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 一种可兴奋的细胞，咽部肌肉细胞。咽是一种管状结构 泵负责将细菌吸入蠕虫，将它们浓缩， 然后把它们磨得粉碎。它由上皮细胞、边缘细胞、 和肌肉细胞。基底膜包裹着整个咽部， 将它与蠕虫的其余部分隔离开来。咽部神经元位于 肌细胞凹陷中的基膜。除了 连接到口腔和肠道，只有两个洞在 基板，一对神经元的突起穿过的基板 连接到咽部神经系统。咽部甚至抽出 当整个咽部神经系统被激光杀死时 显微手术，咽部肌肉运动保持同步。 脊椎动物的心脏，神经系统调节心脏收缩的时间和速度 抽水。乙酰胆碱可能是其中一种神经递质， 由于乙酰胆碱激动剂促进收缩和抑制松弛， 而不能合成乙酰胆碱的突变体只会在 存在乙酰胆碱激动剂。艾弗里博士之前的研究表明 咽肌兴奋性异常的基因可能是 并对其电生理效应进行了研究。咽部 运动速度很快，一些突变的影响也很微妙，所以他 开发了对肌肉运动进行毫秒分析的方法。 有三个假设需要检验。首先，影响 咽部肌肉松弛作用于肌细胞内以控制 膜电流。拟议中的实验将测试Eat-6， Eat-11、Eat-12、EGL-30或EXP-2基因(至少其中两个已知 影响咽肌兴奋性)作用于咽肌，以及 它们的电生理效应是什么。第二个假设是 这些基因中的一些或全部编码信号的组成部分 肌细胞内控制时间的转导通路 放松一下。这将通过确定某些基因是否 编码已知的信号转导蛋白。第三个假设是 通过控制外向电流来调节弛豫时间 这会使膜电位恢复到静止水平。这将是 通过在存在药物和突变的情况下测量电流进行测试 这会影响放松的时机。将测量弛豫电流 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 确定影响咽肌兴奋性的基因是否起作用 在肌肉或神经系统中。第二，两个基因，Eat-6和 EAT-11基因将被克隆，其表达模式将被 确定，突变的等位基因将被测序。第三，空值 这两个基因的表型将被确定。第四，新的突变 增强或抑制现有的咽部兴奋性突变体 表型将被分离。五、咽肌膜 将记录野生型和突变型咽部的潜伏期 细胞内电极或膜片钳技术。 要研究的基因会影响咽部的电特性 以不同的方式锻炼肌肉。例如，Eat-5突变解除了夫妻关系 语料库和终末灯泡运动。EAT-6基因突变抑制咽部 放松，而吃-4会影响放松的时间。Eat-5基因 位于一个3.8kb的基因组片段中，该片段拯救了Eat-5 突变表型。测序正在进行中。Eat-4基因全部下降 在一个已经被线虫基因组测序的区域内 项目。它已经通过RFLP进行了专门的本地化 与Eat-4突变有关，并位于拯救 Eat-4突变表型。GENEFINDER计划已经确定了七个 这个粘粒中的可能基因，其中两个与限制重叠 该片段在EAT-4中发生突变。这两个基因都没有编码一种具有 与已知蛋白质有很大的相似性，进一步的工作旨在 确定哪个基因是Eat-4。