GENETIC ANALYSIS OF FLAGELLAR GENE EXPRESSION

鞭毛基因表达的遗传分析

• 批准号: 3285411
• 负责人: PAUL A. LEFEBVRE
• 金额: $ 10.9万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1987-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3285411
• 关键词: Chlorophyta; affinity chromatography; alleles; autoradiography; autosomal dominant trait; autosomal recessive trait; cytogenetics; eukaryote; flagellum; gel electrophoresis; gene complementation; gene expression; gene mutation; genetic manipulation; genetic regulation; mutagens; radiotracer; regulatory gene

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.

本提案中描述的工作的长期目标是 阐明包含蛋白质的基因的机制 单一的真核细胞器——鞭毛——受到调节。 截肢 衣藻的鞭毛诱导基因表达发生巨大变化， 其中至少 30 个鞭毛蛋白基因被激活。 10以内 分钟后，一些信号从细胞表面传播到细胞核， 特异性刺激鞭毛基因的转录，并且 鞭毛蛋白的合成增加。 在拟议的研究中， 突变体在诱导鞭毛基因表达方面存在缺陷 将分离并表征爆鞭。 的理解 正常真核细胞中调节基因表达的机制 是理解基因表达变化的先决条件 与癌症等疾病有关。 额外的健康相关性 作品的精髓来自于对结构的极端保护和 真核纤毛和鞭毛的生物化学。 获得的见解 衣藻控制纤毛的生物合成和组装 诸如动​​力蛋白之类的蛋白质应该与人类不动的纤毛有关 疾病和精子尾部缺陷。 工作的具体目标是 接下来。 首先，大量不能刺激鞭毛的突变体 将分离衣藻中的蛋白质合成。 在初步 研究表明，这种突变体已在不能 去鞭毛后再生全长鞭毛。 合成缺陷 通过总细胞的体内标记和 SDS 凝胶分析直接进行测试 蛋白质。 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和互补测试 其他突变体。 所寻求数据的一个例子是对 可以产生观察到的可变调控位点的数量 表型。 互补组的数量代表在一个大的 这些突变体的样本可以作为对复杂性的估计 监管途径。 第三个具体目标是生化 表征每个互补组的突变体。 例如，如果一个 发现不能刺激鞭毛蛋白合成的突变体，但是 去鞭毛后，鞭毛蛋白的 mRNA 水平增加 通常，突变体可能存在翻译缺陷 病变。