NOVEL CONTROL MECHANISMS IN ENDOSPORE FORMATION

这是研究酶活性的生化调节的提案 在代谢适应和分化过程中。 两个一般问题是 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) 情况如何 酶的合成受发育调节吗？ 这些现象发生在 大多数细胞类型，但人们对其知之甚少。 实验系统是 区分细菌枯草芽孢杆菌，我们已对其进行了表征 详细介绍了核苷酸生物合成的两种靶酶，天冬氨酸 转氨甲酰酶和谷氨酰胺 PRPP 酰胺转移酶，它们选择性地 在营养匮乏（孢子形成）的细胞中降解。 这些的合成 两种酶在降解之前也会突然关闭。 建议继续深入研究发育规律 通过追求以下特定目标来利用这些酶： 1. 重建纯化组分的体外降解过程 并解释严格响应对降解的调节； 2. 分离和鉴定相关基因的突变体和克隆 退化； 3. 检验酰胺转移酶降解的假设 随后是重要的 Fe-S 簇的氧化失活； 4. 阐明克隆的体外结构和转录 天冬氨酸转氨甲酰酶（pyrB）基因； 5. 构建pyrB基因的突变形式并测试其功能 体内以测试有关pyrB表达调节的假设；和 如果资源充足 6. 研究其他基因的发育和营养调控 通过突变体的克隆、测序和表达研究来构建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.