A structural comparison of lipopolysaccharides from two strains of Helicobacter pylori, of which one strain (442) does and the other strain (471) does not stimulate pepsinogen secretion.

Lipopolysaccharides (LPSs) from strains of Helicobacter pylori (442 and 471), which differed in stimulation of pepsinogen secretion, were isolated as water-soluble material of high-M(r), and as water-insoluble gels of low-M(r). Chemical and spectroscopic analyses of soluble LPS and oligosaccharides liberated from the gels led to proposed structures with Lewis (Le) antigen termini connected to N -acetyllacto-saminoglycans of alternating 3-linked beta-D-Gal and 4-linked beta-D-GlcNAc residues with various laterally attached glycosyl substituents. The LPS of H.pylori 442 was similar to previously examined strains (NCTC 11637 and P466) in having partially glycosylated chains with alpha-L-Fuc units attached to O-3 of the majority of GlcNAc residues in Le(x)units, and in chain termination with Le(x)or Le(y)determinants. In contrast, terminal Le(y)units occurred in LPS of H.pylori 471 and glycosaminoglycan chains carried a smaller proportion of alpha-L-Fuc units, but at O-6 of a majority of nonfucosylated GlcNAc residues, there was a novel type of branching with alpha-D-Gal substituents. Evidence for the branched regions was obtained from(1)H-NMR spectra and from characterization of oligosaccharides formed by the action of endo-beta-galactosidase. Examination of oligosaccharides liberated from water-insoluble LPS gels of H.pylori 442 and 471 provided evidence for similar core OS structures to those from other H.pylori strains but interesting differences were observed.     

Sequence structure and expression of a cloned beta-glucosidase gene from an extreme thermophile

Summary The gene for a -glucosidase from the extremely thermophilic bacterium Caldocellum saccharolyticum has been isolated from a genomic library and sequenced. An open reading frame identified by computer analysis of the sequence could encode a protein of M_r 54400, which is close to the size of the polypeptide experimentally determined using maxicells. Analysis of the amino-terminal residues of the protein produced in Escherichia coli suggests that it is processed by a methionine aminopeptidase. A sequence within C. saccharolyticum DNA upstream of the -glucosidase gene was found to act as a promoter for expression of the thermophile gene in E. coli. The protein has been overproduced in E. coli and Bacillus subtilis where it retains its enzymatic activity and heat stability. There appears to be a single copy of the gene in Caldocellum DNA.     

不对称水解(R,S)-2,6-二甲基苯基氨基丙酸甲酯新菌种的分离鉴定及酯酶基因的克隆、表达

【目的】筛选鉴定1株可以选择性水解农药甲霜灵的中间体(R,S)-2,6-二甲基苯基氨基丙酸甲酯(MAP)的菌株,并克隆、表达该菌株中的酯酶基因。【方法】以MAP为唯一碳源,对活性污泥样品中的微生物进行富集培养,采用罗丹明B平板显色法进行初筛,通过摇瓶复筛得到了1株对MAP具有最高对映体选择性和水解活力的新菌株,根据其形态、生理生化特征及16S rRNA序列分析,确立该菌株的系统发育学地位。构建该菌株的基因文库,筛选获得含目的基因的克隆子,通过序列分析和引物扩增得到酯酶基因,将基因与表达载体pET28a(+)连接后,转化大肠杆菌BL21Gold(DE3)plysS,构建重组菌。【结果】该菌属于革兰氏阴性菌,结合16S rRNA基因、形态特征和生理生化实验结果,鉴定该菌为反硝化无色杆菌。通过基因文库法,找到了该菌中的酯酶基因EHest,并成功构建了重组大肠杆菌EHest-p ET28a(+)-BL21Gold(DE3)plys S,表达了来自Achromobacter denitrificans 1104且具有不对称水解MAP活性的酯酶EHesterase,大小约27 kDa,表达酶活是原始菌株的27.1倍。用EHesterase催化MAP水解,底物浓度50 g/L,反应1 h,底物转化率为29.5%,产物(酸)的ee_p为85.1%,对映体选择性为R型。该酶的最适反应pH和温度分别为pH 9.0和50°C。它水解MAP的活性分别是水解橄榄油和乙酸乙酯活性的333倍和667倍。【结论】筛选到1株具有不对称水解MAP能力的新菌株Achromobacter denitrificans 1104。     

Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which does not have deaminoneuraminic acid (KDN) 9-phosphate synthase activity

A cDNA of the mouse homologue of Escherichia coli N-acetylneuraminic acid (Neu5Ac) synthase (neuB gene product) was cloned by the PCR-based method. The mouse homologue consists of 359 amino acids, and the cDNA sequence displays 33% identity to that of the E. coli Neu5Ac synthase. The recombinant mouse homologue which is transiently expressed in HeLa cells does not exhibit the Neu5Ac synthase activity, which catalyzes condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc) to synthesize Neu5Ac, but the Neu5Ac 9-phosphate (Neu5Ac-9-P) synthase activity, which catalyzes condensation of PEP and ManNAc 6-phosphate (ManNAc-6-P) to synthesize Neu5Ac-9-P. Thus, the mouse homologue of E. coli Neu5Ac synthase is the Neu5Ac-9-P synthase. The Neu5Ac-9-P synthase is a cytosolic enzyme and ubiquitously distributed in mouse various tissues. Notably, the Neu5Ac-9-P synthase can not catalyze the synthesis of deaminoneuraminic acid (KDN) or KDN-9-P from PEP and Man or ManNAc-6-P, thus suggesting that the enzyme is not involved in the synthesis of KDN. This is consistent with the previous observation that only a very low activity to synthesize KDN is found in mouse B16 cells [Angata, T., et al. (1999) Biochem. Biophys. Res. Commun. 261, 326-331].     

The cloning and expression of a gene encoding haemolytic activity from the fish pathogen Renibacterium salmoninarum

A gene encoding haemolytic activity from Renibacterium salmoninarum (strain PPD) was cloned into Escherichia coli using the cosmid vector pHC79, and subsequently subcloned on a 1.6 kbp SAlI fragment into pBR328. Southern blot hybridisation revealed that a homologous sequence is found in other strains of R. salmoninarum.     

Cloning and DNA sequencing of xyaA, a gene encoding an endo-beta-1,4-xylanase from an alkalophilic Bacillus strain (N137).

The gene xyaA encoding an alkaline endo-beta 1,4-xylanase from an alkalophilic Bacillus sp. strain (N137) isolated in our laboratory was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1,656-bp DNA fragment containing xyaA was determined, revealing one open reading frame of 993 bp that encodes a xylanase (XyaA) of 39 kDa. This xylanase lacks a typical putative signal peptide, yet the protein is found in the Bacillus culture supernatant. In Escherichia coli, the active protein is located mainly in the periplasmic space. The xylanase activity of the cloned XyaA is an endo-acting enzyme that shows optimal activity at pH 8 and 40 degrees C. This activity is stable at a pH between 6 and 11. Incubations of XyaA at 40 degrees C for 1 h destroyed 45% of the activity.     

Isolation of halophilic and halotolerant bacteria from a Japanese salt field and comparison of the partial 16S rRNA gene sequence of an extremely halophilic isolate with those of other extreme halophiles

Halophilic and halotolerant bacteria were isolated from soil samples of a Japanese salt field, an environment where salt concentrations vary annually. From 1 g of each of the five samples collected, over 1×10³ bacterial colonies (colony forming units (cfu)g^-1) grew on agar medium containing 2M Na⁺. In contrast, 0–4 bacterial colonies (cfu g^-1) were observed on agar medium containing 4M Na⁺. Two of the five samples contained numerous bacteria (10²–10³ cfu g^-1) capable of growth on a 2M Na⁺ alkaline (pH=9.5) medium, while few bacterial colonies were observed from the other three samples. Only one of the five samples was shown to contain bacteria capable of growth on a 4M Na⁺ alkaline medium. Most of the bacteria isolated on 4M Na⁺ agar were eubacteria, but one extreme halophile (TR-1, already described as Haloarcula japonica JCM7785) was also isolated. The 16S rRNA sequence of TR-1 was determined and shows high homology (94.4–98.5%) to Ha. marismortui and Ha. sinaiiensis. These results suggested that: 1) environments with seasonally varying salinity can harbour halotolerants as well as halophiles and, 2) closely related halophiles can be isolated from geographically distant habitats.     

Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50-96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus, Photorhabdus luminescens W14 P. luminescens TTO1, and Yersinia pestis CO92. The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity.     

Cloning and expression in Escherichia coli of the gene encoding an extracellular deoxyribonuclease (DNase) from Aeromonas hydrophila.

The gene encoding an extracellular DNase from Aeromonas hydrophila CHC-1 has been cloned and sequenced. Following expression of the dns in Escherichia coli, it was revealed that some of the cloned enzyme was present in the cell-free extracellular supernatant fluid, and there was no cell lysis and concurrent release of cytoplasmic or periplasmic proteins. Therefore, results suggest that E. coli cells were capable of secreting the DNase extracellularly, albeit very inefficiently. The dns is transcribed from its own promoter in E. coli, and expressed as a 25-kDa product, as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of the culture supernatant preparations followed by a DNA-hydrolysis assay. Nucleotide sequence analysis predicted a single open reading frame of 690 bp encoding a 230-amino acid (aa) polypeptide, with a potential 20-aa signal peptide located at the N terminus of the predicted protein. The deduced aa sequence of the entire protein is highly homologous with that of the DNase of Vibrio cholerae.     

麻类脱胶高效菌株果胶裂解酶基因克隆与表达

【目的】克隆麻类脱胶高效菌株Dickeya sp.DCE-01的果胶裂解酶基因并进行原核表达,对表达产物进行纯化和酶学性质研究。【方法】根据该菌株全基因组序列预测的果胶裂解酶基因Q59419设计引物,PCR扩增后将该基因连接到pEASY-E1和pACYCDuet-1载体上,导入E.coli BL21(DE3)进行表达。选择酶活力高的阳性克隆子进行大量诱导表达后,采用超滤和Sephadex G-100凝胶层析两步法纯化出果胶裂解酶,研究其酶学性质。【结果】克隆到果胶裂解酶基因pel(GenBank登录号:JX964997),其序列全长1 128 bp,编码375个氨基酸。pACYCDuet-1-pel-BL表达胞外果胶裂解酶活力最高,发酵液粗酶活达298.8 IU/mL。其最适反应温度为50°C,最适pH为9.0;保温1 h,酶活稳定温度≤45°C,稳定pH为9.0?10.0。酶催化作用依赖于Ca2+,其最适作用浓度为2 mmol/L;Zn2+、Ca2+和NH4+促进酶活力,Fe3+和Pb2+严重抑制酶活力;聚半乳糖醛酸钠为该酶的最适底物。【结论】从麻类脱胶高效菌株中发掘到碱性果胶裂解酶基因,其表达产物在生物质加工过程中具有重要工业化应用前景。     

Analysis of the Salmonella fim gene cluster: Identification of a new gene (fimI) encoding a fimbrin-like protein and located downstream from the fimA gene

Abstract The fimA gene coding for the major component (fimbrin) of type 1 fimbriae was mapped within the Salmonella typhi fim gene cluster, and its nucleotide sequence determined. The deduced amino acid sequence of S. typhi fimbrin is highly homologous to that of S. typhimurium type 1 fimbrin and showed similarity to that of other enterobacterial type 1 fimbrins. Downstream of fimA , an open reading frame was found, named fimI , able to encode a fimbrin-like protein. The fimI product could represent the counterpart, in type 1 fimbriae, of the PapH protein involved in cell anchoring and length modulation of Escherichia coli Pap pili. This genetic organization was found to be common to other Salmonella serovars, including S. typhimurium and S. choleraesuis .     

Cloning,expression and evolution of the gene encoding the elongation factor 1alpha from a low thermophilic Sulfolobus solfataricus strain

The gene encoding the elongation factor 1alpha (EF-1alpha) from the archaeon Sulfolobus solfataricus strain MT3 (optimum growth temperature 75 degrees C) was cloned, sequenced and expressed in Escherichia coli. The structural and biochemical properties of the purified enzyme were compared to those of EF-1alpha isolated from S. solfataricus strain MT4 (optimum growth temperature 87 degrees C). Only one amino acid change (Val15-->Ile) was found. Interestingly, the difference was in the first guanine nucleotide binding consensus sequence G(13)HIDHGK and was responsible for a reduced efficiency in protein synthesis, which was accompanied by an increased affinity for both guanosine diphosphate (GDP) and guanosine triphosphate (GTP), and an increased efficiency in the intrinsic GTPase activity. Despite the different thermophilicities of the two microorganisms, only very marginal effects on the thermal properties of the enzyme were observed. Molecular evolution among EF-1alpha genes from Sulfolobus species showed that the average rate of nucleotide substitution per site per year (0.0312x10(-9)) is lower than that reported for other functional genes.     

Pre-mRNA processing enhancer (PPE) element increases the expression of an intronless thymidylate synthase gene but does not affect intron-dependent S phase regulation

The pre-mRNA processing enhancer (PPE) element is an RNA sequence element derived from the intronless HSV-TK gene. Insertion of the element into the highly intron-dependent human β-globin gene leads to efficient expression in the absence of splicing. We have analyzed the effect of the PPE element on the expression of mouse thymidylate synthase (TS) minigenes. We have previously shown that the expression of intronless TS minigenes is moderately (up to 20-fold) stimulated by the inclusion of introns. Furthermore, S phase-specific expression of TS minigenes in growth-stimulated cells depends on the presence of a spliceable intron as well as the TS promoter. The goal of our study was to determine if the PPE element would overcome the dependence on introns for efficient expression and for S phase-specific expression of transfected TS minigenes. We found that insertion of the PPE element into an intronless TS minigene partially overcame intron dependence. However, the increase in expression was much less than that observed for the intronless β-globin gene. We also found that intronless TS or HSV-TK genes that contained the PPE element and that were driven by the TS promoter were expressed at a constant level in serum-stimulated cells. However, when an intron was included in these genes, they were expressed in an S phase-specific manner. Thus the PPE element was not able to overcome the dependence on introns for S phase-specific expression of TS minigenes. J. Cell. Biochem. 69:104–116, 1998. © 1998 Wiley-Liss, Inc.     

Cloning and expression of a gene encoding a root specific nitrate reductase in bean (Phaseolus vulgaris)

A structural gene encoding nitrate reductase (NR) in the legume Phaseolus vulgaris cv. Saxonia has been cloned and sequenced. The NR gene encodes a protein of 881 amino acids with a MW of 99.2 kDa. The coding sequence is interrupted by three introns, which are located in the molybdopterin cofactor binding domain. In the flanking regions the signals of a functional eukaryotic gene are present. The gene is the smallest NR gene so far identified in higher plants. Comparison to other NRs shows homology ranging from 65 to 74% at the amino acid level. The similarity is highest for the three functional domains, and lowest in the N-terminal end of the protein. mRNA studies demonstrate that the gene is nitrate inducible and expressed exclusively in the roots of bean. Southern blot analysis indicates the presence of a second NR gene in bean. This gene may encode a NR enzyme expressed in leaves.     

Inheritance of resistance to potato viruses Y and A in progeny obtained from potato cultivars containing gene Ry:evidence for a new gene for extreme resistance to PVA

Extreme resistance in cultivated potato (Solanum tuberosum) to potato viruses Y and A (PVY and PVA) conditioned by the presence of Ry genes introduced from Solanum stoloniferum was described by Cockerham (1970). Cockerham detailed a number of genes which controlled a variety of reactions, including extreme resistance to both viruses (i.e. little or no visible reaction of plants and no viral replication following graft and manual inoculation) controlled by gene Ry _sto. In the present study, cvs Pirola and Barbara, which contain a Ry gene, were found to have extreme resistance to PVY isolates from the ordinary (PVY°), veinal necrosis (PVY^N) and potato tuber necrotic ringspot (PVY^NTN) subgroups, and PVA. The inheritance of this phenotype was examined in seedling progenies obtained by crossing Barbara and Pirola with susceptible cultivars. Segregation data for resistance to PVY and PVA in a progeny involving cv Pirola best fitted a genetical model of one gene controlling extreme resistance to both PVY and PVA, although the possibility that there are two genes, each controlling resistance to one virus but closely linked, cannot be excluded. Segregation data from progenies involving cv Barbara best fitted a genetical model in which there are two independent genes, one controlling extreme resistance to PVA and PVY and a second gene controlling extreme resistance to PVA but not to PVY. This previously unrecognised gene conferring extreme resistance to PVA only, should be given the notation Ra in keeping with nomenclature used for other resistance genes.     

Molecular cloning of cDNA encoding a ubiquitin-activating enzyme (E1) from goldfish (Carassius auratus) and expression analysis of the cloned gene

Destruction of cyclin B is required to the mitotic and meiotic cycles. A cyclin-specific ubiquitinating system, including ubiquitin-activating enzyme (E1), is thought to be responsible for cyclin B destruction. Here we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, E1 from goldfish ovary. The cloned cDNA is 4069 bp long and encodes 1059 amino acids. The deduced amino acid sequence is highly homologous to E1 from other species. Recombinant goldfish E1 could transfer ubiquitin to cyclin-selective ubiquitin-conjugating enzyme. Tissue distribution revealed a single 4.0-kb message ubiquitous among tissues.