The mouse mdr gene family consists of three distinct genes (mdrl, mdr2, and mdr3), for which we have isolated full-length cDNA clones. cDNA subfragments corresponding to discrete regions showing little sequence conservation among the three mdr genes were used as gene-specific DNA probes in hybridization experiments. Long-range mapping by pulse-field gel electrophoresis indicated that the three mdr genes are closely linked on a genomic DNA segment of approximately 625 kilobases. The gene order and direction of transcription of the three genes were determined and indicate the arrangement (5') mdr3 (3')-(5') mdrl (3')-(3') mdr2 (5'). Southern blotting analyses of genomic DNA from a panel of independently derived multidrug-resistant cell lines identified mdr gene amplification in 10 of 12 cell lines studied. In individual cell lines showing gene amplification, the copy number of each of the three mdr genes was identical, suggesting that the three mdr genes became amplified as part of a single amplicon in these cells. Although increased expression of all three mdr genes was detected in 2 of 12 cell lines tested, multidrug resistance was associated in 10 of 12 lines with the independent overexpression of either mdrl (7 of 12) or mdr3 (3 of 12) but not mdr2. mdrl overexpression was consistently associated with gene amplification, while increased mdr3 expression was detected in certain cell lines that did not show gene amplification. Increased levels of mdrl mRNA were linked to the overexpression of a P glycoprotein of apparent molecular weight 180,000 to 200,000, whereas increased mdr3 expression resulted in increased expression of a P glycoprotein of molecular weight 160,000 to 180,000. Our results suggest that at least two members of the mouse mdr gene family, mdrl and mdr3, can independently confer multidrug resistance in the cell lines examined.In cultured cells, multidrug resistance is associated with the overexpression of a heterogeneous group of antigenically related membrane phosphoglycoproteins, termed P glycoproteins (12). P glycoproteins are encoded by a small family of closely related genes given the appellation pgp or mdr which often become amplified in highly drug-resistant cell lines (reviewed in references 8, 12, and 15). The mdrlpgp gene family is composed of three members in rodents (hamster and mouse) and two members in humans (24 suggest that P glycoprotein functions in drug-resistant cells as an ATP-dependent drug efflux pump.Sequence analysis of full-length cDNA clones encoding mouse mdrl, mdr2, and mdr3 indicates that the three encoded proteins are highly homologous (overall amino acid homology of 75 to 85%) and share the same predicted structural domains (10a, 19, 20
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