The Role of Unc119 in T Cell Antigen Receptor Signaling1

Unc119 在 T 细胞抗原受体信号转导中的作用1

• 批准号: 6612705
• 负责人: Peter Richard Williamson
• 金额: $ 30.27万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6612705
• 关键词: Cryptococcus neoformans; fungal genetics; gel mobility shift assay; gene expression; genetic enhancer element; genetic regulation; laboratory mouse; miscellaneous oxidoreductase; molecular cloning; posttranslational modifications; temperature; transcription factor; virulence

Description (Adapted from Abstract): Cryptococcus neoformans is a major pathogen in immunocompromised patients, causing life-threatening meningoencephalitis in approximately 6% of HIV positive individuals. In vitro melanin production has classically been associated with virulence in C. neoformans. During the previous funding period congenic knockout strains of C. neoformans establish the importance of the CNLAC1 gene in virulence and laccase-dependent catecholamine oxidation products have been identified in the brain of infected mice. Furthermore, a complex pattern of regulatory DNA-binding sites upstream of the CNLAC1 gene have been identified over been identified together with an unusual Sp1 enhancer site. Sp1 consensus sequences have been found in the other virulence genes such as Cap64 and Cap59 suggesting co-regulation of virulence. The hypothesis to be examined is that molecular regulators of CNLAC1 control virulence of Cryptococcus neoformans. The objectives of the proposed research are to identify and characterize genes involved in laccase expression to determine their role in virulence. Aim 1 proposes to identify and characterize important regulatory DNA-binding sites in the upstream region of CNLAC1. The plan is to use pVEW promoter plasmid and electromobility shift assays to determine significant enhancer and repressor regions under conditions of glucose repression and derepression as well as the host temperature of 37 C. Aim 2 proposes to identify and characterize genes involved in transcriptional enhancement of repression of CNLAC1 and determine their role in virulence. Genes will be cloned by homology to genes predicted by CNLAC1 DNA-binding sites and from laccase-deficient insertional mutants. For each gene the plan is to produce knockout and wild-type complemented strains of C. neoformans and test the effects of the gene on laccase production, capsule formation, secreted manno-proteins, urease activity and virulence. Aim 3 will identify and characterize genes involved in post-transcriptional modification of laccase from DL-1 mutants. Initially, the plans are to complement a vacuolar H+-ATPase on laccase secretion and assess for possible co-regulation of CNLAC1 and CNVPH1. It is anticipated that these studies will provide insights into regulation and virulence factors in C. neoformans that may be used to provide novel approaches to the treatment and control of cryptococcosis.

描述（摘自摘要）：新型隐球菌是免疫功能低下患者的主要病原体，导致大约 6% 的 HIV 阳性个体出现危及生命的脑膜脑炎。体外黑色素的产生通常与新型隐球菌的毒力相关。在之前的资助期间，新型隐球菌的同源基因敲除菌株确立了 CNLAC1 基因在毒力中的重要性，并且在受感染小鼠的大脑中发现了漆酶依赖性儿茶酚胺氧化产物。此外，CNLAC1 基因上游的调控 DNA 结合位点的复杂模式已与不寻常的 Sp1 增强子位点一起被识别。在 Cap64 和 Cap59 等其他毒力基因中也发现了 Sp1 共有序列，表明毒力存在共同调节。要检验的假设是 CNLAC1 的分子调节因子控制新型隐球菌的毒力。拟议研究的目的是鉴定和表征参与漆酶表达的基因，以确定它们在毒力中的作用。目标 1 提议鉴定和表征 CNLAC1 上游区域的重要调控 DNA 结合位点。该计划是使用 pVEW 启动子质粒和电迁移率分析来确定在葡萄糖抑制和去抑制以及 37°C 宿主温度条件下的显着增强子和阻遏子区域。目标 2 建议鉴定和表征参与 CNLAC1 抑制转录增强的基因，并确定它们在毒力中的作用。基因将通过与 CNLAC1 DNA 结合位点和漆酶缺陷插入突变体预测的基因同源性进行克隆。对于每个基因，计划生产新型隐球菌的敲除和野生型互补菌株，并测试该基因对漆酶产生、荚膜形成、分泌的甘露糖蛋白、脲酶活性和毒力的影响。目标 3 将鉴定和表征参与 DL-1 突变体漆酶转录后修饰的基因。最初，计划是补充液泡 H+-ATP 酶对漆酶分泌的影响，并评估 CNLAC1 和 CNVPH1 可能的共同调节。预计这些研究将提供对新型隐球菌的调节和毒力因子的见解，可用于提供治疗和控制隐球菌病的新方法。