GENOMIC APPROACHES TO CANDIDA VIRULENCE

这项提案的长期目标是利用后基因组时代的技术和方法，以增加我们对艾滋病毒相关的口腔念珠菌病发病机制的分子基础的理解。我们的近期目标是利用C.白色念珠菌基因组DNA序列数据由斯坦福大学DNA测序和技术中心（斯坦福大学中心）生成。利用该技术，可以对C.将评估各种疾病情况下的白色念珠菌基因表达。 这些信息将有助于开发特定的诊断方法，药物靶点和深入了解C。免疫功能低下个体中的白色念珠菌发病机制。本研究的具体目标是：（1）利用PCR产物技术和寡核苷酸技术制备DNA微阵列。 全基因组DNA微阵列的制作将在本资助终止前完成。 制造将是一个反复的过程，从构建基于PCR的DNA微阵列和使用可用的C的子集立即开始。白色念珠菌基因组序列。 在这些研究的早期阶段，将制定使微阵列普遍适用于念珠菌研究社区的协议。(2)第一代微阵列将用于分析C.从各种环境中获得的白色念珠菌。这将是第一个方法来编目的功能和协调表达的基因序列，已知和未知的。发病机制研究中的许多关键问题之一是感染动物模型的验证，因为这些模型将为进一步分析功能尚不清楚的基因提供基础。因此，我们特别感兴趣的是比较C的表达模式。口腔粘膜中的白色念珠菌基因，与现存啮齿动物模型中表达的那些。(3)启动感兴趣的单个基因的功能分析。使用微阵列比较基因表达模式将确定值得进一步研究的基因序列。那些功能未知的基因将作为进一步遗传分析的目标，包括构建特异性敲除突变和分析随后的表型。以这种方式，将有可能开始一个完整的表征的基因表达的范围，有助于致病念珠菌在不同的主机环境。

The long term goal of this proposal is to use post-genomics era technologies and approaches to increase our understanding of the molecular basis of pathogenesis in HIV-related oral candidiasis. Our immediate goal is to fabricate DNA micro arrays using the C. albicans genomic DNA sequence data generated by the Stanford DNA Sequencing and Technology Center (Stanford Center). Using this technology the patterns of C. albicans gene expression in various disease contexts will be assessed. This information will contribute to the development of specific diagnostics, drug targets and insights into the mechanism of C. albicans pathogenesis in immunocompromised individuals. The Specific Aims of this proposal are to:(1) fabricate DNA micro arrays using both PCR-product based and oligonucleotide-based technologies. Fabrication of whole genome DNA micro arrays will be completed before the termination of this grant. Fabrication will be an iterative process, beginning immediately with the construction of PCR based DNA micro arrays and using a subset of available C. albicans genomic sequences. At an early stage in these studies, protocols to make the micro arrays generally available to the Candida research community will be developed. (2) The first generation of micro arrays will be used to analyze patterns of gene expression in C. albicans obtained from a variety of environments. This will be the first approach to cataloging the function and coordinate expression of gene sequences, both known and unknown. One of the many key issues in the study of pathogenesis is the validation of animal models of infection as these will provide the basis for further analysis of genes whose functions are as yet unknown. We are therefore particularly interested in comparing the patterns of expression of C. albicans genes in the oral mucosa, with those expressed in the extant rodent models. (3) initiate the functional analysis of individual genes of interest. Comparison of the patterns of gene expression using micro arrays will identify genetic sequences which merit further study. Those of unknown function will be targeted for further genetic analysis, including the construction of specific knockout mutations and the analysis of ensuing phenotype. In this way it will be possible to begin a complete characterization of the range of gene expression which contributes to the pathogenesis of Candida in different host contexts.