Isolation and characterization of a seed-specific isoform of microsomal omega-6 fatty acid desaturase gene (FAD2-1B) from soybean.

In plants, the endoplasmic reticulum (ER)-associated oleate desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic tissues. In this study, we report the characterization of a seed-specific isoform of microsomal omega-6 fatty acid desaturase gene (FAD2-1B) sharing high sequence similarity with FAD2-1 from soybean. Several potential promoter elements including seed-specific motifs are found in the 5'-flanking region of FAD2-1B gene. The ORF of FAD2-1B is 1161 bp long and encodes a protein of 387 amino acids. This deduced protein holds three histidine boxes and four putative membrane-spanning helices, and possesses a signal for endoplasmic reticulum retention at C-terminal. Yeast cells transformed with the plasmid construct containing soybean FAD2-1B accumulate an appreciable amount of linoleic acid (18:2), normally not present in wild-type yeast cells, indicating that the cloned gene encodes a functional FAD2 enzyme. Both semi-quantitative RT-PCR and in silico analysis show that FAD2-1B gene is specifically expressed in developing seeds of soybean.     

玉米FAD2基因的克隆及序列分析

高等植物中的A12脂肪酸脱饱和酶是将油酸转化为亚油酸的酶。根据已发表的其他高等植物的FAD2基因的保守序列设计同源引物,通过RT—PCR从玉米幼胚中扩增得到一个特异的cDNA基因片段。通过生物信息学分析,从玉米幼胚cDNA和基因组中均扩增得到1164 bp FAD2基因（GenBank登陆号：DQ496227）,它编码387个氨基酸,含有完整的ORF框,在ORF框内无内含子。序列联配与树状分析结果表明,FAD2推导的氨基酸序列与其他物种的A12脱饱和酶基因具有同源性。它含有3个组氨酸保守域和2段很长的疏水区,是一个跨膜4次的膜结合蛋白。半定量RT—PCR分析显示FAD2基因在玉米幼胚中表达量最高,在叶、茎、根中亦有低水平表达。     

Molecular cloning of the nahG gene encoding salicylate hydroxylase from Pseudomonas fluorescens

A gene encoding the salicylate hydroxylase was cloned from the genomic DNA of Pseudomonas fluorescens SME11. The DNA fragment containing the nahG gene for the salicylate hydroxylase was mapped with restriction endonucleases and sequenced. The DNA fragment contained an ORF of 1,305 bp encoding a polypeptide of 434 amino acid residues. The nucleotide and amino acid sequences of the salicylate hydroxylase revealed several conserved regions with those of the enzyme encoded in P. putida PpG7: The homology of the nucleotide sequence is 83% and that of amino acid sequence is 72%. We found large conserved regions of the amino acid sequence at FAD and NADH binding regions. The FAD binding site is located at the amino terminal region and a lysine residue functions as a NADH-binding site.     

Cloning, sequencing and bacterial expression of human glycine tRNA synthetase.

The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da. The protein sequence has approximately 60% identity with B. mori GlyRS and 45% identity with S. cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. The enzyme was expressed in E. coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa. The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS. Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with [14C]-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.     

Molecular cloning and expression of a fifth muscarinic acetylcholine receptor

A cDNA of 2149 base pairs with an incomplete open reading frame (ORF) encoding amino acids 1-516 of a 531-amino acid protein highly homologous to muscarinic receptors was cloned from a rat brain cDNA library. The complete ORF was then deduced from a DNA fragment cloned from a rat genomic library. This ORF was subcloned into the eukaryotic expression vector p91023(B) under control of the adenovirus major late promoter and co-transfected with the thymidine kinase selection marker into muscarinic receptor-negative, thymidine kinase-negative murine L cells. Stable transformants were selected and tested for acquisition of muscarinic receptors by following appearance of specific binding sites for the muscarinic ligand [3H] N-methylscopolamine. Two cell lines, LM5.36 and LM5.40, were cloned and shown to express typical muscarinic receptor sites, thus confirming that the newly cloned ORF encodes a muscarinic receptor, the rat M5 muscarinic acetylcholine receptor. Tests for activities showed it to stimulate phosphoinositide hydrolysis in intact cells, without affecting positively or negatively adenylyl cyclase activity. The M5 receptor contains two putative glycosylation sites at its amino terminus and, based on hydropathicity analysis, is predicted to span the plasma membrane seven times. Like 17 other receptors of this class, the M5 receptor has 19 conserved amino acids, among which are 4 prolines located in the 4th, 5th, 6th, and 7th predicted transmembrane regions, conferring possible bends to these helices, and 2 cysteines, one in the 1st and the other in the 2nd extracellular loop, possibly providing for a disulfide bond. Similarity in amino acid composition and in patterns of antagonist binding and biologic effects suggest the M5 receptor to be M1-like.     

Cloning and Sequencing of the Gene Encoding the Soluble Fumarate Reductase from Saccharomyces cerevisiae

A gene of the soluble fumarate reductase (FRDS) that binds FADnon-covalently was cloned by polymerase chain reaction (PCR)using degenerate oligonucleotides designed from partial aminoacid sequences of highly purified enzyme. The nucleotide sequenceof a 0.99-kb amplified product was found to be nearly identicalto a partial sequence of an open reading frame (ORF) previouslyreported (EMBL database accession number S-30830). Accordingto the sequence in the EMBL database, we cloned 1.7-kb fragmentcontaining entire sequence of this ORF by PCR and found thatthis fragment contained a perfect match to the 0.99-kb sequenceamplified with the degenerate primers. From these results, weconcluded that this ORF is the FRDS gene. The amino acid sequencesof the regions involved in the non-covalent binding of FAD andthe active site, which are conserved among the flavoproteinsubunits of membrane-bound fumarate reductase and succinatedehydrogenase, were found in FRDS. However, unlike the membrane-boundenzymes, FRDS did not contain the histidine residue that covalentlybinds the isoalloxazine ring of FAD at or near the correspondingposition. FRDS showed high homology to the product of S. cerevisiaeOSM1 gene which was reported to be required for growth in hypertonicmedia.     

Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA

The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.     

Primary structure and processing of the Candida tsukubaensis alpha-glucosidase. Homology with the rabbit intestinal sucrase-isomaltase complex and human lysosomal alpha-glucosidase.

The nucleotide sequence of a 4.39-kb DNA fragment encoding the alpha-glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the alpha-glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N-terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the alpha-glucosidase precursor is proteolytically processed by removal of an N-terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63-65 kDa and 50-52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expression in the heterologous host, the C. tsukubaensis alpha-glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensis alpha-glucosidase, the rabbit sucrase-isomaltase complex (proSI) and human lysosomal acid alpha-glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.     

The role of the Sphingomonas species UG30 pentachlorophenol-4-monooxygenase in p-nitrophenol degradation

Pentachlorophenol-4-monooxygenase is an aromatic flavoprotein monooxygenase which hydroxylates pentachlorophenol and a wide range of polyhalogenated phenols at their para position. The PCP-degrading Sphingomonas species UG30 was recently shown to mineralize p-nitrophenol. In this study, the UG30 pcpB gene encoding the pentachlorophenol-4-monooxygenase gene was cloned for use to study its potential role in p-nitrophenol degradation. The UG30 pcpB gene consists of 1614 bp with a predicted translational product of 538 amino acids and a molecular mass of 59,933 Da. The primary sequence of pentachlorophenol-4-monooxygenase contained a highly conserved FAD binding site at its N-terminus associated with a beta alpha beta fold. UG30 has been shown previously to convert p-nitrophenol to 4-nitrocatechol. We observed that pentachlorophenol-4-monooxygenase catalyzed the hydroxylation of 4-nitrocatechol to 1,2,4-benzenetriol. About 31.2% of the nitro substituent of 4-nitrocatechol (initial concentration of 200 microM) was cleaved to yield nitrite over 2 h, indicating that the enzyme may be involved in the second step of p-nitrophenol degradation. The enzyme also hydroxylated p-nitrophenol at the para position, but only to a very slight extent. Our results confirm that pentachlorophenol-4-monooxygenase is not the primary enzyme in the initial step of p-nitrophenol metabolism by UG30.     

Molecular characterization of a novel glucosyltransferase from Lactobacillus reuteri strain 121 synthesizing a unique,highly branched glucan with alpha-(1-->4) and alpha-(1-->6) glucosidic bonds

Lactobacillus reuteri strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the alpha-(1-->4) glucosidic type. The glucan also contains alpha-(1-->6)-linked glucosyl units and 4,6-disubstituted alpha-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (gtf) genes from lactic acid bacteria, the L. reuteri strain 121 glucosyltransferase gene (gtfA) was isolated. The gtfA open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced M(r) of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The gtfA gene was expressed in Escherichia coli, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the L. reuteri strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the gtfA ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of L. reuteri strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in L. reuteri strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a Lactobacillus strain.     

Isolation and expression in Escherichia coli of a cDNA clone encoding porcine pancreatic elastase

We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.     

解淀粉芽孢杆菌BamHI甲基转移酶基因克隆、功能鉴定与序列分析

从解淀粉芽孢杆菌Baillus amyloliquefaciens CICIM B2125中克隆了BamHI甲基转移酶基因(bamHIM),并在大肠杆菌JM109中得到了成功表达.该基因含有1 271 bp的开放阅读框(ORF),编码423个氨基酸,成熟蛋白分子量为49 kD.该基因在自身启动子引导下,表达了具有活性的BamHI甲基转移酶(M.BamHI).该酶可以将BamHI位点的碱基甲基化.氨基酸序列分析表明该酶存在有NADB_Rossmann结构域.     

一色齿毛菌锰过氧化物酶2基因克隆及生物信息学分析

摘要 锰过氧化物酶（manganese peroxidase,MnP）是由一系列同功酶组成的木质素降解酶。我们前期工作克隆了一色齿毛菌（Cerrena unicolor) MnP1基因序列。在此基础上,本研究采用简并PCR、染色体步移和RACE等技术对C. unicolor mnp2基因(Cu-mnp2)序列进行克隆。同时,采用生物信息学软件对Cu-mnp2的基因结构、Cu-MnP2的蛋白质结构及多物种MnPs蛋白质序列的系统进化关系进行分析。克隆得到3 053 bp的Cu-mnp2 DNA序列(GenBank:JX270806.1)和1 429 bp的Cu-mnp2 cDNA序列(GenBank: JQ782580.1)。序列分析结果显示,Cu-mnp2 DNA序列包含14个外显子和13个内含子,启动子区域包含TATA-BOX、SP1和AP1等作用元件;Cu-mnp2 cDNA序列包含71 bp的5′UTR、230 bp的3′ UTR以及1 128 bp的开放阅读框(ORF)。Cu-mnp2 ORF序列的BLAST比对结果表明,Cu-mnp2与Trametes versicolor FP-101664 SS1 mnp序列覆盖度为53%,序列相似性为65%;与Heterobasidion irregulare mnp、C. unicolor mnp1等cDNA序列都有较高的序列相似性。Cu-mnp2的ORF编码(GenBank:AFK91530.1)由340个氨基酸残基组成的多肽链(Cu-MnP2)。Cu-MnP2蛋白质序列的BLAST比对和蛋白质三维结构均显示,Cu-MnP2包含Mn ²⁺ 、Ga ²⁺ 、血红素及芳香底物结合位点。对包含Cu-MnP1、Cu-MnP2蛋白质序列在内的多物种MnPs蛋白质序列的系统发育分析表明,多物种的MnPs分为两大类群,分别为包含4个二硫键的短MnPs和包含5个二硫键的长MnPs。其中,Cu-MnP1与Cu-MnP2均属于短MnPs,Cu-MnP2与Trametes versicolor MrP 的蛋白质序列亲缘关系最近。通过Cu-mnp2基因的克隆和序列分析,对继续研究C. uniclor的MnP同工酶基因结构和功能奠定基础。     

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT.

4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases.     

Molecular cloning and expression of the hyu genes from Microbacterium liquefaciens AJ 3912, responsible for the conversion of 5-substituted hydantoins to alpha-amino acids, in Escherichia coli

A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to alpha-amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl alpha-amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomyces cerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.     

Cholest-4-en-3-one-delta 1-dehydrogenase, a flavoprotein catalyzing the second step in anoxic cholesterol metabolism

The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Delta(1)-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Delta(1)-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Delta(1)-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.     

Molecular cloning and characterization of the gene encoding squalene epoxidase in Panax notoginseng.

Squalene epoxidase (SE) is one of the rate-limiting enzymes in the triterpene saponins biosynthetic pathway. Panax notoginseng, one of the famous medicinal plants in China, produces bioactive triterpene saponins. Here we report the P. notoginseng SE, which was cloned from the root of P. notoginseng by PCR. The nucleotide sequence of the ORF (GenBank accession no. DQ386734) contains 1611 nucleotides and encodes 537 amino acid residues with molecular weight of 59.14 kDa and pI of 8.81. The gene has 98% identity with P. ginseng but different identities with other SE families. P. notoginseng SE has a FAD function domain, NAD(P)-binding Rossmann-fold domains, hydrophobicity and 4 transmembrane helices. This SE may be a microsomal membrane-associated enzyme. Real time quantitative PCR shows that the cDNA has different expression pattern and is highly expressed in root, especially in 3-year-old root.