PROTEIN(S) INVOLVED IN NEUROTRANSMISSION

性状（改编自申请人摘要）：血清素 5-羟色胺（5-hydroxytryptamine，5-HT）及其受体参与了 精神抑郁症的病因和治疗。 的理解 5-HT受体基因表达的改变对5-HT 介导的神经功能，并将提供深入了解分子基础 精神抑郁症 在上一个融资期， 转录因子复合物，因子B，对人5-HT 2A受体很重要 （5-HT 2AR）基因表达。 NF-AT是一种关键转录因子， 活化的T细胞中产生白细胞介素-2的因子。 的目标 该项目是进一步表征和克隆编码 因子B。具体目标如下： 1. 为了进一步明确B因子复合物的DNA结合序列及其与DNA结合的关系， 通过凝胶阻滞调节5-HT 2AR基因转录的作用， 启动子活性测定。 2. 因子B复合物与转录因子Sp1的相互作用 将进行研究，以确定是否因子B复合物和Sp1结合 是独立的、相互排斥的或协同的。 DNA酶1足迹法 并将在人神经母细胞瘤（SHSY-5 Y）细胞中进行启动子活性测定。 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研究。 3. 将进行UV交联，以确定因子B复合物 由单个或多个多肽组成。 他们的免疫反应 将使用NF 45和NF 90多克隆抗体研究其性质。 4. 鉴定编码具有因子B活性的多肽的cDNA。 如果因子B复合物由一种多肽组成，则克隆其cDNA 通过筛选表达文库。 如果因子B复合物由两个 或多个亚基，它们将通过DNA亲和层析纯化。 然后基于氨基酸序列设计简并寡核苷酸。 序列并用作探针。 5. 表征并研究编码以下基因的cDNA的有效性： 因子B。将通过以下方法研究分离的因子B cDNA克隆的有效性： 观察表达的多肽是否（a）结合0.35kb的 片段和（B）刺激共转染的0.35 kb（或 0.70 kb）构建体（缺乏因子B活性和5-HT 2 AR 表达式）。 将通过以下方法研究因子B的组织分布： 北方分析。 因子B多肽也将在 让Jurkat细胞静止，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.