cDNA cloning, expression and characterization of human prostaglandin F synthase

Nuclear import usually relies on the presence of nuclear localization sequences (NLSs). NLSs are recognized by NLS receptors (importins), which target their substrates to the nuclear pore. We identified the NLSs of the yeast ribosomal proteins S22 and S25 and studied the former by mutational analysis. Furthermore, in S25 the nucleolar targeting information was found to overlap with its NLS. Comparison with previously published data on yeast ribosomal protein NLSs and computer analysis indicates the existence of a novel type of ribosomal protein-specific NLS that differs from the classical Chelsky and bipartite NLSs. The existence of such a ribosomal protein-specific NLS is in accordance with the recent identification of ribosomal protein-specific importins.     

cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type VI

Peptidylarginine deiminase (PAD) catalyzes the post-translational modification of protein through the conversion of arginine to citrulline in the presence of calcium ions. Human, similar to rodents, has four isoforms of PAD (type I, II, III and IV/V), each of which is distinct in substrate specificity and tissue specific expression. In our large-scale sequencing project, we identified a new human PAD cDNA from a human fetal brain cDNA library. The putative protein encoded by this cDNA is designated hPADVI. Expression analysis of hPADVI showed that it is mainly expressed in adult human ovary and peripheral blood leukocytes. We conclude that hPADVI may be orthologous to mouse ePAD, basing on sequence comparison, chromosome localization and exon-intron structure analysis. PAD-mediated deimination of epithelial cell keratin resulting in cytoskeletal remodeling suggests a possible role for hPADVI in cytoskeletal reorganization in the egg and in early embryo development. This study describes a new important member of the human PAD family.     

cDNA cloning and gene expression analysis of human myo-inositol 1-phosphate synthase

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) (IPS) is a key enzyme in myo-inositol biosynthesis pathway. This study describes the molecular cloning of the full length human myo-inositol 1-phosphate synthase (hIPS) cDNA, tissue distribution of its mRNA and characterizes its gene expression in cultured HepG2 cells. Human testis, ovary, heart, placenta, and pancreas express relatively high level of hIPS mRNA, while blood leukocyte, thymus, skeletal muscle, and colon express low or marginal amount of the mRNA. In the presence of glucose, hIPS mRNA level increases 2- to 4-fold in HepG2 cells. hIPS mRNA is also up-regulated 2- to 3-fold by 2.5 microM lovastain. This up-regulation is prevented by mevalonic acid, farnesol, and geranylgeraniol, suggesting a G-protein mediated signal transduction mechanism in the regulation of hIPS gene expression. hIPS mRNA expression is 50% suppressed by 10mM lithium ion in these cells. Neither 5mM myo-inositol nor the three hormones: estrogen, thyroid hormone, and insulin altered hIPS mRNA expression in these cells.     

Molecular cloning, functional expression and pharmacological characterization of a human bradykinin B2 receptor gene.

The gene encoding a putative G protein-coupled receptor (HG10) was cloned from human genomic DNA by low stringency PCR and found to be homologous to the recently described rat bradykinin B2 receptor. The receptor was expressed in xenopus oocytes and stably transfected CHO cell lines. Binding studies demonstrated that HG10 encodes a high affinity BK receptor with an apparent Kd of 150 pM. Displacement by BK agonists and antagonists allowed the characterization of the receptor as a B2 subtype. Functional coupling to the Ca(2+)-phosphatidylinositol cascade was demonstrated in transfected CHO cells where inositol phosphates accumulation and intracellular calcium concentration were elevated in response to BK stimulation. The agonistic and antagonistic properties of BK analogs do not match strictly the pharmacological profile described for the rat or guinea pig B2 receptor subtypes or the putative B3 subtype. This discrepancy is attributed either to species variability or to differences in the coupling efficiency of receptors to the transduction cascade in different cell types. From our results, the existence of B3 receptors and of B2 subtypes appears questionable.     

cDNA cloning and expression of an apoptosis-related gene, human TFAR15 gene

By means of cDNA-RDA method. some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these tragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1218 nueleotides and encodes 212 amino acids. The putative protein protein product of TFAR15 is partially homologous to C. elegans protein C14A4. 11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung. and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-(?) cells after GM-CSF withdrawal. In vitro analysis showed that the recombinant TFAR15 protein co(?)ld inhibit the natural cell death of 293 cells, an embryonic kidney cell     

Identification of a novel human adenylate kinase. cDNA cloning, expression analysis, chromosome localization and characterization of the recombinant protein.

Adenylate kinases have an important role in the synthesis of adenine nucleotides that are required for cellular metabolism. We report the cDNA cloning of a novel 22-kDa human enzyme that is sequence related to the human adenylate kinases and to UMP/CMP kinase of several species. The enzyme was expressed in Escherichia coli and shown to catalyse phosphorylation of AMP and dAMP with ATP as phosphate donor. When GTP was used as phosphate donor, the enzyme phosphorylated AMP, CMP, and to a small extent dCMP. Expression as a fusion protein with the green fluorescent protein showed that the enzyme is located in the cytosol. Northern blot analysis with mRNA from eight different human tissues demonstrated that the enzyme was expressed exclusively in brain, with two mRNA isoforms of 2.4 and 4.0 kb. The gene that encoded the enzyme was localized to chromosome 1p31. Based on the substrate specificity and the sequence similarity with the previously identified human adenylate kinases, we have named this novel enzyme adenylate kinase 5.     

棉铃虫酚氧化酶原基因的克隆、序列分析和组织表达

利用RT-PCR和RACE方法,获得了棉铃虫Helicoverpa armigera酚氧化酶原(prophenoloxidase,PPO)基因一个亚型cDNA的完整序列。该序列全长2 405 bp,含有一个2 097 bp的开放阅读框,编码一个由698个氨基酸残基组成的蛋白质。推导的氨基酸序列与其他鳞翅目昆虫PPO2基因相应氨基酸序列有较高的同源性(76％～80％),同时该序列具有铜离子结合位点等PPO基因所具有的典型特征。组织特异性表达分析表明,该基因在棉铃虫血细胞、体壁和中肠中均有表达。     

cDNA cloning, characterization and expression analysis of peroxiredoxin 5 gene in the ridgetail white prawn Exopalaemon carinicauda

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species. In this study, a full-length of peroxiredoxin 5 (designated EcPrx5) cDNA was cloned from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of the EcPrx5 was of 827 bp, containing a 5′ untranslated region (UTR) of 14 bp, a 3′ UTR of 228 bp with a poly (A) tail, and an open reading frame of 585 bp encoding a polypeptide of 194 amino acids with the predicted molecular weight of 20.83 kDa and estimated isoelectric point of 7.62. BLAST analysis revealed that amino acids of EcPrx5 shared 89, 68, 66, 65, 53 and 51 % identity with that of Macrobrachium rosenbergii, Megachile rotundata, Harpegnathos saltator, Acromyrmex echinatior, Danio rerio, and Homo sapiens counterparts, respectively. The conserved Prx domain and the signature of peroxiredoxin catalytic center identified in EcPrx5 suggested that EcPrx5 belonged to the atypical 2-Cys Prx subgroup. Real time quantitative RT-PCR analysis indicated that EcPrx5 could be detected in all the tested tissues with highest expression level in hepatopancreas. As time progressed, the expression level of EcPrx5 both in hemocytes and hepatopancreas increased in the first 6 h after Vibrio anguillarum and white spot syndrome virus challenge, and showed different expression profiles. The results indicated that EcPrx5 involved in immune response against bacterial and viral infection in E. carinicauda.     

苜蓿夜蛾保幼激素酯酶cDNA的克隆、序列分析及表达研究

【目的】研究苜蓿夜蛾Heliothis viriplaca保幼激素酯酶(Juvenile hormone esterase,JHE)的功能和作用机理。【方法】本试验提取苜蓿夜蛾的总RNA,并利用RT-PCR和RACE技术,扩增得到苜蓿夜蛾保幼激素酯酶基因的全长c DNA序列,命名为Hvjhe(Gen Bank登录号:JQ901384)。【结果】该基因含有3 106 bp,包括一个1 746 bp的开放阅读框,编码一个含581个氨基酸的多肽,分子量约为63.9 ku,多肽的等电点为5.11,且氨基酸序列具含有5个保幼激素酯酶特有的氨基酸保守模块。推导的氨基酸序列与烟芽夜蛾Heliothis virescens和棉铃虫Helicoverpa armigera中获得的保幼激素酯酶相似性较高,分别达到83%和82%。荧光定量PCR结果表明,该基因在苜蓿夜蛾不同发育时期和不同组织中都有m RNA水平的特异性表达,且在预蛹期表达量相对较高,取食期、蛹期期和成虫期表达量相对较低;在中肠和脂肪体内的表达量相对于其它组织较高。该基因在大肠杆菌Escherich coli表达系统中进行了诱导表达,经SDS-PAGE和Western blot检测结果表明,表达出与预测的蛋白分子量相符的融合蛋白。【结论】研究结果表明本试验获得了苜蓿夜蛾中一个新的保幼激素酯酶基因的c DNA序列。     

cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor.

Recent binding studies in the central nervous system and other tissues provide evidence that the mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-B (NMB), exert their numerous physiological effects through at least two different receptors. We describe the structure and expression of a cloned NMB-preferring bombesin receptor (NMB-R) with properties distinct from a GRP-preferring bombesin receptor (GRP-R) reported previously. In particular, the NMB-R shows higher affinity binding to NMB than to GRP in BALB 3T3 fibroblasts expressing the cloned NMB-R. The distinct regional distribution of NMB-R and GRP-R mRNA in the brain suggests that both bombesin receptor subtypes play independent roles in mediating many of the dramatic effects of bombesin-like peptides in the central nervous system.     

Human indolethylamine N-methyltransferase: cDNA cloning and expression, gene cloning, and chromosomal localization.

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K(m) value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon-intron splice junctions conformed to the "GT-AG" rule, and no canonical TATA or CAAT sequences were present within the 5'-flanking region of the gene. Human INMT mapped to chromosome 7p15.2-p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.     

cDNA cloning and expression of human lactosylceramide synthase.

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycolipids in mammals. We have isolated and sequenced the cDNA clone encoding human lactosylceramide synthase. The deduced amino acid sequence of the human lactosylceramide synthase showed 94.2% identity with rat lactosylceramide synthase. Northern blotting analysis revealed that lactosylceramide synthase mRNA was expressed in various tissues, with the highest level in brain and adrenal gland.     

Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii.

A novel ketoreductase isolated from Zygosaccharomyces rouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8-kDa ketoreductase was purified more than 300-fold to > 95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4-methylenedioxyphenyl acetone of 2.9 mM and a Km for NADPH of 23.5 microM. The enzyme is able to effectively reduce alpha-ketolactones, alpha-ketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0-kb ketoreductase gene was cloned and sequenced from a Z. rouxii cDNA library using a degenerate primer to the N-terminal sequence of the purified protein. Furthermore, it was expressed in both Escherichia coli and Pichia pastoris and shown to be active. Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class.