GENOMIC APPROACHES TO CANDIDA VIRULENCE

这项建议的长期目标是利用后基因组时代的技术和方法来增加我们对hiv相关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斯坦福DNA测序和技术中心（斯坦福中心）生成的白色念珠菌基因组DNA序列数据制作DNA微阵列。利用这项技术，白色念珠菌基因在各种疾病背景下的表达模式将被评估。这一信息将有助于开发特异性诊断、药物靶点，并深入了解免疫功能低下个体中白色念珠菌的发病机制。本提案的具体目标是：(1)使用基于pcr产物和基于寡核苷酸的技术制造DNA微阵列。全基因组DNA微阵列的制造将在本资助终止前完成。制作将是一个迭代的过程，立即开始构建基于PCR的DNA微阵列，并使用可用的白色念珠菌基因组序列子集。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界的方案。(2)第一代微阵列将用于分析从各种环境中获得的白色念珠菌的基因表达模式。这将是对已知和未知基因序列的功能和协调表达进行编目的第一种方法。在研究发病机制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未知的基因提供基础。因此，我们对比较口腔黏膜中白色念珠菌基因的表达模式与现有啮齿动物模型中的表达模式特别感兴趣。(3)启动感兴趣的单个基因的功能分析。利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成为进一步遗传分析的目标，包括构建特定的敲除突变和分析随后的表型。通过这种方式，将有可能开始一个完整的基因表达范围的特征，这有助于念珠菌在不同宿主环境中的发病机制。

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.