Genetic Analysis of Pleiotropic Drug Resistance

描述（由申请人提供）：多重耐药是指通过少数位点的遗传改变获得广泛的耐药表型。多药耐药是肿瘤和感染性疾病化疗中的临床难题。我们正在研究酿酒酵母（Saccharomyces cerevisiae）的多效耐药（Pdr）作为真核生物多重耐药模型。atp结合盒转运蛋白Pdr5p的表达在酿酒葡萄球菌的多药耐药中起关键作用。我们最近发现了一种新的信号通路，导致缺乏atp合成酶的Fo亚复合物的特定线粒体突变激活转录因子Pdr3p的功能，进而刺激PDR5的表达。本提案的目标是确定导致核Pdr3p激活的途径中的信号和参与者。初步实验表明，Fo亚基水平是Pdr3p活性的关键调节因子。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。遗传分析将用于鉴定Pdr3p的负调控因子。一个基因破坏突变文库将筛选激活Pdr3p的零突变，同时将采用化学诱变方法来鉴定可以刺激Pdr3p功能的必要基因和获得功能等位基因。我们已经确定了新的蛋白Yp1055cp作为控制Pdr3p的线粒体-核（逆行）途径的参与者。两个杂交和共免疫沉淀实验将用于确定这两个蛋白是否直接相互作用。参与mRNA降解的一种蛋白（Lsm1p）也是Pdr3p正常逆行信号传递所必需的。Lsm1p和Yp1055cp在控制Pdr3p中的作用将通过遗传分析进行研究。我们将通过修复线粒体基因组DNA的破坏文库（rho0）来直接筛选Pdr3p逆行信号的其他参与者，这将导致组成性Pdr3p激活。将对产生的突变体进行测试，以寻找降低环己亚胺耐药性的突变体。引人注目的是，在致病性酵母秃念珠菌中也有类似的现象，即ABC转运蛋白基因在rho0细胞中上调。我们在遗传易处理的酿酒葡萄球菌细胞中对这一现象的研究将使我们能够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.