NOVEL CONTROL MECHANISMS IN ENDOSPORE FORMATION

这是一种研究酶活性的生化调控的建议。 在代谢适应和分化过程中。以下是两个一般性问题 提出：(1)是什么生化机制催化和调节选择性 体内酶的失活和降解？和(2)情况如何？ 发育受控的酶的合成？这些现象发生在 大多数细胞类型，对此知之甚少。该实验系统是 区分细菌枯草芽孢杆菌，我们已经在其中表征了 详细介绍了核苷酸生物合成的两种靶酶--天冬氨酸 转氨甲基酶和谷氨酰胺PRPP氨基转移酶，它们是选择性的 在营养匮乏(产孢子)的细胞中降解。这些化合物的合成 两种酶在降解之前也被突然关闭。 建议继续广泛开展对发育规律的研究 通过追求以下具体目标来获得这些酶： 1.从纯化的成分重建体外降解过程 并用严格的反应来解释降解的调控； 2.分离和鉴定相关基因的突变体和克隆 退化； 3.为了检验氨基转移酶的降解是 在此之前，一个必需的铁-S簇氧化失活； 4.阐明克隆株的结构和体外转录 天冬氨酸氨基转移酶(PyrB)基因； 5.构建突变形式的pyrB基因并检测其功能。 体内实验，以验证有关pyrB表达调控的假说；以及 如果资源充足 6.研究大熊猫其他基因的发育和营养调控 PYR基因的克隆、测序及突变体的表达研究 克隆人。

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.