GENETIC ANALYSIS OF PLEIOTROPIC DRUG RESISTANCE

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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.