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1.
Acylation of anthocyanins with hydroxycinnamic acid derivatives is one of the most important and less under-stood modification reactions during anthocyanin biosynthesis. Anthocyanin aromatic acyltransferase catalyses the transfer of hydroxycinnamic acid moieties from their CoA esters to the glycosyl groups of anthocyanins. A full-length cDNA encoding the anthocyanin 5-aromatic acyltransferase (5AT) (EC 2.3.1.153) that acylates the glucose bound at the 5-position of anthocyanidin 3,5-diglucoside was isolated from petals of Gentiana triflora on the basis of the amino acid sequence of the purified enzyme. The isolated full-length cDNA had an open reading frame of 469 amino acids and the calculated molecular weight was 52,736. The deduced amino acid sequence contains consensus motifs that are conserved among the putative acyl CoA-mediated acyltransferases, and this indicates that 5AT is a member of a proposed superfamily of multi-functional acyltransferases (St-Pierre et al. (1998) Plant J. 14, 703-713). The cDNA was expressed in Escherichia coli and yeast, and confirmed to encode 5AT. The enzymatic characteristics of the recombinant 5AT were consistent with those of the native gentian 5AT. Immunoblot analysis using specific antibodies to 5AT showed that the 5AT protein is present in petals, but not in sepals, stems or leaves of G. triflora. RNA blot analysis showed that the 5AT gene is expressed only in petals and that its expression is temporally regulated during flower development coordinately with other anthocyanin biosynthetic genes. Immunohistochemical analysis demonstrated that the 5AT protein is specifically expressed in the outer epidermal cells of gentian petals and that it is localized mainly in the cytosol.  相似文献   

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Calnexin is a 90-kDa integral membrane protein of the endoplasmic reticulum (ER). Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane. We have purified human calnexin in association with the human interferon-gamma receptor and cloned calnexin cDNA from placenta. Fragments of calnexin have been prepared as glutathione S-transferase fusion proteins and analyzed for their abilities to bind 45Ca2+ and ruthenium red. A subdomain containing four internal repeats binds Ca2+ with the highest affinity. This sequence is highly conserved when compared to calreticulin (a luminal ER protein), an Onchocerca surface antigen, and yeast and plant calnexin homologues. Consequently, this sequence represents a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the "E-F hand" motif. An adjacent subdomain, also highly conserved and containing four internal repeats, fails to bind Ca2+. The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic interactions. The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic. We have also determined the cDNA sequences of mouse and rat calnexins. Comparison of the known mammalian calnexin sequences reveals very high conservation of sequence identity (93-98%), suggesting that calnexin performs important cellular functions. The gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35.  相似文献   

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GMP synthetase is a key enzyme in the de novo synthesis of guanine nucleotides. Human GMP synthetase has been purified to homogeneity, and a cDNA encoding the enzyme has been isolated from the T-lymphoblastoma cell line, A3.01. The open reading frame encodes a protein of 693 amino acids with a predicted molecular weight of 76,725. The cDNA complements a guaA mutant of Escherichia coli, which lacks a functional GMP synthetase and extracts from the transformed E. coli exhibit GMP synthetase activity, which is absent in the parental strain. RNA hybridization analysis shows that human GMP synthetase is encoded by a single 2.4-kilobase message. DNA hybridization analysis suggests that the human GMP synthetase is encoded by one gene. In several human cell lines, the level of mRNA expression is substantially higher in proliferating, transformed cells than in nontransformed cells. In two transformed cell lines, treatment with phorbol ester inhibits proliferation and results in a dramatic down-regulation in the levels of GMP synthetase mRNA and protein.  相似文献   

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A cDNA for a novel human papain-like cysteine protease, designated cathepsin F, has been cloned from a lambdagt10-skeletal muscle cDNA library. The nucleotide sequence encoded a polypeptide of 302 amino acids composed of an 88-residue propeptide and a 214-residue mature protein. Protein sequence comparisons revealed 58% homology with cathepsin W; about 42-43% with cathepsins L, K, S, H, and O; and 38% with cathepsin B. Sequence comparisons of the propeptides indicated that cathepsin F and cathepsin W may form a new cathepsin subgroup. Northern blot analysis showed high expression levels in heart, skeletal muscle, brain, testis, and ovary; moderate levels in prostate, placenta, liver, and colon; and no detectable expression in peripheral leukocytes and thymus. The precursor polypeptide of human recombinant cathepsin F, produced in Pichia pastoris, was processed to its active mature form autocatalytically or by incubation with pepsin. Mature cathepsin F was highly active with comparable specific activities toward synthetic substrates as reported for cathepsin L. The protease had a broad pH optimum between 5.2 and 6.8. Similar to cathepsin L, its pH stability at cytosolic pH (7.2) was short, with a half-life of approximately 2 min. This may suggest a function in an acidic cellular compartment. Transient expression of T7-tagged cathepsin F in COS-7 cells revealed a vesicular distribution of the gene product in the juxtanuclear region of the cells. However, contrary to all known cathepsins, the open reading frame of the cathepsin F cDNA did not encode a signal sequence, thus suggesting that the protease is targeted to the lysosomal compartment via an N-terminal signal peptide-independent lysosomal targeting pathway.  相似文献   

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We have cloned and sequenced the cDNA encoding triadin, a junctional terminal cisternae protein from human skeletal muscle. The cDNA, 2941 base pairs in length, encodes a protein of 729 amino acids with a predicted molecular mass of 81,545 Da. Hydropathy analysis indicates that triadin of human skeletal muscle has the same topology in the myoplasmic, transmembrane and sarcoplasmic reticulum luminal domains as that of triadin from rabbit skeletal muscle. The number and relative position of potential modulation sites are also conserved between the human and rabbit proteins. The cDNA sequence of the predicted sarcoplasmic reticulum luminal domain of human triadin diverged from that of rabbit, with an observed similarity of 82%, translating to an identity of 77% in amino acid sequence. Two insertions of 9 and 12 residues in the amino acid sequence were observed in the predicted luminal domain of triadin, although the structural and functional consequences of such insertions are expected to be minimal. Using fluorescence in situ hybridisation, we have assigned the gene encoding human triadin to the long arm of chromosome 6 in the region 6q22-6q23. Our structural analysis of human triadin supports a central role for this protein in the mechanism of skeletal muscle excitation/contraction coupling.  相似文献   

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A globulin fraction prepared from rice embryos contained polypeptides or polypeptide groups of 49 kDa (designated REG1), 46 kDa (designated REG2), about 35 kDa, 32 kDa and 25 kDa. The amino-terminal sequences of REG1 and the major polypeptide in the 35-kDa group were identical, suggesting that the REG1 polypeptide undergoes partial proteolytic processing that removes a carboxy-terminal region. A cDNA clone, designated pcREG2, encoding REG2 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence of REG2 was found to be 68% identical to that of the maize GLB2 globulin. Reg2 mRNA was present at high levels during embryo development for up to 14 days after flowering (DAF). Lower levels were found 20 DAF when the maturation of embryos was almost completed, and at the dry mature stage. Reg2 mRNA almost disappeared upon imbibition of isolated dry mature embryos but it was re-induced at a low level by further treatment with ABA. The expression of Reg2 was not induced by ABA in suspension-cultured cells, unlike that of Osem, one of the late embryogenesis abundant protein (LEA) genes.  相似文献   

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5-Lipoxygenase of mouse macrophages and bone marrow-derived mast cells (BMMC) was investigated. Indirect immunocytofluorescence combined with confocal microscopy provided evidence for distinct intracellular expression patterns and trafficking of 5-lipoxygenase upon cellular activation. In resting BMMC, 5-lipoxygenase was found within the nucleus co-localizing with the nuclear stain Yo-Pro-1. When BMMC were IgE/antigen-activated the 5-lipoxygenase immunofluorescence pattern was changed from nuclear to perinuclear. The absence of divalent cations in the incubation medium, or calcium ionophore A23187 challenge, altered the predominantly nuclear expression pattern to new sites both cytosolic and intranuclear. The cDNA for murine macrophage 5-lipoxygenase was cloned by the polymerase chain reaction and would predict a 674 amino acid protein. Using control cells obtained from 5-lipoxygenase-deficient mice it was determined that a single isoform accounts for both soluble and membrane-bound and nuclear and cytosolic-localized enzyme in macrophages and BMMC. A mutation at amino acid 672 (Val-->Met) introduced serendipitously during the cloning process was found to completely abolish 5-lipoxygenase enzyme activity when the enzyme was expressed in human embryonic kidney 293 cells. This subtle change is proposed to affect the ability of the COOH-terminal isoleucine to coordinate the essential non-heme iron atom. In macrophages and BMMC obtained from 5-lipoxygenase-deficient mice, compensatory changes in expression of genes involved in the biosynthesis of leukotriene B4 were investigated. 5-Lipoxygenase-activating protein expression was reduced by 50%, while leukotriene A4 hydrolase expression was unaltered. The 5-lipoxygenase gene was mapped to the central region of mouse chromosome 6 in a region that shares homology with human chromosome 10 by interspecific backcross analysis. These studies provide a global picture of the murine 5-lipoxygenase system and raise questions about the role of 5-lipoxygenase and leukotrienes within the nucleus.  相似文献   

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The gene responsible for the second form of autosomal dominant polycystic kidney disease, PKD2, has recently been identified. We now describe the cloning, genomic localization, cDNA sequence, and expression analysis of its murine homologue, Pkd2. The cloned cDNA sequence is 5134 bp long and is predicted to encode a 966-amino-acid integral membrane protein with six membrane-spanning domains and intracellular NH2 and COOH termini. Pkd2 is highly conserved with 91% identity and 98% similarity to polycystin-2 at the amino acid level. Pkd2 mRNA is widely expressed in mouse tissues. Pkd2 maps to mouse Chromosome 5 and is excluded as a candidate gene for previously mapped mouse mutations resulting in a polycystic kidney phenotype.  相似文献   

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A full-length cDNA for horseshoe crab big defensin with a strong antimicrobial activity was obtained from a hemocyte cDNA library. The open reading frame of the cDNA coded for an NH2-terminal signal sequence followed by a propeptide and the mature big defensin. The propeptide is linked to the mature protein through an -Arg-X-Lys/Arg-Arg- motif, the processing site for Kex2-like proteases. Northern blot analysis revealed that big defensin is expressed in all the tissues tested, suggesting that big defensin plays an important role not only in hemocytes but also in other tissues for host defense. The subcellular localization, determined by immunocytochemistry at ultrastructural level, confirmed the previous findings obtained by biochemical analysis that big defensin locates in both small and large granules in hemocytes. Big defensin is the first example to demonstrate the existence of broad tissue distribution in horseshoe crab.  相似文献   

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G alpha q is the alpha subunit of one of the heterotrimeric GTP-binding proteins that mediates stimulation of phospholipase C beta. We report the isolation and characterization of cDNA clones from a frontal cortex cDNA library encoding human G alpha q. The encoded protein is 359 amino acids long and is identical in all but one amino acid residue to mouse G alpha q. Analysis of human genomic DNA reveals an intronless sequence with strong homology to human G alpha q cDNA. In comparison to G alpha q cDNA, this genomic DNA sequence includes several small deletions and insertions that alter the reading frame, multiple single base changes, and a premature termination codon in the open reading frame, hallmarks of a processed pseudogene. Probes derived from human G alpha q cDNA sequence map to both chromosomes 2 and 9 in high-stringency genomic blot analyses of DNA from a panel of human-rodent hybrid cell lines. PCR primers that selectively amplify the pseudogene sequence generate a product only when DNA containing human chromosome 2 is used as the template, indicating that the authentic G alpha q gene (GNAQ) is located on chromosome 9. Regional localization by FISH analysis places GNAQ at 9q21 and the pseudogene at 2q14.3-q21.  相似文献   

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cDNA encoding N(G),N(G)-dimethylarginine dimethylaminohydrolase from rat kidney had been cloned [Kimoto, M., Sasakawa, T., Tsuji, H., Miyatake, S., Oka, T., Nio, N. & Ogawa, T. (1997) Biochim. Biophys. Acta 1337, 6-10]. The enzyme hydrolyzes N(G),N(G)-dimethyl-L-arginine and N(G)-monomethyl-L-arginine, which are known as endogenous inhibitors for the nitric oxide-generating system. In the present study, human N(G),N(G)-dimethylarginine dimethylaminohydrolase has been purified to homogeneity from liver and characterized. The cDNA clone encoding human N(G),N(G)-dimethylarginine dimethylaminohydrolase was isolated from a human kidney lambda gt10 library using a probe prepared from a plasmid containing the entire coding region of rat N(G),N(G)-dimethylarginine dimethylaminohydrolase. Its open reading frame encoded a protein of 285 amino acids with a molecular mass of 31,121 Da. The deduced amino acid sequence exhibits 93% identity with that of rat. The cDNA was expressed as a fusion protein in Escherichia coli and the recombinant protein exhibited enzyme activity which is the same as that of natural enzyme.  相似文献   

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A cDNA for a putative novel serine protease, TLSP, was cloned from human hippocampus cDNA with polymerase chain reaction based strategies. The putative amino acid sequence of TLSP is similar to the trypsin-type serine proteases. TLSP mRNA is expressed in keratinocytes. Overexpressed TLSP protein in neuro2a cells was detected in culture medium.  相似文献   

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Phosphomannose isomerase catalyses the interconversion of fructose-6-P and mannose-6-P and has a critical role in the supply of D-mannose derivatives required for many eukaryotic glycosylation reactions. Three classes of enzymes possessing phosphomannose-isomerase activity have been identified in bacteria and lower eukaryotes. We have purified human phosphomannose isomerase to homogeneity from placental tissue. Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose-isomerase cDNA. A full-length cDNA was isolated from a human testes lambda gt11 library using this fragment as a probe. The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria. Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose-isomerase activity. The cDNA, under the control of the GAL1 promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phosphomannose isomerase had been deleted. The human enzyme was found to be able to functionally substitute for the yeast enzyme. Phosphomannose-isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle. The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies.  相似文献   

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The human FLT3 cDNA was cloned from a pre-B cell line and characterized. The deduced amino acid sequence shows that FLT3 codes for a receptor-type tyrosine kinase of 993 residues, presenting a strong similarity with the corresponding mouse FLT3/FLK2 protein as well as with the receptors for colony-stimulating factor 1 (CSF1R/FMS) and steel locus factor (SLFR/KIT). An analysis of the expression of the gene using amplification of reverse transcribed FLT3 mRNA by polymerase chain reaction shows that FLT3 is expressed in various lymphohematopoietic cells and tissues, including a series of immature cell lines and leukemias of lymphocytic origin.  相似文献   

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