Science.gov

Sample records for characterized clone resource

  1. Final progress report, Construction of a genome-wide highly characterized clone resource for genome sequencing

    SciTech Connect

    Nierman, William C.

    2000-02-14

    At TIGR, the human Bacterial Artificial Chromosome (BAC) end sequencing and trimming were with an overall sequencing success rate of 65%. CalTech human BAC libraries A, B, C and D as well as Roswell Park Cancer Institute's library RPCI-11 were used. To date, we have generated >300,000 end sequences from >186,000 human BAC clones with an average read length {approx}460 bp for a total of 141 Mb covering {approx}4.7% of the genome. Over sixty percent of the clones have BAC end sequences (BESs) from both ends representing over five-fold coverage of the genome by the paired-end clones. The average phred Q20 length is {approx}400 bp. This high accuracy makes our BESs match the human finished sequences with an average identity of 99% and a match length of 450 bp, and a frequency of one match per 12.8 kb contig sequence. Our sample tracking has ensured a clone tracking accuracy of >90%, which gives researchers a high confidence in (1) retrieving the right clone from the BA C libraries based on the sequence matches; and (2) building a minimum tiling path of sequence-ready clones across the genome and genome assembly scaffolds.

  2. Clone DB: an integrated NCBI resource for clone-associated data.

    PubMed

    Schneider, Valerie A; Chen, Hsiu-Chuan; Clausen, Cliff; Meric, Peter A; Zhou, Zhigang; Bouk, Nathan; Husain, Nora; Maglott, Donna R; Church, Deanna M

    2013-01-01

    The National Center for Biotechnology Information (NCBI) Clone DB (http://www.ncbi.nlm.nih.gov/clone/) is an integrated resource providing information about and facilitating access to clones, which serve as valuable research reagents in many fields, including genome sequencing and variation analysis. Clone DB represents an expansion and replacement of the former NCBI Clone Registry and has records for genomic and cell-based libraries and clones representing more than 100 different eukaryotic taxa. Records provide details of library construction, associated sequences, map positions and information about resource distribution. Clone DB is indexed in the NCBI Entrez system and can be queried by fields that include organism, clone name, gene name and sequence identifier. Whenever possible, genomic clones are mapped to reference assemblies and their map positions provided in clone records. Clones mapping to specific genomic regions can also be searched for using the NCBI Clone Finder tool, which accepts queries based on sequence coordinates or features such as gene or transcript names. Clone DB makes reports of library, clone and placement data on its FTP site available for download. With Clone DB, users now have available to them a centralized resource that provides them with the tools they will need to make use of these important research reagents. PMID:23193260

  3. Cloning and characterization of new bioluminescent proteins

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

  5. Cloning and characterization of porcine resistin gene.

    PubMed

    Dai, M H; Xia, T; Chen, X D; Gan, L; Feng, S Q; Qiu, H; Peng, Y; Yang, Z Q

    2006-02-01

    Resistin is a member of resistin-like molecules (RELMs) and a hormone secreted from mature adipocytes in rodents and leukocytes in human. We now report the cloning and characterization of the full-length porcine resistin cDNA and gene. Sequence analysis indicated that the pig resistin cDNA sequence had an open reading frame of 330 bp encoding a 12 kDa protein of 109 amino acids. The deduced amino acid sequence showed 75.2% identity to the human resistin. The porcine resistin gene was composed of four exons and had exactly the same exon structure as the human resistin gene. The tissue distribution of porcine resistin mRNA was assessed by semi-quantitative RT-PCR. Resistin gene expression was the highest in porcine leukocytes and low in adipose tissue. Resistin protein could be detected in porcine serum by western blotting and it circulated in serum as dimers and trimers. We provided the first evidence that resistin was abundantly expressed in porcine leukocytes and had an expression pattern similar to that in human resistin mRNA and protein. This suggests that the pig may be a suitable animal model for studying the function of resistin in human insulin resistance. PMID:16023825

  6. Babesia bovis clones: biochemical and enzymatic characterization

    SciTech Connect

    Rodriguez Camarillo, S.D.

    1985-01-01

    Studies were undertaken to generate additional knowledge of the biochemistry of Babesia bovis. A modified in vitro culture technique used for cloning B. bovis. This technique included a low oxygen concentration atmosphere (2%, O/sub 2/, 5% CO/sub 2/, 93% N/sub 2/) and 4 mm fluid level. Cultures initiated with one infected erythrocyte were maintained until parasitemias of positive wells reached 2% parasitemia. Primary clones were obtained and from these, nine clones were recloned twice and used for subsequent studies. A procedure was developed to concentrate and separate B. bovis merozoites and infected erythrocytes by Percoll density gradients. Merozoites separated at 1.087 g/ml specific density, whereas infected erythrocytes separated at 1.121 g/ml. Viability of purified parasites was not affected. Agarose gel electrophoresis was used to identify metabolic enzyme in B. bovis and B. bigemina. The enzymes LDH, GDH, GPI and HK were detected in both species. Molecular analysis by one and two-dimensional gel electrophoresis of proteins metabolically labeled with /sup 35/S-methionine indicated that two clones, derived from the same field strain, were similar but not identical to the parent. Fewer proteins were observed in the parental strain. Growth of two 60-Co irradiated B. bovis clones indicated a dose-effect relationship. Growth of parasites exposed for the longest period was initially retarded but returned to normal growth after two or three subcultures. Cultures exposed for shorter periods were unaffected with respect to the rate of growth. Analysis of electrophoretic mobility of metabolic enzyme showed a change in migration pattern.

  7. Cloning

    MedlinePlus

    Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. ... named Dolly. There are three different types of cloning: Gene cloning, which creates copies of genes or ...

  8. Molecular cloning and characterization of canine ICOS.

    PubMed

    Lee, Je-Hwan; Joo, Young-Don; Yim, Daesong; Lee, Richard; Ostrander, Elaine A; Loretz, Carol; Little, Marie-Térèse; Storb, Rainer; Kuhr, Christian S

    2004-10-01

    Inducible costimulatory receptor (ICOS) is one recently identified member of the CD28 family of costimulatory molecules. Evidence suggests ICOS functions as a critical immune regulator and, to evaluate these effects, we employed the canine model system that has been used to develop strategies currently in clinical use for hematopoietic stem cell transplantation. To investigate the effects of blocking the ICOS pathway in the canine hematopoietic cell transplantation model, we tested existing murine and human reagents and cloned the full length of the open reading frame of canine ICOS cDNA to allow the development of reagents specific for the canine ICOS. Canine ICOS contains a major open reading frame of 624 nucleotides, encoding a protein of 208 amino acids, and localizes to chromosome 37. Canine ICOS shares 79% sequence identity with human ICOS, 70% with mouse, and 69% with rat. Canine ICOS expression is limited to stimulated PBMC. PMID:15475250

  9. Technological Literacy and Human Cloning. Resources in Technology.

    ERIC Educational Resources Information Center

    Baird, Steven L.

    2002-01-01

    Discusses how technology educators can deal with advances in human genetics, specifically, cloning. Includes a definition and history of cloning, discusses its benefits, and looks at social concerns and arguments for and against human cloning. Includes classroom activities and websites. (Contains 10 references.) (JOW)

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

    PubMed

    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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Cloning

    MedlinePlus

    ... DNA Reproductive cloning, which creates 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

  13. In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

    PubMed

    Wolf, D; Laver-Rudich, Z; Rotter, V

    1985-08-01

    The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons. PMID:3018534

  14. Cloning and characterization of centromeric DNA from Neurospora crassa.

    PubMed Central

    Centola, M; Carbon, J

    1994-01-01

    The centromere locus from linkage group VII of Neurospora crassa has been cloned, characterized, and physically mapped. The centromeric DNA is contained within a 450-kb region that is recombination deficient, A+T-rich, and contains repetitive sequences. Repetitive sequences from within this region hybridize to a family of repeats located at or near centromeres in all seven linkage groups of N. crassa. Genomic Southern blots and sequence analysis of these repeats revealed a unique centromere structure containing a divergent family of centromere-specific repeats. The predominantly transitional differences between copies of the centromere-specific sequence repeats and their high A+T content suggest that their divergence was mediated by repeat-induced point (RIP) mutations. Images PMID:7904723

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

    SciTech Connect

    Wakamatsu, N.; Trasler, J.M.; Gravel, R.A.

    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.

  16. Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana

    PubMed Central

    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

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

    PubMed

    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

  18. The UniTrap resource: tools for the biologist enabling optimized use of gene trap clones

    PubMed Central

    Roma, Guglielmo; Sardiello, Marco; Cobellis, Gilda; Cruz, Pedro; Lago, Giampiero; Sanges, Remo; Stupka, Elia

    2008-01-01

    We have developed a comprehensive resource devoted to biologists wanting to optimize the use of gene trap clones in their experiments. We have processed 300 602 such clones from both public and private projects to generate 28 199 ‘UniTraps’, i.e. distinct collections of unambiguous insertions at the same subgenic region of annotated genes. The UniTrap resource contains data relative to 9583 trapped genes, which represent 42.3% of the mouse gene content. Among the trapped genes, 7 728 have a counterpart in humans, and 677 are known to be involved in the pathogenesis of human diseases. The aim of this analysis is to provide the wet lab researchers with a comprehensive database and curated tools for (i) identifying and comparing the clones carrying a trap into the genes of interest, (ii) evaluating the severity of the mutation to the protein function in each independent trapping event and (iii) supplying complete information to perform PCR, RT-PCR and restriction experiments to verify the clone and identify the exact point of vector insertion. To share this unique resource with the scientific community, we have designed and implemented a web interface that is freely accessible at http://unitrap.cbm.fvg.it/. PMID:17942430

  19. Genomic mapping by end-characterized random clones: A mathematical analysis

    SciTech Connect

    Port, E.; Sun, F.; Martin, D.

    1995-03-01

    Physical maps can be constructed by {open_quotes}fingerprinting{close_quotes} a large number of random clones and inferring overlap between clones when the fingerprints are sufficiently similar. E. Lander and M. Waterman gave a mathematical analysis of such mapping strategies. The analysis is useful for comparing various fingerprinting methods. Recently it has been proposed that ends of clones rather than the entire clone be fingerprinted or characterized. Such fingerprints, which include sequenced clone ends, require a mathematical analysis deeper than that of Lander-Waterman. This paper studies clone islands, which can include uncharacterized regions, and also the islands that are formed entirely from the ends of clones. 23 refs., 11 figs., 4 tabs.

  20. Molecular cloning and characterization of Schistosoma japonicum aldose reductase.

    PubMed

    Liu, Jian; Wang, Jipeng; Wang, Shuqi; Xu, Bin; Liu, Xiufeng; Wang, Xiaoning; Hu, Wei

    2013-02-01

    Antioxidant defense is an essential mechanism for schistosomes to cope with damage from host immune-generated reactive oxygen species. The evaluation of the effects of aldose reductase, an important enzyme that may be involved in this system, has long been neglected. In the present study, aldose reductase of Schistosoma japonicum (SjAR) was cloned and characterized. The activity of SjAR was assessed in vitro and was suppressed by the reported inhibitor, epalrestat. RT-PCR analysis revealed that SjAR was expressed at each of the development stages analyzed with increased levels in cercariae. The results also showed that SjAR was expressed at higher levels in adult male worms than in adult female worms. Indirect enzyme-linked immunosorbent assay and western blot analysis indicated that the purified recombinant SjAR (rSjAR) protein displayed a significant level of antigenicity. Immunolocalization analysis revealed that SjAR was mainly distributed in the gynecophoral canal of adult male worms. BALB/c mice immunized with rSjAR induced a 32.91 % worm reduction compared to the adjuvant group (P < 0.01). Moreover, a 28.27 % reduction in egg development in the liver (P > 0.05) and a 42.75 % reduction in egg development in the fecal samples (P < 0.05) were also observed. These results suggested that SjAR may be a potential new drug target or vaccine candidate for schistosomes. PMID:23160889

  1. Cloning and characterization of a murine SIL gene

    SciTech Connect

    Collazo-Garcia, N.; Scherer, P.; Aplan, P.D.

    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.

  2. Cloning, Characterization, Regulation, and Function of Dormancy-Associated MADS-Box Genes from Leafy Spurge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DORMANCY-ASSOCIATED MADS-BOX (DAM) genes are SHORT VEGETATIVE PHASE–Like MADS box transcription factors linked to endodormancy induction. We have cloned and characterized several cDNA and genomic clones of DAM genes from the model perennial weed leafy spurge (Euphorbia esula). We present evidence fo...

  3. Cloning and functional characterization of carotenoid cleavage dioxygenase 4 genes.

    PubMed

    Huang, Fong-Chin; Molnár, Péter; Schwab, Wilfried

    2009-01-01

    Although a number of plant carotenoid cleavage dioxygenase (CCD) genes have been functionally characterized in different plant species, little is known about the biochemical role and enzymatic activities of members of the subclass 4 (CCD4). To gain insight into their biological function, CCD4 genes were isolated from apple (Malus x domestica, MdCCD4), chrysanthemum (Chrysanthemum x morifolium, CmCCD4a), rose (Rosa x damascena, RdCCD4), and osmanthus (Osmanthus fragrans, OfCCD4), and were expressed, together with AtCCD4, in Escherichia coli. In vivo assays showed that CmCCD4a and MdCCD4 cleaved beta-carotene well to yield beta-ionone, while OfCCD4, RdCCD4, and AtCCD4 were almost inactive towards this substrate. No cleavage products were found for any of the five CCD4 genes when they were co-expressed in E. coli strains that accumulated cis-zeta-carotene and lycopene. In vitro assays, however, demonstrated the breakdown of 8'-apo-beta-caroten-8'-al by AtCCD4 and RdCCD4 to beta-ionone, while this apocarotenal was almost not degraded by OfCCD4, CmCCD4a, and MdCCD4. Sequence analysis of genomic clones of CCD4 genes revealed that RdCCD4, like AtCCD4, contains no intron, while MdCCD, OfCCD4, and CmCCD4a contain introns. These results indicate that plants produce at least two different forms of CCD4 proteins. Although CCD4 enzymes cleave their substrates at the same position (9,10 and 9',10'), they might have different biochemical functions as they accept different (apo)-carotenoid substrates, show various expression patterns, and are genomically differently organized. PMID:19436048

  4. Cloning, overexpression and characterization of Leishmania donovani squalene synthase.

    PubMed

    Bhargava, Prachi; Kumar, Kishore; Chaudhaery, Shailendra S; Saxena, Anil K; Roy, Uma

    2010-10-01

    Squalene synthase (SSN, EC 2.5.1.21), a major enzyme in the sterol biosynthetic pathway, catalyses an unusual head-to-head reductive dimerization of two molecules of farnesyl-pyrophosphate (FPP) in a two-step reaction to form squalene. FPP serves as a metabolic intermediate in the formation of sterols, dolichols, ubiquinones and farnesylated proteins. Here, we report cloning, expression and purification of a catalytically active recombinant squalene synthase of Leishmania donovani (LdSSN). The pH and temperature optima of LdSSN were 7.4 and 37°C, respectively. Biochemical studies revealed that the K(m) and V(max) for the substrate FPP were 3.8 μM and 0.59 nM min(-1) mg(-1) and for NADPH were 43.23 μM and 0.56 nM min(-1) mg(-1). LdSSN was found to be sensitive towards denaturants as manifested by a loss of enzyme activity at the concentration of 1 M urea or 0.25 M guanidine hydrochloride. Zaragozic acid A, a potent inhibitor of mammalian SSN, was also a competitive inhibitor of recombinant LdSSN, with a K(i) of 74 nM. This is the first report on the purification and characterization of full-length recombinant SSN from L. donovani. Studies on recombinant LdSSN will help in evaluating this enzyme as a potential drug target for visceral leishmaniasis. PMID:20722739

  5. Molecular cloning, characterization, and expression of sheep FGF5 gene.

    PubMed

    Zhang, Lihua; He, Sangang; Liu, Mingjun; Liu, Guosong; Yuan, Zheng; Liu, Chenxi; Zhang, Xumei; Zhang, Ning; Li, Wenrong

    2015-01-25

    The fibroblast growth factor 5 gene (FGF5) is a member of the FGF gene family, and represents a candidate gene for hair length because of its role in the regulation of the hair follicle growth cycle. In our current study, we cloned, sequenced, and characterized the full-length FGF5 cDNA of Chinese Merino sheep. We obtained the complete genomic sequence of the FGF5 gene from sheep blood samples, and compared it to other FGF5 sequences in GenBank. We found that the FGF5 gene spanned 21,743bp of genomic DNA, and consisted of 3 exons and 2 introns, both of which differed from those of a previously annotated FGF5 genomic sequence from sheep. We also identified a previously undescribed FGF5 mRNA splicing variant, FGF5S, and the western blot analysis showed that the molecular weights of the FGF5 (34kDa) and FGF5s (17kDa) proteins were consistent with the estimates based on the genomic and cDNA sequence data. We examined the expression of both FGF5 mRNAs in various tissues of sheep, and found that the expression of the FGF5S mRNA was restricted to the brain, spleen, and skin tissue. The single-nucleotide polymorphism analysis of the genomic sequence revealed 72 genetic variants of the FGF5 gene. Our findings provide insight into the functions of the FGF5 gene in Chinese Merino. PMID:25445274

  6. Cloning and characterization of three Eimeria tenella lipid phosphate phosphatases.

    PubMed

    Guo, Aijiang; Cai, Jianping; Luo, Xuenong; Zhang, Shaohua; Hou, Junling; Li, Hui; Cai, Xuepeng

    2015-01-01

    Although lipid phosphate phosphatases (LPPs) play an important role in cellular signaling in addition to lipid biosynthesis, little is thus far known about parasite LPPs. In this study, we characterized three Eimeria tenella cDNA clones encoding LPP named EtLPP1, EtLPP2 and EtLPP3. Key structural features previously described in LPPs, including the three conserved domains proposed as catalytic sites, a single conserved N-glycosylation site, and putative transmembrane domains were discovered in the three resulting EtLPP amino acid sequences. Expression of His6-tagged EtLPP1, -2, and -3 in HEK293 cells produced immunoreactive proteins with variable molecular sizes, suggesting the presence of multiple forms of each of the three EtLPPs. The two faster-migrating protein bands below each of the three EtLPP proteins were found to be very similar to the porcine 35-kDa LPP enzyme in their molecular size and the extent of their N-glycosylation, suggesting that the three EtLPPs are partially N-glycosylated. Kinetic analyses of the activity of the three enzymes against PA, LPA, C1P and S1P showed that Km values for each of the substrates were (in μM) 284, 46, 28, and 22 for EtLPP1; 369, 179, 237, and 52 for EtLPP2; and 355, 83, and 260 for EtLPP3. However, EtLPP3 showed negligible activity on S1P. These results confirmed that the three EtLPPs have broad substrate specificity. The results also indicated that despite structural similarities, the three EtLPPs may play distinct functions through their different models of substrate preference. Furthermore, particularly high expression levels of the three EtLPP genes were detected in the sporozoite stage of the E. tenella life cycle (p<0.001), suggesting that their encoded proteins might play an important biological function in the sporozoite stage. PMID:25861032

  7. Cloning and Characterization of Three Eimeria tenella Lipid Phosphate Phosphatases

    PubMed Central

    Guo, Aijiang; Cai, Jianping; Luo, Xuenong; Zhang, Shaohua; Hou, Junling; Li, Hui; Cai, Xuepeng

    2015-01-01

    Although lipid phosphate phosphatases (LPPs) play an important role in cellular signaling in addition to lipid biosynthesis, little is thus far known about parasite LPPs. In this study, we characterized three Eimeria tenella cDNA clones encoding LPP named EtLPP1, EtLPP2 and EtLPP3. Key structural features previously described in LPPs, including the three conserved domains proposed as catalytic sites, a single conserved N-glycosylation site, and putative transmembrane domains were discovered in the three resulting EtLPP amino acid sequences. Expression of His6-tagged EtLPP1, -2, and -3 in HEK293 cells produced immunoreactive proteins with variable molecular sizes, suggesting the presence of multiple forms of each of the three EtLPPs. The two faster-migrating protein bands below each of the three EtLPP proteins were found to be very similar to the porcine 35-kDa LPP enzyme in their molecular size and the extent of their N-glycosylation, suggesting that the three EtLPPs are partially N-glycosylated. Kinetic analyses of the activity of the three enzymes against PA, LPA, C1P and S1P showed that Km values for each of the substrates were (in μM) 284, 46, 28, and 22 for EtLPP1; 369, 179, 237, and 52 for EtLPP2; and 355, 83, and 260 for EtLPP3. However, EtLPP3 showed negligible activity on S1P. These results confirmed that the three EtLPPs have broad substrate specificity. The results also indicated that despite structural similarities, the three EtLPPs may play distinct functions through their different models of substrate preference. Furthermore, particularly high expression levels of the three EtLPP genes were detected in the sporozoite stage of the E. tenella life cycle (p<0.001), suggesting that their encoded proteins might play an important biological function in the sporozoite stage. PMID:25861032

  8. Characterization of glyphosate resistance in cloned Amaranthus palmeri plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Molecular cloning and characterization of duck interleukin-17

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. Isolation and characterization of human defensin cDNA clones

    SciTech Connect

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

    1988-10-01

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

  12. CLONING AND FUNCTIONAL CHARACTERIZATION OF CHICKEN INTERLEUKIN-17D

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The proinflammatory cytokine IL-17D was cloned from a testis cDNA library prepared from the Korean native chicken. The full-length chicken IL-17D (chIL-17D) cDNA consisted of a 348 nucleotide sequence encoding an open reading frame of 116 amino acids with a predicted molecular mass of 17.0 kDa. Co...

  13. Establishment of clones of Trypanosoma cruzi and their characterization in vitro and in vivo*

    PubMed Central

    Pan, S. Chia-tung

    1982-01-01

    An efficient technique for isolating clones of Trypanosoma cruzi from cultures and from animals has been developed. It is based on the inoculation of one organism, obtained by serial dilutions of cultured epimastigotes or isolated blood stream trypomastigotes, into enriched NNN medium (NNN-F:93). The cloning efficiency (percentage of positive cultures over the number inoculated) was 70% for cultured epimastigotes and 30-40% for blood-stream trypomastigotes. In vitro cultural characteristics of 14 secondary clones of an avirulent strain indicated that 12 clones grew in the F-94 medium primarily as epimastigotes at 27 °C and exclusively as amastigotes at 37 °C; 2 clones grew in F-94 medium primarily as amastigotes regardless of incubation temperature (27 °C or 37 °C). In vivo characterization of 7 clones from 2 virulent strains indicated that the virulence of individual clones was low immediately after isolation in NNN-F:93 medium, but the virulence of some clones returned to the level of the parent strain after more than 8 serial passages in CD-1 mice. PMID:7044587

  14. Characterization of lunar ilmenite resources

    SciTech Connect

    Heiken, G.; Vaniman, D.T.

    1989-01-01

    Ilmenite will be an important lunar resource, to be used mainly for oxygen production but also as a source of iron. Ilmenite abundances in high-Ti basaltic lavas are higher (10--20%) than in high-Ti mare soils (mostly <10%). This factor alone may make crushed high-Ti basaltic lavas most attractive as a target for ilmenite extraction. Concentration of ilmenite from either a crushed basalt or regolith requires sizing to avoid polycrystalline fragments. In coarse-grained high-Ti basaltic lavas, about 60--80% of the ilmenite will consist of relatively ''clean'' single crystals if the rocks are crushed to a size of 0.2 mm. Fine-grained high-Ti basalts, with thin skeletal or hopper-shaped ilmenites, would produce essentially no free or ''clean'' ilmenite grains unless crushed to sizes of less than 0.15 mm, and only /approximately/7% free ilmenite if crushed to sizes smaller than 0.05 mm. Data from the 2.8 m-thick regolith sampled by coring at the Apollo 17 site show that in even the most basalt-clast-rich and least mature stratigraphic intervals, free ilmenite grains make up less than 2% of the 0.02- to 0.2-mm size fraction and a mere 0.3% of the 0.2- to 2-mm size fraction. 23 refs., 9 figs., 1 tab.

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

    PubMed

    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

  16. Cloning and pharmacological characterization of a rat kappa opioid receptor.

    PubMed Central

    Meng, F; Xie, G X; Thompson, R C; Mansour, A; Goldstein, A; Watson, S J; Akil, H

    1993-01-01

    A full-length cDNA was isolated from a rat striatal library by using low-stringency screening with two PCR fragments, one spanning transmembrane domains 3-6 of the mouse delta opioid receptor and the other unidentified but homologous to the mouse delta receptor from rat brain. The novel cDNA had a long open reading frame encoding a protein of 380 residues with 59% identity to the mouse delta receptor and topography consistent with a seven-helix guanine nucleotide-binding protein-coupled receptor. COS-1 cells transfected with the coding region of this clone showed high-affinity binding to kappa opioid receptor-selective ligands such as dynorphin A and U-50,488 and also nonselective opioid ligands such as bremazocine, ethylketocyclazocine, and naloxone. Not bound at all (or bound with low affinity) were dynorphin A-(2-13), enantiomers of naloxone and levophanol [i.e., (+)-naloxone and dextrorphan], and selective mu and delta opioid receptor ligands. Activation of the expressed receptor by kappa receptor agonists led to inhibition of cAMP. Finally, in situ hybridization revealed a mRNA distribution in rat brain that corresponded well to the distribution of binding sites labeled with kappa-selective ligands. These observations indicate that we have cloned a cDNA encoding a rat kappa receptor of the kappa 1 subtype. Images Fig. 3 PMID:8234341

  17. Cloning and characterization of micro-RNAs from moss.

    PubMed

    Arazi, Tzahi; Talmor-Neiman, Mali; Stav, Ran; Riese, Maike; Huijser, Peter; Baulcombe, David C

    2005-09-01

    Micro-RNAs (miRNAs) are one class of endogenous tiny RNAs that play important regulatory roles in plant development and responses to external stimuli. To date, miRNAs have been cloned from higher plants such as Arabidopsis, rice and pumpkin, and there is limited information on their identity in lower plants including Bryophytes. Bryophytes are among the oldest groups of land plants among the earth's flora, and are important for our understanding of the transition to life on land. To identify miRNAs that might have played a role early in land plant evolution, we constructed a library of small RNAs from the juvenile gametophyte (protonema) of the moss Physcomitrella patens. Sequence analysis revealed five higher plant miRNA homologues, including three members of the miR319 family, previously shown to be involved in the regulation of leaf morphogenesis, and miR156, which has been suggested to regulate several members of the SQUAMOSA PROMOTER BINDING-LIKE (SPL) family in Arabidopsis. We have cloned PpSBP3, a moss SPL homologue that contains an miR156 complementary site, and demonstrated that its mRNA is cleaved within that site suggesting that it is an miR156 target in moss. Six additional candidate moss miRNAs were identified and shown to be expressed in the gametophyte, some of which were developmentally regulated or upregulated by auxin. Our observations suggest that miRNAs play important regulatory roles in mosses. PMID:16146523

  18. Cloning and characterization of two flavohemoglobins from Aspergillus oryzae

    SciTech Connect

    Zhou Shengmin; Fushinobu, Shinya; Nakanishi, Yoshito; Kim, Sang-Wan; Wakagi, Takayoshi; Shoun, Hirofumi

    2009-03-27

    Two flavohemoglobin (FHb) genes, fhb1 and fhb2, were cloned from Aspergillus oryzae. The amino acid sequences of the deduced FHb1 and FHb2 showed high identity to other FHbs except for the predicted mitochondrial targeting signal in the N-terminus of FHb2. The recombinant proteins displayed absorption spectra similar to those of other FHbs. FHb1 and FHb2 were estimated to be a monomer and a dimer in solution, respectively. Both of the isozymes exhibit high NO dioxygenase (NOD) activity. FHb1 utilizes either NADH or NADPH as an electron donor, whereas FHb2 can only use NADH. These results suggest that FHb1 and FHb2 are fungal counterparts of bacterial FHbs and act as NO detoxification enzymes in the cytosol and mitochondria, respectively. This study is the first to show that a microorganism contains two isozymes of FHb and that intracellular localization of the isozymes could differ.

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

    PubMed

    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

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

    PubMed

    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

  1. Cloning and Functional Characterization of SAD Genes in Potato

    PubMed Central

    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

  2. Cloning, heterologous expression, and characterization of Thielavia terrestris glucoamylase.

    PubMed

    Rey, Michael W; Brown, Kimberly M; Golightly, Elizabeth J; Fuglsang, Claus C; Nielsen, Bjarne R; Hendriksen, Hanne V; Butterworth, Amy; Xu, Feng

    2003-12-01

    Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/ cellular biology is poorly understood. Only a few genes have been cloned from the Thielavia genus. We detected an extracellular glucoamylase in culture filtrates of T. terrestris and cloned the corresponding glaA gene. The coding region contains five introns. Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase. Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15. The T. terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A. oryzae alpha-amylase promoter and an Aspergillus niger glucoamylase terminator. The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 micromol/(min x mg) with maltose as substrate. With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60 degrees C. The enzyme was stable at 60 degrees C for 30 min. The Km and kcat of the enzyme for maltotriose were determined at various pHs and temperatures. At 20 degrees C and pH 4.0, the enzyme had a Km of 0.33 +/- 0.07 mM and a kcat of (5.5 +/- 0.5) x 103 min(-1) for maltotriose. The temperature dependence of kcat/Km indicated an activation free energy of 2.8 kJ/mol across the range of 20-70 degrees C. Overall, the enzyme derived from the thermophilic fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi. PMID:14665735

  3. Physiological Characterization of Adaptive Clones in Evolving Populations of the Yeast, SACCHAROMYCES CEREVISIAE

    PubMed Central

    Adams, Julian; Paquin, Charlotte; Oeller, Paul W.; Lee, Lester W.

    1985-01-01

    Populations of a diploid strain of S. cerevisiae were grown in glucose-limited continuous culture for more than 260 generations. A series of seven sequential adaptive changes were identified by monitoring the frequency of cycloheximide resistance in these populations. Samples were taken from the continuous cultures following each adaptive shift and characterized physiologically to determine (1) the range of phenotypes that can be selected in a precisely defined constant environment and (2) the order and predictability of the occurrence of the adaptive mutations in evolving populations. The clones were characterized with respect to the growth parameters, maximum growth rate, saturation coefficient and yield, as well as for changes in cell size and geometry and rate of glucose uptake. The maximum growth rates of the seven adaptive clones were very similar, but in contrast the saturation coefficients differed substantially. Surprisingly, not all clones showed reductions in the saturation coefficients, in comparison to the immediately preceding clones, as would be predicted from classical continuous culture kinetics. In addition, yield estimates first increased and then decreased for later isolated adaptive clones. In general, the results suggest epistatic interactions between the adaptive clones, consistent with earlier published results. The rate of glucose uptake, as measured by 14Cxylose uptake, increased dramatically after the selection and fixation of seven adaptive clones. Progressive decreases in cell volume and changes in cell geometry, resulting in increased surface area to volume ratios, were also observed in the adaptive clones, but these changes were not always seen in other haploid and diploid yeast populations evolving under the same conditions. Such changes may be easily explainable in terms of the characteristics of the glucose-limited environment. The significance of the results to the evolution of microorganisms under nutrient-limiting conditions is

  4. Characterization of a forest soil metagenome clone that confers indirubin and indigo production on Escherichia coli.

    PubMed

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

    2005-12-01

    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 to select two antibacterial clones by screening the constructed libraries using Bacillus subtilis as a target organism. Several clones produced purple or brown colonies. One of the selected antibacterial clones, pJEC5, produced purple colonies. Structural analysis of the purified pigments demonstrated that the metagenomic clone produced both the pigment indirubin and its isomer, indigo blue, resulting in purple colonies. In vitro mutational and subclonal analyses revealed that two open reading frames (ORFs) are responsible for the pigment production and antibacterial activity. The ORFs encode an oxygenase-like protein and a putative transcriptional regulator. Mutations of the gene encoding the oxygenase canceled both pigment production and antibacterial activity, whereas a subclone carrying the two ORFs retained pigment production and antibacterial activity. This finding suggests that these forest soil microbial genes are responsible for producing the pigment with antibacterial activity. PMID:16332749

  5. Molecular cloning of tetracycline resistance genes from Streptomyces rimosus in Streptomyces griseus and characterization of the cloned genes.

    PubMed Central

    Ohnuki, T; Katoh, T; Imanaka, T; Aiba, S

    1985-01-01

    Two tetracycline resistance genes of Streptomyces rimosus, an oxytetracycline producer, were cloned in Streptomyces griseus by using pOA15 as a vector plasmid. Expression of the cloned genes, designated as tetA and tetB was inducible in S. griseus as well as in the donor strain. The tetracycline resistance directed by tetA and tetB was characterized by examining the uptake of tetracycline and in vitro polyphenylalanine synthesis by the sensitive host and transformants with the resultant hybrid plasmids. Polyphenylalanine synthesis with crude ribosomes and the S150 fraction from S. griseus carrying the tetA plasmid was resistant to tetracycline, and, by a cross-test of ribosomes and S150 fraction coming from both the sensitive host and the resistant transformant, the resistance directed by tetA was revealed to reside mainly in crude ribosomes and slightly in the S150 fraction. However, the resistance in the crude ribosomes disappeared when they were washed with 1 M ammonium chloride. These results suggest that tetA specified the tetracycline resistance of the machinery for protein synthesis not through ribosomal subunits, but via an unidentified cytoplasmic factor. In contrast, S. griseus carrying the tetB plasmid accumulated less intracellular tetracycline than did the host, and the protein synthesis by reconstituting the ribosomes and S150 fraction was sensitive to the drug. Therefore, it is conceivable that tetB coded a tetracycline resistance determinant responsible for the reduced accumulation of tetracycline. Images PMID:2982781

  6. Cloning and partial characterization of Entamoeba histolytica PTPases

    SciTech Connect

    Herrera-Rodriguez, Sara Elisa; Baylon-Pacheco, Lidia; Talamas-Rohana, Patricia; Rosales-Encina, Jose Luis . E-mail: rosales@cinvestav.mx

    2006-04-21

    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.

  7. Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum.

    PubMed Central

    Leyva, A; Palacios, J M; Mozo, T; Ruiz-Argüeso, T

    1987-01-01

    A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHU1 containing hup genes of Bradyhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHU1 (5.9-kb HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. leguminosarum hup-specific region closely parallels that of B. japonicum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed Tn5 mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the Tn5 insertion strains in symbiosis with peas. Transposon Tn5 insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids. Images PMID:2822654

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

    PubMed

    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

  9. Cloning and characterization of root-specific barley lectin

    SciTech Connect

    Lerner, D.R.; Raikhel, N.V. )

    1989-09-01

    Cereal lectins are a class of biochemically and antigenically related proteins localized in a tissue-specific manner in embryos and adult plants. To study the specificity of lectin expression, a barley (Hordeum vulgare L.) embryo cDNa library was constructed and a clone (BLc3) for barley lectin was isolated. BLc3 is 972 nucleotides long and includes an open reading frame of 212 amino acids. The deduced amino acid sequence contains a putative signal peptide of 26 amino acid residues followed by a 186 amino acid polypeptide. This polypeptide has 95% sequence identity to the antigenically indistinguishable wheat germ agglutinin isolectin-B (WGA-B) suggesting that BLc3 encodes barley lectin. Further evidence that BLc3 encodes barley lectin was obtained by immunoprecipitation of the in vitro translation products of BLc3 RNA transcripts and barley embryo poly(A{sup +}) RNA. In situ hybridizations with BLc3 showed that barley lectin gene expression is confined to the outermost cell layers of both embryonic and adult root tips. On Northern blots, BLc3 hybridizes to a 1.0 kilobyte mRNA in poly(A{sup +}) RNA from both embryos and root tips. We suggest, on the basis of immunoblot experiments, that barley lectin is synthesized as a glycosylated precursor and processed by removal of a portion of the carboxyl terminus including the single N-linked glycosylation site.

  10. [Cloning and characterization of D-113 gene promoter from cotton].

    PubMed

    Luo, Ke-Ming; Guo, Yu-Long; Xiao, Yue-Hua; Hou, Lei; Pei, Yan

    2002-02-01

    To study the expression of late embryogenesis abundant gene in seeds, the 1,024 bp 5' flanking sequence of D-113 gene, a late embryogenesis abundant gene of Gossypium hirsutum cv. Coker 312, was cloned by PCR. The similarity compared with the sequence of Lea protein gene family published was 92.50%. There are three putative ABREs and one enhancer-like which riches A/T in the promoter. The promoter was fused to the beta-glucuronidase gene to form pLD II. Via a particle bombardment, pLD II was introduced into embryogenic calli of cotton and seeds of Brassica napus which were all treated with abscisic acid for 3d before bombardment, also into roots, stems and leafs of cotton. Transient expression was measured histochemically as spot number 24 h after bombardment. GUS sexpression was observed in the seeds of Brassica napus and the embryogenic calli of cotton, but not found in roots and leaves of cotton. Those results indicated that the expression of D-113 gene promoter was embryo specific. PMID:11902000

  11. Cloning, purification and biochemical characterization of dipetarudin, a new chimeric thrombin inhibitor.

    PubMed

    López, M; Mende, K; Steinmetzer, T; Nowak, G

    2003-03-25

    The development of thrombin inhibitors could provide invaluable progress for antithrombotic therapy. In this paper, we report the cloning, purification and biochemical characterization of dipetarudin, a chimeric thrombin inhibitor composed of the N-terminal head structure of dipetalogastin II, the strongest inhibitor from the assassin bug Dipetalogaster maximus, and the exosite 1 blocking segment of hirudin, connected through a five glycine linker. The cloning of dipetarudin was performed by a simple method which had not been used previously to clone chimeras. Biochemical characterization of dipetarudin revealed that it is a slow, tight-binding inhibitor with a molecular mass (M(r)=7560) and a thrombin inhibitory activity (K(i)=446 fM) comparable to r-hirudin. PMID:12651003

  12. Cloning, expression and characterization of sugarcane (Saccharum officinarum L.) transketolase.

    PubMed

    Kalhori, Nahid; Nulit, R; Go, Rusea

    2013-10-01

    Pentose phosphate pathway (PPP) composed of two functionally-connected phases, the oxidative and non-oxidative phase. Both phases catalysed by a series of enzymes. Transketolase is one of key enzymes of non-oxidative phase in which transfer two carbon units from fructose-6-phosphate to erythrose-4-phosphate and convert glyceraldehyde-3-phosphate to xylulose-5-phosphate. In plant, erythrose-4-phosphate enters the shikimate pathway which is produces many secondary metabolites such as aromatic amino acids, flavonoids, lignin. Although transketolase in plant system is important, study of this enzyme is still limited. Until to date, TKT genes had been isolated only from seven plants species, thus, the aim of present study to isolate, study the similarity and phylogeny of transketolase from sugarcane. Unlike bacteria, fungal and animal, PPP is complete in the cytosol and all enzymes are found cytosolic. However, in plant, the oxidative phase found localised in the cytosol but the sub localisation for non-oxidative phase might be restricted to plastid. Thus, this study was conducted to determine subcellular localization of sugarcane transketolase. The isolation of sugarcane TKT was done by reverse transcription polymerase chain reaction, followed by cloning into pJET1.2 vector and sequencing. This study has isolated 2,327 bp length of sugarcane TKT. The molecular phylogenetic tree analysis found that transketolase from sugarcane and Zea mays in one group. Classification analysis found that both plants showed closer relationship due to both plants in the same taxon i.e. family Poaceae. Target P 1.1 and Chloro P predicted that the compartmentation of sugarcane transketolase is localised in the chloroplast which is 85 amino acids are plant plastid target sequence. This led to conclusion that the PPP is incomplete in the cytosol of sugarcane. This study also found that the similarity sequence of sugarcane TKT closely related with the taxonomy plants. PMID:24132392

  13. Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

    PubMed Central

    Yoshikane, Yu; Yokochi, Nana; Ohnishi, Kouhei; Hayashi, Hideyuki; Yagi, Toshiharu

    2006-01-01

    Pyridoxamine–pyruvate aminotransferase is a PLP (pyridoxal 5′-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine–pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the α family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429–432]. The Kd value for pyridoxal determined by means of CD was 100-fold lower than the Km value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed. PMID:16545075

  14. Cloning and characterization of a bovine adeno-associated virus.

    PubMed

    Schmidt, Michael; Katano, Hisako; Bossis, Ioannis; Chiorini, John A

    2004-06-01

    To better understand the relationship between primate adeno-associated viruses (AAVs) and those of other mammals, we have cloned and sequenced the genome of an AAV found as a contaminant in two isolates of bovine adenovirus that was reported to be serologically distinct from primate AAVs. The bovine AAV (BAAV) genome has 4,693 bp, and its organization is similar to that of other AAV isolates. The left-hand open reading frame (ORF) and both inverted terminal repeats (ITRs) have the highest homology with the rep ORF and ITRs of AAV serotype 5 (AAV-5) (89 and 96%, respectively). However, the right-hand ORF was only 55% identical to the AAV-5 capsid ORF; it had the highest homology with the capsid ORF of AAV-4 (76%). By comparing the BAAV cap sequence with a model of an AAV-4 capsid, we mapped the regions of BAAV VP1 that are divergent from AAV-4. These regions are located on the outside of the capsid and are partially located in exposed loops. BAAV was not neutralized by antisera raised against recombinant AAV-2, AAV-4, or AAV-5, and it demonstrated a unique cell tropism profile in four human cancer cell lines, suggesting that BAAV might have transduction activity distinct from that of other isolates. A murine model of salivary gland gene transfer was used to evaluate the in vivo performance of recombinant BAAV. Recombinant BAAV-mediated gene transfer was 11 times more efficient than that with AAV-2. Overall, these data suggest that vectors based on BAAV could be useful for gene transfer applications. PMID:15163744

  15. Cloning, overexpression in Escherichia coli, and characterization of a thermostable fungal acetylxylan esterase from Talaromyces emersonii.

    PubMed

    Waters, Deborah M; Murray, Patrick G; Miki, Yuta; Martínez, Angel T; Tuohy, Maria G; Faulds, Craig B

    2012-05-01

    The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels. PMID:22407679

  16. Molecular Characterization of Kastamonu Garlic: An Economically Important Garlic Clone in Turkey

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to assess genetic relationship of Kastamonu garlic, which is very popular in Turkey due to its high quality features, along with some previously characterized garlic clones collected from different regions of the world using AFLP and locus specific DNA markers. UPGMA cluste...

  17. Cloning, sequencing and characterization of lipase from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipase gene (lip) of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing bacterium P. resinovorans NRRL B-2649 was cloned, sequenced and characterized by using consensus primers and PCR-based genome walking method. The ORF of the putative Lip (314 amino acids) and its active site (Ser111, Asp...

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

    EPA Science Inventory

    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...

  19. Cloning, Overexpression in Escherichia coli, and Characterization of a Thermostable Fungal Acetylxylan Esterase from Talaromyces emersonii

    PubMed Central

    Murray, Patrick G.; Miki, Yuta; Martínez, Angel T.; Tuohy, Maria G.; Faulds, Craig B.

    2012-01-01

    The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels. PMID:22407679

  20. Cloning and characterization of a critical regulator for pre-harvest sprouting in Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sprouting of grains in mature spikes before harvest is a major problem in wheat (Triticum aestivum) production worldwide. We cloned and characterized a gene underlying a wheat quantitative trait locus (QTL) on the short arm of chromosome 3A for pre-harvest sprouting (PHS) resistance in white wheat u...

  1. Characterization of a deep-sea sediment metagenomic clone that produces water-soluble melanin in Escherichia coli.

    PubMed

    Huang, Yali; Lai, Xintian; He, Xiaocui; Cao, Lixiang; Zeng, Zhirui; Zhang, Jiong; Zhou, Shining

    2009-01-01

    To access to the microbial genetic resources of deep-sea sediment by a culture-independent approach, the sediment DNA was extracted and cloned into fosmid vector (pCC1FOS) generating a library of 39,600 clones with inserts of 24-45 kb. The clone fss6 producing red-brown pigment was isolated and characterized. The pigment was identified as melanin according to its physico-chemical characteristics. Subcloning and sequences analyses of fss6 demonstrated that one open reading frame (ORF2) was responsible for the pigment production. The deduced protein from ORF2 revealed significant amino acid similarity to the 4-hydroxyphenylpyruvate dioxygenase (HPPD) from deep-sea bacteria Idiomarina loihiensis. Further study demonstrated that the production of melanin was correlated with homogentistic acid (HGA). The p-hydroxyphenylpyruvate produced by the Escherichia coli host was converted to HGA through the oxidation reaction of introduced HPPD. The results demonstrate that expression of DNA extracted directly from the environment might generate applicable microbial gene products. The construction and analysis of the metagenomic library from deep-sea sediment contributed to our understanding for the reservoir of unexploited deep-sea microorganisms. PMID:18648877

  2. Cloning and Functional Characterization of Chicken Stem Cell Antigen 2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stem cell antigen 2 (SCA2) is a Ly-6 family member whose function is largely unknown. To characterize biological properties and tissue distribution of chicken SCA2, SCA2 protein was expressed and purified in E. coli, and a polyclonal antibody developed. Utilizing the polyclonal antibody SCA2 is a 13...

  3. Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene.

    PubMed Central

    Franco, A A; Mundy, L M; Trucksis, M; Wu, S; Kaper, J B; Sears, C L

    1997-01-01

    Strains of Bacteroides fragilis that produce a ca. 20-kDa heat-labile protein toxin (termed B. fragilis toxin [BFT]) have been associated with diarrheal disease of animals and humans. BFT alters the morphology of intestinal epithelial cells both in vitro and in vivo and stimulates secretion in ligated intestinal segments of rats, rabbits, and lambs. Previous genetic and biochemical data indicated that BFT was a metalloprotease which hydrolyzed G (monomeric) actin, gelatin, and azocoll in vitro. In this paper, the cloning and sequencing of the entire B. fragilis toxin gene (bft) from enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 is reported. The bft gene from this ETBF strain consists of one open reading frame of 1,191 nucleotides encoding a predicted 397-residue holotoxin with a calculated molecular weight of 44,493. Comparison of the predicted BFT protein sequence with the N-terminal amino acid sequence of purified BFT indicates that BFT is most probably synthesized by ETBF strains as a preproprotein. These data predict that BFT is processed to yield a biologically active toxin of 186 residues with a molecular mass of 20.7 kDa which is secreted into the culture supernatant. Analysis of the holotoxin sequence predicts a 20-residue amphipathic region at the carboxy terminus of BFT. Thus, in addition to the metalloprotease activity of BFT, the prediction of an amphipathic domain suggests that oligomerization of BFT may permit membrane insertion of the toxin with creation of a transmembrane pore. Comparison of the sequences available for the bft genes from ETBF 86-5443-2-2 and VPI 13784 revealed two regions of reduced homology. Hybridization of oligonucleotide probes specific for each bft to toxigenic B.fragilis strains revealed that 51 and 49% of toxigenic strains contained the 86-5433-2-2 and VPI 13784 bft genes, respectively. No toxigenic strain hybridized with both probes. We propose that these two subtypes of bft be termed bft-1 (VPI 13784) and bft-2 (86

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

    NASA Technical Reports Server (NTRS)

    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.

  5. Molecular cloning and characterization of potato spindle tuber viroid cDNA sequences

    PubMed Central

    Owens, Robert A.; Cress, Dean E.

    1980-01-01

    Double-stranded cDNA has been synthesized from a polyadenylylated potato spindle tuber viroid (PSTV) template and inserted in the Pst I endonuclease site of plasmid pBR322 by using the oligo(dC)·oligo(dG)-tailing procedure. Tetracycline-resistant ampicillin-sensitive transformants contained sequences complementary to PSTV [32P]cDNA, and one recombinant clone (pDC-29) contains a 460-base-pair insert. This cloned double-stranded PSTV cDNA contains the cleavage sites for six restriction endonucleases predicted by the published primary sequence of PSTV as well as one additional site each for Ava I, Hae III, Hpa II, and Sma I. The additional Ava I, Hpa II, and Sma I sites are explained by the presence of a second C-C-C-G-G-G sequence in the cloned double-stranded cDNA. The largest fragment released by Hae III digestion contains approximately 360 base pairs. These results suggest that we have cloned almost the entire sequence of PSTV, but the sequence cloned differs slightly from that published. Hybridization probes derived from pDC-29 insert have allowed detection and preliminary characterization of RNA molecules having the same size as PSTV but the opposite polarity. This RNA is present during PSTV replication in infected tomato cells. Images PMID:16592877

  6. Cloning, expression, and preliminary structural characterization of RTN-1C

    SciTech Connect

    Fazi, Barbara; Melino, Sonia; Sano, Federica Di; Cicero, Daniel O.; Piacentini, Mauro . E-mail: mauro.piacentini@uniroma2.it; Paci, Maurizio

    2006-04-14

    Reticulons (RTNs) are endoplasmic reticulum-associated proteins widely distributed in plants, yeast, and animals. They are characterized by unique N-terminal parts and a common 200 amino acid C-terminal domain containing two long hydrophobic sequences. Despite their implication in many cellular processes, their molecular structure and function are still largely unknown. In this study, the reticulon family member RTN-1C has been expressed and purified in Escherichia coli and its molecular structure has been analysed by fluorescence and CD spectroscopy in different detergents in order to obtain a good solubility and a relative stability. The isotopically enriched protein has been also produced to perform structural studies by NMR spectroscopy. The preliminary results obtained showed that RTN-1C protein possesses helical transmembrane segments when a membrane-like environment is produced by detergents. Moreover, fluorescence experiments indicated the exposure of tryptophan side chains as predicted by structure prediction programs. We also produced the isotopically labelled protein and the procedure adopted allowed us to plan future NMR studies to investigate the biochemical behaviour of reticulon-1C and of its peptides spanning out from the membrane.

  7. Cloning, expression and characterization of Mycobacterium tuberculosis sirR.

    PubMed

    Namwat, Wises; Somnate, Baramee; Maleehual, Dutsadee; Chareonsudjai, Sorujsiri; Lulitanond, Viraphong; Faksri, Kiatichai

    2014-05-01

    Identification of new drug targets is important for the improvement of chemotherapy for tuberculosis treatment. Metal-associated gene products are candidates for novel drug development. A Mycobacterium tuberculosis (MTB) sirR-encoded protein has been proposed, but the function of MTB SirR has not yet been elucidated. Bioinformatics analysis revealed that MTB SirR contains iron binding domains with 34%-59% similarity to previously described metal-dependent gene regulators and that the gene lies in Rv2787-sirR operon. RT-PCR revealed that the Rv2787-sirR operon is transcribed a single bicistronic mRNA. Heterologous expression, purification and characterization of recombinant MTB His-tagged SirR demonstrated a 25 kDa protein (by SDS-PAGE and immunoblotting) that exists as a dimer (native PAGE). Based on electrophoretic mobility shift assay, MTB SirR bound a cis element located at -85 bp upstream of its operon. As Rv2787-sirR operon is unique only to MTB (and M. bovis), further studies on its regulation and other functions of the encoded proteins should provide leads towards the discovery of novel anti-TB drugs. PMID:24974654

  8. Cloning and Characterization of a Novel Drosophila Stress Induced DNase

    PubMed Central

    Seong, Chang-Soo; Varela-Ramirez, Armando; Tang, Xiaolei; Anchondo, Brenda; Magallanes, Diego; Aguilera, Renato J.

    2014-01-01

    Drosophila melanogaster flies mount an impressive immune response to a variety of pathogens with an efficient system comprised of both humoral and cellular responses. The fat body is the main producer of the anti-microbial peptides (AMPs) with anti-pathogen activity. During bacterial infection, an array of secreted peptidases, proteases and other enzymes are involved in the dissolution of debris generated by pathogen clearance. Although pathogen destruction should result in the release a large amount of nucleic acids, the mechanisms for its removal are still not known. In this report, we present the characterization of a nuclease gene that is induced not only by bacterial infection but also by oxidative stress. Expression of the identified protein has revealed that it encodes a potent nuclease that has been named Stress Induced DNase (SID). SID belongs to a family of evolutionarily conserved cation-dependent nucleases that degrade both single and double-stranded nucleic acids. Down-regulation of sid expression via RNA interference leads to significant reduction of fly viability after bacterial infection and oxidative stress. Our results indicate that SID protects flies from the toxic effects of excess DNA/RNA released by pathogen destruction and from oxidative damage. PMID:25083901

  9. Chondroitin sulfate synthase-3. Molecular cloning and characterization.

    PubMed

    Yada, Toshikazu; Sato, Takashi; Kaseyama, Hiromi; Gotoh, Masanori; Iwasaki, Hiroko; Kikuchi, Norihiro; Kwon, Yeon-Dae; Togayachi, Akira; Kudo, Takashi; Watanabe, Hideto; Narimatsu, Hisashi; Kimata, Koji

    2003-10-10

    Recently, it has become evident that chondroitin sulfate (CS) glycosyltransferases, which transfer glucuronic acid and/or N-acetylgalactosamine residues from each UDP-sugar to the nonreducing terminus of the CS chain, form a gene family. We report here a novel human gene (GenBank trade mark accession number AB086062) that possesses a sequence homologous with the human chondroitin sulfate synthase-1 (CSS1) gene, formerly known as chondroitin synthase. The full-length open reading frame consists of 882 amino acids and encodes a typical type II membrane protein. This enzyme contains a beta 3-glycosyltransferase motif and a beta 4-glycosyltransferase motif similar to that found in CSS1. Both the enzymes were expressed in COS-7 cells as soluble proteins, and their enzymatic natures were characterized. Both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, CS polymer, and their corresponding oligosaccharides were used as the acceptor substrates, but no polymerization reaction was observed as in the case of CSS1. The new enzyme was thus designated chondroitin sulfate synthase-3 (CSS3). However, the specific activity of CSS3 was much lower than that of CSS1. The reaction products were shown to have a GlcUA beta 1-3GalNAc linkage and a GalNAc beta 1-4GlcUA linkage in the nonreducing terminus of chondroitin resulting from glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcript level of CSS3 was much lower than that of CSS1, although it was ubiquitously expressed in various human tissues. These results indicate that CSS3 is a glycosyltransferase having both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. It may make a contribution to CS biosynthesis that differs from that of CSS1. PMID:12907687

  10. Molecular cloning, characterization, and expression of pannexin genes in chicken.

    PubMed

    Kwon, Tae-Jun; Kim, Dong-Bin; Bae, Jae Woong; Sagong, Borum; Choi, Soo-Young; Cho, Hyun-Ju; Kim, Un-Kyung; Lee, Kyu-Yup

    2014-09-01

    Pannexins (Panx) are a family of proteins that share sequences with the invertebrate gap junction proteins, innexins, and have a similar structure to that of the vertebrate gap junction proteins, connexins. To date, the Panx family consists of 3 members, but their genetic sequences have only been completely determined in a few vertebrate species. Moreover, expression of the Panx family has been reported in several rodent tissues: Panx1 is ubiquitously expressed in mammals, whereas Panx2 and Panx3 expressions are more restricted. Although members of the Panx family have been detected in mammals, their genetic sequences in avian species have not yet been fully elucidated. Here, we obtained the full-length mRNA sequences of chicken PANX genes and evaluated the homology of the amino acids from these sequences with those of other species. Furthermore, PANX gene expression in several chicken tissues was investigated based on mRNA levels. PANX1 was detected in the brain, cochlea, chondrocytes, eye, lung, skin, and intestine, and PANX2 was expressed in the brain, eye, and intestine. PANX3 was observed in the cochlea, chondrocytes, and bone. In addition, expression of PANX3 was higher than PANX1 in the cochlea. Immunofluorescent staining revealed PANX1 in hair cells, as well as the supporting cells, ganglion neurons, and the tegmentum vasculosum in chickens, whereas PANX3 was only detected in the bone surrounding the cochlea. Overall, the results of this study provide the first identification and characterization of the sequence and expression of the PANX family in an avian species, and fundamental data for confirmation of Panx function. PMID:25002553

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  12. Construction and Characterization of an Infectious Molecular Clone of Koala Retrovirus

    PubMed Central

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

    2013-01-01

    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 106 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. PMID:23427161

  13. Cloning and characterization of polyphenol oxidase cDNAs of Phytolacca americana.

    PubMed Central

    Joy, R W; Sugiyama, M; Fukuda, H; Komamine, A

    1995-01-01

    Two cDNA clones encoding polyphenol oxidases were isolated from a cDNA library constructed from a log-phase suspension culture of Phytolacca americana (pokeweed) producing betalains. The clones exhibit 93 and 86% sequence identity at the nucleotide and deduced amino acid levels, respectively. Both clones contain two copper-binding domains characterized by histidine-rich regions, which are found ubiquitously in all polyphenol oxidases/tyrosinases, and a putative third histidine-rich, copper-binding region, which is common to all plant polyphenol oxidases. One of the Phytolacca cDNA deduced amino acid sequences contains the ubiquitous transit peptide for all proteins targeted to the internal lumen of thylakoid membranes of plastids and is considered to be 98 residues in length based on a proposed sequence cleavage site motif. This would produce a processed peptide of approximately 54 kD. In addition to common features of transit peptides, it was found that an additional conserved region for polyphenol oxidases was located between the hydroxy amino acid-rich region and the thylakoid transfer domain. Spatial and temporal expression was investigated by northern blot analysis of total RNA from various organs of Phytolacca plants. Transcripts of the two clones were found to be 2.1 and 2.3 kb, respectively. Both transcripts were present only at substantial levels in ripening, betalain-containing fruit. PMID:7539531

  14. Cloning and characterization of the aroA gene from Mycobacterium tuberculosis.

    PubMed Central

    Garbe, T; Jones, C; Charles, I; Dougan, G; Young, D

    1990-01-01

    The aroA gene from Mycobacterium tuberculosis has been cloned by complementation of an aroA mutant of Escherichia coli after lysogenization with a recombinant DNA library in the lambda gt11 vector. Detailed characterization of the M. tuberculosis aroA gene by nucleotide sequencing and by immunochemical analysis of the expressed product indicates that it encodes a 5-enolpyruvylshikimate-3-phosphate synthase that is structurally related to analogous enzymes from other bacterial, fungal, and plant sources. The potential use of the cloned gene in construction of genetically defined mutant strains of M. tuberculosis by gene replacement is proposed as a novel approach to the rational attenuation of mycobacterial pathogens and the possible development of new antimycobacterial vaccines. Images PMID:2123856

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

    SciTech Connect

    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.

  16. Identification and characterization of mutant clones with enhanced propagation rates from phage-displayed peptide libraries.

    PubMed

    Nguyen, Kieu T H; Adamkiewicz, Marta A; Hebert, Lauren E; Zygiel, Emily M; Boyle, Holly R; Martone, Christina M; Meléndez-Ríos, Carola B; Noren, Karen A; Noren, Christopher J; Hall, Marilena Fitzsimons

    2014-10-01

    A target-unrelated peptide (TUP) can arise in phage display selection experiments as a result of a propagation advantage exhibited by the phage clone displaying the peptide. We previously characterized HAIYPRH, from the M13-based Ph.D.-7 phage display library, as a propagation-related TUP resulting from a G→A mutation in the Shine-Dalgarno sequence of gene II. This mutant was shown to propagate in Escherichia coli at a dramatically faster rate than phage bearing the wild-type Shine-Dalgarno sequence. We now report 27 additional fast-propagating clones displaying 24 different peptides and carrying 14 unique mutations. Most of these mutations are found either in or upstream of the gene II Shine-Dalgarno sequence, but still within the mRNA transcript of gene II. All 27 clones propagate at significantly higher rates than normal library phage, most within experimental error of wild-type M13 propagation, suggesting that mutations arise to compensate for the reduced virulence caused by the insertion of a lacZα cassette proximal to the replication origin of the phage used to construct the library. We also describe an efficient and convenient assay to diagnose propagation-related TUPS among peptide sequences selected by phage display. PMID:24952360

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

    PubMed

    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

  18. Cloning and characterization of farnesyl pyrophosphate synthase from the highly branched isoprenoid producing diatom Rhizosolenia setigera

    PubMed Central

    Ferriols, Victor Marco Emmanuel N.; Yaginuma, Ryoko; Adachi, Masao; Takada, Kentaro; Matsunaga, Shigeki; Okada, Shigeru

    2015-01-01

    The diatom Rhizosolenia setigera Brightwell produces highly branched isoprenoid (HBI) hydrocarbons that are ubiquitously present in marine environments. The hydrocarbon composition of R. setigera varies between C25 and C30 HBIs depending on the life cycle stage with regard to auxosporulation. To better understand how these hydrocarbons are biosynthesized, we characterized the farnesyl pyrophosphate (FPP) synthase (FPPS) enzyme of R. setigera. An isolated 1465-bp cDNA clone contained an open reading frame spanning 1299-bp encoding a protein with 432 amino acid residues. Expression of the RsFPPS cDNA coding region in Escherichia coli produced a protein that exhibited FPPS activity in vitro. A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis. Product analysis by gas chromatography-mass spectrometry also revealed that RsFPPS produced small amounts of the cis-isomers of geranyl pyrophosphate and FPP, candidate precursors for the cis-isomers of HBIs previously characterized. Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed. Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom. PMID:25996801

  19. Establishment and Characterization of Molecular Clones of Porcine Endogenous Retroviruses Replicating on Human Cells

    PubMed Central

    Czauderna, Frank; Fischer, Nicole; Boller, Klaus; Kurth, Reinhard; Tönjes, Ralf R.

    2000-01-01

    The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication-competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of the env gene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restored env start codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p.t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic env transcript of 3.1 kb. PERV-B(33) and PERV-B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals. PMID:10756014

  20. Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus.

    PubMed

    Li, Xundong; Zhuo, Kan; Luo, Mei; Sun, Longhua; Liao, Jinling

    2011-06-01

    The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2-70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. xylophilus. PMID:21371475

  1. Angucyclines Sch 47554 and Sch 47555 from Streptomyces sp. SCC-2136: cloning, sequencing, and characterization.

    PubMed

    Basnet, Devi Bahdur; Oh, Tae-Jin; Vu, Thi Thu Hang; Sthapit, Basundhara; Liou, Kwangkyoung; Lee, Hei Chan; Yoo, Jin-Cheol; Sohng, Jae Kyung

    2006-10-31

    The entire gene cluster involved in the biosynthesis of angucyclines Sch 47554 and Sch 47555 was cloned, sequenced, and characterized. Analysis of the nucleotide sequence of genomic DNA spanning 77.5-kb revealed a total of 55 open reading frames, and the deduced products exhibited strong sequence similarities to type II polyketide synthases, deoxysugar biosynthetic enzymes, and a variety of accessory enzymes. The involvement of this gene cluster in the pathway of Sch 47554 and Sch 47555 was confirmed by genetic inactivation of the aromatase, including a portion of the ketoreductase, which was disrupted by inserting the thiostrepton gene. PMID:17085966

  2. Molecular cloning and biochemical characterization of carbonic anhydrase from Populus tremula x tremuloides.

    PubMed

    Larsson, S; Björkbacka, H; Forsman, C; Samuelsson, G; Olsson, O

    1997-07-01

    A leaf cDNA library from hybrid aspen, Populus tremula x tremuloides, was constructed. From this two different cDNA clones, denoted CA1a and CA1b, encoding a chloroplastic carbonic anhydrase (CA) were isolated and DNA sequenced. Analysis of the deduced amino acid sequences showed that the isolated CAs belong to the beta-CA family, and have identities around 70% to other dicotyledonous plant CAs. The two hybrid aspen cDNA clones display a high nucleotide sequence identity, only 12 nucleotides differ. Since only one gene copy of this soluble chloroplastic CA is present in the nuclear genome, we postulate that the two isolated cDNA clones are alleles. Northern blot hybridization revealed a CA transcript of ca. 1300 bases, 140 bases shorter than in pea. Western and northern blot hybridizations on crude protein extracts and on total RNA, respectively, isolated from stem and leaves, showed that hybrid aspen CA is expressed specifically in the leaf under the growth conditions used. Based on the deduced amino acid sequence, the mature hybrid aspen CA enzyme subunit has a molecular mass of 24.8 kDa. The enzyme was over-expressed in Escherichia coli, and purified by affinity chromatography. Biochemical characterization showed that the protein structure and the CO2-hydration activity are similar to the pea enzyme. Molecular characterization of a CA from a perennial plant has not previously been performed, and it demonstrates that both the structure and activity of hybrid aspen CA resembles CAs from annual plants. PMID:9247540

  3. Map-based cloning and characterization of BPH29, a B3 domain-containing recessive gene conferring brown planthopper resistance in rice

    PubMed Central

    Wang, Ying; Cao, Liming; Zhang, Yuexiong; Cao, Changxiang; Liu, Fang; Huang, Fengkuan; Qiu, Yongfu; Li, Rongbai; Lou, Xiaojin

    2015-01-01

    Rice (Oryza sativa L.) production, essential for global food security, is threatened by the brown planthopper (BPH). The breeding of host-resistant crops is an economical and environmentally friendly strategy for pest control, but few resistance gene resources have thus far been cloned. An indica rice introgression line RBPH54, derived from wild rice Oryza rufipogon, has been identified with sustainable resistance to BPH, which is governed by recessive alleles at two loci. In this study, a map-based cloning approach was used to fine-map one resistance gene locus to a 24kb region on the short arm of chromosome 6. Through genetic analysis and transgenic experiments, BPH29, a resistance gene containing a B3 DNA-binding domain, was cloned. The tissue specificity of BPH29 is restricted to vascular tissue, the location of BPH attack. In response to BPH infestation, RBPH54 activates the salicylic acid signalling pathway and suppresses the jasmonic acid/ethylene-dependent pathway, similar to plant defence responses to biotrophic pathogens. The cloning and characterization of BPH29 provides insights into molecular mechanisms of plant–insect interactions and should facilitate the breeding of rice host-resistant varieties. PMID:26136269

  4. Cloning and Characterization of a Critical Regulator for Preharvest Sprouting in Wheat

    PubMed Central

    Liu, Shubing; Sehgal, Sunish K.; Li, Jiarui; Lin, Meng; Trick, Harold N.; Yu, Jianming; Gill, Bikram S.; Bai, Guihua

    2013-01-01

    Sprouting of grains in mature spikes before harvest is a major problem in wheat (Triticum aestivum) production worldwide. We cloned and characterized a gene underlying a wheat quantitative trait locus (QTL) on the short arm of chromosome 3A for preharvest sprouting (PHS) resistance in white wheat using comparative mapping and map-based cloning. This gene, designated TaPHS1, is a wheat homolog of a MOTHER OF FLOWERING TIME (TaMFT)-like gene. RNA interference-mediated knockdown of the gene confirmed that TaPHS1 positively regulates PHS resistance. We discovered two causal mutations in TaPHS1 that jointly altered PHS resistance in wheat. One GT-to-AT mutation generates a mis-splicing site, and the other A-to-T mutation creates a premature stop codon that results in a truncated nonfunctional transcript. Association analysis of a set of wheat cultivars validated the role of the two mutations on PHS resistance. The molecular characterization of TaPHS1 is significant for expediting breeding for PHS resistance to protect grain yield and quality in wheat production. PMID:23821595

  5. The cloning and characterization of a second brain enzyme with NAAG peptidase activity.

    PubMed

    Bzdega, Tomasz; Crowe, Samantha L; Ramadan, Epolia R; Sciarretta, Kathryn H; Olszewski, Rafal T; Ojeifo, Olumide A; Rafalski, Victoria A; Wroblewska, Barbara; Neale, Joseph H

    2004-05-01

    The peptide neurotransmitter N-acetylaspartylglutamate is inactivated by extracellular peptidase activity following synaptic release. It is speculated that the enzyme, glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse enzyme (tentatively identified as glutamate carboxypeptidase III or GCPIII) that is homologous to an enzyme identified in a human lung carcinoma. The mouse peptidase was cloned from two non-overlapping EST clones and mouse brain cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild-type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound peptidase activity with similar V(max) and K(m) values (1.4 micro m and 54 pmol/min/mg; 3.5 micro m and 71 pmol/min/mg, respectively). Both enzymes are activated by a similar profile of metal ions and their activities are blocked by EDTA. GCPIII message was detected in brain and spinal cord by RT-PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous enzymes with similar pharmacological properties and affinity for NAAG. PMID:15086519

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

    PubMed

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

    2014-01-01

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

  7. Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase

    SciTech Connect

    Kwangmu Yen; Karl, M.R.; Blatt, L.M.; Simon, M.J.; Winter, R.B.; Fausset, P.R.; Lu, H.S.; Harcourt, A.A.; Chen, K.K. )

    1991-09-01

    Pseudomonas mendocina KR1 metabolizes toluene as a carbon source by a previously unknown pathway. The initial step of the pathway is hydroxylation of toluene to form p-cresol by a multicomponent toluene-4-monooxygenase (T4MO) system. The authors have cloned and characterized a gene cluster from KR 1 that determines the T4MO activity. To clone the T4MO genes, KR1 DNA libraries were constructed in Escherichia coli HB 101 by using a broad-host-range vector and transferred to a KR1 mutant able to grow on p-cresol but no on toluene. An insert consisting of two SacI fragments of identical size was shown to complement the mutant for growth on toluene. One of the SacI fragments, when cloned into the E. coli vector pUC19, was found to direct the synthesis of indigo dye. The indigo-forming property was correlated with the presence of T4MO activity. The T4MO genes were mapped to a 3.6-kb region, and the direction of transcription was determined. DNA sequencing and N-terminal amino acid determination identified a five-gene cluster, tmoABCDE, within this region. Expression of this cluster carrying a single mutation in each gene demonstrated that each of the five genes is essential for T4MO activity. Other evidence presented indicated that none of the tmo genes was involved in the regulation of the tmo gene cluster, in the control of substrate transport of the T4MO system, or in major processing of the products of the tmo genes. It was tentatively concluded that the tmoABCDE genes encode structural polypeptides of the T4MO enzyme system. One of the tmo genes was tentatively identified as a ferredoxin gene.

  8. Cloning and characterization of the first GH10 and GH11 xylanases from Rhizopus oryzae.

    PubMed

    Xiao, Zhizhuang; Grosse, Stephan; Bergeron, Hélène; Lau, Peter C K

    2014-10-01

    The only available genome sequence for Rhizopus oryzae strain 99-880 was annotated to not encode any β-1,4-endoxylanase encoding genes of the glycoside hydrolase (GH) family 10 or 11. Here, we report the identification and cloning of two such members in R. oryzae strain NRRL 29086. Strain 29086 was one of several selected fungi grown on wheat or triticale bran and screened for xylanase activity among other hydrolytic actions. Its high activity (138 U/ml) in the culture supernatant led to the identification of two activity-stained proteins, designated Xyn-1 and Xyn-2 of respective molecular masses 32,000 and 22,000. These proteins were purified to electrophoretic homogeneity and characterized. The specific activities of Xyn-1 and Xyn-2 towards birchwood xylan were 605 and 7,710 U/mg, respectively. Kinetic data showed that the lower molecular weight Xyn-2 had a higher affinity (K m=3.2 ± 0.2 g/l) towards birchwood xylan than Xyn-1 by about 4-fold. The melting temperature (T m) of the two proteins, estimated to be in the range of 49.5-53.7 °C indicated that they are rather thermostable proteins. N-terminal and internal peptide sequences were obtained by chemical digestion of the purified xylanases to facilitate cloning, expression in Escherichia coli, and sequencing of the respective gene. The cloned Rhizopus xylanases were used to demonstrate release of xylose from flax shives-derived hemicellulose as model feedstock. Overall, this study expands the catalytic toolbox of GH10 and 11 family proteins that have applications in various industrial and bioproducts settings. PMID:24760228

  9. Cloning, expression, and functional characterization of the rat Pax6 5a orthologous splicing variant.

    PubMed

    Wei, Fei; Li, Min; Cheng, Sai-Yu; Wen, Liang; Liu, Ming-Hua; Shuai, Jie

    2014-08-15

    Pax6 functions as a pleiotropic regulator in eye development and neurogenesis. Its splice variant Pax6 5a has been cloned in many vertebrate species including human and mouse, but never in rat. This study focused on the cloning and characterization of the Pax6 5a orthologous splicing variant in rat. It was cloned from Sprague-Dawley rats 10 days post coitum (E10) by RT-PCR and was sequenced for comparison with Pax6 sequences in the GenBank by BLAST. The rat Pax6 5a was revealed to contain an additional 42 bp insertion at the paired domain. At the nucleotide level, the rat Pax6 5a coding sequence (1,311 bp) had a higher degree of homology to the mouse (96% identical) than to the human (93% identical) sequence. At the amino acid (aa) level, rat PAX6 5a shares 99.8% identity with the mouse sequence and 99.5% with the human sequence. The splice variant is preferentially expressed in the rat E10 embryonic headfolds and not in the trunk of neurula. Its effects on the proliferation of rat mesenchymal stem cells (rMSCs) were preliminarily evaluated by the MTT assay. Both pLEGFP-Pax6 5a-transfected cells and pLEGFP-Pax6-transfected cells exhibited a similar growth curve (P>0.05), suggesting that the Pax6 5a has a similar effect on the proliferation of rMSCs as Pax6. PMID:24952136

  10. Cloning, characterization, and tissue distribution of prolactin receptor in the sea bream (Sparus aurata).

    PubMed

    Santos, C R; Ingleton, P M; Cavaco, J E; Kelly, P A; Edery, M; Power, D M

    2001-01-01

    The prolactin receptor (PRLR) was cloned and its tissue distribution characterized in adults of the protandrous hermaphrodite marine teleost, the sea bream (Sparus aurata). An homologous cDNA probe for sea bream PRLR (sbPRLR) was obtained by RT-PCR using gill mRNA. This probe was used to screen intestine and kidney cDNA libraries from which two overlapping clones (1100 and 2425 bp, respectively) were obtained. These clones had 100% sequence identity in the overlapping region (893 bp) and were used to deduce the complete amino acid sequence of sbPRLR. The receptor spans 2640 bp and encodes a protein of 537 amino acids. Features characteristic of PRLR, two pairs of cysteines, WS box, hydrophobic transmembrane domain, box 1, and box 2, were identified and showed a high degree of sequence identity to PRLRs from other vertebrate species. SbPRLR is 29 and 32% identical to tilapia (Oreochromis niloticus) and goldfish (Carassius auratus) PRLRs, respectively. In the sea bream two PRLR transcripts of 2.8 and 3.2 kb were detected in the intestine, kidney, and gills and a single transcript of 2.8 kb was detected in skin and pituitary by Northern blot. Spermiating gonads (more than 95% male tissue; gonado-somatic index of 0.6) contained, in addition to the 2.8-kb transcript, three more transcripts of 1.9, 1.3, and 1.1 kb. RT-PCR, which is a far more sensitive method than Northern blot, detected PRLR mRNA in gills, intestine, brain, pituitary, kidney, liver, gonads, spleen, head-kidney, heart, muscle, and bone. Immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR detected PRLR in several epithelial tissues of juvenile sea bream, including the anterior gut, renal tubule, choroid membrane of the third ventricle, saccus vasculosus, branchial chloride cells, and branchial cartilage. PMID:11161768

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

    SciTech Connect

    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.

  12. Characterization and Comparison of 2 Distinct Epidemic Community-Associated Methicillin-Resistant Staphylococcus aureus Clones of ST59 Lineage

    PubMed Central

    Chen, Chih-Jung; Unger, Clemens; Hoffmann, Wolfgang; Lindsay, Jodi A.; Huang, Yhu-Chering; Götz, Friedrich

    2013-01-01

    Sequence type (ST) 59 is an epidemic lineage of community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) isolates. Taiwanese CA-MRSA isolates belong to ST59 and can be grouped into 2 distinct clones, a virulent Taiwan clone and a commensal Asian-Pacific clone. The Taiwan clone carries the Panton–Valentine leukocidin (PVL) genes and the staphylococcal chromosomal cassette mec (SCCmec) VT, and is frequently isolated from patients with severe disease. The Asian-Pacific clone is PVL-negative, carries SCCmec IV, and a frequent colonizer of healthy children. Isolates of both clones were characterized by their ability to adhere to respiratory A549 cells, cytotoxicity to human neutrophils, and nasal colonization of a murine and murine sepsis models. Genome variation was determined by polymerase chain reaction of selected virulence factors and by multi-strain whole genome microarray. Additionally, the expression of selected factors was compared between the 2 clones. The Taiwan clone showed a much higher cytotoxicity to the human neutrophils and caused more severe septic infections with a high mortality rate in the murine model. The clones were indistinguishable in their adhesion to A549 cells and persistence of murine nasal colonization. The microarray data revealed that the Taiwan clone had lost the ø3-prophage that integrates into the β-hemolysin gene and includes staphylokinase- and enterotoxin P-encoding genes, but had retained the genes for human immune evasion, scn and chps. Production of the virulence factors did not differ significantly in the 2 clonal groups, although more α-toxin was expressed in Taiwan clone isolates from pneumonia patients. In conclusion, the Taiwan CA-MRSA clone was distinguished by enhanced virulence in both humans and an animal infection model. The evolutionary acquisition of PVL, the higher expression of α-toxin, and possibly the loss of a large portion of the β-hemolysin-converting prophage likely contribute to

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

    SciTech Connect

    Steiner, Konstanze; Hagenbuch, Bruno; Dietrich, Daniel R.

    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.

  14. Human T-cell clones with reactivity to Mycobacterium leprae as tools for the characterization of potential vaccines against leprosy.

    PubMed Central

    Emmrich, F; Kaufmann, S H

    1986-01-01

    T-cell clones with the T4 phenotype were established from patients with tuberculoid leprosy. The antigen reactivity of these clones ranged from stringent specificity for Mycobacterium leprae to broad cross-reactivity with other mycobacteria. Killed M. leprae had a weak stimulatory capacity which could be enhanced by ultrasonication. Among the three candidate antileprosy vaccines, M. leprae, M. bovis BCG, and the ICRC (Indian Cancer Research Center) strain, the last was superior in stimulating cross-reactive T4 clones. This finding argues for a differential masking of similar or identical membrane antigens in various mycobacterial species. T-cell clones with defined reactivity patterns for mycobacterial antigens could be helpful tools for the characterization of an antileprosy vaccine. PMID:3081446

  15. Quantitative and qualitative characterization of expanded CD4+ T cell clones in rheumatoid arthritis patients

    PubMed Central

    Ishigaki, Kazuyoshi; Shoda, Hirofumi; Kochi, Yuta; Yasui, Tetsuro; Kadono, Yuho; Tanaka, Sakae; Fujio, Keishi; Yamamoto, Kazuhiko

    2015-01-01

    Rheumatoid arthritis (RA) is an autoimmune destructive arthritis associated with CD4+ T cell-mediated immunity. Although expanded CD4+ T cell clones (ECs) has already been confirmed, the detailed characteristics of ECs have not been elucidated in RA. Using combination of a single-cell analysis and next-generation sequencing (NGS) in TCR repertoire analysis, we here revealed the detailed nature of ECs by examining peripheral blood (PB) from 5 RA patients and synovium from 1 RA patient. When we intensively investigated the single-cell transcriptome of the most expanded clones in memory CD4+ T cells (memory-mECs) in RA-PB, senescence-related transcripts were up-regulated, indicating circulating ECs were constantly stimulated. Tracking of the transcriptome shift within the same memory-mECs between PB and the synovium revealed the augmentations in senescence-related gene expression and the up-regulation of synovium-homing chemokine receptors in the synovium. Our in-depth characterization of ECs in RA successfully demonstrated the presence of the specific immunological selection pressure, which determines the phenotype of ECs. Moreover, transcriptome tracking added novel aspects to the underlying sequential immune processes. Our approach may provide new insights into the pathophysiology of RA. PMID:26245356

  16. Cloning and characterization of Pseudomonas sp. strain DNT genes for 2,4-dinitrotoluene degradation.

    PubMed

    Suen, W C; Spain, J C

    1993-03-01

    The degradation of 2,4-dinitrotoluene (DNT) by Pseudomonas sp. strain DNT is initiated by a dioxygenase attack to yield 4-methyl-5-nitrocatechol (MNC) and nitrite. Subsequent oxidation of MNC by a monooxygenase results in the removal of the second molecule of nitrite, and further enzymatic reactions lead to ring fission. Initial studies on the molecular basis of DNT degradation in this strain revealed the presence of three plasmids. Mitomycin-derived mutants deficient in either DNT dioxygenase only or DNT dioxygenase and MNC monooxygenase were isolated. Plasmid profiles of mutant strains suggested that the mutations resulted from deletions in the largest plasmid. Total plasmid DNA partially digested by EcoRI was cloned into a broad-host-range cosmid vector, pCP13. Recombinant clones containing genes encoding DNT dioxygenase, MNC monooxygenase, and 2,4,5-trihydroxytoluene oxygenase were characterized by identification of reaction products and the ability to complement mutants. Subcloning analysis suggests that the DNT dioxygenase is a multicomponent enzyme system and that the genes for the DNT pathway are organized in at least three different operons. PMID:8449889

  17. Construction and characterization of an infectious cDNA clone of Echovirus 25.

    PubMed

    Hou, Wangheng; Yang, Lisheng; Li, Shuxuan; Yu, Hai; Xu, Longfa; He, Delei; Chen, Mengyuan; He, Shuizhen; Ye, Xiangzhong; Que, Yuqiong; Shih, James Wai Kuo; Cheng, Tong; Xia, Ningshao

    2015-07-01

    Echovirus 25 (E-25) is a member of the enterovirus family and a common pathogen that induces hand, foot, and mouth disease (HFMD), meningitis, skin rash, and respiratory illnesses. In this study, we constructed and characterized an infectious full-length E-25 cDNA clone derived from the XM0297 strain, which was the first subgenotype D6 strain isolated in Xiamen, China. The 5'-Untranslated Regions (5'-UTR), P3 (3A-3B, 3D) and P3 (3C) regions of this E-25 (XM0297) strain were highly similar to EV-B77, E-16 and E-13, respectively. Our data demonstrate that the rescued E-25 viruses exhibited similar growth kinetics to the prototype virus strain XM0297. We observed the rescued viral particles using transmission electron microscope (TEM) and found them to possess an icosahedral structure, with a diameter of approximately 30 nm. The cross neutralization test demonstrated that the E-25 (XM0297) strain immune serum could not neutralize EV-A71, CV-A16 or CV-B3; likewise, the EV-A71 and CV-A16 immune serum could not neutralize E-25 (XM0297). The availability of this infectious clone will greatly enhance future virological investigations and possible vaccine development against E-25. PMID:26004198

  18. Characterization of four human YAC libraries for clone size, chimerism and X chromosome sequence representation.

    PubMed Central

    Nagaraja, R; Kere, J; MacMillan, S; Masisi, M J; Johnson, D; Molini, B J; Halley, G R; Wein, K; Trusgnich, M; Eble, B

    1994-01-01

    Four collections of human X-specific YACs, derived from human cells containing supernumerary X chromosomes or from somatic cell hybrids containing only X human DNA were characterized. In each collection, 80-85% of YAC strains contained a single X YAC. Five thousand YACs from the various libraries were sized, and cocloning was assessed in subsets by the fraction of YAC insert-ends with non-X sequences. Cocloning was substantial, ranging up to 50% for different collections; and in agreement with previous indications, in all libraries the larger the YACs, the higher the level of cocloning. In libraries made from human-hamster hybrid cells, expected numbers of clones were recovered by STS-based screening; but unexpectedly, the two collections from cells with 4 or 5 X chromosomes yielded numbers of YACs corresponding to an apparent content of only about two X equivalents. Thus it is possible that the DNA of inactive X chromosomes is poorly cloned into YACs, speculatively perhaps because of its specialized chromatin structure. Images PMID:8078777

  19. Molecular cloning and functional characterization of borneol dehydrogenase from the glandular trichomes of Lavandula x intermedia.

    PubMed

    Sarker, Lukman S; Galata, Mariana; Demissie, Zerihun A; Mahmoud, Soheil S

    2012-12-15

    Several varieties of Lavandula x intermedia (lavandins) are cultivated for their essential oils (EOs) for use in cosmetic, hygiene and personal care products. These EOs are mainly constituted of monoterpenes including camphor, which contributes an off odor reducing the olfactory appeal of the oil. We have recently constructed a cDNA library from the glandular trichomes (the sites of EO synthesis) of L. x intermedia plants. Here, we describe the cloning of a borneol dehydrogenase cDNA (LiBDH) from this library. The 780 bp open reading frame of the cDNA encoded a 259 amino acid short chain alcohol dehydrogenase with a predicted molecular mass of ca. 27.5 kDa. The recombinant LiBDH was expressed in Escherichia coli, purified by Ni-NTA agarose affinity chromatography, and functionally characterized in vitro. The bacterially produced enzyme specifically converted borneol to camphor as the only product with K(m) and k(cat) values of 53 μM and 4.0 × 10(-4) s(-1), respectively. The LiBDH transcripts were specifically expressed in glandular trichomes of mature flowers indicating that like other Lavandula monoterpene synthases the expression of this gene is regulated in a tissue-specific manner. The cloning of LiBDH has far reaching implications in improving the quality of Lavandula EOs through metabolic engineering. PMID:23058847

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

    PubMed

    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

  1. Cloning, characterization, localization, and mutational screening of the human BARX1 gene.

    PubMed

    Gould, D B; Walter, M A

    2000-09-15

    The Bar subclass of homeodomain proteins was first identified for its role in Drosophila eye development. The Bar subclass homolog, Barx1, has since been cloned in mouse and in chick. The expression of Barx1 in developing teeth and craniofacial mesenchyme of neural crest origin makes it a strong candidate for the related human disorders of Axenfeld-Reiger syndrome (ARS) and iridogoniodysgenesis syndrome (IGDS). Here we report the cloning and characterization of a novel human Bar class gene, human BARX1. Screening of a human fetal craniofacial library resulted in the isolation of a 1.6-kb full-length transcript. Sequence analysis indicated that human BARX1, mouse Barx1, and chick Barx1 show 100% identity at the amino acid level within their homeodomains. Human BARX1 is expressed in a number of tissues including testis and heart by Northern analysis and in iris and craniofacial tissues by PCR of cDNA libraries. BARX1 chromosomal localization to 9q12 was determined by radiation hybrid mapping. Intron/exon boundaries were established, and primers were generated to PCR amplify all four exons. A mutation screen was conducted in 55 patients affected with ARS, IGDS, or related ocular malformations. While six sequence polymorphisms were detected, no disease-causing mutations of BARX1 were observed. PMID:10995576

  2. Molecular cloning and characterization of copper amine oxidase from Huperzia serrata.

    PubMed

    Sun, Jieyin; Morita, Hiroyuki; Chen, Guoshen; Noguchi, Hiroshi; Abe, Ikuro

    2012-09-15

    A cDNA encoding a novel copper amine oxidase (CAO) was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae), which produces the Lycopodium alkaloid huperzine A. A 2043-bp open reading frame encoded an Mr 76,854 protein with 681 amino acids. The deduced amino acid sequence shared 44-56% identity with the known CAOs of plant origin, and contained the active site consensus sequence of Asn-Tyr-Asp/Glu. The phylogenetic tree analysis revealed that HsCAO from the primitive vascular plant H. serrata is closely related to Physcomitrella patens subsp CAO. The recombinant enzyme, heterologously expressed in Escherichia coli, catalyzed the oxidative deamination of aliphatic and aromatic amines. Among them, the enzyme accepted cadaverine as the best substrate to catalyze the oxidative deamination to Δ(1)-piperideine, which is the precursor of the Lycopodium alkaloids. Furthermore, a homology modeling and site-directed mutagenesis studies predicted the active site architecture, which suggested the crucial active site residues for the observed substrate preference. This is the first report of the cloning and characterization of a CAO enzyme from the primitive Lycopodium plant. PMID:22901898

  3. Cloning, molecular characterization, and expression pattern of FGF5 in Cashmere goat (Capra hircus).

    PubMed

    Bao, W L; Yao, R Y; He, Q; Guo, Z X; Bao, C; Wang, Y F; Wang, Z G

    2015-01-01

    Fibroblast growth factor 5 (FGF5) is a secreted signaling protein that belongs to the FGF family, and was found to be associated with hair growth in humans and other animals. The Inner Mongolia Cashmere goat (Capra hircus) is a goat breed that provides superior cashmere; this breed was formed by spontaneous mutation in China. Here, we report the cloning, molecular characterization, and expression pattern of the Cashmere goat FGF5. The cloned FGF5 cDNA was 813 base pairs (KM596772), including an open reading frame encoding a 270-amino-acid polypeptide. The nucleotide sequence shared 99% homology with Ovis aries FGF5 (NM_001246263.1). Bioinformatic analysis revealed that FGF5 contained a signal peptide, an FGF domain, and a heparin-binding growth factor/FGF family signature. There was 1 cAMP- and cGMP-dependent protein kinase phosphorylation site, 11 protein kinase C phosphorylation sites, 4 casein kinase II phosphorylation sites, 1 amidation site, 1 N-glycosylation site, and 1 tyrosine kinase phosphorylation site in FGF5. Real-time polymerase chain reaction showed that FGF5 mRNA levels were higher in testis than in the pancreas and liver. These data suggest that FGF5 may play a crucial role in Cashmere goat hair growth. PMID:26400346

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

    SciTech Connect

    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).

  5. Cloning, sequencing, expression and characterization of DNA photolyase from Salmonella typhimurium.

    PubMed Central

    Li, Y F; Sancar, A

    1991-01-01

    We have cloned the phr gene that encodes DNA photolyase from Salmonella typhimurium by in vivo complementation of Escherichia coli phr gene defect. The S.typhimurium phr gene is 1419 base pairs long and the deduced amino acid sequence has 80% identity with that of E. coli photolyase. We expressed the S.typhimurium phr gene in E.coli by ligating the E.coli trc promoter 5' to the gene, and purified the enzyme to near homogeneity. The apparent molecular weight of S.typhimurium photolyase is 54,000 dalton as determined by SDS-polyacrylamide gel electrophoresis, which is consistent with the calculated molecular weight of 53,932 dalton from the deduced phr gene product. S.typhimurium photolyase is purple-blue in color with near UV-visible absorption peaks at 384, 480, 580, and 625 nm and a fluorescence peak at 470 nm. From the characteristic absorption and fluorescence spectra and reconstitution experiments, S.typhimurium photolyase appears to contain flavin and methenyltetrahydrofolate as chromophore-cofactors as do the E.coli and yeast photolyases. Thus, S.typhimurium protein is the third folate class photolyase to be cloned and characterized to date. The binding constant of S.typhimurium photolyase to thymine dimer in DNA is kD = 1.6 x 10(-9) M, and the quantum yield of photorepair at 384 nm is 0.5. Images PMID:1840665

  6. Characterization of an infectious molecular clone of human T-cell leukemia virus type I.

    PubMed Central

    Zhao, T M; Robinson, M A; Bowers, F S; Kindt, T J

    1995-01-01

    An infectious molecular clone of human T-cell leukemia virus type I (HTLV-I) was derived from an HTLV-I-transformed rabbit T-cell line, RH/K30, obtained by coculture of rabbit peripheral blood mononuclear cells (PBMC) with the human HTLV-I-transformed cell line MT-2. The RH/K30 cell line contained two integrated proviruses, an intact HTLV-I genome and an apparently defective provirus with an in-frame stop codon in the env gene. A genomic DNA fragment containing the intact HTLV-I provirus was cloned into bacteriophage lambda (K30 phi) and subcloned into a plasmid vector (K30p). HTLV-I p24gag protein was detected in culture supernatants of human and rabbit T-cell and fibroblast lines transfected with these clones, at levels comparable to those of the parental cell line RH/K30. Persistent expression of virus was observed in one of these lines, RL-5/K30p, for more than 24 months. Biologic characterization of this cell line revealed the presence of integrated HTLV-I provirus, spliced and unspliced mRNA transcripts, and typical extracellular type C retrovirus particles. As expected, these virus particles contained HTLV-I RNA and reverse transcriptase activity. The transfected cells also expressed surface major histocompatibility complex class II, whereas no expression of this molecule was detected in the parental RL-5 cell line. Virus was passaged by cocultivation of irradiated RL-5/K30p cells with either rabbit PBMC or human cord blood mononuclear cells, demonstrating in vitro infectivity. The virus produced in these cells was also infectious in vivo, since rabbits injected with RL-5/K30p cells became productively infected, as evidenced by seroconversion, amplification of HTLV-I-specific sequences by PCR from PBMC DNA, and virus isolation from PBMC. Availability of infectious molecular clones will facilitate functional studies of HTLV-I genes and gene products. PMID:7884847

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

    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. Copyright 2000 Academic Press.

  8. Cloning, expression, purification and preliminary crystallographic characterization of a shikimate dehydrogenase from Corynebacterium glutamicum

    SciTech Connect

    Schoepe, Jan Niefind, Karsten; Chatterjee, Shivani; Schomburg, Dietmar

    2006-07-01

    The crystallization and preliminary X-ray characterization of a shikimate dehydrogenase from C. glutamicum is presented. The shikimate dehydrogenase from Corynebacterium glutamicum has been cloned into an Escherichia coli expression vector, overexpressed and purified. Native crystals were obtained by the vapour-diffusion technique using 2-methyl-2,4-pentanediol as a precipitant. The crystals belong to the centred monoclinic space group C2, with unit-cell parameters a = 118.77, b = 63.17, c = 35.67 Å, β = 92.26° (at 100 K), and diffract to 1.64 Å on a synchrotron X-ray source. The asymmetric unit is likely to contain one molecule, corresponding to a packing density of 2.08 Å{sup 3} Da{sup −1} and a solvent content of about 41%.

  9. Molecular cloning and characterization of human papilloma virus DNA derived from a laryngeal papilloma.

    PubMed Central

    Gissmann, L; Diehl, V; Schultz-Coulon, H J; zur Hausen, H

    1982-01-01

    Papilloma virus DNA from a laryngeal papilloma was cloned in phage lambda L 47 and characterized after cleavage with different restriction enzymes. Hybridization with the DNAs of human papilloma virus types 1, 2, 3, 4, 5, and 8 showed no homology under stringent hybridization conditions. Human papilloma virus type 6 DNA, however, was partially identical to laryngeal papilloma virus DNA; different restriction enzyme fragments hybridizing with the other DNA were identified on each genome. The degree of homology was determined by reassociation kinetics to be 25%. According to the present nomenclature, laryngeal papilloma virus therefore represents a different type of human papilloma virus and is tentatively designated as human papilloma virus type 11. Sequences homologous to laryngeal papilloma virus DNA were also found in four of nine additional laryngeal papillomas. Attempt to detect homologous DNA in 12 carcinomas of the larynx were negative. Images PMID:6292500

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis.

    PubMed

    Zhu, Longbao; Cui, Wenjing; Fang, Yueqin; Liu, Yi; Gao, Xinxing; Zhou, Zhemin

    2013-05-01

    The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg(-1) and the k cat/K m was 1.9 × 10(4) mM(-1) s(-1), exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry. PMID:23338700

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

    PubMed

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

    PubMed

    Steiner, Konstanze; Hagenbuch, Bruno; Dietrich, Daniel R

    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 2772bp containing a 2115bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80kDa. 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 13.9μM and 13.4μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a Km value of 103μM. 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. PMID:25218291

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

    PubMed Central

    Steiner, Konstanze; Hagenbuch, Bruno; Dietrich, Daniel R.

    2014-01-01

    Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrains 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 homologs 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. PMID:25218291

  15. Molecular cloning and characterization of genistein 4'-O-glucoside specific glycosyltransferase from Bacopa monniera.

    PubMed

    Ruby; Santosh Kumar, R J; Vishwakarma, Rishi K; Singh, Somesh; Khan, Bashir M

    2014-07-01

    Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49% identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism. PMID:24664316

  16. TWRS privatization support project waste characterization resource dictionary

    SciTech Connect

    Patello, G.K.; Wiemers, K.D.

    1996-09-01

    A single estimate of waste characteristics for each underground storage tanks at the Hanford Site is not available. The information that is available was developed for specific programmatic objectives and varies in format and level of descriptive detail, depending on the intended application. This dictionary reflects an attempt to define what waste characterization information is available. It shows the relationship between the identified resource and the original data source and the inter-relationships among the resources; it also provides a brief description of each resource. Developed as a general dictionary for waste characterization information, this document is intended to make the user aware of potenially useful resources.

  17. Development of novel SCAR markers for genetic characterization of Lonicera japonica from high GC-RAMP-PCR and DNA cloning.

    PubMed

    Cheng, J L; Li, J; Qiu, Y M; Wei, C L; Yang, L Q; Fu, J J

    2016-01-01

    Sequence-characterized amplified region (SCAR) markers were further developed from high-GC primer RAMP-PCR-amplified fragments from Lonicera japonica DNA by molecular cloning. The four DNA fragments from three high-GC primers (FY-27, FY-28, and FY-29) were successfully cloned into a pGM-T vector. The positive clones were sequenced; their names, sizes, and GenBank numbers were JYHGC1-1, 345 bp, KJ620024; YJHGC2-1, 388 bp, KJ620025; JYHGC7-2, 1036 bp, KJ620026; and JYHGC6-2, 715 bp, KJ620027, respectively. Four novel SCAR markers were developed by designing specific primers, optimizing conditions, and PCR validation. The developed SCAR markers were used for the genetic authentication of L. japonica from its substitutes. This technique provides another means of developing DNA markers for the characterization and authentication of various organisms including medicinal plants and their substitutes. PMID:27173286

  18. Molecular cloning and characterization of a Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae.

    PubMed

    Kinsella, B T; Larkin, A; Bolton, M; Cantwell, B A

    1991-07-01

    The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S. cerevisiae transformants, was developed. PMID:1934116

  19. Molecular cloning with a pMEA300-derived shuttle vector and characterization of the Amycolatopsis methanolica prephenate dehydratase gene.

    PubMed Central

    Vrijbloed, J W; van Hylckama Vlieg, J; van der Put, N M; Hessels, G I; Dijkhuizen, L

    1995-01-01

    An efficient restriction barrier for methylated DNA in the actinomycete Amycolatopsis methanolica could be avoided by using a nonmethylating Escherichia coli strain for DNA isolations. The A. methanolica prephenate dehydratase gene was cloned from a gene bank in a pMEA300-derived shuttle vector in E. coli and characterized. PMID:7592448

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

    PubMed Central

    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

  1. Cloning and molecular characterization of telomerase reverse transcriptase (TERT) and telomere length analysis of Peromyscus leucopus.

    PubMed

    Zhao, Xin; Ueda, Yasutaka; Kajigaya, Sachiko; Alaks, Glen; Desierto, Marie J; Townsley, Danielle M; Dumitriu, Bogdan; Chen, Jichun; Lacy, Robert C; Young, Neal S

    2015-08-15

    Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase complex that regulates telomerase activity to maintain telomere length for all animals with linear chromosomes. As the Mus musculus (MM) laboratory mouse has very long telomeres compared to humans, a potential alternative animal model for telomere research is the Peromyscus leucopus (PL) mouse that has telomere lengths close to the human range and has the wild counterparts for comparison. We report the full TERT coding sequence (pTERT) from PL mice to use in the telomere research. Comparative analysis with eight other mammalian TERTs revealed a pTERT protein considerably homologous to other TERTs and preserved all TERT specific-sequence signatures, yet with some distinctive features. pTERT displayed the highest nucleotide and amino acid sequence homology with hamster TERT. Unlike human but similar to MM mice, pTERT expression was detected in various adult somatic tissues of PL mice, with the highest expression in testes. Four different captive stocks of PL mice and wild-captured PL mice each displayed group-specific average telomere lengths, with the longest and shortest telomeres in inbred and outbred stock mice, respectively. pTERT showed considerable numbers of synonymous and nonsynonymous mutations. A pTERT proximal promoter region cloned was homologous among PL and MM mice and rat, but with species-specific features. From PL mice, we further cloned and characterized ribosomal protein, large, P0 (pRPLP0) to use as an internal control for various assays. Peromyscus mice have been extensively used for various studies, including human diseases, for which pTERT and pRPLP0 would be useful tools. PMID:25962353

  2. Cloning, Characterization, and Expression of a 200-Kilodalton Diagnostic Antigen of Babesia bigemina†

    PubMed Central

    Tebele, N.; Skilton, R. A.; Katende, J.; Wells, C. W.; Nene, V.; McElwain, T.; Morzaria, S. P.; Musoke, A. J.

    2000-01-01

    Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from infected erythrocytes. One of the major drawbacks of these tests is that antigen quality can vary from batch to batch. Since the quality of the antigen contributes to the sensitivity and specificity of serological tests, the use of standardized recombinant antigens should ensure consistency in assay quality. Previously, a 200-kDa merozoite antigen (p200) was identified as a candidate diagnostic antigen for use in a serological assay for the detection of B. bigemina antibodies in infected cattle. In this study, we have cloned, characterized, and expressed p200. A 3.5-kbp cDNA clone encoding p200 was isolated and shown to be almost full length, lacking approximately 300 bp at the 5′ end. The predicted amino acid sequence shows that p200 consists of a long, highly charged central repeat region of an uninterrupted α helix, indicative of a fibrous protein. Immunoelectron microscopy localized p200 to the merozoite cytoplasm, suggesting that the antigen may be a structural protein involved in forming filament structures within the cytoskeleton. The 3.5-kbp cDNA was expressed in bacteria as a fusion protein with glutathione S-transferase (GST), but the yield was poor. To improve the yield, cDNA fragments encoding antigenic domains of p200 were expressed as fusions with GST. One of these fusion proteins, C1A-GST, is composed of a 7-kDa fragment of the p200 repeat region and contains epitopes that react strongly with sera from cattle experimentally infected with B. bigemina. Recombinant C1A-GST should permit the development of an improved enzyme-linked immunosorbent assay for the detection of antibodies against B. bigemina. PMID:10834983

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

    PubMed

    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

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

    PubMed Central

    Lu, Deshun; Yuan, Xiu-juan; Evans, Robert J; Pappas, Amy T; Wang, He; Su, Eric W; Hamdouchi, Chafiq; Venkataraman, Chandrasekar

    2005-01-01

    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. PMID:16001983

  5. Molecular cloning and characterization of a 20q13.2 amplicon in breast carcinoma

    SciTech Connect

    Collins, C.; Froula, J.; Kowbel, D.

    1994-09-01

    Comparative genomic hybridization (CGH) has identified an amplification event involving chromosome band 20q13.2 in 15-20% of primary breast carcinomas. The application of FISH to the study of tumor interphase nuclei using 33 locus specific cosmid and P1 probes revealed amplification of band 20q13.2 in 35% of breast cancer cell lines and 8% of primary tumors. Moreover, this study localized the amplification event to the 1.5Mb interval defined by (Flpter 0.80-0.84.) and excluded all known genes in the region as candidates for the putative oncogene(s). To both identify the putative oncogene(s) and characterize the amplicon, a 12 member 4Mb YAC contig has been assembled by STS mapping that spans the core of the amplicon. The YAC contig is now being converted to a P1 contig to facilitate sequencing, exon trapping and direct selection of cDNAs. This is being accomplished by performing interAlu PCR reactions on individual YACs and sequencing the reaction products to create 5-10 new STSs per megaYAC. The DuPont P1 library is then screened for these STSs by the PCR. To date 21 P1 clones, forming 6 contigs, have been isolated by screening the DuPont P1 library for existing and/or newly created STSs. The ends of the 21 P1 clones are being sequenced to facilitate contig alignment and to enable chromosome walking. In collaboration with the Human Genome Center at the Lawrence Berkeley Laboratory we have initiated the directed sequencing of two P1 contigs, localized within the amplicon core, and ultimately will sequence the entire 1-2Mb amplicon.

  6. Cloning and molecular characterization of telomerase reverse transcriptase (TERT) and telomere length analysis of Peromyscus leucopus

    PubMed Central

    Zhao, Xin; Ueda, Yasutaka; Kajigaya, Sachiko; Alaks, Glen; Desierto, Marie J; Townsley, Danielle M.; Dumitriu, Bogdan; Chen, Jichun; Lacy, Robert C.; Young, Neal S.

    2015-01-01

    Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase complex that regulates telomerase activity to maintain telomere length for all animals with linear chromosomes. As the Mus musculus (MM) laboratory mouse has very long telomeres compared to humans, a potential alternative animal model for telomere research is the Peromyscus leucopus (PL) mouse that has telomere lengths close to the human range and has the wild counterparts for comparison. We report the full TERT coding sequence (pTERT) from PL mice to use in the telomere research. Comparative analysis with eight other mammalian TERTs revealed a pTERT protein considerably homologous to other TERTs and preserved all TERT specific-sequence signatures, yet with some distinctive features. pTERT displayed the highest nucleotide and amino acid sequence homology with hamster TERT. Unlike human but similar to MM mice, pTERT expression was detected in various adult somatic tissues of PL mice, with the highest expression in testes. Four different captive stocks of PL mice and wild-captured PL mice each displayed group-specific average telomere lengths, with the longest and shortest telomeres in inbred and outbred stock mice, respectively. pTERT showed considerable numbers of synonymous and nonsynonymous mutations. A pTERT proximal promoter region cloned was homologous among PL and MM mice and rat, but with species-specific features. From PL mice, we further cloned and characterized ribosomal protein, large, P0 (pRPLP0) to use as an internal control for various assays. Peromyscus mice have been extensively used for various studies, including human diseases, for which pTERT and pRPLP0 would be useful tools. PMID:25962353

  7. Cloning and characterization of HsfA2 from Lily (Lilium longiflorum).

    PubMed

    Xin, Haibo; Zhang, Hua; Chen, Li; Li, Xiaoxin; Lian, Qinglong; Yuan, Xue; Hu, Xiaoyan; Cao, Li; He, Xiuli; Yi, Mingfang

    2010-08-01

    Heat shock transcription factors (Hsfs) are the terminal components of the signal transduction chain mediating the activation of genes responsive to both heat stress and a large number of chemical stressors. This paper aims to clone Hsf from lily and characterize its function by analyses of mRNA expression, transactivation activity and thermotolerance of transgenic Arabidopsis. In this study, the gene encoding HsfA2 with 1,053 bp open reading frame (ORF) was cloned by rapid amplification of cDNA ends (RACE) technique from Lilium longiflorum 'White heaven'. Multiple alignment and phylogenetic analyses showed that the deduced protein was a novel member of the Hsf class A2. Expression analyses by RT-PCR indicated that LlHsfA2 expression was induced by heat shock and H(2)O(2) treatment, but not by NaCl. It was also found that the expression of LlHsfA2 correlated with thermotolerance in Lilium longiflorum 'White heaven' and Oriental hybrid 'Acapulco' under heat stress. Furthermore, yeast one-hybrid assay showed that LlHsfA2 had transactivation activity. In addition, overexpression of LlHsfA2 activated the downstream genes including Hsp101, Hsp70, Hsp25.3 and Apx2 and enhanced the thermotolerance of transgenic Arabidopsis plants. Taken together, our data suggest that LlHsfA2 is a novel and functional HsfA2, involved in heat signaling pathway in lily and useful for improvement of thermotolerance in transgenic plants. PMID:20499070

  8. Molecular cloning and characterization of beta-expansin gene related to root hair formation in barley.

    PubMed

    Kwasniewski, Miroslaw; Szarejko, Iwona

    2006-07-01

    Root hairs are specialized epidermal cells that play a role in the uptake of water and nutrients from the rhizosphere and serve as a site of interaction with soil microorganisms. The process of root hair formation is well characterized in Arabidopsis (Arabidopsis thaliana); however, there is a very little information about the genetic and molecular basis of root hair development in monocots. Here, we report on isolation and cloning of the beta-expansin (EXPB) gene HvEXPB1, tightly related to root hair initiation in barley (Hordeum vulgare). Using root transcriptome differentiation in the wild-type/root-hairless mutant system, a cDNA fragment present in roots of wild-type plants only was identified. After cloning of full-length cDNA and genomic sequences flanking the identified fragment, the subsequent bioinformatics analyses revealed homology of the protein coded by the identified gene to the EXPB family. Reverse transcription-PCR showed that expression of HvEXPB1 cosegregated with the root hair phenotype in F2 progeny of the cross between the hairless mutant rhl1.a and the wild-type Karat parent variety. Expression of the HvEXPB1 gene was root specific; it was expressed in roots of wild-type forms, but not in coleoptiles, leaves, tillers, and spikes. The identified gene was active in roots of two other analyzed root hair mutants: rhp1.a developing root hair primordia only and rhs1.a with very short root hairs. Contrary to this, a complete lack of HvEXPB1 expression was observed in roots of the spontaneous root-hairless mutant bald root barley. All these observations suggest a role of the HvEXPB1 gene in the process of root hair formation in barley. PMID:16679418

  9. Molecular cloning and characterization of novel cathelicidin-derived myeloid antimicrobial peptide from Phasianus colchicus.

    PubMed

    Wang, Yipeng; Lu, Zekuan; Feng, Feifei; Zhu, Wei; Guang, Huijuan; Liu, Jingze; He, Weiyu; Chi, Lianli; Li, Zheng; Yu, Haining

    2011-03-01

    Cathelicidins were initially characterized as a family of antimicrobial peptides. Now it is clear that they fulfill several immune functions in addition to their antimicrobial activity. In the current work, three cDNA sequences encoding pheasant cathelicidins were cloned from a constructed bone marrow cDNA library of Phasianus colchicus, using a nested-PCR-based cloning strategy. The three deduced mature antimicrobial peptides, Pc-CATH1, 2 and 3 are composed of 26, 32, and 29 amino acid residues, respectively. Unlike the mammalian cathelicidins that are highly divergent even within the same genus, Pc-CATHs are remarkably conserved with chicken fowlicidins with only a few of residues mutated according to the phylogenetic analysis result. Synthetic Pc-CATH1 exerted strong antimicrobial activity against most of bacteria and fungi tested, including the clinically isolated (IS) drug-resistant strains. Most MIC values against Gram-positive bacteria were in the range of 0.09-2.95 μM in the presence of 100mM NaCl. Pc-CATH1 displayed a negligible hemolytic activity against human erythrocytes, lysing 3.6% of erythrocytes at 3.15 μM (10 μg/ml), significantly higher than the corresponding MIC. Pc-CATH1 was stable in the human serum for up to 72 h, revealing its extraordinary serum stability. These specific features of Pc-CATH1 may make its applications much wider given the potency and breadth of the peptide's bacteriocidal capacity and its resistance towards serum and high-salt environments. PMID:20955730

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

    PubMed Central

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

    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 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. PMID:23658852

  11. Lunar science measurements and instruments for resource characterization

    SciTech Connect

    Vaniman, D.

    1992-12-31

    Resource characterization is a requirement for effective production of any product from planetary materials, whether that product is to be used locally or exported. The characterization required is not necessarily costly or extensive; for example, our current knowledge of lunar regolith is probably sufficient for it to be used immediately for shielding purposes. However, other products from regolith (e.g., oxygen and solar-wind gases) will require more thorough and particularly site-specific resource characterization before actual production commences. If global maps of the Moon are obtained by some combination of gamma ray, reflectance, X-ray fluorescence, and/or imaging methods, the task of targeting resource sites will be considerably improved. Once these sites are selected, however, they must be characterized on the ground. The product of this characterization should be useable maps that will maximize the product output and minimize wasted energy and effort.

  12. Lunar science measurements and instruments for resource characterization

    SciTech Connect

    Vaniman, D.

    1992-01-01

    Resource characterization is a requirement for effective production of any product from planetary materials, whether that product is to be used locally or exported. The characterization required is not necessarily costly or extensive; for example, our current knowledge of lunar regolith is probably sufficient for it to be used immediately for shielding purposes. However, other products from regolith (e.g., oxygen and solar-wind gases) will require more thorough and particularly site-specific resource characterization before actual production commences. If global maps of the Moon are obtained by some combination of gamma ray, reflectance, X-ray fluorescence, and/or imaging methods, the task of targeting resource sites will be considerably improved. Once these sites are selected, however, they must be characterized on the ground. The product of this characterization should be useable maps that will maximize the product output and minimize wasted energy and effort.

  13. Cloning, expression, and characterization of a cellobiose dehydrogenase from Thielavia terrestris induced under cellulose growth conditions.

    PubMed

    Langston, James A; Brown, Kimberly; Xu, Feng; Borch, Kim; Garner, Ashley; Sweeney, Matt D

    2012-06-01

    The enzyme cellobiose dehydrogenase (CDH) is of considerable interest, not only for its biotechnological applications, but also its potential biological role in lignocellulosic biomass breakdown. The enzyme catalyzes the oxidation of cellobiose and other cellodextrins, utilizing a variety of one- and two-electron acceptors, although the electron acceptor employed in nature is still unknown. In this study we show that a CDH is present in the secretome of the thermophilic ascomycete Thielavia terrestris when grown with cellulose, along with a mixture of cellulases and hemicellulases capable of breaking down lignocellulosic biomass. We report the cloning of this T. terrestris CDH gene (cbdA), its recombinant expression in Aspergillus oryzae, and purification and characterization of the T. terrestris CDH protein (TtCDH). The TtCDH shows spectral properties and enzyme activity similar to other characterized CDH enzymes. Substrate specificity was determined for a number of carbohydrate electron donors in the presence of the two-electron acceptor 2,6-dichlorophenol-indophenol. The TtCDH also shows dramatic synergy with Thermoascus aurantiacus glycoside hydrolase family 61A protein in the presence of a β-glucosidase for the cleavage of cellulose. PMID:22484439

  14. Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase.

    PubMed Central

    Arnau, J; Jørgensen, F; Madsen, S M; Vrang, A; Israelsen, H

    1997-01-01

    The Lactococcus lactis pfl gene, encoding pyruvate formate-lyase (PFL), has been cloned and characterized. The deduced amino acid sequence of the L. lactis PFL. protein showed high similarity to those of other bacterial PFL proteins and included the conserved glycine residue involved in posttranslational activation of PFL. The genetic organization of the chromosomal pfl region in L. lactis showed differences from other characterized pfl loci, with an upstream open reading frame independently transcribed in the same orientation as the pfl gene. The gene coding for PFL-activase (act), normally found downstream of pfl, was not identified in L. lactis. Analysis of pfl expression showed a strong induction under anaerobiosis at the transcriptional level independent of the growth medium used. During growth with galactose, pfl showed the highest levels of expression. Constructed L. lactis pfl strains were unable to produce formate under anaerobic growth. Higher levels of diacetyl and acetoin were produced anaerobically in the constructed Lactococcus lactis subsp. lactis biovar diacetylactis pfl strain. PMID:9294449

  15. Cloning and Characterization of Two Bistructural S-Layer-RTX Proteins from Campylobacter rectus

    PubMed Central

    Braun, Martin; Kuhnert, Peter; Nicolet, Jacques; Burnens, André P.; Frey, Joachim

    1999-01-01

    Campylobacter rectus is an important periodontal pathogen in humans. A surface-layer (S-layer) protein and a cytotoxic activity have been characterized and are thought to be its major virulence factors. The cytotoxic activity was suggested to be due to a pore-forming protein toxin belonging to the RTX (repeats in the structural toxins) family. In the present work, two closely related genes, csxA and csxB (for C. rectus S-layer and RTX protein) were cloned from C. rectus and characterized. The Csx proteins appear to be bifunctional and possess two structurally different domains. The N-terminal part shows similarity with S-layer protein, especially SapA and SapB of C. fetus and Crs of C. rectus. The C-terminal part comprising most of CsxA and CsxB is a domain with 48 and 59 glycine-rich canonical nonapeptide repeats, respectively, arranged in three blocks. Purified recombinant Csx peptides bind Ca2+. These are characteristic traits of RTX toxin proteins. The S-layer and RTX domains of Csx are separated by a proline-rich stretch of 48 amino acids. All C. rectus isolates studied contained copies of either the csxA or csxB gene or both; csx genes were absent from all other Campylobacter and Helicobacter species examined. Serum of a patient with acute gingivitis showed a strong reaction to recombinant Csx protein on immunoblots. PMID:10198015

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

    PubMed Central

    Kanmani, Palanisamy; Kumaresan, Kuppamuthu; Aravind, Jeyaseelan

    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 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. PMID:26691486

  17. Molecular Cloning, Overexpression and Characterization of a Novel Water Channel Protein from Rhodobacter sphaeroides

    PubMed Central

    Erbakan, Mustafa; Shen, Yue-xiao; Grzelakowski, Mariusz; Butler, Peter J.; Kumar, Manish; Curtis, Wayne R.

    2014-01-01

    Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, expression and characterization of a novel aquaporin, RsAqpZ, from a purple photosynthetic bacterium, Rhodobacter sphaeroides ATCC 17023. The protein was expressed homologously at a high yield (∼20 mg/L culture) under anaerobic photoheterotrophic growth conditions. Stopped-flow light scattering experiments demonstrated its high water permeability (0.17±0.05 cm/s) and low energy of activation for water transport (2.93±0.60 kcal/mol) in reconstituted proteoliposomes at a protein to lipid ratio (w/w) of 0.04. We developed a fluorescence correlation spectroscopy based technique and utilized a fluorescent protein fusion of RsAqpZ, to estimate the single channel water permeability of RsAqpZ as 1.24 (±0.41) x 10−12 cm3/s or 4.17 (±1.38)×1010 H2O molecules/s, which is among the highest single channel permeability reported for aquaporins. Towards application to water purification technologies, we also demonstrated functional incorporation of RsAqpZ in amphiphilic block copolymer membranes. PMID:24497982

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

    PubMed Central

    Cheng, Yuan-Yuan; Qian, Yun-Kai; Li, Zhi-Feng; Wu, Zhi-Hong; Liu, Hong; Li, Yue-Zhong

    2011-01-01

    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. PMID:22072918

  19. Cloning and functional characterization of the Lymantria dispar initiator caspase dronc.

    PubMed

    Kitaguchi, Koji; Hamajima, Rina; Yamada, Hayato; Kobayashi, Michihiro; Ikeda, Motoko

    2013-06-28

    Ld652Y cells from the gypsy moth, Lymantria dispar, are extremely sensitive to various apoptotic stimuli, whereas BM-N cells from the silkworm, Bombyx mori, are relatively resistant to apoptotic stimuli. We previously cloned and characterized a B. mori homologue (bm-dronc) of Drosophila melanogaster dronc. In the present study, we cloned and characterized an L. dispar homologue of dronc (ld-dronc) comparatively with Bm-Dronc. The open reading frame of ld-dronc consisted of 1329bp that was predicted to encode a 443 amino-acid polypeptide with a molecular mass of 50,706Da and 54-57% amino acid sequence identity with Dronc homologues from other lepidopteran insects identified to date. Ld-Dronc had a long prodomain, large p20 domain, and small p10 domain, and a catalytic site composed of (308)QTCRG(312), which was distinct from the sites QACRG in Bm-Dronc and QMCRG in Dronc homologues of several other lepidopteran insects. Transiently expressed Ld-Dronc underwent proteolytic processing in the lepidopteran cell lines L. dispar Ld652Y, Spodoptera frugiperda Sf9, and B. mori BM-N, and dipteran D. melanogaster S2, but only triggered apoptosis in the lepidopteran cell lines. Endogenous Ld-Dronc underwent processing in Ld652Y cells upon infection with vAcΔp35, but not in mock-infected Ld652Y cells, supporting the involvement of Ld-Dronc in apoptosis induction. In vAcΔp35-infected apoptotic cells, Ld-Dronc underwent proteolytic processing more rapidly and extensively than Bm-Dronc. Similar results were obtained for Ld-Dronc and Bm-Dronc expressed transiently in S2, Ld652Y, Sf9, and BM-N cells. Taken together, these findings suggest that the intrinsic properties of Dronc proteinsare responsible, at least in part, for the differing sensitivity of Ld652Y and BM-N to apoptosis induction upon NPV infection. PMID:23743202

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

    SciTech Connect

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

    1991-10-22

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

  1. Isolation and characterization of cDNA clones for human apolipoprotein A-I.

    PubMed Central

    Breslow, J L; Ross, D; McPherson, J; Williams, H; Kurnit, D; Nussbaum, A L; Karathanasis, S K; Zannis, V I

    1982-01-01

    We have isolated cDNA clones encoding human apolipoprotein (apo) A-I. Twenty putative apo A-I cDNA clones were selected by screening 10,000 clones of an adult human liver cDNA library with an oligonucleotide probe. The probe was a mixture of synthetic 14-base-long DNA oligomers constructed to correspond to the codons for apo A-I amino acids 105-109. Four of these clones were examined further and showed 600- to 800-base-pair (bp) inserts. Preliminary restriction mapping and partial DNA sequence analysis indicated that the shorter inserts were a subset of the longer DNA inserts. DNA sequence analysis of the clone with an insert of approximately equal to 600 bp, designated pAI-113, revealed that it contained a DNA sequence corresponding to apo A-I amino acids 94-243. The DNA base sequence of this clone also contained a standard termination codon, polyadenylylation signal, and poly(A) tail. Partial DNA sequence of a second clone that contained an 800-bp insert, designated pAI-107, showed that it corresponded to apo A-I amino acids 18-243 and also included the 3' untranslated region. Isolation of these cDNA clones will facilitate molecular analyses of apolipoproteins in normal and disease states. PMID:6294659

  2. Characterization and pharmacological properties of in vitro propagated clones of Echinacea tennesseensis (Beadle) small

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tissue culture techniques have been used to establish and maintain a repository of medicinal Echinacea. In vitro clones obtained from hypocotyls of germinated seeds, varied macroscopically, microscopically and exhibited variation in immune enhancing activity. Two in vitro produced clones of Echinace...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. Cloning and Characterization of the Antiviral Activity of Feline Tetherin/BST-2

    PubMed Central

    Fukuma, Aiko; Abe, Masumi; Morikawa, Yuko; Miyazawa, Takayuki; Yasuda, Jiro

    2011-01-01

    Human Tetherin/BST-2 has recently been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. In this study, we cloned a cDNA fragment encoding a feline homolog of Tetherin/BST-2 and characterized the protein product. The degree of amino acid sequence identity between human Tetherin/BST-2 and the feline homolog was 44.4%. Similar to human Tetherin/BST-2, the expression of feline Tetherin/BST-2 mRNA was inducible by type I interferon (IFN). Exogenous expression of feline Tetherin/BST-2 efficiently inhibited the release of feline endogenous retrovirus RD-114. The extracellular domain of feline Tetherin/BST-2 has two putative N-linked glycosylation sites, N79 and N119. Complete loss of N-linked glycosylation by introduction of mutations into both sites resulted in almost complete abolition of its antiviral activity. In addition, feline Tetherin/BST-2 was insensitive to antagonism by HIV-1 Vpu, although the antiviral activity of human Tetherin/BST-2 was antagonized by HIV-1 Vpu. Our data suggest that feline Tetherin/BST-2 functions as a part of IFN-induced innate immunity against virus infection and that the induction of feline Tetherin/BST-2 in vivo may be effective as a novel antiviral strategy for viral infection. PMID:21479233

  5. Enolase from the ectomycorrhizal fungus Tuber borchii Vittad.: biochemical characterization, molecular cloning, and localization.

    PubMed

    Polidori, Emanuela; Saltarelli, Roberta; Ceccaroli, Paola; Buffalini, Michele; Pierleoni, Raffaella; Palma, Francesco; Bonfante, Paola; Stocchi, Vilberto

    2004-02-01

    Enolase from Tuber borchii mycelium was purified to electrophoretical homogeneity using an anion-exchange and a gel permeation chromatography. Furthermore, the corresponding gene (eno-1) was cloned and characterized. The purified enzyme showed a higher affinity for 2-PGA (0.26 mM) with respect to PEP; the stability and activity of enolase were dependent of the divalent cation Mg2+. T. borchii eno-1 has an ORF of 1323 bp coding for a putative protein of 440 amino acids and Southern blotting analysis revealed that the gene is present as a single copy in T. borchii. The enzymatic activity and the mRNA expression level evaluated in mycelia grown either in different carbon sources, in pyruvate or during starvation were the same in all the conditions tested, while biochemical and Northern blotting analyses performed with mycelia at different days of growth showed T. borchii eno-1 regulation in response to the growth phase. Finally, Western blotting analysis demonstrated that enolase is localized only in the cytosolic fraction confirming its important role in glycolysis. PMID:14732262

  6. Cloning, expression and characterization of a eukaryotic cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011.

    PubMed

    Leipold, Friedemann; Wardenga, Rainer; Bornscheuer, Uwe T

    2012-05-01

    In this study, we have cloned and characterized a cycloalkanone monooxygenase (CAMO) from the ascomycete Cylindrocarpon radicicola ATCC 11011 (identical to Cylindrocarpon destructans DSM 837). The primary structure of this Baeyer-Villiger monooxygenase (BMVO) revealed 531 residues with around 45% sequence identity to known cyclohexanone monooxygenases. The enzyme was functionally overexpressed in Escherichia coli and investigated with respect to substrate spectrum and kinetic parameters. Substrate specificity studies revealed that a large variety of cycloaliphatic and bicycloaliphatic ketones are converted by this CAMO. A high catalytic efficiency against cyclobutanone was observed and seems to be a particular property of this BVMO. The thus produced butyrolactone derivatives are valuable building blocks for the synthesis of a variety of natural products and bioactive compounds. Furthermore, the enzyme revealed activity against open-chain ketones such as cyclobutyl, cyclopentyl and cyclohexyl methyl ketone which have not been reported to be accepted by typical cyclohexanone monooxygenases. These results suggest that the BVMO from C. radicicola indeed might be rather unique and since no BVMOs originating from eukaryotic organisms have been produced recombinantly so far, this study provides the first example for such an enzyme. PMID:22075635

  7. Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene

    PubMed Central

    Sheng, Yanmin; Wang, Yingdian; Capell, Teresa; Shi, Lianxuan; Ni, Xiuzhen; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

    2015-01-01

    The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity. PMID:26030746

  8. Cloning, sequencing and characterization of the biosynthetic gene cluster of sanglifehrin A, a potent cyclophilin inhibitor.

    PubMed

    Qu, Xudong; Jiang, Nan; Xu, Fei; Shao, Lei; Tang, Gongli; Wilkinson, Barrie; Liu, Wen

    2011-03-01

    Sanglifehrin A (SFA), a potent cyclophilin inhibitor produced by Streptomyces flaveolus DSM 9954, bears a unique [5.5] spirolactam moiety conjugated with a 22-membered, highly functionalized macrolide through a linear carbon chain. SFA displays a diverse range of biological activities and offers significant therapeutic potential. However, the structural complexity of SFA poses a tremendous challenge for new analogue development via chemical synthesis. Based on a rational prediction of its biosynthetic origin, herein we report the cloning, sequencing and characterization of the gene cluster responsible for SFA biosynthesis. Analysis of the 92 776 bp contiguous DNA region reveals a mixed polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) pathway which includes a variety of unique features for unusual PKS and NRPS building block formation. Our findings suggest that SFA biosynthesis requires a crotonyl-CoA reductase/carboxylase (CCR) for generation of the putative unusual PKS starter unit (2R)-2-ethylmalonamyl-CoA, an iterative type I PKS for the putative atypical extender unit (2S)-2-(2-oxo-butyl)malonyl-CoA and a phenylalanine hydroxylase for the NRPS extender unit (2S)-m-tyrosine. A spontaneous ketalization of significant note, may trigger spirolactam formation in a stereo-selective manner. This study provides a framework for the application of combinatorial biosynthesis methods in order to expand the structural diversity of SFA. PMID:21416665

  9. Cloning and characterization of calreticulin and its association with salinity stress in P. trituberculatus.

    PubMed

    Lv, Jianjian; Wang, Yu; Zhang, Dening; Gao, Baoquan; Liu, Ping; Li, Jian

    2015-09-01

    Calreticulin (CRT) is a highly conserved and multifunctional endoplasmic reticulum (ER) chaperone protein and plays important roles in salinity stress response. Portunus trituberculatus is a commercially important fishery species, and water salinity conditions influence its commercial farming significantly. In order to research the function of calreticulin under salinity stress, the full-length cDNA sequence of calreticulin from P. trituberculatus (PtCRT) was firstly cloned and characterized. The complete cDNA sequence of PtCRT is 1676 bp with 1218 bp open reading frame (ORF), encoding a polypeptide of 405 amino acids. Multiple sequence alignments showed that the deduced acid amino sequences of PtCRT shared the highest homology to CRT of Fenneropenaeus chinensis (89%). Fluorescent quantitative real-time PCR analysis indicated that PtCRT was expressed in all detected tissues and showed the highest expression level in hepatopancreas. In addition, salinity challenge significantly influenced the expression level of PtCRT in gill. Six single nucleotide polymorphisms (SNPs) were detected in cDNA sequence of PtCRT, and one SNP was associated with the salt tolerant trait. All results indicated that PtCRT plays an important role in mediating the salinity adaption of P. trituberculatus. PMID:25995067

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

    PubMed

    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.

    PubMed

    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 characterization of the actin gene from Puccinia striiformis f. sp. tritici.

    PubMed

    Liu, Jie; Zhang, Qiong; Chang, Qing; Zhuang, Hua; Huang, Li-Li; Kang, Zhen-Sheng

    2012-06-01

    The fungus Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, is an obligate biotrophic basidiomycete. Urediniospores are the most common spore type involved in the epidemiology of this disease. Tip growth of germ tubes of germinated urediniospores is a key step during infection of wheat, but few studies have investigated it so far. Recent research has found that actin is closely associated with hyphal tip growth. In this study, we have cloned and obtained the full-length actin cDNA from P. striiformis f. sp. tritici and characterized its expression. Furthermore, actin filament (F-actin) patterns were visualized microscopically during germ tube formation. The most conspicuous actin-containing structures were actin patches. They were mainly concentrated near the hyphal tip and scattered throughout the cortex. By using cytochalasin B, we observed that depolymerization of F-actin greatly reduced the germination rate of urediniospores and disrupted the transport of vesicles to the germ tube tip, indicating that F-actin played a key role in the tip growth of P. striiformis f. sp. tritici. This work helps us to understand the tip growth mechanism of P. striiformis f. sp. tritici, and may provide a theoretical framework for designing novel pesticides. PMID:22806107

  13. Isolation and characterization of cDNA clones for human erythrocyte. beta. -spectrin

    SciTech Connect

    Prchal, J.T.; Morley, B.J.; Yoon, S.H.; Coetzer, T.L.; Palek, J.; Conboy, J.G.; Kan, Y.W.

    1987-11-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical ..cap alpha.. (M/sub r/ 240,000) and ..beta.. (M/sub r/ 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. The authors report here the isolation and characterization of a human erythroid-specific ..beta..-spectrin cDNA clone that encodes parts of the ..beta..-9 through ..beta..-12 repeat segments. This cDNA was used as a hybridization probe to assign the ..beta..-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte ..beta..-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the ..beta..-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities.

  14. Cloning and characterization of p8 homolog cDNA in the Atlantic halibut (Hippoglossus hippoglossus).

    PubMed

    Wang, Jia-Qing; Li, Lin-Chun; Lin, Sheng-Guo; Wang, Zhi-Ping

    2010-06-01

    The p8 gene encodes a transcription factor known to modulate cell growth, division, and apoptosis and influences gene expression. In this study, an Atlantic halibut (Hippoglossus hippoglossus) homolog of the p8 gene was cloned, sequenced, and characterized. The full-length p8 cDNA consists of 601 bp and encodes 76 amino acids with a molecular mass of 9 kD. The bHLH region is well conserved between Atlantic halibut and other animals. Analysis by RT-PCR showed that the p8 transcript is constitutively expressed in 9 of the 12 tissues tested: pancreas, intestine, stomach, gill, head kidney, heart, liver, ovary, and spleen. A predicted microRNA target site was found in the 3'UTR of Atlantic halibut p8 mRNA. We speculate that the target site may pair to microRNA molecules because the target site resides in a big loop, a space large enough for the binding of microRNA molecules. PMID:20454949

  15. Molecular cloning, characterization and expression analysis of the complement component C6 gene in grass carp.

    PubMed

    Shen, Yu-Bang; Zhang, Jun-Bin; Xu, Xiao-Yan; Li, Jia-Le

    2011-05-15

    The complement system, as a representative of innate immunity, plays a key role in the host defense against infections. C6 is the member of complement components creating the membrane attack complex (MAC). In this study, we cloned and characterized the grass carp complement component C6 (gcC6) gene. Our data showed that gcC6 gene contained a 2724bp open reading frame (ORF), a 237bp 5'-untranslated region (UTR) and a 219bp 3'-UTR. The deduced amino acid sequence of gcC6 showed 77.6% and 58.9% identity to zebrafish C6 and rainbow trout C6, respectively. GcC6 gene was expressed in a wide range of grass carp tissues, and the highest expression level of gcC6 was detected in the spleen and liver. Upon challenge with Aeromonas hydrophila, its expression was significantly up-regulated in muscle, trunk kidney, liver, head kidney, spleen, heart and intestine, whereas it was down-regulated in the brain and skin. The expression level of gcC6 was high at the unfertilized egg stage. It was significantly increased at 1 day post-hatching, but it was decreased at 10 days post-hatching. This result suggested that the complement C6 transcripts in early embryos were of maternal origin. PMID:21353312

  16. Molecular cloning, sequence characterization, and tissue expression analysis of Hi-Line Brown chicken Akirin2.

    PubMed

    Man, Chaolai; Li, Xiang; Lee, Jongeun

    2011-10-01

    Akirins are novel important nuclear proteins able to modulate transcriptional activities in a gene-specific manner. Akirin2 is an important gene related to immune responses, it is necessary to isolate the akirin2 gene from chicken because it may be associated with vaccine and enhancement of immune response. In this study, a Hi-Line Brown chicken homolog of the vertebrate akirin2 gene was cloned, sequenced, and characterized. The akirin2 full-length coding sequence (CDS) consisted of 576nt and encoded 191 amino acids with a molecular weight of 21.58 kD. The COOH-terminal alpha-helix region was well conserved between chicken and other animals. RT-PCR analysis showed that the akirin2 transcripts were constitutively expressed in 16 tissues tested. Several microRNA target sites were predicted in the CDS of chicken akirin2 gene. We presume that Akirin2 protein may be used as a new-type immunopotentiator in poultry immune system in the future. PMID:21858694

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

    PubMed

    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

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

    PubMed

    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

  19. Cloning and partial characterization of the mouse glutamine:fructose-6-phosphate amidotransferase (GFAT) gene promoter.

    PubMed Central

    Sayeski, P P; Wang, D; Su, K; Han, I O; Kudlow, J E

    1997-01-01

    Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the enzyme that is rate limiting in the synthesis of glucosamine and hexosamines. Glucosamine has been proposed to contribute to the glucotoxicity of diabetes. Evidence that the gene encoding GFAT is transcriptionally regulated prompted us to clone and characterize its promoter. The position of the mouse GFAT promoter relative to the translational start site was located by primer extension and found to be 149 bp upstream of the translational start site. A 1.9 kb SacI fragment of the GFAT gene was found to contain the promoter and 88 bp of sequence downstream of the transcriptional start site. This promoter segment could drive expression of a luciferase reporter gene, could confer correct transcriptional initiation to the reporter and could confer the EGF-responsiveness previously observed in the native gene. The mouse GFAT promoter lacks a canonical TATA box and has several GC boxes within a highly GC-rich region. Deletional analysis of the promoter indicated that a proximal element extending to -120 relative to the transcriptional start site could confer reporter expression at a level of 57% of the 1.9 kb construct. Detailed analysis of this proximal region by DNase I footprinting, electrophoretic mobility shift assays and site-directed mutagenesis indicated that Sp1 binds to three elements in this proximal promoter segment and plays a vital role in regulation of transcription from this gene. PMID:9060444

  20. Cloning, expression and characterization of a novel esterase from a South China Sea sediment metagenome

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Li, Fuchao; Chen, Huaxin; Zhao, Jin; Yan, Jinfei; Jiang, Peng; Li, Ronggui; Zhu, Baoli

    2015-07-01

    Lipolytic enzymes, including esterases and lipases, represent a group of hydrolases that catalyze the cleavage and formation of ester bonds. A novel esterase gene, scsEst01, was cloned from a South China Sea sediment metagenome. The scsEst01 gene consisted of 921 bp encoding 307 amino acid residues. The predicted amino acid sequence shared less than 90% identity with other lipolytic enzymes in the NCBI nonredundant protein database. ScsEst01 was successfully co-expressed in Escherichia coli BL21 (DE3) with chaperones (dnaK-dnaJ-grpE) to prevent the formation of inclusion bodies. The recombinant protein was purified on an immobilized metal ion affinity column containing chelating Sepharose charged with Ni2+. The enzyme was characterized using p -nitrophenol butyrate as a substrate. ScsEst01 had the highest lipolytic activity at 35°C and pH 8.0, indicative of a meso-thermophilic alkaline esterase. ScsEst01 was thermostable at 20°C. The lipolytic activity of scsEst01 was strongly increased by Fe2+, Mn2+ and 1% Tween 80 or Tween 20.

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

    PubMed

    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

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

    PubMed

    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

  3. Molecular cloning, expression, and characterization of a novel endo-alpha-N-acetylgalactosaminidase from Enterococcus faecalis.

    PubMed

    Goda, Hatsumi M; Ushigusa, Kota; Ito, Hiromi; Okino, Nozomu; Narimatsu, Hisashi; Ito, Makoto

    2008-10-31

    We report here the molecular cloning, expression and characterization of a novel endo-alpha-N-acetylgalactosaminidase, classified into the GH101 family, from Enterococcus faecalis (endo-EF). The recombinant endo-EF was found to catalyze the liberation of core1-disaccharides (Galbeta1-3GalNAc) from core1-pNP (Galbeta1-3GalNAcalpha-pNP) like other GH101 family enzymes. However, endo-EF seems to differ in specificity from the GH101 enzymes reported to date, because it was able to release trisaccharides from core2-pNP (Galbeta1-3[GlcNAcbeta1-6]GalNAcalpha-pNP) and tetrasaccharides from Gal-core2-pNP (Galbeta1-3[Galbeta1-3GlcNAcbeta1-6]GalNAcalpha-pNP). Interestingly, the enzyme could transfer not only core1-disaccharides but also core2-trisaccharides to alkanols generating alkyl-oligosaccharides. Endo-EF should facilitate O-glycoprotein research. PMID:18725192

  4. Cloning, expression and characterization of a novel thermophilic polygalacturonase from Caldicellulosiruptor bescii DSM 6725.

    PubMed

    Chen, Yanyan; Sun, Dejun; Zhou, Yulai; Liu, Liping; Han, Weiwei; Zheng, Baisong; Wang, Zhi; Zhang, Zuoming

    2014-01-01

    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. PMID:24705464

  5. Cloning and characterization of glyceraldehyde-3-phosphate dehydrogenase encoding gene in Gracilaria/Gracilariopsis lemaneiformis

    NASA Astrophysics Data System (ADS)

    Ren, Xueying; Sui, Zhenghong; Zhang, Xuecheng

    2006-04-01

    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.

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

    PubMed

    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

    2015-01-01

    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. PMID:26782459

  7. Cloning and characterization of a potentially protective chitinase-like recombinant antigen from Wuchereria bancrofti.

    PubMed Central

    Raghavan, N; Freedman, D O; Fitzgerald, P C; Unnasch, T R; Ottesen, E A; Nutman, T B

    1994-01-01

    While there is no direct evidence demonstrating the existence of protective immunity to Wuchereria bancrofti infection in humans, the presence of individuals, in populations in areas where infection is endemic, with no clinical evidence of past or current infection despite appreciable exposure to the infective larvae, suggests that protective immunity to filarial parasites may occur naturally. Earlier work indicated that such putatively immune individuals generated antibodies to a 43-kDa antigen from larval extracts of the related filarial parasite Brugia malayi that was recognized by only 8% of the infected population. With rabbit antiserum raised against this 43-kDa antigen, this current study identified a recombinant clone, WbN43, with an insert size of 2.3 kb, from a W. bancrofti genomic expression library. The recombinant fusion protein was differentially recognized by the putatively immune individuals but not by the infected patients. The coding sequence (684 bp) from the 5' end had significant sequence similarity to chitinases from Serratia marcescens, Bacillus circulans, Streptomyces plicatus, and B. malayi. Peptide sequencing of the expressed product also defined a chitinase-like sequence. Molecular characterization indicated WbN43 to be a low-copy-number gene, with expression predominantly in infective larvae and microfilariae but not in adult parasites. Images PMID:8168956

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

    PubMed

    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 characterizing of the murine IRF-3 gene promoter region.

    PubMed

    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

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

    PubMed Central

    Moraga, Ángela Rubio; Mozos, Almudena Trapero; Ahrazem, Oussama; Gómez-Gómez, Lourdes

    2009-01-01

    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. PMID:19695093

  11. Molecular cloning and characterization of procirsin, an active aspartic protease precursor from Cirsium vulgare (Asteraceae).

    PubMed

    Lufrano, Daniela; Faro, Rosário; Castanheira, Pedro; Parisi, Gustavo; Veríssimo, Paula; Vairo-Cavalli, Sandra; Simões, Isaura; Faro, Carlos

    2012-09-01

    Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well-known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards κ-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet. The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases. PMID:22727116

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

    PubMed

    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

  13. Cloning and characterization of a Loa loa-specific repetitive DNA.

    PubMed

    Egwang, T G; Ajuh, P M; Akue, J P

    1992-12-01

    A Loa loa EcoRI genomic library in lambda gt11 was screened with 32P-labeled L. loa DNA and 1 repetitive clone, LL20, was isolated. An 800-bp Rsa I fragment of LL20, which is L. loa specific, was subcloned into pUC19 and the recombinant plasmid was designated pRsa4. While the 3.8-kb Eco RI fragment of LL20 cross-hybridized to other filarial DNA under low stringency conditions, the 800-bp fragment of pRsa4 was L. loa specific under the same conditions. Further characterization of the insert of pRsa4 was therefore carried out. Its lower limit of detection is 800 pg of L. loa genomic DNA, it has a low copy number (50-100) and an interspersed distribution in the genome. As a probe it does not distinguish between simian and human L. loa DNA. The nucleotide sequence contains 69% A + T and 31% G + C and shows no notable internal repeats. PMID:1484545

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

    PubMed

    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

  15. Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

    PubMed Central

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

    2005-01-01

    Two xyloglucan-specific endo-β-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 β-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

  16. Cloning and characterization of a PI-like MADS-box gene in Phalaenopsis orchid.

    PubMed

    Guo, Bin; Hexige, Saiyin; Zhang, Tian; Pittman, Jon K; Chen, Donghong; Ming, Feng

    2007-11-30

    The highly evolved flowers of orchids have colorful sepals and fused columns that offer an opportunity to discover new genes involved in floral development in monocotyledon species. In this investigation, we cloned and characterized the homologous PISTALLATA-like (PI-like) gene PhPI15 (Phalaenopsis PI STILLATA # 15), from the Phalaenopsis hybrid cultivar. The protein sequence encoded by PhPI15 contains a typical PI-motif. Its sequence also formed a subclade with other monocot PI-type genes in phylogenetic analysis. Southern analysis showed that PhPI15 was present in the Phalaenopsis orchid genome as a single copy. Furthermore, it was expressed in all the whorls of the Phalaenopsis flower, while no expression was detected in vegetative organs. The flowers of transgenic tobacco plants ectopically expressing PhPI15 showed male-sterile phenotypes. Thus, as a Class-B MADS-box gene, PhPI15 specifies floral organ identity in orchids. PMID:18047777

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

    PubMed Central

    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

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

    PubMed

    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

  19. Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae.

    PubMed

    Satake, Ryoko; Ichiyanagi, Atsushi; Ichikawa, Keiichi; Hirokawa, Kozo; Araki, Yasuko; Yoshimura, Taro; Gomi, Keiko

    2015-11-01

    Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 s(-1) and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels. PMID:25912449

  20. Virulence Potential and Genome-Wide Characterization of Drug Resistant Streptococcus pneumoniae Clones Selected In Vivo by the 7-Valent Pneumococcal Conjugate Vaccine

    PubMed Central

    Frazão, Nelson; Hiller, N. Luisa; Powell, Evan; Earl, Josh; Ahmed, Azad; Sá-Leão, Raquel; de Lencastre, Hermínia; Ehrlich, Garth D.; Tomasz, Alexander

    2013-01-01

    We used mouse models of pneumococcal colonization and disease combined with full genome sequencing to characterize three major drug resistant clones of S. pneumoniae that were recovered from the nasopharynx of PCV7-immunized children in Portugal. The three clones – serotype 6A (ST2191), serotype 15A (ST63) and serotype 19A (ST276) carried some of the same drug resistance determinants already identified in nasopharyngeal isolates from the pre-PCV7 era. The three clones were able to colonize efficiently the mouse nasopharyngeal mucosa where populations of these pneumococci were retained for as long as 21 days. During this period, the three clones were able to asymptomatically invade the olfactory bulbs, brain, lungs and the middle ear mucosa and established populations in these tissues. The virulence potential of the three clones was poor even at high inoculum (105 CFU per mouse) concentrations in the mouse septicemia model and was undetectable in the pneumonia model. Capsular type 3 transformants of clones 6A and 19A prepared in the laboratory produced lethal infection at low cell concentration (103 CFU per mouse) but the same transformants became impaired in their potential to colonize, indicating the importance of the capsular polysaccharide in both disease and colonization. The three clones were compared to the genomes of 56 S. pneumoniae strains for which sequence information was available in the public databank. Clone 15A (ST63) only differed from the serotype 19F clone G54 in a very few genes including serotype so that this clone may be considered the product of a capsular switch. While no strain with comparable degree of similarity to clone 19A (ST276) was found among the sequenced isolates, by MLST this clone is a single locust variant (SLV) of Denmark14-ST230 international clone. Clone 6A (ST2191) was most similar to the penicillin resistant Hungarian serotype 19A clone. PMID:24069360

  1. Sodium-dependent methotrexate carrier-1 is expressed in rat kidney: cloning and functional characterization.

    PubMed

    Kneuer, Carsten; Honscha, Kerstin U; Honscha, Walther

    2004-03-01

    Previous Northern blot studies suggested strong expression of a homolog to the sodium-dependent hepatocellular methotrexate transporter in the kidneys. Here, we report on the cloning of the cDNA for the renal methotrexate carrier isoform-1 (RK-MTX-1) and its functional characterization. Sequencing revealed 97% homology to the rat liver methotrexate carrier with an identical open reading frame. Differences were located in the 5'-untranslated region and resulted in the absence of putative regulatory elements (Barbie box, Ah/ARNT receptor) identified in the cDNA for the hepatocellular carrier. For functional characterization, MTX-1 cDNA was stably expressed in Madin-Darby canine kidney (MDCK) cells. A sodium-dependent transport of methotrexate with a K(m) of 41 microM and a V(max) of 337 pmol.mg protein(-1).min(-1) was observed. This uptake was blocked by the reduced folates dihydro- and tetrahydrofolate as well as by methotrexate itself. Folate was inhibiting only weakly, whereas 5-methyltetrahydrofolate was a strong inhibitor. Further inhibitors of the methotrexate transport included the bile acids cholate and taurocholate and xenobiotics like bumetanide and BSP. PAH, ouabain, bumetanide, cholate, taurocholate, and acetyl salicylic acid were tested as potential substrates. However, none of these substances was transported by MTX-1. Furthermore, expression of RK-MTX-1 in MDCK cells enhanced methotrexate toxicity in these cells fivefold. Analysis of a fusion protein of RK-MTX-1 and the influenza virus hemagglutinin epitope by immunoblotting revealed a major band at 72 kDa within the cell membrane but not in the soluble fraction of transfected MDCK. Indirect immunofluorescence staining revealed an exclusive localization of the carrier in the plasma membrane, and by confocal laser-scanning microscopy we were able to demonstrate that the protein is expressed in the serosal region of MDCK tubules grown in a morphogenic collagen gel model. PMID:14612385

  2. Cloning and pharmacological characterization of the dog P2X7 receptor

    PubMed Central

    Roman, S; Cusdin, FS; Fonfria, E; Goodwin, JA; Reeves, J; Lappin, SC; Chambers, L; Walter, DS; Clay, WC; Michel, AD

    2009-01-01

    Background and purpose: Human and rodent P2X7 receptors exhibit differences in their sensitivity to antagonists. In this study we have cloned and characterized the dog P2X7 receptor to determine if its antagonist sensitivity more closely resembles the human or rodent orthologues. Experimental approach: A cDNA encoding the dog P2X7 receptor was isolated from a dog heart cDNA library, expressed in U-2 OS cells using the BacMam viral expression system and characterized in electrophysiological, ethidium accumulation and radioligand binding studies. Native P2X7 receptors were examined by measuring ATP-stimulated interleukin-1β release in dog and human whole blood. Key results: The dog P2X7 receptor was 595 amino acids long and exhibited high homology (>70%) to the human and rodent orthologues although it contained an additional threonine at position 284 and an amino acid deletion at position 538. ATP possessed low millimolar potency at dog P2X7 receptors. 2′-&3′-O-(4benzoylbenzoyl) ATP had slightly higher potency but was a partial agonist. Dog P2X7 receptors possessed relatively high affinity for a number of selective antagonists of the human P2X7 receptor although there were some differences in potency between the species. Compound affinities in human and dog blood exhibited a similar rank order of potency as observed in studies on the recombinant receptor although absolute potency was considerably lower. Conclusions and implications: Dog recombinant and native P2X7 receptors display a number of pharmacological similarities to the human P2X7 receptor. Thus, dog may be a suitable species for assessing target-related toxicity of antagonists intended for evaluation in the clinic. PMID:19814727

  3. Production, characterization, cloning and sequence analysis of a monofunctional catalase from Serratia marcescens SYBC08.

    PubMed

    Zeng, Hua-Wei; Cai, Yu-Jie; Liao, Xiang-Ru; Zhang, Feng; Zhang, Da-Bing

    2011-04-01

    A monofunctional catalase from Serratia marcescens SYBC08 produced by liquid state fermentation in 7 liter fermenter was isolated and purified by ammonium sulfate precipitation (ASP), ion exchange chromatography (IEC), and gel filtration (GF) and characterized. Its sequence was analyzed by LC-MS/MS technique and gene cloning. The highest catalase production (20,289 U · ml(-1)) was achieved after incubation for 40 h. The purified catalase had an estimated molecular mass of 230 kDa, consisting of four identical subunits of 58 kDa. High specific activity of the catalase (199,584 U · mg(-1) protein) was 3.44 times higher than that of Halomonas sp. Sk1 catalase (57,900 U · mg(-1) protein). The enzyme without peroxidase activity was found to be an atypical electronic spectrum of monofunctional catalase. The apparent K(m) and V(max) were 78 mM and 188, 212 per µM H(2) O(2) µM heme(-1) s(-1), respectivly. The enzyme displayed a broad pH activity range (pH 5.0-11.0), with optimal pH range of 7.0-9.0: It was most active at 20 °C and had 78% activity at 0 °C. Its thermo stability was slightly higher compared to that of commercial catalase from bovine liver. LC-MS/MS analysis confirmed that the deduced amino acid sequence of cloning gene was the catalase sequence from Serratia marcescens SYBC08. The sequence was compared with that of 23 related catalases. Although most of active site residues, NADPH-binding residues, proximal residues of the heme, distal residues of the heme and residues interacting with a water molecule in the enzyme were well conserved in 23 related catalases, weakly conserved residues were found. Its sequence was closely related with that of catalases from pathogenic bacterium in the family Enterobacteriaceae. This result imply that the enzyme with high specific activity plays a significant role in preventing those microorganisms of the family Enterobacteriaceae against hydrogen peroxide resulted in cellular damage. Calalase yield by Serratia

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

    PubMed Central

    Tóth, Ákos; Barna, Terézia; Szabó, Erna; Elek, Rita; Hubert, Ágnes; Nagy, István; Nagy, István; Kriszt, Balázs; Táncsics, András; Kukolya, József

    2016-01-01

    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

  5. Should the World Stop Cloning Around? 12th Grade Lesson. Schools of California Online Resources for Education (SCORE): Connecting California's Classrooms to the World.

    ERIC Educational Resources Information Center

    MacDonald, David R.; Karayan, Michael

    This lesson for grade 12 is designed to raise student awareness of the potential of human cloning and of the effects it could have on the present, naturally born population. Students work in teams to research the issue and are provided with background information, detailed instructions, on-line resources, and reflection questions. The teacher's…

  6. Characterization of sphere-forming HCT116 clones by whole RNA sequencing

    PubMed Central

    Chung, Eunkyung; Oh, Inkyung

    2016-01-01

    Purpose To determine CD133+ cells defined as cancer stem cells (CSCs) in colon cancer, we examined whether CD133+ clones in HCT116 demonstrate known features of CSCs like sphere-forming ability, chemodrug-resistance, and metastatic potential. Methods Magnetic cell isolation and cell separation demonstrated that <1% of HCT116 cells expressed CD133, with the remaining cells being CD133- clones. In colon cancer cells, radioresistance is also considered a CSC characteristic. We performed clonogenic assay using 0.4 Gy γ-irradiation. Results Interestingly, there were no differences between HCT116 parental and HCT116 CD133+ clones when the cells comprised 0.5% of the total cells, and CD133- clone demonstrated radiosensitive changes compared with parental and CD133+ clones. Comparing gene expression profiles between sphere-forming and nonforming culture conditions of HCT116 subclones by whole RNA sequencing failed to obtain specific genes expressed in CD133+ clones. Conclusion Despite no differences of gene expression profiles in monolayer attached culture conditions of each clone, sphere-forming conditions of whole HCT116 subclones, parental, CD133+, and CD133- increased 1,761 coding genes and downregulated 1,384 genes related to CSCs self-renewal and survival. Thus, spheroid cultures of HCT116 cells could be useful to expand colorectal CSCs rather than clonal expansion depending on CD133 expressions. PMID:27073788

  7. Isolation and partial characterization of cDNA clone of human ceruloplasmin receptor.

    PubMed

    Sasina, L K; Tsymbalenko, N V; Platonova, N A; Puchkova, L V; Voronina, O V; Gyulikhandanova, N E; Gaitskhoki, V S

    2000-05-01

    An individual clone, presumably carrying a 3 bp fragment of ceruloplasmin receptor cDNA was isolated from the expression library of human placenta cDNA using polyclonal specific antibodies to ceruloplasmin receptors. EcoR1-hydrolysate of isolated DNA was cloned in a pTZ19 bacterial vector and sequenced in the forward and reverse direction. The comparison of the revealed sequence with known sequences of human genome revealed its high similarity to ceruloplasmin cDNA. PMID:10977961

  8. Cloning, characterization and expression analysis of coagulation factor II gene in grass carp (Ctenopharyngodon idella).

    PubMed

    Xu, B H; Chen, K J; Yao, Y B; Liu, Q L; Xiao, T Y; Su, J M; Peng, H Z

    2015-01-01

    Here, we characterized the structure and function of the coagulation factor II (FII) gene in grass carp and determined its role in coagulation mechanisms. The FII gene EST was obtained using a constructed splenic transcriptome database; the full-length FII gene sequence was obtained by 3' and 5' RACE. The open reading frame (ORF) of FII was cloned and the full-length gene was found to be 1718 bp, with an ORF of 1572 bp; the gene contained a 25 bp 5'-untranslated region (UTR) and 108 bp 3'-UTR. The ORF encoded 524 amino acids, including 74 alkaline amino acids (arginine and lysine) and 69 acidic amino acids (aspartic acid and glutamic acid). The theoretical pI was 6.22. The calculated instability index (II) was 39.81, indicating that FII was a stable protein; the half-life period was predicted to be approximately 30 h. Amino acid sequence comparisons indicated that grass carp FII showed most similarity (71%) to FII of Takifugu rubripes, followed by Oplegnathus fasciatus (48% similarity) and Larimichthys crocea (47% similarity). A real-time reverse transcription PCR analysis showed that under normal circumstances, FII was most highly expressed in the liver, followed by the gill, spleen, thymus, and head-kidney (P < 0.001). After injection of the grass carp reovirus 873 (GCRV873), the pattern of FII expression was significantly altered (P < 0.001); gene expression was high after injection, suggesting a response involving the initiation of the coagulation system and defense of the body in combination with the platelet and complement system. PMID:26535692

  9. Cloning, functional characterization and heterologous expression of TaLsi1, a wheat silicon transporter gene.

    PubMed

    Montpetit, Jonatan; Vivancos, Julien; Mitani-Ueno, Namiki; Yamaji, Naoki; Rémus-Borel, Wilfried; Belzile, François; Ma, Jian Feng; Bélanger, Richard R

    2012-05-01

    Silicon (Si) is known to be beneficial to plants, namely in alleviating biotic and abiotic stresses. The magnitude of such positive effects is associated with a plant's natural ability to absorb Si. Many grasses can accumulate as much as 10% on a dry weight basis while most dicots, including Arabidopsis, will accumulate less than 0.1%. In this report, we describe the cloning and functional characterization of TaLsi1, a wheat Si transporter gene. In addition, we developed a heterologous system for the study of Si uptake in plants by introducing TaLsi1 and OsLsi1, its ortholog in rice, into Arabidopsis, a species with a very low innate Si uptake capacity. When expressed constitutively under the control of the CaMV 35S promoter, both TaLsi1 and OsLsi1 were expressed in cells of roots and shoots. Such constitutive expression of TaLsi1 or OsLsi1 resulted in a fourfold to fivefold increase in Si accumulation in transformed plants compared to WT. However, this Si absorption caused deleterious symptoms. When the wheat transporter was expressed under the control of a root-specific promoter (a boron transporter gene (AtNIP5;1) promoter), a similar increase in Si absorption was noted but the plants did not exhibit symptoms and grew normally. These results demonstrate that TaLsi1 is indeed a functional Si transporter as its expression in Arabidopsis leads to increased Si uptake, but that this expression must be confined to root cells for healthy plant development. The availability of this heterologous expression system will facilitate further studies into the mechanisms and benefits of Si uptake. PMID:22351076

  10. Cloning, expression, purification and characterization of UMP kinase from Staphylococcus aureus.

    PubMed

    Hari Prasad, O; Nanda Kumar, Y; Reddy, O V S; Chaudhary, Abhijit; Sarma, P V G K

    2012-04-01

    Uridine monophosphate kinase (UMPK) an enzyme of de novo biosynthesis catalyses the formation of UDP and it is involved in cell wall and RNA biosynthesis. In the present study UMPK of Staphylococcus aureus ATCC12600 was characterized. Analysis of purified UMPK by gel filtration chromatography on Sephadex G-200 indicated a molecular weight of 150 kDa and exhibited monomeric form with molecular weight of 25 kDa in SDS-PAGE confirming homohexamer nature of UMPK in solution. The enzyme kinetics of UMPK showed K(m) of 2.80 ± 0.1 μM and Vmax 51.38 ± 1.39 μM of NADH/min/mg. The enzyme exhibited cooperative kinetics with ATP as substrate, as GTP decreased this cooperativity and increased affinity for ATP. The UMPK gene was amplified, sequenced (Accession number: FJ415072), cloned in pQE30 vector and overexpressed in Escherichia coli DH5α. The purified recombinant UMPK showed similar properties of native UMPK. The UMPK gene sequence showed complete homology with pyrH gene sequence of all S. aureus strains reported in the database, the 3D structure of S. aureus UMPK built from the deduced amino acid sequence was super imposed with human UMPK (PDB ID: 1TEV) to find out the structural identity using the MATRAS programme gave an RMSD value 4.24 Å indicating very low homology and extensive structural variations with human UMPK structure. Thus, UMPK may be a potential drug target in the development of antimicrobials. PMID:22528139

  11. Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus.

    PubMed

    Ma, Jun-Yan; Cao, Hai-Long; Tan, Hai-Dong; Hu, Xue-Jun; Liu, Wu-Jun; Du, Yu-Guang; Yin, Heng

    2016-01-01

    Inulinase is an enzyme that belongs to glycoside hydrolase family 32. It converts inulin into high-fructose syrups and fructoligosaccharides, both of which are widely used in pharmaceutical and food industries. In this study, the kcINU1 gene (GenBank accession number AF178979) encoding an exoinulinase was cloned from Kluyveromyces cicerisporus CBS4857 and expressed in Pichia pastoris X-33, yielding a maximum of 45.2 ± 0.6 U mL(-1) of inulinase activity of culture supernatant. The expressed inulinase was purified and characterized. The enzyme had an optimum temperature of 55 °C and an optimum pH of 4.5. It had a K m of 0.322 mM and a V max of 4317 μM min(-1) mg(-1) protein when inulin was used as a substrate. It retained nearly 90 % of the maximal activity after pre-incubation at 50 °C for 1 h or at pH ranging from 3.0 to 6.0 at 4 °C for 24 h, demonstrating that KcINU1 was stable at high temperature and low pH. Moreover, we constructed two KcINU1 mutants, Asp30Ala and Glu215Ala, by site-directed mutagenesis and confirmed via zymogram analysis that Asp-30 and Glu-215 of the enzyme were the catalytic active center. The present study has provided important information for understanding the catalytic mechanism of exoinulinase. PMID:26446826

  12. Cloning, expression, and characterization of thermophilic L-asparaginase from Thermococcus kodakarensis KOD1.

    PubMed

    Hong, Sung-Jun; Lee, Yun-Ha; Khan, Abdur Rahim; Ullah, Ihsan; Lee, Changhee; Park, Choi Kyu; Shin, Jae-Ho

    2014-06-01

    The present study demonstrates cloning, expression, and characterization of hyperthermostable L-asparaginase from Thermococcus kodakarensis KOD1 in Escherichia coli BLR(DE3). The recombinant 6× His-tagged protein L-asparaginase from T. kodakarensis (TkAsn), was purified to homogeneity by heat treatment followed by affinity chromatography using a nickel-nitrilotriacetic acid (Ni-NTA) column. The molecular mass of the purified enzyme was found to be approximately 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzymatic properties, such as optimum temperature and pH, were 90 °C and 8.0, respectively. Its appearent Km , Vmax , and Kcat values were 2.6 mM, 1121 µmol min(-1)  mg(-1) , and 694 S(-1) , respectively. The enzyme displayed high thermal stability at optimum temperature with an insignificant loss in enzymatic activity, retaining almost 90% of its activity over a time period of 32 h. The relative activity of the enzyme was significantly inhibited by the supplementation of Cu(2+) and Ni(2+) ions, while moderately inhibited by other ions. In contrast, Mg(2+) ions enhanced the relative activity compared to the control. The acrylamide contents in baked dough were reduced to sixty percent after treatment with recombinant TkAsn as compared to the untreated control. Results of the present study revealed that the enzyme was highly active at broader range of temperatures and pH, which reflect the potential of recombinant TkAsn in the food processing industry. In addition, the high thermal stability of the enzyme may facilitates its handling, storage, and transportation. PMID:24442710

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

    PubMed

    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. PMID:12846842

  14. Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici

    PubMed Central

    Feng, Bao Zhen; Li, Peiqian

    2014-01-01

    Laccases are blue copper oxidases (E.C. 1.10.3.2) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. A novel laccase gene pclac2 and its corresponding full-length cDNA were cloned and characterized from Phytophthora capsici for the first time. The 1683 bp full-length cDNA of pclac2 encoded a mature laccase protein containing 560 amino acids preceded by a signal peptide of 23 amino acids. The deduced protein sequence of PCLAC2 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. In order to achieve a high level secretion and full activity expression of PCLAC2, expression vector pPIC9K with the Pichia pastoris expression system was used. The recombinant PCLAC2 protein was purified and showed on SDS-PAGE as a single band with an apparent molecular weight ca. 68 kDa. The high activity of purified PCLAC2, 84 U/mL, at the seventh day induced with methanol, was observed with 2,2′-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate. The optimum pH and temperature for ABTS were 4.0 and 30 °C, respectively. The reported data add a new piece to the knowledge about P. Capsici laccase multigene family and shed light on potential function about biotechnological and industrial applications of the individual laccase isoforms in oomycetes. PMID:24948955

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Cloning and characterization of a new multi-stress inducible metallothionein gene in Tetrahymena pyriformis.

    PubMed

    Fu, Chengjie; Miao, Wei

    2006-06-01

    A new multi-stress-inducible metallothionein (MT) gene isoform has been cloned and characterized from the ciliate Tetrahymena pyriformis. Both the 5'- and 3'-UT regions of the Tp-MT2 gene are very different from the previously reported Tp-MT1 isoform in this organism and from other described MT genes in Tetrahymena pigmentosa and Tetrahymena thermophila. The putative protein sequence of Tp-MT2 contains cysteine clusters with characteristics of the typical Tetrahymena Cd-inducible MT genes. However, the sequence has a special feature of four intragenic tandem repeats within its first half, with a conserved structural pattern x(5/8)CCCx(6)CCx(6)CxCxNCxCCK. To investigate the transcriptional activities of both Tp-MT2 and Tp-MT1 genes toward heavy metals (Cd, Hg, Cu, Zn) and H(2)O(2), the mRNA levels of these two isoforms were evaluated by means of real-time quantitative PCR. Results showed that Tp-MT2 had a higher basal expression level than Tp-MT1 and both genes were induced by Cd, Hg, Cu, and Zn ions after short exposure (1h), although to different extents. Cd was the most effective metal inducer of both two isoforms, but the relative expression level of Tp-MT2 was much lower than that of Tp-MT1. Different expression patterns were also shown between the two genes when treated with Cd over a period of 24h. We suggest that TpMT-1 plays the role of a multi-inducible stress gene, while TpMT-2 may have a more specific function in basal metal homeostasis although it may have undergone a functional differentiation process. The putative functional significance and evolutionary mode of the TpMT-2 isoform are discussed. PMID:16621695

  17. Molecular cloning and characterization of crustin from mud crab Scylla paramamosain.

    PubMed

    Imjongjirak, Chanprapa; Amparyup, Piti; Tassanakajon, Anchalee; Sittipraneed, Siriporn

    2009-05-01

    Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In the present study, we report the identification and characterization of a crustin (CrusSp) from the hemocyte of mud crab, Scylla paramamosain using an expressed sequence tag (EST) and rapid amplification cDNA end (RACE) approaches. Analysis of the nucleotide sequence revealed seven different variances of the CrusSp cDNA in mud crab. The open reading frame encodes a protein of 111 amino acids with 21 residues signal sequence. The predicted molecular mass of the mature protein (90 amino acids) is 10.27 kDa with an estimated pI of 8.54. Analysis of the protein domain features indicated typical conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A neighbour-joining tree showed that S. paramamosain crustin is closely related to other crustin homologues, and displays the highest similarity to crustin antimicrobial peptide in shore crab Carcinus maenas. Four exons and three introns were identified within the 999 bp genomic DNA sequence of CrusSp. Tissue distribution analysis showed that CrusSp was highly expressed in hemocytes, gills, intestines and muscle but it was not expressed in hepatopancreas and eyestalks. To gain insight into the in vitro antimicrobial activities of CrusSp, the mature peptide coding region was cloned into E. coli for heterologous expression. The recombinant CrusSp could inhibit the growth of gram-positive bacteria but had no inhibition activity against gram-negative bacteria. These results indicated the involvement of CrusSp in the innate immunity of S. paramamosain. PMID:18425600

  18. Cloning and characterization of the genes encoding nitrilotriacetate monooxygenase of Chelatobacter heintzii ATCC 29600.

    PubMed Central

    Knobel, H R; Egli, T; van der Meer, J R

    1996-01-01

    A 6.2-kb DNA fragment containing the genes for the nitrilotriacetate (NTA) monooxygenase of Chelatobacter heintzii ATCC 29600 was cloned and characterized by DNA sequencing and expression studies. The nucleotide sequence contained three major open reading frames (ORFs). Two of the ORFs, which were oriented divergently with an intergenic region of 307 bp, could be assigned to the NTA monooxygenase components A and B. The predicted N-terminal amino acid sequences of these ORFs were identical with those determined for the purified components. We therefore named these genes ntaA (for component A of NTA monooxygenase) and ntaB (for component B). The ntaA and ntaB genes could be expressed in Escherichia coli DH5alpha, and the gene products were visualized after Western blotting (immunoblotting) and incubation with polyclonal antibodies against component A or B. By mixing overproduced NtaB from E. coli and purified component A from C. heintzii ATCC 29600, reconstitution of a functional NTA monooxygenase complex was possible. The deduced gene product of ntaA showed only significant homology to SoxA (involved in dibenzothiophene degradation) and to SnaA (involved in pristamycin synthesis); that of ntaB shared weak homologies in one domain with other NADH:flavine mononucleotide oxidoreductases. These homologies provide no conclusive answer as to the possible evolutionary origin of the NTA monooxygenase. The deduced gene product of the third ORF (ORF1) had homology in the N-terminal region with the GntR class of bacterial regulator proteins and therefore may encode a regulator protein, possibly involved in regulation of ntaA and ntaB expression. PMID:8892809

  19. Molecular cloning and functional characterization of an antifungal PR-5 protein from Ocimum basilicum.

    PubMed

    Rather, Irshad Ahmad; Awasthi, Praveen; Mahajan, Vidushi; Bedi, Yashbir S; Vishwakarma, Ram A; Gandhi, Sumit G

    2015-03-01

    Pathogenesis-related (PR) proteins are involved in biotic and abiotic stress responses of plants and are grouped into 17 families (PR-1 to PR-17). PR-5 family includes proteins related to thaumatin and osmotin, with several members possessing antimicrobial properties. In this study, a PR-5 gene showing a high degree of homology with osmotin-like protein was isolated from sweet basil (Ocimum basilicum L.). A complete open reading frame consisting of 675 nucleotides, coding for a precursor protein, was obtained by PCR amplification. Based on sequence comparisons with tobacco osmotin and other osmotin-like proteins (OLPs), this protein was named ObOLP. The predicted mature protein is 225 amino acids in length and contains 16 cysteine residues that may potentially form eight disulfide bonds, a signature common to most PR-5 proteins. Among the various abiotic stress treatments tested, including high salt, mechanical wounding and exogenous phytohormone/elicitor treatments; methyl jasmonate (MeJA) and mechanical wounding significantly induced the expression of ObOLP gene. The coding sequence of ObOLP was cloned and expressed in a bacterial host resulting in a 25kDa recombinant-HIS tagged protein, displaying antifungal activity. The ObOLP protein sequence appears to contain an N-terminal signal peptide with signatures of secretory pathway. Further, our experimental data shows that ObOLP expression is regulated transcriptionally and in silico analysis suggests that it may be post-transcriptionally and post-translationally regulated through microRNAs and post-translational protein modifications, respectively. This study appears to be the first report of isolation and characterization of osmotin-like protein gene from O. basilicum. PMID:25550044

  20. Cloning, characterization, and expression analysis of LGP2 cDNA from goose, Anser cygnoides.

    PubMed

    Wei, L; Song, Y; Cui, J; Qu, N; Wang, N; Ouyang, G; Liao, M; Jiao, P

    2016-10-01

    Laboratory of genetics and physiology 2 ( LGP2: ) is a homologue of the retinoic acid inducible gene-I and melanoma differentiation associated gene 5 that lacks the caspase activation and recruitment domain required for signaling. It plays a pivotal role in host immune response. In this study, we cloned and characterized the full-length open reading frame ( ORF: ) sequence of LGP2 in the Qingyuan goose (Anser cygnoides) and evaluated the mRNA expression of this gene post infection with an H5N1 highly pathogenic avian influenza virus ( HPAIV: ). The full-length goose LGP2 ORF (2,028 bp) encoded a polypeptide of 675 amino acids. The deduced amino acid sequence contained 5 main overlapping structural domains-2 DEAD/DEAH box helicase domains, one conserved restriction domain of bacterial type III restriction enzyme, one helicase superfamily C-terminal domain and one C-terminal regulatory domain. Quantitative real-time PCR analysis indicated that goose LGP2 was constitutively expressed in all 19 investigated tissues, but the expression level was different among them. It was high expressed in the trachea, jejunum, bursa, kidney and heart, but low in the glandular stomach, lung, liver, spleen, crop and muscular stomach. A significant increase in the transcription of LGP2 was detected in the brain, spleen and lungs of geese post infection with H5N1 HPAIV versus uninfected tissues. These findings indicated that goose LGP2 was an important receptor that is involved in the host antiviral innate immune defense to H5N1 HPAIV in geese. PMID:27143779

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

    PubMed

    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

  2. Molecular cloning and pharmacological characterization of rat multidrug resistance protein 1 (mrp1).

    PubMed

    Nunoya, Kenichi; Grant, Caroline E; Zhang, Dawei; Cole, Susan P C; Deeley, Roger G

    2003-08-01

    Multidrug resistance protein 1 (MRP1) transports a wide range of structurally diverse conjugated and nonconjugated organic anions and some peptides, including oxidized and reduced glutathione (GSH). The protein confers resistance to certain heavy metal oxyanions and a variety of natural product-type chemotherapeutic agents. Elevated levels of MRP1 have been detected in many human tumors, and the protein is a candidate therapeutic target for drug resistance reversing agents. Previously, we have shown that human MRP1 (hMRP1) and murine MRP1 (mMRP1) differ in their substrate specificity despite a high degree of structural conservation. Since rat models are widely used in the drug discovery and development stage, we have cloned and functionally characterized rat MRP1 (rMRP1). Like mMRP1 and in contrast to hMRP1, rMRP1 confers no, or very low, resistance to anthracyclines and transports the two estrogen conjugates, 17beta-estradiol-17-(beta-d-glucuronide) (E217betaG) and estrone 3-sulfate, relatively poorly. Mutational studies combined with vesicle transport assays identified several amino acids conserved between rat and mouse, but not hMRP1, that make major contributions to these differences in substrate specificity. Despite the fact that the rodent proteins transport E217betaG poorly and the GSH-stimulated transport of estrone 3-sulfate is low compared with hMRP1, site-directed mutagenesis studies indicate that different nonconserved amino acids are involved in the low efficiency with which each of the two estrogen conjugates is transported. Our studies also suggest that although rMRP1 and mMRP1 are 95% identical in primary structure, their substrate specificities may be influenced by amino acids that are not conserved between the two rodent proteins. PMID:12867490

  3. Molecular Cloning and Characterization of Taurocyamine Kinase from Clonorchis sinensis: A Candidate Chemotherapeutic Target

    PubMed Central

    Tokuhiro, Shinji; Nagataki, Mitsuru; Jarilla, Blanca R.; Nomura, Haruka; Kim, Tae Im; Hong, Sung-Jong; Agatsuma, Takeshi

    2013-01-01

    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. Methology/Principal findings 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, creatine. PMID:24278491

  4. Comparative study of two thioredoxins from common cutworm (Spodoptera litura): cloning, expression, and functional characterization.

    PubMed

    Kang, Tinghao; Wan, Hu; Zhang, Yashu; Shakeel, Muhammad; Lu, Yanhui; You, Hong; Lee, Kwang Sik; Jin, Byung Rae; Li, Jianhong

    2015-04-01

    Thioredoxins (Trxs) are a ubiquitous family of antioxidant enzymes that are involved in protecting organisms against various oxidative stresses. Here, we cloned and characterized two thioredoxins, named SlTrx1 and SlTrx2, from the common cutworm Spodoptera litura. SlTrx1 and SlTrx2, respectively, consist of 988 and 606 bp full-length cDNA with 318 and 447 bp open reading frames encoding 106 and 149 amino acid residues. Furthermore, the N-terminal region of SlTrx2 contains a predicted mitochondrial localization signal (33 amino acids). A phylogenetic relationship analysis revealed that SlTrx1 is in the cytosolic thioredoxin Trx1 cluster, whereas SlTrx2 is in the mitochondrial thioredoxin Trx2 cluster. Recombinant SlTrx1 (14 kDa) and SlTrx2 (16 kDa), expressed in baculovirus-infected insect Sf9 cells, demonstrated insulin disulfide reductase activity at the same optimum temperature and pH value of 35 °C and 7.0, respectively, in vitro. During S. litura development, we found that SlTrx1 and SlTrx2 had similar transcript expression patterns and were constitutively expressed in the epidermis, fat body, and midgut, with the highest expression occurring in the sixth-instar larval stage in the epidermis and midgut. In addition, both SlTrx1 and SlTrx2 mRNA were up-regulated in S. litura after injection with H2O2, cumene hydroperoxide, indoxacarb, and metaflumizone. These results suggest that SlTrx1 and SlTrx2 function as potent antioxidant enzymes, and provide a molecular basis for the roles SlTrx1 and SlTrx2 during development and the oxidative stress response of S. litura. PMID:25542738

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

    PubMed

    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

  6. Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1.

    PubMed

    Saa, Laura; Jaureguibeitia, Arrate; Largo, Eneko; Llama, María J; Serra, Juan L

    2010-03-01

    Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His(6)PheA1 and His(6)PheA2 were purified and its catalytic activity characterized. His(6)PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His(6)PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His(6)PheA1 and His(6)PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction. PMID:19787347

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

    PubMed

    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

  8. Cloning and characterization of a SnRK2 gene from Jatropha curcas L.

    PubMed

    Chun, J; Li, F-S; Ma, Y; Wang, S-H; Chen, F

    2014-01-01

    Although the SnRK2 class of Ser/Thr protein kinases is critical for signal transduction and abiotic stress resistance in plants, there have been no studies to examine SnRK2 in Jatropha curcas L. In the present study, JcSnRK2 was cloned from J. curcas using the rapid amplification of cDNA end technique and characterized. The JcSnRK2 genomic sequence is 2578 base pairs (bp), includes 10 exons and 9 introns, and the 1017-bp open reading frame encodes 338 amino acids. JcSnRK2 was transcribed in all examined tissues, with the highest transcription rate observed in the roots, followed by the leaves and stalks; the lowest rate was observed in flowers and seeds. JcSnRK2 expression increased following abscisic acid treatment, salinity, and drought stress. During a 48-h stress period, the expression of JcSnRK2 showed 2 peaks and periodic up- and downregulation. JcSnRK2 was rapidly activated within 1 h under salt and drought stress, but not under cold stress. Because of the gene sequence and expression similarity of JcSnRK2 to AtSnRK2.8, primarily in the roots, an eukaryotic expression vector containing the JcSnRK2 gene (pBI121-JcSnRK2) was constructed and introduced to the Arabidopsis AtSnRK2.8 mutant snf2.8. JcSnRK2-overexpressing plants exhibited higher salt and drought tolerance, further demonstrating the function of JcSnRK2 in the osmotic stress response. J. curcas is highly resistant to extreme salt and drought conditions and JcSnRK2 was found to be activated under these conditions. Thus, JcSnRK2 is potential candidate for improving crop tolerance to salt and drought stress. PMID:25526217

  9. Molecular cloning, characterization, and expression of Cuc m 2, a major allergen in Cucumis melo

    PubMed Central

    Sankian, Mojtaba; Mahmoudi, Mahmoud; Varasteh, Abdol-Reza

    2013-01-01

    Background: Several studies reported the clinical features of IgE-mediated hypersensitivity after ingestion of melon. Melon allergy is a common IgE-mediated fruit allergy in Iran. This prompted us to investigate immunochemical and molecular properties of the major allergen in melon fruit, to compare the IgE-binding capacity of the natural protein with the recombinant allergen, and to determine cross-reactivity of the major allergen with closely-related allergens from other plants displaying clinical cross-reactivity with melon. Methods: Identification and molecular characterization of the major melon allergen were performed using IgE immunoblotting, allergen-specific ELISA, affinity-based purifications, cross-inhibition assays, cloning, and expression of the allergen in Escherichia coli. Results: Melon profilin was identified and isolated as a major IgE-binding component and designated as Cuc m 2. Sequencing corresponding cDNA revealed an open reading frame of 363 bp coding for 131 amino acid residues and two fragments of 171 bp and 383 bps for the 5’and 3’ UTRs, respectively. Significant cross-reactivity was found between melon profilin and Cynodon dactylon, tomato, peach, and grape profilins in cross-inhibition assays. Although the highest degree of amino acid identity was revealed with watermelon profilin, there was no significant cross-reactivity between melon and watermelon profilins. Conclusion: Melon profilin is the major IgE-binding component in melon extract, and the recombinant and natural forms exhibited similar IgE-binding capacities. A part of the fruit-fruit and pollen-fruit cross-reactions could be explained by the presence of this conserved protein; however, sequence homology provides insufficient information to predict IgE cross-reactivity of profilins. PMID:26989709

  10. [Cloning, prokaryotic expression and characterization of lysine decarboxylase gene from Huperzia serrata].

    PubMed

    Di, Ci; Li, Jing; Tang, Yuntao; Peng, Qingzhong

    2014-08-01

    Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures. PMID:25423760

  11. [Cloning, prokaryotic expression and characterization of lysine decarboxylase gene from Huperzia serrata].

    PubMed

    Di, Ci; Li, Jing; Tang, Yuntao; Peng, Qingzhong

    2014-08-01

    Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures. PMID:25507483

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

    PubMed

    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

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

    NASA Technical Reports Server (NTRS)

    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. Purification and partial characterization of the multicomponent dextranase complex of Streptococcus sobrinus and cloning of the dextranase gene.

    PubMed Central

    Barrett, J F; Barrett, T A; Curtiss, R

    1987-01-01

    The presence of proteases in culture supernatant fluids and on the cell surface of Streptococcus sobrinus and the aggregation of multicomponent enzyme complexes make the isolation and characterization of cell surface proteins difficult. We report a simple purification procedure for dextranase and the cloning of the dextranase structural gene. S. sobrinus culture supernatant fluids were precipitated with 70% ammonium sulfate, and the precipitate was dialyzed against sodium acetate buffer and loaded onto a hemoglobin-Sepharose 4B column connected to a blue dextran-agarose column at 4 degrees C. After being washed with low concentrations of salt, the dextranase and the dextran-binding proteins were eluted with 5 M KI and further purified by gel filtration. Two dextranases (molecular weights, 175,000 and 160,000) were purified and partially characterized. The structural gene for the dextranase of S. sobrinus 6715 strain UAB66, serotype g, was cloned into the cosmid vector, pHC79. Clones were selected for expression of dextranase activity by detection of zones of enzyme-mediated hydrolysis of a blue dextran substrate incorporated into minimal medium agar plates. Release of dextranase was achieved by induction of thermoinducible, excision-defective Escherichia coli K-12 lysogens containing recombinant cosmid molecules of S. sobrinus DNA. Recombinant cosmid molecules were repackaged simultaneously into infectious lambdoid particles. Recombinant clones expressing dextranase activity which varied in size from the high-molecular-weight protein produced by S. sobrinus (i.e., 175,000) to lower-molecular-weight forms expressed by S. sobrinus have been identified and partially characterized. Images PMID:3546141

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

  17. Gas-Fired Distributed Energy Resource Technology Characterizations

    SciTech Connect

    Goldstein, L.; Hedman, B.; Knowles, D.; Freedman, S. I.; Woods, R.; Schweizer, T.

    2003-11-01

    The U. S. Department of Energy (DOE) Office of Energy Efficiency and Renewable Energy (EERE) is directing substantial programs in the development and encouragement of new energy technologies. Among them are renewable energy and distributed energy resource technologies. As part of its ongoing effort to document the status and potential of these technologies, DOE EERE directed the National Renewable Energy Laboratory to lead an effort to develop and publish Distributed Energy Technology Characterizations (TCs) that would provide both the department and energy community with a consistent and objective set of cost and performance data in prospective electric-power generation applications in the United States. Toward that goal, DOE/EERE - joined by the Electric Power Research Institute (EPRI) - published the Renewable Energy Technology Characterizations in December 1997.As a follow-up, DOE EERE - joined by the Gas Research Institute - is now publishing this document, Gas-Fired Distributed Energy Resource Technology Characterizations.

  18. Molecular characterization of methicillin-resistant Staphylococcus aureus: characterization of major clones and emergence of epidemic clones of sequence type (ST) 36 and ST 121 in Tehran, Iran.

    PubMed

    Ohadian Moghadam, Solmaz; Pourmand, Mohammad Reza; Mahmoudi, Mahmood; Sadighian, Hooman

    2015-04-01

    Information about the molecular structure of MRSA strains provides significant insights into the epidemiology of this important pathogen. To investigate the molecular characteristics of MRSA isolates, MRSA isolates were subjected to molecular typing by means of spa typing, multilocus sequence typing, Staphylococcal Cassette Chromosome mec (SCCmec) grouping and to phenotypic antimicrobial susceptibility testing by means of disk diffusion assay. Then the presence of pvl genes was evaluated. Cluster analysis by eBURSTv3 showed that MRSA isolates belonged to two major clonal complexes (CC); CC8 (ST239, ST585, ST2732, ST1294) and CC30 (ST30, ST36, ST1163) and four singletons. Subsequent analysis of MRSA isolates revealed that the most prevalent SCCmec type was type III (55.8%) followed by type IV (34.9%) and type II (2.3%). Totally 11 different spa types were discriminated among which types t037 and t030 were predominant. The prevalence of Panton-Valentine leukocidin (PVL)-positive MRSA strains was high (20%), which is a matter of great concern, because the PVL is frequently associated with severe and recurrent SSTIs. ST239-III- t037 represented the most predominant MRSA clone. The presence of sequence type (ST) 36 and ST 121 are being reported for the first time in Iran. PMID:25795589

  19. 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.

    PubMed

    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. PMID:26505312

  20. Characterization of in vivo mutated T cell clones from patients with systemic lupus erythematosus.

    PubMed

    Theocharis, S; Sfikakis, P P; Lipnick, R N; Klipple, G L; Steinberg, A D; Tsokos, G C

    1995-02-01

    Patients with systemic lupus erythematosus (SLE) have increased percentages of activated T cells and increased numbers of cells with mutations in their hypoxanthineguanine phosphoribosyltransferase (hprt) gene, as judged by growth in the presence of 6-thioguanine. To study the relevance of these mutant T cells to disease pathogenesis, we have assessed the phenotype and functional capabilities of such cells from 21 patients with SLE who never had received cytotoxic drugs. The frequency of T cells with mutations in hprt in the blood of these patients ranged from normal to 25 times normal (mean +/- SEM [21.1 +/- 6.1] x 10(-6) versus [4.8 +/- 0.8] x 10(-6), in 15 age-matched normal individuals, P < 0.001) and correlated significantly with disease duration. CD4+ and CD8+ phenotypes were comparable among mutated and nonmutated clones from both patients and normals. Although the frequency of CD3+CD4-CD8- cells was low, it was increased among SLE-derived T cells (mutated and wild-type) compared with clones derived from normals (5% for SLE vs 1% for normals). A substantial percentage of all clones were able to help autologous B cells to produce anti-ssDNA, 11 of 68 (16%) selected clones and 3 of 28 (11%) nonselected clones. Help for autoantibody production was confined to CD4+ SLE-derived T cell clones. It could be blocked using an anti-HLA-DR mAb, suggesting that classical cognate help was operative. This represents the first estimate of the frequency of T cells able to drive autoantibody production in SLE. PMID:7828367

  1. Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

    PubMed

    Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

    2015-10-01

    A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time. PMID:25817696

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

    PubMed

    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

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

    PubMed Central

    Karnaouri, Anthi; Paschos, Thomas; Taouki, Ioanna; Christakopoulos, Paul

    2013-01-01

    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. PMID:23638383

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed Central

    Zhou, Tian-Shan; Zhou, Rui; Yu, You-Ben; Xiao, Yao; Li, Dong-Hua; Xiao, Bin; Yu, Oliver; Yang, Ya-Jun

    2016-01-01

    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. PMID:26907264

  7. Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110

    PubMed Central

    2010-01-01

    Background Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110. Results The gene encoding a novel ER was isolated from an osmophilic yeast C. magnoliae JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from Trichosporonoides megachiliensis SNG-42. The intact coding region of ER from C. magnoliae JH110 was cloned, functionally expressed in Escherichia coli using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42°C and 5.5, respectively. Among various aldoses, the C. magnoliae JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme

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

    SciTech Connect

    Midha, Shuchi; Mishra, Rajeev; Aziz, M.A.; Sharma, Meenakshi; Mishra, Ashish; Khandelwal, Puneet; Bhatnagar, Rakesh . E-mail: rakbhat01@yahoo.com

    2005-10-14

    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 {sup {omega}}-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

  9. Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus.

    PubMed

    Ewis, Hosam E; Abdelal, Ahmed T; Lu, Chung-Dar

    2004-03-31

    Screening of the genomic libraries of Geobacillus stearothermophilus ATCC12980 and ATCC7954 for esterase/lipase activity led to the isolation of two positive clones. The results of subclonings and sequence analyses identified two genes, est30 and est55, encoding two different carboxylesterases, and genetic rearrangement in the est55 locus was revealed from genomic comparison. The est30 gene encodes a polypeptide of 248 amino acids with a calculated molecular mass of 28338 Da, and the est55 gene encodes a polypeptide of 499 amino acids with a calculated molecular mass of 54867 Da. Both enzymes were purified to near homogeneity from recombinant strains of Escherichia coli. The results of enzyme characterization showed that while both enzymes possess optimal activities with short chain acyl derivatives, Est55 has a broader pH tolerance (pH 8-9) and optimal temperature range (30-60 degrees C) than Est30. The activation energy of Est55 (35.7 kJ/mol) was found to be significantly lower than that of Est30 (101.9 kJ/mol). Both enzymes were stable at 60 degrees C for more than 2 h; at 70 degrees C, the half-life for thermal inactivation was 40 and 180 min for Est55 and Est30, respectively. With p-nitrophenyl caproate as the substrate and assayed at 60 degrees C, Est55 had K(m) and k(cat) values of 0.5 microM and 39758 s(-1) while Est30 exhibited values of 2.16 microM and 38 s(-1). Inhibition studies indicated that both Est30 and Est55 were strongly inhibited by phenylmethanesulfonyl fluoride, p-hydroxymercuribenzoate, and tosyl-l-phenylalanine, consistent with the proposed presence of Ser-His-Glu catalytic triad of the alpha/beta hydrolase family. The enzymatic properties of Est30 and Est55 reported here warrant the potential applications of these enzymes in biotechnological industries. PMID:15033540

  10. CLONING AND CHARACTERIZATION OF CDNA ENCODING GIARDIA LAMBLIA d-GIARDIN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed

    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

  12. Cloning, Expression and Characterization of a Glycoside Hydrolase Family 39 Xylosidase from Bacillus Halodurans C-125

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene encoding a glycoside hydrolase family 39 xylosidase (BH1068) from the alkaliphile Bacillus halodurans strain C-125 was cloned with a C-terminal His-tag and the recombinant gene product termed XylBH1068 was expressed in E. coli. Of the artificial substrates tested, XylBH1068 hydrolyzed nitro...

  13. Cloning, sequencing and characterization of lipase genes from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipase (lip) and lipase-specific foldase (lif) genes of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans NRRL B-2649 were cloned using primers based on consensus sequences, followed by PCR-based genome walking. Sequence analyses showed a putative Lip gene-product (...

  14. Cloning and Characterization of a Putative GS3 Ortholog Involved in Maize Kernel Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A single nucleotide polymorphism (SNP) in the second exon of GS3 is the causal mutation for grain size and weight variation in rice. This gene has been cloned and is hypothesized to be involved in the evolution of grain size during domestication. In this study, we isolated the maize homolog, ZmGS3, ...

  15. Cloning and characterization of an alpha-glucuronidase from a mixed microbial population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alpha-Glucuronidase enzymes play an essential role in the full enzymatic hydrolysis of hemicellulose. Up to this point, all genes encoding alpha-glucuronidase enzymes have been cloned from individual, pure culture strains. Using a high-throughput screening strategy, we have isolated the first alph...

  16. Molecular cloning and functional characterization of the diapause hormone receptor in the corn earworm Helicoverpa zea

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The diapause hormone (DH) in the heliothine moth has shown its activity in termination of pupal diapause, while the orthology in the silkworm is known to induce embryonic diapause. In the current study, we cloned the diapause hormone receptor from the corn earworm Helicoverpa zea (HzDHr) and tested ...

  17. Cloning and characterization of a recombinant family 5 endoglucanase from Bacillus licheniformis strain B-41361

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene encoding a family 5 endoglucanase, cel5A, was cloned from the moderate thermophile Bacillus licheniformis strain B-41361. The primary structure of the translated cel5A gene predicts a 49 amino acid putative secretion signal and a 485 residue endoglucanase consisting of an N-terminal family...

  18. Resources

    MedlinePlus

    ... 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 - ...

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

    PubMed Central

    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 purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound role in the pathogenesis. PMID:23519063

  20. Development and characterization of an in vivo pathogenic molecular clone of equine infectious anemia virus.

    PubMed

    Cook, R F; Leroux, C; Cook, S J; Berger, S L; Lichtenstein, D L; Ghabrial, N N; Montelaro, R C; Issel, C J

    1998-02-01

    An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAV(PV)) of the cell culture-adapted strain of EIAV (EIAV(PR)). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of tat), S2, and S3 (encoding the second exon of rev) open reading frames, the complete env gene (including the first exon of rev), and the 3' long terminal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAV(PV3.3), and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). As a consequence of this initial study, a single molecular clone, EIAV(PV3.3#3) (redesignated EIAV(UK)), was selected for further study and inoculated into two ponies (613 and 614) and two horses (700 and 764). Pony 614 and the two horses developed febrile responses by 12 dpi, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6 degrees C) until 76 dpi. EIAV could be isolated from the plasma of these animals by 5 to 7 dpi, and all became seropositive for antibodies to this virus by 21 dpi. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3' fragment of EIAV(UK) differed from the consensus sequence of EIAV(PV) by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAV(PV) consensus sequence was observed in the hypervariable region of the LTR. However, EIAV(UK) was found to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstrate that substitution of a 3.3-kbp fragment from the EIAV(PV) strain into the infectious nonpathogenic

  1. Cloning and Characterization of an Intracellular Esterase from the Wine-Associated Lactic Acid Bacterium Oenococcus oeni▿ †

    PubMed Central

    Sumby, Krista M.; Matthews, Angela H.; Grbin, Paul R.; Jiranek, Vladimir

    2009-01-01

    We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium, Oenococcus oeni. The published sequence for O. oeni strain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned into Escherichia coli using an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 with p-nitrophenyl (pNP)-linked substrates ranging in chain length from C2 to C18. EstB28 exhibited greatest specificity for C2 to C4 pNP-linked substrates. PMID:19734337

  2. Characterization of cDNA clones for the human c-yes gene.

    PubMed Central

    Sukegawa, J; Semba, K; Yamanashi, Y; Nishizawa, M; Miyajima, N; Yamamoto, T; Toyoshima, K

    1987-01-01

    Three c-yes cDNA clones were obtained from poly(A)+ RNA of human embryo fibroblasts. Sequence analysis of the clones showed that they contained inserts corresponding to nearly full-length human c-yes mRNA, which could encode a polypeptide of 543 amino acids with a relative molecular weight (Mr) of 60,801. The predicted amino acid sequence of the protein has no apparent membrane-spanning region or suspected ligand binding domain and closely resembles pp60c-src. Comparison of the sequences of c-yes and v-yes revealed that the v-yes gene contains most of the c-yes coding sequence except the region encoding its extreme carboxyl terminus. The region missing from the v-yes protein is the part that is highly conserved in cellular gene products of the protein-tyrosine kinase family. PMID:2436037

  3. Molecular characterization of cloned variants of Coxiella burnetii isolated in China.

    PubMed

    Ning, Z; Yu, S R; Quan, Y G; Xue, Z

    1992-03-01

    To study the molecular properties of Coxiella burnetii phase variants we cloned the phase variants of C. burnetii Qiyi (CBQY) strain by the red plaque technique. Three cloned strains, CBQYIC3 (phase I), CBQYIIC7 (phase II) and CBQYIIC5 (semirough-phase) were analysed by SDS-PAGE, immunoblot assay, plasmid isolation and agarose gel electrophoresis of DNA restriction fragments. The results suggest that the unique phase-dependent substance is a lipopolysaccharide and that most protein components of phase I and phase II cells are shared. No significant differences of DNA restriction fragments were found between clonal isolates of phase I and phase II C. burnetii CBQY strains. A plasmid of approximately 56 Kb was isolated from both phase I and phase II variants indicating that phase variation probably could not be attributed to its presence or absence. PMID:1359769

  4. Cloning and characterization of the Bacillus licheniformis gene coding for alkaline phosphatase.

    PubMed Central

    Hulett, F M

    1984-01-01

    The structural gene for alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1) of Bacillus licheniformis MC14 was cloned into the Pst1 site of pMK2004 from chromosomal DNA. The gene was cloned on an 8.5-kilobase DNA fragment. A restriction map was developed, and the gene was subcloned on a 4.2-kilobase DNA fragment. The minimum coding region of the gene was localized to a 1.3-kilobase region. Western blot analysis was used to show that the gene coded for a 60,000-molecular-weight protein which cross-reacts with anti-alkaline phosphatase prepared against the salt-extractable membrane alkaline phosphatase of B. licheniformis MC14 . Images PMID:6327655

  5. Cloning and characterization of the Bacillus licheniformis gene coding for alkaline phosphatase.

    PubMed

    Hulett, F M

    1984-06-01

    The structural gene for alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1) of Bacillus licheniformis MC14 was cloned into the Pst1 site of pMK2004 from chromosomal DNA. The gene was cloned on an 8.5-kilobase DNA fragment. A restriction map was developed, and the gene was subcloned on a 4.2-kilobase DNA fragment. The minimum coding region of the gene was localized to a 1.3-kilobase region. Western blot analysis was used to show that the gene coded for a 60,000-molecular-weight protein which cross-reacts with anti-alkaline phosphatase prepared against the salt-extractable membrane alkaline phosphatase of B. licheniformis MC14 . PMID:6327655

  6. Cloning and characterization of the gene for Escherichia coli seryl-tRNA synthetase.

    PubMed Central

    Härtlein, M; Madern, D; Leberman, R

    1987-01-01

    Seryl-tRNA synthetase is the gene product of the serS locus in Escherichia coli. Its gene has been cloned by complementation of a serS temperature sensitive mutant K28 with an E. coli gene bank DNA. The resulting clones overexpress seryl-tRNA synthetase by a factor greater than 50 and more than 6% of the total cellular protein corresponds to the enzyme. The DNA sequence of the complete coding region and the 5'- and 3' untranslated regions was determined. Protein sequence comparison of SerRS with all available aminoacyl-tRNA synthetase sequences revealed some regions of significant homology particularly with the isoleucyl- and phenylalanyl-tRNA synthetases from E. coli. Images PMID:3029694

  7. Functional Cloning and Characterization of Antibiotic Resistance Genes from the Chicken Gut Microbiome

    PubMed Central

    Zhou, Wei; Wang, Ying

    2012-01-01

    Culture-independent sampling in conjunction with a functional cloning approach identified diverse antibiotic resistance genes for different classes of antibiotics in gut microbiomes from both conventionally raised and free-range chickens. Many of the genes are phylogenetically distant from known resistance genes. Two unique genes that conferred ampicillin and spectinomycin resistance were also functional in Campylobacter, a distant relative of the Escherichia coli host used to generate the genomic libraries. PMID:22286984

  8. Molecular and biological characterization of a neurovirulent molecular clone of simian immunodeficiency virus.

    PubMed Central

    Flaherty, M T; Hauer, D A; Mankowski, J L; Zink, M C; Clements, J E

    1997-01-01

    To identify the molecular determinants of neurovirulence, we constructed an infectious simian immunodeficiency virus (SIV) molecular clone, SIV/17E-Fr, that contained the 3' end of a neurovirulent strain of SIV, SIV/17E-Br, derived by in vivo virus passage. SIV/17E-Fr is macrophage tropic in vitro and neurovirulent in macaques. In contrast, a molecular clone, SIV/17E-Cl, that contains the SU and a portion of the TM sequences of SIV/17E-Br is macrophage tropic but not neurovirulent. To identify the amino acids that accounted for the replication differences between SIV/17E-Fr and SIV/17E-Cl in primary macaque cells in vitro, additional infectious molecular clones were constructed. Analysis of these recombinant viruses revealed that changes in the TM portion of the envelope protein were required for the highest level of replication in primary macaque macrophages and brain cells derived from the microvessel endothelium. In addition, a full-length Nef protein is necessary for optimum virus replication in both of these cell types. Finally, viruses expressing a full-length Nef protein in conjunction with the changes in the TM had the highest specific infectivity in a sMAGI assay. Thus, changes in the TM and nef genes between SIV/17E-Cl and SIV/17E-Fr account for replication differences in vitro and correlate with replication in the central nervous system in vivo. PMID:9223467

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

    PubMed

    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

  10. Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1.

    PubMed Central

    Cavicchioli, R; Watson, K

    1991-01-01

    A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0. Images PMID:2014986