PROTEIN(S) INVOLVED IN NEUROTRANSMISSION

描述(摘自申请者摘要)：5-羟色胺 (5-羟色胺，5-羟色胺)及其受体参与了 精神抑郁的病因及治疗。一种理解 5-羟色胺受体基因的表达对5-羟色胺起着至关重要的作用 并将提供对分子基础的洞察力 精神抑郁的症状。在前一个资助期内，像 转录因子复合体B因子对人5-HT2a受体的重要作用 (5-HT2AR)基因的表达。核因子-AT是一种关键转录因子 活化T细胞产生白介素2的因子。的目标是 本项目旨在进一步鉴定和克隆S编码基因 因素b.具体目标如下： 1.进一步确定B因子复合体的DNA结合序列及其 凝胶滞留在5-HT2AR基因转录调控中的作用 启动子活性分析。 2.B因子复合体与转录因子Sp1的相互作用 将进行研究，以确定因子b复合体和Sp1结合 是独立的、互斥的或协同的。DNA酶1足迹 人神经母细胞瘤(SHSY-5Y)细胞启动子活性检测 已执行。转录因子之间的相互作用也将是 通过体内基因组足迹进行研究。 3.进行紫外光交联以确定b因子是否复合 由单一或多个多肽组成(S)。它们的免疫反应 将使用NF45和NF90多克隆抗体研究其特性。 4.鉴定编码具有B因子活性的多肽(S)的基因(S)。 如果b因子复合体由一种多肽组成，则其cdna将被克隆。 通过筛选表达文库。如果因子b复数由两个 或更多的亚基，则用DNA亲和层析进行纯化。 然后将根据氨基酸设计简并的寡核苷酸 序列(S)，用作探针。 5.S编码基因的鉴定和有效性研究 B因子。分离的B因子cDNA克隆(S)的有效性将通过 观察表达的多肽是否会(A)与0.35kb结合 并(B)刺激共转染0.35kb(或 0.70kb)构建于NCI-H460细胞中(缺乏B因子活性和5-HT2AR 表达)。B因子的组织分布将通过以下方法研究 北方分析。因子b多肽(S)也将在 让Jurkat细胞休息，看看它是否能刺激IL-2的产生。

DESCRIPTION (Adapted from applicant's abstract): Serotonin (5-hydroxytryptamine, 5-HT) and its receptors have been implicated in the etiology and therapeutic treatment of mental depression. An understanding of 5-HT receptor gene expression is fundamentally important for 5-HT mediated neural function and will provide insights into the molecular basis of mental depression. In the previous funding period a NF-AT like transcription factor complex, factor b, important for human 5-HT2A receptor (5-HT2AR) gene expression was identified. NF-AT is a key transcription factor for interleukin-2 production in activated T-cells. The objective of this project is to further characterize and clone the cDNA(s) encoding factor b. Specific aims are as follows: 1. To further define the DNA binding sequence of factor b complex and its role in regulating transcription of the 5-HT2AR gene by gel retardation and promotor activity assays. 2. The interaction between factor b complex and transcription factor Sp1 will be studied in order to determine if factor b complex and Sp1 binding are independent, mutually exclusive, or synergistic. DNase 1 footprinting and promotor activity assays in human neuroblastoma (SHSY-5Y) cells will be performed. The interaction between transcription factors will also be studied by in vivo genomic footprinting. 3. UV cross-linking will be performed to determine whether factor b complex consists of a single or multiple polypeptide(s). Their immuno-reactive properties will be studied using NF45 and NF90 polyclonal antibodies. 4. To identify the cDNA(s) encoding polypeptide(s) with factor b activity. If factor b complex is composed of one polypeptide, its cDNA will be cloned by screening an expression library. If factor b complex is composed of two or more subunits, they will be purified by DNA affinity chromatography. Degenerate oligonucleotides will then be designed based on amino acid sequence(s) and used as probes. 5. To characterize and investigate the validity of the cDNA(s) encoding factor b. The validity of isolated factor b cDNA clone(s) will be studied by observing if the expressed polypeptides will (a) bind to the 0.35 kb fragment and (b) stimulate promotor activity of co-transfected 0.35 kb (or 0.70 kb) construct in NCI-H460 cells (lacking factor b activity and 5-HT2AR expression). The tissue distribution of factor b will be studied by Northern analysis. Factor b polypeptide(s) will also be expressed in resting Jurkat cells to see if it can stimulate IL-2 production.