NOVEL CONTROL MECHANISMS IN ENDOSPORE FORMATION

这是一个研究酶活性的生化调节的建议 在代谢适应和分化过程中。 两个一般性问题是 提出：（1）什么生化机制催化和调节选择性 体内酶的失活和降解？ （2）如何 酶的合成受发育调控？ 这些现象发生在 大多数细胞类型，并且知之甚少。 实验系统是 区分细菌枯草芽孢杆菌，其中我们已经表征 详细描述了核苷酸生物合成两种靶酶，天冬氨酸 转氨甲酰酶和谷氨酰胺PRPP酰胺转移酶，其选择性地 在营养缺乏（孢子形成）的细胞中降解。 合成这些 两种酶在降解之前也会突然关闭。 建议继续广泛研究发展规律 这些酶通过追求以下具体目标： 1.从纯化组分中重建体外降解过程 并解释了严格响应对降解的调节作用; 2.分离和表征参与的基因的突变体和克隆， 降解; 3.为了验证酰胺转移酶降解是 在此之前，必需的Fe-S簇的氧化失活; 4.目的：阐明克隆的重组蛋白的结构和体外转录 天冬氨酸转氨甲酰酶基因; 5.为了构建pyrB基因的突变形式并测试它们在细胞中的功能， 体内以测试关于pyrB表达调节的假设;和 如果资源充足 6.为了研究其他基因在发育和营养调控中的作用， 通过克隆、测序和突变体的表达研究pyr簇 克隆

This is a proposal to study the biochemical regulation of enzyme activity during metabolic adaptation and differentiation. Two general questions are posed: (1) what biochemical mechanisms catalyze and regulate the selective inactivation and degradation of enzymes in vivo? and (2) how is the synthesis of enzymes developmentally regulated? These phenomena occur in most cell types and are poorly understood. The experimental system is the differentiating bacterium Bacillus subtilis, in which we have characterized in detail two target enzymes of nucleotide biosynthesis, aspartate transcarbamylase and glutamine PRPP amidotransferase, which are selectively degraded in nutrient-starved (sporulating) cells. The synthesis of these two enzymes is also abruptly shut off prior to their degradation. It is proposed to continue extensive study of the developmental regulation of these enzymes by pursuing the following specific aims: 1. To reconstruct the degradation process in vitro from purified components and to explain the regulation of degradation by the stringent response; 2. To isolate and characterize mutants and clones of genes involved in degradation; 3. To test the hypotheses that degradation of the amidotransferase is preceded by oxidative inactivation of an essential Fe-S cluster; 4. To elucidate the structure and transcription in vitro of the cloned aspartate transcarbamylase (pyrB) gene; 5. To construct mutant forms of the pyrB gene and test their functioning in vivo so as to test hypotheses concerning regulation of pyrB expression; and if resources are adequate 6. To study developmental and nutritional regulation of other genes of the pyr cluster by cloning, sequencing and expression studies with mutant clones.