Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.     

大肠杆菌ubiC基因的克隆及序列分析

研究以克隆得到正确序列的大肠杆菌ubiC基因目的,实验通过PCR方法从大肠杆菌基因组中扩增得到了ubiC基因,扩增产物克隆到pUC118载体,转化大肠杆菌JM109,DNA序列分析结果表明克隆得到的大肠杆菌ubiC基因碱基序列正确。     

Overproduction of the Bacillus sphaericus R modification methylase in Escherichia coli and its purification to homogeneity

A DNA fragment containing the information coding for the GGCC-specific Bacillus sphaericus R modification methylase, BspR, was inserted into plasmid vector pKK223-3 under the control of the strong and inducible tac promoter, and transformed into Escherichia coli HB101. Upon induction this strain accumulated the methylase enzyme (while cell growth was inhibited) up to several percent of total cellular protein. Homogeneous methylase could be prepared in three purification steps.     

嘌呤核苷磷酸化酶基因的克隆及原核表达载体的构建

通过PCR方法从产气肠杆菌、胡萝卜软腐欧文氏菌、大肠杆菌扩增嘌呤核苷磷酸化酶(PNPase)基因,然后将扩增的约720bp的基因片段克隆到pET-28b表达载体上,构建重组PNPase的表达载体。核苷酸及推导的氨基酸序列分析表明,该基因在三个菌株之间有很高的同源性。SDS-PAGE电泳结果显示出明显的特异性蛋白质条带,其分子量约为29.8kDa.该载体的构建为进一步研究核苷及其类似物的生物合成奠定基础。     

Molecular cloning and expression of an extracellular nuclease of Serratia marcescens in Escherichia coli

Abstract The gene encoding an extracellular nuclease of Serratia marcescens was cloned in Escherichia coli using the vector pBR322. Transformants were selected by their ability to grow in the presence of ampicillin, and nuclease-positive clones were detected on a commercially available DNase test agar. The production of a nuclease could be detected in recombinant strains and enzyme activity was found in culture supernatants of such strains. Deletion derivatives of the parental recombinant plasmid were constructed to define the region of DNA encoding the expression of the nuclease. The smallest DNA fragment found to produce the nuclease was determined to be 2.2 kb in length, although a somewhat smaller fragment appeared to be partially active.     

酿酒酵母海藻糖合成酶基因的克隆和在大肠村菌中的表达

用PCR方法克隆了1．5kb的酿酒母Sacchromyces cerevisiae海藻糖合成酶基因TPSI,将该片段连接到pUC19载体,通过转化分别引入海藻糖合成酶基因缺失和缺陷的大肠杆菌Escherichia coli FF4169 和FF4050,对转化株的质粒DNA酶切分析表明均含有1．5kb PCR克隆片段,生长曲线实验证明,带有克隆片段的转化株在含0．5mol/L NaCl的高渗透压基础培养基中生长良好;用高效液相色谱（HPLC）结合蒸发散射（ELSD）技术测定细胞内海藻糖实验证明转化株能够合成海藻糖。     

稻胚凝集素基因的克隆,序列分析及表达

以水稻基因组ＤＮＡ为模板,以特异引物经聚合酶链式反应方法扩增出稻胚凝集素基因并克隆到Ｅ．ｃｏｌｉ质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）的ＳｍａⅠ位点。序列分析表明,克隆到的基因片段大小为７８１ｂｐ,没有内含子,编码１条长２２７个氨基酸、分子量约２３ｋＤ的肽链,其中Ｎ－端２８个氨基酸是信号肽。与报道的稻胚凝集素ｃＤＮＡ序列进行顺序同源性比较,发现它们之间有很高的同源性（９９．７４％）,其编码区第１６７     

灵杆菌非特异性核酸酶的原核表达、纯化及活性分析

以灵杆菌基因组DNA为模板,PCR扩增非特异性核酸酶 (Non-specific nuclease,NU) 基因,并克隆到pMAL-c4X载体上构建重组表达载体pMAL-c4X-NU。经测序及 BLASTN发现其与灵杆菌Serratia marcescens核酸酶基因的同源性为97％。将构建的表达载体pMAL-c4X-NU转入大肠杆菌BL21,经IPTG诱导实现了胞内表达78 kDa的麦芽糖结合蛋白-NU融合蛋白 (Maltose-binding protein-NU,MBP-NU),其最佳诱导表达条件为37 ℃,0.75 mmol/L IPTG诱导1.5 h。用Amylose resin纯化得到了目的蛋白。活性检测表明MBP-NU具有同时降解DNA和RNA的活性,在37 ℃、pH 8.0时活性最高,比活力为1.11×106 U/mg,目标蛋白的纯化效率可达10.875 mg/L。纯化的目标蛋白中无蛋白酶活性存在。0.5 mmol/L乙二胺四乙酸 (Ethylene diamine tetraacetic acid,EDTA)、1 mmol/L苯甲基磺酰氟 (Phenylmethanesulfonyl fluoride,PMSF) 以及150 mmol/L KCl对MBP-NU的活性几乎无影响,因此MBP-NU可作为蛋白质纯化过程中核酸的高效降解酶。     

Construction of a human full-length cDNA bank

We aimed to construct a full-length cDNA bank from an entire set of human genes and to analyze the function of a protein encoded by each cDNA. To achieve this purpose, a multifunctional phagemid shuttle vector, pKAl, was constructed for preparing a high-quality cDNA library composed of full-length cDNA clones which can be sequenced and expressed in vitro and in mammalian cells without subcloning the cDNA fragment into other vectors. Using this as a vector primer, we have prepared a prototype of the bank composed of full-length cDNAs encoding 236 human proteins whose amino acid sequences are identical or similar to known proteins. Most cDNAs contain a putative cap site sequence, some of which show a pyrimidine-rich conserved sequence exhibiting polymorphism. It was confirmed that the vector permits efficient in vitro translation, expression in mammalian cells and the preparation of nested deletion mutants.     

Molecular cloning and physical map of bacteriophage PM2 DNA

The entire genome and the DNA fragments of the lipid-containing bacteriophage pM2 were cloned in the pBR322 plasmid vector. A physical map including the sites for the following restriction enzymes was obtained: HpaII, HaeIII, TthI, Sau96I, AvaII, PstI, BstNI, AccI, HincII, HpaI and HindIII. No restriction sites on PM2 DNA were found for BalI, BamHI, BclI, BglI, BglII, BstEII, KpnI, PvuII, SacI, SalI, Sau3A, XbaI and XhoI.     

丁酸梭菌1，3-丙二醇氧化还原酶基因dhaT的克隆及在大肠杆菌中的表达

以丁酸梭菌（Clostridium butyricum）基因组DNA为模板,利用PCR技术扩增得到1,3-丙二醇氧化还原酶基因dhaT,将它连接到pMD18一T载体上,得到重组质粒pMD—dhaT,对此重组质粒进行序列测定,对其DNA序列分析表明,dhaT基因全长为1 158bp。将dhaT基因插入表达载体pSE-380中,构建成重组子pSE—dhaT,并在大肠杆菌JMl09中进行诱导表达。研究表明,以1,3-丙二醇为底物时,基因工程菌在37℃下,以1．0mmol／L IPTG诱导14h,酶活力达到16．28U／mL,比原始菌株提高5、6倍。     

烟实夜蛾触角化学感受蛋白cDNA的克隆、序列分析及在大肠杆菌中的表达

利用RT-PCR技术扩增了编码烟实夜蛾 Helicoverpa assulta 触角化学感受蛋白（chemosensory protein）的全长cDNA。克隆和测序结果表明,烟实夜蛾化学感受蛋白基因核苷酸序列全长384 bp(GenBank序列号： DQ285667),编码127个氨基酸残基,预测N-末端包含16个氨基酸组成的信号肽序列,因此估测其成熟蛋白分子量为12.97 kD,等电点为5.32。将该基因重组到表达载体pGEX-4T₂中,并转入原核细胞中进行表达。SDS-PAGE和Western印迹分析表明,经IPTG诱导后,烟实夜蛾化学感受蛋白基因能在大肠杆菌BL21中表达,电泳检测到一条约39 kD的外源蛋白,与预测的融合蛋白分子量大小相符。     

Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12

We have cloned genes responsible for enterobactin synthesis (entD) and transport (fepA,fes) from Escherichia coli K-12. Relevant recombinant plasmids enabled EntD- and transport-defective mutants to grow on iron-limiting medium. Subcloning and deletion analysis demonstrated that the gene order is entD-fepA-fes. Protein synthesis studies in minicells suggest that FepA is first translated as an Mr 84 000 precursor, which is subsequently cleaved to the active Mr 81 000 receptor; the fes gene product is an Mr 44 000 protein; no polypeptide has been identified as the entD gene product.     

Clostridium difficile Hfq can replace Escherichia coli Hfq for most of its function

A gene for the Hfq protein is present in the majority of sequenced bacterial genomes. Its characteristic hexameric ring-like core structure is formed by the highly conserved N-terminal regions. In contrast, the C-terminal forms an extension, which varies in length, lacks homology, and is predicted to be unstructured. In Gram-negative bacteria, Hfq facilitates the pairing of sRNAs with their mRNA target and thus affects gene expression, either positively or negatively, and modulates sRNA degradation. In Gram-positive bacteria, its role is still poorly characterized. Numerous sRNAs have been detected in many Gram-positive bacteria, but it is not yet known whether these sRNAs act in association with Hfq. Compared with all other Hfqs, the C. difficile Hfq exhibits an unusual C-terminal sequence with 75% asparagine and glutamine residues, while the N-terminal core part is more conserved. To gain insight into the functionality of the C. difficile Hfq (Cd-Hfq) protein in processes regulated by sRNAs, we have tested the ability of Cd-Hfq to fulfill the functions of the E. coli Hfq (Ec-Hfq) by examining various functions associated with Hfq in both positive and negative controls of gene expression. We found that Cd-Hfq substitutes for most but not all of the tested functions of the Ec-Hfq protein. We also investigated the role of the C-terminal part of the Hfq proteins. We found that the C-terminal part of both Ec-Hfq and Cd-Hfq is not essential but contributes to some functions of both the E. coli and C. difficile chaperons.     

Expression of human dihydrofolate reductase cDNA and its induction by chloramphenicol in Bacillus subtilis

A bifunctional plasmid (pMP358) able to replicate and to express cloned human dihydrofolate reductase cDNA (cDHFR) in both Escherichia coli and Bacillus subtilis was constructed. The expression of cDHFR in B. subtilis was the result of a deletion that placed the cDNA fragment under the control of the chloramphenicol acetyltransferase (CAT) gene promoter of Staphylococcus aureus plasmid pC194. By sequence analysis of plasmid pMP358, we observed a gene fusion occurring between the cDHFR and the 32nd codon of the CAT gene. We report that such a “hybrid” gene is able to direct the synthesis of a 25-kDal “hybrid” protein, which was found to be inducible by supplementing B. subtilis cells with sublethal doses of chloramphenicol.     

Characterization of a small cryptic plasmid from Salmonella enteritidis that affects the growth of Escherichia coli

Abstract We examined the plasmid content of 25 clinical isolates of Salmonella enteritidis , and detected the presence of small plasmids (3–5.3 kb) in 9 of them, alone, or in addition to the large, so-called virulence plasmid. A 5.3-kb plasmid isolated as unique extrachromosomal DNA from a strain responsible for a high-mortality outbreak was characterized by restriction mapping and cloning. The plasmid replicon was localized in a 1.7-kb fragment, that hybridized with three of the small plasmids detected in S. enteritidis , and with another small plasmid from Salmonella typhimurium . A strain of Escherichia coli carrying this plasmid, or a cloned 3.7-kb Pvu II restriction fragment, showed a slower growth rate, especially in minimal medium, as well as a noticeable increase in DNA methyltransferase activity.