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1.
TouchdownPCR扩增溶藻弧菌依赖ATP的DNA解旋酶rep基因部分序列,得一1.2kb片段,再以反向PCR和巢式PCR联合扩增其侧翼序列,拼接得一由2016bp组成,共编码671aa的完整基因。该基因演绎的氨基酸序列与几种弧菌的同源性都比较高,与副溶血弧菌RIMD2210633、溶藻弧菌12G01、Vibriosp.Ex25、创伤弧菌YJ016、霍乱弧菌RC385、灿烂弧菌12801、费氏弧菌ES114及矿angustumS14分别为96%、95%、95%、94%、92%、91%、89%、86%。  相似文献   

2.
Vibrio fischeri strain Y-1 (ATCC 33715) emits light with a lambda max of 545 nm rather than the 485-nm emission typical of other strains of V. fischeri. The yellow emission is due to the interaction of the enzyme luciferase with a yellow fluorescent protein (YFP). On the basis of the N-terminal amino acid sequence of YFP, a mixed-sequence oligonucleotide probe was synthesized and used to isolate a 1.6-kbp HindIII fragment containing the first 208 bases of the gene that codes for YFP (luxY). Another synthetic oligonucleotide complementary to bases 167-184 of the YFP coding sequence was used to isolate a second (ca. 1.9 kbp) DNA fragment generated by digestion with both EcoRI and ClaI that contained the remainder of the luxY gene. The intact luxY gene, which encoded a 22,211-dalton polypeptide composed of 194 amino acid residues, was reconstructed from the two primary clones and is contained within a 765-bp SspI-XhoII fragment. Both strands of the entire luxY coding sequence were determined from the reconstructed gene, while the region surrounding the junction used in the reconstruction was also determined from the original partial clones. As with other genes that have been studied from V. fischeri, the luxY gene was unusually AT-rich. The sequence of luxY did not bear any apparent similarity to any of the sequences contained in the current GenBank database. Escherichia coli containing a plasmid with the luxY gene expresses a protein that reacts with antibody raised to authentic YFP.  相似文献   

3.
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   

4.
The gene encoding NADH dehydrogenase from an alkalophile, Bacillus sp., was cloned and sequenced. The cloned DNA fragment contained an open reading frame of 1,557 nucleotides which encodes a polypeptide composed of 519 amino acid residues (Mr 55,830). The predicted amino acid sequence was consistent with the partial amino acid sequences including the N-terminal and C-terminal sequences determined in a previous study. Sequence comparison with other flavoenzymes revealed high homology between the present dehydrogenase and Escherichia coli thioredoxin reductase.  相似文献   

5.
Degenerate primers were designed based on all possible sequences of the N-terminal and C-terminal regions of Delonix regia trypsin inhibitor (DrTI). Five hundred sixty-one bp of polymerase chain reaction (PCR) product was amplified using the above degenerate primers and genomic DNA and cDNA of Delonix regia as a template. The amplified PCR products were cloned and sequenced. DNA sequence analysis of cDNA and genomic clones of DrTI have the same nucleotide sequence in the coding region, and manifested a genomic clone without intervening sequences in the coding region. The amino acid sequence deduced from the DrTI genomic and cDNA clones agreed with that identified via amino acid sequencing analysis, except that two amino acid residues, Ser and Lys, existed between residues Lys141 and Ser142. DrTI open reading frame was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in Escherichia coli to yield a glutathione S-transferase (GST)-fusion protein with a calculated molecular mass of about 45 kDa. The recombinant DrTI (reDrTI) was derived by treating the GST-DrTI fusion protein with thrombin. Both the reDrTI and GST-DrTI fusion protein exhibited a strong identical inhibitory effect on trypsin activity.  相似文献   

6.
Transposon mutants of Pseudomonas putida P8 were generated by applying a mini-Tn5 mutagenesis system. The mutants obtained were checked for their ability to tolerate increased temperatures and elevated phenol concentrations. Approximately 5,800 transposon mutants were used to generate a pool of 600 temperature-sensitive strains; one of these strains was identified as being damaged in its ability to perform cis-trans isomerization of fatty acids. A gene library of P. putida P8 was constructed and screened by using as a probe sequences immediately adjacent to the mini-Tn5 insertion. A DNA fragment that complemented the mutation was isolated and cloned. The corresponding gene, termed cti, is located close to the methionine synthase locus (metH) in P. putida P8. A cti-carrying fragment integrated into a plasmid also conferred the ability for cis-trans isomerization to Escherichia coli; the cti gene was completely sequenced, and the amino acid sequence was deduced.  相似文献   

7.
Enterobacter sp. G-1 is a bacterium isolated previously as a chitinase-producing bacterium. We found this bacterium also produced N-acetylglucosaminidase and characterized that in this study. Extracellular N-acetylglucosaminidase of 92.0 kDa was purified near homogeneity by 8.57-fold from Enterobacter sp. G-1. The optimum temperature and the optimum pH of the purified N-acetylglucosaminidase was 45 degrees C and 6.0, respectively. The N-terminal amino acid sequence of 23 residues of N-acetylglucosaminidase was identified. Based on the N-terminal sequence, we amplified pieces of the DNA fragments by PCR. Using these PCR products as probes, we screened the genomic library and successfully isolated the entire N-acetylglucosaminidase gene (designated nag1) from Enterobacter sp. G-1. The nucleotide sequence of the nag1 gene was found to consist of 2,655 bp encoding a protein of 885 amino acid residues. Comparison of the deduced amino acid sequence from the nag1 gene found 97.3% identity with chitobiase from Serratia marcescens, 54.4% identity with N,N'-diacetylchitobiase from Vibrio harveyi, and 42.7% identity with N-acetylglucosaminidase (ExoI) from Vibrio furnissii. Enzymatic activity assay of N-acetylglucosaminidase indicated stronger activity toward PNP-GlcNAc than PNP-(GlcNAc)2 or PNP-(GlcNAc)3.  相似文献   

8.
The gene encoding the thermostable direct hemolysin of Vibrio parahaemolyticus was characterized. This gene (designated tdh) was subcloned into pBR322 in Escherichia coli, and the functional tdh gene was localized to a 1.3-kilobase HindIII fragment. This fragment was sequenced, and the structural gene was found to encode a mature protein of 165 amino acid residues. The mature protein sequence was preceded by a putative signal peptide sequence of 24 amino acids. A putative tdh promoter, determined by its similarity to concensus sequences, was not functional in E. coli. However, a promoter that was functional in E. coli was shown to exist further upstream by use of a promoter probe plasmid. A 5.7-kilobase SalI fragment containing the structural gene and both potential promoters was cloned into a broad-host-range plasmid and mobilized into a Kanagawa phenomenon-negative V. parahaemolyticus strain. In contrast to E. coli, where the hemolysin was detected only in cell lysates, introduction of the cloned gene into V. parahaemolyticus resulted in the production of extracellular hemolysin.  相似文献   

9.
Currently, there is no effective therapy for cryptosporidiosis and it is unclear why antifolate drugs which are effective treatments for infections caused by closely related parasites are not also effective against Cryptosporidium parvum. In protozoa, the target of these drugs, dihydrofolate reductase (DHFR), exists as a bifunctional enzyme also manifesting thymidylate synthase (TS) activity and is encoded by a fused DHFR-TS gene. In order to prepare a probe to isolate the C. parvum DHFR-TS gene we have used degenerate oligonucleotides whose sequences are based on strongly conserved regions of TS protein sequence to prime the polymerase chain reaction (PCR) with C. parvum DNA. The PCR amplified a 375-bp DNA fragment which was cloned and sequenced; the deduced amino acid sequence had significant identity with known TS sequences, including strict conservation of all phylogenetically invariant TS amino acid residues. The cloned PCR fragment was used as a probe to isolate a number of overlapping clones from a C. parvum genomic library which were definitively shown to be of cryptosporidial origin by genomic Southern and molecular karyotype analyses. The deduced protein sequence of C. parvum TS was most similar to the bifunctional TS enzymes of Plasmodium chabaudi and Plasmodium falciparum.  相似文献   

10.
A 7.1-kbp DNA fragment isolated from a wild strain of Klebsiella oxytoca was sequenced, leading to the identification of 10 open-reading frames (ORFs), including a 504-bp Pad gene. The Pad gene of the Gram-negative bacterium was subsequently expressed in Escherichia coli as a chimeric Pad. The deduced amino acid (AA) sequence of the Pad gene from wild-type K. oxytoca showed approximately 50% homology to those of other bacterial PADs from Gram-positive bacilli plus a coccus. These data and a genomic library search of some gamma-proteobacteria, including E. coli and Vibrio sp., indicated that PAD of K. oxytoca is a member of the bacterial PAD family characteristic of Gram-negative bacteria. Using Pad-specific PCR primers designed from the Gram-negative bacterial Pad of K. oxytoca, Pad genes of two further strains of K. oxytoca, another wild isolate and JCM 1665 and two PAD-positive Enterobacter spp. were successfully amplified for specific Pad detection.  相似文献   

11.
Abstract A DNA fragment of 550 bp was specifically amplified by PCR with primers based on the N-terminal sequence of the purified 3-hexulose-6-phosphate synthase from Methylomonas aminofaciens 77a and on that of a lysyl endopep(idase-derived peptide. Using this PCR product as a probe, a gene coding for 3-hexulose-6-phosphate synthase in M. aminofaciens 77a chromosomal DNA was cloned in Escherichia coli JM109. Sequencing analysis revealed that the gene encoding 3-hexulose-6-phosphate synthase contained a 624-bp open reading frame, encoding a protein composed of 208 amino acid residues with a calculated relative molecular mass of 21 224.  相似文献   

12.
13.
在完成小花棘豆毒素 95 %氨基酸序列的基础上 ,根椐已知的氨基酸序列 ,设计合成了特异简并引物 .以小花棘豆总RNA为模板 ,逆转录合成cDNA第一链 ,用置换法合成双链cDNA .用特异引物对此双链cDNA进行PCR扩增 ,将扩增后的目的基因与用SmaⅠ酶切的质粒pUC 18连接 ,转化大肠杆菌JM10 7.筛选阳性克隆进行序列分析 ,获得了OXY基因的全部序列 .经测序后测得基因序列与原氨基酸序列对照完全一致 .GenBnak数据检索说明 ,OXY基因编码序列确定是一个从未报道的序列 .此研究结果对该毒素的应用研究奠定了基础 .  相似文献   

14.
Bacillus subtilis WHNB02植酸酶phyC基因的克隆及序列分析   总被引:1,自引:0,他引:1  
采用PCR法获得产植酸酶芽孢杆菌(Bacillus subtilis)WHNB02株植酸酶的全长phyc基因,并将其克隆到pUC18-T载体。序列分析表明该基因全长1152bp,编码一个383个氨基酸的多肽,信号肽切割位点位于第26个氨基酸残基之后。系统进化树表明,来源于7株芽孢杆菌的植酸酶在遗传上分为两大类。将Bacillus subtilis WHNB02植酸酶phyC基因序列及其氨基酸序列在GenBank中登录,登录号分别为AF220075和AA043434.1。  相似文献   

15.
The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17 x b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp-Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-AspAsp-Val-Leu -Leu-Val- Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.  相似文献   

16.
霍乱弧菌zot基因的克隆及其在大肠杆菌中的表达   总被引:2,自引:0,他引:2  
从霍乱疫苗菌中抽提基因组DNA,用PCR的方法扩增zot基因。序列分析表明,zot基因编码399个氨基酸,其中4个氨基酸与文献报道有差异。将zot基因插入含T7启动子的质粒pET-28(a+)构建表达质粒pET-ZOT,转化大肠檑菌BL21(DE3)筛有达菌株BLZOT。表达菌株经1mmol/LT IPTG诱导表达3-5h后,表达大量ZOT蛋白,并形成包涵体,经SDS-PAGE分析重组ZOT蛋白分  相似文献   

17.
Monoacylglycerol lipase [MGLP, EC 3.1.1.23] is produced intracellularly by the moderately thermophilic Bacillus sp. strain H-257. The gene encoding MGLP was cloned, sequenced, and expressed in Escherichia coli. A genomic library of Bacillus sp. strain H-257, prepared in the plasmid vector pACYC184, was screened with a 0.2-kbp DNA fragment amplified by the polymerase chain reaction (PCR) with oligonucleotide primers designed based on the amino acid sequence of a purified MGLP. The plasmid pMGLP31, identified by hybridization with the amplified DNA fragment, contained a 5.3-kbp insert from Bacillus sp. strain H-257 DNA. Sequence analysis of the MGLP gene revealed an open reading frame encoding MGLP consisting of 250 amino acids, with a calculated molecular mass of 27.4 kDa. The deduced amino acid sequence of MGLP contained the consensus pentapeptide (-Gly-Xaa-Ser-Xaa-Gly-), which is conserved among lipases, esterases, and serine proteases. The MGLP is homologous to a putative esterase/lipase from Streptomyces coelicolor (41.8% homology). When pMGLP31 was introduced into E. coli DH1, the transformants produced MGLP intracellularly as an active form to an approximately 13.8-fold greater extent than Bacillus sp. strain H-257. The purified recombinant MGLP was shown to be identical to the native enzyme in terms of chromatographic behavior, isoelectric point, and physicochemical and catalytic properties.  相似文献   

18.
Arthrobacter oxydans P52 isolated from soil samples was found to degrade the phenylcarbamate herbicides phenmedipham and desmedipham cometabolically by hydrolyzing their central carbamate linkages. The phenylcarbamate hydrolase (phenmedipham hydrolase) responsible for the degradative reaction was purified to homogeneity. The enzyme was shown to be a monomer with a molecular weight of 55,000. A 41-kb wild-type plasmid (pHP52) was identified in A. oxydans P52, but not in a derivative of this strain that had spontaneously lost the ability to hydrolyze phenylcarbamates, indicating that the gene for phenylcarbamate degradation (pcd) is plasmid encoded. Determination of two partial amino acid sequences allowed the localization of the coding sequence of the pcd gene on a 3.3-kb PstI restriction fragment within pHP52 DNA by hybridization with synthetic oligonucleotides. The phenylcarbamate hydrolase was functionally expressed in Escherichia coli under control of the lacZ promoter after the 3.3-kb PstI fragment was subcloned into the vector pUC19. A stretch of 1,864 bases within the cloned Pst fragment was sequenced. Sequence analysis revealed an open reading frame of 1,479 bases containing the amino acid partial sequences determined for the purified enzyme. Sequence comparisons revealed significant homology between the pcd gene product and the amino acid sequences of esterases of eukaryotic origin. Subsequently, it was demonstrated that the esterase substrate p-nitrophenylbutyrate is hydrolyzed by phenmedipham hydrolase.  相似文献   

19.
K Inatomi 《DNA research》1998,5(6):365-371
The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.  相似文献   

20.
平胸龟Sox基因的克隆和序列分析   总被引:9,自引:1,他引:9  
本文采用PCR技术扩增和克隆了平胸龟的Sox基因,并通过银染测序进行了序列分析。结果显示,平胸龟雌雄个体均能扩增出相同长度的片段,其DNA序列与人SRY基因的HMG-box同源性达72.7%,推测的氨基酸序列与SRY基因的HMG-box序列同源性为46.5%,充分显示出Sox基因在系统进化上的保守性。本文为Sox基因的起源和进化研究提供了资料。  相似文献   

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