GENETIC ANALYSIS OF FLAGELLAR GENE EXPRESSION

本提案中所述工作的长期目标是 阐明了蛋白质的基因的机制， 一个真核细胞器--鞭毛--受到调控。 截肢 衣原体的鞭毛诱导基因表达的巨大变化， 其中至少30个鞭毛蛋白基因被激活。 在10 分钟，一些信号从细胞表面传递到细胞核， 特异性刺激鞭毛基因的转录， 鞭毛蛋白的合成增加。 在拟议的研究中， 在诱导鞭毛基因表达缺陷的突变体后， 爆燃将被分离和表征。 了解 调节正常真核细胞中基因表达的机制将 是理解基因表达改变的先决条件 与癌症等疾病有关。 额外的健康相关性 的工作来自于极端保守的结构， 真核生物纤毛和鞭毛的生物化学。 获得的见解 衣原体参与纤毛合成和组装的调控 诸如动力蛋白的蛋白质应该与人类不动纤毛相关 以及精子尾部缺陷。 工作的具体目标是 如下 首先，大量不能刺激鞭毛的突变体 衣原体中的蛋白质合成将被分离。 初步 研究中，这种突变体已经在细胞中鉴定出来， 脱气后再生全长鞭毛。 综合缺陷 通过体内标记和总细胞的SDS凝胶分析直接测试 蛋白 第二个目标是通过以下方式对这些突变体进行遗传表征： 野生型等位基因的显性测试，以及 其他变种人 所寻求的数据的一个例子是对 可以产生观察到的 表型。 在一个大的互补群中， 这些突变体的样本作为对这些突变体的复杂性的估计。 调控途径 第三个具体目标是生物化学 表征来自每个互补组的突变体。 比如一个 发现了一种不能刺激鞭毛蛋白合成， 去纤维化后鞭毛蛋白的mRNA水平增加 通常，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.