GENETICS OF NEMATODE PHARYNGEAL MUSCLE EXCITABILITY

线虫咽肌兴奋性的遗传学

• 批准号: 2378765
• 负责人: Leon Avery
• 金额: $ 24.89万
• 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/2378765
• 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，咽肌细胞。 咽是管状的 泵负责将细菌吸入蠕虫体内并浓缩它们， 并将它们磨碎。它由上皮细胞、边缘细胞、 和肌肉细胞。 基底层包围整个咽部， 将其与蠕虫的其余部分隔离。 咽神经元位于 肌细胞凹陷处的基底层。 除了 连接口腔和肠道的地方只有两个孔 基底层，一对神经元的突起通过基底层 连接到咽神经系统。 咽部泵动均匀 当整个咽部神经系统被激光杀死时 显微手术和咽部肌肉运动保持同步。 脊椎动物的心脏，神经系统调节心脏的时间和速率 泵送。 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促进收缩并抑制松弛， 不能合成乙酰胆碱的突变体只能泵入 乙酰胆碱激动剂的存在。 艾弗里博士之前的工作表明 咽肌兴奋性异常的基因可能是 确定了它们，并研究了它们的电生理效应。 咽部 运动很快，一些突变的影响很微妙，所以他有 开发了肌肉运动的毫秒分析方法。 共有三个假设需要检验。 首先，影响基因 咽部肌肉松弛作用在肌细胞内进行控制 膜电流。 拟议的实验将测试 eat-6、 eat-11、eat-12、egl-30 或 exp-2 基因（其中至少两个是已知的） 影响咽部肌肉的兴奋性）作用于咽部肌肉，并且 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 肌肉细胞内控制时间的转导途径 松弛。 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 使膜电位恢复到静息水平。 这将是 通过测量药物和突变存在下的电流进行测试 影响放松时间。 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，将被克隆，其表达模式将被 确定，并对突变等位基因进行测序。 三、零 这两个基因的表型将被确定。 四、新突变 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 将从野生型和突变体咽部记录电位 细胞内电极或膜片钳技术。 待研究的基因影响咽部的电特性 肌肉以各种方式。 例如，eat-5 突变使 语料库和末端灯泡运动。 eat-6突变抑制咽 放松，而 eat-4 影响放松的时间。 eat-5基因 已位于拯救 eat-5 的 3.8 kb 基因组片段内 突变表型。 测序正在进行中。 eat-4基因全部掉落 位于已被秀丽隐杆线虫基因组测序的区域内 项目。它已通过 RFLP 进行了专门本地化 与 eat-4 突变相关，位于拯救的粘粒内 eat-4 突变体表型。 GENEFINDER 计划已确定七项 该粘粒内可能存在的基因，其中两个与限制重叠 eat-4 中的片段发生突变。 这两个基因都不编码具有 与已知蛋白质显着相似，进一步的工作旨在 确定哪个基因是 eat-4。