MUTATIONAL ANALYSIS OF AMOEBOID CHEMOTAXIS

变形虫趋化性是一种重要的现象， 转导和细胞运动相互作用。 它可能对运动至关重要 癌细胞在转移中远离原始细胞， 白色血细胞对感染迅速反应的能力。 盘基网骨藻为植物的生长提供了一个强有力的模式系统， 用遗传学方法分析变形虫运动和趋化性。 他们有一个habloid基因组，可以分析其表达 基因上，很容易培养。 这项研究的最终目的是 是一个解剖变形虫运动和趋化性，通过分析 突变体的生化和行为特性。 将通过化学诱变产生突变体。 Dictyostelium 将使用已建立的选择室选择突变体， 筛选试验 所选的突变体将在 它们的生物化学和行为。 化学引诱剂结合， 产生细胞内信使（如IP 3、钙和cGMP）， 并测定肌动蛋白/肌球蛋白与细胞骨架的结合。 计算机控制的图像分析将用于确定单个细胞 速度，转向，方向和形状，以响应两者的稳定 化学引诱物梯度和浓度的快速变化。 遗传 研究将确定互补组，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 用网骨藻基因组DNA文库筛选突变体， 变换向量 趋化性转化体将在 形态学的基础或使用选择室。 复苏 来自趋化转化体的转化载体将导致 鉴定可以补充突变的野生型序列。 长期目标是确定生物化学过程 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.