MOLECULAR ANALYSIS OF TRANSCRIPTION IN NEUROSPORA

基因调控是细胞问题的基础 分化和组织形态发生，包括癌症。 粗糙脉孢菌具有发达的遗传系统， 包括DNA转化 这使得广泛的 对几种受调控的N. crassa基因，提供 更复杂的转录调控的详细模型 真核生物系统 最近我开发了一种RNA聚合酶II N. crassa，使生物化学 分析将扩展到真菌转录调控， 第一次 该项目的目的是确定和 在功能上使基本蛋白质和DNA成分 的N. crassa RNA聚合酶II转录装置，在 为了提供一个框架， 遗传特征的真菌调节蛋白调节 转录。 具体而言，该项目的目标是： (i)利用强表达的“管家”基因， （谷氨酸脱氢酶）为模型，N. crassa RNA聚合酶 II转录因子将从现有的粗品中分离出来， 体外培养系统 这些因素将被确定，然后进行分析 在分离过程中通过它们与特定DNA序列的结合和/或 通过它们在转录测定中的需要， 蛋白质级分。 (ii)AM转录所需的启动子DNA序列 将被确定。 这些序列将通过其 与特定转录因子的相互作用， 启动子突变的系统集，其影响将是 使用体内和体外转录测定进行测试。 (iii)N.粗RNA 聚合酶II转录起始复合物，以及 每个步骤所需的量，将由添加顺序决定 实验，并通过使用特定步骤的抑制剂， 转录起始 (iv)开发体外转录系统的可能性 其他真菌如S. cerevisiae，S. pombe和A. nidulans将使用N. crassa转录因子 单独或与来自这些其它蛋白质组合 真菌

Gene regulation is fundamental to the problems of cell differentiation and tissue morphogenesis, including cancer. Neurospora crassa possesses a well-developed genetic system, including DNA transformation. These have enabled extensive genetic analysis of several regulated N. crassa genes, providing detailed models of transcriptional regulation for more complex eukaryotic systems. Recently I developed an RNA polymerase II in vitro transcription system for N. crassa, enabling biochemical analysis to be extended to fungal transcriptional regulation for the first time. The aim of this project is to identify and functionally characterise the basic protein and DNA components of the N. crassa RNA polymerase II transcription apparatus, in order to provide a framework for defining biochemically how genetically characterized fungal regulatory proteins modulate transcription. Specifically, the aims of the project will be: (i) Using the strongly expressed 'housekeeping' gene, am (glutamate dehydrogenase) as a model, N. crassa RNA polymerase II transcription factors will be isolated from the existing crude in vitro system. The factors will be identified and then assayed during isolation by their binding to specific DNA sequences and/or by their requirement in a transcription assay containing separated protein fractions. (ii) The promoter DNA sequences required for am transcription will be determined. These sequences will be identified by their interaction with specific transcription factors, and by creating systematic sets of promoter mutations whose effects will be tested using in vivo and in vitro transcription assays. (iii) The different steps in the formation of an N. crassa RNA polymerase II transcription initiation complex, and the factors required for each step, will be determined by order-of-addition experiments, and by using inhibitors of specific steps in transcription initiation. (iv) The possibility of developing in vitro transcription systems for genes from other fungi such as S. cerevisiae, S. pombe and A. nidulans will be explored using N. crassa transcription factors either alone or in combination with proteins from these other fungi.