GENETIC ANALYSIS OF PLEIOTROPIC DRUG RESISTANCE

多效性耐药性的遗传分析

基本信息

批准号：
2187366
负责人：
W Scott Moye-Rowley
金额：
$ 15.42万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-08-01 至 1997-07-31
项目状态：
已结题

项目摘要

Multiple drug resistance (Mdr) refers to the ability of tumor cells to avoid the toxic action of anticancer drugs with unrelated modes of action. Animal cells often acquire this property by amplifying the expression of a P-glycoprotein-encoding gene, MDR1. Yeast cells also possess a phenotype, known as pleiotropic drug resistance (Pdr), analogous to mammalian Mdr. The yeast genes that confer Pdr inactivate the toxic effects of a variety of unrelated drugs. The goal of this work is to study the genes that elicit Pdr in yeast as a model for the more complicated mammalian system. PDR5 is a yeast gene that encodes a protein with homology to the MDR1 gene product. Antisera will be prepared against the PDR5 protein and used to determine the location of this protein. Fractionation studies will determine the membrane region that this protein is likely to be associated with. The promoter region of PDR5 will be subjected to deletion mutagenesis in order to identify DNA elements important in the control of the expression of this gene. Yeast mutants that lack PDR5 are hypersensitive to several drugs, indicating the importance of this protein in drug detoxification. PDR4 and PDR7 are genes that affect PDR5 mRNA levels. The site of action of these gene products at PDR5 will be determined. PDR1 and PDR3 are zinc finger transcription factors that can give rise to semi-dominant Pdr mutants. Both of these factors affect PDR5 expression but their mode of action is unknown. The ability of these regulatory proteins to bind to the PDR5 promoter will be determined. We will produce antisera against PDR3 and assess whether the localization of this factor changes in semi-dominant PDR3 mutant strains. The nature of the amino acid change in the semi-dominant PDR3 mutants will be determined to gain insight into how this protein functions. PDR3 stimulates drug resistance in a PDR5-independent fashion. Other downstream target genes provide are regulated by PDR3 and give rise to Pdr. These other drug resistance pathways will be identified by screening a yeast high copy plasmid library for genes that confer Pdr only in the presence of PDR3. Glutathione S-transferases (GST) are believed to be important in drug detoxification in a variety of organisms. Yeast GST genes will be cloned and mutant strains of yeast produced that lack these proteins. Viability and drug resistance will be assayed for the GST-less mutants. Yeast and mammalian cells share a great deal of functional homology as has already been seen in the study of protein localization and transcription. The homology between PDR5 and MDR1 indicates that multiple drug resistance is also likely to be conserved between yeast and animals. The use of genetics to study Pdr in yeast provides a unique advantage not available in mammalian cells.

多重耐药性（Mdr）是指肿瘤细胞对多种药物产生耐药性的能力。避免与不相关模式的抗癌药物产生毒性作用行动。动物细胞通常通过放大 P-糖蛋白编码基因 MDR1 的表达。酵母细胞也具有一种表型，称为多效耐药性（Pdr），类似于哺乳动物 Mdr。赋予 Pdr 失活的酵母基因各种不相关药物的毒性作用。这项工作的目标是研究在酵母中引发 Pdr 的基因，作为更多研究的模型复杂的哺乳动物系统。 PDR5 是一种酵母基因，编码与 MDR1 同源的蛋白质基因产物。将针对 PDR5 蛋白制备抗血清并用于确定该蛋白质的位置。分馏研究将确定该蛋白质可能存在的膜区域与.相关联。 PDR5 的启动子区域将受到缺失诱变为了识别在表达控制中重要的DNA元件这个基因的。缺乏 PDR5 的酵母突变体对多种物质过敏药物，说明该蛋白在药物解毒中的重要性。 PDR4 和 PDR7 是影响 PDR5 mRNA 水平的基因。行动地点将确定 PDR5 处的这些基因产物。 PDR1和PDR3是锌指转录因子，可以产生半显性 Pdr 突变体。这两个因素都会影响 PDR5 表达但其作用方式尚不清楚。这些能力将确定与 PDR5 启动子结合的调节蛋白。我们将产生针对 PDR3 的抗血清并评估是否本地化该因子在半显性 PDR3 突变株中发生变化。大自然半显性 PDR3 突变体中的氨基酸变化将是决心深入了解这种蛋白质的功能。 PDR3 以不依赖于 PDR5 的方式刺激耐药性。其他下游靶基因受 PDR3 调节并产生博士。这些其他耐药途径将通过筛选酵母高拷贝质粒库中赋予 Pdr 的基因仅在 PDR3 存在的情况下。谷胱甘肽 S-转移酶 (GST) 被认为在药物中很重要多种生物体的解毒作用。酵母GST基因将被克隆以及缺乏这些蛋白质的酵母突变株。生存能力并对无 GST 的突变体进行耐药性分析。酵母和哺乳动物细胞在功能上有很多同源性在蛋白质定位的研究中已经看到转录。 PDR5 和 MDR1 之间的同源性表明多重耐药性也可能在酵母和动物。利用遗传学研究酵母中的 Pdr 提供了独特的方法哺乳动物细胞所不具备的优势。