Home About us Contact
|
Home About us Contact | ||
|
|
||
Cloning
Kinds of Cloning Terms modified by Cloning Selected AbstractsCLONING AND COMPARISON OF THE GENES ENCODING PREPROAPAMIN FROM THE VENOM OF 2 HONEYBEE AND 4 WASP SPECIESINSECT SCIENCE, Issue 1 2003ZHANG Su-fang Abstract Preproapamin genes were amplified by RT-PCR from total RNA from the venom glands of 2 honeybee species, Apis mellifera, A. cerana cerana, and 4 wasp species, Vespa magnifica, V. velutina nigrothorax and Polistes hebraeus, respectively. Their PCR products were ligated into pGEM® -T easy vector and the nucleotide sequences analyzed. The six fragments were all 141 bp in length and contained an ORF coding the precursor of apamin. The apamin precursors of V. magnifica, V. velutina nigrothorax and P. hebraeus had 95% and 93% similarity with that of A. melliera in nucleotide and amino acid sequences, respectively. That of Vespula maculifrons was identical to that of A. mellifera in nucleotide and amino acid sequences. Apamin precursors of V. magnifica, P. hebraeus and V. velutina nigrothorax also had the same nucleotide sequences. The nucleotide sequences of preproapamin genes from the Chinese honeybee, A. cerana cerana and 4 wasp species are described for the first time. A notable discovery was that the wasps species had exactly same apamins as the honeybees despite the fact they belong to different insect families. [source] CLONING AND EXPRESSION OF SPODOPTERA LITURA UBIQUITIN GENEINSECT SCIENCE, Issue 1 2003LI Zhao-fei Abstract Ubiquitin (UBI) plays a very important role in regulated non-lysosomal ATP dependent protein degradation. In the present work, the coding sequence of Spodoptera litura UBI gene was isolated (GenBank Accession No. AF436066). The length of this ORF is 228bp, encoding a protein with Mr of 8.56 kD and isoelectric point of 6.56. Multiple sequence alignment indicated that S. litura UBI is very similar to the homologous proteins of other eukaryotic species and it has 84% identity with S. litura nucleopolyhedrovirus (SpltMNPV) UBI at amino acid level. RT-PCR analysis showed that S. litura UBI gene is ubiquitously expressed in larva tissues which are susceptible to SpltMNPV infection. By constructing E. coli expression vector, S. litura UBI was highly expressed and the recombinant protein was purified using Ni-NTA resin column, and currently further study on the function of S. litura UBI in SpltMNPV infection is underway. [source] CLONING AND SEQUENCING OF THE ,-AMYLASE GENE FROM BACILLUS SUBTILIS US116 STRAIN ENCODING AN ENZYME CLOSELY IDENTICAL TO THAT FROM BACILLUS AMYLOLIQUEFACIENS BUT DISTINCT IN THERMAL STABILITYJOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2010EZZEDINE BEN MESSAOUD ABSTRACT The gene encoding for the ,-amylase AMYUS116 was cloned and sequenced. The amino acid sequence of AMYUS116 exhibited an almost perfect homology with the ,-amylase BACAAM, excluding the residues N205 and N217 of AMYUS116 that were changed to H205 and I217 into BACAAM. Three mutant derivatives from AMYUS116 (N205H, N217I and N205H/N217I) were created by site-directed mutagenesis and their physicochemical and kinetic properties were compared with those of the wild-type enzymes. Therefore, the undertaken amylases mainly generated maltohexaose from starch and had radically the same kinetic parameters and optimum pH and temperature. They, however, were significantly distinct in thermal stability; AMYUS116 was more thermosensible as its half-life time at 80C was 13 min, while those of BACAAM and the double mutant were likewise 38 min. The single-mutant amylases exhibited an identically intermediate thermal stability as their half-life times at 80C were roughly 22 min. PRACTICAL APPLICATIONS Of particular interest to the current search is that the different thermal stability between AMYUS116 and BACAAM can lead to novel findings pertaining to protein stability, which can bring about new strategies for protein engineering. Basically, the comparative study of closely related amylases and the protein engineering of already existing ones are certainly important because they offer opportunities to understand the structure,function relationships of these biocatalysts. [source] IDENTIFICATION AND CLONING OF AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS LINKED TO THE MATING TYPE LOCUS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)JOURNAL OF PHYCOLOGY, Issue 3 2001Ralf Werner Amplified fragment length polymorphism (AFLP) markers have been widely used to generate molecular maps of plant species, including crops and cereals. We report on a useful protocol to identify AFLPs from Chlamydomonas reinhardtii Dangeard with digoxigenin labeled primers. Although Chlamydomonas has a small genome with a high GC content, we could detect polymorphic bands that led to the identification of several AFLP markers linked to the mating type locus of Chlamydomonas. Three of these markers were isolated from the gel, reamplified, and cloned. The clones were sequenced, and the insertion of the correct fragment was verified in AFLP gels and in Southern blots. One marker showed sequence identity to parts of the fus1 gene, known to be unique in the plus mating type. We also converted some of the AFLP markers into sequence tagged site markers, which allows a fast and convenient screening of progeny of crosses. This procedure will be a useful and fast alternative to the conventional generation of maps for the positional cloning of genes from Chlamydomonas. [source] Cloning and characterization of cDNA for syndecan core protein in sea urchin embryosDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2000Kazuo Tomita The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription,polymerase chain reaction (RT-PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan-1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae. [source] Cloning a novel developmental regulating gene, Xotx5: Its potential role in anterior formation in Xenopus laevisDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2000Hiroki Kuroda The vertebrate Otx gene family is related to otd, a gene contributing to head development in Drosophila. In Xenopus, Xotx1, Xotx2, and Xotx4 have already been isolated and analyzed. Here the cloning, developmental expression and functions of the additional Otx Xenopus gene, Xotx5 are reported. This latter gene shows a greater degree of homology to Xotx2 than Xotx1 and Xotx4. Xotx5 was initially expressed in Spemann's organizer and later in the anterior region. Ectopic expression of Xotx5 had similar effects to other Xotx genes in impairing trunk and tail development, and especially similar effects to Xotx2 in causing secondary cement glands. Taken together, these findings suggest that Xotx5 stimulates the formation of the anterior regions and represses the formation of posterior structures similar to Xotx2. [source] Cloning and characterization of voltage-gated calcium channel alpha1 subunits in Xenopus laevis during developmentDEVELOPMENTAL DYNAMICS, Issue 11 2009Brittany B. Lewis Abstract Voltage-gated calcium channels play a critical role in regulating the Ca2+ activity that mediates many aspects of neural development, including neural induction, neurotransmitter phenotype specification, and neurite outgrowth. Using Xenopus laevis embryos, we describe the spatial and temporal expression patterns during development of the 10 pore-forming alpha1 subunits that define the channels' kinetic properties. In situ hybridization indicates that CaV1.2, CaV2.1, CaV2.2, and CaV3.2 are expressed during neurula stages throughout the neural tube. These, along with CaV1.3 and CaV2.3, beginning at early tail bud stages, and CaV3.1 at late tail bud stages, are detected in complex patterns within the brain and spinal cord through swimming tadpole stages. Additional expression of various alpha1 subunits was observed in the cranial ganglia, retina, olfactory epithelium, pineal gland, and heart. The unique expression patterns for the different alpha1 subunits suggests they are under precise spatial and temporal regulation and are serving specific functions during embryonic development. Developmental Dynamics 238:2891,2902, 2009. © 2009 Wiley-Liss, Inc. [source] Cloning and functional characterization of two key enzymes of glycosphingolipid biosynthesis in the amphibian Xenopus laevisDEVELOPMENTAL DYNAMICS, Issue 3 2008Melchor E. Luque No abstract is available for this article. [source] Cloning of vertebrate Protogenin (Prtg) and comparative expression analysis during axis elongationDEVELOPMENTAL DYNAMICS, Issue 10 2006Christine Vesque Abstract A murine cDNA encoding Protogenin, which belongs to the DCC/Neogenin family, was cloned in a screen performed to identify novel cDNAs regionally expressed in the neural plate. Isolation of the putative zebrafish orthologues allowed a comparative analysis of the expression patterns of Protogenin genes during embryogenesis in different vertebrate species. From mid-gastrulation to early somite stages, Protogenin expression is restricted to posterior neural plate and mesoderm, with an anterior limit at the level of the rhombencephalon in mouse, chicken, and zebrafish. During somitogenesis, the expression profiles in the three species share features in the neural tube but present also species-specific characteristics. The initiation of Protogenin expression just before somitogenesis and its maintenance in the neural tube and paraxial mesoderm during this process suggest a conserved role in axis elongation. Developmental Dynamics 235:2836,2844, 2006. © 2006 Wiley-Liss, Inc. [source] Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevisDEVELOPMENTAL DYNAMICS, Issue 3 2005Teun P. De Boer Abstract Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (,62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals. Developmental Dynamics 233:864,871, 2005. © 2005 Wiley-Liss, Inc. [source] Prep2: Cloning and expression of a new prep family memberDEVELOPMENTAL DYNAMICS, Issue 3 2002Klaus Haller Abstract We describe Prep2, a new murine homeobox-containing gene closely related to Prep1. The PREP2 protein belongs to the three amino acid loop extension (TALE) superclass of homeodomain-containing proteins and encodes a polypeptide of 462 residues. As for PREP1, PREP2 binds an appropriate site on DNA as a heterodimer with PBX1A. Northern analysis, immunoblotting, immunohistochemistry, and in situ hybridization show widespread Prep2 expression during organogenesis and in the adult. The data suggest that Prep2 functions to varying degrees in a broad array of tissues and developmental processes. © 2002 Wiley-Liss, Inc. [source] Cloning and expression of three zebrafish roundabout homologs suggest roles in axon guidance and cell migrationDEVELOPMENTAL DYNAMICS, Issue 2 2001Jeong-Soo Lee Abstract We report the cloning and expression patterns of three novel zebrafish Roundabout homologs. The Roundabout (robo) gene encodes a transmembrane receptor that is essential for axon guidance in Drosophila and Robo family members have been implicated in cell migration. Analysis of extracellular domains and conserved cytoplasmic motifs shows that zebrafish Robo1 and Robo2 are orthologs of mammalian Robo1 and Robo2, respectively, while zebrafish Robo3 is likely to be an ortholog of mouse Rig-1. The three zebrafish robos are expressed in distinct but overlapping patterns during embryogenesis. They are highly expressed in the developing nervous system, including the olfactory system, visual system, hindbrain, cranial ganglia, spinal cord, and posterior lateral line primordium. They are also expressed in several nonneuronal tissues, including somites and fin buds. The timing and patterns of expression suggest roles for zebrafish robos in axon guidance and cell migration. Wiley-Liss, Inc. © 2001 Wiley-Liss, Inc. [source] Purification and cDNA Cloning of Lysozyme II from Cabbage Butterfly, Artogeia rapae LarvaeENTOMOLOGICAL RESEARCH, Issue 4 2005BANG In Seok ABSTRACT Last instar larvae of cabbage butterfly Artogeia rapae respond to injection of bacteria with a set of inducible antibacterial peptides/proteins. The inducible peptides/proteins are related to the known hinnavins (I and II) and lysozymes (I and II). The lysozyme II has been isolated by heat treatment, cation exchange, and reversed-phase chromatography from immunized hemolymph of last instar larvae. The lysozyme II gene of A. rapae was isolated and its nucleotide sequence was determined by the RACE-PCR from immunized fat body with E. coli. It has an open reading frame of 414 bp nucleotide corresponding to 138 amino acids including an 18 amino acid signal sequence. The molecular weight and the isoelectric point of Artogeia lysozyme II without a signal peptide were 13,649.38 Da and 9.11, respectively. It is great similarity with Manduca lysozyme among other lepidopteran. [source] Cloning and Characterization of the cDNA Encoding the Masquerade-like Serine Proteinase Homologue Gene of the Silkworm, Bombyx moriENTOMOLOGICAL RESEARCH, Issue 3 2002Doo-Sang PARK ABSTRACT From Bombyx mori larvae, RT-PCR and cDNA library screening isolated masquerade-like serine proteinase homologue cDNA gene, proposed to be related to insect immunity and its characteristics were examined. The isolated gene is composed of 1.3 kb of nucleotide and 420 amino acid residues were encoded. According to the results of database search, the isolated gene showed high sequence homology with Holotrichia and Tenebrio's 45 kDa protein, Drosophila CG5390 gene. Moreover, it is composed of regulatory domain and catalytic domain, which is characteristic of serine proteinase that can be found in the insect immune reaction and embryonic development processes. Enzyme activation site by proteolytic cleavage and the sequence of three amino acids participate in the catalytic triad of enzyme and 14 cystein residues used in disulfide bridges are well conserved with the compared genes. The mRNA expression was increased following E. coli injection and constitutive expression was also observed before injection by Northern blot analysis. [source] Nitrogenase gene diversity and microbial community structure: a cross-system comparisonENVIRONMENTAL MICROBIOLOGY, Issue 7 2003Jonathan P. Zehr Summary Biological nitrogen fixation is an important source of fixed nitrogen for the biosphere. Microorganisms catalyse biological nitrogen fixation with the enzyme nitrogenase, which has been highly conserved through evolution. Cloning and sequencing of one of the nitrogenase structural genes, nifH, has provided a large, rapidly expanding database of sequences from diverse terrestrial and aquatic environments. Comparison of nifH phylogenies to ribosomal RNA phylogenies from cultivated microorganisms shows little conclusive evidence of lateral gene transfer. Sequence diversity far outstrips representation by cultivated representatives. The phylogeny of nitrogenase includes branches that represent phylotypic groupings based on ribosomal RNA phylogeny, but also includes paralogous clades including the alternative, non-molybdenum, non-vanadium containing nitrogenases. Only a few alternative or archaeal nitrogenase sequences have as yet been obtained from the environment. Extensive analysis of the distribution of nifH phylotypes among habitats indicates that there are characteristic patterns of nitrogen fixing microorganisms in termite guts, sediment and soil environments, estuaries and salt marshes, and oligotrophic oceans. The distribution of nitrogen-fixing microorganisms, although not entirely dictated by the nitrogen availability in the environment, is non-random and can be predicted on the basis of habitat characteristics. The ability to assay for gene expression and investigate genome arrangements provides the promise of new tools for interrogating natural populations of diazotrophs. The broad analysis of nitrogenase genes provides a basis for developing molecular assays and bioinformatics approaches for the study of nitrogen fixation in the environment. [source] Cloning, distribution and functional analysis of the type III sodium channel from human brainEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2000Yu Hua Chen Abstract The type III voltage-gated sodium channel was cloned from human brain. The full-length cDNA has 89% identity with rat type III, and the predicted protein (1951 amino acids) has 55 differences. The expression pattern of human type III mRNA was determined in adult brain tissue and, in contrast to rat, was detected in many regions, including caudate nucleus, cerebellum, hippocampus and frontal lobe. The human type III channel was stably expressed in Chinese hamster ovary (CHO) cells and its biophysical properties compared to the human type II channel using identical conditions. The voltage dependence and kinetics of activation were found to be similar to that of type II. The kinetics of inactivation of the two human subtypes were also similar. However, type III channels inactivated at more hyperpolarized potentials and were slower to recover from inactivation than type II. When expressed in human embryonic kidney (HEK293T) cells, type III channels produced currents with a prominent persistent component, which were similar to those reported for rat type II [Ma et al. (1997) Neuron, 19, 443,452]. However, unlike type II, this was prominent even in the absence of coexpressed G-proteins, suggesting type III may adopt this gating mode more readily. The distinct properties of the channel, together with its wide distribution in adult brain, suggest that in humans, type III may have important physiological roles under normal, and perhaps also pathological conditions. [source] Cloning and characterization of SDF-1,, a novel SDF-1 chemokine transcript with developmentally regulated expression in the nervous systemEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2000Marc Gleichmann Abstract The cytokines SDF-1, and -1, are two alternatively spliced variants of the CXC (,) chemokines that are highly conserved among species. SDF-1, was shown to function as a B-cell maturation factor, a ligand for the CXCR4 (LESTR/fusin) chemokine receptor, thereby inhibiting replication of T cell-tropic HIV-1 strains and inducing cell death in human neuronal cell lines. In this report the cloning of the rat SDF-1, cDNA and a new SDF-1 isoform, SDF-1,, are presented. Using Northern blot analysis, the expression pattern of both isoforms was studied in different tissues and it is shown that during postnatal development of the central and peripheral nervous system SDF-1,- and SDF-1,-mRNA expression is inversely regulated. Whilst SDF-1,-mRNA is the predominant isoform in embryonic and early postnatal nerve tissue, SDF-1,-mRNA is expressed at higher levels in adulthood. After peripheral nerve lesion a transient increase in SDF-1,-mRNA expression is observed. As revealed by in situ hybridization, neurons and Schwann cells are the main cellular sources of both SDF-1, and SDF-1, mRNAs in the nervous system. Computer-assisted analysis revealed that both transcripts encode secreted peptides with putative proteolytic cleavage sites which might generate novel neuropeptides. [source] Characterization of porcine dentin sialoprotein (DSP) and dentin sialophosphoprotein (DSPP) cDNA clonesEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 1 2003Yasuo Yamakoshi Dentin sialophosphoprotein (DSPP) is a chimeric glycoprotein with dentin sialoprotein (DSP) on its N -terminus and dentin phosphoprotein (DPP) on its C -terminus. We have constructed and screened a unidirectional cDNA library derived from the pulp organ of developing pig teeth, and isolated cDNA clones encoding DSP-only, as well as two DSPP clones with alternative sequences in their 3, coding regions. The DSP-only transcript has an open reading frame of 386 codons, and is generated through the use of a polyadenylation signal within intron 4, immediately following the DSP coding region. the use of this polyadenylation signal deletes the DPP coding region and places a TGA translation termination signal as the fourth codon following the exon 4-encoded segment. The DSPP cDNAs contain open reading frames of 593 and 600 codons. Northern blots hybridized to radiolabeled DSP probes showed bands at 1.4, 2.5, 4.4, and 4.8 kb. Cloning and characterization of reverse transcriptase polymerase chain reaction products confirmed the existence of mRNA encoding pDSP386, pDSPP593, and pDSPP600in vivo, but also suggested that DNA sequence redundancies in the DSPP coding region make it prone to cloning artifacts. [source] Cloning of Xenopus orthologs of Ctf7/Eco1 acetyltransferase and initial characterization of XEco2FEBS JOURNAL, Issue 24 2008Masatoshi Takagi Sister chromatid cohesion is important for the correct alignment and segregation of chromosomes during cell division. Although the cohesin complex has been shown to play a physical role in holding sister chromatids together, its loading onto chromatin is not sufficient for the establishment of sister chromatid cohesion. The activity of the cohesin complex must be turned on by Ctf7/Eco1 acetyltransferase at the replication forks as the result of a specific mechanism. To dissect this mechanism in the well established in vitro system based on the use of Xenopus egg extracts, we cloned two Xenopus orthologs of Ctf7/Eco1 acetyltransferase, XEco1 and XEco2. Both proteins share a domain structure with known members of Ctf7/Eco1 family proteins. Moreover, biochemical analysis showed that XEco2 exhibited acetyltransferase activity. We raised a specific antibody against XEco2 and used it to further characterize XEco2. In tissue culture cells, XEco2 gradually accumulated in nuclei through the S phase. In nuclei formed in egg extract, XEco2 was loaded into the chromatin at a constant level in a manner sensitive to geminin, an inhibitor of the pre-replication complex assembly, but insensitive to aphidicolin, an inhibitor of DNA polymerases. In both systems, no specific localization was observed during mitosis. In XEco2-depleted egg extracts, DNA replication occurred with normal kinetics and efficiency, and the condensation and sister chromatid cohesion of subsequently formed mitotic chromosomes was unaffected. These observations will serve as a platform for elucidating the molecular function of Ctf7/Eco1 acetyltransferase in the establishment of sister chromatid cohesion in future studies, in which XEco1 and XEco2 should be dissected in parallel. [source] Cloning, characterization and localization of a novel basic peroxidase gene from Catharanthus roseusFEBS JOURNAL, Issue 5 2007Santosh Kumar Catharanthus roseus (L.) G. Don produces a number of biologically active terpenoid indole alkaloids via a complex terpenoid indole alkaloid biosynthetic pathway. The final dimerization step of this pathway, leading to the synthesis of a dimeric alkaloid, vinblastine, was demonstrated to be catalyzed by a basic peroxidase. However, reports of the gene encoding this enzyme are scarce for C. roseus. We report here for the first time the cloning, characterization and localization of a novel basic peroxidase, CrPrx, from C. roseus. A 394 bp partial peroxidase cDNA (CrInt1) was initially amplified from the internodal stem tissue, using degenerate oligonucleotide primers, and cloned. The full-length coding region of CrPrx cDNA was isolated by screening a leaf-specific cDNA library with CrInt1 as probe. The CrPrx nucleotide sequence encodes a deduced translation product of 330 amino acids with a 21 amino acid signal peptide, suggesting that CrPrx is secretory in nature. The molecular mass of this unprocessed and unmodified deduced protein is estimated to be 37.43 kDa, and the pI value is 8.68. CrPrx was found to belong to a ,three intron' category of gene that encodes a class III basic secretory peroxidase. CrPrx protein and mRNA were found to be present in specific organs and were regulated by different stress treatments. Using a ,-glucuronidase,green fluorescent protein fusion of CrPrx protein, we demonstrated that the fused protein is localized in leaf epidermal and guard cell walls of transiently transformed tobacco. We propose that CrPrx is involved in cell wall synthesis, and also that the gene is induced under methyl jasmonate treatment. Its potential involvement in the terpenoid indole alkaloid biosynthetic pathway is discussed. [source] Cloning and expression of murine enzymes involved in the salvage pathway of GDP- l -fucoseFEBS JOURNAL, Issue 1 2004GDP- l -fucose pyrophosphorylase, l -fucokinase In the salvage pathway of GDP- l -fucose, free cytosolic fucose is phosphorylated by l -fucokinase to form l -fucose-1-phosphate, which is then further converted to GDP- l -fucose in the reaction catalyzed by GDP- l -fucose pyrophosphorylase. We report here the cloning and expression of murine l -fucokinase and GDP- l -fucose pyrophosphorylase. Murine l -fucokinase is expressed as two transcripts of 3057 and 3270 base pairs, encoding proteins of 1019 and 1090 amino acids with predicted molecular masses of 111 kDa and 120 kDa respectively. Only the longer splice variant of l -fucokinase was enzymatically active when expressed in COS-7 cells. Murine GDP- l -fucose pyrophosphorylase has an open reading frame of 1773 base pairs encoding a protein of 591 amino acids with a predicted molecular mass of 65.5 kDa. GDP- l -fucose, the reaction product of GDP- l -pyrophosphorylase, was identified by HPLC and MALDI-TOF MS analysis. The tissue distribution of murine l -fucokinase and GDP- l -fucose pyrophosphorylase was investigated by quantitative real time PCR, which revealed high expression of l -fucokinase and GDP- l -fucose pyrophosphorylase in various tissues. The wide expression of both enzymes can also be observed from the large amount of data collected from a number of expressed sequence tag libraries, which indicate that not only the de novo pathway alone, but also the salvage pathway, could have a significant role in the synthesis of GDP- l -fucose in the cytosol. [source] Cloning, expression and characterization of a gene encoding nitroalkane-oxidizing enzyme from Streptomyces ansochromogenesFEBS JOURNAL, Issue 24 2002Jihui Zhang A nitroalkane-oxidizing enzyme gene (naoA) was cloned from a genomic DNA library of Streptomyces ansochromogenes 7100. The deduced protein (NaoA) of this gene contains 363 amino acids and has high similarity to several nitroalkane-oxidizing enzymes from various micro-organisms. The naoA gene was subcloned into an expression vector pET23b and overexpressed in Escherichia coli BL21(DE3). The protein was then purified, and its characteristics were studied. Experimental results showed that NaoA can convert 1-nitropropane, 2-nitropropane and nitroethane into the corresponding carbonyl compounds. The optimal pH and temperature for NaoA was found to be pH 7,8 and 48,56 °C, respectively. The Km of NaoA for nitroethane is ,,26.8 mm. NADH and nitro blue tetrazolium are strong inhibitors of NaoA, and thiol compounds and superoxide dismutase partially inhibit the enzyme activity. Therefore, superoxide may be an essential intermediate in the oxidation of nitroalkane by NaoA. [source] Recombinant human glucose-6-phosphate dehydrogenaseFEBS JOURNAL, Issue 14 2002Evidence for a rapid-equilibrium random-order mechanism Cloning and over-expression of human glucose 6-phosphate dehydrogenase (Glc6P dehydrogenase) has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady-state kinetics of the recombinant enzyme, studied by fluorimetric initial-rate measurements, gave converging linear Lineweaver,Burk plots as expected for a ternary-complex mechanism. Patterns of product and dead-end inhibition indicated that the enzyme can bind NADP+ and Glc6P separately to form binary complexes, suggesting a random-order mechanism. The Kd value for the binding of NADP+ measured by titration of protein fluorescence is 8.0 µm, close to the value of 6.8 µm calculated from the kinetic data on the assumption of a rapid-equilibrium random-order mechanism. Strong evidence for this mechanism and against either of the compulsory-order possibilities is provided by repeating the kinetic analysis with each of the natural substrates replaced in turn by structural analogues. A full kinetic analysis was carried out with deaminoNADP+ and with deoxyglucose 6-phosphate as the alternative substrates. In each case the calculated dissociation constant upon switching a substrate in a random-order mechanism (e.g. that for NADP+ upon changing the sugar phosphate) was indeed constant within experimental error as expected. The calculated rate constants for binding of the leading substrate in a compulsory-order mechanism, however, did not remain constant when the putative second substrate was changed. Previous workers, using enzyme from pooled blood, have variously proposed either compulsory-order or random-order mechanisms. Our study appears to provide unambiguous evidence for the latter pattern of substrate binding. [source] Cloning, chromosomal localization and characterization of the murine mucin gene orthologous to human MUC4FEBS JOURNAL, Issue 13 2002Jean-Luc Desseyn We report here the full coding sequence of a novel mouse putative membrane-associated mucin containing three extracellular EGF-like motifs and a mucin-like domain consisting of at least 20 tandem repeats of 124,126 amino acids. Screening a cosmid and a BAC libraries allowed to isolate several genomic clones. Genomic and cDNA sequence comparisons showed that the gene consists of 25 exons and 24 introns covering a genomic region of ,,52 kb. The first intron is ,,16 kb in length and is followed by an unusually large exon (, 9.5 kb) encoding Ser/Thr-rich tandemly repeated sequences. Radiation hybrid mapping localized this new gene to a mouse region of chromosome 16, which is the orthologous region of human chromosome 3q29 encompassing the large membrane-anchored mucin MUC4. Contigs analysis of the Human Genome Project did not reveal any other mucin on chromosome 3q29 and, interestingly, our analysis allowed the determination of the genomic organization of the human MUC4 and showed that its exon/intron structure is identical to that of the mouse gene we cloned. Furthermore, the human MUC4 shares considerable homologies with the mouse gene. Based on these data, we concluded that we isolated the mouse ortholog of MUC4 we propose as Muc4. Expression studies showed that Muc4 is ubiquitous like SMC and MUC4, with highest levels of expression in trachea and intestinal tract. [source] Cloning and characterization of novel snake venom proteins that block smooth muscle contractionFEBS JOURNAL, Issue 11 2002Yasuo Yamazaki In this study, we isolated a 25-kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino-acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage-gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K+ -induced depolarization in the 0.1,1 µm range, but did not block caffeine-stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K+ -induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity. [source] Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylaseFEBS JOURNAL, Issue 23 2000Evidence that GDP-mannose, GDP-Glc pyrophosphorylases are different proteins GDP-Man, the mannosyl donor for most Man-containing polymers is formed by the transfer of Man-1- P to GTP to form GDP-Man and PPi. This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP). The pig liver GMPP consists of an , subunit (43 kDa) and a , subunit (37 kDa). Purified pig GMPP catalyzes the synthesis of GDP-Glc (from Glc-1- P and GTP) and GDP-Man (from Man-1- P and GTP), but has higher activity for the formation of GDP-Glc than for synthesis of GDP-Man. In the present study, we report the cloning of the cDNA for the , subunit of GMPP, and its expression in a bacterial system resulting in the formation of active enzyme. The full length cDNA encoding the , subunit was isolated from a porcine cDNA library, and its predicted gene product showed high amino-acid sequence homology to GMPPs from other species. The gene was expressed in Escherichia coli cells, and a 37-kDa protein was over-produced in these cells. This gene product reacted strongly with antibody reactive to the native , subunit of pig GMPP. Most interestingly, this recombinant protein had high activity for synthesizing GDP-Man (from Man-1- P and GTP), but very low activity for the formation of GDP-Glc (from Glc-1- P and GTP). Other properties of the recombinant protein were also analyzed. This study suggests that the , subunit is the GMPP, whereas the , subunit, or a combination of both subunits, may have the GDP-Glc pyrophosphorylase activity. [source] Cloning of MMP-26FEBS JOURNAL, Issue 11 2000A novel matrilysin-like proteinase A cDNA encoding a novel human matrix metalloproteinase (MMP), named MMP-26, was cloned from fetal cDNA. The deduced 261-amino-acid sequence is homologous to macrophage metalloelastase (51.8% identity). It includes only the minimal characteristic features of the MMP family: a signal peptide, a prodomain and a catalytic domain. As with MMP-7, this new MMP does not comprise the hemopexin domain, which is believed to be involved in substrate recognition. A study of MMP-26 mRNA steady states levels reveals, among the tissue examined, a specific expression in placenta. MMP-26 mRNA could also be detected in several human cell lines such as HEK 293 kidney cells and HFB1 lymphoma cells. Recombinant MMP-26 was produced in mammalian cells and used to demonstrate a proteolytic activity of the enzyme on gelatin and ,-casein. [source] Analysis of the thyrotropin-releasing hormone-degrading ectoenzyme by site-directed mutagenesis of cysteine residuesFEBS JOURNAL, Issue 9 2000Cys68 is involved in disulfide-linked dimerization Thyrotropin-releasing hormone-degrading ectoenzyme is a member of the M1 family of Zn-dependent aminopeptidases and catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH2). Cloning of the cDNA of this enzyme and biochemical studies revealed that the large extracellular domain of the enzyme with the catalytically active site contains nine cysteine residues that are highly conserved among species. To investigate the functional role of these cysteines in TRH-DE we used a site-directed mutagenesis approach and replaced individually each cysteine by a serine residue. The results revealed that the proteolytically truncated and enzymatically fully active enzyme consists of two identical subunits that are associated noncovalently by protein,protein interactions but not via interchain S-S bridges. The eight cysteines contained within this region are all important for the structure of the individual subunit and the enzymatic activity, which is dramatically reduced in all mutant enzymes. This is even true for the four cysteines that are clustered within the C-terminal domain remote from the Zn-binding consensus sequence HEICH. In contrast, Cys68, which resides within the stalk region seven residues from the end of the hydrophobic membrane-spanning domain, can be replaced by serine without a significant change in the enzymatic activity. Interestingly, this residue is involved in the formation of an interchain disulfide bridge. Covalent dimerization of the subunits, however, does not seem to be essential for efficient biosynthesis, enzymatic activity and trafficking to the cell surface. [source] Cloning of the guanylate kinase homologues AGK-1 and AGK-2 from Arabidopsis thaliana and characterization of AGK-1FEBS JOURNAL, Issue 2 2000Vinod Kumar Guanylate kinase is an essential enzyme for nucleotide metabolism, phosphorylating GMP to GDP or dGMP to dGDP. The low molecular mass cytosolic forms of guanylate kinase are implicated primarily in the regulation of the supply of guanine nucleotides to cell signalling pathways. The high molecular mass and membrane-associated forms of guanylate kinase homologues, notably found in neuronal tissues, are assigned roles in cell junction organization and transmembrane regulation. Here, we describe the first plant guanylate kinase-encoding genes, AGK1 and AGK2, from Arabidopsis thaliana. The nucleotide sequences of their genomic and cDNA clones predict proteins that carry N-terminal and C-terminal extensions of the guanylate kinase-like domain. The amino acid sequences of this domain share 46,52% identity with guanylate kinases from yeast, Escherichia coli, human, mouse and Caenorhabditis elegans. Arabidopsis guanylate kinases (AGKs) exhibit a high degree of conservation of active site residues and sequence motifs in common with other nucleoside monophosphate kinases, which suggests overall structural similarity of the plant proteins. Although bacterially expressed AGK-1 is enzymatically much less active than yeast guanylate kinase, its kinase domain is shown to complement yeast GUK1 recessive lethal mutations. AGKs are expressed ubiquitously in plant tissues with highest transcriptional activity detected in roots. The identification of AGKs provides new perspectives for understanding the role of guanylate kinases in plant cell signalling pathways. [source] Methane assimilation and trophic interactions with marine Methylomicrobium in deep-water coral reef sediment off the coast of NorwayFEMS MICROBIOLOGY ECOLOGY, Issue 2 2008Sigmund Jensen Abstract Deep-water coral reefs are seafloor environments with diverse biological communities surrounded by cold permanent darkness. Sources of energy and carbon for the nourishment of these reefs are presently unclear. We investigated one aspect of the food web using DNA stable-isotope probing (DNA-SIP). Sediment from beneath a Lophelia pertusa reef off the coast of Norway was incubated until assimilation of 5 ,mol 13CH4 g,1 wet weight occurred. Extracted DNA was separated into ,light' and ,heavy' fractions for analysis of labelling. Bacterial community fingerprinting of PCR-amplified 16S rRNA gene fragments revealed two predominant 13C-specific bands. Sequencing of these bands indicated that carbon from 13CH4 had been assimilated by a Methylomicrobium and an uncultivated member of the Gammaproteobacteria. Cloning and sequencing of 16S rRNA genes from the heavy DNA, in addition to genes encoding particulate methane monooxygenase and methanol dehydrogenase, all linked Methylomicrobium with methane metabolism. Putative cross-feeders were affiliated with Methylophaga (Gammaproteobacteria), Hyphomicrobium (Alphaproteobacteria) and previously unrecognized methylotrophs of the Gammaproteobacteria, Alphaproteobacteria, Deferribacteres and Bacteroidetes. This first marine methane SIP study provides evidence for the presence of methylotrophs that participate in sediment food webs associated with deep-water coral reefs. [source] | |||||||||||||||||||||||||||||||||||||||||||