小鼠干扰素调节因子3启动子质粒的构建及其启动子活性分析

目的构建包含小鼠干扰素调节因子3(mIRF-3)基因启动子的质粒,并评价其启动子活性。方法以小鼠全血细胞总DNA为模板,PCR扩增mIRF-3目的片段,亚克隆此片段至pGL3-basic荧光素酶报告基因的多克隆位点,构建含mIRF-3启动子的重组报告质粒pmIRF-3。将pmIRF-3、pGL3-basic和pGL3-control质粒分别转染小鼠胚胎成纤维细胞NIH3T3后,行荧光素酶活性检测,计算相对活性单位(RLU)。生物信息学分析转录因子结合位点。结果成功构建了重组报告质粒pmIRF-3。与pGL3-basic组相比,pmIRF-3、pGL3-control组RLU明显增加(0.443±0.113vs.18.907±3.335、25.704±5.850)(P<0.01)。mIRF-3启动子区域内存在AML-1a、E2F、MZF1、CdxA、OCT-x等多个转录因子结合位点。结论 mIRF-3转录起始位点附近1479bp序列在NIH3T3细胞中具有较强的启动子活性。     

Baculovirus ETL promoter acts as a shuttle promoter between insect cells and mammalian cells

AIM: To identify a shuttle promoter that can mediate gene expression in both insect cells and mammalian cells to facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle. METHODS: Recombinant baculoviruses carrying the beta-galactosidase reporter gene under the control of an early to late (ETL) promoter of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) or a cytomegalovirus immediate early promoter (CMV promoter) were constructed. COS1, HeLa, CHO-K1, hFob1.19, and MCF-7 mammalian cells were tested for the expression of b-galactosidase. RESULTS: ETL promoter activity was higher in bone-derived hFob1.19 than in COS1, HeLa, CHO-K1, or MCF-7 mammalian cells. The transient plasmid transfection assay indicated that ETL promoter activity in mammalian cells was dependent on baculovirus gene expression. CONCLUSION: ETL promoter activity in mammalian cells is baculovirus gene expression-dependent, and the shuttle promoter will facilitate the application of baculovirus expression vectors in mammalian cell expression systems and for gene therapy.     

一种新的链霉菌表达载体启动子

eryA基因直接控制着红霉素母环6-脱氧-红霉内酯B的合成,在红霉素生物合成过程中具有重要作用。本文克隆了eryA基因的启动子PeryA,以绿色荧光蛋白基因为报告基因,构建了大肠埃希菌-糖多孢红霉菌穿梭型质粒。PEG介导原生质体转化法将穿梭型质粒分别转入糖多孢红霉菌A226与变铅青链霉菌JT46,荧光显微镜检测发现,此启动子在两菌株中都具有功能。随后,以变铅青链霉菌JT46为宿主,对PeryA启动子区域进行了深入研究,结果发现该启动子的-35区并不是必需的,仅有-10区、长度为41bp的该启动子在链霉菌中仍具有功能。定点突变证明-10区对于该启动子是必不可少的。因此,41bp的该启动子片段可作为链霉菌的有效启动子,这是迄今为止所发现的最短的启动子之一,可用于构建新的链霉菌表达载体。     

CD2AP启动子的克隆和活性分析

目的 构建表达CD2AP基因转录起始位点上游启动子的表达质粒,转染人类胚胎肾(HEK)-293T细胞,评价其启动子活性.方法 以人全血细胞总DNA为模板,PCR扩增CD2AP转录起始位点上游2082 bp的启动子区片段.亚克隆此片段至无启动子活性的pGL-3基本载体荧光素酶报告基因上游的多克降位点,构建含CD2AP启动子的重组报告质粒.转染HEK-293T细胞,行荧光素酶活性检测,计算相对活性单位(RLU).生物信息学分析转录因子结合位点.结果 酶切,测序鉴定证实成功构建含有CD2AP基因转录起始位点上游2082 bp的启动区的表达质粒.CD2AP的启动子与正常的pGL-3基本质粒比较,其RLU增加了74.8倍.其上游启动子区序列中含多个转录因子结合序列如AP1、Sp1、CREB和GATA-1等.结论 CD2AP转录起始位点上游序列在HEK-293T细胞中具有较强的启动活性.     

Cloning of DNA fragments containing Streptomyces promoter activity

The thiostrepton-resistance gene is expressed in Streptomyces jumonjinensis [16]-8 SANK 61185 carrying the plasmid pIJ702 but not the tyrosinase gene. We have isolated DNA fragments from various streptomycetes that restore expression of the tyrosinase gene on pIJ702 in this organism. The nucleotide sequences of two of these DNA fragments show that they contain putative ribosome binding sites and- 10 regions of possible promoters.     

以Insig-2基因启动子为靶点的药物筛选模型的建立

目的通过双荧光素酶报告基因检测系统建立以胰岛素诱导基因2(insulin-induced gene 2,Insig-2)启动子为靶点的药物筛选模型。方法将人Insig-2基因启动子序列克隆入荧光素酶报告基因载体pGL3-basic中,构建重组质粒pGL3-Insig-2并与内参质粒pRL-TK瞬时共转染工具细胞,通过检测荧光素酶报告基因表达水平的变化反映Insig-2基因启动子启动转录的活性,并对共转染质粒比例、工具细胞选择等条件进行探索和优化,相关药物处理进行验证。结果成功构建了重组质粒pGL3-Insig-2;确认pGL3-Insig-2:pRL-TK共转染比例为4∶1,确定3T3-L1细胞为工具细胞;1,25-(OH)2D3、小檗碱和姜黄素均能明显增强Insig-2基因启动子活性。结论成功建立了以Insig-2基因启动子为靶点的药物筛选模型,为筛选新型调脂药奠定基础。     

小鼠Gas6启动子的克隆与功能鉴定

目的:探讨软骨分化的负性转录因子Gas6基因启动子调控的分子机制。方法:采用5′RLM-RACE技术克隆Gas6的转录起始位点,利用生物信息学的方法对Gas6潜在启动子区域在小鼠、人与大鼠之间进行同源性比较,对Gas6上游序列进行系列缺失突变,构建Luciferase基因报告载体,转染细胞后检测Luciferase的表达水平。结果:Gas6的转录起始位点为碱基C,在小鼠、人与大鼠之间存在两个高度保守序列A-box与B-box,缺失A-box,B-box的后1/2区域以及NF-Y结合位点后,潜在启动子的转录活性明显下降。结论:A-box,B-box及NF-Y结合位点与Gas6的转录活性有关。     

Transcriptional suppression of the HIV promoter by natural compounds

Tannins and lignins are natural compounds contained in plants such as tea leaves. Previously, we demonstrated that tannic acid represses 12-o-tetra-decanoyl phorbol-13-acetate (TPA)-induced human immunodeficiency virus (HIV) promoter activity. Furthermore, we demonstrated that a 30-bp element located just downstream of the NF-kappaB element in the HIV promoter responds negatively to tannic acid. However, the kinds of molecules responsible for this suppressive effect have remained unknown, because tannic acid is a mixture of various galloylglucoses. Here, we examined structure-defined natural compounds for HIV promoter-suppressive effects. We found that ellagitannins suppress TPA-induced HIV promoter activity to the same extent as tannic acid. 3-phenylcoumarins, isoflavone and chalcones have more suppressive effects than ellagitannins. On the other hand, other flavonoids and acetogenins have no suppressive effect. 3-phenylcoumarins and chalcones showed no suppressive effect on the cytomegalovirus (CMV) promoter, suggesting that they act specifically on the HIV promoter. These results suggest that 3-phenylcoumarin or chalcone compounds could be used to develop novel anti-HIV drugs with an action targeted at HIV promoter activity.     

三乙烯四胺对c-myc启动区转录活性的调节作用

目的探讨三乙烯四胺(TETA)如何调节c-myc基因表达。方法利用圆二色谱分析TETA对c-myc启动区核酸酶超敏元件Ⅲ1(c-mycNHEⅢ1)碱基序列形成的G-四链体DNA(G4-DNA)结构稳定性的影响。同时,分别构建含c-mycNHEⅢ1的野生型和突变型的c-myc启动区荧光报告质粒,并分别转染HEK293细胞24h后,再接种到96孔板,TETA以终浓度0,0.1,1.0,10及100μmol·L-1处理8h,测定荧光素酶活性,计算TETA对其转录活性抑制率。结果圆二色谱实验结果表明,TETA5μmol·L-1就能够进一步增强c-myc NHEⅢ1在240nm处的负峰和260nm处的正峰的形成,即增强G4-DNA结构的稳定性。报告基因分析结果表明,TETA能够浓度依赖性地抑制野生型c-myc启动区荧光素酶基因的表达,但对突变型c-myc启动区荧光素酶基因的抑制能力明显下降。在TETA1nmol·L-1作用时,对突变型c-myc启动区荧光素酶基因抑制率仅为4.3%,而对野生型的抑制率还高达30.4%。结论TETA可能通过稳定c-mycNHEⅢ1序列形成的G4-DNA结构调节基因的表达。     

人中脑星形胶质来源的神经营养因子基因启动子区域的克隆及活性检测

目的克隆人中脑星形胶质来源的神经营养因子MANF(mesencephalic astrocyte-derived neurotrophie factor)基因的启动子区域,并鉴定其转录活性。方法利用TESS在线软件分析MANF基因上游序列中潜在的顺时作用元件。以HepG2细胞的总基因组为模板,通过PCR反应扩增了MANF基因5'非翻译区1 415 bp片段,并将此片段插入到不含有启动子的pEGFP-1载体中构建了pEGFP-1-MANF-5F重组质粒。用脂质体介导的方法将pEGFP-1-MANF-5F质粒转染到293T细胞,在倒置显微镜下观察绿色荧光的表达。同时,将上述MANF基因5'非翻译区1 415 bp片段插入到荧光素酶报告基因载体pGL3-Basic中,以构建质粒pGL3-MANF-5F,并通过共转染内参质粒pRL-TK到N2 A细胞中,24 h后检测双荧光素酶的表达。结果重组质粒pEGFP-1-MANF-5F转染293T细胞后,在细胞中可见绿色荧光蛋白;pGL3-MANF-5F重组质粒在N2 A细胞中检测到双荧光素酶的活性,且在Tunicamycin诱导时表达明显升高;结论已成功克隆了MANF基因的5'端非翻译区,并证实该区域具有启动子活性,且内质网应激可调节MANF的启动子活性,这为进一步研究MANF基因在疾病中的表达调控机制奠定了基础。     

Glial-cell-line-derived neurotrophic factor: gene promoter and receptors

These patents claim the structure of the glial-cell-line-derived neurotrophic factor (GDNF) gene promoter and of novel GDNF receptors. Since GDNF is a potent survival and growth factor for various neuronal (and non-neuronal) cell populations, knowledge of these molecules may have significant diagnostic and therapeutic applications.     

Diversity of class 1 integron gene cassette Pc promoter variants in clinical Escherichia coli strains and description of a new P2 promoter variant

Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant as well as occasionally from a second promoter located downstream of Pc, named P2. So far, the distribution of the variants has only been described in an in silico study. In this study, the prevalence of these variants in vivo was analysed in a population of 85 Escherichia coli strains from a variety of phylogenetic groups isolated from healthy subjects and clinical samples in Spain and France from 2004 to 2007. The weakest variants (PcW and PcH1) prevailed (variants associated with the integrase having the most efficient excision activity), whilst the two strongest variants, PcW_TGN-10 and PcS, were less frequent. Furthermore, a new variant of P2 associated with PcW was characterised in one integron (harbouring the gene cassette bla_OXA-1–aadA1) from a French strain of a healthy subject. This variant was hereafter named P2m3 and shows a G → A substitution in its −10 element (TACAGT to TACAAT), a mutation that doubled the strength of P2 and approached the level of expression of the strong PcW_TGN-10 variant. When the correlation between the Pc variants and the origin of the strains was analysed, no significant difference (P < 0.05) was observed in the Pc variant distribution according to the geographic origin or clinical setting.     

Effects of serotonin transporter promoter polymorphisms on serotonin function.

The serotonin transporter promoter polymorphism (5-HTTLPR) has been associated with vulnerability to stress-induced depressive symptoms and with the speed and rate of response to antidepressant treatment. The goal of the present study was to evaluate the association between the 5-HTTLPR and the functional response of the serotonin system as measured by the neuroendocrine and cerebral metabolic response to intravenous administration of the selective serotonin reuptake inhibitor citalopram in normal control subjects. Genotyping was performed for 5-HTTLPR insertion/deletion polymorphism long (l) and short (s) variant alleles. The ll genotype was compared with the combined sl+ss and with the ss genotype alone. Citalopram plasma concentrations did not differ significantly between groups. The s allele was associated with a less of an increase in prolactin and cortisol than the ll genotype. The s allele was associated with greater decreases in left frontal, precentral and middle temporal gyri compared to the ll genotype. The ll genotype was associated with greater decreases in right frontal, insula and superior temporal gyrus compared to the ss genotype. These findings suggest that 5-HTTLPR is associated with an altered functional response of the serotonin system, which may represent a neurobiologic substrate for the differential response to antidepressant treatment in late life and the emergence of neuropsychiatric symptoms in neurodegenerative disorders.