生物信息学辅助克隆和分析人GLUT-9基因启动子

目的:对人GLUT-9基因5′-非翻译区进行克隆,并对其启动子和转录调控区进行生物信息学分析,为后续的GLUT-9基因的转录调控研究奠定基础。方法:通过搜索网络数据库,得到GLUT-9基因翻译起始点上游约2 kb的序列。以人总DNA为模板,采用PCR方法进行序列克隆。运用BDGP:Neural Network Promoter Prediction软件对上述序列进行分析,预测出该段序列可能的启动子区域;运用TFSEARCH软件分析潜在的转录因子结合位点。结果:人GLUT-9基因的启动子可能位于转录起始点上游反义链的-550到-503区,序列上存在包括CdxA、SRY和Lyf-1等12个转录因子的潜在结合位点。结论:克隆得到人GLUT-9基因5′-非翻译区2 kb的序列,对人GLUT-9基因启动子和5′-非翻译区调控区序列进行了初步的生物信息学分析,为后续试验提供了研究基础。     

转录因子Sp1与AP-2的研究进展

正转录因子是能与位于转录起始位点上游50~5 000bp的顺式作用元件、沉默子或增强子结合并参与调节靶基因转录效率的一组蛋白,并能将来自细胞表面的信息传递至核内基因。转录因子通常有几个功能域,可分为DNA结合域、转录调控域及自身活性调控域,DNA结合域可与特定的DNA序列(一般长8~20bp)相互作用,使转录因子与靶基因中特定转录因子结合位点结合起来,进而转录调控域就可发挥其激活或抑制     

核因子Κb与Th细胞分化

核因子κB是一种广泛存在于体内多种细胞的转录因子,由一个复杂的体系组成,目前已发现它调节着100多种靶基因的表达。能与调控免疫应答、炎症反应、细胞分化和生长、细胞粘附和细胞凋亡所必需的许多细胞因子,粘附因子等基因启动子或增强子部位的κB位点发生特异性结合,启动和调节这些基因的转录,在机体的免疫应答、炎症反应和细胞的生长生育等方面发挥重要作用。本文复习相关文献,对NF-κB及其对Th细胞分化的调节作一综述。1核因子-κB的生物学特性1.1核因子-κB的组成核因子-κB是一种多向性核转录因子,是Sen[1]等1986年首次在成熟B细…     

人PNPLA3基因启动子转录调控的初步分析

目的构建人PNPLA3基因不同长度的上游启动子荧光素酶报告基因裁体,在LO2细胞中比较不同长度启动子片段的活性,为研究PNPLA3基因的转录调控机制提供初步依据。方法对PNPLA3基因5’侧翼区约1 400个碱基进行生物信息学分析,预测其转录调控区域;利用PCR及酶切方法,以人外周血中全基因组DNA为模版扩增不同长度的PNPLA3基因上游启动子区序列,分别构建荧光素酶基因报告载体。瞬时转染LO2细胞,利用双荧光素酶报告基因分析系统检测各启动子的转录活性。利用在线分析软件预测PNPLA3基因主要转录调控区的潜在转录因子结合位点。结果成功构建5段不同长度的PNPLA3基因上游启动子区的报告载体;在LO2细胞内启动子活性随片段长度变化,表现为当PNPLA3启动子片段从-1015截短到-647,从-647截短至-553,从-553截短至-333时活性变化不大,而从-333截短至-8时活性显著下降(P<0.05);Match分析显示-333～-8片段具有多个转录因子结合位点。结论初步判断-333～-8区是PNPLA3基因的主要转录调控区。     

MTS1基因ARF启动子基础转录调控区转录活性研究

目的　研究MTS1基因ARF启动子基础转录调控区的转录激活及其与E2 F1转录因子之间的关系。方法　以含ARF启动子基础转录调控区E2 F1结合位点野生型序列的W6重组质粒为模板 ,设计该片段中E2 F1A、B、C结合位点序列突变或缺失的引物 ,用聚合酶链反应构建该区域中E2 F1A、B、C结合位点序列突变或缺失的ZE、ZF、ZG重组质粒。再将W6、ZE、ZF、ZG重组质粒转染进Jurkat细胞 ,检测其荧光素酶报告基因的表达。结果　构建的ZE、ZF、ZG重组质粒经SacⅠ或NaeⅠ酶切鉴定和DNA序列分析得到证实。与E2 F1位点野生型重组质粒W6比较 ,突变型重组质粒ZE、ZF、ZG在Jurkat细胞中荧光素酶报告基因的表达量减少 ,以E2 F1A位点突变的重组质粒减少明显。结论　构建ARF启动子E2 F1A、B、C结合位点序列突变的重组质粒成功 ;MTS1基因ARF启动子基础转录调控区的转录激活可能与E2 F1转录因子的作用有关。     

人Toll样受体4基因5′正调控区的鉴定

目的初步确定Toll样受体4(Toll like receptor 4,TLR4)基因5′远端髓系细胞特异性的转录调控区域。方法采用分子克隆结合报告基因分析的方法,构建TLR4基因5′旁侧-2413～ 66序列与荧光素酶报告基因载体pGL-3的重组质粒及其缺失质粒,通过DEAE-Dextran介导的转染技术导入人K562细胞,检测各报告基因质粒荧光素酶活性。结果TLR4基因5′旁侧转录起始位点上游-1337～-683 bp区域在K562细胞中,能够增强报告基因转录活性。结论TLR4基因5′旁侧存在远端细胞特异性的调控区域,定位并鉴定参与调控的转录因子,可望阐明TLR4基因表达调控及其组织和细胞特异性表达的机制。     

NF-κB与肾脏疾病的研究进展

核因子-KappaB(NF-κB)是细胞中一个重要的核转录因子,NF-κB是1986年从B细胞核抽提物中找到的转录因子,能与免疫球蛋白κ轻链基因增强子B序列GGGACTTTCC特异性结合,并能促进κ轻链基因表达[1]。现已证明NF-KB是一种具有转录激活功能的蛋白质,它能与多种基因启动子或增强子部位κB位点发生特异性结合并促进其转录,是细胞中一个重要的二聚化合物转录因子,NF-κB可以调控多种促炎因子、趋化因子、黏附分子及其受体的基因表达,在炎症损伤、肿瘤、凋亡、细胞再生方面均有重要作用。近年来发现NF-κB在肾脏疾病的发生、发展过程中起…     

人类pax—5基因外显子1B的5′侧翼区碱基序列分析

pax-5基因是B细胞生成和B细胞发育中的重要转录因子。为了更好的理解pax-5基因表达的调节机制，对pax-5基因外显子1B的5′侧翼区进行了分离克隆及序列测定。整段碱基序列（6671bp）分析表明，无TATAbox NFAT,2 LPOLYA-B,3GATA1,2AP4,10MZF1,1ETS1-B,1GATA3,1NKX25,2RORA1,1LYF1,2Ikaros2,2TCF11,1GATA-C和1FREAC7，并确定在序列上，因此，pax-5基因外显子1B的5′侧翼区与pax-5表达的调节有关，且对保证B细胞谱系和B细胞发育起重要调节作用。     

Major depression and heat shock protein 70-1 gene

BACKGROUND: Heat shock protein (HSP) expression can be induced by any stress such as with adrenocorticotropic hormones and catecholamines. It has been reported that patients with major depression have a 162-base deletion in the 5'-flanking region of heat shock protein 70 (HSP70)-1 gene mRNA. METHODS: To detect the HSP70-1 gene mRNA, total RNA was isolated and amplified by RT-PCR, and the sequence was confirmed in all five patients by DNA direct sequencing analysis. RESULTS: RT-PCR produced was no deletions of 162 bp in the human heat shock protein 70-1 gene in any of the patients with major depression or the nine controls. CONCLUSION: This finding is inconsistent with previous reports. We suggest that the 162-base deletion in the 5'-flanking region of the HSP70-1 gene mRNA is not associated with major depression. Further studies are required to determine the amounts of HSP70 and its mRNA in stress disorders such as major depression.     

Genetic polymorphisms and functional characterization of the 5'-flanking region of the human CYP2C9 gene: in vitro and in vivo studies

OBJECTIVE: Genetic polymorphisms were identified in the 5'-flanking region of the human CYP2C9 gene, and their effects on the phenotype were evaluated on the basis of the luciferase reporter gene assay and the in vivo pharmacokinetics of phenytoin. METHODS: Genetic polymorphisms were screened by polymerase chain reaction-single-strand conformational polymorphism analysis, following sequencing with DNA samples obtained from 50 healthy volunteers and 133 adult epileptic patients. HepG2 hepatoma cells were cotransfected with various sequence patterns of 5'-flanking region-luciferase reporter gene constructs. Pharmacokinetic parameters of phenytoin in relation to the corresponding sequence patterns were estimated by the Bayesian method, and the results were compared with in vitro activities. RESULTS: Genetic analysis revealed the existence of 7 single nucleotide polymorphisms (SNPs). Allele frequencies of T-->C transition at position -1912 (T-1912C), C-1886G, C-1566T, G-1538A, C-1189T, G-982A, and A-162G were 0.019, 0.019, 0.077, 0.019, 0.579, 0.019, and 0.003, respectively. Some mutations occurred simultaneously, and a total of 6 sequence patterns (patterns 1-6) were observed. The luciferase reporter gene assay indicated that the presence of mutation(s) resulted in a reduction in luciferase activity of 41.4% (pattern 2) to 86.8% (pattern 5) compared with the activity of the wild-type construct. The calculated intrinsic clearance of phenytoin was also lower (up to a 40% reduction for pattern 2) when a mutation(s) was present. CONCLUSION: In addition to the two major mutations in the coding region (CYP2C9*2 and CYP2C9*3 ), mutations in the 5'-flanking region of the human CYP2C9 gene appear to contribute to the large interindividual variability in drug metabolism activity.     

Novel single nucleotide polymorphisms in the specific protein 1 binding site of the bovine PRNP promoter in Japanese Black cattle: impairment of its promoter activity

Susceptibility to transmissible spongiform encephalopathy and different alleles of the prion protein gene (PRNP) of humans and sheep are associated. A tentative association between PRNP promoter polymorphisms and bovine spongiform encephalopathy (BSE) susceptibility has been reported in German cattle, whereas none of the known polymorphisms within the bovine PRNP-coding sequence affect BSE susceptibility. In the present study, novel single nucleotide polymorphisms located in the 5'-flanking region of bovine PRNP affecting its expression were demonstrated in Japanese Black cattle. We sequenced exon 1, and the approximately 200-bp 5'-flanking region of the PRNP translation initiation site containing the proximal promoter of PRNP was harvested. We identified 7 single nucleotide polymorphisms: -184A-->G, -141T-->C, -85T-->G, -47C-->A, -6C-->T, +17C-->T and +43C-->T. Six segregated haplotypes in the population were cloned into luciferase-expressing plasmids, transfected into N2a cells, and their reporter activities were measured 48 h after transfection. Six haplotypes showed a decreased expression level including -6C-->T in specific protein 1 binding site (p < 0.05) or -141T-->C (p < 0.01) at 48 h compared with the wild-type haplotype. These results advocate that certain polymorphisms such as specific protein 1 binding site polymorphisms in the bovine PRNP promoter region in Japanese Black cattle could influence promoter activity, suggesting that breeding cattle with such substitutions may be a useful approach in reducing BSE risk.     

CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12: evidence for an allelic variant with altered catalytic activity

OBJECTIVE: To determine the existence of mutant and variant CgammaP3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression. METHODS: A bacterial artificial chromosome that contains the complete CgammaP3A4 gene was isolated and the exons and surrounding introns were directly sequenced to develop primers to polymerase chain reaction (PCR) amplify and sequence the gene from lymphocyte DNA. DNA samples from Chinese, black, and white subjects were screened. Mutating the affected amino acid in the wild-type cDNA and expressing the variant enzyme with use of the baculovirus system was used to functionally evaluate the variant allele having a missense mutation. RESULTS: To investigate the existence of mutant and variant CgammaP3A4 alleles in humans, all 13 exons and the 5'-flanking region of the human CgammaP3A4 gene in three racial groups were sequenced and four alleles were identified. An A-->G point mutation in the 5'-flanking region of the human CgammaP3A4 gene, designated CgammaP3A4*1B, was found in the three different racial groups. The frequency of this allele in a white population was 4.2%, whereas it was 66.7% in black subjects. The CgammaP3A4*1B allele was not found in Chinese subjects. A second variant allele, designated CgammaP3A4*2, having a Ser222Pro change, was found at a frequency of 2.7% in the white population and was absent in the black subjects and Chinese subjects analyzed. Baculovirus-directed cDNA expression revealed that the CYP3A4*2 P450 had a lower intrinsic clearance for the CYP3A4 substrate nifedipine compared with the wild-type enzyme but was not significantly different from the wild-type enzyme for testosterone 6beta-hydroxylation. Another rare allele, designated CgammaP3A4*3, was found in a single Chinese subject who had a Met445Thr change in the conserved heme-binding region of the P450. CONCLUSIONS: These are the first examples of potential function polymorphisms resulting from missense mutations in the CgammaP3A4 gene. The CgammaP3A4*2 allele was found to encode a P450 with substrate-dependent altered kinetics compared with the wild-type P450.