Amplification of the Murine mdr 2 Gene and a Reconsideration of the Structure of the Murine mdr Gene Locus

A common feature of cells selected in vitro for the multidrug resistance (MDR) phenotype is the amplification and concomitant overexpression of the mdr genes. In murine macrophage-like J774.2-derived MDR cell lines, there is a good correlation between levels of amplification and expression for the mdr1b gene, but not for the other two gene family members, mdr1a and mdr2. To understand this phenomenon better, a study of the amplification and expression of the mdr2 gene was undertaken. Southern blotting of genomic DNAs from a series of six MDR cell lines revealed that five of these lines had 5′-end amplification of mdr2, whereas only three contained 3′-end amplification. The analysis also suggested the involvement of a recombination hot-spot in this phenomenon. Despite the observation that the ratio between the number of copies of the 5′ and 3′ ends of the gene differs among cell lines, the ratio of 5′ to 3′ end transcription of mdr2 was approximately 1 in all cell lines. An analysis of promoter methylation in MDR cell lines demonstrated that this mechanism may play a role in regulating the transcription of mdr2, but not of mdr1b. Long-range mapping of the mdr locus in parental and amplified cell lines suggested that the three mdr genes are oriented in the same direction, and also revealed the presence of a number of rearrangement events. Models for the murine mdr gene locus in wild-type cells and in a cell line containing a rearrangement are presented.