Cloning and sequence analysis of the cDNA encoding a snake neurotoxin precursor

A recombinant plasmid has been constructed containing a sequence of 186 nucleotides encoding a potent neurotoxin found in the venom of the sea-snake Laticauda semifasciata and designated as erabutoxin a. This sequence is flanked, in the upstream region, by a sequence of 60 nucleotides encoding a hydrophobic peptide fragment presumably involved in the secretion process of the neurotoxin. The sequence coding for the toxin ends with a termination codon which is followed by a 3'-untranslated sequence of approximately 240 nucleotides (excluding the poly(A) tract).     

Cloning and sequence analysis of flaA, a gene encoding a Spirochaeta aurantia flagellar filament surface antigen.

Spirochaeta aurantia DNA that coded for an antigenic determinant of the flagellin associated with the filament surface of the periplasmic flagella was isolated. When expressed in Escherichia coli, the antigenic polypeptide had an apparent molecular weight of 37,000. Sequence analysis of the antigen-encoding DNA revealed the presence of an open reading frame that determined a polypeptide with a predicted molecular weight of 31,241. This polypeptide showed a region of identity with the N-amino-terminal region of the 39,000- and 37,000-dalton flagellins of the distantly related spirochetes Treponema phagedenis and Treponema pallidum, respectively (S. J. Norris, N. W. Charon, R. G. Cook, M. D. Fuentes, and R. J. Limberger, J. Bacteriol. 170:4072-4082, 1988). The region of identity in the deduced S. aurantia polypeptide was preceded by a possible signal sequence and signal peptidase cleavage site.     

Analysis of responses to sarafotoxin 6a and sarafotoxin 6c in the pulmonary vascular bed of the cat.

Pulmonary vascular responses to sarafotoxins 6a and 6c (S6a and S6c) were investigated in the intact-chest cat under constant flow conditions. Injections of S6a and S6c into the perfused lobar artery caused dose-related increases in lobar arterial pressure, increased left atrial pressure, and produced biphasic changes in systemic arterial (aortic) pressure. When left atrial pressure was maintained constant, injections of S6a, S6c, and endothelin 1 (ET-1) caused dose-related increases in lobar arterial pressure. The increases in lobar arterial pressure in response to S6a and S6c were not altered by treatment with a cyclooxygenase inhibitor or a thromboxane receptor blocking agent. Increases in lobar arterial pressure in response to S6a and S6c were not altered when airflow to the left lower lung lobe was interrupted by bronchial occlusion, and pressor responses were not diminished when the left lower lobe was perfused with low-molecular-weight dextran. Under conditions of controlled blood flow and constant left atrial pressure, S6a, S6b, S6c, and ET-1 had similar pressor activity, whereas the thromboxane A2 mimic, U-46619, had far greater activity when compared on a nanomolar basis. The present studies demonstrate that S6a and S6c have significant vasoconstrictor activity in the feline pulmonary vascular bed. These data suggest that pulmonary vasoconstrictor responses to the endothelin peptides are not dependent on release of cyclooxygenase products and the activation of thromboxane A2 receptors, alterations in bronchomotor tone, or interaction with formed elements in blood.     

Cloning and sequence analysis of the ces10 gene encoding a Sphingomonas paucimobilis esterase

Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals; and, in plants, they have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein is shown to be synthesized as a preprotein with both a N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, using an analytical system of transgenically cultured tobacco cells (Nicotiana tabacum, BY2). Here, we report that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. We also demonstrate that HRP C1a derived from a preprotein lacking CTPP is shunted into the secretory pathway.     

白花柽柳质膜水孔蛋白基因克隆及序列分析

植物水孔蛋白在植物体内形成水选择性运输通道,在植物种子萌发、细胞伸长、气孔运动、受精等过程中调节水分的快速跨膜运输。有的水孔蛋白还在干旱胁迫应答中起重要作用。本文根据白花柽柳的PEG6000胁迫处理构建的SSH消减文库的水孔蛋白基因表达序列标签(EST),设计基因特异性引物进行5′RACE,克隆出一个1 043 bp的核苷酸序列。应用生物信息学软件进行分析,预测该序列编码287个氨基酸,具有6个跨膜区,有MIP家族信号序列SGXHXNPAVT,高等植物PIP高度保守序列GGGANXXXXGY和TGI/TNPARSL/FGAAI/VI/VF/YN,这是质膜水孔蛋白基因的典型的结构特征。经NCBI比对,与Arabidopsis thaliana (MIP-C),同源性达到95%,预测该蛋白的相对分子量是30.9KD,理论等电点是8.84。     

甜叶悬钩子(Rubus suavissimus S.Lee)中UDP-糖基转移酶基因的克隆、表达及序列分析

糖基化是植物次生代谢产物生物合成中重要的修饰反应。目前已报道的以二萜为底物的UDP-糖基转移酶(UGT)数量稀少。本研究基于甜叶悬钩子叶片的转录组数据,利用生物信息学分析手段,选定可能具有二萜类催化活性的UDP-糖基转移酶基因进行克隆。最终克隆得到18条UGT基因序列,在大肠杆菌中进行了异源表达,并对克隆的基因进行了初步的序列分析。本研究为进一步挖掘和验证甜叶悬钩子中UGT的功能奠定了基础。     

Cloning and sequence analysis of the gene encoding Methylophilus methylotrophus cytochrome c", a unique protein with a perpendicular orientation of the histidinyl ligands.

Cytochrome c" from Methylophilus methylotrophus is an unusual monohaem protein that undergoes a major redox-linked spin-state transition: one of the two axial histidines bound to the iron in the oxidised form is detached upon reduction and a proton is taken up. A 3.5-kb DNA fragment, containing the gene encoding cytochrome c" (cycA), has been cloned and sequenced. The cytochrome c" gene codes for a pre-protein with a typical prokaryotic 20-residue signal sequence, suggesting that the protein is synthesised as a precursor which is processed during its secretion into the periplasm. The C-terminus of cytochrome c" has homology with the corresponding region of an oxygen-binding haem protein (SHP) from phototrophically grown Rhodobacter sphaeroides. SHP is similar in size and in the location of its haem-binding site. Immediately downstream from cytochrome c" a second open reading frame (ORF) codes for a 23-kDa protein with similarity to the cytochrome b-type subunit of Ni-Fe hydrogenase. The possibility of coordinated expression of cycA and this ORF is discussed.     

Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase.

The colH gene encoding a collagenase was cloned from Clostridium histolyticum JCM 1403. Nucleotide sequencing showed a major open reading frame encoding a 116-kDa protein of 1,021 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, HEXXH. A 116-kDa collagenase and a 98-kDa gelatinase were copurified from culture supernatants of C. histolyticum. While the former degraded both native and denatured collagen, the latter degraded only denatured collagen. Peptide mapping with V8 protease showed that all peptide fragments, except a few minor ones, liberated from the two enzymes coincided with each other. Analysis of the N-terminal amino acid sequence of the two enzymes revealed that their first 24 amino acid residues were identical and coincided with those deduced from the nucleotide sequence. These results indicate that the 98-kDa gelatinase is generated from the 116-kDa collagenase by cleaving off the C-terminal region, which could be responsible for binding or increasing the accessibility of the collagenase to native collagen fibers. The role of the C-terminal region in the functional and evolutional aspects of the collagenase was further studied by comparing the amino acid sequence of the C. histolyticum collagenase with those of three homologous enzymes: the collagenases from Clostridium perfringens and Vibrio alginolyticus and Achromobacter lyticus protease I.     

Cloning and sequence of a gene encoding macrotetrolide antibiotic resistance from Streptomyces griseus.

A gene (nonR) conferring tetranactin resistance on the macrotetrolide-sensitive strain, Streptomyces lividans TK64, was isolated during a shotgun cloning experiment, in which chromosomal fragments from Streptomyces griseus were ligated into the vector pIJ699 and then introduced by transformation into S. lividans TK64. The sequence (3326 bp) of the cloned DNA revealed three complete open reading frames (ORFs) and one incomplete ORF encoded on one strand of the DNA. The nonR gene (designated here ORFA) encodes a polypeptide of 279 amino acids (Mr 30610) and contains a putative active site motif, GXSXG, characteristic of serine proteases and esterases. A functional role for the nonR gene product may involve the inactivation of the antibiotic through hydrolysis of one or more ester linkages in the macrotetrolide ring. The deduced product of the incomplete ORFX lying adjacent to ORFA showed 27.9% sequence identity with the C-terminal region of rat mitochondrial enoyl-CoA hydratase, and is possibly a macrotetrolide biosynthetic enzyme.     

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coli. The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.     

嗜热古菌S-腺苷甲硫氨酸合成酶的序列分析、基因克隆与异源表达

将Methanopyrus sp.SNP6进行功能基因组测序,获得S-腺苷甲硫氨酸合成酶基因(sam)序列,并进行序列分析。将sam基因扩增后连接至表达质粒p ET-28b(+),转化E.coli BL21(DE3),获得重组菌后进行诱导表达。生物信息学分析表明,sam基因编码蛋白的理论分子量为44 086.4Da,三级结构为同源四聚体,与其他相关古菌来源的S-腺苷甲硫氨酸合成酶的蛋白序列较保守。实验结果显示,构建的重组表达质粒p ET28b(+)-sam可在E.coli BL21(DE3)宿主菌中高水平表达。重组蛋白的分子量与预期值基本一致,部分为胞内可溶性表达,另一部分以包涵体形式存在。本研究首次实现了Methanopyrus sp.SNP6菌株S-腺苷甲硫氨酸合成酶的异源表达,为后期的蛋白纯化、酶学性质研究和酶促转化法生产S-腺苷甲硫氨酸奠定了理论基础。     

Cloning and sequence analysis of cDNA encoding Rhizopus niveus lipase.

Complementary DNA encoding Rhizopus niveus lipase (RNL) was isolated from the R. niveus IF04759 cDNA library using a synthetic oligonucleotide corresponding to the amino acid sequence of the enzyme. A clone, which had an insert of 1.0 kilobase pairs, was found to contain the coding region of the enzyme. The lipase gene was expressed in Escherichia coli as a lacZ fusion protein. The mature RNL consisted of 297 amino acid residues with a molecular mass of 32 kDa. The RNL sequence showed significant overall homology to Rhizomucor miehei lipase and the putative active site residues were strictly conserved.