Identification and expression analysis of a novel splice variant of human Sprouty1 gene

Sprouty (SPRY) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor (FGF) and epidermal growth factor (EGF) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. During large scale DNA sequencing of the human fetal brain cDNA library, we cloned a novel splice variant of human Sprouty1 gene, and termed it human Sprouty1b (SPRY1b). It has the same deduced protein as Sprouty1a, which has been reported. And like other members of Sprouty family, SPRY1b deduced protein also has a C-terminal cysteine-rich region. According to the search against human genome database, SPRY1b was mapped to 4q25-28. Expression analysis of SPRY1a and SPRY1b shows that they are hardly expressed in adult human, but have different expression patterns in fetus, which confirmed that SPRY1 is an important gene during fetal development.     

Molecular cloning and chracterization of a c-Rel induced lectin-like receptor and its alternative splice isoforms

ThemoleculesintheC typelectinfamilyarecha acterizedbythepresenceofcarbohydraterecogn tiondomain(CRD)andclassifiedinto7grou accordingtotheoverallarchitectureoftheprotein thepositionofCRDrelativetootherdomainsan thedegreeofsimilarityofCRDsamongeachmol cule[1].MostC typelectinmoleculesbindwisugarmoleculesthroughCRDsinaCa2+depen dentfashion.However,someofthenewlyidenti fiedC typelectinsdon′tbindtosugarmolecules duetolackoftheconservedresiduesrequiredfor Ca2+coordinationinCRDs[2].According…     

Molecular cloning and expression of a novel klotho-related protein

Klotho protein is a novel beta-glucosidase-like protein produced predominantly in the kidney. The klotho mouse, which genetically lacks klotho gene expression, manifests various systemic phenotypes resembling aging. In the present study we succeeded in isolating a novel human protein structurally related to klotho protein. The protein possesses one beta-glucosidase-like domain and is 42% identical with klotho protein at the amino acid level. Unlike klotho protein, it possesses neither a signal sequence nor a transmembrane domain, suggesting that it is a cytosolic protein, and thus was termed cytosolic beta-glucosidase-like protein-1 (cBGL1). By Northern blot analysis cBGL1 mRNA was expressed most abundantly in the liver, followed by the small intestine, colon, spleen, and kidney. When klotho and cBGL1 gene expression was examined in renal cell carcinoma tissues, both klotho and cBGL1 mRNA levels in tumors were lower than those in nontumor regions, suggesting that renal epithelial cells may lose klotho and cBGL1 gene expression during the course of malignant transformation. In conclusion, we describe the primary structure and gene expression of a novel protein related to klotho protein.     

新基因hKCNE4的克隆、表达及功能

KCNE4是KCNE家族中的一员,编码电压依赖性钾通道的B亚基。KCNE1、KCNE2、KCNE3均在人类基因组发现,并且有重要的生理功能,而KCNE4只是在鼠文库中得到,在人类cDNA文库中没有相关报道。本研究应用生物信息学分析,通过RT-PCR和RACE方法,从人类心脏cDNA文库中获取人类hKCNE4全长cDNA序列。其全长1188bp,编码170个氨基酸,和鼠KCNE4蛋白具有90％同源性。Northern Blot结果表明该基因在心脏、骨骼肌和肾脏高度表达。通过电子PCR方法将hKCNE4基因定位在2q35—36。亚细胞定位表明该基因的编码产物定位在细胞膜上,但体外电生理实验证明HERG基因同hKCNE4没有相互作用。     

Ectopic expression of new alternative splice variant of Smac/DIABLO increases mammospheres formation

Smac-α is a mitochondrial protein that, during apoptosis, is translocated to the cytoplasm, where it negatively regulates members of the inhibitor of apoptosis (IAP) family via the IAP-binding motif (IBM) contained within its amino-terminus. Here, we describe a new alternative splice variant from Smac gene, which we have named Smac-ε. Smac-ε lacks both an IBM and a mitochondrial-targeting signal (MTS) element. Smac-ε mRNA exhibits a tissue-specific expression pattern in healthy human tissues as well as in several cancer cell lines. The steady-state levels of endogenous Smac-ε protein is regulated by the proteasomal pathway. When ectopically expressed, this isoform presents a cytosolic localization and is unable to associate with or to regulate the expression of X-linked Inhibitor of apoptosis protein, the best-studied member of IAP family. Nevertheless, over-expression of Smac-ε increases mammosphere formation. Whole genome expression analyses from these mammospheres show activation of several pro-survival and growth pathways, including Estrogen-Receptor signaling. In conclusion, our results support the functionality of this new Smac isoform.     

一种新的FR4剪切变异体的确认和鉴定

目的 确认并鉴定一个新发现的小鼠FR4剪切变异体,并在mRNA水平检测其在小鼠脾淋巴细胞亚群中的表达.方法 PCR克隆FR4 CDS区时发现存在一个新的剪切变异体,酶切和测序予以鉴定,Western blot检测脾细胞中变异体的表达,流式分选脾细胞中CD8+、CD4+CD25-、CD4+CD25+T淋巴细胞,RT-PCR检测新的变异体在各细胞亚群中的表达.结果 凝胶电泳显示存在一个CDS区碱基数大于野生型FR4的条带,测序表明此条带在野生型外显子基础上包含一个内含子成分,Westernblotting证实新发现的剪切体在蛋白水平也存在,脾细胞中不同T细胞亚群均表达此新型变异体.结论 mRNA和蛋白水平均证实存在一种新型FR4剪切变异体,不同T细胞亚群均表达此变异体暗示其在淋巴细胞摄取叶酸功能上不可或缺.     

Molecular cloning and characterization of a novel lactate dehydrogenase gene from Clonorchis sinensis

From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel lactate dehydrogenase (LDH) gene which encoded a putative protein with a predicted molecular weight of 35.6 kDa. The optimum pH and temperature for the enzyme were 7.5 and 50°C in the pyruvate reduction while 11 and 80°C in the lactate oxidation reaction, respectively. CsLDH showed no substrate inhibition by high lactate and NAD⁺ concentration, and the optimal pyruvate and optimal NADH concentrations were 10 and 0.5 mmol/l, respectively. The relative activities of these 2-oxocarboxylic acids were pyruvic acid>2-ketobutyrate>oxalacetic acid>α-ketoglutaric acid>phenylpyruvate. The cofactor 3-acetylpyridine adenine dinucleotide was much more effective than NAD⁺. The cofactor analogs in which the nicotinamide ring is replaced by 3-pyridinealdehyde were lower activity cofactors, while the nicotinamide ring is replaced by nicotinic acid or thionicotinamide which is not a cofactor to CsLDH. The succinic acid and malic acid are not substrates of CsLDH. Cu²⁺, Fe²⁺, and Zn²⁺ greatly inhibited the CsLDH activity both in the direction of pyruvate reduction and in the direction of lactate oxidation. The inhibition of CsLDH by gossypol may make gossypol a potential therapy drug or a lead compound for C. sinensis. Accordingly, the CsLDH may be a novel potential drug target.     

纳豆激酶酶原基因的克隆、融合表达及活性测定

目的:利用基因工程技术, 构建表达具溶栓活性的纳豆激酶的大肠杆菌工程菌。方法:利用PCR方法扩增纳豆激酶酶原(pro-NK)基因, 并克隆到表达载体pET3c上, 构建表达pro-NK和载体上22氨基酸短肽的融合蛋白的表达质粒pENK;表达质粒pENK分别转化溶源化宿主菌BL21(DE3)pLysS^-和BL21(DE3)pLysS⁺, 获得表达菌pENK-(DE3)pLysS^-和pENK-(DE3)pLysS⁺。利用SDS-PAGE和纤维蛋白平板法检测目的蛋白的表达及活性。结果:SDS-PAGE显示两株菌株均表达42kD的目的蛋白。纤维蛋白平板法显示表达产物具溶栓的活性。在pENK-(DE3)pLysS^-中融合蛋白不需异丙基硫代-β-D-半乳糖苷(IPTG)诱导就有基础表达, 融合蛋白的表达使表达菌细胞溶解, 菌落中空, 表明表达产物具细胞毒作用。结论:本研究成功实现了在大肠杆菌表达具有溶栓活性的pro-NK融合蛋白, 为开发纳豆激酶成为新一代溶栓药物的研究奠定基础。     

Exome sequencing revealed a novel splice site variant in the ALX1 gene underlying frontonasal dysplasia

Frontonasal dysplasia (FND) is a heterogeneous group of disorders characterized by hypertelorism, telecanthus, broad nasal root, wide prominent nasal bridge, short and wide nasal ridge, broad columella and smooth philtrum. To date one X‐linked and three autosomal recessive forms of FND have been reported in different ethnic groups. We sought to identify the gene responsible for FND in a consanguineous Pakistani family segregating the disorder in autosomal recessive pattern. Genome‐wide homozygosity mapping using 250KNsp array revealed five homozygous regions in the selected affected individuals. Exome sequencing found a novel splice acceptor site variant (c.661‐1G>C: NM_006982.2) in ALX1. Sanger sequencing confirmed the correct segregation of the pathogenic variant in the whole family. Our study concludes that the splice site variant identified in the ALX1 gene causes mild form of FND.     

人TRAF2新剪切体的鉴定与表达分析

目的:对TRAF2的新剪切体进行鉴定与表达分析.方法:以人脑库为模板,利用PCR扩增,电泳确定新剪切体的存在.再提取不同细胞与组织的总RNA,逆转录得到cD-NA后作为模板对两种不同剪切体的表达进行比较分析.结果:利用P1与P2引物对从人脑库扩增出1条约1 500 bp的条带,而利用P3与P4引物对则得到2条明显电泳带,通过测序表明其中1条包含TRAF2的第6外显子,另1条缺失了TRAF2的第6外显子.通过对新剪切体TRAF2Δ6与全长剪切体TRAF2的表达量比较表明在T47D中全长剪切体表达占优势;而在Hep3B、GC-1与MCF7中TRAT△6剪切体表达占优势;在HepG2、HBL100、A549与HeLa细胞中未发现两种剪切体的明显表达;在PANCI与SW480中两种剪切体表达量相近.在胎大脑与一级神经胶质瘤中TRAF2Δ6表达占优,而二级与三级神经胶质瘤中则全长剪切体TRAF2表达占优势.结论:人体TRAF2基因除可表达全长TRAF2 mRNA外,还可通过可变剪切表达缺失第6外显子的TRAF2Δ6 mRNA.而且这两种剪切体的表达存在细胞与组织特异性.     

Molecular cloning, tissue expression, and chromosomal assignment of a novel gene encoding a subunit of the human signal-recognition particle

Human cancers derived from breast, esophageal, or ovarian tissues frequently show allelic losses on chromosome band 17q25. Moreover, a locus responsible for hereditary focal nonepidermolytic palmoplantar keratoderma, a condition associated with esophageal cancer (TOC; tylosis with oesophageal cancer), has been mapped to the same band. During efforts to sequence, by shotgun methods, a 1-Mb target region that we had defined as the DNA segment harboring the putative tumor suppressor gene(s) involved in these events, we identified a novel cDNA. The full-length cDNA is 2495 bp long and is expressed predominantly in skeletal muscle, heart, kidney, and placenta. The predicted product, a 627-amino-acid protein, exhibited significant sequence homology to the canine 68-kd subunit of the signal recognition particle that has been implicated in the transport of secreted and membrane proteins to the endoplasmic reticulum for proper processing. We confirmed the location of this gene at chromosome 17q25.1 by radiation-hybrid mapping and by fluorescence in situ hybridization. Received: September 18, 2000 / Accepted: November 10, 2000     

Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis

Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in living cells. AK catalyzes reversible high energy phosphoryl transfer reactions between ATP (or GTP) and AMP to generate ADP (or GDP). From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel AK3 isozyme. The 956 bp cDNA encodes a putative protein of 228 amino acids with a predicted molecular mass of 26.2 kDa. The recombinant CsAK3 protein produced in Escherichia coli can be refolded into a functional protein with AK3 activity. The optimum pH and temperature for the enzyme are 8.5 and 40°C, respectively. The calculated activation energy is 56.04 kJ mol^–1. The K_m of the CsAK3 for AMP and GTP are 118 M and 359 M, respectively. CsAK3 is inhibited by Ap₅A (>70% inhibition by 2.0 mM AP₅A). Ap₅A may be a potential lead compound acting on C. sinensis in which AK3 as a drug target.     

Identification and characterization of a novel splice variant of rhesus macaque MHC IA

Major histocompatibility complex class I (MHC I) molecules play a pivotal role in the immune recognition to intracellular pathogens. A number of important splice variants have already been characterized for these molecules in different species, suggesting their important roles in modulation of immune responses. In this study, we have identified and characterized a novel alternatively spliced form of rhesus macaque MHC IA (designated MHC IA-sv2) that lacks exons coding for the α2 and α3 domains. Despite lacking the α2 and α3 domains, MHC IA-sv2 is targeted to the cell surface, as a 23-kDa glycoprotein that is totally susceptible to endoglycosidase-H digestion and is reduced to 18kDa after deglycosylation with PNGase F. In contrast, the full-length MHC IA reaches the cell surface as a 43-kDa protein of form with complex-type N-glycosylation (endoglycosidase-H resistant). Moreover, we provide evidence here that MHC IA-sv2 can self-associate, forming homodimers, or associate with the fully mature MHC IA molecule, forming a heterodimeric structure in mammalian cells. These data demonstrate that the formation of heterodimers may have some functional implications in the fine tuning of MHC IA-mediated innate and adaptive immune responses.     

Identification and characterization of a novel splice variant of rhesus macaque MHC IA

Major histocompatibility complex class I (MHC I) molecules play a pivotal role in the immune recognition to intracellular pathogens. A number of important splice variants have already been characterized for these molecules in different species, suggesting their important roles in modulation of immune responses. In this study, we have identified and characterized a novel alternatively spliced form of rhesus macaque MHC IA (designated MHC IA-sv2) that lacks exons coding for the α2 and α3 domains. Despite lacking the α2 and α3 domains, MHC IA-sv2 is targeted to the cell surface, as a 23-kDa glycoprotein that is totally susceptible to endoglycosidase-H digestion and is reduced to 18 kDa after deglycosylation with PNGase F. In contrast, the full-length MHC IA reaches the cell surface as a 43-kDa protein of form with complex-type N-glycosylation (endoglycosidase-H resistant). Moreover, we provide evidence here that MHC IA-sv2 can self-associate, forming homodimers, or associate with the fully mature MHC IA molecule, forming a heterodimeric structure in mammalian cells. These data demonstrate that the formation of heterodimers may have some functional implications in the fine tuning of MHC IA-mediated innate and adaptive immune responses.