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We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial cDNA clones were isolated which were truncated at the 5' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topoisomerase purified to homogeneity. The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg2+. In the presence of Mg2+ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei. 相似文献
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SY Liu FC Gherardini M Matuschek H Bahl J Wiegel 《Canadian Metallurgical Quarterly》1996,178(6):1539-1547
The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface. 相似文献
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Matrix metalloproteinases (MMPs) play key roles in tissue remodeling during physiological and pathological processes by degrading various extracellular matrix (ECM) components. Although nine distinct MMPs have been characterized by cDNA cloning, there are thought to be more corresponding to the complexity of the ECM. MMP genes expressed in human tissues and cell lines were analyzed by the polymerase chain reaction (PCR) using degenerate primers that corresponded to the conserved amino acid (aa) sequences of the MMPs. One isolated complementary DNA (cDNA) fragment had sequence homology to the reported MMPs, but was otherwise unique. A human placenta cDNA library (Clontech) was screened using the fragment as a probe and a 3.4-kb cDNA fragment containing a long open reading frame (potentially encoding 582 aa) was isolated. The putative gene product had a common domain structure and the conserved sequence of a MMP, but it had a unique transmembrane (TM)-like structure at the C terminus. It should, therefore, be an TM protein, whereas all the other reported MMPs are secretory proteins. Thus, the gene is thought to be the first of a new subclass of MMPs whose products are potentially expressed on the cell surface. 相似文献
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ME Suykerbuyk PJ Schaap H Stam W Musters J Visser 《Canadian Metallurgical Quarterly》1995,43(5):861-870
Rhamnogalacturonase was purified from culture filtrate of Aspergillus aculeatus after growth in medium with sugar-beet pulp as carbon source. Purified protein was used to raise antibodies in mice and with the antiserum obtained a gene coding for rhamnogalacturonase (rhgA) was isolated from a lambda cDNA expression library. The cloned rhgA gene has an open-reading frame of 1320 base pairs encoding a protein of 440 amino acids with a predicted molecular mass of 45 962 Da. The protein contains a potential signal peptidase cleavage site behind Gly-18 and three potential sites for N-glycosylation. Limited homology with A. niger polygalacturonase amino acid sequences is found. A genomic clone of rhgA was isolated from a recombinant phage lambda genomic library. Comparison of the genomic and cDNA sequences revealed that the coding region of the gene is interrupted by three introns. Furthermore, amino acid sequences of four different peptides, derived from purified A. aculeatus rhamnogalacturonase, were also found in the deduced amino acid sequence of rhgA. A. aculeatus strains overexpressing rhamnogalacturonase were obtained by cotransformation using either the A. niger pyrA gene or the A. aculeatus pyrA gene as selection marker. For expression of rhamnogalacturonase in A. awamori the A. awamori pyrA gene was used as selection marker. Degradation patterns of modified hairy regions, determined by HPLC, show the recombinant rhamnogalacturonase to be active, and the enzyme was found to have a positive effect in the apple hot-mash liquefaction process. 相似文献
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M Kagawa N Murakoshi Y Nishikawa G Matsumoto Y Kurata T Mizobata Y Kawata J Nagai 《Canadian Metallurgical Quarterly》1999,362(2):346-355
We have purified a heat-stable catalase from a thermophilic bacterium, Thermus species strain YS 8-13. The enzyme was purified 160-fold from crude cellular extracts and possessed a specific activity of 8000 units/mg at 65 degrees C. The purified enzyme displayed the highest activity at pH 7 to 10 and temperatures around 85 degrees C. The catalase was determined to be a manganese catalase, based on results from atomic absorption spectra and inhibition experiments using sodium azide. The enzyme was composed of six identical subunits of molecular weight 36,000. Amino acid sequences determined from the purified protein were used to design oligonucleotide primers, which were in turn used to clone the coding gene. The nucleotide sequence of a 1.4-kb fragment of Thermus sp. YS 8-13 genomic DNA containing a 909-bp open reading frame was determined. The gene encoded a 302-residue polypeptide of deduced molecular weight 33,303. The deduced amino acid sequence displayed a region-specific homology with the sequences of the manganese catalase from a mesophilic organism, Lactobacillus plantarum. 相似文献
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Methionine synthase catalyzes cobalamin-dependent methyl transfer reaction from 5-methyltetrahydrofolate to homocysteine, forming methionine. Rat methonine synthase cDNA was cloned and analyzed by RT-PCR, 3'- and 5'-RACE techniques. The cDNA consists of a 0.3-kb upstream untranslated region, a 3.8-kb coding region, and a 0.4-kb downstream untranslated region. The open reading frame encoded a polypeptide of 1,253 amino acid residues with a calculated molecular weight of 139,162. This molecular weight was in good agreement with the observed one (143,000) of the purified rat liver enzyme. The deduced amino acid sequence was 53, 92, and 64% identical with those of the Escherichia coli, human, and presumptive Caenorhabditis elegans enzymes, respectively. All the fingerprint sequences, forming parts of the cobalamin- and S-adenosylmethionine-binding sites, were completely conserved in the rat methionine synthase. A high-level expression of catalytically active enzyme in insect cells was done by infection with a baculovirus containing the rat methionine synthase cDNA. 相似文献
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We have cloned a genomic DNA encoding the alkaline protease (Alp) of Fusarium sp. S-19-5 from a genomic DNA library and sequenced the nucleotides. Complementary DNA encoding Alp was also isolated from the cDNA library after amplifying the gene by PCR using partial sequences of the Alp genomic DNA as primers. The Alp gene has an open reading frame of 1137 nucleotides containing three introns. A TATA box (TAAATA) was observed 112 base pairs upstream from the translation initiation codon in the 5'-non coding region. The Alp protein has a pre region consisting of 14 amino acids and a pro region of 85 amino acids preceding the mature region, which consists of 280 amino acids. The amino acid sequence of Fusarium Alp has 52% homology with that of Aspergillus oryzae and 51% homology with that of Acremonium chrysogenum. The entire cDNA encoding Fusarium Alp was introduced into Saccharomyces cerevisiae, which then secreted enzymatically active Alp into the culture medium. 相似文献
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Cloning and characterization of a Choristoneura fumiferana ultraspiracle (Cfusp) cDNA are described. First, a PCR fragment and then a cDNA clone (4.4 kb) were isolated from spruce budworm cDNA libraries. Comparison of the deduced amino acid sequence of this cDNA with the sequences in Genbank showed that this sequence had high homology with the ultraspiracle cDNAs cloned from Drosophila melanogaster (Dmusp), Bombyx mori (Bmusp), Manduca sexta (Msusp), and Aedes aegypti (Aausp). The Cfusp cDNA contained all the regions that are typical for a steroid/thyroid hormone receptor superfamily member. The DNA binding domain or C region was the most conserved sequence among all the usps. The A/B, D, and E regions also showed high amino acid identity with the amino acid sequences of Dmusp, Msusp, Bmusp, and Aausp. The Cfusp 4.5-kb mRNA was present in the embryos, in all larval stages, and in the pupae. The Cfusp mRNA levels in the midgut increased during the sixth-instar larval development and reached peak levels during the ecdysteroid raises for the pupal molt. However, Cfusp mRNA levels remained unchanged in the midgut of fifth-instar larvae, and in the epidermis and fat body of sixth-instar larvae indicating both a tissue- and stage-specific regulation of Cfusp mRNA expression. 相似文献
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A 39-base oligonucleotide "guessmer" probe, based on partial N-terminal sequence analysis of the aspartate aminotransferase purified from Thermus aquaticus strain YT1, was used to screen a genomic library prepared from T. aquaticus DNA. A 1842 bp DNA fragment was isolated that proved to contain the coding sequence for the aspartate aminotransferase. The gene is 1152 bases long and codes for a protein of 383 amino acid residues. The amino acid sequence obtained showed 88.7%, 45.1% and 32.9% identity of sequence with those of thermostable aspartate aminotransferases from T. thermophilus, Bacillus YM2, and Sulfolobus solfataricus, respectively. It showed 39.1% identity with one of the gene products tentatively identified as aspartate aminotransferase from the methanogenic archaebacterium Methanococcus jannaschii. Neither the amino acid compositions nor the aligned amino acid sequences provides any obvious clue as to the origin of thermal stability in this group of enzymes. 相似文献
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Disproportionating enzyme (D-enzyme, 4-alpha-glucanotransferase; EC 2.4.1.25) has been purified to homogeneity from potato tubers and its activity characterized. The enzyme catalyzes the transfer of maltooligosaccharides from one 1,4-alpha-D-glucan molecule to another, or to glucose. Maltooligosaccharides are effective donor molecules, but short chain amylose and amylopectin may also function as donors. Enzyme activity is not affected by inorganic phosphate, 3-phosphoglycerate, or hexose phosphates. A cDNA clone encoding the enzyme was isolated using oligonucleotide probes derived from partial peptide sequences of the purified enzyme. The identity of the cDNA clone was confirmed by expression in Escherichia coli resulting in D-enzyme activity. The amino acid sequence deduced from the cDNA shows significant homology with a 4-alpha-glucanotransferase from Streptococcus. The deduced sequence indicates the presence of an amino-terminal plastid transit peptide of 52 amino acid residues and a mature polypeptide of 524 residues. D-enzyme mRNA is present in leaves, stems, roots, and stolons but is most abundant in developing and mature tubers. The amount of mRNA in leaves increases in response to light and to sucrose added to the medium. These results are discussed in terms of the function of D-enzyme in potato starch metabolism. 相似文献
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A cDNA encoding glutamine-dependent asparagine synthetase was isolated from dark-adapted Glycine max cell culture. The deduced amino acid sequence showed 76-89% identity with other plant sequences. The gene for asparagine synthetase is expressed predominantly in shoots as compared to roots of etiolated plants and the level of expression decreases following light treatment, suggesting that the gene expression is down-regulated by light. 相似文献
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The prtT gene, coding for trypsinlike proteolytic activity, has been isolated from Porphyromonas gingivalis ATCC 53977. This gene is present immediately downstream from the sod gene on a 5.9-kb DNA fragment from the organism isolated in Escherichia coli. The complete nucleotide sequence of the gene was determined, and the deduced amino acid sequence of the enzyme corresponds to a 53.9-kDa protein with an estimated pI of 11.85. Gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography also indicated a similar molecular size for the protease. The enzyme was purified to near homogeneity following anion-exchange and gel-filtration chromatography. The purified enzyme also exhibited a single protein species with a size of approximately 53 kDa. Enzyme activity was strongly dependent upon the presence of reducing agents (dithiothreitol, cysteine, and 2-mercaptoethanol) and was also stimulated in the presence of calcium ions. A comparison of the properties of the prtT gene product with comparable parameters of proteases previously purified from different strains of P. gingivalis suggested that the cloned protease represents a previously uncharacterized enzyme. 相似文献