THE MOLECULAR BIOLOGY OF ORGANELLAR TRANSLOCATION

本项目的长期目标是确定 细胞器在细胞中移动。 重要的是要了解这些 机制，因为这些生理上重要的现象，如轴突 运输、酶和激素分泌是细胞器的形式 易位 结合遗传学、形态学和生物化学方法 将用于研究丝状真菌中的细胞器易位 构巢曲霉，这是唯一适合这样的研究。 突变 已在α-和β-微管蛋白基因以及其他七种基因中分离出来 细胞器转位所必需的基因。 识别 这些基因的产物，基因将通过互补克隆， 测序 抗原将由克隆的基因产生，并将被 用来产生抗体 这些抗体将用于识别和 纯化细胞器易位基因的产物。 新细胞器 易位突变体将被分离为现有细胞器的回复突变体， 易位突变体和随机分离的条件致死 变种人 将对新分离的突变体进行等位性测试， 现有的突变体和彼此，并将映射到基因座。 这些 突变体将在光学显微镜下进行形态学表征 水平，以确定每个突变是否特异性地阻断一种形式的 细胞器易位，如核运动，或者如果它有更多的 对细胞器移位或微管功能的一般影响。 他们 也将通过荧光显微镜检查，以确定它们是否 影响微管或微丝的组装或分布。

The long-term goal of this project is to determine the mechanisms by which organelles move through cells. It is important to understand these mechanisms because such physiologically important phenomena as axonal transport and enzyme and hormonal secretion are forms of organellar translocation. Combined genetic, morphological, and biochemical approaches will be used to study organellar translocation in the filamentous fungus Aspergillus nidulans, which is uniquely suited for such studies. Mutations have been isolated in the Alpha- and Beta-tubulin genes and in seven other genes that are essential to organellar translocation. To identify the products of these genes, the genes will be cloned by complementation and sequenced. Antigens will be produced from the cloned genes and will be used to raise antibodies. These antibodies will be used to identify and purify the products of the organellar translocation genes. New organellar translocation mutants will be isolated as revertants of existing organellar translocation mutants and among randomly isolated conditionally lethal mutants. The newly isolated mutants will be tested for allelism to existing mutants and to each other and will be mapped to locus. These mutants will be characterized morphologically at the light microscopic level to determine if each mutation specifically blocks one form of organellar translocation, such as nuclear movement, or if it has more general effects on organellar translocation or microtubule function. They will also be examined by fluorescence microscopy to determine if they affect microtubule or microfilament assembly or distribution.