Structural analysis of the murinemdr2 gene and its transcription unit in multidrug resistant cells and normal tissue

Abstract

The mouse mdr (P-glycoprotein) gene family consists of three members, mdr1a, mdr1b (Class I), and mdr2 (Class II). The work performed in this thesis was undertaken to understand the role of the mdr2 gene in multidrug resistance (MDR) and in normal physiology. Analysis of mdr2 amplification in a series of highly amplified MDR J774.2 mouse macrophage-like cell lines revealed that some cell lines have amplified a truncated copy of the gene. In these cell lines, the amplicon terminates non-randomly within the gene, and only those cell lines amplifying the entire gene overexpress its message. Even when copy number at the 5{dollar}\sp\prime{dollar}- and 3{dollar}\sp\prime{dollar}-ends of the gene are unequal, transcription across the gene is molar, and initiation is modulated by promoter methylation. Cloning and analysis of the 5{dollar}\sp\prime{dollar}-end of the gene revealed the presence of a complex transcriptional unit, which gives rise to a tissue-specific pattern of 5{dollar}\sp\prime{dollar}-end formation. One of the transcription initiation was mapped to a TATA-less promoter within the cloned fragment. A rearrangement was detected in the 5{dollar}\sp\prime{dollar}-end of the mdr2 gene in the J7.V2-1 (V2) cell line. Cloning and characterization of this deletion showed that it was due to the replacement of a 1.8 kb segment containing the normal 4th exon with an (AT){dollar}\sb{lcub}\rm n{rcub}{dollar} tract. In V2, the normal allele and this rearranged allele were equally amplified, but the rearranged allele was preferentially transcribed, generating a non-functional message.