ROLE OF MDR-RELATED GENES IN DICTYOSTELIUM DEVELOPMENT

耐多药相关基因在盘基网柄菌发育中的作用

• 批准号: 2910170
• 负责人: ADAM KUSPA
• 金额: $ 20.56万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910170
• 关键词: Dictyostelium; affinity labeling; autoradiography; cell differentiation; cell type; cytogenetics; developmental genetics; fungal genetics; fusion gene; gene complementation; gene expression; genetic mapping; immunoprecipitation; molecular cloning; multidrug resistance; nucleic acid hybridization; nucleic acid sequence; polymerase chain reaction; protein structure function; reporter genes; site directed mutagenesis; transport proteins

In multicellular organisms the proportioning of differentiated cell-types must be carefully controlled such that the ultimate tissue is of the appropriate size for optimal function within the mature organism. While the development of Dictyostelium is simpler than that of metazoans, resulting in a ball of spores held on a cellular stalk, proportioning of the major cell types is tightly controlled in this organism. Dictyostelium will be studied as a model organism to gain an understanding of cell differentiation and the proportioning of cell types during multicellular development. The tagB gene is required for the differentiation of one of the three main prestalk cell subtypes, Pst A cells, from an established pool of prestalk cells. The predicted tagB protein product is very similar in its primary structure to the product of the human multidrug resistance gene, MDRI, which encodes P-glycoprotein (P-gp). P-gp was originally described in drug resistant tumor cells as an ATP-dependent efflux pump whose overexpression leads to reduced drug accumulation in cells. A developmentally regulated a rhodamine (Rh) transporter is also expressed in prespore cells with properties similar to human P-gp. Physical genomic mapping techniques identified six additional loci in Dictyostelium which containing tagB-related genes. The role of tagB in cell differentiation will be determined by a variety of experiments. First w will determine whether tagB encodes an ATP-dependent transporter. We will also attempt to functionally substitute the tagB gene with the human MDRI gene which would indicate the tagB is an MDR homolog. The TagB protein will be altered, by site-directed mutagenesis of its gene, in amino acid residues known to be important for mammalian P-gp function, followed by a functional analysis of the altered tagB gene in tagB mutant cells. Genetic tests of tagB function will be carried out in an attempt to determine the cell autonomous function of tagB in specifying the pstA cell fate. The precise localization of tabB expression within the developing organism will be determined using reporter genes fused to the tagB promoter. Ectopic expression of tagB will also provide useful information regarding tagB function. Using reporter gene constructs and selected double mutants, we will determine the epistatic relationships of tagB to other genes involved in PstA cell formation. The tagB-related genes will be cloned by PCR or by positional cloning in hopes of characterizing additional MDR-related genes. The expression patterns will be determined for these genes with the goal of finding a candidate gene for the prespore Rh transporter. If found, it will be deleted from the genome by homologous recombination in order to determine its role in development and in Rh transport in prespore cells. The prespore Rh transporter will be characterized biochemically to clarify its role in development ad to aid in the identification of its gene. Additional genes which regulate cell-type proportioning in Dictyostelium will be isolated and characterized with respect to tagB and the other MDR-related genes. Multidrug resistance is a major obstacle in the chemotherapy of human cancer where cross resistance of tumor cells to a large variety of functionally unrelated drugs is mediated by P-gp. The normal function of p-gp is poorly understood. The study of this gene family in a simple eukaryote should help to further our understanding of the roles of human MDR genes in normal and malignant cell physiology.

在多细胞生物中，分化细胞类型的比例 必须仔细控制，以便最终的组织是 成熟有机体内最佳功能的适当大小。 尽管 盘基网柄菌的发育比后生动物简单， 产生一个固定在细胞茎上的孢子球， 主要细胞类型在该生物体中受到严格控制。 网柄菌属 将作为模式生物进行研究，以了解细胞 多细胞过程中细胞类型的分化和比例 发展。 tagB 基因是分化其中之一所必需的 三种主要的前茎细胞亚型，Pst A 细胞，来自已建立的 前茎细胞池。 预测的 tagB 蛋白产物非常相似 其一级结构是人类多重耐药性的产物 基因MDRI，编码P-糖蛋白(P-gp)。 P-gp最初是 在耐药肿瘤细胞中被描述为 ATP 依赖性外排泵 其过度表达导致细胞内药物积累减少。一个 发育调节的罗丹明 (Rh) 转运蛋白也表达于 前孢子细胞具有与人类 P-gp 相似的特性。 物理基因组 作图技术在盘基网柄菌中确定了另外六个基因座 含有tagB相关基因。 tagB在细胞分化中的作用 将通过各种实验来确定。 首先 w 将确定 tagB 是否编码 ATP 依赖性转运蛋白。 我们也将尝试 在功能上用人类 MDRI 基因替代 tagB 基因，这将 表明 tagB 是 MDR 同系物。 TagB 蛋白将被改变， 其基因的定点突变，在已知的氨基酸残基中 对哺乳动物 P-gp 功能很重要，随后进行功能分析 tagB突变细胞中改变的tagB基因。 tagB功能的基因测试 将尝试确定细胞自主功能 tagB 指定 pstA 细胞命运。 tabB的精准定位 发育中的生物体内的表达将使用报告基因确定 与 tagB 启动子融合的基因。 tagB 的异位表达也会 提供有关 tagB 功能的有用信息。 使用报告基因 构建体和选择的双突变体，我们将确定上位性 tagB 与参与 PstA 细胞形成的其他基因的关系。 这 希望通过PCR或定位克隆来克隆tagB相关基因 表征其他 MDR 相关基因。 表达模式 将确定这些基因，目的是找到候选基因 前孢子 Rh 转运蛋白基因。 一经发现将删除 通过同源重组来确定基因组的作用 前孢子细胞中的发育和 Rh 运输。 前孢子Rh 将对转运蛋白进行生化表征，以阐明其在 开发广告以帮助识别其基因。 额外的基因 将分离出调节盘基网柄菌中细胞类型比例的物质 并针对 tagB 和其他 MDR 相关基因进行了表征。 多药耐药性是人类化疗的一大障碍 癌症中肿瘤细胞对多种药物具有交叉耐药性 功能无关的药物由 P-gp 介导。 正常功能为 p-gp 的了解甚少。 对该基因家族的简单研究 真核生物应该有助于进一步了解人类的作用 正常和恶性细胞生理学中的 MDR 基因。