Expression of an endoglucanase gene fromClostridium cellulolyticum inEscherichia coli

Summary A gene coding for an endoglucanase from the anaerobic cellulolytic bacteriumClostridium cellulolyticum has been cloned by direct selection inEscherichia coli, using the carboxymethyl cellulose-Congo Red assay. The cloned gene has been subcloned in the two possible orientations in pUC plasmids. One of the two resulting constructs exhibited a higher level of expression, which was associated with a high level of plasmid instability. The enzyme synthesized inE. coli from the cloned gene has been characterized by two procedures, maxicells and gel filtration chromatography, as a polypeptide of approximately 40 kilodaltons.     

Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor alpha

A gene encoding human tumour necrosis factor alpha (TNF-alpha) has been chemically synthesized, cloned and expressed to yield a biologically active protein in Escherichia coli. The 480-bp gene was assembled by enzymic ligation of 32 oligonucleotides, cloned directly into M13mp18 for sequence verification and expressed in the broad host range high-level expression vector pMMB66EHST. Expressed recombinant TNF-alpha was shown to have the correct molecular weight, processed N-terminal sequence, antibody cross-reactivity and tumour cell killing activity. The expression product of the synthetic gene has been purified to homogeneity by a two-step ion-exchange procedure and the purified material shown to be active.     

Synthesis of a gene for the protein kinase domain of the epidermal growth factor receptor and its expression in Escherichia coli

A gene encoding the protein kinase domain of the epidermal growth factor receptor has been chemically synthesised, cloned and expressed in Escherichia coli. The 942-base-pair gene was constructed by enzymatic ligation of 56 oligonucleotides and cloned into an expression vector downstream of the E. coli trp promoter. Production of active gene product was confirmed by means of a protein kinase assay, demonstrating that the enzymatic activity of the protein kinase domain of the epidermal growth factor receptor is retained after expression in E. coli.     

Specific DNA probe for detecting Legionella pneumophila

A fragment of the gene for cytolysin has been cloned. The product of the gene has been earlier identified as an immunoserological marker of Legionella pneumophila. Clones were selected by immunodetection of cytolysin gene product expression. An EcoRI 3.8 kb fragment of the genomic DNA from Legionella pneumophila strain Philadelphia-1 was used as a DNA probe that hybridized with the bacterial colonies of 20 Legionella pneumophila strains but not with the colonies of 10 other Legionella species or 8 other bacterial genera. The cloned fragment has been shown to be unique for Legionella pneumophila. The region of homology is suggested to be longer than a possible dimension of the cytolysin gene.     

杂色云芝漆酶基因(Lcc1)的克隆及在甲醇毕赤酵母中的表达

以白腐菌杂色云芝Coriolus versicolor RNA为模板,通过RT-PCR获得漆酶Leel基因的cDNA片段。构建了甲醇酵母表达质粒pMETA-Lccl载体,并将其线性化后用电穿孔法导入Pichia methabolica PMAD16,部分阳性克隆的PCR结果表明Lccl基因已经整合到甲醇毕赤酵母染色体上,经摇瓶培养筛选出表达水平较高的酵母工程菌株。漆酶酶活力达53U/L     

Cloning, mapping and gene product identification of rhaT from Escherichia coli K12

Rhamnose utilization requires the function of a specific rhamnose transport system. Rhamnose transport mutants have been isolated and characterized. The structural gene, rhaT, encoding the rhamnose permease has been cloned from Escherichia coli. rhaT has been mapped in the rha locus (87.7 min) by analysis of cotransduction with glpK and other rha markers. The precise location of the gene has been determined by complementation analysis of rhamnose transport mutants transformed with recombinant plasmids containing different fragments of the cloned region. Gene order (counterclockwise) is established as glpK . . . rhaT-rhaR-rhaS-rhaB-rhaA-rhaD. The gene product has been identified by expression of rhaT in a T7 RNA polymerase/promoter system. This 23 kDa protein has been assigned to the rhaT product and has been shown to be located in the cell membrane.     

Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1.

Deletions or insertions in the copB gene of plasmid R1 result in a copy mutant phenotype. The wild-type copB gene has been cloned on various plasmid vectors. The presence of such chimeric plasmids reduced the copy number of R1 copB mutant plasmids to normal or subnormal levels, indicating the expression of a trans-acting inhibitor activity from the copB chimeras. However, the cloned copB gene did not affect the copy number of wild-type R1, and no incompatibility was exerted by the cloned copB gene against wild-type R1 (or R100). Although the copB gene is not normally required for the incompatibility exerted by copA, it is shown that the CopB function is required for expression of incompatibility by the copA gene from some types of chimeric plasmids. Mutant plasmids that have lost both Cop functions replicate in an uncontrolled fashion.     

天花粉蛋白基因的克隆及序列分析

本文应用ＤＮＡ多聚酶链式反应（ＰＣＲ）技术,从括楼基因组ＤＮＡ中扩增并克隆了天花粉蛋白（ＴＣＳ）基因。核酸序列分析结果表明,克隆片段包括ＴＣＳ的前原蛋白的编码序列和５＇一侧翼区段。其编码序列与已发表的不同来源的３种ＴＣＳ基因的核苷酸序列的同源性分别为９９．２０％,９８．７４％和９８．６４％。推导出的氨基酸序列与已发表的４种ＴＣＳ的氨基酸序列的同源性分别为９８．６２％、９８．６２％、９７．４１％和９     

A single human keratin 18 gene is expressed in diverse epithelial cells of transgenic mice

The expression of keratin 18 (K18) is restricted in humans primarily to a variety of single layered or simple epithelia. However, direct introduction of a cloned K18 gene into cultured, somatic cells by DNA transfection has been shown to result in the promiscuous expression of K18 even while the endogenous mouse form of K18 (Endo B) remains silent. To determine if the cloned K18 genomic DNA fragment contains sufficient information to be regulated appropriately when subjected to a normal developmental environment, and to determine if the cloned gene is expressed in diverse epithelia, the K18 gene, including 2.5 kb of 5' flanking sequence and 3.5 kb of 3' flanking sequence, has been introduced into the germ line of mice. Mice from all three resulting K18 transgenic lines express the gene in an appropriate tissue-specific pattern that includes hepatocytes, simple epithelia of the intestinal tract, ductal cells of several glands and epithelial cells of the thymus. No expression of K18 was found in muscle, heart, or in most of the brain even in mice carrying 18 copies of the K18 gene. In most tissues, the level of K18 RNA was directly proportional to copy number and was as efficiently expressed as the endogenous Endo B gene. The K18 protein was identified by both protein blotting methods and indirect immunofluorescence staining. No pathological consequences of overexpression of the K18 gene were observed. The cloned K18 gene appears to contain all cis-acting DNA sequences necessary for appropriate expression. In addition, diverse epithelial cell types are able to express this single human gene.     

Barnase and barstar. Expression of its cloned inhibitor permits expression of a cloned ribonuclease

Barnase is the extracellular ribonuclease of Bacillus amyloliquefaciens and barstar its specific intracellular inhibitor. The gene for barstar has now been cloned and sequenced. When the wild-type gene for barnase is reconstructed from its previously cloned parts on the same plasmid as the barstar gene, the lethal effect of its expression is suppressed. A plasmid has been devised which directs the secretion of 100 mg per active barnase liter by Escherichia coli and another which provides large (500 to 1000 mg/l) yields of barstar. The structure of these plasmids and the derived 89 amino acid sequence of barstar are reported.     

木质素过氧化物酶基因（LipH8）的克隆及在甲醇毕赤酵母中的表达

以黄孢原毛平革菌 (Phanerochaetechrysosporium)RNA为模板 ,克隆LipH8基因片段 ,研究LipH8基因在甲醇毕赤酵母中的表达。构建了甲醇酵母表达质粒pMETA_LipH8载体 ,并将其线性化后用电穿孔法导入PichiamethabolicaPMAD16 ,部分阳性克隆的PCR结果表明LipH8基因已经整合到甲醇毕赤酵母染色体上 ,经摇瓶培养筛选出表达水平较高的酵母工程菌株。胞外木质素过氧化物酶活力达 932U L。     

植酸酶酵母工程菌的构建*

从无花果曲霉(Aspergillus ficuum)3．4322中用RT-PCR方法扩增出一条约1．4kb的特异性条带,DNA序列测定表明,目的片段为不含信号肽的植酸酶编码序列,全长1347bp。无花果曲霉(Aspergillus ficuum)3．4322phyA基因序列已在GenBank注册(注册号为：AF537344)。将该基因克隆到酵母表达载体pYES2中,构建成不带信号肽phyA基因的重组表达载体pYPA2。用醋酸锂法将pYPA2转进urd缺陷型的酿酒酵母(s．oeraisiae INVSc1),筛选获得含植酸酶基因的酵母转化子。经半乳糖诱导表达后,用磷钼蓝显色(AMES)法对酵母菌体进行酶活测定,测出了明显的植酸酶活性,pYPA2胞内植酸酶活性约11．55IU／mL,表明无花果曲霉(Aspergillus ficuum)3．4322phyA基因能在酿酒酵母中表达。     

Expression of tissue-specific genes in transgenic mice

The ability to introduce cloned genes into mouse germ line has been used for analyzing cis-acting DNA sequences involved in tissue-specific and developmental regulation of the introduced gene. Using this system we have attempted to produce a transgenic mouse model for human dominantly inherited disease, familial amyloidotic polyneuropathy. Recently the mutant transthyretin gene which is considered to be responsible for this disease has been cloned and well characterized at molecular level. We have produced transgenic mice by microinjecting human mutant gene. Amyloid deposition was observed in the mucosa of alimentary tract and renal glomeruli, suggesting that this approach is successful in establishing the mouse model for human genetic disease. In addition, these experiments suggest that the expression of the mutant gene is regulated normally during developmental process and that the cause of adult onset is not due to the dysregulation of this gene expression.