起始识别复合物结合位点对沉默子的辅助作用

背景：沉默子由起始识别复合物结合位点,阻遏活化蛋白RAP1等元件组成,人们对于沉默子的功能及相互关系有较多研究,但对组成沉默子的元件与基因沉默之间关系的研究很少。目的：研究起始识别复合物结合位点对沉默子基因沉默的辅助作用。方法：用右侧带有报告基因URA3的起始识别复合物结合位点,以及不带任何结合位点的序列替代酵母基因组HML区域的HML-I沉默子后,比较URA3基因表达量,并使用DNA拓扑法分析染色质的结构变化。结果与结论：HML-E沉默子单独存在的情况下,酵母无法在FOA平板上生长,但在起始识别复合物结合位点的辅助下,能够在FOA平板上生长,两者之间区域的染色质大多为负超螺旋。因此可知起始识别复合物结合位点能增加HML-E沉默子作用范围,产生这种作用的原因是两者相互作用下形成紧密的染色质结构。     

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     

染色质免疫沉淀技术的应用及进展

真核生物细胞状态是受内源性和外源性因素共同影响的，真核生物的基因组DNA以染色质的形式存在。所有信号传递途径的终点都是DNA。DNA通过核蛋白复合物组成染色质，染色质是基因调控的一个重要作用位点。过去10年，染色质免疫沉淀技术（chromatin immunoprecipitation assay，Chip）已经成为进行表观遗传信息研究的主要方法，它可以灵敏地检测目标蛋白与特异DNA片段的结合情况，还可以用来研究组蛋白与基因表达的关系，有助于判断何种组蛋白修饰会出现在细胞核中基因组的某一特定位置。因此，Chip是目前研究体内DNA与蛋白质相互作用最好的方法。     

全反式维甲酸诱导小鼠碱性磷酸酶基因启动子区染色体重构

背景:碱性磷酸酶基因是成骨细胞分化和骨形成的重要标志。在C3H10T1/2细胞中,全反式维甲酸可通过核受体上调小鼠碱性磷酸酶的表达,与MAPK通路无关。目的:从染色体结构调控方面揭示全反式维甲酸上调碱性磷酸酶表达的分子机制。方法:10-6mol/L全反式维甲酸处理C3H10T1/2细胞0,1,6,12h,DNA酶Ⅰ超敏感实验确定全反式维甲酸调控区域的位置,染色质免疫共沉淀实验检验全反式维甲酸处理细胞后一系列转录相关因子与全反式维甲酸调控区域结合的量效关系以及时相分布。结果与结论:DNA聚合酶Ⅰ超敏感实验表明,小鼠碱性磷酸酶启动子转录起始位点上游约520bp附近为潜在的全反式维甲酸调控区域;染色质免疫共沉淀实验表明,全反式维甲酸对小鼠碱性磷酸酶的上调作用是通过一系列转录相关因子的时序性共同作用来实现。表明全反式维甲酸诱导小鼠碱性磷酸酶基因转录的过程中伴随着染色质重构和组蛋白的修饰作用。     

全反式维甲酸诱导小鼠碱性磷酸酶基因启动子区染色体重构

背景：碱性磷酸酶基因是成骨细胞分化和骨形成的重要标志。在C3H10T1/2细胞中,全反式维甲酸可通过核受体上调小鼠碱性磷酸酶的表达,与MAPK通路无关。目的：从染色体结构调控方面揭示全反式维甲酸上调碱性磷酸酶表达的分子机制。方法：10-6mol/L全反式维甲酸处理C3H10T1/2细胞0,1,6,12h,DNA酶Ⅰ超敏感实验确定全反式维甲酸调控区域的位置,染色质免疫共沉淀实验检验全反式维甲酸处理细胞后一系列转录相关因子与全反式维甲酸调控区域结合的量效关系以及时相分布。结果与结论：DNA聚合酶Ⅰ超敏感实验表明,小鼠碱性磷酸酶启动子转录起始位点上游约520bp附近为潜在的全反式维甲酸调控区域;染色质免疫共沉淀实验表明,全反式维甲酸对小鼠碱性磷酸酶的上调作用是通过一系列转录相关因子的时序性共同作用来实现。表明全反式维甲酸诱导小鼠碱性磷酸酶基因转录的过程中伴随着染色质重构和组蛋白的修饰作用。     

染色质重塑复合物与基因表达调控的研究进展

染色质重塑复合物能够通过与转录因子等多种蛋白质结合实现染色质重塑,在组织特异性表达、细胞核内信号传导、细胞分化、增殖等方面起重要作用.本文对近年来关于染色质重塑复合物家族构成、调控机制、主要方式、启动子区的特异性靶定以及以基因的转录激活或抑制作用等进行综述,以利于对基因调控、细胞因子应答、肿瘤发生、发育和分化等过程中表...     

转录因子GATA-2对iASPP基因的转录调控研究

癌基因iASPP促进细胞增殖、抑制细胞凋亡,其转录起始位点上游序列有转录因子GATA-2的假定结合位点,GATA-2在造血干/祖细胞的增殖和分化中起重要作用。本研究旨在探讨GATA-2对iASPP的转录调控作用。首先检测了部分白血病细胞系中iASPP和GATA-2蛋白的表达,然后构建带有GATA-2开放读码框的真核表达载体与带有iASPP转录起始位点上游3000 bp至下游500 bp序列中GATA-2假定结合位点的荧光素酶报告载体,共转染HEK293和CV-1细胞,检测荧光素酶活性,并对荧光素酶活性上调的结合位点区进行染色质免疫共沉淀实验。结果显示,白血病细胞系中iASPP高表达的细胞大多存在GATA-2的高表达;GATA-2对iASPP报告基因荧光素酶活性有显著影响,并呈现明显的＂剂量-效应＂关系。当pCMV5-GATA2转染剂量上升至100 ng时,iASPP荧光素酶活性在HEK293细胞中上调到2倍;此转录激活作用在CV-1细胞中尤为显著,荧光素酶活性上调可达6.7。染色质免疫共沉淀结果证实GATA-2与iASPP转录起始区nt-361～-334有特异性结合。结论：转录因子GATA-2可以与iASPP的转录调控区结合,并促进iASPP基因转录。     

核共抑制复合物与急性髓系白血病M2b型关系的研究进展

急性髓系白血病 (AML) M2b型是急性白血病中较多见的类型之一 ,其特征为染色体t(8;2 1)而致AML1 ETO融合基因 ,该融合基因在白血病发病中起重要的作用。近年来发现AML1 ETO融合基因在白血病发病中需要结合核共抑制复合物 ,包括N CoR (nuclearco repressor)、SMRT(silencingmediatorforretinoidandthyroid hormonereceptors)及mSin3A(mammalianhomologofyeastrepressorSin3) ,并可通过募集组蛋白去乙酰化酶HDAC(histonedeacetylase) ,一方面使AML1所调控靶基因启动子或增强子染色质区域组蛋白去乙酰化 ,使局部染色质空间结构…     

阴沟肠杆菌染色质ampD基因的研究

目的研究阴沟肠杆菌染色质ampD基因及其高概率突变位点。方法从医院感染患者临床标本中分离阴沟肠杆菌,经头孢西丁初筛试验和头孢西丁三维试验确定是否产AmpCβ-内酰胺酶及其产酶类型,抽提产酶菌株染色质DNA,PCR扩增ampD基因并连接入pMD19-T载体,双链测序后与同种非突变标准菌株的ampD基因序列比对,并得到高概率突变位点。结果大部分产酶阴沟肠杆菌含有ampD基因,其中18株产持续高产型酶菌株的染色质中均扩增出ampD基因;阴沟肠杆菌ampD基因上有62个突变率高于50%的位点。结论产AmpCβ-内酰胺酶的阴沟肠杆菌染色质上均含有ampD基因;阴沟肠杆菌染色质ampD基因的高概率突变位点可能成为致持续高产酶的关键位点。     

阴沟肠杆菌染色质AmpD基因的研究

目的研究阴沟肠杆菌染色质AmpD基因及其高概率突变位点。方法从医院感染患者临床标本中分离阴沟肠杆菌,经头孢西丁初筛试验和头孢西丁三维试验确定是否产AmpCβ-内酰胺酶及其产酶类型,抽提产酶菌株染色质DNA,PCR扩增AmpD基因并连接入pMD19-T载体,双链测序后与同种非突变标准菌株的AmpD基因序列比对,并得到高概率突变位点。结果大部分产酶阴沟肠杆菌含有AmpD基因,其中18株产持续高酶菌株的染色质中均扩增出AmpD基因;阴沟肠杆菌AmpD基因上有62个突变率高于50%的位点。结论产AmpCβ-内酰胺酶的阴沟肠杆菌染色质上均含有AmpD基因;阴沟肠杆菌染色质AmpD基因的高概率突变位点可能成为致持续高产酶的关键位点。     

Long-term silencing of retroviral vectors is resistant to reversal by trichostatin A and 5-azacytidine

One problem limiting the development of long-term gene replacement therapy is gene silencing. A variety of experiments have implicated DNA methylation and histone deacetylation in gene silencing and shown that the agents 5-azacytidine (5-Aza) and trichostatin A (TSA) are able to reverse these effects. To begin to investigate clinically relevant strategies to reverse silencing with these drugs, we transduced the MEL and FDCP-1 hematopoietic cell lines with Moloney murine leukemia virus (MMLV) and Harvey murine sarcoma virus (HMSV)-based retroviral vectors carrying the beta-galactosidase/neomycin resistance fusion gene (beta-geo). Fifty-one clones were isolated under G418 selection over 2 weeks and then allowed to grow without selection as beta-gal activity was monitored over time. More than 80% of these clones showed significant silencing over a period of 70-80 days. The clones were then exposed to a wide range of 5-Aza and TSA concentrations, both alone and in combination, in an effort to reverse silencing. Despite demonstration that the agents were able to decrease DNA methylation and increase histone acetylation, significant reversal of long-term silencing was not seen under any experimental condition. These results suggest that long-term retroviral silencing involves mechanisms in addition to DNA methylation and histone acetylation and that new pharmacologic strategies are needed to overcome the silencing process.     

Pentraxin-chromatin interactions: serum amyloid P component specifically displaces H1-type histones and solubilizes native long chromatin

Pure serum amyloid P component (SAP) and native long chromatin, mixed together at wt/wt ratios between 1:1 and 1:2 in the presence of physiological concentrations of NaCl and calcium, both remained in solution, whereas each alone precipitates rapidly under these conditions. This solubilization accompanies the binding of SAP to chromatin and the displacement of H1-type histones, which are essential for condensation and higher order folding of chromatin. Such binding of SAP to chromatin is remarkable since displacement of H1 and H5 by salt alone requires approximately 0.5 M NaCl. SAP also bound to nucleosome core particles forming soluble complexes with an apparent stoichiometry of 1:2, a result that is compatible with attachment of SAP at the nucleosome dyad, the site of H1 in intact chromatin. SAP thus undergoes a specific, avid interaction with chromatin that promotes its solubilization and may thereby contribute to the physiological handling of chromatin released from cells in vivo. In contrast, C-reactive protein (CRP) did not bind significantly to either chromatin or to core particles at physiological ionic strength. Incubation of chromatin with either normal serum, or acute phase human serum containing raised levels of CRP, did not induce complement activation regardless of the presence of added SAP or CRP, nor was any cleavage of DNA observed.     

Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo

We previously demonstrated that sustainable enhanced levels of transgene products could be expressed from a bacterial DNA-free expression cassette either formed from a fragmented plasmid in mouse liver or delivered as a minicircle vector. This suggested that bacterial DNA sequences played a role in episomal transgene silencing. To further understand the silencing mechanism, we systematically altered the DNA components in both the expression cassette and the bacterial backbone, and compared the gene expression profiles from mice receiving different DNA forms. In nine vectors tested, animals that received the purified expression cassette alone always expressed persistently higher levels of transgene compared to 2fDNA groups. In contrast, animals that received linearized DNA by a single cut in the bacterial backbone had similar expression profiles to that of intact plasmid groups. All three linear DNAs formed large concatemers and small circles in mouse liver, while ccDNA remained intact. In all groups, the relative amount of vector DNA in liver remained similar. Together, these results further established that the DNA silencing effect was mediated by a covalent linkage of the expression cassette and the bacteria DNA elements.     

细胞周期蛋白A对培养大鼠血管平滑肌细胞起始识别复合物1表达水平的影响

目的 探讨细胞周期蛋白A （Cyclin A）对培养大鼠血管平滑肌细胞（VSMCs）周期起始识别复合物1（ORC1）表达水平的影响.方法 采用组织块贴片法原代培养的大鼠胸主动脉血管平滑肌细胞,利用双胸苷使VSMCs达到细胞周期同步化,应用逆转录聚合酶链反应技术和流式细胞仪检测不同细胞周期VSMCs的ORC1和Cyclin A mRNA和蛋白表达.结果 经鉴定培养的细胞为VSMCs,采用血清饥饿法、双胸苷阻断法和秋水仙素阻抑法分别获得了处于不同细胞周期的同步化的VSMCs：G0/G1期（89.22 ±3.54）％,G1/S交界期（66.74±7.16）％,S期（63.24±4.06）％,G2/M期（51.64±11.18）％,各期比较差异有统计学意义（P均＜0.01）.Cyclin A在静止状态对VSMCs的ORC1 mRNA和蛋白无影响,在G2/M期达到高峰[（52.133 3 ±2.122 1）％],与G1/S期[（10.9167±0.531 1）％]、S期[（7.656 7±0.412 4）％]比较,差异有统计学意义（P均＜0.01）.ORC1在G2/M期达到最低值[（1.276 7 ±0.161 7）％],与G1/S期[（13.371 0±1.057 3）％]、S期[（3.043 3 ±0.538 0）％]比较,差异有统计学意义（P均＜0.01）,可能原因是Cyclin A阻止ORC1结合在VSMCs染色质上.结论 CyclinA可能调控ORC1在VSMCs细胞周期的启动.     

构建人csp-B核基质结合区克隆及其介导的反转录载体

背景:核基质结合区是染色质被限制酶消化后仍附着在核基质上的DNA序列.大量实验表明,核基质结合区可以作为DNA复制的起始点或调控基因的转录,构建核基质结合区表达载体能提高外源基因表达水平,增强外源基因表达的稳定性及提高转化细胞稳定株的频率等.目的:通过克隆人基因组不同的核基质结合区片段,构建核基质结合区介导的包含氯霉素乙酰转移酶(CAT)报告基因的反转录病毒载体pLXSN-MAR,以探索核基质结合区对基因表达的影响.方法:开放性实验于2007-01/12在新乡医学院生物化学与分子生物学实验室及分子研究室完成.自行构建含氯霉素乙酰转移酶报告基因的质粒PLXSN-CAT载体.TaqDNA聚合酶、T_4 DNA连接酶、DNA Marker、限制性内切酶BamH I、凝胶纯化试剂盒、质粒提取试剂盒均购于大连TaKaRa公司;引物由上海生工生物工程技术服务有限公司合成.以人基因组DNA为模板,采用聚合酶链反应扩增csp-B核基质结合区序列,克隆入反转录载体PLXSN-CAT中,经限制性内切酶酶切及DNA序列分析鉴定目的基因.结果与结论:聚合酶链反应扩增的特异性片段长度为931 bp,以此构建的重组质粒命名为PLXSN-CAT-MAR,经BamH I酶切后显示5.9 kb和931 bp左右的两条片段,测序结果与Gen-bank中的人csp-B核基质结合区(Genbank序列号M62716)序列一致,证明人核基质结合区片段已成功克隆到了反转录载体PLXSN-CAT中.提示成功构建了PLXSN-CAT-MAR反转录表达载体.