权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

MOLECULAR CHARACTERIZATION OF PERFORIN GENE REGULATION

穿孔素基因调控的分子特征

基本信息

批准号：
2619435
负责人：
Mathias Georg Lichtenheld
金额：
$ 2.79万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-09-01 至 1998-11-30
项目状态：
已结题

项目摘要

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells lyse virus-infected cells and possibly emerging tumor cells but they also contribute to transplant rejection and perhaps to autoimmune tissue destruction. One of the lytic molecules of CTL and NK cells is a pore-forming protein known as perforin. Perforin gene expression is restricted to CTL and NK cells. While NK cells express perforin constitutively, CTL express perforin upon their activation. Our objective is to characterize relevant molecular mechanisms 1) that determine the CTL and NK-cell restricted expression of perforin; 2) that determine the induction of perforin in CTL upon their activation; and we propose 3) to identify the transcription factors involved. Within the first objective, we will characterize a cell-type specific cis-acting element in the 5' flanking region of the perforin gene (specific aim 1. 1), determine the role of the proximal perforin promoter (specific aim 1.2) and analyze and determine the role of the chromatin structure of the perforin gene locus (specific aim 1.3). To achieve these aims we will analyze fragments of the perforin 5' flanking and promoter region for their control over the "CAT" reporter gene after transient transfection into different cell types and will compare DNase I hypersensitivity sites of the perforin gene locus between different cell types. Within the second objective, we will determine whether perforin mRNA induction in CTL occurs primarily at the transcriptional level or the posttranscriptional level (specific aim 2.1). We anticipate that the regulation event takes place at the transcriptional level based on our preliminary data and therefore propose to identify regulatory elements involved (specific aim 2.2). To achieve these aims we will analyze, by Northern blot analysis and nuclear run-off assay, perforin gene induction or upregulation in cloned CTL in response to IL-2, T-cell receptor signals or second-messenger pathway activating agents. We will identify the cis-acting responsive regions by reporter gene analysis as outlined above. Within the third objective, we propose to identify putative transcription factors acting on relevant regulatory elements defined in the previous aims (specific aim 3). We will apply in vitro DNase I footprinting and will analyze a particular DNA binding site by gel shift analysis.

细胞毒性T淋巴细胞(CTL)和自然杀伤(NK)细胞溶解病毒感染的细胞和可能出现的肿瘤细胞，但它们也有助于移植排斥反应，可能还会导致自身免疫组织毁灭。CTL和NK细胞的裂解分子之一是一种称为穿孔素的成孔蛋白。穿孔素基因的表达是仅限于CTL和NK细胞。NK细胞表达穿孔素从结构上讲，CTL在激活后表达穿孔素。我们的目的是刻画相关的分子机制：1) 测定穿孔素的CTL和NK细胞限制性表达；2) 确定CTL中穿孔素在激活时的诱导；提出3)鉴定涉及的转录因子。在第一个目标，我们将描述一种细胞类型的特定顺式作用穿孔素基因5‘侧翼区的元件(特定目标1。 1)，确定近端穿孔素启动子的作用(特定目的 1.2)，并分析和确定染色质结构的作用穿孔素基因座(特异靶1.3)。为了实现这些目标，我们将分析穿孔素5‘侧翼和启动子区域的片段它们在瞬时转染后对“猫”报告基因的控制不同的细胞类型，并将比较DNase I过敏性部位穿孔素基因座在不同细胞类型之间的差异。在第二个目标，我们将确定穿孔素mRNA是否诱导 CTL主要发生在转录水平或转录后水平(特定目标2.1)。我们预计，调控事件发生在转录水平上，基于我们的初步数据，因此建议确定管制要素涉及(具体目标2.2)。为了实现这些目标，我们将通过以下方式分析 Northern印迹分析和核径流分析，穿孔素基因诱导克隆的CTL对IL-2、T细胞受体的反应上调信号或第二信使途径激活剂。我们将确定报告基因分析显示的顺式反应区域上面。在第三个目标中，我们建议确定假定的作用于相关调控元件的转录因子前述目标(具体目标3)。我们将在体外应用DNase I 足迹，并将通过凝胶位移分析特定的DNA结合位置分析。