GENETIC ANALYSIS OF FLAGELLAR GENE EXPRESSION

本提案中描述的工作的长期目标是 阐明了蛋白质的基因组成的机制 只有一个真核细胞器--鞭毛--受到调控。截肢 衣藻的鞭毛引起了基因表达的巨大变化， 其中至少有30个鞭毛蛋白基因被激活。10分钟内 几分钟后，一些信号从细胞表面传递到细胞核， 特异性地刺激鞭毛基因的转录，以及 鞭毛蛋白合成增加。在这项拟议的研究中， 在诱导鞭毛基因表达方面存在缺陷的突变体 爆燃将被分离和表征。一种对 调节正常真核细胞中基因表达的机制将 是了解基因表达变化的先决条件 与癌症等疾病相关的。另一个与健康相关的问题 的工作来自于对结构的极端保护 真核生物纤毛和鞭毛的生物化学。在以下方面获得了见解 衣藻在控制纤毛生物合成和组装中的作用 动力蛋白等蛋白质应该与人类静止纤毛有关 紊乱和精子尾部缺陷。这项工作的具体目标如下 下面是。第一，大量不能刺激鞭毛的突变体 衣藻的蛋白质合成将被分离出来。在预赛中 研究发现，这种突变体存在于细胞中，不能 羽化后长出全长鞭毛。综合缺陷 直接通过体内标记和总细胞的SDS凝胶分析进行检测 蛋白。第二个目标是通过以下方式确定这些突变体的基因特征 野生型等位基因的显性检验，以及野生型等位基因的互补检验 其他变种人。所寻求的数据的一个例子是对 可产生观察到的 表型。代表的互补基团的数量很大 这些突变体的样本可以用来估计 调控途径。第三个具体目标是在生物化学上 描述来自每个互补组的突变体的特征。例如，如果一个 发现了不能刺激鞭毛蛋白质合成的突变体，但 羽化后鞭毛蛋白的mRNAs水平升高。 正常情况下，突变体可能存在翻译缺陷。 损伤。

The long term goal of the work described in this proposal is the elucidation of the mechanism by which the genes for the proteins comprising a single eukaryotic organelle--the flagellum--are regulated. Amputating the flagella of Chlamydomonas induces a massive change in gene expression, in which at least 30 genes for flagellar proteins are activated. Within 10 minutes, some signal travels from the cell surface to the nucleus and specifically stimulates transcription from the flagellar genes, and increased synthesis of the flagellar proteins. In the proposed research, mutants defective in the induction of flagellar gene expression after deflagellation will be isolated and characterized. An understanding of mechanisms which regulate gene expression in normal eukaryotic cells will be a prerequisite for understanding alterations in gene expression associated with diseases such as cancer. An additional health relatedness of the work comes from the extreme conservation of the structure and biochemistry of eukaryotic cilia and flagella. Insights gained in Chlamydomonas into the control of biosynthesis and assembly of ciliary proteins such as the dyneins should have relevance to human immotile cilia disorders, and sperm tail defects. The specific aims of the work are as follows. First, a large number of mutants which cannot stimulate flagellar protein synthesis in Chlamydomonas will be isolated. In preliminary studies, such mutants have been identified among cells which cannot regenerate full length flagella after deflagellation. The synthesis defect is tested directly by in vivo labeling and SDS gel analysis of total cell protein. The second aim is to genetically characterize these mutants by dominance tests with wild type alleles, and complementation tests with other mutants. An example of the data sought would be an estimate of the number of mutable regulatory loci which can generate the observed phenotype. The number of complementation groups represented in a large sample of these mutants serves as an estimate of the complexity of the regulatory pathway. The third specific aim is to biochemically characterize mutants from each complementation group. For example, if a mutant is found which cannot stimulate flagellar protein synthesis, but after deflagellation the levels of mRNAs for flagellar proteins increase normally, a possible translational defect would be suggested for the mutant lesion.