Ras signaling in C. neoformans virulence and development

超出所提供的空间。Ras分子是高度保守的信号元件，介导细胞对各种细胞外信号的反应。Ras通路已在哺乳动物系统中被广泛研究，并与许多人类恶性肿瘤的发病机制有关。在微生物中，Ras元件调节分化事件，包括真菌酵母-菌丝体转变。担子菌新隐球菌提供了一个强大的遗传模型系统，其中解剖Ras信号在人类病原体中的分子机制。Alspaugh博士之前已经证明，这种生物中的Ras途径通过MAP激酶途径控制菌丝分化，通过cAMP途径控制琼脂粘附，以及通过肌动蛋白极化的温度依赖性调节来控制高温生长。在这项提议中，Alspaugh实验室将使用几种无偏见的遗传方法来进一步表征C.新生生物中调节高温生长和细胞分化的Ras通路。首先，将通过酵母双杂交筛选确定与Rasl物理相互作用的蛋白质。期望通过这种方法可以识别编码Rasl信号转导途径的上游和下游成分的基因。此外，Ras蛋白在高温下调节生长的下游组分将通过两种互补的方法进行鉴定。Rasl信号的积极效应将通过多拷贝抑制因子分析来确定。利用Rasl突变体背景下的随机插入诱变技术和高温生长恢复的筛选，将确定参与Rasl控制高温生长的抑制或调节蛋白。最后，在与杜克大学其他真菌学实验室的合作下，Atspaugh实验室正在构建基于新生C.基因组计划的基因微阵列。通过这些基因微阵列可以识别受Rasl转录调控的基因。通过这些方法鉴定的基因的生物学相关性将通过基因破坏和对隐球菌病动物模型中体外和体内产生的突变株的分析来评估。网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. Ras molecules are highly conserved signaling elements that mediate cellular responses to a variety of extracellular signals. Ras pathways have been extensively studied in mammalian systems and implicated in the pathogenesis of many human malignancies. Among microorganisms, Ras elements regulate differentiation events including fungal yeast-mycelium transitions. The basidiomycete Cryptococcus neoformans provides a powerful genetic model system in which to dissect the molecular mechanisms of Ras signaling in a human pathogen. Dr. Alspaugh has previously demonstrated that Ras pathways in this organism control hyphal differentiation through a MAP kinase pathway, agar adherence through a cAMP pathway, and high temperature growth by a temperature-dependent regulation of actin polarization. In this proposal, the Alspaugh laboratory will use several unbiased genetic methods to further characterize the Ras pathways in C. neoformans that regulate high temperature growth and cellular differentiation. First, proteins that physically interact with Rasl will be identified by a yeast two-hybrid screen. It is expected that genes encoding both upstream and downstream components of the Rasl signal transduction pathway will be identified by this method. Additionally, downstream components of Ras proteins that regulate growth at elevated temperature will be identified by two complementary approaches. Positive effectors of Rasl signaling will be identified by multicopy suppressor analysis. Inhibitory or regulatory proteins involved in Rasl control of high temperature growth will be identified using random insertional mutagenesis in the rasl mutant background and screening for the restoration of high temperature growth. Finally, in collaboration with other mycology laboratories at Duke University, the Atspaugh lab is constructing gene rnicroarrays based on the C. neoformans genome project. Genes that are transcriptionally regulated by Rasl will be identified using these gene microarrays. The biological relevance of genes identified by any of these approaches will be assessed by gene disruption and analysis of the resulting mutant strains both in vitro as well as in vivo in animal models of cryptococcosis. PERFORMANCE SITE ========================================Section End===========================================