Comparative and functional genomics of C. neoformans

新型隐球菌的比较和功能基因组学

• 批准号: 6850675
• 负责人: JAMES W KRONSTAD
• 金额: $ 24.3万
• 依托单位: UNIVERSITY OF BRITISH COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6850675
• 关键词: Cryptococcus neoformans; artificial chromosomes; comparative genomic hybridization; enzyme activity; functional /structural genomics; fungal genetics; fungal proteins; gene mutation; genetic mapping; genetic strain; genetic transcription; laboratory mouse; microarray technology; serial analysis of gene expression; serotyping; temperature sensitive mutant; virulence

DESCRIPTION (provided by applicant): The fungal pathogen Cryptococcus neoformans causes a high incidence of life-threatening infections in AIDS patients and this fungus therefore poses a major threat to the estimated 40 million people worldwide who are infected with HIV. In this context, our long-term objectives are to develop a detailed understanding of virulence in C. neoformans through a combination of genomic and molecular genetic approaches and to contribute the knowledge that we acquire to strategies to combat fungal infections. Our first specific aim is to determine the gene content in the two capsular serotypes of C. neoformans (A and D) that represent the global population of isolates that infect AIDS patients. To achieve this goal, we will perform comparative DNA hybridization experiments with whole genome arrays of bacterial artificial chromosome (BAC) clones. The arrays will be developed from physical maps of BAC clones generated by a fingerprinting method; maps have already been constructed with this method for three isolates of C. neoformans. Comparative hybridization will identify regions of difference between strains and these regions will be sequenced and compared to existing genomic sequence information to develop a view of variability in gene content within and between serotypes. We will also examine the conservation of a gene cluster with a putative role in iron acquisition. The second specific aim is to characterize the transcriptomes of two strains of C. neoformans representing the A and D serotypes during pulmonary infection in a murine model and after phagocytosis by a macrophage-like cell line. We will use serial analysis of gene expression (SAGE) to identify the relative abundance of transcripts on a genomic scale. The new data that we obtain will be compared between the serotypes and compared with SAGE data that we have already collected from C. neoformans cells that are responding to different temperatures (25 degrees C vs. 37 degrees C) and iron levels. The third specific aim is to perform a molecular genetic analysis of the cluster of iron-regulated genes that was identified by our SAGE analysis in a serotype D strain. These genes encode a putative multicopper oxidase (CnFET3) and a putative iron permease (CnFTR1). We will compare the organization and transcriptional regulation of these genes (and a third iron-regulated gene CnURF1) between serotypes. We will also disrupt the genes to examine their role in virulence.

描述（由申请人提供）：真菌病原体新型隐球菌在艾滋病患者中引起危及生命的感染的发病率很高，因此这种真菌对全世界估计有4000万艾滋病毒感染者构成重大威胁。在此背景下，我们的长期目标是详细了解C.通过基因组和分子遗传学方法的结合，我们将获得的知识用于对抗真菌感染的策略。我们的第一个具体目标是确定两种荚膜血清型C.代表感染艾滋病患者的全球分离株群体的新型人（A和D）。为了实现这一目标，我们将用细菌人工染色体（BAC）克隆的全基因组阵列进行比较DNA杂交实验。该阵列将开发从物理地图的BAC克隆产生的指纹方法，地图已经构建了三个分离的C。新人类比较杂交将鉴定菌株之间的差异区域，并对这些区域进行测序，并与现有的基因组序列信息进行比较，以了解血清型内和血清型之间基因含量的变异性。我们还将研究一个基因簇的保守性，该基因簇在铁的获得中具有假定的作用。第二个具体目标是表征两株C.代表A和D血清型的新生儿在鼠模型中肺部感染期间和被巨噬细胞样细胞系吞噬后。我们将使用基因表达系列分析（SAGE）来确定基因组规模上转录本的相对丰度。我们获得的新数据将在血清型之间进行比较，并与我们已经从C.新生儿细胞对不同的温度（25摄氏度对37摄氏度）和铁水平做出反应。第三个具体的目的是进行分子遗传学分析的铁调节基因的集群，确定了我们的SAGE分析在血清型D菌株。这些基因编码推定的多铜氧化酶（CnFET 3）和推定的铁通透酶（CnFTR 1）。我们将比较血清型之间这些基因（和第三个铁调节基因CnURF1）的组织和转录调控。我们还将破坏这些基因，以研究它们在毒力中的作用。