人FAM33A基因启动子的克隆与鉴定

目的 鉴定人FAM33A基因的启动子,为进一步研究其转录调控机制奠定基础.方法 采用5'RACE技术(5'端cDNA快速扩增)鉴定FAM33A的转录起始位点.采用PCR定向克隆、酶切亚克隆等策略,构建FAM33A启动子荧光素酶报告基因.采用LipofectamineTM 2000转染H1299细胞,并通过Dual-LuciferaseGA16A Reporter Assay System进行荧光素酶报告基因活性检测.结果 确定了FAM33A的转录起始位点,构建了覆盖FAM33A 5'端ATG附近约2 kb区域的一系列FAM33A启动子荧光素酶报告基因.启动子活性分析表明,这些重组体均具有较高的启动子活性,同时含有典型的GC盒以及Sp1、E2F和GATA-1等潜在的转录因子结合位点.结论 FAM33A启动子区域主要定位于转录起始位点附近约590 bp的区域内.     

人LAIR-1/CD305基因启动子的生物信息学分析

目的:研究人LAIR-1/CD305启动子及其5′上游调控序列。方法:通过检索NCBI中的人类基因组数据库,获得LAIR-1的转录本序列及翻译起始位点上游2500bp的序列。利用Promoter2.0等软件预测LAIR-1的启动子序列,然后利用MatInspector等软件对启动子序列的转录因子结合位点和启动子功能模块进行预测。结果:LAIR-1基因的核心启动子区位于翻译起始密码子上游的600～200bp区域内。在转录调控区发现了一些重要的转录因子结合位点,并预测到13种启动子功能模块。结论:LAIR-1基因的表达可能受到多种转录因子和5′端上游调控序列的调控。     

人大麻素受体启动子真核报告质粒的构建及活性分析

目的:利用双荧光素酶报告基因体系,根据大麻素受体1(cannabinoid receptor 1,cnr1)启动子序列不同部位的转录活性来初步确定其活性区域,为脊髓缺血耐受保护中cnr1高表达的转录调控的研究奠定基础。方法:从NCBI中获取cnr1基因转录起始点5’端向上约1800 bp的核苷酸序列,按照300 bp的距离间隔,设计6个不同长度的截短体PCR引物,以人全血基因组DNA为模板,分别扩增cnr1启动子区域的截短体片段,并克隆入荧光素酶报告基因pGL3-Basic质粒中。将含有不同截短体的重组质粒分别转染Hela、Jurket和A549细胞后行荧光素酶活性检测。根据不同截短体转录活性检测结果,确定cnr1启动子活性区域。结果:成功将cnr1启动子区6个不同长度(1800、1500、1200、900、600和300 bp)的截短体克隆入荧光素酶报告基因pGL3-Basic质粒。在3种细胞系Hela、Jurket和A549的荧光素酶活性检测均显示600 bp的截短体转录活性最强。结论:成功构建了cnr1启动子的报告基因重组质粒,初步证实-600 bp到-200 bp区为cnr1的启动子的活性区域,从而为进一步研究cnr1的转录调控奠定了基础。     

利用同源序列确定人GIPC2核心启动子和寻找可能的顺式作用元件

目的利用同源序列确定人GIPC2核心启动子区和寻找可能存在的基因调控区域。方法 1)利用Vista比对不同物种GIPC2基因编码区和非编码区序列,得到GIPC2同源序列;2)分别构建插入人和大鼠GIPC2翻译起始位点ATG上游不同长度截短片段的重组质粒,转染人HEK293T细胞和大鼠PC12细胞;3)根据报告基因荧光强度推断人和大鼠GIPC2的核心启动子区;4)将人GIPC2内含子区同源序列置于核心启动子前面,构建重组质粒,转染HEK293T,HT-29和PC12细胞,检测启动子活性。结果人GIPC2核心启动子区确定为+32至+190序列区域;人和大鼠GIPC2 ATG上游一段同源序列有抑制GIPC2启动子活性的功能;人和大鼠GIPC2第一个内含子内的同源序列有促进启动子活性的功能。结论利用同源序列方法确定了人GIPC2基因的核心启动子区,并且找到能抑制和促进GIPC2启动子活性的可能的顺式作用元件。     

第1类整合子中aadA2基因翻译起始位点的确定

目的 确定第1类整合子中aadA2基因能否从上游无核糖体结合位点的密码子ATG起始翻译并合成有功能的蛋白.方法 定点突变含有不同翻译起始密码子的aadA2基因盒,并连同上游的可变区启动子分别克隆入质粒pACYC184中,转化大肠埃希菌JM109,免疫印迹检测含有不同翻译起始密码子的aadA2基因的翻译产物,用微量肉汤稀释法检测链霉素对含有不同翻译起始密码子aadA2基因的大肠埃希菌JM109的最小抑菌浓度.结果 aadA2基因可从上游无核糖体结合位点的密码子ATG及上游具有核糖体结合位点的密码子GTG起始翻译,同时在GTG密码子下游还存在着起始翻译密码子,其翻译产物在免疫印迹中均可与抗氨基糖苷-3″-腺苷酰基转移酶多克隆抗体产生特异性杂交条带,并赋予宿主细菌对链霉素不同水平的耐药.结论 当位于第1类整合子第1位基因盒中,aadA2基因可从上游无核糖体结合位点的密码子ATG起始翻译并合成有功能的蛋白,这一结构特点使得整合入第1类整合子的基因盒,不需带有核糖体结合位点即可起始基因盒中相应读码框的翻译,从而有利于第1类整合子表达从外界环境中捕获的基因.     

人类XBP1基因5′上游DNA序列的转录激活功能分析

目的分析人类转录因子X-盒结合蛋白1（X-box binding protein Ⅰ，XBP1）基因启动子在不同细胞系中转录活性的差别，研究其转录调控机制。方法在对XBP1基因进行生物信息学分析的基础上，设计6种XBP1启动子缺失突变体，分别包括剧XBP1基因5’上游-1039～66bp、-859～66bp、-623～66bp、-351～66bp，-227—66bp、-227～-45bp，针对每一种缺失突变体构建相应的氯霉素乙酰转移酶（chloramphenicol acetyltansferase，CAT）报告基因载体，再分别瞬时转染K562、HepG2、NIH-3T3和LO2细胞，应用报告基因CAT瞬时表达系统，检测XBP1每一种缺失突变体在K562、HepG2、NIH-3T3和LO2 4种不同细胞中的活性差异，并进一步确定脚，基因启动子的具体调节部位。结果成功获得XBP1基因启动子6种缺失突变体的正确克隆和6种相应的报告基因载体p1—XBP1p，p2-XBP1p，p3-XBP1p，p4-XBP1p，p5-XBP1p，p6-XBP1p，每一种报告基因转染K562、HepG2、NIH-313和LO2 4种不同细胞系后，p4-XBP1p和p5-XBP1p在K562、HepG2细胞中的转录活性均高于其它报告载体，p5-XBP1p在HepG2细胞中转录活性最高；而6种报告基因载体在NIH-3T3小鼠成纤维细胞中不具有转录活性。结论。XBP1基因的转录激活能力在不同细胞中有所差异，其表达活性具有一定的细胞类型特异性，与细胞类型和细胞状态密切相关；XBP1基因启动子的-227～66bp区域是该启动子的核心部位，而且核心启动子区包括ATG翻译起始密码子的下游部位，这一部位是转录活性的重要部位，一旦缺失，XBP1基因启动子会丧失活性。     

人B细胞活化因子基因启动子活性研究

为探索与自身免疫病发病密切相关的B细胞活化因子(BAFF)基因高水平转录的启动序列及IFN-γ等炎性细胞因子对其活性的影响,以人BAFF基因转录起始点上游-1 349 bp至-329 bp的片段为靶序列,取长度不等的片段作为启动子与含氯霉素乙酰基转移酶(CAT)报告基因的质粒组成5个重组体,脂质体转染法转染上述重组体至人骨髓白血病细胞株:HL-60,CAT-ELISA测定细胞CAT表达水平以比较各重组体的启动子活性;同时加入细胞因子IL-10、IFN-γ、IL-4、重组人BAFF蛋白(rBAFF),以测定其对BAFF启动序列的影响。结果表明,-1 349~-329 bp、-1 349~-743 bp序列具有强启动调控活性,而-1 349~-1 099 bp片段启动活性最低。细胞因子IFN-γ、IL-10和rBAFF均能在一定程度上调人BAFF基因的启动活性,IL-4则一定程度地抑制BAFF基因的启动活性。研究提示,人BAFF基因-1349~-743 bp片段是进一步研究BAFF基因转录相关DNA结合蛋白的重要调控靶序列。细胞因子IL-10、IFN-γ、IL-4、rBAFF可以一定程度的在转录水平影响BAFF合成和分泌。     

人间皮素基因中Wnt/β-catenin通路调控元件分析及在卵巢癌细胞中启动子区鉴定

目的分析人间皮素基因启动子区中Wnt/β-catenin信号通路相关转录因子T细胞因子/淋巴细胞增强因子(T cell factor/lymphoid enhancer factor,TCF/LEF)结合位点,鉴定间皮素基因在卵巢癌细胞表达相关核心启动子区。方法采用生物信息学方法分析人间皮素基因启动子区域内可能的TCF/LEF转录因子结合位点。克隆人间皮素基因5'端附近1 764 bp启动子序列,对该片段做5'端的不同截短,克隆至p GL3-basic报告基因载体,分别转染人卵巢癌细胞系SKOV3和3-AO,双荧光酶报告基因系统检测不同启动子片段活性。结果人间皮素基因启动子区含有多个潜在的TCF/LEF结合位点。成功克隆扩增间皮素基因启动子区-1456～+308、-164～+308、+47～+308三个片段,经测序证明无误,构建p GL3载体。转染SKOV-3与3-AO细胞后,双荧光酶报告基因系统检测显示-1456～+308与-164～+308片段在两个细胞系中均有较高启动活性,+47～+308片段活性较前两者显著下降(P均0. 01)。结论含有TCF/LEF转录因子结合位点的-164～+47序列是卵巢癌中间皮素基因表达的核心启动子区,为进一步观察卵巢癌中间皮素基因的表达调控机制奠定基础。     

人干扰素调节因子3基因启动子的初步鉴定

子活性分析表明,IRF3启动子区域定位于主要转录起始位点区域前111-167 bp的区域内.采用转录因子结合位点预测分析软件分析表明,该区域内存在E2F转录因子结合位点.结论 -167～-111 bp区存在IRF3启动子的核心调控元件,转录因子E2F可能参与IRF3的转录调控.     

NF-κB结合元件缺失对NOX1启动子转录活性的影响

目的:构建含NADPH氧化酶1(NOX1)近端启动子区的pGL3萤光素酶报告基因载体及缺失NF-κB结合元件的相应载体,分别测定其相应活性,探讨NF-κB结合元件缺失对NOX1启动子区转录活性的影响。方法:采用PCR法克隆NOX1启动子区序列(1 415 bp),将目的片段与pGL3萤光素酶载体分别双酶切、纯化后进行连接(pGL3-NOX1-1415),测序鉴定;应用Alibaba 2.1软件分析NOX1近端启动子区,获取NF-κB结合元件;重叠PCR法将含该元件的启动子区域(88 bp)缺失,并构建相应载体(pGL3-NOX1-1327)。将两载体分别与pRL-TK内参质粒瞬时转染进入A549细胞,采用TNF-α(10μg/L)刺激细胞24 h,萤光酶标仪检测A549细胞的萤光素酶活性。结果:测序鉴定结果提示pGL3-NOX1-1415及NF-κB结合元件缺失的pGL3-NOX1-1327载体构建成功;细胞实验显示,TNF-α刺激后,转染pGL3-NOX1-1415的A549细胞萤光素酶活性明显强于对照组(P0.05),而转染NF-κB结合元件缺失的pGL3-NOX1-1327的细胞萤光素酶活性明显低于转染pGL3-NOX1-1415组(P0.05)。结论:TNF-α诱导A549细胞NOX1基因活化与NF-κB密切相关,NF-κB参与了TNF-α诱导的NOX1基因启动子的转录调控。     

Analysis of 11q21-24 loss of heterozygosity candidate target genes in breast cancer: indications of TSLC1 promoter hypermethylation

Loss of heterozygosity (LOH) at the distal half of chromosome arm 11q is frequent in a variety of human tumors, including breast cancer, and is often associated with poor prognosis. In an ongoing attempt to locate and characterize the main target genes within this chromosome region, we first looked for aberrations in known genes either suggested to be involved in tumorigenesis or shown to suppress tumor formation. We examined 31 primary breast tumors showing LOH in 11q21-24 for mutations in the MRE11A, CHK1, PPP2R1B, and TSLC1 genes. The absence of intragenic alterations related to cancer led us next to evaluate possible gene silencing resulting from promoter region CpG hypermethylation, using the bisulfite sequencing technique. In addition to the four genes mentioned above, we also analyzed the ATM gene, which had been investigated for certain germline mutations in an earlier study. Only the TSLC1 promoter region exhibited aberrant methylation patterns, and altogether 33% (10/30) of the successfully analyzed tumors showed evidence of elevated levels of TSLC1 CpG methylation. Ten percent (3/30) of the tumors showed significantly increased methylation. Thus, as has been shown in lung and some other forms of cancer, hypermethylation of the TSLC1 promoter region is also frequently a second hit along with LOH in breast cancer.     

人血管细胞中hCREG1基因启动子对血清饥饿发生应答

目的： 为揭示E1A 激活基因阻遏子（CREG1）在人血管平滑肌细胞(VSMCs)和人脐静脉内皮细胞（HUVECs）中表达的调控机制，构建人CREG1（hCREG1）启动子报告基因载体，探讨CREG1在不同血管细胞中的表达。方法: 在对hCREG1进行生物信息学分析的基础上，以人基因组DNA为模板，PCR方法扩增含有hCREG1基因上游-3 677 bp序列，构建了hCREG1 5′上游-3 677 bp、-2 310 bp和-945 bp序列片段，分别将这3个序列克隆到pMD18-T载体上并定向亚克隆到pEGFP-1报告基因载体-pEGFP-hCREG1-P3677、pEGFP-hCREG1-P2310和pEGFP-hCREG1-P945。将重组质粒瞬时转染VSMCs株HITASY和HUVECs，检测绿色荧光蛋白（GFP）的表达。结果: 经测序鉴定证实，3个报告基因载体中插入的PCR扩增序列均与hCREG1基因上游DNA序列完全匹配。瞬时转染体外培养的人VSMCs和HUVECs后，经荧光观察和蛋白质印迹（Western blotting）证实，细胞内存在GFP的表达，并且0.5%FBS培养的HITASY细胞中GFP表达较10%FBS培养的细胞中明显增加。HUVECs中的GFP表达较人VSMCs明显增加。结论: hCREG1基因启动子报告基因载体构建成功，核心启动子存在于5′上游序列的-945 bp-0 bp区域，为进一步研究hCREG1基因表达提供了条件。     

Expression of the TSLC1 adhesion molecule in pulmonary epithelium and its down-regulation in pulmonary adenocarcinoma other than bronchioloalveolar carcinoma

TSLC1 (tumor suppressor in lung cancer-1) is an adhesion molecule of the Ig superfamily that binds homophilically and mediates cell-cell interactions. Originally, TSLC1 was cloned as a candidate tumor suppressor from the genomic region that frequently exhibits loss of heterogeneity in human non-small-cell lung cancer (NSCLC). However, there have been no studies on TSLC1 expression in normal lungs or NSCLC. Here we show that pulmonary epithelial cells express TSLC1 and its expression levels are often decreased or lost in primary pulmonary adenocarcinoma, a major histologic type of NSCLC. Immunohistochemistry revealed that TSLC1 was localized at cell-cell boundaries of all columnar epithelial cells in mouse embryonic lungs of 10.5 and 13 days postcoitus. Similar staining patterns were observed in bronchiolar and alveolar epithelial cells of adult human lungs, suggesting a physiologic role for TSLC1 in interactions of these cells. Next we performed Western blot analyses of TSLC1 in 47 primary pulmonary adenocarcinomas and judged each tumor as either decreased or nondecreased by comparing TSLC1 expression levels of the tumor with the levels of normal lungs. The expression profiles had a significant relation to histologic subtypes but not to other clinicopathologic parameters. Sixteen bronchioloalveolar carcinomas (BACs) were all judged nondecreased, while 19 of 31 (63%) adenocarcinomas other than BAC were judged decreased (p < 0.0001). Immunohistochemistry of tumors judged nondecreased revealed that not only BAC cells but also tumor cells in lepidic growth components of adenocarcinomas other than BAC expressed TSLC1 on their lateral plasma membranes. These tumor cells are considered less invasive because they proliferate in a lepidic growth pattern along alveolar walls. Thus, the present results not only support the hypothesis that TSLC1 is a tumor suppressor of NSCLC but also suggest that preserved integrity of TSLC1 may contribute to less invasive phenotypes of lepidic growth tumor cells.     

Construction of eukaryotic expression vector of TSLC1 gene

Introduction

To construct a eukaryotic expression vector of the tumour suppressor in lung cancer 1 (TSLC1) gene, so as to explore the mechanisms of tumour suppression of the gene theoretically.

Material and methods

The open reading frame (ORF) of TSLC1 gene was amplified with RT-PCR from normal human foreskin acrobystia, and cloned to pMD19-T simple vector (TA Clone method). The resultant plasmid was transformed into Escherichia coli JM109 for amplification. The TA Clone recombinant was digested by double restriction enzyme (Bgl II/EcoR I) and analysed with agarose gel electrophoresis. The positive one was sequenced. The inserted DNA fragment was recovered, and then it was mounted into the eukaryotic expression vector pIRES2-EGFP, transformed into E. coli JM109 for amplification. A positive recombinant plasmid named pIRES2-EGFP-TSLC1 was confirmed by Bgl II/EcoR I double-enzyme digestion analysis.

Results

RT-PCR amplified the ORF of the TSLC1 gene. It was approximately 1400 base pairs. The obtained DNA was confirmed a high degree of homology with the sequence of TSLC1 cDNA sequence ({"type":"entrez-nucleotide","attrs":{"text":"AY358334","term_id":"37181792"}}AY358334) stored at GenBank.

Conclusions

Construction of a TSLC1 eukaryotic expression vector was successful, and it has established a solid foundation for further study.     

产气肠杆菌组成型启动子的筛选及其活性测定

目的：从产气肠杆菌基因组中筛选组成型启动子,并检测其活性。方法用Sau3 AⅠ限制性内切酶将产气杆菌的基因组酶切到500 bp大小,连接到pCMR8 a载体上,转化DH5α感受态后涂布氯霉素和卡那抗生素的双抗平板上筛选启动子,经SDS－PAGE分析启动子的蛋白表达能力,筛选较强启动子并对其进行截短和顺反性实验,最后利用截短启动子表达不同的蛋白。结果成功筛选到了5个启动子序列,获得到了表达能力最强启动子的截短核心序列,验证了其具有反向启动外源蛋白表达的能力,成功表达了其他外源蛋白。结论本实验通过大肠埃希菌表达系统成功的筛选并验证了产气杆菌的一种组成型启动子,具有较强的启动外源蛋白表达能力,对工业上蛋白的生产及实验室表达某种蛋白具有重要意义。     

乙型肝炎病毒核心启动子及前C区突变检测基因芯片的制备及临床应用

目的 建立检测乙型肝炎病毒核心启动子及前C区突变基因芯片并探讨其临床应用的价值.方法 设计并合成针对核心启动子1762/1764、1814及前C区1896位点突变的特异性探针,制备寡核苷酸芯片.采用不对称PCR对该区域进行扩增,扩增产物与芯片杂交后分析结果,并评价该方法的特异性、灵敏度.采用该方法检测138例HBV DNA阳性血清标本.结果 该方法能够特异地检测乙型肝炎病毒核心启动子1762/1764、1814及前C区1896位点,灵敏度达1×101拷贝/μl.138例HBV DNA阳性血清标本中,T1762/A1764突变40例（28.99%）,C1814突变11例（7.97%）,A1896突变16例（11.59%）.前C区A1896突变在高拷贝组中明显高于低拷贝组（P＜0.01）.结论 本研究建立的基因芯片能够准确同时检测HBVV核心启动子区突变和前C区突变,前C区A1896突变可能与HBV复制状态有关.     

慢性HBV C基因启动子变异的检测

目的 了解慢性乙型肝炎病毒（ＨＢＶ）Ｃ基因启动子变异的情况以及对病毒血清学的影响。方法 应用错配聚合酶链反应特异性扩增含有ＨＢＶ Ｃ基因启动子的片段,扩增产物用限制性内切酶Ｂｃｌ Ｉ酶切,琼脂糖凝胶电泳后,观察酶切后的限制性片段长度多态性（ＲＦＬＰ）图谱,分析Ｃ基因启动子区段的核苷酸（ｎｔ）１７６２由碱基Ａ→Ｔ和（ｎｔ）１７６４碱基由Ｇ→Ａ的变异,并经直接测序分析证实ＲＦＬＰ结果。结果 １１０例慢     

Tumor suppressor in lung cancer-1 as a novel ameloblast adhesion molecule and its downregulation in ameloblastoma

Tumor suppressor in lung cancer-1 (TSLC1) is an intercellular adhesion molecule of the immunoglobulin superfamily. There is little information regarding the developmental expression profiles. In an attempt to clarify the distribution of TSLC1 proteins in mouse embryos tissue by immunohistochemistry, it was found that the TSLC1-specific signals were detected in the tooth germ as early as bud stage. The signals of TSLC1 were in the enamel epithelium at the cap stage, and became restricted to ameloblasts during the transition to and throughout the bell stage. In contrast, the signals for E-cadherin, which is important in odontogenesis, were distributed in all the components of the ectoderm-derived germ at any stage. In addition, E-cadherin preferred to locate on the basal membrane of ameloblasts, whereas TSLC1 preferred the lateral. And in further contrast, all the ameloblastomas examined were positive for E-cadherin (18/18) whereas all but one was negative for TSLC1 (1/18). These results indicate that TSLC1 is a novel interameloblast adhesion molecule that may be downregulated during ameloblastic tumorigenesis.