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Clone Encoding (clone + encoding)
Kinds of Clone Encoding Selected AbstractsCloning, Expression and Characterization of Protein Elicitors from the Soyabean Pathogenic Fungus Phytophthora sojaeJOURNAL OF PHYTOPATHOLOGY, Issue 3 2000J. Becker The oomycete Phytophthora sojae is a severe pathogen of soybean. Several resistance genes against races of P. sojae exist in soybean but the nature of corresponding avirulence genes is unknown. Clones encoding four different isoforms of a protein elicitor from P. sojae (sojein 1,4) belonging to the class of acidic ,-elicitins have been isolated. These 98 amino acid proteins show high homology to elicitins from other Phytophthora species. The different sojein isoforms were expressed in Escherichia coli as His-tagged fusion proteins. Purified sojein as well as recombinant sojein isoforms induce hypersensitive reaction (HR)-like lesions in tobacco but are not active as race-specific elicitors in soybean. However all sojein isoforms induce defence-related genes like those encoding phenylalanine ammonia lyase, glutathione-S-transferase and chalcone synthase in tobacco and soybean plants and cell cultures. It is concluded that sojeins contribute to the induction of defence responses but that they are not involved in race specific recognition of the P. sojae races by soybean plants. Zusammenfassung Klonierung, Expression und Charactier von Proteinelictoren aus dem Soyabohnenpathogen Phytophthora sojae Der Oomycet Phytophthora sojae ist ein ernstes Pathogen der Sojabohne. In der Sojabohne gibt es mehrere Resistenzgene gegen verschiedene Rassen von P. sojae, jedoch ist die Natur der korrespondierenden Avirulenzgene unbekannt. Wir haben 4 verschiedene Isoformen eines Protein-Elicitors aus P. sojae (Sojein 1,4) kloniert, die zur Klasse der sauren ,-Elicitine gehören. Sie kodieren für Proteine mit 98 Aminosäuren und zeigen hohe Homologie zu Elicitinen aus anderen Phytophthora Spezies. Aus genomischer DNA und aus revers-transkribierter mRNA wurden die gleichen 4 Isoformen erhalten. Die verschiedenen Sojeine wurden in Escherichia coli als His-markierte Fusionproteine exprimiert. Sowohl gereinigtes als auch rekombinantes Sojein induziert HR-ähnliche Läsionen in Tabak. In der Sojabohne sind sie allerdings nicht als rassenspezifische Elicitoren aktiv. Dagegen induzieren alle Sojein-Isoformen Abwehrgene wie die Phenylalanin Ammonium-Lyase, Glutathion-S-Transferase und Chalkonsynthase in Tabak-und Sojabohnenpflanzen und Zellkulturen. Die Sojeine tragen also zur Induktion von Abwehrreaktionen bei, sind aber nicht in die rassenspezifische Erkennung von P. sojae durch Sojabohnenpflanzen involviert. [source] Functional characterization of a neuropeptide F-like receptor from Drosophila melanogasterEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2003Guoping Feng Abstract A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (NPFR76F, also called GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62,66% similarity and 32,34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein G,16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila. [source] Fusion of farnesyldiphosphate synthase and epi -aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacumFEBS JOURNAL, Issue 14 2002Maria Brodelius A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi -aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-,- d -thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi- aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi -aristolochene than the corresponding amount of single enzymes. [source] Cloning, sequencing, heterologous expression, and characterization of murine cytochrome P450 3a25*(Cyp3a25), a testosterone 6,-hydroxylaseJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2001Ding Dai Abstract A full-length cDNA clone encoding a novel form of the cytochrome P450 3A subfamily (Cyp3a-25) has been isolated from a mouse liver cDNA library. The sequence contained 2010 base pairs and encoded a protein with 503 amino acids. The amino acid sequence shared greater identities with rat CYP3A18 (90%) and golden hamster CYP3A10 (81%) sequences than with known mouse sequences (Cyp3a-11, Cyp3a-13, Cyp3a-16, and Cyp3a-41 [68,70%]). CYP3A25 was expressed in the Escherichia coli PCWori+ expression vector following slight modifications of the N- and C-terminals of the cDNA. The purified CYP3A25 was recognized on an immunoblot by CYP3A1 antibody and has a molecular weight of 50 kD. CYP3A25 was catalytically active in the 6,-hydroxylation of testosterone and the N-demethylation of benzphetamine and erythromycin. It was demonstrated by RT-PCR that the CYP3A25 mRNA is present in both fetal and adult tissues, including liver, lung, intestines, kidney, and brain. Northern blotting demonstrated that expression is greatest in the liver and small intestine. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:90,99, 2001 [source] Discovery of the hepatitis C virusLIVER INTERNATIONAL, Issue 2009Michael Houghton Abstract After nearly 6 years of intensive investigations between 1982 and 1988 in my laboratory at Chiron corporation, in which numerous molecular biological methods were used to investigate the viral aetiology of parenterally transmitted non-A, non-B viral hepatitis (NANBH), a single cDNA clone (5-1-1) was isolated that was shown to be derived from a new flavi-like virus, termed the hepatitis C virus (HCV). After screening hundreds of millions of bacterial cDNA clones derived from different liver and plasma samples obtained from experimentally infected chimpanzees, a single HCV clone was eventually isolated using a novel, blind immunoscreening method in which antibodies derived from a clinically diagnosed NANBH patient were used to identify a cDNA clone encoding an immunodominant epitope within HCV nonstructural protein 4. Its viral origin was demonstrated by its specific hybridization to a large single-stranded RNA molecule of ,10 000 nucleotides found only in NANBH-infected samples that shared distant sequence identity with flaviviruses. Further, HCV clone 5-1-1 was shown to be extrachromosomal and to encode an antigen eliciting antibody seroconversion only in NANBH-infected chimpanzees and humans. Subsequent work demonstrated that HCV was the principal cause of parenterally transmitted NANBH around the world, with an estimated 170 million global carriers and that blood screening tests detecting circulating HCV antibodies and viral RNA could effectively eradicate the transmission of transfusion-associated NANBH. Key viral-encoded enzymes essential to its life cycle are now the targets of vigorous, ongoing drug development activities, and the feasibility of successful vaccination strategies has been demonstrated using the valuable chimpanzee model, without which any progress on HCV would not have been possible. My colleagues and coworkers who made essential contributions to the discovery of HCV were George Kuo, who had his own laboratory at Chiron and who provided intellectual and practical input, Dan Bradley of the Centers for Disease Control and Prevention, who provided a large supply of well-characterized chimpanzee samples and knowledge of the NANBH field, and Qui-Lim Choo, in my own laboratory, who provided many years of outstandingly dedicated and precise molecular biology expertise. [source] Expression of a transcription factor (FsERF1) involved in ethylene signalling during the breaking of dormancy in Fagus sylvatica seedsPHYSIOLOGIA PLANTARUM, Issue 3 2005Jesús Angel Jiménez By means of reverse transcriptase-polymerase chain reaction, using degenerate oligonucleotides conserved among ethylene-responsive transcription factors, we have isolated and characterized a cDNA clone encoding a protein involved in ethylene signalling during the breaking of dormancy in Fagus sylvatica L. seeds. This clone, named FsERF1, exhibits high homology to ethylene-responsive factors (ERFs) from several plant species. The expression of FsERF1 as a fusion protein in Escherichia coli confirmed that it was able to bind to the GCC box, a cis element present in the promoters of several ethylene-responsive genes, corroborating its role as a DNA-binding protein. Northern analysis showed that the transcript levels increased when dormancy was broken by ethephon (an ethylene-releasing compound), or by moist prechilling pretreatment at restricted water content, and were almost undetectable when seeds remained dormant by the addition of abscisic acid, aminooxyacetic acid (an ethylene biosynthesis inhibitor) or warm pretreatment, and when seeds were artificially dried, suggesting that FsERF1 function may be more closely related with the transition from seed dormancy to germination than with responses to drought stress mediated by ethylene. [source] Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virensPLANT BIOTECHNOLOGY JOURNAL, Issue 5 2003Chandrakanth Emani Summary Mycoparasitic fungi are proving to be rich sources of antifungal genes that can be utilized to genetically engineer important crops for resistance against fungal pathogens. We have transformed cotton and tobacco plants with a cDNA clone encoding a 42 kDa endochitinase from the mycoparasitic fungus, Trichoderma virens. Plants from 82 independently transformed callus lines of cotton were regenerated and analysed for transgene expression. Several primary transformants were identified with endochitinase activities that were significantly higher than the control values. Transgene integration and expression was confirmed by Southern and Northern blot analyses, respectively. The transgenic endochitinase activities were examined in the leaves of transgenic tobacco as well as in the leaves, roots, hypocotyls and seeds of transgenic cotton. Transgenic plants with elevated endochitinase activities also showed the expected 42 kDa endochitinase band in fluorescence, gel-based assays performed with the leaf extracts in both species. Homozygous T2 plants of the high endochitinase-expressing cotton lines were tested for disease resistance against a soil-borne pathogen, Rhizoctonia solani and a foliar pathogen, Alternaria alternata. Transgenic cotton plants showed significant resistance to both pathogens. [source] Expression of Arabidopsis SR-like splicing proteins confers salt tolerance to yeast and transgenic plantsTHE PLANT JOURNAL, Issue 5 2002Javier Forment Summary Searching for novel targets of salt toxicity in eukaryotic cells, we have screened an Arabidopsis thaliana cDNA library to isolate genes conferring increased tolerance to salt stress when expressed in the yeast Saccharomyces cerevisiae. Here we show that expression of the ,alternating arginine-rich' (or RS) domains of two different SR-like, putative splicing proteins from Arabidopsis allows yeast cells to tolerate higher lithium and sodium concentrations. Protection against salt stress appears to require the in vivo phosphorylation of these plant polypeptides, since the yeast SR protein kinase Sky1p, which was able to phosphorylate in vitro at least one of them, also proved to be essential for the observed salt tolerance phenotype. In addition, a clone encoding the U1A protein, a previously characterised Arabidopsis splicing factor, was also isolated in the screening. No significant decrease in the intracellular concentration of lithium was observed in yeast cells incubated in the presence of LiCl upon expression of any of the Arabidopsis proteins, suggesting that their effects are not mediated by the stimulation of ion transport. In support of the general significance of these data, we also show that the expression of the RS domain of one of the SR-like proteins in transgenic Arabidopsis plants increases their tolerance to LiCl and NaCl. These results point to an important role of pre-mRNA splicing and SR-like proteins in the salt tolerance of eukaryotic cells, offering a novel route to improve this important trait in crop plants. [source] Three pheromone-binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyiFEBS JOURNAL, Issue 10 2000Rosario Maida Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone-binding proteins (PBPs) could be identified in pheromone-sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI-TOF MS of sensillum lymph droplets from pheromone-sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant-binding proteins, immunoreactivity was found only with an anti-(Apol PBP) serum. Free-flow IEF, ion-exchange chromatography and Western blot analyses revealed at least three anti-(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N-Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K., Krieger, J. & Breer, H. (1989) FEBS Lett.256, 2215,2218] and a new PBP having only 57% identity with this amino-acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N-terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin-labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta1088, 277,284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)-6,11-hexadecadienyl acetate (AC1) and the (E,Z)-6,11-hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H-labelled acetate, whereas no binding of the 3H-labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively. [source] Identification and isolation of cDNA clones encoding the abundant secreted proteins in the saliva proteome of Culicoides nubeculosusINSECT MOLECULAR BIOLOGY, Issue 3 2009C. L. Russell Abstract Culicoides spp. are vectors of several infectious diseases of veterinary importance and a major cause of allergy in horses and other livestock. Their saliva contains a number of proteins which enable blood feeding, enhance disease transmission and act as allergens. We report the construction of a novel cDNA library from Culicoides nubeculosus linked to the analysis of abundant salivary gland proteins by mass spectrometry. Fifty-four novel proteins sequences are described including those of the enzymes maltase, hyaluronidase and two serine proteases demonstrated to be present in Culicoides salivary glands, as well as several members of the D7 family and protease inhibitors with putative anticoagulant activity. In addition, several families of abundant proteins with unknown function were identified including some of the major candidate allergens that cause insect bite hypersensitivity in horses. [source] Continuous expression in tobacco leaves of a Brassica napus PEND homologue blocks differentiation of plastids and development of palisade cellsTHE PLANT JOURNAL, Issue 1 2005Paul Wycliffe Summary Brassica napus complementary deoxyribonucleic acid (cDNA) clones encoding a DNA-binding protein, BnPEND, were isolated by Southwestern screening. A distinctive feature of the protein was a bZIP-like sequence in the amino-terminal portion, which, after expression in Escherichia coli, bound DNA. BnPEND transcripts were present in B. napus roots and flower buds, and to a lesser extent in stems, flowers and young leaves. Treatment in the dark for 72 h markedly increased the amount of BnPEND transcript in leaves of all ages. Sequence comparison showed that BnPEND was similar to a presumed transcription factor from B. napus, GSBF1, a protein deduced from an Arabidopsis thaliana cDNA (BX825084) and the PEND protein from Pisum sativum, believed to anchor the plastid DNA to the envelope early during plastid development. Homology to expressed sequence tag (EST) sequences from additional species suggested that BnPEND homologues are widespread among the angiosperms. Transient expression of BnPEND fused with green fluorescent protein (GFP) in Nicotiana benthamiana epidermal cells showed that BnPEND is a plastid protein, and that the 15 amino acids at the amino-terminal contain information about plastid targeting. Expression of BnPEND in Nicotiana tabacum from the Cauliflower Mosaic Virus 35S promoter gave stable transformants with different extents of white to light-green areas in the leaves, and even albino plants. In the white areas, but not in adjacent green tissue, the development of palisade cells and chloroplasts was disrupted. Our data demonstrate that the BnPEND protein, when over-expressed at an inappropriate stage, functionally blocks the development of plastids and leads to altered leaf anatomy, possibly by preventing the release of plastid DNA from the envelope. [source] | |||||||||||||