MUTATIONAL ANALYSIS OF AMOEBOID CHEMOTAXIS

阿米巴趋化性是一种重要的现象，其中信号 转导和细胞运动相互作用。它可能对运动很关键 癌细胞在转移过程中远离原始细胞，以及 白血球等对感染作出快速反应的能力。 盘基鞭毛虫提供了一个强大的模型系统 用遗传学方法分析变形虫的运动性和趋化性。 他们有一个类似于HB的基因组，所有的表达都需要分析 从基因上讲，而且很容易培养。这项研究的最终目的是 是对变形虫运动和趋化性的剖析，通过分析 突变体的生化和行为特性。 突变将通过化学诱变产生。网柄网柄菌属 将使用已建立的选择小室选择突变体 筛选试验。选定的突变体将在 关于它们的生物化学和行为。化学诱导剂结合， 细胞内信使(如IP3、钙和cGMP)的产生， 并检测肌动蛋白/肌球蛋白与细胞骨架的关系。 将使用计算机控制的图像分析来确定单个细胞 速度、转向、方向和形状对两者的稳定响应 化学吸引剂的梯度和浓度的快速变化。遗传 研究将确定互补小组并确保 生化缺陷与行为改变有关。 突变基因的野生型拷贝将通过互补的方式被克隆 网柄网柄菌基因组DNA文库的选育突变体 变换向量。趋化转化子将在 根据形态或使用选择小室。追回 来自趋化转化子的转化载体将导致 鉴定可以补充突变的野生型序列。 长期目标是确定生化过程 负责特定的行为反应。

Amoeboid chemotaxis is an important phenomenon in which signal transduction and cell motility interact. It may be critical to movement of cancer cells away from the original in metastasis as well as the ability of white blood cells such as to respond rapidly to infection. Dictyostelium discoideum amoebae provide a powerful model system for analyzing amoeboid motility and chemotaxis using a genetic approach. They have a habloid genome which allbws expression of be analyzed genetically, and are easily cultivited. The ultimate goal of this study is a dissection of amoeboid motility and chemotaxis by analyzing the biochemical and behavioral properties of mutants. Mutants will be generated by chemical mutagenesis. Dictyostelium mutants will be selected using an established selection chamber and screening assay. The mutants that are selected will be analyzed in terms of their biochemistry and behavior. Chemoattractant binding, production of intracellular messengers (such as IP3, calcium, and cGMP), and actin/myosin association with the cytoskeleton will assayed. Computer-controlled image analysis will be used to determine single cell speed, turning, orientation and shape in response to both stable chemoattractant gradients and rapid changes in concentration. Genetic studies will determine complementation groups and insure that the biochemical defect is associated with the behavioral alteration. The wild-type copies of the mutant genes will be cloned by complementing selected mutants with a library of genomic DNA in a Dictyostelium transformation vector. Chemotactic transformants will be selected on the basis of morphology or using the selection chamber. Recovery of the transformation vector from chemotactic transformants will lead to the identification of wild-type sequences that can complement the mutation. Tbe long term objective is to identify the biochemical processes responsible for particular behavioral responses.