EST ANALYSIS OF A BASIDIOMYCETE CRYPTOCOCCUS NEOFORMANS

担子菌新型隐球菌的EST分析

• 批准号: 6374446
• 负责人: JUNEANN W MURPHY
• 金额: $ 47.33万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6374446
• 关键词: Cryptococcus neoformans; computer assisted sequence analysis; fungal genetics; genetic library; nucleic acid sequence; sequence tagged sites

DESCRIPTION (Adapted from applicants' abstract): Cryptococcus neoformans is a ubiqutious fungal pathogen that causes life-threatening disease in man. Althogh AIDS patients and immunodeficient individuals are the most susceptible, normal individuals acquire this life-threatening disease as well. Without T lymphocyte function, currently available antifungal drugs do not cure the disease. Moreover, multiple drug resistance is appearing on clinical isolates. Even though several virulence factors have been identified, those factors do not account for all aspects fo the pathogenesis. C. neoformans is an encapsulated Basidiomycete, making it unique genetically among human pathogens. Consequently, gene databases for the other fungi do not serve as guides for studying C. neoformans. A thorough understandind of the genome of C. neoformans would be an extremely valuable resource for moving forward research on C. neoformans. The most efficient, econominal approach to generating a useful genetic inventory is through a combination of expressed seuqence tags (ESTs) and genomic sequencing. Although low density seuqencing of the C. neoformans genome is being done, the annotation with be completely dependent on reliable gene-fingidn software. Based on preliminarydata, it is hypothesized that C. neformans intron/exon junctions are sufficiently different from known eukaryotic seuqneces that gene-finding software will have to be trained for C. neoformans before the software will be useful. We proposed to: 1) generate ESTs from a primary JEV21 cDNA library, 2) prepare a JEC21 normalized library, 3) sequence 4000 cDNAs from the normalized library, 4) fully sequence by primer walking PCR products from selected cDNA clones and genomic DNA and identify the intron/exon boundaries, and 5) train gene-finding sofltware to recongize intron/exon junctions of C. neoformans. ESTs will be useful to identify ORFs, new drug targets, new virulence markers, cell surface adherence factors, and metabolic and signaling pathways.

描述（改编自申请人摘要）：新型隐球菌是一种 一种普遍存在的真菌病原体，它会导致危及人类生命的疾病。 艾滋病患者和免疫缺陷者是最易感的， 个体也会获得这种危及生命的疾病。无T淋巴细胞 功能，目前可用的抗真菌药物不能治愈这种疾病。 此外，临床分离株出现多重耐药性。甚至 虽然已经鉴定了几种毒力因子，但这些因子不 解释了发病机制的各个方面。C.新生儿是一种 担子菌，使其在人类病原体中具有独特的遗传特性。 因此，其他真菌的基因数据库不能作为指导， 学习C。新人类对C.新生 将是一个非常宝贵的资源，推动研究C。 新人类最有效，最经济的方法来产生有用的 基因清单是通过表达的序列标签（EST）的组合， 和基因组测序。虽然C.新生 基因组正在做，完全依赖可靠的注释 gene-fingidn软件。根据实验数据，推测C. neformans内含子/外显子连接与已知的 真核生物认为，基因发现软件必须针对C. 新人类之前的软件将是有用的。我们建议：1）产生EST 从初级JEV 21 cDNA文库，2）制备JEC 21标准化文库，3） 从标准化文库中测序4000个cDNA，4）通过引物完全测序 从选择的cDNA克隆和基因组DNA的步移PCR产物，并鉴定 内含子/外显子边界，和5）训练基因发现软件以重新定位 C.内含子/外显子连接新人类EST将有助于鉴定ORF， 新的药物靶点、新的毒力标志物、细胞表面粘附因子，以及 代谢和信号通路。