Multiple proteins bind to the P2 promoter region of the zein gene pMS1 of maize

Summary A 216 by promoter fragment of the 19 kDa protein zein gene pMS1, containing the CCAAT and TATA boxes, was analysed by a variety of techniques for in vitro interactions with nuclear proteins from endosperm tissue. HMG proteins were found to form stable complexes with these A/T-rich promoter sequences and several specific DNA-binding proteins appear to be involved in the formation of DNA-protein complexes with this fragment. A 29 bp region spanning the two CCAAT boxes was protected from DNase I digestion in footprinting experiments.     

LRP16基因启动子克隆及特征分析

克隆LRP16基因启动子分子,并对启动子特征进行分析,预测启动子区调控元件,为深入研究LRP16基因的表达调控机制奠定基础。在NCBI的人类基因组数据库中截取并下载LRP16基因转录起始位点5′侧翼区2.7kb的基因组序列,设计PCR引物,从健康外周血单个核细胞中扩增,利用Genomatix程序对5′侧翼区近1000bp进行启动子特征分析,获得了与GenBank序列一致,长度为2.7kb的LRP16基因启动子DNA序列,该序列具有典型的真核生物RNA聚合酶Ⅱ启动子特征及多个核受体结合位点,如α视黄酸受体及RAR相关孤生受体。     

小鼠T-bet启动子荧光素酶报告基因载体的构建及活性鉴定

为了研究T-bet在T细胞中的转录调控机制,并研究其在多发性硬化症中的信号通路,本研究构建小鼠TBX21(编码T-bet)基因启动子区和增强子区萤火虫荧光素酶报告基因载体。在对小鼠TBX21基因5?侧翼区进行详尽生物信息学特征分析后,设计相应引物,用PCR的方法从小鼠基因组中扩增出TBX21基因5?侧翼区–1 000 bp-28 bp片段长为1 028 bp的启动子区(以翻译起始点ATG为+1)和–3 308 bp-–2 000 bp片段长为1308 bp的非编码区保守序列(No-coding conserved sequence,CNS),再用定向克隆的方法将这两个片段定向重组入专门用于启动子活性研究的萤火虫荧光素酶报告基因载体(p GL4.10)中,构建出包含小鼠TBX21基因启动子区和CNS区的萤火虫荧光素酶报告基因载体(p GL4.10-TBX21pr-CNS),电泳与测序鉴定,最后再将p GL4.10-TBX21pr-CNS与内参p RL-TK用lipofectamine 2000共转染293T细胞和Jurkat细胞中,通过双荧光素酶报告基因检测系统鉴定p GL4.10-TBX21pr-CNS的启动子和增强子活性,并用独立样本t检验方法进行统计分析。对照组共转染p GL4.10与内参p RL-TK。结果表明,成功构建出荧光素酶报告基因重组质粒p GL4.10-TBX21pr-CNS。与转染空质粒p RL-TK组相比,293T细胞(P=0.012 2)和Jurkat细胞(P=0.002 2)中转染p GL4.10-TBX21pr-CNS组荧光素酶活性升高。研究结果表明在293T细胞和Jurkat细胞中p GL4.10-TBX21pr-CNS可以表现出启动子活性,为后续小鼠T-bet转录调控研究提供了基本材料。     

Structural and dynamic differences of the estrogen receptor DNA-binding domain, binding as a dimer and as a monomer to DNA: molecular dynamics simulation studies

Molecular dynamics (MD) simulations of the estrogen receptor DNA-binding domain (ERDBD) as a dimer in complex with its DNA response element (ERE) show a significant difference in both structure and dynamics, compared to a MD simulation of monomeric ERDBD bound to its half-site response element (EREH). The C-terminal zinc binding domain (Zn_II), including a region (helix II) which is in a helical conformation in ERE-(ERDBD)₂, is considerably more flexible in EREH-ERDBD than in the dimeric complex. In EREH-ERDBD, all helical hydrogen bonds in helix II are broken and the entire Zn_II region is detached from a hydrogen bonding network that in ERE-(ERDBD)₂ connects to other parts of the protein as well as to the DNA. The regions that become flexible in EREH-ERDBD are identical to the regions where the NMR solution structure of free ERDBD is poorly ordered. This strongly suggests that dimerisation of ERDBD is required for ordering of the Zn_II region and that monomeric binding to DNA is not sufficient for the ordering. This contrasts to the glucocorticoid receptor DNA-binding domain (GRDBD) which has essentially the same mobility (uniform and limited), regardless of whether it is free as a monomer in solution, bound as a monomer to its half-site response element or in a dimeric complex with the full response element. The hydrogen bonding network that connects Zn_II with other parts of the protein and to DNA is almost identical in ERDBD and GRDBD. However, in GRDBD there is also a serine (in the N-terminal zinc coordinating region) with a central role in this network, connecting to the Zn_II region. This serine is replaced by a glycine in ERDBD and we suggest that this substitution is sufficient for destabilisation of the network, thus leading to a more flexible Zn_II region, which becomes ordered first upon forming a complex with another ERDBD and DNA. Received: 6 March 1998 / Revised version: 22 June 1998 / Accepted: 2 September 1998     

Immunoreactivities and Levels of Mineralocorticoid and Glucocorticoid Receptors in the Hippocampal CA1 Region and Dentate Gyrus of Adult and Aged Dogs

Corticosteroids are important factors in the maintenance of homeostasis in the brain. They are regulated via the interaction with two corticosteroid receptor systems—the mineralocorticoid (MR) and glucocorticoid receptor (GR). In the present study, we observed age-related changes in serum cortisol levels, and immunoreactivities and protein levels of MR and GR in the hippocampal CA1 region and dentate gyrus. The serum cortisol levels were significantly high (about twofold) in the aged group compared to that in the adult group. In the adult dog (2–3 years old), MR and GR immunoreactivity was detected in neurons in the pyramidal layer of the CA1 region, and in the granular and multiform layers of the dentate gyrus. In the aged dog (10–12 years old), MR immunoreactivity in the CA1 region was significantly decreased, especially, in the dentate multiform layer. In contrast, GR immunoreactivity in the aged dog was slightly decreased in the CA1 region and dentate gyrus. In the Western blot analysis, MR protein level in the aged dog was significantly lower compared to that of the adult dog; GR protein level in the aged dog was not significantly decreased. This result indicates that the reduction of MR immunoreactivity and protein level in the hippocampus of the aged dog may be associated with neural dysfunction in the aged hippocampus.