权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Joint study on human genomic analysis of drug resistance

人类耐药性基因组分析联合研究

基本信息

批准号：
03044118
负责人：
KUWANO Michihiko
金额：
$ 5.12万
依托单位：
Kyushu University Faculty of Medicine (1993)大分医科大学 (1991-1992)
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

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

利用克隆入相的基因组DNA或酵母人工染色体(YAC)载体，研究了人多药耐药1(MDR1)基因在获得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基因启动子区域，以明确其转录调控结构域的结构。通过缺失分析已经确定了对抗癌药物和紫外线有反应的调控元件。我们认为mdr1基因是应激诱导基因中的一员，诱导性是细胞在化疗后获得耐药的潜在机制。2.我们试图构建该区域的图谱，以确定mdr区的远程基因组组织，并分离出耐药的诊断探针。从全人类和华盛顿大学构建的7号染色体特异性文库中分离到mdr1基因周围的20个YAC克隆。为…构建了1.5Mb的Contigu通过STS内容映射程序从这些克隆中获得更多信息。利用稀有切割酶构建了包含mdr1基因的600kb物理图谱。3.用聚乙二醇介导球形体融合的方法将含有mdr1基因的YAC克隆人转入小鼠细胞系。融合株的脉冲场凝胶电泳法和聚合酶链式反应分析表明，导入的YAC克隆结构完整。在获得对长春新碱的阶段性耐药过程中，观察到人MDR1基因的阶段性扩增和表达，表明克隆的功能是完整的。相反，没有观察到内源性小鼠mdr1基因的扩增和表达，这可能是一种未知的机制，允许选择性表达人mdr1基因。4.还研究了拓扑异构酶靶向药物耐药的机制。在依托泊苷耐药细胞中，拓扑异构酶II水平降低。我们还发现拓扑异构酶II基因是热诱导的。分离了人拓扑异构酶II的启动子区域，为进一步研究拓扑异构酶II基因的表达奠定了基础。较少