Cloning of NHE-1 gene fragment from human lung can.cer cells and construction of its antisense expression vector

Objective: To clone the partial sequence of Na^ /H^ exchanger- 1 (NHE- 1) gene of human lung cancer cells and insert it reversely into the multiclone site of pLXSN in order to construct an antisense expression vector for tumor gene therapy it~ vivo. Methods: With use of the upstream and downstream primers containing Barn H I and EcoR I in their 5‘ ends respectively, a partial sequence of the first exon of NHE-1 gene was cloned in a length of 454 bp from genomic DNA of human lung cancer cell A549 with PCR method. The product was then direetionally and reversely insert into the multiclone site of pLXSN. Finally, the constructed recombinant was identified with agarose gel electrophoresis and DNA sequencing. Results: The cloned fragment was 461 bp in length and successfully ligated to pLXSN with the identification by agarose gel etectrophoresis. DNA sequencing confirmed that the fragment cloned and inserted into the vector was identical with the targeted one. Conclusion: The targeted fragment is successfully cloned and reversely inserted into pLXSN in our experiment. The antisense expression vector of NHE-1, pNHE-1, was constructed successfully.     

Cloning and Identification of frc Gene from Oxalobacter Frmigenes

The cloning and identification of frc gene from Oxalobacter formigenes in the intestines of Chinese people were conducted. The genomic DNA of Oxalobacter formigenes was extracted. frc gene fragment was amplified by polymerase chain reaction (PCR) and linked with pEGFP-C1. The recombinant plasmid was designated pEGFP-frc and was identified by restriction-enzyme digestion and sequencing. Human embryo kidney 293 cells were transfected with pEGFP-frc, then RT-PCR and Western blotting were performed to detect the expression of frc gene. The length of frc gene was found to be 1287 bp, and the homology of nucleotides and amino-acid residue with the sequence in GenBank was 95.88% and 99.07%. Bright green fluorescent light could be observed in 293 cells transfected with the pEGFP-frc. frc mRNA and fusion protein FCoAT-EGFP were detected in the cells. It is concluded that frc gene cloned from the Oxalobacter formigenes in the intestines of Chi- nese people can be expressed in eucaryotic 293 cells and keep its enzyme activity.     

MEK AND p38 MAPK-DEPENDANT PATHWAYS ARE INVOLOVED IN THE POSITIVE EFFECT OF INTERLEUKIN-6 ON HUMAN GROWTH HORMONE GENE EXPRESSION IN RAT MtT/S SOMATOTROPH CELLS

Objective To investigate the effect of interleukin-6（IL-6） on the human growth hormone （hGH） gene expression in a rat somatotropic pituitary cell line MtT/S. Methods The plasmids containing various lengths of hGH gene 5＇-promoter fragments were constructed. Stably transfected MtT/S cells were created by cotransfecting the above plasmids and pcDNA3.1 （＋） with DMRIE-C transfection reagent. After the administration of these cells with IL-6 and/or various inhibitors of signaling transduction path-ways, the luciferase activities in MtT/S cells lysis were assayed to demonstrate the effects of IL-6 on hGH gene promoter activity and possibly involved mechanism. Results The 10^3U/mL IL-6 stimulated GH secretion and synthesis, and promoted the 5＇-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.69 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase （MAPKK/MEK） inhibitor PD98059 （40μmol/L） and p38 mitogen-activated protein kinase （MAPK） inhibitor SB203580 （5μmol/L） completely blocked the stimulatory effect of IL-6. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit-1 nor inhibition of Pit-1 expression affected IL-6 induction of hGH promoter activity. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-6. The results showed that the stimulatory effect of IL-6 was abolished following deletion of the - 196 to - 132 bp fragment. Conclusions IL-6 promotes GH secretion and synthesis by rat MtT/S somatotroph cells. The stimulatory effect of IL-6 on hGH gene promoter appears to require the activation of MEK and p38 MAPK, and a fragment of promoter se- quence that spans the - 196 to - 132 bp of the gene, but may be unlinked with Pit-1 protein.     

CHANGES IN EXPRESSION AND STRUCTURE OF SOME PROTOONCOGENES IN TPA-INDUCED HL-60 AND RAJI CELLS

Different expression levels of c-myc, c-H-ras, c-sis and c-erb were observed in HL-60 and Raji cells. Induction of HL-60 cells with TPA (10ng/ml) for two days elicited differentiation together with a decreased expression level of c-myc and c-H-ras. No significant changes in morphological structure in Raji cells were seen to occur over a 4-day induction period with TPA (1ng/ml), but the level of expression of c-H-ras declined slightly over the period whereas that of c-myc remained unchanged. From the EcoRI and SacI restriction fragment patterns, it can be seen that the c-myc oncogene was amplified in HL-60 and different in structure in Raji as compared to WBC. After induction the patterns of the two restriction endonuclease fragments for HL-60 cells showed no change. The pattern of Sacl fragments of c-H-ras gene of Raji cells induced with TPA for 4 days displayed an additional fragment of 12.7 Kb. Possible roles of oncogene expression in cell differentiation are discussed.     

EFFECT OF INTERLEUKIN-1β ON GROWTH HORMONE GENE EXPRESSION AND ITS POSSIBLE MOLECULAR MECHANISM IN RAT MtT/S SOMATOTROPH CELLS

Objective To elucidate the effect of interleukin-1β （IL- 1β） on human growth hormone （hGH） gene expression in a rat somatotropic pituitary cell line MtT/S.
Methods Stably transfected MtT/S cells were firstly established by transfecting 484-Lucl plasmid which contained hGH gene promoter --484 to ＋30 bp and luciferase reporter gene. The effect of IL-1β on hGH gene expression was determined by assaying the luciferase activities. RT-PCR method was also used to determine whether IL-1 recepor mRNA was expressed in MtT/S cells.
Results The 10^3 U/mL IL-1β stimulated secretion and synthesis of GH, and promoted the 5＇-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.38 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase （MAPKK/MEK） inhibitor PD98059 （40 μmol/L） and p38 mitogen-activated protein kinase （MAPK） inhibitor SB203580 （5 μmol/L） completely blocked the stimulatory effect of IL-1μ, and phosphatidylinositol-3-kinase （PI3-K） inhibitor LY294002 partly abolished the effect of IL-1μ. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit- 1 nor inhibition of Pit- 1 expression affected induction of hGH promoter activity by IL-1μ. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-1β, and results showed that the stimulatory effect of IL-1β was abolished following deletion of the --196 to -- 132 bp fragment.
Conclusions IL-1β promotes GH secretion and synthesis in rat MtT/S somatotroph cells. The stimulatory effect of IL-1β on hGH gene promoter appears to require the activation of MEK, p38 MAPK, PI3-K, and a fragment of promoter sequence that spans the -196 to -132 bp of the gene, but it may be unlinked with Pit-1 protein.     

Mutations of mitochondrion DNA in mouse tumors

Objective： To ascertain the variations of mitochondrion DNA （mtDNA） in mouse tumors and to inquire into the relationship between mutations of mtDNA and carcinogenesis Methods： The variations of D-loop, ND3 and tRNA^Met＋Glu＋Ile gene fragments of mtDNA from six tumor cell lines of mice were analyzed by PCR technology with restriction fragment length polymorphism analysis （polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP） and single strand conformation polymorphism analysis （SSCP-PCR） method. Results： ND3 and tRNA^Met＋Glu＋Ile gene fragments ofmtDNA from the tumors showed no variation in 27 endonuclease sites; D-loop ofmtDNA from Hca-F had an additional endonuclease sites of Hinf I in contrast to that of the inbred mouse. Deeply analyzed by PCR-SSCP, the D-loop ofmtDNA was found to possess mutations in 4 of 6 tumors. Conclusion： D-loop is the hot spot of tumor mtDNA mutation which can act as contributors to the carcinogenic     

EXPRESSION AND DELETION ANALYSIS OF EcoRII ENDONUCLEASE AND METHYLASE GENE

INTRODUCTIONEcoRIIrestrictionendonuclease（EcoRII．R）andEcoRIIDNA－methyltransferase（EcoRII．M）constitutearestriction－modificationsysteminE．coliR，whichrecog－nizethesamesequenceCCWGGasDNAcytosinemethylase（Dcm）existinggenerallyinE．coli．CleavagewithEcoRII．RinrecognizedsequencescanbeinhibitedbyeitherEcoRII．MmethylationorDcmmethylation．EcoRII．Risthefirsttype－IIrestrictionendonucleaseofwhichendonucleolyticactivitywasfoundtodependonthesimultaneousinteractionwit…     

EcoRⅡ限制性内切酶和甲基化酶基因克隆,表达和缺失突？…

目的 同时克隆ＥｃｏＲⅡ限制性内切酶基因和ＥｃｏＲⅡ甲基化酶基因,初步探讨该限制－修饰系统的协同表达机制。方法 采用外切酶Ⅲ删除法构建单向缺失亚克隆,根据亚克隆的酶活性对上述两种基因进行初步定位,经核酸序列测定确定亚克隆的缺失部位,再以Ｓ１核酸酶保护分析法测定基因的转录起始点。     

EOLAl基因启动子序列的鉴定

目的 鉴定EOLA1基因启动子序列,为深入研究EOLAl的转录调控机制奠定基础.方法 通过PCR反应进一步缺火突变EOLAI基因5'侧翼区-1672～+51(1723 bp)序列,将产物插入到含荧光素酶报告基因的载体pGL3-Basic中,转染ECV304细胞,瞬时表达后测定荧光素酶活性.采用生物信息学分析该1 723 bp片段可能存在的转录因子结合位点.结果 缺失到785 bp片段仍具有肩动子活性,进一步缺欠到717 bp片段后活件消失,从而确定启动子位于-738～ -676 bp序列,其附近含Spl和Myf顺式作用元件.结论 确定了EOLAI启动子范围和转录因子结合位点.     

成纤维细胞生长因子19转录活性及上游结合元件分析

目的 构建FGF19基因启动子区荧光素酶报告基因质粒,并研究分析其启动子区的转录活性和结合元件。方法 设计特异性引物PCR扩增基因组DNA,得到长为3323bp的FGF19基因启动子区片段,将此PCR产物插入荧光素酶报告基因载体pGL3-Basic Vector,获得全长为3323bp的FGF19基因启动子区荧光素酶报告基因质粒。在此基础上设计特异性引物,构建5''端系列缺失荧光素酶报告基因质粒。通过瞬时转染实验检测所构建质粒的相对荧光素酶活性,分析不同启动子区片段对FGF19基因转录活性的影响,并用软件预测影响启动子转录活性的关键转录因子。结果 构建了7个FGF19基因启动子区荧光素酶报告基因质粒,经双酶切和测序验证均正确。瞬时转染及荧光素酶报告基因分析实验发现启动子区-2351~-2316是调控FGF19启动子转录活性的重要序列,且在线软件预测该序列存在潜在的转录因子位点。结论 FGF19基因启动子区-2351~-2316是调控其启动子转录活性的关键位置。     

间日疟原虫深圳株红内期SSUrDNA基因片段的克隆与序列分析

目的 体外扩增间日疟原虫深圳株红内期小亚单位核糖体核糖核酸编码基因(SSUrDNA)片段，研究其结构与功能。方法 设计一对特异性引物，采用聚合酶链反应(PCR)从间日疟原虫患者血样中扩增出间日疟原虫SSUrDNA片段，以PUC19质粒T载体构建重组子导入大肠杆菌JM109；阳性克隆双酶切鉴定后，双脱氧末端终止法测定序列。结果 间日疟原虫SSUrDNA扩增片段大小为341bp；阳性克隆双酶切及PCR扩增均得到预期大小的片段；序列测定插入片段为341bp，与Sal I株顺序相比，仅在第151位处缺失一个碱基C。结论 成功克隆了间日疟原虫SSUrDNA片段．该序列在间日疟原虫虫株间高度保守。     

EOLA1基因启动子序列的鉴定

目的 鉴定EOLA1基因启动子序列,为深入研究EOLAl的转录调控机制奠定基础.方法 通过PCR反应进一步缺火突变EOLAI基因5'侧翼区-1672～+51(1723 bp)序列,将产物插入到含荧光素酶报告基因的载体pGL3-Basic中,转染ECV304细胞,瞬时表达后测定荧光素酶活性.采用生物信息学分析该1 723 bp片段可能存在的转录因子结合位点.结果 缺失到785 bp片段仍具有肩动子活性,进一步缺欠到717 bp片段后活件消失,从而确定启动子位于-738～ -676 bp序列,其附近含Spl和Myf顺式作用元件.结论 确定了EOLAI启动子范围和转录因子结合位点.     

人肺癌细胞NHE-1基因部分正调控序列的克隆

目的 克隆人肺癌细胞Na∧ /H∧ 交换泵-1（NHE-1）基因上游正调控序列部分,为进一步将该片段导入肺癌细胞株,竞争性结合转录因子,达到抑制细胞内NHE-1基因的表达奠定基础。方法 采用PCR技术从人肺癌A549细胞基因组中扩增长约170bp的NHE-1基因调控序列中起正调控作用的片段。在上、下游引物的5′-端带上BanHI和EcoRI酶切位点。然后将该片段连接到pUC18载体上,最后对产生的重组子进行酶切、PCR和测序鉴定。结果 经酶切及PCR鉴定,所克隆的目的片段大约为180bp,而NDA测序证实克隆并插入到载体中的片段为目的的片段,长度为168bp,与报道的序列比较缺失2个t。结论 本实验已成功地克隆了人肺癌细胞NHE-1基因调控序列中正调控序列片段。     

小鼠Reelin 基因启动子克隆鉴定及生物信息学分析

目的克隆小鼠Reelin基因启动子区并分析其潜在DNA甲基化位点和转录因子结合位点。方法根据NCBI上小鼠Reelin 5’非翻译区序列设计引物,利用高保真PCR方法克隆昆明小鼠Reelin启动子区序列,并进行TA克隆,测序鉴定。利用亚硫酸氢钠处理基因组DNA,通过PCR技术和测序技术对Reelin启动子-636 bp至-135bp序列甲基化情况分析。应用Methyl Primer Express software V1.0和TFSEARCH软件分析该区域甲基化位点和转录因子结合位点。结果成功克隆了小鼠Reelin基因启动子区0至-450片段。甲基化测序分析小鼠Reelin启动子区-636 bp至-135 bp序列没有CpG二核苷酸被甲基化。小鼠Reelin翻译起始点0至-450片段利用CpG岛序列分析软件分析显示,该区域C+G含量高达78.71%,CpG含量为14.44%。该区域有120多个潜在转录因子结合位点。结论在小鼠Reelin基因启动子区0～-450发现有一个CpG岛和多个转录因子结合位点,该区域可能在小鼠Reelin基因表达调控起重要作用。     

survivin启动子有效片段的克隆及其在胃癌细胞AGS中的转录活性研究

目的 克隆survivin启动子的有效片段，并检测它在人胃癌细胞株AGS和人正常胃上皮细胞GES-1中的转录活性，为该启动子在胃癌的靶向性基因治疗中奠定基础。方法 用PCR扩增survivin基因的启动子片段，克隆入荧光素酶报告质粒pGL3-Basic，构建pGL3-sur-pro重组质粒，用脂质体法瞬时转染AGS及GES-1中，检测survivin启动子在细胞中的转录活性。同时构建含CMV启动子的pGL3-CMV-pro重组质粒作为阳性对照。结果 琼脂糖凝胶电泳显示PCR扩增的survivin启动子片段长约440bp，测序结果与GenBank中survivin启动子序列一致。成功构建了pGL3-sur-pro重组质粒。荧光素酶活性检测显示，survivin启动子片段在细胞株AGS中有较高转录活性，而在GES-1中几无转录活性。结论 本实验克隆的survivin启动子有效片段在胃癌细胞株AGS中有转录活性，在正常胃上皮细胞中无活性。其有可能作为调控元件用于胃癌的靶向性基因治疗。