CONTROL OF ARG-2 GENE EXPRESSION IN NEUROSPORA

这个项目的长期目标是了解分子 不同调控途径相互作用控制的机制 粗面脉孢菌Arg-2基因的表达该基因编码 精氨酸特异性氨甲酰的线粒体定位小亚基 磷酸合成酶。这项工作的主要目标是定义 调控Arg-2的基因内外元件 按Arg可用性。Arg-2受至少三个不同的 机制：(1)Arg特异性调控负性调控 对Arg的回应。(2)交叉路径控制正向调节 表达对多种氨基酸的限制， 包括Arg。(3)发育调控调控Arg-2在脑内的表达 对尚不确定的发育信号的反应。这其中的每一个 途径影响Arg-2转录本的水平。有证据表明 对于转换组件到特定于Arg的控制，通过 在转录本中有一个24密码子的上游开放阅读框(UORF)。 具体目标如下： 1.转录和翻译部分对 将定义Arg的监管。现有突变对人类免疫系统的影响 Arg-2和调控基因座将被检测。 2.影响基因表达的新突变对Arg Will的响应 并对其进行表征。可以使用以下命令完成选择 含有重组基因的细胞，在重组基因中，大肠杆菌HPH编码 潮霉素磷酸转移酶已融合到Arg-2调控中 序列。这些细胞对抗生素潮霉素具有抗药性。 不含精氨酸的培养基对抗生素敏感 包含Arg. 3.将在体外将突变引入Arg-2调控 序列及其对调控的影响将通过以下方式在体内进行分析 分析报告基因的表达-例如大肠杆菌lacZ- 序列被融合到其中。 4.因子与Arg-2序列的相互作用将是 通过体外DNA和RNA结合研究，表征为 体内研究的补充方法。 了解Arg-2的表达是如何调控的具有实际意义。 作为基本利益，因为N crassa与 致病真菌和用于生产抗生素的真菌。

The long-term goal of this program is to understand the molecular mechanisms by which different regulatory pathways interact to control expression of the Neurospora crassa arg-2 gene. This gene encodes the mitochondrially localized small subunit of arginine-specific carbamoyl phosphate synthetase. The major goal of this work is to define the intragenic and extragenic elements responsible for regulation of Arg-2 by Arg availability. arg-2 is regulated by at least three distinct mechanisms: (1)Arg-specific control negatively regulates expression in response to Arg. (2)Cross-pathway control positively regulates expression in response to limitation for a variety of amino acids, including Arg. (3) Developmental control regulates arg-2 expression in response to as yet unidentified developmental signals. Each of these pathways influences the level of arg-2 transcript. There is evidence for a translational component to Arg-specific control mediated through a 24 codon upstream open reading frame (uORF) in the transcript. Specific aims are as follows: 1. The contributions of transcriptional and translational components to regulation by Arg will be defined. The effects of existing mutations in arg-2 and at regulatory loci will be examined. 2. Novel mutations that affect gene expression in response to Arg will be obtained and characterized. Selection can be accomplished using cells harboring a recombinant gene in which E coli hph, which encodes hygromycin phosphotransferase, has been fused to arg-2 control sequences. These cells are resistant to the antibiotic hygromycin in medium lacking Arg and are sensitive to the antibiotic in medium containing Arg. 3. Mutations will be introduced in vitro into arg-2 regulatory sequences, and their effects on regulation will be analyzed in vivo by analyzing the expression of reporter genes - for example E coli lacZ - to which the sequences are fused. 4. The interactions of factors with arg-2 sequences will be characterized through in vitro DNA and RNA-binding studies as a complementary approach to in vivo studies. An understanding of how arg-2 expression is controlled is of practical as will as fundamental interest because N crassa is related to pathogenic fungi and to fungi used for the production of antibiotics.