The Analysis of Human MDR Gene Family Using YAC Clones.

体外多药耐药（MDR）的获得通常与p糖蛋白的表达增加有关，p糖蛋白是由位于人类7号染色体上的相关基因的小家族编码的。分析整个大DNA片段的功能和调控的一种途径可以转移到其他哺乳动物细胞中。利用酵母抗人工染色体（YAC）介导转染完成了如此大的基因座转移。我们使用MDR1启动子区的引物对作为探针，筛选了从人类DNA中制备的YAC文库，分离出含有MDR基因的YAC克隆。我们还完成了MDR位点周围1.5Mb的YAC配置图。我们将含有整个人MDR基因座的修饰YAC克隆导入小鼠细胞，建立了一系列耐药细胞系。我们展示了人类MDR基因的功能性表达。我们还使用STS在MDR细胞系中展示了扩增单元。我们克隆了与位于MDR1启动子区域的反向CCAATbox结合的交易因子。该克隆与Y-box结合蛋白（YB-1）相同。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在我们已经建立的所有顺铂耐药细胞系中都过表达。因此，YB-1可能保护细胞免受诱导DNA交联剂的细胞毒性作用。我们还分离出了YB-1的基因组克隆，并在染色体1p34上确定了染色体位点。我们分离到了一个新的ATP结合盒超家族的cDNA。人类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10q24上。我们分析了表观藻毒素抗性细胞系的多药耐药相关蛋白（MRP），发现对表观藻毒素抗性的选择优先诱导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.