GENETICS OF CAENORHABDITIS ELEGANS SPERM MORPHOGENESIS

秀丽隐杆线虫精子形态发生的遗传学

• 批准号: 2180520
• 负责人: STEVEN W. L'HERNAULT
• 金额: $ 16.58万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180520
• 关键词: Caenorhabditis elegans; alleles; cell differentiation; cytogenetics; developmental genetics; electrophysiology; gene expression; gene mutation; genetic mapping; genetic recombination; genetically modified animals; histogenesis; immunocytochemistry; molecular cloning; site directed mutagenesis; spermatogenesis; suppressor mutations; voltage /patch clamp

DESCRIPTION:Dr. Steven L'Hernault requests funding to continue his studies of the genetics of C. elegans morphogenesis, which has been supported by a FIRST Award.The goals of this project are to understand how gene products are localized to specialize morphological structures during cellular differentiation. The development of the C. elegans sperm is being studied. Sperm are not required for viability of the animal, so it has been easy to identify genes that have defective sperm; over 58 genes that affect spermatogenesis have been identified. Homogenous sperm can be obtained for biochemical and cell biological studies. The goals of this project are to understand the targeting of specific proteins to organelles and specific regions of cells, the morphogenesis of those organelles, and the genetic control of organelle structure. The FB-MO complex is segregated exclusively into the spermatid during its formation and excluded from the residual body that is left behind at the end of spermatogenesis. Thus, the FB-MO complex behavior depends on an asymmetric division that gives rise to the spermatids and the residual body. In the spermatids, the membranous body eventually fuses with the plasma membrane. The major sperm proteins are associated with the fibrous body.By contrast, all ribosomes and virtually all of the actin, tubulin, and myosin of the spermatocyte end up in the residual body. Disruption of the FB-MO structure appears to occur in many spermatogenesis defective mutants. Three genes involved in spermatogenesis will be studied. Those known as spe-4 and spe-5 appear to encode integral membrane proteins based on sequence. Immunocytochemistry will be used to determine which sperm membranes contain spe-4. spe-4 antisera will be used to examine other spe mutants to determine which genes regulate spe-4 expression or distribution. Ultrastructural studies of spe-5 mutants will be conducted to examine morphogenesis of the sperm and antisera will be generated to the spe-5 gene product. New alleles of spe-4 and spe-5 will be sought, particularly temperature sensitive or conditional alleles that could be used to dissect morphological events of spermatogenesis. Site directed mutagenesis may be conducted to find new alleles if conventional genetics is unsuccessful. Suppressors of spe-4 mutations will be sought, either by reverting putative temperature sensitive mutations or by reverting missense mutations. Allele-specific suppressors will be studied in most detail. The gene spe-17 encodes a novel hydrophilic protein. Antisera will be used to localize the spe-17 gene product in developing sperm.New spe-17 alleles will also be sought, mainly by site-directed mutagenesis since spe-17 is a small gene. The spe-17 phenotype will be analyzed. In spe-17 mutants, ribosomes segregate to the spermatids in association with the FB-MO (they are excluded in the normal case) and the spermatids are abnormal in their structure. Developing spe-17 spermatids will be treated with puromycin to see whether spermatids are rescued if ribosomes are released from the FB- MO. Double mutants between spe-17 and other sperm morphogenesis mutants will also be made. Patch clamp analysis of ion channels in sperm will be conducted. Ion channels are found preferentially in the residual body and not in the spermatid. Several pharmacological treatments affect the development of sperm. The patch clamp analysis will be used to determine which treatment is most analogous to the normal sperm activation program. In addition, electrophysiological analysis of some mutant sperm will be conducted. Maturation of the spermatid into the spermatozoon, the last step of sperm development, will be examined.

描述：DR。史蒂文·埃尔纳特（Steven L'Hernault）要求资助继续他的 秀丽隐杆线虫形态发生的遗传学的研究， 在第一个奖项的支持下。该项目的目标是 了解基因产品的本地化如何专业化 细胞分化过程中的形态结构。 这 正在研究秀丽隐杆线虫精子的发育。 精子不是 动物生存能力所必需的，因此很容易识别 精子有缺陷的基因；超过58个影响的基因 已经确定了精子发生。 同质精子可以是 用于生化和细胞生物学研究。 该项目的目标是了解特定的目标 细胞器和特定区域的蛋白质， 这些细胞器的形态发生，以及 细胞器结构。 FB-MO综合体仅隔离 在形成过程中进入精子，排除在 残留物体在精子发生结束时留下。 因此，FB-MO复合行为取决于不对称的分裂 这引起了精子和残留体。 在 精子，膜体最终与等离子体融合 膜。 主要的精子蛋白与纤维有关 相反，所有核糖体和几乎所有肌动蛋白， 微型细胞和精子细胞的肌球蛋白最终变为残留体。 FB-MO结构的破坏似乎发生在许多 精子发生有缺陷的突变体。 将研究参与精子发生的三个基因。 那些已知的 由于SPE-4和SPE-5似乎编码基于膜蛋白的整体膜蛋白 顺序。 免疫细胞化学将用于确定哪个 精子膜含有SPE-4。 SPE-4抗血清将用于 检查其他SPE突变体以确定哪些基因调节SPE-4 表达或分布。 SPE-5突变体的超微结构研究 将进行检查精子和抗血清的形态发生 将生成SPE-5基因产物。 SPE-4的新等位基因 将寻求SPE-5，特别是温度敏感或 有条件的等位基因可用于剖析形态事件 精子发生。 可以将定向诱变的位点进行 如果常规遗传学不成功，请找到新等位基因。 将寻求SPE-4突变的抑制器，要么通过恢复 推定的温度敏感突变或通过恢复失误 突变。 高等位基因特异性抑制剂将在大多数 细节。 基因SPE-17编码一种新型的亲水蛋白。 Antisera会 用于将SPE-17基因产物定位在开发精子中。 SPE-17等位基因也将主要通过现场定向 诱变以来，SPE-17是一个小基因。 SPE-17表型将 可以分析。 在SPE-17突变体中，核糖体隔离到 与FB-MO相关的精子（在 正常情况）和精子的结构异常。 开发SPE-17的精子将用呼毒素治疗 如果核糖体从FB-释放 莫。 SPE-17和其他精子形态发生之间的双突变体 也将制造突变体。 将进行精子中离子通道的斑块夹分析。 离子 在残留物体中优先找到通道，而不是在 精子。 几种药理治疗会影响发展 精子。 贴片夹分析将用于确定哪个 治疗与普通精子激活程序最类似。 此外，某些突变精子的电生理分析将 进行。 精子成熟到精子中 将检查精子发育的最后一步。