GENETIC ANALYSIS OF PLEIOTROPIC DRUG RESISTANCE

多药耐药(MDR)指的是肿瘤细胞对 以无关的方式避免抗癌药物的毒性作用 行动。动物细胞通常通过放大 表达P-糖蛋白编码基因mdr1。酵母细胞也 拥有一种表型，称为多效性耐药(PDR)， 类似于哺乳动物的MDR。导致PDR失活的酵母基因 各种无关药物的毒性作用。这项工作的目标是 是研究在酵母中引发PDR的基因，作为更多 复杂的哺乳动物系统。 PDR5是一种酵母基因，编码一种与mdr1同源的蛋白质。 基因产物。将制备针对PDR5蛋白的抗血清和 用来确定这种蛋白质的位置。分馏研究 将决定这种蛋白质可能所在的膜区域 关联到。 PDR5启动子区域将受到缺失突变 以确定在表达控制中重要的DNA元件 这种基因的基因。缺乏PDR5的酵母突变株对几种 药物，表明该蛋白在药物解毒中的重要性。 PDR4和PDR7是影响PDR5基因表达水平的基因。行动地点 在PDR5的这些基因产物将被确定。 PDR1和PDR3是锌指转录因子，可以引起 半显性PDR突变体。这两个因素都会影响PDR5 但他们的行为模式尚不清楚。这些人的能力 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定位 该因子在半显性PDR3突变株中的变化。大自然 半显性PDR3突变体的氨基酸变化将是 决心深入了解这种蛋白质是如何发挥作用的。 PDR3以不依赖于PDR5的方式刺激耐药性。其他 提供的下游靶基因受PDR3调控，并导致 PDR。这些其他耐药途径将通过 酵母高拷贝质粒库中PDR相关基因的筛选 仅在PDR3存在的情况下。 谷胱甘肽S转移酶(GST)在药物中起重要作用 在各种生物体中的解毒作用。酵母GST基因将被克隆 突变的酵母菌产生缺乏这些蛋白质的菌株。生存能力 并将对GST含量较低的突变体进行抗药性检测。 酵母和哺乳动物细胞有大量的功能同源性，如 已经在蛋白质定位和研究中看到 抄写。PDR5和MDR1的同源性表明 多重耐药性也可能在酵母菌和 动物。利用遗传学来研究酵母中的PDR提供了一种独特的 这是哺乳动物细胞所没有的优势。

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.