The Analysis of Human MDR Gene Family Using YAC Clones.

多药耐药（multidrug resistance，MDR）的产生通常与P-糖蛋白表达增加有关，P-糖蛋白由位于人类7号染色体上的连锁基因小家族编码。分析整个大DNA片段的功能和调节的一种途径可以转移到其他哺乳动物细胞中。这种大的基因座的转移已经使用酵母抗染色体（YAC）介导的转染完成。我们已经分离出的YAC克隆的MDR基因筛选后，从总的人DNA制备的YAC库使用引物对的MDR 1启动子区作为探针。我们还完成了MDR位点周围1.5Mb的YAC重叠群图谱。我们将含有完整的人MDR基因位点的修饰YAC克隆导入小鼠细胞，并建立了一系列的长春新碱耐药细胞系。我们展示了人MDR基因的功能表达。我们克隆了与位于MDR 1启动子区的反向CCAAT盒结合的反式作用因子。该克隆对Y-box结合蛋白（YB-1）具有特异性。我们制备了特异性抗体并检测了其在各种耐药细胞系中的表达。YB-1在我们建立的所有顺铂耐药细胞系中过表达。因此，YB-1可以保护细胞免受诱导DNA交联的药物的细胞毒性作用。我们还分离了YB-1的基因组克隆，并鉴定了染色体1 p34上的染色体位点。我们分离了一个新的ATP结合盒超家族的cDNA。人克隆与大鼠小管多特异性有机阴离子转运蛋白（cMOAT）同源。MOAT基因位于染色体10 q24。我们分析了表鬼臼毒素耐药细胞系中的多药耐药相关蛋白（MRP），发现表鬼臼毒素耐药选择优先诱导MRP基因的过表达。

The acquistion of multidrug resistance (MDR) in vitro is commonly associated with the increased expression of the P-glycoproteins which are encoded by small families of the linked genes, located on human chromosome 7. One route to analyzes the function and regulation of the entire large DNA segment could be transferred into other mammalian cells. Transfer of loci this large has been accomplished using yeast antificial chromosome (YAC)-mediated transfection. We have isolated YAC clones containing the MDR genes after screening a YAC library prepared from total human DNA using primer pairs of the MDR1 promoter region as a probe. We have also completed the 1.5Mb YAC contig map around MDR loci. We introduced a modified YAC clone containing the entire human MDR locus into mouse cells and established a series of bincristine-resistant cell lines. We showed the functional expression of human MDR genes. We also showed the amplification unit in MDR cell lines using STS.We cloned the transacting factor, which bind to the inverted CCAATbox located at the MDR1 promoter region. This clone was idenfical to the Y-box binding protein (YB-1). We generated the specific antibodies and examined the expression in various drug resistant cell lines. YB-1 was overexpressed in all cisplatin-resistant cell lines, which we had established. Thus, YB-1 may protect cells from the cytotoxic effects of agents that induce cross-linking of DNA.We also isolated the genomic clones for YB-1, and identified the chromosomal locus on chromosome 1p34. We isolated the cDNA of a new ATP binding cassette superfamily. A human clone is homologous to rat canalicular multispecific organic anion transporter (cMOAT). MOAT gene is located on chromosome 10q24. We analyzed the multidrug resistance associated protein (MRP) in epipodophyllotoxins resistant cell lines and found that selection for resistance to epipodophyllotoxins preferentially induces overexpression of MRP gene.