified using an affinity column constructed from C. frondosa collagen-fibrils. The protein migrates as a single band on SDS-PAGE (Mr approximately 33 kDa) and has an isoelectric point of 5.8. Equilibrium sedimentation experiments suggest a molecular mass of approximately 28.5-29.4 kDa. Carbohydrate analysis of tensilin revealed no measurable sugar content. The molar amount of tensilin was determined to be 0.38% that of collagen and 47% that of stiparin, a constitutive matrix glycoprotein. A full-length cDNA clone for tensilin was obtained from a C. frondosa inner dermis cDNA expression library. Predicted properties derived from the deduced peptide sequence were in agreement with those of the native protein. A noted feature of tensilin's deduced peptide sequence, particularly in its N-terminal domain, is its homology to tissue inhibitor of metalloproteinases. Tensilin's C-terminal tail has no known homology to other proteins but contains a putative collagen-fibril binding site. PMID:12524049

  13. Cloning and characterization of human RTVP-1b, a novel splice variant of RTVP-1 in glioma cells

    International Nuclear Information System (INIS)

    Here, we report the cloning and characterization of RTVP-1b, a novel splice variant of human RTVP-1, which was isolated from the U87 glioma cell line. Sequence analysis revealed that RTVP-1b contains an additional 71 base exon between exons 2 and 3 that is missing in RTVP-1, leading to a frame-shift and a different putative protein. The deduced protein was 237 amino acids in length, sharing the N-terminal 141 amino acids with RTVP-1. RT-PCR analysis demonstrated that RTVP-1b was expressed in a wide range of tissues and that its expression was different from that of RTVP-1. In contrast, RTVP-1 and RTVP-1b showed similar patterns of expression in astrocytic tumors; highly expressed in glioblastomas as compared to normal brains, low-grade astrocytomas and anaplastic oligodendrogliomas. Overexpression of RTVP-1b increased glioma cell proliferation but did not affect cell migration. Our results suggest that RTVP-1b represents a potential prognostic marker and therapeutic target in gliomas

  14. Cloning and molecular characterization of an ornithine decarboxylase gene and its expression during embryonic development of the housefly, Musca domestica.

    Science.gov (United States)

    Toutges, Michelle J; Santoso, Adi

    2011-10-01

    We are interested in identifying targets that may be used to develop new control products for the common housefly, Musca domestica, a vector of disease for many vertebrates. One such target, ornithine decarboxylase (ODC), is an embryonic enzyme involved in the regulation of polyamines and is a critical enzyme during M. domestica development. In this study, the cDNA for ODC from M. domestica was cloned, sequenced, and characterized. The full-length cDNA was 1,337-bp, consistent with a single band of approximately 1.35 kb obtained by northern analysis. The open-reading frame contains 1,191 bp, yielding a deduced polypeptide of 396 amino acid residues with a predicted mass of 44,618 Da. The deduced M. domestica ODC protein was homologous to other ODC proteins. mRNA expression profiles analyzed by real-time PCR indicated that the ODC transcript is temporally regulated throughout embryogenesis. Sequence data and Southern blot analysis suggests that there were likely only one or two closely linked copies of the M. domestica ODC gene.

  15. Characterization of an abaecin-like antimicrobial peptide identified from a Pteromalus puparum cDNA clone.

    Science.gov (United States)

    Shen, Xiaojing; Ye, Gongyin; Cheng, Xiongying; Yu, Chunyan; Altosaar, Illimar; Hu, Cui

    2010-09-01

    Abaecin is a major antimicrobial peptide, initially identified from the honeybee. In our effort to discover new antimicrobial peptides from the endoparasitoid wasp Pteromalus puparum, we identified an antibacterial cDNA clone that codes a fragment with high amino acid sequence similarity to abaecin. The proline-rich peptide (YVPPVQKPHPNGPKFPTFP, named PP30) was chemically synthesized and characterized in this study. Antimicrobial assays indicated that the cationic peptide was active against both Gram-negative and positive bacteria, but not active against fungi tested. No hemolytic activity was observed against human erythrocytes after 1h incubation at concentration of 125 microM or below. The antibacterial activity of PP30 against Escherichia coli was attenuated in the presence of increasing concentrations of NaCl. Transmission electron microscopic (TEM) examination of PP30-treated E. coli cells showed morphological changes in the cells and extensive damage to the cell membranes. The circular dichroism (CD) spectroscopy studies indicated that PP30 formed random coil structures in phosphate buffer (pH 7.4), 50% TFE and 25 mM SDS solution. Expression analysis of the gene coding for the peptide indicated that its expression was upregulated upon bacterial infection, indicating that the gene may play a role in preventing potential infection by microorganisms during parasitization in Pieris rapae. PMID:20466006

  16. Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas

    Directory of Open Access Journals (Sweden)

    Xing-Xia Li

    2016-01-01

    Full Text Available The shell of the pearl oyster (Pinctada fucata mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization.

  17. Cloning and characterization of a gene encoding phage-related tail protein (PrTP) of endosymbiont Wolbachia

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Wolbachia is an obligatory, maternally inherited intracellular bacterium, known to infect a wide range of arthropods. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, the feminization of genetic males and male-killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these reproductive abnormalities have not yet been determined. In this study, we report on the cloning and characterization of the gene encoding phage-related tail protein (PrTP) from Wolbachia in Drosophila melanogaster CantonS (wMelCS) and from Wolbachia in Drosophila melanogaster yw67c23 (wMel) by representational difference analysis (RDA) and ligation-mediated PCR (LM-PCR). The functionality of a bipartite nuclear localization signal sequence (NLS) of the gene was also successfully tested in Drosophila S2 cells. PrTP expression in various strains of Wolbachia was investigated. Our results suggest that PrTP may not induce CI directly. However, the existence of prtp provided direct evidence of phage-mediated horizontal gene transfer (HGT) that might play an important role in a variety of reproductive abnormalities of Wolbachia.

  18. Cloning and characterization of a new laccase from Lactobacillus plantarum J16 CECT 8944 catalyzing biogenic amines degradation.

    Science.gov (United States)

    Callejón, S; Sendra, R; Ferrer, S; Pardo, I

    2016-04-01

    In our search for degrading activities of biogenic amines (BAs) in lactic acid bacteria, a protein annotated as laccase enzyme was identified in Lactobacillus plantarum J16 (CECT 8944). In this study, the gene of this new laccase was cloned and heterologously overexpressed in Escherichia coli. The recombinant laccase protein was purified and characterized biochemically. The purified laccase showed characteristic spectroscopic properties of blue multicopper oxidases. The enzyme has a molecular weight of ∼ 62.5 kDa and activity toward typical laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (2,6-DMP). The pH optima on ABTS and 2,6-DMP were 3.5 and 7.0, respectively. Kinetic constants Km and Vmax were of 0.21 mM and 0.54 U/mg for ABTS and 1.67 mM and 0.095 U/mg for 2,6-DMP, respectively. The highest oxidizing activity toward 2,6-DMP was obtained at 60 °C. However, after a preincubation step at 85 °C for 10 min, no residual activity was detected. It has been demonstrated that recombinant L. plantarum laccase oxidizes biogenic amines, mainly tyramine, and thus presents new biotechnological potential for the enzyme in eliminating toxic compounds present in fermented food and beverages. PMID:26590586

  19. Molecular Cloning and Characterization of a P-Glycoprotein from the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae

    Directory of Open Access Journals (Sweden)

    Lixia Tian

    2013-11-01

    Full Text Available Macrocyclic lactones such as abamectin and ivermectin constitute an important class of broad-spectrum insecticides. Widespread resistance to synthetic insecticides, including abamectin and ivermectin, poses a serious threat to the management of diamondback moth, Plutella xylostella (L. (Lepidoptera: Plutellidae, a major pest of cruciferous plants worldwide. P-glycoprotein (Pgp, a member of the ABC transporter superfamily, plays a crucial role in the removal of amphiphilic xenobiotics, suggesting a mechanism for drug resistance in target organisms. In this study, PxPgp1, a putative Pgp gene from P. xylostella, was cloned and characterized. The open reading frame (ORF of PxPgp1 consists of 3774 nucleotides, which encodes a 1257-amino acid peptide. The deduced PxPgp1 protein possesses structural characteristics of a typical Pgp, and clusters within the insect ABCB1. PxPgp1 was expressed throughout all developmental stages, and showed the highest expression level in adult males. PxPgp1 was highly expressed in midgut, malpighian tubules and testes. Elevated expression of PxPgp1 was observed in P. xylostella strains after they were exposed to the abamectin treatment. In addition, the constitutive expressions of PxPgp1 were significantly higher in laboratory-selected and field-collected resistant strains in comparison to their susceptible counterpart.

  20. Cloning, characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea.

    Science.gov (United States)

    De Luca, Viviana; Vullo, Daniela; Del Prete, Sonia; Carginale, Vincenzo; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-02-15

    We have cloned, purified and characterized the γ-carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Antarctic bacterium Colwellia psychrerythraea, which is an obligate psychrophile. The enzyme shows a significant catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with the following kinetic parameters: kcat of 6.0×10(5)s(-1) and a kcat/Km of 4.7×10(6)M(-1)×s(-1). This activity was inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a KI of 502nM. A range of anions was also investigated for their inhibitory action against the new enzyme CpsCA. Perchlorate, tetrafluoroborate, fluoride and bromide were not inhibitory, whereas cyanate, thiocyanate, cyanide, hydrogensulfide, carbonate and bicarbonate showed KIs in the range of 1.4-4.4mM. Diethyldithiocarbamate was a better inhibitor (KI of 0.58mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with KIs ranging between 8 and 38μM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here.

  1. Molecular cloning and characterization of high mobility group box1 (Ls-HMGB1) from humphead snapper, Lutjanus sanguineus.

    Science.gov (United States)

    Cai, Jia; Xia, Hongli; Huang, Yucong; Lu, Yishan; Wu, Zaohe; Jian, Jichang

    2014-10-01

    High mobility group box1 (HMGB1) is a kind of chromatin-associated nonhistone protein important for nucleosome formation, transcriptional regulation and inflammation. However, the reports about HMGB1 of marine fish were still limited. Here, we cloned and characterized a HMGB1 gene from humphead snapper, Lutjanus sanguineus (Ls-HMGB1). The Ls-HMGB1 cDNA composed of 1199 bp with a 70 bp of 5'-UTR, 630 bp open reading frame (ORF) and 499 bp 3'-UTR, encoded a polypeptide of 210 amino acids (GenBank Accession No: KJ783442). Sequence alignment of Ls-HMGB1 showed the highest similarity of 91% with Sciaenops ocellatus HMGB1 protein. Quantitative real-time PCR (qRT-PCR) analysis revealed that Ls-HMGB1 had relatively high expression level in skin, kidney and heart. After Vibrio harveyi and poly I:C stimulation, transcripts of Ls-HMGB1 were significantly increased and reached to peak at 18 h p.i. The L. sanguineus interleukin-6 (Ls-IL6) transcription in HK leukocytes was significantly induced by recombinant LsHMGB1 (rLsHMGB1). These results indicated that Ls-HMGB1 may play an important role in immune response of L. sanguineus during pathogen challenge.

  2. Cloning and characterization of two thermo- and salt-tolerant oligoalginate lyases from marine bacterium Halomonas sp.

    Science.gov (United States)

    Yang, Xuemei; Li, Shangyong; Wu, Ying; Yu, Wengong; Han, Feng

    2016-05-01

    Two new alginate lyase genes, oalY1 and oalY2, have been cloned from the newly isolated marine bacterium Halomonas sp. QY114 and expressed in Escherichia coli The deduced alginate lyases, OalY1 and OalY2, belonged to polysaccharide lyase (PL) family 17 and showed less than 45% amino acid identity with all of the characterized oligoalginate lyases. OalY1 and OalY2 exhibited the highest activities at 45°C and 50°C, respectively. Both of them showed more than 50% of the highest activity at 60°C, and 20% at 80°C. In addition, they were salt-dependent and salt-tolerant since both of them showed the highest activity in the presence of 0.5 M NaCl and preserved 63% and 68% of activity in the presence of 3 M NaCl. Significantly, OalY1 and OalY2 could degrade both polyM and polyG blocks into alginate monosaccharides in an exo-lytic type, indicating that they are bifunctional alginate lyases. In conclusion, our study indicated that OalY1 and OalY2 are good candidates for alginate saccharification application, and the salt-tolerance may present an exciting new concept for biofuel production from native brown seaweeds. PMID:27030725

  3. Cloning and functional characterization of Chondrichthyes, cloudy catshark, Scyliorhinus torazame and whale shark, Rhincodon typus estrogen receptors.

    Science.gov (United States)

    Katsu, Yoshinao; Kohno, Satomi; Narita, Haruka; Urushitani, Hiroshi; Yamane, Koudai; Hara, Akihiko; Clauss, Tonya M; Walsh, Michael T; Miyagawa, Shinichi; Guillette, Louis J; Iguchi, Taisen

    2010-09-15

    Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor-ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark (Scyliorhinus torazame) and whale shark (Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17beta-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor-ligand interactions and estrogen disrupting mechanisms in Elasmobranchii. PMID:20600039

  4. A new lipase as a pharmaceutical target for battling infections caused by Staphylococcus aureus: Gene cloning and biochemical characterization.

    Science.gov (United States)

    Ünlü, Aişe; Tanriseven, Aziz; Sezen, I Yavuz; Çelik, Ayhan

    2015-01-01

    Staphylococcus aureus lipases along with other cell-wall-associated proteins and enzymes (i.e., catalase, coagulase, protease, hyaluronidase, and β-lactamase) play important roles in the pathogenesis of S. aureus and are important subject of drug targeting. The appearance of antibiotic-resistant types of pathogenic S. aureus (e.g., methicillin-resistant S. aureus, MRSA) is a worldwide medical problem. In the present work, a novel lipase from a newly isolated MRSA strain from a cow with subclinical mastitis was cloned and biochemically characterized. The mature part of the lipase was expressed in Escherichia coli and purified by nickel affinity chromatography. It displays a high lipase activity at pH 8.0 and 25 °C using p-nitrophenyl palmitate and has a preference for medium-long-chain substrates of p-nitrophenyl esters (pNPC10-C16). Furthermore, in search of inhibitors, the effect of farnesol on the growth of S. aureus and the lipase activity was also studied. Farnesol inhibits the growth of S. aureus and is a mixed-type inhibitor with Ki and Ki (') values of 0.2 and 1.2 mmol L(-1), respectively. A lipase with known properties could not only serve as a template for developing inhibitors for S. aureus but also a valuable addition to enzyme toolbox of biocatalysis. The discovery of this lipase can be potentially important and could provide a new target for pharmaceutical intervention against S. aureus infection. PMID:25385356

  5. Cloning, Identification, and Characterization of the rpoS-Like Sigma Factor rpoX from Vibrio alginolyticus

    Directory of Open Access Journals (Sweden)

    Jing-jing Zhao

    2009-01-01

    Full Text Available Vibrio alginolyticus ZJ-51 displays phase variation between opaque/rugose colonies (Op and translucent/smooth colonies (Tr. These colony variants show great differences in biofilm formation and motility. In this study, a gene encoding for an rpoS-like sigma factor, rpoX, has been cloned and characterized. The absence of rpoX did not affect colony switching rate but did decrease biofilm formation in both the Op and the Tr variants. When challenged with hydrogen peroxide, the ΔrpoX in the Op background showed a slightly higher survival rate compared with the wild type, whereas survival was decreased in the Tr background. Deletion of rpoX in the Tr background resulted in a higher ability to resist ethanol challenges and to survive hyperosmolarity challenges, and in the Op background the opposite phenotype was observed. This indicates that the rpoX gene is involved in biofilm formation and stress response but the effects are controlled by colony phase variation in V. alginolyticus.

  6. Purification, characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17.

    Science.gov (United States)

    Shin, J-S; Yun, H; Jang, J-W; Park, I; Kim, B-G

    2003-06-01

    A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37 degrees C, respectively. Pyruvate and pyridoxal 5'-phosphate increased enzyme stability whereas (S)-alpha-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues) showed significant homology with omega-amino acid:pyruvate transaminases (omega-APT) from various bacterial strains (80 identical residues with four omega-APTs). However, of 159 conserved residues in the four omega-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward beta-alanine (a typical amino donor for the omega-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date. PMID:12687298

  7. Cloning, characterization, and expression of Cytochrome b (Cytb)-a key mitochondrial gene from Prorocentrum donghaiense

    Institute of Scientific and Technical Information of China (English)

    ZHAO Liyuan; MI Tiezhu; ZHEN Yu; YU Zhigang

    2012-01-01

    Mitochondrial cytochrome b (Cytb),one of the few proteins encoded by the mitochondrial DNA,plays an important role in transferring electrons.As a mitochondrial gene,it has been widely used for phylogenetic analysis.Previously,a 949-bp fragment of the coding gene and mRNA editing were characterized from Prorocentrum donghaiense,which might prove useful for resolving P.donghaiense from closely related species.However,the full-length coding region has not been characterized.In this study,we used rapid amplification of cDNA ends (RACE) to obtain full-length,1124 bp cDNA.Cytb transcript contained a standard initiation codon ATG,but did not have a recognizable stop codon.Homology comparison showed that the P.donghaiense Cytb had a high sequence identity to Cytb sequences from other dinoflagellate species.Phylogenetic analysis placed Cytb from P.donghaiense in the clade of dinofiagellates and it clustered together strongly with that from P.minimum.Based on the full-length sequence,we inferred 32 editing events at different positions,accounting for 2.93% of the Cytb gent.34.4% (11) of the changes were A to G,25% (8) were T to C,and 25% (8) were C to U,with smaller proportions of G to C and G to A edits (9.4% (3) and 6.2% (2),respectively).The expression level of the Cytb transcript was quantified by real-time PCR with a TaqMan probe at different times during the whole growth phase.The average Cytb transcript was present at 39.27±7.46 copies of cDNA per cell during the whole growth cycle,and the expression of Cytb was relatively stable over the different phases.These results deepen our understanding of the structure and characteristics of Cytb in P.donghaiense,and confirmed that Cytb in P.donghaiense is a candidate reference gene for studying the expression of other genes.

  8. Reproductive cloning : can cloning harm the clone?

    OpenAIRE

    Pattinson, S.D.

    2002-01-01

    Since the creation of Dolly the sheep was reported in February 1997, the possibility of a cloned child has elicited powerful declarations of condemnation. A widely held view is that cloning a human being would be immoral and ought to be prohibited by legislation. This paper outlines the regulatory approaches taken in the EU countries (with particular reference to the UK), Canada and the US, before examining the claim that creating a clone would be a wrong to the resultant clone. It is argued ...

  9. Optimal depletion of exhaustible resources: existence and characterization results

    Energy Technology Data Exchange (ETDEWEB)

    Mitra, T.

    1980-09-01

    A model of intertemporal allocation is considered in which there is a produced good (which can be used for consumption or for further production), and an exhaustible resource (which is essential for production), the total initial stock of which is given. The use of the resource over the (infinite) planning horizon must not exceed this available stock. A planner is assumed to evaluate consumption in each period, in terms of a utility function, and to maximize the undiscounted sum of these one-period utilities, to obtain, simultaneously, the optimal depletion of the exhaustible resource, and the optimal investment pattern. 20 references.

  10. Cloning, expression, and functional characterization of a Ca(2+)-dependent endoplasmic reticulum nucleoside diphosphatase.

    Science.gov (United States)

    Failer, Bernd U; Braun, Norbert; Zimmermann, Herbert

    2002-10-01

    We have isolated and characterized the cDNA encoding a Ca(2+)-dependent nucleoside diphosphatase (EC ) related to two secreted ATP- and ADP-hydrolyzing apyrases of the bloodsucking insects, Cimex lectularius and Phlebotomus papatasi. The rat brain-derived cDNA has an open reading frame of 1209 bp encoding a protein of 403 amino acids and a calculated molecular mass of 45.7 kDa. The mRNA was expressed in all tissues investigated, revealing two major transcripts with varying preponderance. The immunohistochemical analysis of the Myc-His-tagged enzyme expressed in Chinese hamster ovary cells revealed its association with the endoplasmic reticulum and also with pre-Golgi intermediates. Ca(2+)-dependent nucleoside diphosphatase is a membrane protein with its catalytic site facing the organelle lumen. It hydrolyzes nucleoside 5'-diphosphates in the order UDP >GDP = IDP >CDP but not ADP. Nucleoside 5'-triphosphates were hydrolyzed to a minor extent, and no hydrolysis of nucleoside 5'-monophosphates was observed. The enzyme was strongly activated by Ca(2+), insensitive to Mg(2+), and had a K(m) for UDP of 216 microm. Ca(2+)-dependent nucleoside diphosphatase may support glycosylation reactions related to quality control in the endoplasmic reticulum. PMID:12167635

  11. The ARF-like 2 (ARL2)-binding protein, BART. Purification, cloning, and initial characterization.

    Science.gov (United States)

    Sharer, J D; Kahn, R A

    1999-09-24

    ARF-like proteins (ARLs) comprise a functionally distinct group of incompletely characterized members in the ARF family of RAS-related GTPases. We took advantage of the GTP binding characteristics of human ARL2 to develop a specific, high affinity binding assay that allowed the purification of a novel ARL2-binding protein. A 19-kDa protein (BART, Binder of Arl Two) was identified and purified from bovine brain homogenate. BART binding is specific to ARL2.GTP with high affinity but does not interact with ARL2.GDP or activated ARF or RHO proteins. Based on peptide sequences of purified bovine BART, the human cDNA sequence was determined. The 489-base pair BART open reading frame encodes a novel 163-amino acid protein with a predicted molecular mass of 18,822 Da. Recombinant BART was found to bind ARL2.GTP in a manner indistinguishable from native BART. Northern and Western analyses indicated BART is expressed in all tissues sampled. The lack of detectable membrane association of ARL2 or BART upon activation of ARL2 is suggestive of actions quite distinct from those of the ARFs. The lack of ARL2 GTPase-activating protein activity in BART led us to conclude that the specific interaction with ARL2.GTP is most consistent with BART being the first identified ARL2-specific effector. PMID:10488091

  12. Cloning and Characterization of a Lycium chinense Carotenoid Isomerase Gene Enhancing Carotenoid Accumulation in Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    李招娣; 季静; 王罡

    2015-01-01

    Carotenoid isomerase(CRTISO)is a key enzyme that catalyzes the conversion of cis-lycopene to all-trans lycopene. In this study, we isolated and characterized the CRTISO gene from Lycium chinense (LcCRTISO) for the first time. The open reading frame of LcCRTISO was 1 815 bp encoding a protein of 604 amino acids with a molecular mass of 66.24 kDa. Amino acid sequence analysis revealed that the LcCRTISO had a high level of simi-larity to other CRTISO. Phylogenetic analysis displayed that LcCRTISO kept a closer relationship with the CRTISO of plants than with those of other species. Semi-quantitative PCR analysis indicated that LcCRTISO gene was expressed in all tissues tested with the highest expression in maturing fruits. The overexpression of LcCRTISO gene in transgenic tobacco resulted in an increase of total carotenoids in the leaves withβ-carotene and lutein being the predominants. The results obtained here clearly suggested that the LcCRTISO gene was a promising candidate for carotenoid production.

  13. Purification, cloning, and immunological characterization of arginine kinase, a novel allergen of Octopus fangsiao.

    Science.gov (United States)

    Shen, Hai-Wang; Cao, Min-Jie; Cai, Qiu-Feng; Ruan, Mi-Mi; Mao, Hai-Yan; Su, Wen-Jin; Liu, Guang-Ming

    2012-03-01

    Arginine kinase (AK) is an important enzyme participating in energy metabolism in invertebrates, but, to date, there have been no reports that AK from octopus is an allergen. In this study, octopus AK was purified, and its molecular biological, immunological, and physicochemical characterizations were analyzed. The results showed that octopus AK was purified and confirmed by mass spectrometry for the first time, and its molecular mass was 38 kDa. The full-length gene sequence of octopus AK encompassed 1209 bp and was predicted to encode a protein with 348 amino acid residues. The homology of octopus AK and crustacean AK was about 54%, but the similarity between their three-dimensional structures was high. Octopus AK could react with mouse anti-shrimp AK and rabbit anti-crab AK polyclonal antibody singly. Octopus AK could also react with specific IgE of the sera from octopus-allergic patients effectively, whereas crab AK could inhibit the reaction between them. Finally, the IgE-binding activity of octopus AK could be reduced in the processes of thermal or acid-alkali treatment. In summary, AK was identified as a novel allergen in octopus, which had a sensitizing ability similar to that of crustacean AK. This is significant in allergy diagnosis and the treatment of octopus-allergic disorders.

  14. Molecular cloning and characterization of the von Hippel-Lindau-like protein.

    Science.gov (United States)

    Qi, Heng; Gervais, Michelle L; Li, Wei; DeCaprio, James A; Challis, John R G; Ohh, Michael

    2004-01-01

    von Hippel-Lindau (VHL) tumor suppressor protein-inactivated in VHL disease and sporadic kidney cancer-is a component of an E3 ubiquitin ligase complex that selectively ubiquitinates the alpha subunit of the hypoxia-inducible factor (HIF) transcription factor for subsequent destruction by the 26S proteasome. Here, we report the identification and characterization of the first VHL homologue, VHL-like protein (VLP), located on chromosome 1q21.2. A 676-bp partial cDNA encoding a 139-amino acid protein that is 78% similar to VHL was isolated by reverse transcription-PCR from human brain cerebellum and several cancer cell lines. The expression of VLP transcript is most abundant in the placenta. Like VHL, VLP contains a beta domain capable of binding HIFalpha. However, unlike VHL, it does not contain a recognizable alpha domain, which is required for nucleating the multiprotein E3 ubiquitin ligase complex. The increased expression of VLP in the presence of VHL attenuated the ubiquitination of HIFalpha and led to the accumulation of downstream HIF target genes. These results taken together indicate that VLP functions as a dominant-negative VHL to serve as a protector of HIFalpha. PMID:14757845

  15. Molecular Cloning and Functional Characterization of Porcine MyD88 Essential for TLR Signaling

    Institute of Scientific and Technical Information of China (English)

    Masanori Tohno; Tomoyuki Shimazu; Hisashi Aso; Yasushi Kawai; Tadao Saito; Haruki Kitazawa

    2007-01-01

    We isolated cDNA encoding porcine MyD88 (poMyD88) from Peyer's patches (Pps) of GALT. The complete open reading frame (ORF) of poMyD88 contains 879 bp encoding a deduced 293 aa residues. The amino acid sequence of poMyD88 was characterized by N-terminal death, intermediate and C-terminal Toll/IL-1 receptor (TIR)domains. The putative poMyD88 protein shares a higher level of homology with its human (87.2% amino acid identity) than with its mouse (77.4% amino acid identity) counterpart. Overexpression of poMyD88 participated in the further enhanced activation of NF-κB in human embryonic kidney (HEK) 293 cells expressing porcine TLR2 and porcine TLR4/MD-2, but not porcine RP105/MD-1 after stimulation with the corresponding ligands. The expression levels of MyD88 were highest in the spleen and mesenteric lymph nodes (MLNs), and lower in digestive tissues of newborn swine. In adult swine, the expression levels in the digestive tissues were lower than those in MLNs and the spleen. These results suggest that an MyD88-dependent signaling pathway is present in newborn as well as in adult swine and that it is involved in the innate immune system of these animals.

  16. Cloning and Characterization of a Weissella confusa Dextransucrase and Its Application in High Fibre Baking

    Science.gov (United States)

    Kajala, Ilkka; Shi, Qiao; Nyyssölä, Antti; Maina, Ndegwa Henry; Hou, Yaxi; Katina, Kati; Tenkanen, Maija; Juvonen, Riikka

    2015-01-01

    Wheat bran offers health benefits as a baking ingredient, but is detrimental to bread textural quality. Dextran production by microbial fermentation improves sourdough bread volume and freshness, but extensive acid production during fermentation may negate this effect. Enzymatic production of dextran in wheat bran was tested to determine if dextran-containing bran could be used in baking without disrupting bread texture. The Weissella confusa VTT E-90392 dextransucrase gene was sequenced and His-tagged dextransucrase Wc392-rDSR was produced in Lactococcus lactis. Purified enzyme was characterized using 14C-sucrose radioisotope and reducing value-based assays, the former yielding Km and Vmax values of 14.7 mM and 8.2 μmol/(mg∙min), respectively, at the pH optimum of 5.4. The structure and size of in vitro dextran product was similar to dextran produced in vivo. Dextran (8.1% dry weight) was produced in wheat bran in 6 h using Wc392-rDSR. Bran with and without dextran was used in wheat baking at 20% supplementation level. Dextran presence improved bread softness and neutralized bran-induced volume loss, clearly demonstrating the potential of using dextransucrases in bran bioprocessing for use in baking. PMID:25603169

  17. Cloning and characterization of a Weissella confusa dextransucrase and its application in high fibre baking.

    Directory of Open Access Journals (Sweden)

    Ilkka Kajala

    Full Text Available Wheat bran offers health benefits as a baking ingredient, but is detrimental to bread textural quality. Dextran production by microbial fermentation improves sourdough bread volume and freshness, but extensive acid production during fermentation may negate this effect. Enzymatic production of dextran in wheat bran was tested to determine if dextran-containing bran could be used in baking without disrupting bread texture. The Weissella confusa VTT E-90392 dextransucrase gene was sequenced and His-tagged dextransucrase Wc392-rDSR was produced in Lactococcus lactis. Purified enzyme was characterized using (14C-sucrose radioisotope and reducing value-based assays, the former yielding K(m and V(max values of 14.7 mM and 8.2 μmol/(mg ∙ min, respectively, at the pH optimum of 5.4. The structure and size of in vitro dextran product was similar to dextran produced in vivo. Dextran (8.1% dry weight was produced in wheat bran in 6 h using Wc392-rDSR. Bran with and without dextran was used in wheat baking at 20% supplementation level. Dextran presence improved bread softness and neutralized bran-induced volume loss, clearly demonstrating the potential of using dextransucrases in bran bioprocessing for use in baking.

  18. Cloning and characterization of cDNA encoding an elicitor of Phytophthora colocasiae.

    Science.gov (United States)

    Mishra, Ajay Kumar; Sharma, Kamal; Misra, Raj Shekhar

    2010-02-28

    The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. A cDNA encoding elicitor, the major secreted extracellular glycoprotein of Phytophthora colocasiae, a pathogen of taro (Colocasia esculenta) plants, was isolated, sequenced and characterized. The expression of the corresponding elicitor gene during the disease cycle of P. colocasiae was analyzed. Elicitor was shown to be expressed in mycelium grown in culture media, whereas it was not expressed in sporangiospores and zoospores. In planta, during infection of taro, particularly during the biotrophic stage, expression of elicitor was down-regulated compared to in vitro. The highest levels of expression of elicitor were observed in in vitro grown mycelium and in late stages of infection when profuse sporulation and leaf necrosis occur. The elicitation of the suspension-cultured taro cells was effective in the induction of the enzyme activity of l-phenylalanine-ammonia lyase, peroxidase and lipoxygenase as well as the expression of defense-related endochitinase gene. All these biological activities were exerted within a low concentration range. The glycoprotein represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance. It may also be useful to engineer broad disease protection in taro plant against Phytophthora leaf blight. PMID:19230634

  19. Cloning, Expression and Characterization of Recombinant, NADH Oxidase from Giardia lamblia.

    Science.gov (United States)

    Castillo-Villanueva, Adriana; Méndez, Sara Teresa; Torres-Arroyo, Angélica; Reyes-Vivas, Horacio; Oria-Hernández, Jesús

    2016-02-01

    The NADH oxidase family of enzymes catalyzes the oxidation of NADH by reducing molecular O2 to H2O2, H2O or both. In the protozoan parasite Giardia lamblia, the NADH oxidase enzyme (GlNOX) produces H2O as end product without production of H2O2. GlNOX has been implicated in the parasite metabolism, the intracellular redox regulation and the resistance to drugs currently used against giardiasis; therefore, it is an interesting protein from diverse perspectives. In this work, the GlNOX gene was amplified from genomic G. lamblia DNA and expressed in Escherichia coli as a His-Tagged protein; then, the enzyme was purified by immobilized metal affinity chromatography, characterized, and its properties compared with those of the endogenous enzyme previously isolated from trophozoites (Brown et al. in Eur J Biochem 241(1):155-161, 1996). In comparison with the trophozoite-extracted enzyme, which was scarce and unstable, the recombinant heterologous expression system and one-step purification method produce a stable protein preparation with high yield and purity. The recombinant enzyme mostly resembles the endogenous protein; where differences were found, these were attributable to methodological discrepancies or artifacts. This homogenous, pure and functional protein preparation can be used for detailed structural or functional studies of GlNOX, which will provide a deeper understanding of the biology and pathogeny of G. lamblia. PMID:26685698

  20. Isolation, cloning and characterization of an azoreductase from the halophilic bacterium Halomonas elongata.

    Science.gov (United States)

    Eslami, Maryam; Amoozegar, Mohammad Ali; Asad, Sedigheh

    2016-04-01

    Azo dyes are a major class of colorants used in various industries including textile, paper and food. These dyes are regarded as pollutant since they are not readily reduced under aerobic conditions. Halomonas elongata, a halophilic bacterium, has the ability to decolorize different mono and di-azo dyes in anoxic conditions. In this study the putative azoreductase gene of H. elongata, formerly annotated as acp, was isolated, heterologously expressed in Escherichia coli, purified and characterized. The gene product, AzoH, was found to have a molecular mass of 22 kDa. The enzyme requires NADH, as an electron donor for its activity. The apparent Km was 63 μM for NADH and 12 μM for methyl red as a mono-azo dye substrate. The specific activity for methyl red was 0.27 μmol min(-1)mg(-1). The optimum enzyme activity was achieved in 50mM sodium phosphate buffer at pH 6. Although increased salinity resulted in reduced activity, AzoH could decolorize azo dye at NaCl concentrations up to 15% (w/v). The enzyme was also shown to be able to decolorize remazol black B as a representative of di-azo dyes. This is the first report describing the sequence and activity of an azo-reducing enzyme from a halophilic bacterium. PMID:26724685

  1. Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing

    International Nuclear Information System (INIS)

    Treatment of mouse cells with lipopolysaccharide (LPS) potently initiates an inflammatory response, but the underlying mechanisms are unclear. We therefore sought to characterize cDNA sequences of a new mouse LPS-responsive gene, and to evaluate the effects of MLrg. Full-length cDNAs were obtained from LPS-treated NIH3T3 cells. We report that the MLrg gene produces two alternative splice products (GenBank Accession Nos. (DQ316984) and (DQ320011)), respectively, encoding MLrgW and MLrgS polypeptides. Both proteins contain zinc finger and leucine zipper domains and are thus potential regulators of transcription. Expression of MLrgW and MLrgS were robustly upregulated following LPS treatment, and the proteins were localized predominantly in the nuclear membrane and cytoplasm. In stable transfectants over-expressing MLrgW the proportion of cells in G1 phase was significantly reduced, while in cells over-expressing MLrgS the proportion of cells in G2 was significantly increased; both proteins are thus potential regulators of cell cycle progression. Upregulation of MLrgW and MLrgS may be an important component of the LPS inflammatory pathway and of the host response to infection with GNB.

  2. Molecular cloning and catalytic characterization of a recombinant tropine biosynthetic tropinone reductase from Withania coagulans leaf.

    Science.gov (United States)

    Kushwaha, Amit K; Sangwan, Neelam S; Tripathi, Sandhya; Sangwan, Rajender S

    2013-03-10

    Tropinone reductases (TRs) are small proteins belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes. TR-I and TR-II catalyze the conversion of tropinone into tropane alcohols (tropine and pseudotropine, respectively). The steps are intermediary enroute to biosynthesis of tropane esters of medicinal importance, hyoscyamine/scopolamine, and calystegins, respectively. Biosynthesis of tropane alkaloids has been proposed to occur in roots. However, in the present report, a tropine forming tropinone reductase (TR-I) cDNA was isolated from the aerial tissue (leaf) of a medicinal plant, Withania coagulans. The ORF was deduced to encode a polypeptide of 29.34 kDa. The complete cDNA (WcTRI) was expressed in E. coli and the recombinant His-tagged protein was purified for functional characterization. The enzyme had a narrow pH range of substantial activity with maxima at 6.6. Relatively superior thermostability of the enzyme (30% retention of activity at 60 °C) was catalytic novelty in consonance with the desert area restricted habitat of the plant. The in vitro reaction kinetics predominantly favoured the forward reaction. The enzyme had wide substrate specificity but did not cover the substrates of other well-known plant SDR related to menthol metabolism. To our knowledge, this pertains to be the first report on any gene and enzyme of secondary metabolism from the commercially and medicinally important vegetable rennet species.

  3. Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Wei, Shina; Huang, Youhua; Cai, Jia; Huang, Xiaohong; Fu, Jing; Qin, Qiwei

    2012-08-01

    Lysozymes are key proteins of the host innate immune system against pathogen infection. In this study, a c-type lysozyme gene (Ec-lysC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysC cDNA is composed of 533 bp and encodes a polypeptide of 144-residue protein with 94% identity to lysC of Kelp grouper, Epinephelus bruneus. The genomic DNA of Ec-lysC consists of 4 exons and 3 introns, with a total length of 1897 bp. Amino acid sequence alignment showed that Ec-lysC possessed conserved catalytic residues (Glu50 and Asp67) and "GSTDYGIFQINS" motif. RT-PCR results showed that Ec-lysC transcript was most abundant in head kidney and less in muscle. The expression of Ec-lysC was differentially up-regulated in head kidney after stimulation with lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-lysC was distributed predominantly in the cytoplasm. The recombinant Ec-lysC (rEc-lysC) had lytic activities against Gram-positive bacteria Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus iniae and Gram-negative bacteria V. alginolyticus. The lysozyme acted on M. lysodeikticus cell walls as shown by scanning electron microscopy (SEM). Furthermore, overexpression of Ec-lysC in grouper cells delayed the occurrence of CPE induced by SGIV and inhibited the viral gene transcription significantly. Taken together, Ec-lysC might play an important role in grouper innate immune responses to invasion of bacterial and viral pathogens. C-type lysozyme gene from E. coioides (Ec-lysC) was identified and characterized.

  4. Molecular cloning and functional characterization of an ATP-binding cassette transporter OtrC from Streptomyces rimosus

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    Yu Lan

    2012-08-01

    Full Text Available Abstract Background The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production. Results In order to investigate OtrC’s function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877 and 1.4-fold in SR16 (P = 0.00973 duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively. Conclusions The results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.

  5. Molecular cloning and characterization of a cassava translationally controlled tumor protein gene potentially related to salt stress response.

    Science.gov (United States)

    Santa Brígida, Ailton Borges; dos Reis, Sávio Pinho; Costa, Carinne de Nazaré Monteirou; Cardoso, Cristina Michiko Yokoyama; Lima, Aline Medeiros; de Souza, Cláudia Regina Batista

    2014-03-01

    Cassava (Manihot esculenta Crantz) is one of the most important tropical crops showing tolerance to abiotic stress and adaptations to a wide range of environmental conditions. Here, we aimed to isolate and characterize the full-length cDNA and genomic sequences of a cassava translationally controlled tumor protein gene (MeTCTP), and evaluate its potential role in response to salt stress. The MeTCTP full-length cDNA sequence encodes for a deduced protein with 168 amino acid residues, with theoretical isoelectric point and molecular weight of 4.53 and 19 kDa, respectively, containing two putative signatures of TCTP family and one site for myristoylation. The MeTCTP genomic sequence includes four introns and five exons within a 1,643 bp coding region, and a 264 bp partial promoter sequence containing several putative cis-acting regulatory elements, among them, two putative GT-1 motifs, which may be related to response to sodium chloride (NaCl) and pathogen infection. Semi-quantitative RT-PCR assays showed that MeTCTP transcripts were higher in roots than leaves, and were significantly increased in detached leaves treated with NaCl. Furthermore, the recombinant MeTCTP conferred a protective function against salt stress in bacterial cells. We report for the first time the molecular cloning and characterization of a cassava TCTP with potential role in salt-stress response. Since salinity is one the most important abiotic factors affecting the production of crops worldwide, the MeTCTP gene could be a candidate gene for generation of salt tolerant crops.

  6. Cloning, functional characterization and nutritional regulation of Δ6 fatty acyl desaturase in the herbivorous euryhaline teleost Scatophagus argus.

    Science.gov (United States)

    Xie, Dizhi; Chen, Fang; Lin, Siyuan; Wang, Shuqi; You, Cuihong; Monroig, Óscar; Tocher, Douglas R; Li, Yuanyou

    2014-01-01

    Marine fish are generally unable or have low ability for the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, with some notable exceptions including the herbivorous marine teleost Siganus canaliculatus in which such a capability was recently demonstrated. To determine whether this is a unique feature of S. canaliculatus or whether it is common to the herbivorous marine teleosts, LC-PUFA biosynthetic pathways were investigated in the herbivorous euryhaline Scatophagus argus. A putative desaturase gene was cloned and functionally characterized, and tissue expression and nutritional regulation were investigated. The full-length cDNA was 1972 bp, containing a 1338 bp open-reading frame encoding a polypeptide of 445 amino acids, which possessed all the characteristic features of fatty acyl desaturase (Fad). Functional characterization by heterologous expression in yeast showed the protein product of the cDNA efficiently converted 18:3n-3 and 18:2n-6 to 18:4n-3 and 18:3n-6, respectively, indicating Δ6 desaturation activity. Quantitative real-time PCR showed that highest Δ6 fad mRNA expression was detected in liver followed by brain, with lower expression in other tissues including intestine, eye, muscle, adipose, heart kidney and gill, and lowest expression in stomach and spleen. The expression of Δ6 fad was significantly affected by dietary lipid and, especially, fatty acid composition, with highest expression of mRNA in liver of fish fed a diet with a ratio of 18:3n-3/18:2n-6 of 1.72:1. The results indicated that S. argus may have a different LC-PUFA biosynthetic system from S. canaliculatus despite possessing similar habitats and feeding habits suggesting that LC-PUFA biosynthesis may not be common to all marine herbivorous teleosts.

  7. Cloning and characterization of a wheat homologue of apurinic/apyrimidinic endonuclease Ape1L.

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    Botagoz Joldybayeva

    Full Text Available BACKGROUND: Apurinic/apyrimidinic (AP endonucleases are key DNA repair enzymes involved in the base excision repair (BER pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which includes E. coli Xth, human APE1 and Arabidopsis thaliana AtApe1L has been isolated and its protein product purified and characterized. METHODOLOGY/PRINCIPAL FINDINGS: We report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 3'-repair phosphodiesterase, 3'-phosphatase and 3' → 5' exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg(2+ and Ca(2+ (5-10 mM to the reaction mixture inhibited TaApe1L whereas the presence of Mn(2+, Co(2+ and Fe(2+ cations (0.1-1.0 mM strongly stimulated all its DNA repair activities. Optimization of the reaction conditions revealed that the wheat enzyme requires low divalent cation concentration (0.1 mM, mildly acidic pH (6-7, low ionic strength (20 mM KCl and has a temperature optimum at around 20 °C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 3'-blocking sugar-phosphate and 3'-phosphate groups with good efficiency (kcat/KM = 630 and 485 μM(-1 · min(-1, respectively but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1. CONCLUSIONS/SIGNIFICANCE: Taken together, these data establish the DNA substrate specificity of the wheat AP endonuclease and suggest its possible role in the repair of DNA damage generated by endogenous and environmental factors.

  8. Cloning and characterization of the gene for L-amino acid oxidase in hybrid tilapia.

    Science.gov (United States)

    Shen, Yubang; Fu, Gui Hong; Liu, Feng; Yue, Gen Hua

    2015-12-01

    Tilapia is the common name for a group of cichlid fishes. Identification of DNA markers significantly associated with important traits in candidate genes may speed up genetic improvement. L-Amino acid oxidase (LAO) plays a crucial role in the innate immune defences of animals. Previously, whether LAO variants were associated with economic traits had not been studied in fish. We characterized the cDNA sequence of the LAO gene of hybrid tilapia (Oreochromis spp.). Its ORF was 1536 bp, encoding a flavoenzyme of 511 amino acids. This gene consisted of seven exons and six introns. Its expression was detected in the intestine, blood, kidney, skin, liver. It was highly expressed in the intestine. After a challenge with a bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the liver, intestine and spleen (P < 0.05). We identified one SNP in the genomic sequence of the gene and found that this SNP was associated significantly with body length (P < 0.05), but not with resistance to S. agalactiae. The results of this study suggest that the LAO gene plays an important role in innate immune responses to the bacterial pathogen in tilapia. The investigation of relationship between polymorphism of LAO gene and disease resistance and growth in tilapia showed that one SNP was associated significantly with body length. Further experiments on whether SNPs in the LAO gene are associated with growth in tilapia and other populations could be useful in understanding more functions of the LAO gene. PMID:26546307

  9. Cloning and Characterization of Spliced Variants of the Porcine G Protein Coupled Receptor 120

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    Tongxing Song

    2015-01-01

    Full Text Available The polyunsaturated fatty acids (PUFAs receptor GPR120 exerts a significant impact on systemic nutrient homeostasis in human and rodents. However, the porcine GPR120 (pGPR120 has not been well characterized. In the current study, we found that pGPR120 had 3 spliced variants. Transcript 1 encoded 362-amino acids (aa wild type pGPR120-WT, which shared 88% homology with human short form GPR120. Transcript 1 was the mainly expressed transcript of pGPR120. It was expressed predominantly in ileum, jejunum, duodenum, spleen, and adipose. Transcript 3 (coding 320-aa isoform was detected in spleen, while the transcript 2 (coding 310-aa isoform was only slightly expressed in spleen. A selective agonist for human GPR120 (TUG-891 and PUFAs activated SRE-luc and NFAT-luc reporter in HEK293T cells transfected with construct for pGPR120-WT but not pGPR120-V2. However, 320-aa isoform was not a dominant negative isoform. The extracellular signal-regulated kinase 1/2 (ERK1/2 phosphorylation levels in cells transfected with construct for pGPR120-WT were well activated by PUFAs, especially n-3 PUFA. These results showed that although pGPR120 had 3 transcripts, transcript 1 which encoded pGPR120-WT was the mainly expressed transcript. TUG-891 and PUFAs, especially n-3 PUFA, well activated pGPR120-WT. The current study contributed to dissecting the molecular regulation mechanisms of n-3 PUFA in pigs.

  10. Identification, cloning and functional characterization of novel beta-defensins in the rat (Rattus norvegicus

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    French Frank S

    2006-02-01

    Full Text Available Abstract Background beta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation. Methods In silico rat genomic analyses were used to identify novel beta-defensins related to human defensins 118–123. RNAs isolated from male reproductive tract tissues of rat were reverse transcribed and PCR amplified using gene specific primers for defensins. PCR products were sequenced to confirm their identity. RT-PCR analysis was performed to analyze the tissue distribution, developmental expression and androgen regulation of these defensins. Recombinant defensins were tested against E. coli in a colony forming unit assay to analyze their antimicrobial activities. Results Novel beta-defensins, Defb21, Defb24, Defb27, Defb30 and Defb36 were identified in the rat male reproductive tract. Defb30 and Defb36 were the most restricted in expression, whereas the others were expressed in a variety of tissues including the female reproductive tract. Early onset of defensin expression was observed in the epididymides of 10–60 day old rats. Defb21-Defb36 expression in castrated rats was down regulated and maintained at normal levels in testosterone supplemented animals. DEFB24 and DEFB30 proteins showed potent dose and time dependent antibacterial activity. Conclusion Rat Defb21, Defb24, Defb27, Defb30 and Defb36 are abundantly expressed in the male reproductive tract where they most likely protect against microbial invasion. They are developmentally regulated and androgen is required for full expression in the adult epididymis.

  11. Molecular cloning and characterization of a flavanone 3-Hydroxylase gene from Artemisia annua L.

    Science.gov (United States)

    Xiong, Shuo; Tian, Na; Long, Jinhua; Chen, Yuhong; Qin, Yu; Feng, Jinyu; Xiao, Wenjun; Liu, Shuoqian

    2016-08-01

    Flavonoids were found to synergize anti-malaria and anti-cancer compounds in Artemisia annua, a very important economic crop in China. In order to discover the regulation mechanism of flavonoids in Artemisia annua, the full length cDNA of flavanone 3-hydroxylase (F3H) were isolated from Artemisia annua for the first time by using RACE (rapid amplification of cDNA ends). The completed open read frame of AaF3H was 1095 bp and it encoded a 364-amino acid protein with a predicted molecular mass of 41.18 kDa and a pI of 5.67. The recombinant protein of AaF3H was expressed in E. coli BL21(DE3) as His-tagged protein, purified by Ni-NTA agrose affinity chromatography, and functionally characterized in vitro. The results showed that the His-tagged protein (AaF3H) catalyzed naringenin to dihydrokaempferol in the present of Fe(2+). The Km for naringenin was 218.03 μM. The optimum pH for AaF3H reaction was determined to be pH 8.5, and the optimum temperature was determined to be 35 °C. The AaF3H transcripts were found to be accumulated in the cultivar with higher level of flavonoids than that with lower level of flavonoids, which implied that AaF3H was a potential target for regulation of flavonoids biosynthesis in Artemisia annua through metabolic engineering. PMID:27070290

  12. Cloning, Expression, and Characterization of Siamese Crocodile (Crocodylus siamensis) Hemoglobin from Escherichia coli and Pichia pastoris.

    Science.gov (United States)

    Anwised, Preeyanan; Jangpromma, Nisachon; Temsiripong, Theeranan; Patramanon, Rina; Daduang, Sakda; Jitrapakdee, Sarawut; Araki, Tomohiro; Klaynongsruang, Sompong

    2016-08-01

    Recombinant Crocodylus siamensis hemoglobin (cHb) has been constructed and expressed using Escherichia coli as the expression system in conjunction with a trigger factor from the Cold-shock system as the fusion protein. While successful processing as soluble protein in E. coli was achieved, the net yields of active protein from downstream purification processes remained still unsatisfactory. In this study, cHb was constructed and expressed in the eukaryotic expression system Pichia pastoris. The results showed that cHb was excreted from P. pastoris as a soluble protein after 72 h at 25 °C. The amino acid sequence of recombinant cHb was confirmed using LC-MS/MS. Indeed, the characteristic of Hb was investigated by external heme incorporation. The UV-Vis profile showed a specific pattern of the absorption at 415 nm, indicating the recombinant cHb was formed complex with heme, resulting in active oxyhemoglobin (OxyHb). This result suggests that the heme molecules were fully combined with heme binding site of the recombinant cHb, thus producing characteristic red color for the OxyHb at 540 and 580 nm. The results revealed that the recombinant cHb was prosperously produced in P. pastoris and exhibited a property as protein-ligand binding. Thus, our work described herein offers a great potential to be applied for further studies of heme-containing protein expression. It represents further pleasing option for protein production and purification on a large scale, which is important for determination and characterization of the authenticity features of cHb proteins. PMID:27301987

  13. Molecular and immunological characterization of a DNA-launched yellow fever virus 17D infectious clone.

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    Jiang, Xiaohong; Dalebout, Tim J; Lukashevich, Igor S; Bredenbeek, Peter J; Franco, David

    2015-04-01

    Yellow fever virus (YFV)-17D is an empirically developed, highly effective live-attenuated vaccine that has been administered to human beings for almost a century. YFV-17D has stood as a paradigm for a successful viral vaccine, and has been exploited as a potential virus vector for the development of recombinant vaccines against other diseases. In this study, a DNA-launched YFV-17D construct (pBeloBAC-FLYF) was explored as a new modality to the standard vaccine to combine the commendable features of both DNA vaccine and live-attenuated viral vaccine. The DNA-launched YFV-17D construct was characterized extensively both in cell culture and in mice. High titres of YFV-17D were generated upon transfection of the DNA into cells, whereas a mutant with deletion in the capsid-coding region (pBeloBAC-YF/ΔC) was restricted to a single round of infection, with no release of progeny virus. Homologous prime-boost immunization of AAD mice with both pBeloBAC-FLYF and pBeloBAC-YF/ΔC elicited specific dose-dependent cellular immune response against YFV-17D. Vaccination of A129 mice with pBeloBAC-FLYF resulted in the induction of YFV-specific neutralizing antibodies in all vaccinated subjects. These promising results underlined the potential of the DNA-launched YFV both as an alternative to standard YFV-17D vaccination and as a vaccine platform for the development of DNA-based recombinant YFV vaccines. PMID:25516543

  14. Cloning and functional characterization of three branch point oxidosqualene cyclases from Withania somnifera (L.) dunal.

    Science.gov (United States)

    Dhar, Niha; Rana, Satiander; Razdan, Sumeer; Bhat, Wajid Waheed; Hussain, Aashiq; Dhar, Rekha S; Vaishnavi, Samantha; Hamid, Abid; Vishwakarma, Ram; Lattoo, Surrinder K

    2014-06-13

    Oxidosqualene cyclases (OSCs) positioned at a key metabolic subdividing junction execute indispensable enzymatic cyclization of 2,3-oxidosqualene for varied triterpenoid biosynthesis. Such branch points present favorable gene targets for redirecting metabolic flux toward specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for the desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full-length OSC cDNAs, β-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS), and cycloartenol synthase (WsOSC/CS), having open reading frames of 2289, 2268, and 2277 bp, were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses, and kinetic studies confirmed their monofunctionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript profiling under methyl jasmonate, gibberellic acid, and yeast extract elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by Western blotting. Negative regulation by yeast extract resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like WsOSC/BS and WsOSC/LS possibly leads to diversion of substrate pool toward WsOSC/CS for increased withanolide production.

  15. Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

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    Tang Xian-Lai

    2010-11-01

    Full Text Available Abstract Background Fumarase catalyzes the reversible hydration of fumarate to L-malate and is a key enzyme in the tricarboxylic acid (TCA cycle and in amino acid metabolism. Fumarase is also used for the industrial production of L-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications. Results A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from Bacteroides sp. 2_1_33B and Parabacteroides distasonis ATCC 8503 (26% identical and 43% similar. The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in E. coli BL21(DE3pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form L-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg2+. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: Km= 0.48 mM, Vmax = 827 μM/min/mg, and kcat/Km = 1900 mM/s. Conclusions We isolated a novel fumarase gene, fumF, from a sequence-based screen of a plasmid metagenomic library from uncultivated

  16. Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi

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    Karan Ram

    2013-01-01

    Full Text Available Abstract Background Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica. Its genome sequencing project was recently completed, providing access to many genes predicted to encode polyextremophilic enzymes active in both extremely high salinity and cold temperatures. Results Analysis of the genome sequence of H. lacusprofundi showed a gene cluster for carbohydrate utilization containing a glycoside hydrolase family 42 β-galactosidase gene, named bga. In order to study the biochemical properties of the β-galactosidase enzyme, the bga gene was PCR amplified, cloned, and expressed in the genetically tractable haloarchaeon Halobacterium sp. NRC-1 under the control of a cold shock protein (cspD2 gene promoter. The recombinant β-galactosidase protein was produced at 20-fold higher levels compared to H. lacusprofundi, purified using gel filtration and hydrophobic interaction chromatography, and identified by SDS-PAGE, LC-MS/MS, and ONPG hydrolysis activity. The purified enzyme was found to be active over a wide temperature range (−5 to 60°C with an optimum of 50°C, and 10% of its maximum activity at 4°C. The enzyme also exhibited extremely halophilic character, with maximal activity in either 4 M NaCl or KCl. The polyextremophilic β-galactosidase was also stable and active in 10–20% alcohol-aqueous solutions, containing methanol, ethanol, n-butanol, or isoamyl alcohol. Conclusion The H. lacusprofundi β-galactosidase is a polyextremophilic enzyme active in high salt concentrations and low and high temperature. The enzyme is also active in aqueous-organic mixed solvents, with potential applications in synthetic chemistry. H. lacuprofundi proteins represent a significant biotechnology resource and for developing insights into enzyme catalysis under water limiting conditions. This study provides a system for better understanding how H. lacusprofundi is

  17. The rheology, degradation, processing, and characterization of renewable resource polymers

    Science.gov (United States)

    Conrad, Jason David

    Renewable resource polymers have become an increasingly popular alternative to conventional fossil fuel based polymers over the past couple decades. The push by the government as well as both industrial and consumer markets to go "green" has provided the drive for companies to research and develop new materials that are more environmentally friendly and which are derived from renewable materials. Two polymers that are currently being produced commercially are poly-lactic acid (PLA) and polyhydroxyalkanoate (PHA) copolymers, both of which can be derived from renewable feedstocks and have shown to exhibit similar properties to conventional materials such as polypropylene, polyethylene, polystyrene, and PET. PLA and PHA are being used in many applications including food packaging, disposable cups, grocery bags, and biomedical applications. In this work, we report on the rheological properties of blends of PLA and PHA copolymers. The specific materials used in the study include Natureworks RTM 7000D grade PLA and PHA copolymers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Blends ranging from 10 to 50 percent PHA by weight are also examined. Shear and extensional experiments are performed to characterize the flow behavior of the materials in different flow fields. Transient experiments are performed to study the shear rheology over time in order to determine how the viscoelastic properties change under typical processing conditions and understand the thermal degradation behavior of the materials. For the blends, it is determined that increasing the PHA concentration in the blend results in a decrease in viscosity and increase in degradation. Models are fit to the viscosity of the blends using the pure material viscosities in order to be able to predict the behavior at a given blend composition. We also investigate the processability of these materials into films and examine the resultant properties of the cast films. The mechanical and thermal properties of the

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

    OpenAIRE

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

    2009-01-01

    Abstract Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information an...

  19. Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana: ENZYMES CATALYZING UP TO 10 REARRANGEMENT STEPS YIELDING FRIEDELIN AND OTHER TRITERPENOIDS*

    OpenAIRE

    Wang, Zhonghua; Yeats, Trevor; Han, Hong; Jetter, Reinhard

    2010-01-01

    The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C30H50O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that...

  20. NADPH-cytochrome P450 reductase: molecular cloning and functional characterization of two paralogs from Withania somnifera (L. dunal.

    Directory of Open Access Journals (Sweden)

    Satiander Rana

    Full Text Available Withania somnifera (L. Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in

  1. Cloning and characterization of the dehydration-responsive element-binding protein 2A gene in Eruca vesicaria subsp sativa.

    Science.gov (United States)

    Huang, B L; Zhang, X K; Li, Y Y; Li, D Y; Ma, M Y; Cai, D T; Wu, W H; Huang, B Q

    2016-08-05

    Eruca vesicaria subsp sativa is one of the most tolerant Cruciferae species to drought, and dehydration-responsive element-binding protein 2A (DREB2A) is involved in responses to salinity, heat, and particularly drought. In this study, a gene encoding EvDREB2A was cloned and characterized in E. vesicaria subsp sativa. The full-length EvDREB2A cDNA sequence contained a 388-bp 5'-untranslated region (UTR), a 348-bp 3'-UTR, and a 1002-bp open reading frame that encoded 334 amino acid residues. The theoretical isoelectric point of the EvDREB2A protein was 4.80 and the molecular weight was 37.64 kDa. The genomic sequence of EvDREB2A contained no introns. Analysis using SMART indicated that EvDREB2A contains a conserved AP2 domain, similar to other plant DREBs. Phylogenetic analysis revealed that EvDREB2A and DREB2As from Brassica rapa, Eutrema salsugineum, Arabidopsis thaliana, Arabidopsis lyrata, and Arachis hypogaea formed a small subgroup, which clustered with DREB2Bs from A. lyrata, A. thaliana, Camelina sativa, and B. rapa to form a larger subgroup. EvDREB2A is most closely related to B. rapa DREB2A, followed by DREB2As from E. salsugineum, A. thaliana, A. hypogaea, and A. lyrata. A quantitative real-time polymerase chain reaction indicated that EvDREB2A expression was highest in the leaves, followed by the roots and hypocotyls, and was lowest in the flower buds. EvDREB2A could be used to improve drought tolerance in crops.

  2. PorcineLEM domain-containing 3:Molecular cloning, functional characterization, and polymorphism associated with ear size

    Institute of Scientific and Technical Information of China (English)

    LIANG Jing; SHI Hui-bi; ZHANG Qin; WANGLi-xian; LI Na; ZHANG Long-chao; WANGLi-gang; LIU Xin; ZHAO Ke-bin; YAN Hua; PU Lei; ZHANG Yue-bo

    2016-01-01

    Ear size exhibits remarkable diversity in pig breeds.LEM domain-containing 3 (LEMD3) on chromosome 5 is considered as an important candidate for porcine ear size. This is the ifrst study on cloning and characterization ofLEMD3 cDNA. The complete cDNA contains 4843 bp, including a 2736-bp open reading frame (ORF), a 37-bp 5´-untranslated region (UTR) and a 2070-bp 3´-UTR. The completeLEMD3 gene is 126241-bp and contains 13 exons and 12 introns. The ORF encodes a deduced LEMD3 protein of 911 amino acids, which shares 82–94% nucleic acid and 51–96% amino acid identity with other species. A phylogenetic tree constructed based on the amino acid sequences revealed that the porcine LEMD3 protein was closely related with cattle LEMD3. Resequencing of the ORF and promoter ofLEMD3 from Minzhu pig and Large White revealed three single nucleotide polymorphisms (SNPs): L964C>A in the complete coding region, L4625A>G in the 3´ UTR, and L-394T>C in the promoter region. Genome-wide association study (GWAS) revealed that al of SNPs were shown signiifcant association with ear size in Large White×Minzhu pig intercross population. With conditional GWAS, –log10(P-value) decreased by more than 80% when each of three SNPs was included as a ifxed effect. These results suggested direct involvement ofLEMD3 or close linkage to the causative mutation for ear size. The ifndings of this study might form the basis for understanding the genetic mechanism of ear size variation in pigs and provide potential molecular markers for screening ear size diversity in pig breeds.

  3. Molecular cloning and characterization of the MsHSP17.7 gene from Medicago sativa L.

    Science.gov (United States)

    Li, Zhen-Yi; Long, Rui-Cai; Zhang, Tie-Jun; Yang, Qing-Chuan; Kang, Jun-Mei

    2016-08-01

    Heat shock proteins (HSPs) are ubiquitous protective proteins that play crucial roles in plant development and adaptation to stress, and the aim of this study is to characterize the HSP gene in alfalfa. Here we isolated a small heat shock protein gene (MsHSP17.7) from alfalfa by homology-based cloning. MsHSP17.7 contains a 477-bp open reading frame and encodes a protein of 17.70-kDa. The amino acid sequence shares high identity with MtHSP (93.98 %), PsHSP17.1 (83.13 %), GmHSP17.9 (74.10 %) and SlHSP17.6 (79.25 %). Phylogenetic analysis revealed that MsHSP17.7 belongs to the group of cytosolic class II small heat shock proteins (sHSP), and likely localizes to the cytoplasm. Quantitative RT-PCR indicated that MsHSP17.7 was induced by heat shock, high salinity, peroxide and drought stress. Prokaryotic expression indicated that the salt and peroxide tolerance of Escherichia coli was remarkably enhanced. Transgenic Arabidopsis plants overexpressing MsHSP17.7 exhibited increased root length of transgenic Arabidopsis lines under salt stress compared to the wild-type line. The malondialdehyde (MDA) levels in the transgenic lines were significantly lower than in wild-type, although proline levels were similar between transgenic and wild-type lines. MsHSP17.7 was induced by heat shock, high salinity, oxidative stress and drought stress. Overexpression analysis suggests that MsHSP17.7 might play a key role in response to high salinity stress. PMID:27193169

  4. Molecular cloning, characterization and expression profiling of a ryanodine receptor gene in Asian corn borer, Ostrinia furnacalis (Guenee.

    Directory of Open Access Journals (Sweden)

    Li Cui

    Full Text Available Ryanodine receptor (RyR Ca(2+ release channel is the target of diamide insecticides, which show selective insecticidal activity against lepidopterous insects. To study the molecular mechanisms underlying the species-specific action of diamide insecticides, we have cloned and characterized the entire cDNA sequence of RyR from Ostrinia furnacalis (named as OfRyR. The OfRyR mRNA has an Open Reading Frame of 15324 bp nucleotides and encodes a 5108 amino acid polypeptide that displays 79-97% identity with other insects RyR proteins and shows the greatest identity with Cnaphalocrocis medinalis RyR (97%. Quantitative real-time PCR showed that the OfRyR was expressed at the lowest level in egg and the highest level in adult. The relative expression level of OfRyR in first, third and fifth-instar larva were 1.28, 1.19 and 1.99 times of that in egg. Moreover, two alternative splicing sites were identified in the OfRyR gene. One pair of mutually exclusive exons (a/b were present in the central part of the predicted SPRY domain, and an optional exon (c was located between the third and fourth RyR domains. Diagnostic PCR demonstrated that exons a and b existed in all developmental stages of OfRyR cDNA, but exon c was not detected in the egg cDNA. And the usage frequencies of these exons showed a significant difference between different developmental stages. These results provided the crucial basis for the functional expression of OfRyR and for the discovery of compound with potentially selective insect activtity.

  5. Immunoglobulin Tau Heavy Chain (IgT) in Flounder, Paralichthys olivaceus: Molecular Cloning, Characterization, and Expression Analyses.

    Science.gov (United States)

    Du, Yang; Tang, Xiaoqian; Zhan, Wenbin; Xing, Jing; Sheng, Xiuzhen

    2016-01-01

    Immunoglobulin tau (IgT) is a new teleost immunoglobulin isotype, and its potential function in adaptive immunity is not very clear. In the present study, the membrane-bound and secreted IgT (mIgT and sIgT) heavy chain genes were cloned for the first time and characterized in flounder (Paralichthys olivaceus), and found the nucleic acid sequence were exactly same in the Cτ1-Cτ4 constant domains of mIgT and sIgT, but different in variable regions and the C-terminus. The amino acid sequence of mIgT shared higher similarity with Bovichtus diacanthus (51.2%) and Dicentrarchus labrax (45.0%). Amino acid of flounder IgT, IgM, and IgD heavy chain was compared and the highest similarity was found between IgT Cτ1 and IgM Cμ1 (38%). In healthy flounder, the transcript levels of IgT mRNA were the highest in gill, spleen, and liver, and higher in peripheral blood leucocytes, skin, and hindgut. After infection and vaccination with Edwardsiella tarda via intraperitoneal injection and immersion, the qRT-PCR analysis demonstrated that the IgT mRNA level was significantly upregulated in all tested tissues, with similar dynamic tendency that increased firstly and then decreased, and higher in gill, skin, hindgut, liver, and stomach in immersion than in the injection group, but no significant difference existed in spleen and head kidney between immersion and injection groups. These results revealed that IgT responses could be simultaneously induced in both mucosal and systemic tissues after infection/vaccination via injection and immersion route, but IgT might play a more important role in mucosal immunity than in systemic immunity. PMID:27649168

  6. Cloning and characterization of the dehydration-responsive element-binding protein 2A gene in Eruca vesicaria subsp sativa.

    Science.gov (United States)

    Huang, B L; Zhang, X K; Li, Y Y; Li, D Y; Ma, M Y; Cai, D T; Wu, W H; Huang, B Q

    2016-01-01

    Eruca vesicaria subsp sativa is one of the most tolerant Cruciferae species to drought, and dehydration-responsive element-binding protein 2A (DREB2A) is involved in responses to salinity, heat, and particularly drought. In this study, a gene encoding EvDREB2A was cloned and characterized in E. vesicaria subsp sativa. The full-length EvDREB2A cDNA sequence contained a 388-bp 5'-untranslated region (UTR), a 348-bp 3'-UTR, and a 1002-bp open reading frame that encoded 334 amino acid residues. The theoretical isoelectric point of the EvDREB2A protein was 4.80 and the molecular weight was 37.64 kDa. The genomic sequence of EvDREB2A contained no introns. Analysis using SMART indicated that EvDREB2A contains a conserved AP2 domain, similar to other plant DREBs. Phylogenetic analysis revealed that EvDREB2A and DREB2As from Brassica rapa, Eutrema salsugineum, Arabidopsis thaliana, Arabidopsis lyrata, and Arachis hypogaea formed a small subgroup, which clustered with DREB2Bs from A. lyrata, A. thaliana, Camelina sativa, and B. rapa to form a larger subgroup. EvDREB2A is most closely related to B. rapa DREB2A, followed by DREB2As from E. salsugineum, A. thaliana, A. hypogaea, and A. lyrata. A quantitative real-time polymerase chain reaction indicated that EvDREB2A expression was highest in the leaves, followed by the roots and hypocotyls, and was lowest in the flower buds. EvDREB2A could be used to improve drought tolerance in crops. PMID:27525923

  7. Cloning and characterization of equine CD89 and identification of the CD89 gene in chimpanzees and rhesus macaques.

    Science.gov (United States)

    Morton, H Craig; Pleass, Richard J; Storset, Anne K; Brandtzaeg, Per; Woof, Jenny M

    2005-05-01

    Immunoglobulin A (IgA) is the major antibody class present in external secretions of mammals. At the vulnerable mucosal surfaces, IgA provides a crucial first-line defence by neutralizing pathogens. Primates also have a substantial level of IgA in serum and although not well understood, the biological role of this IgA depends, at least partly, on its ability to interact with specific receptors (FcalphaRs) on the surface of leucocytes. The human FcalphaR, CD89, was the first IgA Fc receptor to be identified and binding of IgA-coated particles to CD89 triggers numerous cellular effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, all of which may play an important role in both systemic and mucosal immunity. For many years humans were the only species known to express CD89, however, it has recently been cloned from cows and rats. Here, we describe the identification of the CD89 gene in three additional species: horses, chimpanzees, and Rhesus macaques. Equine CD89 was identified at the cDNA level, whereas the chimpanzee and Rhesus macaque genes were identified from the available draft genomic sequence. Interestingly, when compared with humans and other primates, horses, cows and rats have a relatively low concentration of serum IgA, so the role of CD89 in these species is of particular interest. The identification and characterization of CD89 in different species will contribute to a greater understanding of the biological role of IgA and CD89 in mucosal and systemic immunity throughout evolution. PMID:15819699

  8. Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome

    Directory of Open Access Journals (Sweden)

    Ritland Carol

    2009-08-01

    Full Text Available Abstract Background Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs and full-length (FLcDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. Results We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR and a cytochrome P450 (CYP720B4 from a non-arrayed genomic BAC library of white spruce (Picea glauca. Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR and 94 kbp (CYP720B4 long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs, high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. Conclusion We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene

  9. Cloning and characterization of interferon stimulated genes Viperin and ISG15,and their promoters from snakehead Channa argus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    By suppression subtractive hybridization,rapid amplification of cDNA ends and gene walking methods,interferon stimulated genes (ISGs),Viperin and ISG15,and their promoters have been cloned and characterized from snakehead Channa argus.The Viperin cDNA was found to be 1474 nt and contain an open reading frame(ORF)of 1059 nt that translates into a putative peptide of 352amino acid(aa).The putative peptide of Viperin shows high identity to that in teleosts and mamals except for the N-terminal 70 aa.The ISG15 cDNA was found to be 758 nt and contain an ORF of 468 nt that translates into a putative peptide of 155 aa.The putative peptide of ISG15 is composed of two tandem repeats of ubiquitin-like(UBL)domains,and a canonical conjugation motif(LRGG)at C-terminal.Viperin and ISG15 promoter regions were characterized by the presence of interferon stimulating response elements(ISRE)and γ-IFN activation sites (GAS).ISRE is a feature of IFN-induced gene promoter and partially overlaps interferon regulatory factor (IRF)1 and IRF2 recognition sites.GAS is responsible for the γ-IFN mediated transcription.One conserved site for NF-kB was found in the promoter region of Viperin.This is the first report of conservative binding motif for NF-kB in accordance with the consensus sequence(GGGRNNYYCC)among teleost ISG promoters.Moreover,there were also TATA,CAAT and Spl transcription factor sites in Viperin and ISG15 promoters.In 5'untranslated region (UTR),snakehead ISG15 gene contains a single intron,which differs from Viperin gene.The transcripts of Vipeirn and ISG15 mRNA were mainly expressed in head kidney,posterior kidney,spleen and gill.The expression levels in liver were found to increase obviously in response to induction by IFN-inducer poly I:C.

  10. Characterization and normalization factors of abiotic resource depletion for life cycle impact assessment in China

    Institute of Scientific and Technical Information of China (English)

    GAO Feng; NIE ZuoRen; WANG ZhiHong; GONG XianZheng; ZUO TieYong

    2009-01-01

    The availability of resources for economic activities differs between regions, and the importance of the resources is consequently observed to be different within regions compared to a global scale. With the current situation in Chinese mining industry and its statistic characteristics, the characterization pro-cedures of abiotic resource in life cycle impact assessment (LCIA) have demonstrated certain limita-tions in the Chinese materials industry. The aim of this paper is to propose new characterization and normalization factors for abiotic resource depletion categories such as metals and non-renewable en-ergy resources in a Chinese context. The actual production of abiotic resources calculated by a modi-fied model is compared to the reserve base in line with the new national standard to determine char-acterization factors in equivalence units, with antimony as the reference mineral. The normalization factors are based on the total base reserves of the most important minerals in China. A case study on primary magnesium production using the Pidgeon process is used to compare LCIA results for abiotic resource categories that are between current LCIA factors and the new Chinese factors. These factors not only reflect the importance of abiotic resource with respect to region-specific resource depletion, but also can compare with the global factors.

  11. A Genome-Scale Resource for the Functional Characterization of Arabidopsis Transcription Factors

    Directory of Open Access Journals (Sweden)

    Jose L. Pruneda-Paz

    2014-07-01

    Full Text Available Extensive transcriptional networks play major roles in cellular and organismal functions. Transcript levels are in part determined by the combinatorial and overlapping functions of multiple transcription factors (TFs bound to gene promoters. Thus, TF-promoter interactions provide the basic molecular wiring of transcriptional regulatory networks. In plants, discovery of the functional roles of TFs is limited by an increased complexity of network circuitry due to a significant expansion of TF families. Here, we present the construction of a comprehensive collection of Arabidopsis TFs clones created to provide a versatile resource for uncovering TF biological functions. We leveraged this collection by implementing a high-throughput DNA binding assay and identified direct regulators of a key clock gene (CCA1 that provide molecular links between different signaling modules and the circadian clock. The resources introduced in this work will significantly contribute to a better understanding of the transcriptional regulatory landscape of plant genomes.

  12. Molecular cloning and characterization of the structural gene for protein I, the major outer membrane protein of Neisseria gonorrhoeae.

    OpenAIRE

    Carbonetti, N H; Sparling, P F

    1987-01-01

    Protein I (P.I) is the major outer membrane protein of Neisseria gonorrhoeae and serves as a porin. By using oligonucleotide probes derived from the known amino-terminal sequence of the mature protein, we have cloned the gene encoding the P.I of gonococcal strain FA19 in three overlapping fragments and determined the DNA sequence. The gene sequence predicts a protein with characteristics typical of the porins of other Gram-negative bacteria. A clone expressing P.I in Escherichia coli was obta...

  13. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea.

    OpenAIRE

    Staskawicz, B; Dahlbeck, D; Keen, N; Napoli, C.

    1987-01-01

    A wide-host-range cosmid cloning vector, pLAFR3, was constructed and used to make cosmid libraries of partially digested Sau3A DNA from race 0 and race 1 of Pseudomonas syringae pv. glycinea. Two avirulence genes, avrB0 and avrC, cloned from race 0, elicited the hypersensitivity reaction (HR) on specific cultivars of soybean. Race 4 transconjugants containing avrB0 induced a dark brown necrotic HR within 24 h on the soybean cultivars Harosoy and Norchief, whereas race 4 transconjugants contai...

  14. Molecular cloning and characterization of two types of IκBα orthologues in orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Gao, Ren; Huang, Youhua; Huang, Xiaohong; Guan, Liya; Wei, Shina; Zhou, Yongcan; Qin, Qiwei

    2014-05-01

    Inhibitors of kappa B (IκBs) are the members of primary regulators of NF-κB, which can inhibit NF-κB activity by blocking the NF-κB in an inactive state in the cytoplasm. In this study, two types of IκBα orthologues (EcIκBαA and EcIκBαB) from orange-spotted grouper, Epinephelus coioides, were cloned and characterized. EcIκBαA and EcIκBαB encoded putative proteins containing 308 and 318 amino acids, which shared 59% and 53% identity to IκBαA and IκBαB of Danio rerio, respectively. Amino acid sequence alignment showed that both EcIκBαA and EcIκBαB contained a conserved degradation motif DSGLDS in the N-terminal region and a PEST sequence in the C-terminal region. In addition, EcIκBαA and EcIκBαB contained 5 and 6 ankyrin repeats, respectively. The genomic DNA of EcIκBαA and EcIκBαB consisted of 6 exons and 5 introns. Both of their transcripts were widely distributed in different tissues, and the expression levels were different in response to various stimuli, including lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Dual-luciferase reporter assay suggested that both EcIκBαA and EcIκBαB were able to inhibit Ecc-Rel and Ecp65 induced NF-κB promoter activity in grouper spleen (GS) cells. Subcellular localization analysis showed that EcIκBαB was present predominantly in the cytoplasm, while EcIκBαA was distributed throughout both the nucleus and the cytoplasm. Furthermore, overexpression of EcIκBαA and EcIκBαB in GS cells inhibited the viral gene transcriptions of MCP, ORF019 and ORF162 of SGIV. Taken together, our findings suggested that both EcIκBαA and EcIκBαB were involved in grouper innate immunity against virus.

  15. Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene.

    Science.gov (United States)

    Sujiwattanarat, Penporn; Pongsanarakul, Parinya; Temsiripong, Yosapong; Temsiripong, Theeranan; Thawornkuno, Charin; Uno, Yoshinobu; Unajak, Sasimanas; Matsuda, Yoichi; Choowongkomon, Kiattawee; Srikulnath, Kornsorn

    2016-01-01

    Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD. PMID:26523498

  16. Cloning and characterization of mr-s, a novel SAM domain protein, predominantly expressed in retinal photoreceptor cells

    Directory of Open Access Journals (Sweden)

    Koike Chieko

    2006-03-01

    Full Text Available Abstract Background Sterile alpha motif (SAM domains are ~70 residues long and have been reported as common protein-protein interaction modules. This domain is found in a large number of proteins, including Polycomb group (PcG proteins and ETS family transcription factors. In this work, we report the cloning and functional characterization of a novel SAM domain-containing protein, which is predominantly expressed in retinal photoreceptors and the pineal gland and is designated mouse mr-s (major retinal SAM domain protein. Results mr-s is evolutionarily conserved from zebrafish through human, organisms through which the mechanism of photoreceptor development is also highly conserved. Phylogenetic analysis suggests that the SAM domain of mr-s is most closely related to a mouse polyhomeotic (ph ortholog, Mph1/Rae28, which is known as an epigenetic molecule involved in chromatin modifications. These findings provide the possibility that mr-s may play a critical role by regulating gene expression in photoreceptor development. mr-s is preferentially expressed in the photoreceptors at postnatal day 3–6 (P3-6, when photoreceptors undergo terminal differentiation, and in the adult pineal gland. Transcription of mr-s is directly regulated by the cone-rod homeodomain protein Crx. Immunoprecipitation assay showed that the mr-s protein self-associates mainly through the SAM domain-containing region as well as ph. The mr-s protein localizes mainly in the nucleus, when mr-s is overexpressed in HEK293T cells. Moreover, in the luciferase assays, we found that mr-s protein fused to GAL4 DNA-binding domain functions as a transcriptional repressor. We revealed that the repression activity of mr-s is not due to a homophilic interaction through its SAM domain but to the C-terminal region. Conclusion We identified a novel gene, mr-s, which is predominantly expressed in retinal photoreceptors and pineal gland. Based on its expression pattern and biochemical analysis

  17. Characterization of the coal resources of South Africa

    Energy Technology Data Exchange (ETDEWEB)

    Jeffrey, L.S. [CSIR, Auckland (New Zealand). Division of Mining Technology

    2005-02-01

    Estimates for South Africa's coal recoverable reserves made in 1999 range from nine to 59 billion tons; latest estimates by the Minerals Bureau suggest that 33 billion tons is a more likely figure. As much as 70% of that coal is located in the Waterberg, Witbank, and Highveld coalfields, as well as lesser amounts in the Ermelo, Free State and Springbok Flats coalfields. However, the Witbank and Highveld coalfields are approaching exhaustion (estimated 9 billion tons of recoverable coal remaining in each), while the coal quality or mining conditions in the Waterberg, Free State and Springbok Flats coalfields are significant barriers to immediate, conventional exploitation. New extraction technologies, technologies exploiting the energy content of the coal in situ, as well as suitable uses and markets for low-grade, high-ash coal are required before the country can utilize its admittedly vast coal resources. Major challenges for exploiting some Limpopo province coalfields are severe water shortages, insufficiently developed infrastructure, fragile environments and poor roof conditions due to the depth and complex geology. In the Central Basin (Witbank, Highveld and Ermelo coalfields) technical innovations for thin seam extraction, economic mining of both pillar coal and intrusion-fragmented resource blocks and the utilization of lower-grade coals are required. The success of the fluidized bed combustion technology is necessary to utilize the low-grade coals of the Free State and Molteno coalfields. Clean coal technologies, coal cost and quality, environmental considerations, sustainable development, the growth of the South African economy and Government's regulation of the electricity industry are the main challenges to the continued use of coal as South Africa's primary energy source.

  18. Characterization and normalization factors of abiotic resource depletion for life cycle impact assessment in China

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The availability of resources for economic activities differs between regions, and the importance of the resources is consequently observed to be different within regions compared to a global scale. With the current situation in Chinese mining industry and its statistic characteristics, the characterization procedures of abiotic resource in life cycle impact assessment (LCIA) have demonstrated certain limita-tions in the Chinese materials industry. The aim of this paper is to propose new characterization and normalization factors for abiotic resource depletion categories such as metals and non-renewable en- ergy resources in a Chinese context. The actual production of abiotic resources calculated by a modi- fied model is compared to the reserve base in line with the new national standard to determine char- acterization factors in equivalence units, with antimony as the reference mineral. The normalization factors are based on the total base reserves of the most important minerals in China. A case study on primary magnesium production using the Pidgeon process is used to compare LCIA results for abiotic resource categories that are between current LCIA factors and the new Chinese factors. These factors not only reflect the importance of abiotic resource with respect to region-specific resource depletion, but also can compare with the global factors.

  19. cDNA cloning and characterization of a gibberellin-responsive gene in hypocotyls of Cucumis sativus L.

    Science.gov (United States)

    Chono, M; Yamauchi, T; Yamaguchi, S; Yamane, H; Murofushi, N

    1996-07-01

    A cDNA clone corresponding to a gibberellin-responsive gene (CRG16) was isolated from cucumber hypocotyls. CRG16 was deduced to encode an extremely hydrophobic protein of 65 amino acids. The deduced sequence exhibited no significant homology to other proteins. Levels of CRG16 mRNA reflected the gibberellin-induced elongation of cucumber hypocotyls.

  20. Cloning and verification of the Lactococcus lactis pyrG gene and characterization of the gene product, CTP synthase

    DEFF Research Database (Denmark)

    Wadskov-Hansen, Steen Lyders Lerche; Willemoës, M.; Martinussen, Jan;

    2001-01-01

    The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactis strains harboring disrupted...

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

    Science.gov (United States)

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

    1988-07-01

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

  2. Cloning and expression of the catalase-peroxidase gene from the hyperthermophilic archaeon Archaeoglobus fulgidus and characterization of the enzyme

    NARCIS (Netherlands)

    Kengen, S.W.M.; Bikker, F.; Vos, de W.M.; Oost, van der J.

    2001-01-01

    A putative perA gene from Archaeoglobus fulgidus was cloned and expressed in Escherichia coli BL21(DE3), and the recombinant catalase-peroxidase was purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 85 kDa. UV-visible spectroscopic analysis indicated the presence of

  3. Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage.

    Science.gov (United States)

    Dopico, B; Lowe, A L; Wilson, I D; Merodio, C; Grierson, D

    1993-02-01

    Differential screening of a cDNA library made from RNA extracted from avocado (Persea americana Mill cv. Hass) fruit stored at low temperature (7 degrees C) gave 23 cDNA clones grouped into 10 families, 6 of which showed increased expression during cold storage and normal ripening. Partial DNA sequencing was carried out for representative clones. Database searches found homologies with a polygalacturonase (PG), endochitinase, cysteine proteinase inhibitor and several stress-related proteins. No homologies were detected for clones from six families and their biological role remains to be elucidated. A full-length cDNA sequence for avocado PG was obtained and the predicted amino acid sequence compared with those from other PGs. mRNA encoding PG increased markedly during normal ripening, slightly later than mRNAs for cellulase and ethylene-forming enzyme (EFE). Low-temperature storage delayed ripening and retarded the appearance of mRNAs for enzymes known to be involved in cell wall metabolism and ethylene synthesis, such as cellulase, PG and EFE, and also other mRNAs of unknown function. The removal of ethylene from the atmosphere surrounding stored fruit delayed the appearance of the mRNAs encoding cellulase and PG more than the cold storage itself, although it hardly affected the expression of the EFE mRNA or the accumulation of mRNAs homologous to some other unidentified clones.

  4. Molecular cloning and characterization of two hypersensitive induced reaction genes from wheat infected by stripe rust pathogen

    Science.gov (United States)

    A novel gene induced during hypersensitive reaction (HIR) in wheat was identified using in silico cloning and designated as TaHIR2. The TaHIR2 gene was deduced to encode a 284-amino acid protein, whose molecular mass and isoelectric point (pI) were 31.05 kD and 5.18, respectively. Amino acid sequenc...

  5. Impact Assessment of Abiotic Resources in LCA: Quantitative Comparison of Selected Characterization Models

    DEFF Research Database (Denmark)

    Rørbech, Jakob Thaysen; Vadenbo, Carl; Hellweg, Stefanie;

    2014-01-01

    Resources have received significant attention in recent years resulting in development of a wide range of resource depletion indicators within life cycle assessment (LCA). Understanding the differences in assessment principles used to derive these indicators and the effects on the impact assessment...... results is critical for indicator selection and interpretation of the results. Eleven resource depletion methods were evaluated quantitatively with respect to resource coverage, characterization factors (CF), impact contributions from individual resources, and total impact scores. We included 2247...... may be critical for impact assessment results. Although no consistent correlations between methods applying similar assessment models could be observed, all methods showed relatively high correlation regarding the assessment of energy resources. Finally, we classify the existing methods into three...

  6. 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

  7. A comparison of parallel pyrosequencing and sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1.

    Directory of Open Access Journals (Sweden)

    Binhua Liang

    Full Text Available BACKGROUND: Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution. METHODOLOGY/PRINCIPAL FINDINGS: HIV-1 gag gene was amplified from 96 patients using nested PCR. The PCR products were cloned and sequenced using capillary based Sanger fluorescent dideoxy termination sequencing. The same PCR products were also directly sequenced using the 454 pyrosequencing technology. The two sequencing methods were evaluated for their ability to characterize quasispecies variation, and to reveal sites under host immune pressure for their putative functional significance. A total of 14,034 variations were identified by 454 pyrosequencing versus 3,632 variations by Sanger clone-based (SCB sequencing. 11,050 of these variations were detected only by pyrosequencing. These undetected variations were located in the HIV-1 Gag region which is known to contain putative cytotoxic T lymphocyte (CTL and neutralizing antibody epitopes, and sites related to virus assembly and packaging. Analysis of the positively selected sites derived by the two sequencing methods identified several differences. All of them were located within the CTL epitope regions. CONCLUSIONS/SIGNIFICANCE: Ultra-deep pyrosequencing has proven to be a powerful tool for characterization of HIV-1 genetic diversity with enhanced sensitivity, efficiency, and accuracy. It also improved reliability of downstream evolutionary and functional analysis of HIV-1 quasispecies.

  8. Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid

    OpenAIRE

    Pollmann, Stephan; Neu, Daniel; Weiler, Elmar W.

    2003-01-01

    Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAM...

  9. Identification and characterization of cDNA clones encoding hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase from tobacco.

    Science.gov (United States)

    Farmer, M J; Czernic, P; Michael, A; Negrel, J

    1999-08-01

    The sequences of three cDNA clones that include the complete coding region of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase (THT) from tobacco are reported. The three cDNAs were isolated by antibody screening of a cDNA expression library produced from poly(A)+RNA purified from tobacco leaves (Nicotiana tabacum cv. Bottom Special), previously infiltrated with an incompatible strain of Ralstonia solanacearum. The identity of these clones was confirmed by the detection of THT activity in extracts of transformed Escherichia coli and by matching the translated polypeptides with tryptic enzyme sequences. cDNA clones tht4 and tht11 differ only by their 5' leader and 3' UTRs and therefore encode the same protein, whereas tht10 and tht11 exhibit 95 and 99% sequence identity at the DNA and deduced amino acid levels, respectively. The three clones encode proteins of 226 amino acids with calculated molecular masses of 26 kDa. The deduced amino acid sequences show no similarity with the sequence of anthranilate hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus, the only enzyme exhibiting hydroxycinnamoyltransferase activity to be cloned so far in plants. In contrast, comparison of the THT amino acid sequence with protein sequence databases revealed substantial homology with mammalian diamine acetyltransferases. The THT clones hybridized to a 0.95-kb mRNA from elicited tobacco cell-suspension cultures and also to a mRNA of similar size from wound-healing potato tubers. The messengers for THT were also found to be expressed at relatively high levels in tobacco root tissues. Southern hybridization of tobacco genomic DNA with THT cDNA suggests that several copies of the THT gene occur in the tobacco genome. Inhibition experiments using amino-acid-specific reagents demonstrated that both histidyl and cysteyl residues are required for THT activity. In the course of these experiments THT was also found to be inhibited by (2-hydroxyphenyl) amino sulfinyl

  10. Extraction and characterization of lignin from different biomass resources

    Directory of Open Access Journals (Sweden)

    Dereca Watkins

    2015-01-01

    Full Text Available Lignocellulosic biomass has been acknowledged for potential use to produce chemicals and biomaterials. Lignin is the second most abundant natural polymer with cellulose being number one, making up to 10–25% of lignocellulosic biomass. Lignin is a three-dimensional, highly cross-linked macromolecule composed of three types of substituted phenols, which include: coniferyl, sinapyl, and p-coumaryl alcohols by enzymatic polymerization, yielding a vast number of functional groups and linkages. There is a wide range of lignin sources available, including: jute, hemp, cotton, and wood pulp. Hence, the lignin's physical and chemical behavior will be different with respect to the original source and extraction method used. The objective of this research is to extract lignin from nonwood cellulosic biomass (Wheat straw, Pine straw, Alfalfa, Kenaf, and Flax fiber by formic acid treatment followed by peroxyformic acid treatment for the potential use as a partial replacement for the phenol precursor in resole phenolic systems. Isolated lignins were purified to remove impurities and characterized by Fourier transform infrared spectroscopy (FTIR, Thermogravimetric analysis (TGA and Differential scanning calorimetry (DSC analysis to compare thermal properties and chemical composition. It was found that lignin obtained from alfalfa provided the greatest yield of the various sources. Enthalpy measurements were higher for lignin from flax fiber and alfalfa at 190.57 and 160.90 J/g, respectively. The source of lignin samples was seen to affect the thermal properties. Overall, lignin extracted from wheat straw had the greatest thermal stability followed very closely by that obtained from flax fiber.

  11. Cloning and characterization of an actin gene of Chlamys farreri and the phylogenetic analysis of mollusk actins

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    An actin gene (CfACT1) was cloned by using RT-PCR, 3' and 5'RACE from hemocytes of the sea scallop Chlamys farreri. The full length of the transcript is 1535 bp, which contains a long 3' un-translated region of 436bp and 59bp of a 5' un-translated sequence. The open reading frame encodes a polypeptide of 376 amino acids. Sequence comparisons indicated that CfACT1 is more closely related to vertebrate cytoplasmic actins than muscle types. Phylogenetic analysis showed that molluscan actins could be generally divided into two categories: muscle and cytoplasmic, although both are similar to vertebrate cytoplasmic actins. It was also inferred that different isotypes existed in muscle or cytoplasma in mollusks. The genomic sequence of CfACT1 was cloned and sequenced. Only one intron was detected:it was located between codons 42 and 43 and different from vertebrate actin genes.

  12. A novel cold-active β-D-galactosidase from the Paracoccus sp. 32d - gene cloning, purification and characterization

    Directory of Open Access Journals (Sweden)

    Wierzbicka-Woś Anna

    2011-12-01

    Full Text Available Abstract Background β-D-Galactosidases (EC 3.2.1.23 catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. Cold-active β-D-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (i eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (ii convert lactose to glucose and galactose which increase the sweetness of milk and decreases its hydroscopicity, and (iii eliminate lactose from dairy industry pollutants associated with environmental problems. Moreover, in contrast to commercially available mesophilic β-D-galactosidase from Kluyveromyces lactis the cold-active counterparts could make it possible both to reduce the risk of mesophiles contamination and save energy during the industrial process connected with lactose hydrolysis. Results A genomic DNA library was constructed from soil bacterium Paracoccus sp. 32d. Through screening of the genomic DNA library on LB agar plates supplemented with X-Gal, a novel gene encoding a cold-active β-D-galactosidase was isolated. The in silico analysis of the enzyme amino acid sequence revealed that the β-D-galactosidase Paracoccus sp. 32d is a novel member of Glycoside Hydrolase Family 2. However, owing to the lack of a BGal_small_N domain, the domain characteristic for the LacZ enzymes of the GH2 family, it was decided to call the enzyme under study 'BgaL'. The bgaL gene was cloned and expressed in Escherichia coli using the pBAD Expression System. The purified recombinant BgaL consists of two identical subunits with a combined molecular weight of about 160 kDa. The BgaL was optimally active at 40°C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and o-nitrophenyl-β-D-galactopyranoside at 10°C with Km values of 2.94 and 1.17 mM and kcat values 43.23 and 71.81 s-1, respectively. One U of the recombinant BgaL would thus be capable

  13. Cloning, functional characterization, and mechanism of action of the B-cell-specific transcriptional coactivator OCA-B.

    OpenAIRE

    Luo, Y.; Roeder, R. G.

    1995-01-01

    Biochemical purification and cognate cDNA cloning studies have revealed that the previously described transcriptional coactivator OCA-B consists of a 34- or 35-kDa polypeptide with sequence relationships to known coactivators that function by protein-protein interactions. Studies with a recombinant protein have proved that a single OCA-B polypeptide is the main determinant for B-cell-specific activation of immunoglobulin (Ig) promoters and provided additional insights into its mechanism of ac...

  14. Cloning, Characterization and Heterologous Expression of the Indolocarbazole Biosynthetic Gene Cluster from Marine-Derived Streptomyces sanyensis FMA

    OpenAIRE

    Wenli Li; Kui Hong; Weiming Zhu; Jingtao Zhang; Qiu Cui; Yuanyuan Du; Tong Li

    2013-01-01

    The indolocarbazole (ICZ) alkaloids have attracted much attention due to their unique structures and potential therapeutic applications. A series of ICZs were recently isolated and identified from a marine-derived actinomycete strain, Streptomyces sanyensis FMA. To elucidate the biosynthetic machinery associated with ICZs production in S. sanyensis FMA, PCR using degenerate primers was carried out to clone the FAD-dependent monooxygenase gene fragment for ICZ ring formation, which was used as...

  15. Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost.

    Science.gov (United States)

    Yasir, Muhammad; Khan, Haji; Azam, Syed Sikander; Telke, Amar; Kim, Seon Won; Chung, Young Ryun

    2013-06-01

    In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25-55°C) and pH (5.5-8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future. PMID:23812813

  16. Molecular cloning and characterization of the aklavinone 11-hydroxylase gene of Streptomyces peucetius subsp. caesius ATCC 27952.

    OpenAIRE

    Hong, Y S; Hwang, C K; Hong, S. K.; Kim, Y.H.(Center for Underground Physics, Institute for Basic Science (IBS), Daejon, 305-811, Korea); Lee, J. J.

    1994-01-01

    The gene encoding aklavinone 11-hydroxylase of Streptomyces peucetius subsp. caesius ATCC 27952 was cloned and sequenced. The deduced amino acid sequence of the gene contains at least two common motifs of well-conserved amino acid sequences of several flavin-type bacterial hydroxylases. The hydroxylase gene is apparently transcribed from a single transcriptional start point. The phenotype of a dnrF mutant generated by gene disruption supports the idea that the dnrF gene encodes aklavinone 11-...

  17. Molecular cloning and characterization of transgelin-like proteins mainly transcribed in newborn larvae of Trichinella spp.

    Science.gov (United States)

    Nagano, Isao; Wu, Zhiliang; Asano, Kazunobu; Takahashi, Yuzo

    2011-05-31

    A cDNA library was constructed from Trichinella pseudospiralis muscle larvae. One cDNA clone, designated Tp4, contained a cDNA transcript of 783 bp in length, with a single open reading frame that encoded 153 amino acids (16,793 Da as the estimated molecular mass). The predicted amino acid sequence of Tp4 showed that the clone had a calponin homology domain and was approximately 50% identical to the transgelin-like proteins (calponin-family members) present in Bombyx mori or Tribolium castaneum. A homologue of the Tp4 clone was also present in cDNA from Trichinella spiralis, and this clone was designated Ts4. A comparison of the amino acid sequence of the transgelin-like proteins from T. spiralis (Ts4 protein) with the Tp4 protein indicated that the two proteins are very similar (about 94% homology). Real time quantitative polymerase chain reaction results showed that the transcription level of the Tp4 and Ts4 genes was highest in newborn larvae. On Western blot, the recombinant Tp4 and Ts4 proteins migrated at 20 kDa when reacted to an antibody against the recombinant Tp4 and Ts4 proteins, respectively. An antibody against the recombinant Tp4 and Ts4 proteins strongly stained two bands migrating at approximately 9 and 8 kDa in the crude extracts from adult worms and newborn larvae, but only weakly stained proteins in muscle larvae. However, an immunocytochemical study showed that the Tp4 protein was present within the muscle of the muscle larvae of T. pseudospiralis. The antibody level against the recombinant Tp4 antigens in infected mice began to increase from 8 days post-infection, was highest in 13 days post-infection, and then slowly decreased. PMID:21242032

  18. Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola.

    OpenAIRE

    Brandl, M. T.; Lindow, S E

    1996-01-01

    Erwinia herbicola 299R synthesizes indole-3-acetic acid (IAA) primarily by the indole-3-pyruvic acid pathway. A gene involved in the biosynthesis of IAA was cloned from strain 299R. This gene (ipdC) conferred the synthesis of indole-3-acetaldehyde and tryptophol upon Escherichia coli DH5 alpha in cultures supplemented with L-tryptophan. The deduced amino acid sequence of the gene product has high similarity to that of the indolepyruvate decarboxylase of Enterobacter cloacae. Regions within py...

  19. Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

    OpenAIRE

    Dong, G.; Vieille, C; Zeikus, J G

    1997-01-01

    The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three protei...

  20. Cloning and characterization of a gene (msdA) encoding methylmalonic acid semialdehyde dehydrogenase from Streptomyces coelicolor.

    OpenAIRE

    Zhang, Y. X.; Tang, L.; Hutchinson, C R

    1996-01-01

    A homolog of the mmsA gene of Pseudomonas aeruginosa, which encodes methylmalonic acid semialdehyde dehydrogenase (MSDH) and is involved in valine catabolism in pseudomonads and mammals, was cloned and sequenced from Streptomyces coelicolor. Of the two open reading frames (ORFs) found, which are convergently transcribed and separated by a 62-nucleotide noncoding region, the deduced amino acid sequence of the msdA ORF (homologous to mmsA) is similar to a variety of prokaryotic and eukaryotic a...

  1. Molecular cloning, nucleotide sequence, and characterization of lppB, encoding an antigenic 40-kilodalton lipoprotein of Haemophilus somnus.

    OpenAIRE

    Theisen, M.; Rioux, C R; Potter, A A

    1993-01-01

    Haemophilus somnus is a facultative intracellular pathogen which causes a wide range of diseases in cattle. To identify putative virulence determinants, a genomic library of H. somnus in Escherichia coli was screened for Congo red binding, a property associated with virulence in pathogenic bacteria, and subsequently with bovine hyperimmune sera raised against H. somnus HS25. A Congo red-binding clone carrying a 1.8-kb DNA insert was found to encode a strongly seroreactive LppB protein with an...

  2. Asymmetric quantum cloning machines

    International Nuclear Information System (INIS)

    A family of asymmetric cloning machines for quantum bits and N-dimensional quantum states is introduced. These machines produce two approximate copies of a single quantum state that emerge from two distinct channels. In particular, an asymmetric Pauli cloning machine is defined that makes two imperfect copies of a quantum bit, while the overall input-to-output operation for each copy is a Pauli channel. A no-cloning inequality is derived, characterizing the impossibility of copying imposed by quantum mechanics. If p and p' are the probabilities of the depolarizing channels associated with the two outputs, the domain in (√p,√p')-space located inside a particular ellipse representing close-to-perfect cloning is forbidden. This ellipse tends to a circle when copying an N-dimensional state with N→∞, which has a simple semi-classical interpretation. The symmetric Pauli cloning machines are then used to provide an upper bound on the quantum capacity of the Pauli channel of probabilities px, py and pz. The capacity is proven to be vanishing if (√px, √py, √pz) lies outside an ellipsoid whose pole coincides with the depolarizing channel that underlies the universal cloning machine. Finally, the tradeoff between the quality of the two copies is shown to result from a complementarity akin to Heisenberg uncertainty principle. (author)

  3. Cloning and characterization of the Bg/II restriction-modification system reveals a possible evolutionary footprint.

    Science.gov (United States)

    Anton, B P; Heiter, D F; Benner, J S; Hess, E J; Greenough, L; Moran, L S; Slatko, B E; Brooks, J E

    1997-03-10

    Bg/II, a type II restriction-modification (R-M) system from Bacillus globigii, recognizes the sequence 5'-AGATCT-3'. The system has been cloned into E. coli in multiple steps: first the methyltransferase (MTase) gene, bglIIM, was cloned from B. globigii RUB561, a variant containing an inactivated endonuclease (ENase) gene (bglIIR). Next the ENase protein (R.BglII) was purified to homogeneity from RUB562, a strain expressing the complete R-M system. Oligonucleotide probes specific for the 5' end of the gene were then synthesized and used to locate bglIIR, and the gene was isolated and cloned in a subsequent step. The nucleotide sequence of the system has been determined, and several interesting features have been found. The genes are tandemly arranged, with bglIIR preceding bglIIM. The amino acid sequence of M.BglII is compared to those of other known MTases. A third gene encoding a protein with sequence similarity to known C elements of other R-M systems is found upstream of bglIIR. This is the first instance of a C gene being associated with an R-M system where the R and M genes are collinear. In addition, open reading frames (ORFs) resembling genes involved with DNA mobility are found in close association with BglII. These may shed light on the evolution of the R-M system. PMID:9073062

  4. Cloning and characterization of a gene (UVR3) required for photorepair of 6-4 photoproducts in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    UV radiation induces two major classes of pyrimidine dimers: the pyrimidine [6-4] pyrimidone photoproduct (6-4 product) and the cyclobutane pyrimidine dimer (CPD). Many organisms produce enzymes, termed photolyases, that specifically bind to these damage products and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. A gene that expresses a protein with 6-4 photolyase activity in vitro was recently cloned from Drosophila melanogaster and Xenopus laevis. We report here the isolation of a homolog of this gene, cloned on the basis of sequence similarity, from the higher plant Arabidopsis thaliana. This cloned gene produces a protein with 6-4 photolyase activity when expressed in Escherichia coli. We also find that a previously described mutant of Arabidopsis (uvr3) that is defective in photoreactivation of 6-4 products carries a nonsense mutation in this 6-4 photolyase homolog. We have therefore termed this gene UVR3. Although homologs of this gene have previously been shown to produce a functional 6-4 photolyase when expressed in heterologous systems, this is the first demonstration of a requirement for this gene for photoreactivation of 6-4 products in vivo

  5. A carboxymethyl cellulase from a marine yeast ( Aureobasidium pullulans 98): Its purification, characterization, gene cloning and carboxymethyl cellulose digestion

    Science.gov (United States)

    Rong, Yanjun; Zhang, Liang; Chi, Zhenming; Wang, Xianghong

    2015-10-01

    We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase (CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U (mg protein)-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0 kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40°C, much lower than that of the CMCases from other fungi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity (pH 5.0-6.0). The enzyme was activated by Na+, Mg2+, Ca2+, K+, Fe2+ and Cu2+, however, it was inhibited by Fe3+, Ba2+, Zn2+, Mn2+ and Ag+. K m and V max values of the purified enzyme were 4.7 mg mL-1 and 0.57 µmol L-1 min-1 (mg protein)-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose (CMC) after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading frame of 954 bp (EU978473). The protein deduced contained the conserved domain of cellulase superfamily (glucosyl hydrolase family 5). The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

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

    OpenAIRE

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

    1988-01-01

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

  7. Characterization of the mRNA and cloned cDNA specifying the third component of mouse complement.

    OpenAIRE

    Domdey, H; Wiebauer, K; Kazmaier, M; Müller, V.; Odink, K.; Fey, G

    1982-01-01

    Eighteen cDNA clones containing inserts specific for the third component of complement (C3) have been derived from high molecular weight mouse liver mRNA. The inserts span 4,600 nucleotides of the C3 coding sequence, including the 3' end of C3 mRNA. The length of C3 mRNA was determined to be 5,100 +/- 200 nucleotides, including a poly(A)-containing tail of mean length 170 nucleotides. From cDNA sequence analysis of the 5'-proximal region of C3 mRNA, the NH2-terminal amino acid sequence of the...

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

    OpenAIRE

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

    1984-01-01

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

  9. Cloning and characterization of a novel barley gene,HvORG4,induced by Fusarium graminearum infection

    Institute of Scientific and Technical Information of China (English)

    Theo; Van-Der; Lee

    2007-01-01

    Barley Fusarium head blight(FHB),caused by species of the Fusarium fungus,is a devastating disease that is reemerging worldwide in recent years.In this study,a novel gene,HvORG4,was cloned from barley by using cDNA library and suppression subtractive hybridization(SSH) library strategies.The SSH library and cDNA library were constructed from the Chinese barley cultivar Jing02-461(resistance to FHB) infected by Fusarium graminearum isolate Huanggang-1.For the SSH analysis,more than 120 differentially express...

  10. A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme

    OpenAIRE

    Dong-Ming Lan; Yong-Hua Wang; Bo Yang; Ning Yang; Wen-Kai Wang; Yan-Fei Shen

    2011-01-01

    A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86–34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM...

  11. Cloning and Characterization of a Gene (mspA) Encoding the Major Sheath Protein of Treponema maltophilum ATCC 51939T

    OpenAIRE

    Heuner, Klaus; Choi, Bong-Kyu; Schade, Rüdiger; Moter, Annette; Otto, Albrecht; Göbel, Ulf B.

    1999-01-01

    The major sheath protein-encoding gene (mspA) of the oral spirochete Treponema maltophilum ATCC 51939T was cloned by screening a genomic library with an anti-outer membrane fraction antibody. The mspA gene encodes a precursor protein of 575 amino acids with a predicted molecular mass of 62.3 kDa, including a signal peptide of 19 amino acids. The native MspA formed a heat-modifiable, detergent- and trypsin-stable complex which is associated with the outer membrane. Hybridization with an mspA-s...

  12. A novel cytochrome P450 gene from Catharanthus roseus cell line C20hi: cloning and characterization of expression

    OpenAIRE

    Lihong He; Shujuan Zhao; Zhibi Hu

    2012-01-01

    An expressed sequence tag (EST) obtained from a subtractive-suppression hybridization cDNA library constructed using Catharanthus roseus cell line C20hi and its parental cell line C20D was used to clone a full-length cytochrome P450 cDNA of cyp71d1. The encoded polypeptide contained 507 amino acids with 39–56% identity to other CYP71D subfamily members at the amino acid level. Expression characteristics of cyp71d1 were determined using semi-quantitative RT-PCR. The cyp71d1 transcript was expr...

  13. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression.

    OpenAIRE

    Gambello, M J; Iglewski, B H

    1991-01-01

    We report the discovery of the lasR gene, which positively regulates elastase expression in Pseudomonas aeruginosa PAO1. The lasR gene was cloned by its ability to restore a positive elastase phenotype in strain PA103, a strain which possesses the elastase structural gene (lasB) but fails to synthesize the enzyme. Nucleotide sequence analysis revealed an open reading frame of 716 nucleotides encoding a protein of approximately 27 kDa. A labeled LasR protein of 27 kDa was detected in Escherich...

  14. Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease

    OpenAIRE

    Mulligan, Elizabeth A.; Dunn, John J.

    2008-01-01

    Expression strains of Escherichia coli BL21(DE3) overproducing the E. coli m5C McrA restriction protein were produced by cloning the mcrA coding sequence behind a T7 promoter. The recombinant mcrA minus BL21 (DE3) host produces active McrA as evidenced by its acquired ability to selectively restrict the growth of T7 phage containing DNA methylated in vitro by HpaII methylase. The mcrA coding region contains several non-optimal E. coli triplets. Addition of the pACYC-RIL tRNA encoding plasmid ...

  15. Clones above the unary clone

    OpenAIRE

    Goldstern, Martin; Sági, Gábor; Shelah, Saharon

    2011-01-01

    Let c be the cardinality of the continuum. We give a family of pairwise incomparable clones (on a countable base set) 2^c members, all with the same unary fragment, namely the set of all unary operations. We also give, for each n, a family of 2^c clones all with the same n-ary fragment, and all containing the set of all unary operations.

  16. Landscape characterization integrating expert and local spatial knowledge of land and forest resources.

    Science.gov (United States)

    Fagerholm, Nora; Käyhkö, Niina; Van Eetvelde, Veerle

    2013-09-01

    In many developing countries, political documentation acknowledges the crucial elements of participation and spatiality for effective land use planning. However, operative approaches to spatial data inclusion and representation in participatory land management are often lacking. In this paper, we apply and develop an integrated landscape characterization approach to enhance spatial knowledge generation about the complex human-nature interactions in landscapes in the context of Zanzibar, Tanzania. We apply an integrated landscape conceptualization as a theoretical framework where the expert and local knowledge can meet in spatial context. The characterization is based on combining multiple data sources in GIS, and involves local communities and their local spatial knowledge since the beginning into the process. Focusing on the expected information needs for community forest management, our characterization integrates physical landscape features and retrospective landscape change data with place-specific community knowledge collected through participatory GIS techniques. The characterization is established in a map form consisting of four themes and their synthesis. The characterization maps are designed to support intuitive interpretation, express the inherently uncertain nature of the data, and accompanied by photographs to enhance communication. Visual interpretation of the characterization mediates information about the character of areas and places in the studied local landscape, depicting the role of forest resources as part of the landscape entity. We conclude that landscape characterization applied in GIS is a highly potential tool for participatory land and resource management, where spatial argumentation, stakeholder communication, and empowerment are critical issues. PMID:23934059

  17. Detection and characterization of a mouse α-spectrin cDNA clone by its expression in Escherichia coli

    International Nuclear Information System (INIS)

    A cloned segment of mouse α-spectrin mRNA has been identified by radioimmunoassay and immunoautoradiography. Double-stranded cDNA derived from spleens of anemic mice was introduced into a bacterial expression vector, pUC, and transformed Escherichia coli colonies were screened by using an 125I-labeled antiserum to erythrocyte membrane ghost proteins. Of 17 positive colonies, 2 bound antibody to mouse spectrin, and these 2 colonies contained 750-base-pair inserts that cross-hybridized. Transfer of the 750-base-pair insert to an expression vector containing the P/sub L/ promoter of phage lambda produced larger amounts of peptides that were bound by antibody to mouse spectrin. The spectrin-like peptides made in E. coli elicited antibody that reacted only with the α-spectrin subunit of erythrocyte membranes. This clone will be useful for the study of the structure and expression of the spectrin gene, particularly in understanding the role of spectrin in human inherited hemolytic anemias

  18. Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum.

    Science.gov (United States)

    Wankhede, Dhammaprakash Pandhari; Biswas, Dipul Kumar; Rajkumar, Subramani; Sinha, Alok Krishna

    2013-12-01

    Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.

  19. Cloning and characterization of a novel oligoalginate lyase from a newly isolated bacterium Sphingomonas sp. MJ-3.

    Science.gov (United States)

    Park, Hwan Hee; Kam, Natania; Lee, Eun Yeol; Kim, Hee Sook

    2012-04-01

    A bacterium possessing alginate-degrading activity was isolated from marine brown seaweed soup liquefied by salted and fermented anchovy. The isolated strain was designated as Sphingomonas sp. MJ-3 based on the analyses of 16S ribosomal DNA sequences, 16S-23S internal transcribed spacer region sequences, biochemical characteristics, and cellular fatty acid composition. A novel alginate lyase gene was cloned from genomic DNA library and then expressed in Escherichia coli. When the deduced amino acid sequence was compared with the sequences on the databases, interestingly, the cloned gene product was predicted to consist of AlgL (alginate lyase L)-like and heparinase-like protein domain. The MJ-3 alginate lyase gene shared below 27.0% sequence identity with exolytic alginate lyase of Sphingomonas sp. A1. The optimal pH and temperature for the recombinant MJ-3 alginate lyase were 6.5 and 50°C, respectively. The final degradation products of alginate oligosaccharides were analyzed by electrospray ionization mass spectrometry and proved to be alginate monosaccharides. Based on the results, the recombinant alginate lyase from Sphingomonas sp. MJ-3 is regarded as an oligoalginate lyase that can degrade oligoalginate and alginate into alginate monosaccharides.

  20. Cloning and characterization of a novel deletion mutant of heterogeneous nuclear ribonucleoprotein M4 from human dendritic cells

    Institute of Scientific and Technical Information of China (English)

    黄欣; 赵忠良; 袁正隆; 张明徽; 朱学军; 陈国友; 曹雪涛

    2000-01-01

    To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to- be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant mRNA transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-a. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.