MOLECULAR ANALYSIS OF TRANSCRIPTION IN NEUROSPORA

基因调控是细胞问题的根本 分化和组织形态发生，包括癌症。 粗糙脉孢菌拥有发达的遗传系统， 包括DNA转化。这些都使广泛的 对几个受调控的粗糙奈瑟氏菌基因的遗传分析，提供了 更复杂的转录调控的详细模型 真核系统。最近，我开发了一种RNA聚合酶II 粗枝杆菌的体外转录系统，使其能够进行生化 分析将扩展到真菌转录调控 第一次。该项目的目标是确定和 基本蛋白质和DNA组分的功能特征 在粗面核糖核酸聚合酶II转录装置中 以便提供一个框架，用于定义生物化学如何 具有遗传特征的真菌调节蛋白调节 抄写。 具体而言，该项目的目标将是： (I)使用强烈表达的‘家务’基因， (谷氨酸脱氢酶)为模型，粗糙核糖核酸聚合酶 II转录因子将从现有的原油中分离出来 体外系统。将确定这些因素，然后进行分析 在分离过程中通过与特定DNA序列和/或 根据他们在转录测试中的要求，包含分离的 蛋白质组分。 (Ii)AM转录所需的启动子DNA序列 将会被确定。这些序列将通过它们的 与特定转录因子的相互作用，并通过创建 启动子突变的系统性集合，其影响将是 使用体内和体外转录试验进行测试。 (3)粗面核糖核酸形成的不同步骤 聚合酶II转录起始复合体及其影响因素 每一步所需的，将由添加顺序确定 实验，并通过使用特定步骤的抑制剂 转录启动。 (4)开发体外转录系统的可能性 其他真菌的基因，如酿酒酵母、金黄色葡萄球菌和金黄色葡萄球菌。 将使用粗毛拟青霉转录因子来研究尼杜兰。 单独或与来自这些另一个的蛋白质组合 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.