GENETICS OF NEMATODE PHARYNGEAL MUSCLE EXCITABILITY

线虫咽肌兴奋性的遗传学

• 批准号: 2222692
• 负责人: Leon Avery
• 金额: $ 18.85万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-05 至 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2222692
• 关键词: Caenorhabditis elegans; alleles; biological signal transduction; drug design /synthesis /production; electrophysiology; gene expression; gene interaction; gene mutation; lasers; membrane potentials; molecular cloning; muscle cells; muscle relaxation; northern blottings; nucleic acid sequence; oral pharyngeal; phenotype; restriction fragment length polymorphism

DESCRIPTION: The long-term objective is to understand the function of an excitable cell, the pharyngeal muscle cell. The pharynx is a tubular pump responsible for sucking bacteria into the worm, concentrating them, and grinding them up. It consists of epithelial cells, marginal cells, and muscle cells. A basal lamina surrounds the entire pharynx and isolates it from the rest of the worm. Pharyngeal neurons lie under the basal lamina in indentations of the muscle cells. Aside from the connections to the mouth and the intestine, there are only two holes in the basal lamina, through which the processes of a pair of neurons pass to connect to the pharyngeal nervous system. The pharynx pumps even when the entire pharyngeal nervous system has been killed by laser microsurgery, and pharyngeal muscle motions remain synchronized.As in the vertebrate heart, the nervous system regulates the timing and rate of pumping. Acetylcholine is probably one of the neurotransmitters used, since acetylcholine agonists promote contraction and inhibit relaxation, and a mutant unable to synthesize acetylcholine will only pump in the presence of acetylcholine agonists. Dr. Avery's previous work has shown that genes with abnormal pharyngeal muscle excitability can be identified, and their electrophysiological effects studied. Pharyngeal motions are fast and the effects of some mutations are subtle, so he has developed the means for millisecond analysis of muscle motions. There are three hypotheses to be tested. First, genes that affect pharyngeal muscle relaxation act within the muscle cell to control membrane currents. The proposed experiments will test whether the eat-6, eat-11, eat-12, egl-30, or exp-2 genes (at least two of which are known to affect pharyngeal muscle excitability) act on pharyngeal muscle, and what their electrophysiological effects are. The second hypothesis is that some or all of these genes encode components of a signal transduction pathway within the muscle cell that controls the timing of relaxation. This will be tested by determining whether certain genes encode known signal transduction proteins. The third hypothesis is that the timing of relaxation is modulated by controlling the outward current that returns the membrane potential to resting levels. This will be tested by measuring the current in the presence of drugs and mutations that influence relaxation timing. Relaxation current will be measured by extracellular or intracellular recording. There are five specific aims. First, laser microsurgery experiments will determine whether genes that affect pharyngeal muscle excitability act in the muscle or in the nervous system. Second, two genes, eat-6 and eat-11, are to be cloned, their expression patterns are to be determined, and mutant alleles are to be sequenced. Third, the null phenotypes of these two genes will be determined. Fourth, new mutations that enhance or suppress existing pharyngeal excitability mutant phenotypes will be isolated. Fifth, pharyngeal muscle membrane potential will be recorded from wild type and mutant pharynxes using intracellular electrodes or patch-clamp techniques. The genes to be studied affect the electrical properties of pharyngeal muscle in various ways. For example, the eat-5 mutation uncouples corpus and terminal bulb motions. eat-6 mutations inhibit pharyngeal relaxation, and eat-4 affects the timing of relaxation. The eat-5 gene has been located within a 3.8 kb genomic fragment that rescues the eat-5 mutant phenotype. Sequencing is in progress. The eat-4 gene all falls within a region that has already been sequenced by the C. elegans genome project. It has been specifically localized by means of an RFLP associated with an eat-4 mutation, and lies within a cosmid that rescues the eat-4 mutant phenotype. The GENEFINDER program has identified seven possible genes within this cosmid, two of which overlap the restriction fragment mutated in eat-4. Neither gene encodes a protein with significant similarity to known proteins, and further work is aimed at pinning down which gene is eat-4.

描述：长期目标是了解 一个可激发的细胞，咽肌肉细胞。 咽是管状 负责将细菌吸入蠕虫的泵，将其集中到 并把它们磨碎。它由上皮细胞，边缘细胞组成， 和肌肉细胞。 基础薄片围绕整个咽部和 将其与其他蠕虫的其余部分隔离。 咽神经元在 肌肉细胞凹痕中的基底层。 除了 与嘴和肠道的连接，其中只有两个孔 基底层，一对神经元的过程通过 连接到咽神经系统。 咽泵甚至 当整个咽神经系统被激光杀死时 显微外科手术和咽部肌肉运动保持同步。 脊椎动物心脏，神经系统调节 抽水。 乙酰胆碱可能是使用的神经递质之一， 由于乙酰胆碱激动剂会促进收缩和抑制松弛，因此 一个无法合成乙酰胆碱的突变体只会在 乙酰胆碱激动剂的存在。 艾弗里博士以前的工作已经表明 具有异常咽部肌肉兴奋性的基因可能是 鉴定出来，其电生理效应研究了。 咽 动作很快，某些突变的影响是微妙的，所以他有 开发了毫秒分析肌肉运动的手段。 有三个假设要检验。 首先，影响影响的基因 肌肉细胞内的咽肌肉松弛行动 膜电流。 拟议的实验将测试EAT-6是否 EAT-11，EAT-12，EGL-30或EXP-2基因（其中至少两个已知 影响咽肌肉兴奋性）对咽肌的作用，并 他们的电生理作用是什么。 第二个假设是 这些基因中的某些或全部编码信号的组件 控制时间的肌肉细胞内的转导途径 松弛。 这将通过确定某些基因是否来测试 编码已知的信号转导蛋白。第三个假设是 放松的时机通过控制向外电流来调节 这将膜的潜力恢复到静止水平。 这将是 通过在存在药物和突变的情况下测量电流测试 这会影响放松时机。 放松电流将被测量 通过细胞外或细胞内记录。 有五个具体目标。 首先，激光显微外科实验将 确定影响咽肌肉兴奋性法的基因是否 在肌肉或神经系统中。 第二，两个基因，吃6和 EAT-11，要克隆，它们的表达方式应为 确定的和突变等位基因应进行测序。 第三，零 将确定这两个基因的表型。 第四，新突变 增强或抑制现有的咽兴奋性突变体 表型将被隔离。 第五，咽肌膜 使用使用野生型和突变咽的潜力 细胞内电极或斑块钳技术。 要研究的基因会影响咽的电性能 肌肉以各种方式。 例如，Eat-5突变uncouples 语料库和末端灯泡运动。 EAT-6突变抑制咽 放松，Eat-4会影响放松的时机。 EAT-5基因 位于3.8 kb的基因组片段之内，可挽救EAT-5 突变表型。 测序正在进行中。 Eat-4的基因全部瀑布 在已经由秀丽隐杆线虫基因组测序的区域内 项目。它是通过RFLP专门定位的 与EAT-4突变相关，并位于宇宙中拯救 EAT-4突变表型。 GeneFinder计划已经确定了七个 该宇宙中的可能基因，其中两个与限制重叠 碎片在EAT-4中突变。 两者都不编码具有的蛋白质 与已知蛋白质的显着相似性，进一步的工作针对 固定哪个基因是吃4。