Filamentous Fungi Genetic Analysis

本项目的目的是开发和利用模式丝状真菌粗糙脉孢菌的寡核苷酸微阵列技术。这些研究将为丝状真菌的基因表达谱提供坚实的基础，从而促进这一组内以及真菌与其他生物之间的比较研究。脉孢子虫现有丰富的生物学和分子工具基础，加上存档的高质量基因组序列，从而创造了在全球范围内提出基因组调控问题的机会。 本提案中描述的由微阵列技术产生的基因表达数据可以显著地增强对该生物体基因的功能分析。我们的第一个目标是生成包含代表粗糙奈瑟氏菌每个预测基因的70-MET寡核苷酸的幻灯片，并使用这些幻灯片来标准化我们的技术，以开发对微阵列数据的合理统计分析。我们的第二个目标是对暴露在各种生长条件和环境压力下的脉孢子虫测序菌株进行基线转录图谱。我们的第三个目标将使用本提案的项目#1产生的基因敲除菌株来测试来自微阵列实验的假设。预期的基因敲除将包括(1)已在粗糙诺氏杆菌中注释的基因 这些基因包括：(1)酿酒酵母中参与转录调控过程并已被转录分析的基因；(2)被注释为推测的调控因子的基因，这些基因在酿酒酵母中缺失；(3)在酿酒酵母和庞贝酵母中缺失且在粗毛酵母中具有未知生物学功能的基因的同源基因；以及(4)在其他生物中没有同源基因的丝状真菌特异基因。 该项目概述的工作将为在各种条件下生长的野生型脉孢菌提供程序、计算工具和基线转录图谱数据，并将为未来的合作研究铺平道路。除了在其他丝状真菌中促进类似的研究外，这些数据还将加快对真菌的功能分析，包括重要的人类病原体。

The aim of this Project is to develop and utilize oligonucleotide microarray technology for the model filamentous fungus Neurospora crassa. These studies will provide a solid foundation of gene expression profiles for the filamentous fungi that will promote comparative studies within this group and between the fungi and other organisms. The rich foundation of biological and molecular tools available for Neurospora has been complemented with an archival quality genomic sequence, thus creating the opportunity for asking questions of genome regulation on a global scale. Gene expression data generated by microarray technology as described in this proposal can measurably enhance the functional analysis of this organism's genes. Our first objective is to generate slides containing 70-met oligonucleotides representing each of the predicted genes of N. crassa and to use these slides to standardize our techniques to develop sound statistical analysis of microarray data. Our second objective is to carry out base-line transcriptional profiling on the sequenced strain of Neurospora following exposure to a variety of growth conditions and environmental stresses. Our third objective will test hypotheses derived from microarray experiments using knockout strains generated from Project #1 of this proposal. Anticipated knockouts will include (1) genes that have been annotated in N. crassa that are involved in transcriptional regulatory processes in S. cerevisiae and that have been transcriptionally profiled, (2) genes that are annotated as putative regulatory factors that are absent from S. cerevisiae, (3) homologs of genes that are absent from S. cerevisiae and S. pombe and are of unknown biological function in N. crassa, and (4) apparent filamentous fungal-specific genes that do not have homologs in other organisms. The work outlined in this project will provide procedures, computational tools, and baseline transcriptional profiling data for wild type Neurospora grown under a variety of conditions and will pave the way for collaborative studies in the future. In addition to fostering similar studies in other filamentous fungi, these data will expedite the functional analysis of fungi in general including important human pathogens.