EST ANALYSIS OF A BASIDIOMYCETE CRYPTOCOCCUS NEOFORMANS

简介(摘自申请者摘要)：新生隐球菌是一种 在人类中引起危及生命的疾病的普遍存在的真菌病原体。尽管如此 艾滋病患者和免疫缺陷者是最易感染、正常的 个人也会患上这种危及生命的疾病。没有T淋巴细胞 功能，目前可用的抗真菌药物并不能治愈这种疾病。 此外，临床分离株也出现了多重耐药。连 虽然已经确定了几个毒力因子，但这些因子并没有 说明了病机的方方面面。新生革兰氏杆菌是一种囊化的 担子菌，使其在人类病原体中具有独一无二的基因。 因此，其他真菌的基因数据库不能作为指导 研究新生隐球菌属。对新生隐孢子虫基因组的全面了解 将是推进C。 新形态主义者。最有效、最经济的方法来产生有用的 基因盘点是通过表达序列标签(EST)的组合来实现的 和基因组测序。尽管新生葡萄球菌的低密度测序 基因组正在做，完全依靠可靠的注解 Gene-fingidn软件。根据初步数据，推测C. Neformans内含子/外显子连接与已知的完全不同 真核生物需要对基因发现软件进行培训，以便为C. 新式之前的软件将是有用的。我们提出：1)生成EST 从初级JEV21 cDNA文库中，2)制备JEC21归一化文库，3) 从归一化文库中测序4000个cDNA，4)用引物进行全序列测定 从选定的cDNA克隆和基因组DNA中获得步行PCR产物，并鉴定 内含子/外显子边界，以及5)训练基因发现软件以重新匹配 新生葡萄球菌的内含子/外显子连接。EST将有助于识别ORF， 新的药物靶点，新的毒力标记，细胞表面黏附因子，以及 代谢和信号通路。

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.