共查询到20条相似文献,搜索用时 203 毫秒
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In-Hye Park Jie Chang Yong-Seok Lee Shu-Jun Fang Yong-Lark Choi 《The protein journal》2012,31(3):238-245
The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 (trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal
peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase
was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight
of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C
and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained
66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl
β-d-cellobiose, but no activity was detected against p-nitrophenyl β-d-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography,
while enzyme did not hydrolyze cellobiose and cellotriose. 相似文献
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Identification of Three <Emphasis Type="Italic">Superoxide dismutase</Emphasis> Genes from a <Emphasis Type="Italic">Geobacillus</Emphasis> sp. 总被引:1,自引:0,他引:1
We report the characterization of three Superoxide dismutase (sod) genes isolated from a bacterium in the Geobacillus genus. We isolated the bacterium from high-temperature pond mud and used 16S rRNA gene sequence to confirm its identity in
the Geobacillus genus. The three genes Mn-sod, Fe/Mn-sod, and Cu/Zn-sod were cloned and analyzed. Their open reading frames are Mn-sod: 615 bp encoding a 204 amino acid protein; Fe/Mn-sod: 1,236 bp encoding a 411 amino acid protein; Cu/Zn-sod: 522 bp encoding a 173 amino acid protein. When these sod genes were expressed in Escherichia coli, only Mn-SOD was able to be purified. The activity of the purified Mn-SOD we got was about 2,730 U/mg. Studies of this Mn-SOD
showed that it was thermostable at 60°, had 70% activity at 80° after 2.5 h, and still had 30% activity at 90° after 2.5 h.
Mn-SOD activity required the ion Mn2+. Based on gel electrophoresis, we deduced that this Mn-SOD was a homotetramer. No activity was detected after the other two
genes (Fe/Mn-sod, Cu/Zn-sod) were expressed in Escherichia coli, but activities were detected when expressed in Pichia pastoris. 相似文献
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A chalcone reductase (CHR) gene was isolated from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). The full-length cDNA of A. mongholicus CHR, designated as Amchr (GenBank accession No. HM357239), was 1196 bp long. It had a 957 bp open reading frame encoding a 318-amino acid protein
of 35 kDa, a 67 bp 5′ non-coding region and a 172 bp 3′-untranslated region. The putative AmCHR protein showed striking similarity
to CHR from other leguminous species. Two-dimensional structure modeling showed that AmCHR consisted of 45.28% α-helix, 10.38%
extended strand and 44.34% random coil. Prediction showed that three-dimensional AmCHR was a global protein containing an
aldo-ket-red domain, with a putative Asp-Tyr-Lys-His catalytic tetrad in the center. The AmCHR gene was 1251 bp long, consisting
of three exons and two introns. Intron I was 125 bp and intron II was 169 bp long. Southern blot analysis indicated that Amchr belonged to a small multigene family. Under natural conditions, Amchr was expressed differentially in the root, stem and leaf tissues of A. mongholicus, with a preferential expression in the root. The recombinant AmCHR protein was successfully expressed in Escherichia coli strain BL21 with pET42a vector. The result showed that the expressed AmCHR protein had molecular weight of about 35 kDa,
which matched the size of the predicted protein by bioinformatic analysis. This study opened avenues towards understanding
of the function of AmCHR protein and the role of the Amchr gene in the calycosin-7-O-β-d-glucoside branch pathway in A. mongholicus. 相似文献
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Jiawen Yong Julia von Bremen Sabine Groeger Gisela Ruiz-Heiland Sabine Ruf 《Journal of cellular and molecular medicine》2021,25(20):9710-9723
Hypoxia-induced apoptosis of cementoblasts (OCCM-30) may be harmful to orthodontic treatment. Hypoxia-inducible factor 1-alpha (HIF-1α) mediates the biological effects during hypoxia. Little is known about the survival mechanism capable to counteract cementoblast apoptosis. We aimed to investigate the potential roles of HIF-1α, as well as the protein-protein interactions with ERK1/2, using an in-vitro model of chemical-mimicked hypoxia and adipokines. Here, OCCM-30 were co-stimulated with resistin, visfatin or ghrelin under CoCl2-mimicked hypoxia. In-vitro investigations revealed that CoCl2-induced hypoxia triggered activation of caspases, resulting in apoptosis dysfunction in cementoblasts. Resistin, visfatin and ghrelin promoted the phosphorylated ERK1/2 expression in OCCM-30 cells. Furthermore, these adipokines inhibited hypoxia-induced apoptosis at different degrees. These effects were reversed by pre-treatment with ERK inhibitor (FR180204). In cells treated with FR180204, HIF-1α expression was inhibited despite the presence of three adipokines. Using dominant-negative mutants of HIF-1α, we found that siHIF-1α negatively regulated the caspase-8, caspase-9 and caspase-3 gene expression. We concluded that HIF-1α acts as a bridge factor in lengthy hypoxia-induced apoptosis in an ERK1/2-dependent pathway. Gene expressions of the caspases-3, caspase-8 and caspase-9 were shown to be differentially regulated by adipokines (resistin, visfatin and ghrelin). Our study, therefore, provides evidence for the role of ERK1/2 and HIF-1α in the apoptotic response of OCCM-30 cells exposed to CoCl2-mimicked hypoxia, providing potential new possibilities for molecular intervention in obese patients undergoing orthodontic treatment. 相似文献
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Lee Y De Zoysa M Whang I Lee S Kim Y Oh C Choi CY Yeo SY Lee J 《Fish & shellfish immunology》2011,30(2):480-487
The caspase family represents aspartate-specific cysteine proteases that play key roles in apoptosis and immune signaling. In this study, we cloned the first death effector domain (DED)-containing molluscan caspase-8 gene from disk abalone (Haliotis discus discus), which is named as hdCaspase-8. The full-length hdCaspase was 2855 bp, with a 1908 bp open reading frame encoding 636 amino acids. The hdCaspase-8 had 72 kDa predicted molecular mass with an estimated isoelectric point (PI) of 6.0. The hdCaspase-8 amino acid sequence contained the characteristic feature of an N-terminal two DED, a C-terminal catalytic domain and the caspase family cysteine active site 513KPKLFFLQACQG524. Phylogenetic analysis results showed that hdCaspase-8 is more similar to the invertebrate Tubifex tubifex (sludge worm) caspase-8.Real-time RT-PCR results showed that hdCaspase-8 constitutively and ubiquitously expressed in all tested tissue of unchallenged disk abalone. The basal expression level of hdCaspase-8 in gill tissue was higher than all other tested tissues. The hdCaspase-8 mRNA expression in gill and hemocytes was significantly up-regulated by exposure to bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Listeria monocytogenes) and VHSV (viral hemorrhagic septicemia virus), as compared to control animals. These results suggest that hdCaspase-8 may be involved in immune response reactions in disk abalone. 相似文献
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A cDNA clone encoding a putative RTE1-like protein (Rh-RTH1) was obtained from total RNA isolated from senescing rose (Rosa hybrida cv. Tineke) petals using RT-PCR and RACE techniques. The cDNA (1,061 bp) contained an open reading frame of 684 bp corresponding
to 227 amino acids. The amino acid sequence had 60.0, 49.6, 61.2, 42.5 and 39.8% identity with that of Arabidopsis RTH, RTE1,
tomato GRL2, GRL1 and GR, respectively. Northern hybridization indicated that Rh-RTH1 expression is enhanced by endogenous and exogenous ethylene and inhibited by 1-MCP in petals and gynoecia. Rh-RTH1 expression partly correlated with sites of the ethylene receptor gene Rh-ETR1 and Rh-ETR3 expression, such as the petals, gynoecia, roots, and buds. The induction of Rh-RTH1 and Rh-ETR3 expression was substantially suppressed by 1-MCP treatment, while Rh-ETR1 expression was not reduced by 1-MCP treatment. Following treatment of flowers with sucrose, the level of Rh-RTH1 and Rh-ETR3 mRNA was only slightly decreased in petals and gynoecia. Upon wounding treatment, Rh-RTH1, Rh-ETR1 and Rh-ETR3 showed a quick increase in mRNA accumulation which was positively correlated with the increase in ethylene production. The
expression of Rh-RTH1 showed partial correlation with that of Rh-ETR1 and Rh-ETR3. 相似文献
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Mating-type genes were cloned and sequenced in the heterothallic phytopathogenic fungus Exserohilum monoceras, a candidate bioherbicide for the control of Echinochloa weeds in rice fields. Using PCR-based methods, we determined both idiomorphs and flanking regions of these genes. The structural
organization of the E. monoceras
Mat locus was similar to that of other heterothallic ascomycetes. The MAT1-1-1 gene was 1191 bp and encoded a predicted protein of 379 amino acids with an α box domain. The MAT1-2-1 gene was 1093 bp and encoded a predicted protein of 345 amino acids with a high mobility group box domain. Expression of
both MAT genes was detected under vegetative and invasive growth conditions. MAT-specific primers were developed to assess the mating-type frequency of E. monoceras field isolates. Both mating types were observed in Japanese field isolates. Analysis of mating types and sexual hybridization of E. monoceras could provide useful approaches for the conventional genetic manipulation of this fungus to produce a more efficient bioherbicide. 相似文献
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Kano R Yamada T Makimura K Kawasaki M Mochizuki T Kamata H Hasegawa A 《Mycopathologia》2011,171(5):333-337
This study first report to identify the mating type (−)-specific gene of alpha-box and the mating type (+)-specific gene of
the high-mobility-group (HMG) DNA-binding domain in zoophilic dermatophytes of Arthroderma benhamiae in an effort to understand the epidemiological characteristics of Trichophyton mentagrophytes. The sequence of the alpha-box gene (1,387 bp) was found to contain two exons, from 184 to 475 bp and from 525 to 1,387 bp,
coding a protein of 384 amino acids, beginning with a putative initiating methionine (ATG). The sequence of the HMG gene (1,910 bp) contained two exons, from 234 to 415 bp and from 479 to 1,457 bp, coding a protein of 386 amino acids, beginning
with a putative initiating methionine (ATG). 相似文献
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The RT PCR and RACE methods were used to obtain the cDNA sequence of an APX gene of muskmelon after the leaves were induced
with powdery mildew. The cDNA length of the APX gene is 1,047 bp with a 750 bp ORF encoded a 249 amino acid and the molecular
weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns. The identity of the amino acid sequence deduced from the cDNA with the
APX family of other homologous members was about 74–97%. A Full-length of ORF was sub-cloned into prokaryotic expression vector
pET24a. The recombinant proteins had high expression level in E. coli. Analysis of expression at mRNA level showed that CmAPX exhibited highly tissue-specific patterns of expression. The mRNA level and enzyme activities assays showed that CmAPX might play an important role in the pathogenesis of powdery mildew. 相似文献
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Xiaoying Cao Zhimin Zong Xiuyun Ju Yong Sun Chuanchao Dai Qun Liu Jihong Jiang 《Molecular biology reports》2010,37(3):1559-1567
A new full-length cDNA encoding 3-hydroxy-3-methylglutoryl-Coenzyme A reductase (HMGR; EC1.1.1.34), which catalyzes the first
committed step of isoprenoids biosynthesis in MVA pathway, was isolated from young leaves of Euphorbia Pekinensis Rupr. by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of HMGR (designated as EpHMGR, GenBank Accession NO. EF062569) was 2,200 bp containing a 1,752 bp ORF encoding 583 amino acids. Bioinformatic analyzes
revealed that the deduced EpHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and
a catalytic domain. The predicted 3-D model of EpHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated
that at most two copies of EpHMGR gene existed in E. Pekinensis genome. Tissue expression analysis revealed that EpHMGR expressed strongly in roots, weakly in stems and leaves. The functional
colour complementation assay indicated that EpHMGR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that EpHMGR plays an influential role in isoprenoid biosynthesis. 相似文献
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Loong AM Chng YR Chew SF Wong WP Ip YK 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2012,182(3):367-379
This study aimed to obtain the full sequence of carbamoyl phosphate synthetase III (cps
III) from, and to determine the mRNA expression of cps
III in, the liver of P. annectens during aestivation in air, hypoxia or mud, or exposure to environmental ammonia (100 mmol l−1 NH4Cl). The complete coding cDNA sequence of cps
III from the liver of P. annectens consisted of 4530 bp, which coded for 1,510 amino acids with an estimated molecular mass of 166.1 kDa. The Cps III of P. annectens consisted of a mitochondrial targeting sequence of 44 amino acid residues, a GAT domain spanning from tyrosine 45 to isoleucine
414, and a methylglyoxal synthase-like domain spanning from valine 433 to arginine 1513. Two cysteine residues (cysteine 1337
and cysteine 1347) that are characteristic of N-acetylglutamate dependency were also present. The critical Cys-His-Glu catalytic triad (cysteine 301, histidine 385 and glutamate
387) together with methionine 302 and glutamine 305 affirmed that P. annectens expressed Cps III and not Cps I. A comparison of the translated amino acid sequence of Cps III from P. annectens with CPS sequences from other animals revealed that it shared the highest similarity with elasmobranch Cps III. A phylogenetic
analysis indicates that P. annectens CPS III could have evolved from Cps III of elasmobranchs. Indeed, Cps III from P. annectens used mainly glutamine as the substrate, and its activity decreased significantly when glutamine and ammonia were included
together in the assay system. There were significant increases (9- to 12-fold) in the mRNA expression of cps III in the liver of fish during the induction phase (days 3 and 6) of aestivation in air. Aestivation in hypoxia or in mud had
a delayed effect on the increase in the mRNA expression of cps III, which extended beyond the induction phase of aestivation, reiterating the importance of differentiating effects that are
intrinsic to aestivation from those intrinsic to hypoxia. Furthermore, results from this study confirmed that environmental
ammonia exposure led to a significant increase in the mRNA expression of cps III in the liver of P. annectens, alluding to the important functional role of urea not only as a product of ammonia detoxification but also as a putative
internal cue for aestivation. 相似文献
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Sebastián A. Trejo Laura M. I. López Néstor O. Caffini Claudia L. Natalucci Francesc Canals Francesc X. Avilés 《Planta》2009,230(2):319-328
Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs
to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another
one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by
RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues
belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZα
vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated
from plant latex, confirming the presence of the preprocysteine peptidase in the latex. 相似文献
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Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Antarctic ice algae Chlamydomonas sp. ICE-L 总被引:1,自引:0,他引:1
Shenghao Liu Pengying Zhang Bailin Cong Chenlin Liu Xuezheng Lin Jihong Shen Xiaohang Huang 《Extremophiles : life under extreme conditions》2010,14(3):329-337
A cDNA encoding heat shock protein 70 of Antarctic ice algae Chlamydomonas sp. ICE-L (designated as CiHsp70) was identified by RT-PCR and rapid amplification of cDNA ends approaches. The full-length
cDNA of CiHsp70 was 2,232 bp, consisting of a 5′-terminal untranslated region (UTR) of 76 bp, a 3′-terminal UTR of 203 bp
with a poly (A) tail, and an open reading frame of 1,953 bp. The CiHsp70 cDNA encoded a polypeptide of 651 amino acids with
an ATPase domain of 388 amino acids, the substrate peptide binding domain of 246 amino acids and a C-terminus domain of 17
amino acids. The inducible CiHsp70 cDNA was highly homologous to other plant cytosolic Hsp70 genes and clustered together
with green algae and higher plant rather than brown algae, diatom and Cryptophyta. Antarctic ice algae were treated with different
stress conditions and messenger RNA (mRNA) expression levels of CiHsp70 were quantified by quantitative RT-PCR. The results
showed that both cold and heat shock treatments could stimulate CiHsp70 mRNA expression. Meanwhile, CiHsp70 mRNA expression
level increased 2.9-fold in response to UV-B radiation for 6 h, while the expression levels of CiHsp70 were remarkably increased
after removing the UV-B radiation and immediately providing additional 6 h visible light. Furthermore, treating with 62 or
93‰ NaCl for 2 h, CiHsp70 mRNA expression level increased 3.0- and 2.1-fold, respectively. Together, our observations revealed
that CiHsp70 as a molecular chaperone might play an important role in Antarctic ice algae Chlamydomonas sp. ICE-L acclimatizing to polar environment. 相似文献
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A full-length metallothionein-1(MT-1) cDNA was cloned from the Chinese mitten crab, Eriocheir sinensis, based upon the hepatopancreas cDNA library. The full-length cDNA contained a single 180 bp open reading frame that encoded
a 59 amino acid protein. The deduced amino acid sequence was cysteine (Cys)-rich, with residues observed in patterns characteristic
of other reported MTs: Cys–X–Cys, Cys–X–X–Cys, or Cys–X–X–X–Cys. Gene structure obtained via PCR yielded a 3816 bp gene, which
was comprised of three exons and two introns arranged in a “3 + 2” pattern. The cloned 5′flanking region (1,735 bp) contained
several predicted binding sites, which included MREs, AP-1, SP1, USF, GATA, HNF-1, and HSF. MT-1 mRNA expression analysis
revealed that while levels were highest in the hepatopancreas, expression was abundant in testis and thoracic ganglia, moderate
in intestine (P < 0.05), and weak in other tissues (P < 0.05). MT-1 mRNA expression exhibited reproductive variation in the male, with levels approximately tenfold greater in
August, during seasonal gonadal maturation, compared to other times of the year. Cu2+ exposure via tank water (0–1 mg/l for 7 days) resulted in a dose-dependent bell curve response in MT-1 mRNA expression, with
peak expression observed after exposure to 0.1 mg/l Cu2+. A time course experiment (0.1 mg/l Cu2+ over 9 days) revealed MT-1 mRNA expression peaked sharply on day 5 before gradually decreasing with prolonged exposure. In
the present report, we provide sequence analysis of the first MT-1 gene cloned in E. sinensis, and evidence that its physiological and toxicological regulation is evolutionary conserved. 相似文献