Joint study on human genomic analysis of drug resistance

本研究采用基因组DNA克隆技术和酵母人工染色体（YAC）载体技术，研究了人多药耐药基因MDR1在耐药过程中的基因扩增和表达机制.从噬菌体文库中分离MDR1基因启动子区，以阐明其转录调控结构域的结构。已通过缺失分析确定了对抗癌剂和UV作出反应的调节元件。我们认为MDR1基因是应激诱导基因的一员，而应激诱导是化疗后细胞获得耐药的潜在机制。2.我们尝试构建该区域的图谱，以确定MDR区域的长距离基因组结构，并筛选耐药诊断探针。从华盛顿大学构建的人全基因文库和7号染色体特异性文库中分离到20个MDR1基因区的YAC克隆。1.5Mb contigu已建成 ...更多信息 通过STS内容映射过程从这些克隆中分离。利用稀有切割酶构建了包括MDR1基因在内的600kb的物理图谱。3.利用聚乙二醇介导的原生质球融合法将含MDR1基因的人YAC克隆转入小鼠细胞系。融合系的脉冲场凝胶电泳和PCR分析显示转移的YAC克隆结构完整。在获得长春新碱逐步耐药的过程中，观察到人MDR 1基因的逐步扩增和表达，表明克隆的功能完整。与此相反，小鼠内源性MDR1基因未扩增和表达，提示人MDR1基因选择性表达的机制尚不清楚。4.对拓扑异构酶靶向药物的耐药机制进行了探讨。拓扑异构酶II水平下降，观察到在依托泊苷耐药细胞。我们还发现拓扑异构酶II基因是热诱导的。分离人拓扑异构酶II的启动子区域用于进一步研究拓扑异构酶II基因表达。少

The mechanisms of gene amplification and expression of human multidrug resistance 1(MDR1) gene have been studied during acquisition of drug resistance by using genomic DNA cloned into phase or yeast artificial chromosome(YAC)vector.1. The promoter region of MDR1 gene was isolated from phage library to clarify the structure of transcriptional regulatory domain. Regulatory elements which respond to anticancer agents and to UV have been identified by deletion analysis. We propose that MDR1 gene is a member of the stress inducible genes and the inducibility is the underlying mechanism by which cells acquire drug resistance after chemotherapy.2.We tried to construct a map of this region to determine the long range genomic organization of MDR region and to isolate diagnostic probe for drug resistance. Twenty YAC clones around MDR1 gene region were isolated from the total human and the chromosome 7 specific library which was constructed at Washington University. 1.5Mb contigu has been built f … More rom these clones by STS content mapping procedure. Physical map spanning 600kb including MDR1 gene was also constructed using rare cutter enzymes.3.The YAC clone human containing MDR1 gene was transferred to mouse cell line by polyethylene glycol mediated spheroplast fusion method. Pulsed-field gel electrophoresis and PCR analysis of the fusion line showed intact structure of the YAC clone transferred. The step-wise gene amplification and expression of human MDR1 gene were observed during acquisition of step-wise resistance to vincristine, indicating functional intactness of the clone. In contrast, amplification and expression of endogenous mouse MDRI gene were not observed, suggesting an unknown mechanism which permit, selective expression of human MDR1 gene.4.The mechanisms of drug resistance to topoisomerase targeting agents were also investigated. Decrease of level of topoisomerase II was observed in etoposide resistant cells. We also found that the topoisomerase II gene is heat inducible. The promoter region of the human topoisomerase II was isolated for further study of topoisomerase II gene expression. Less