Genetic Analysis of Pleiotropic Drug Resistance

描述（申请人提供）：多重耐药是指通过少数基因座的遗传变化获得广泛的耐药表型。多药耐药是肿瘤和感染性疾病化疗治疗中的一个临床问题。我们正在研究多效性耐药（Pdr）在酿酒酵母作为真核多药耐药模型。ATP结合盒转运蛋白Pdr5p的表达在沙门氏菌多药耐药中起关键作用。啤酒。我们最近发现了一个新的信号通路，导致特定的线粒体突变体缺乏ATP合酶的Fo亚复合物激活转录因子Pdr3p的功能，这反过来又刺激PDR 5的表达。该提案的目标是确定导致核Pdr3p激活的信号和参与者。初步实验表明，Fo亚基的水平是Pdr3p活性的关键调节剂。为了直接探索这种关系，我们将使用Fo突变体和过表达载体来评估Fo组分与Pdr3p活性之间的联系。将采用遗传分析来鉴定Pdr3p的负调节子。将筛选基因破坏突变文库中激活Pdr3p的无效突变，同时将采用化学诱变方法来鉴定可刺激Pdr3p功能的必需基因和功能获得性等位基因。我们已经确定了新的蛋白质Yp1055cp作为参与者的髓核（逆行）途径控制Pdr3p。两个杂交和免疫共沉淀实验将用于确定这两种蛋白质是否直接相互作用。参与mRNA降解的蛋白质（Lsm1p）也是Pdr3p正常逆行信号传导所必需的。通过遗传分析研究Lsm1p和Yp1055cp在控制Pdr3p中的作用。我们将通过修复线粒体基因组DNA（rho0）的破坏文库来直接筛选Pdr3p逆行信号传导中的其他参与者，这将导致组成性Pdr3p激活。将检测所得突变体中降低放线菌酮抗性的突变体。引人注目的是，一个类似的现象，其中ABC转运蛋白基因在rho0细胞中上调已被描述在致病性酵母光滑念珠菌。我们在遗传上易处理的S.酿酒酵母细胞将使我们更快地了解多药耐药的这一重要方面。

DESCRIPTION (provided by applicant): Multiple drug resistance refers to acquisition of broad range of resistance phenotypes through genetic changes at a small number of loci. Multidrug resistance is a clinical problem in chemotherapeutic treatment of tumors and infectious disease. We are studying pleiotropic drug resistance (Pdr) in Saccharomyces cerevisiae as a model for eukaryotic multiple drug resistance. Expression of an ATP-binding cassette transporter protein, Pdr5p, is critical in multidrug resistance of S. cerevisiae. We have recently discovered a new signaling pathway causing particular mitochondrial mutants lacking the Fo subcomplex of the ATPsynthase to activate function of the transcription factor Pdr3p, that in turn stimulates PDR5 expression. The goal of this proposal is to identify the signal and participants in the pathway leading to activation of nuclear Pdr3p. Preliminary experiments indicate that the level of Fo subunits is a key modulator of Pdr3p activity. To directly explore this relationship, we will use Fo mutants and overexpression vectors to assess the link between Fo components and Pdr3p activity. Genetic analysis will be employed to identify negative regulators of Pdr3p. A library of gene disruption mutations will be screened for null mutations that activate Pdr3p while a chemical mutagenesis approach will be undertaken to identify essential genes and gain-of function alleles that can stimulate Pdr3p function. We have identified the novel protein Yp1055cp as a participant in the mitochondrial-nuclear (retrograde) pathway controlling Pdr3p. Two hybrid and co-immunoprecipitation experiments will be used to determine if these two proteins directly interact. A protein (Lsm1p) involved in mRNA degradation is also required for normal retrograde signaling to Pdr3p. The role of Lsm1p and Yp1055cp in control of Pdr3p will be investigated by genetic analysis. We will directly screen for other participants in Pdr3p retrograde signaling by curing the disruption library of mitochondrial genomic DNA (rho0) which will result in constitutive Pdr3p activation. The resulting mutants will be tested for mutants that reduce cycloheximide resistance. Strikingly, a similar phenomenon in which ABC transporter genes are up-regulated in rho0 cells has been described in the pathogenic yeast Candida glabrata. Our study of this phenomenon in the genetically-tractable S. cerevisiae cell will allow us to more rapidly understand this important facet of multidrug resistance.