TRANSDUCTION OF THE A SIGNAL IN MYXOCOCCUS DEVELOPMENT

粘球菌xanthus使用细胞间的相互作用，以协调 细胞聚集形成有组织的发育程序 多细胞子实体，并从杆状细胞分化 变成卵圆形孢子 这一发展计划是由营养素启动的。 在高细胞密度下的限制。 去除营养物质后，M. xanthus细胞通过以下方式感知它们的营养状态和细胞密度： 监测细胞外A信号浓度的增加，和 随后激活A信号转导通路。 激活 只有当细胞外A信号浓度在 特定中间范围。 使用灵敏的传感机制， 一个信号通路指导M的早期阶段的进展。 xanthus发育 我们的长期目标是确定：1）细胞如何感知 细胞外A信号浓度，2）这些信息是如何 转导，导致基因表达的变化和3）什么是 基因表达的变化与复合物之间的联系 多细胞子实体形成的行为反应。 回答 这些问题，我们将描述的监管网络的A 信号转导途径 该电路的几个组件具有 已被确定，包括细胞外信号，A信号，和 特异性反应，基因表达增加，基因4521。 一 已经确定了映射到sasA（用于 A信号抑制子）基因座。 sasA基因座是 通过早期发展和差异调节 不同A信号依赖基因的表达。 本研究的重点是利用经典和分子遗传学方法， 技术，结合蛋白质生物化学，充分表征 sasA基因座及其编码的调控元件。 分子遗传 方法将被用来分析sasA基因座结构，通过定义 基因座的物理限制，分析转录本，测序 识别开放阅读框架的区域，以及结构， 这是一个无效突变。 研究sasA基因的功能 我们将确定sasA基因产物是否直接或 间接调节基因4521的表达。 第一，顺式作用 将确定控制基因4521表达的调控元件 删除分析。 然后，将使用生物化学技术来测试 纯化的sasA基因产物与基因4521调节区的结合。 将详细表征任何结合活性。 如果sasA基因 产品似乎间接调节基因4521的表达，生化 技术以及基因筛选将用于识别可能的 中间调节元件。 此外，基因筛查将 开发用于识别A信号转导的其他组分 途径，可能包括受体和额外的 转导元件

Myxococcus xanthus uses cell-cell interactions to coordinate a developmental program in which cells aggregate to form organized multicellular fruiting bodies and differentiate from rod-shaped cells into ovoid spores. This developmental program is initiated by nutrient limitation at a high cell density. After the removal of nutrients, M. xanthus cells sense their nutritional status and cell density by monitoring the increase in the extracellular A signal concentration, and subsequently activate the A signal transduction pathway. Activation occurs only when the extracellular A signal concentration is within a specific intermediate range. Using a sensitive sensing mechanism, the A signal pathway directs the progression through the early stages of M. xanthus development. Our long term goals are to determine: 1) how the cells sense the extracellular A signal concentration, 2) how this information is transduced, resulting in a change in gene expression and 3) what is the connection between the change in gene expression and the complex behavioral response of multicellular fruiting body formation. To answer these questions we will characterize the regulatory network of the A signal transduction pathway. Several components of this circuitry have been identified, including the extracellular signal, A signal, and a specific response, an increase in expression of a gene, gene4521. A regulatory element has been identified which maps to the sasA (for suppressor of A signal) locus. The sasA locus is required for the progression through early development and differentially regulates the expression of distinct A signal-dependent genes. The focus of this research is to use classical and molecular genetic techniques, combined with protein biochemistry, to fully characterize the sasA locus and its encoded regulatory element. Molecular genetic approaches will be used to analyze the sasA locus structure by defining the physical limits of the locus, analyzing the transcripts, sequencing the region, identifying the open reading frame(s), and construction and characterizing a null mutation. To study the function of the sasA gene product we will determine if the sasA gene product directly or indirectly regulates gene4521 expression. First, the cis-acting regulatory elements controlling gene4521 expression will be determined by deletion analysis. Then, biochemical techniques will be used to test binding of purified sasA gene product to the gene4521 regulatory region. Any binding activity will be characterized in detail. If the sasA gene product appears to indirectly regulate gene4521 expression, biochemical techniques as well as genetic screens will be used to identify possible intermediate regulatory elements. In addition, genetic screens will be developed to identify other components of the A signal transduction pathway, which will likely include a receptor and additional transduction elements.