Cell Cycle Regulation In C. Elegans

线虫的细胞周期调控

• 批准号: 6540969
• 负责人: ANDY GOLDEN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6540969
• 关键词: Caenorhabditis elegans; antibody; cell cycle; developmental genetics; egg /ovum; embryogenesis; fertilization; gene expression; genetic screening; green fluorescent proteins; meiosis; sperm; stainings; temperature sensitive mutant; tissue /cell culture

We have been studying a class of temperature-sensitive (ts) embryonic lethal mutants from C. elegans that arrest in metaphase of meiosis I. In wildtype animals, oocytes in prophase of meiosis I are fertilized by sperm. Following fertilization, the oocyte chromosomes undergo two meiotic divisions, discarding the extra chromosomes in the polar bodies. These first meiotic divisions are important as any errors in chromosome segregation at this stage can lead to embryos with an abnormal number of chromosomes, which would likely lead to lethality. In our mutants, the oocyte chromosomes arrest in metaphase of meiosis I and never separate their chromosome homologs and never extrude polar bodies. In order to molecularly identify the genes required for the first meiotic division, we have mapped our mutants and sequenced candidate genes. Five of the six genes have now been identified and they encode subunits of the Anaphase Promoting Complex or Cyclosome (APC/C). This complex serves as an E3 ubiquitin ligase that target proteins for destruction (by the 26S proteosome) during the metaphase to anaphase transition of the cell cycle. We have named our mutants "mat" for their defects in the metaphase to anaphase transition during meiosis I. These ts mutants also display defects in spermatocyte meiosis; primary spermatocytes arrest in metaphase of meiosis I with a normal meiotic spindle, yet fail to separate chromosome homologs. Thus, these mutants disrupt meiosis in both oocytes and spermatocytes. To address the role of the mat genes in mitosis, we have performed shift-up experiments during embryogenesis and larval development. Temperature shift-up experiments during embryogenesis do not result in embryonic phenotypes, however, somatic defects in the gonad, vulva, and male tail are apparent in adults. This observation suggests that mitotic divisions in the soma are affected by the mat mutants. For many of the alleles, these shift-up experiments also result in sterility, suggesting mitotic defects in germline proliferation. We are currently constructing double mutants of many of these APC/C mutants and are finding that some combinations are synthetic lethal at the permissive temperature. We are currently using some of the double mutants that are still viable at the permissive temperature to re-examine the role of APC/C during the mitotic cell cycles during embryogenesis and larval development. Specifically, we hope to observe more severe mitotic defects during the development of the germline and various tissues. To further understand the role of these mat genes during development, we are characterizing their expression patterns using antibody staining and GFP transgenic lines. We have also initiated a genetic suppression screen in order to identify regulators or substrates of these APC/C subunits. One such suppressor mutation is dominant. We anticipate finding novel molecules that shed light on how APC/C functions and is regulated in different tissues and at different times during the development of a multicellular organism.

我们一直在研究一类来自秀丽隐杆线虫的温度敏感（ts）胚胎致死突变体，它们在减数分裂 I 中期停滞。在野生型动物中，减数分裂 I 前期的卵母细胞由精子受精。受精后，卵母细胞染色体经历两次减数分裂，丢弃极体中多余的染色体。这些第一次减数分裂很重要，因为此阶段染色体分离的任何错误都可能导致胚胎染色体数量异常，这可能会导致死亡。在我们的突变体中，卵母细胞染色体停滞在减数分裂 I 的中期，永远不会分离它们的染色体同源物，也永远不会挤出极体。为了从分子上鉴定第一次减数分裂所需的基因，我们绘制了突变体图谱并对候选基因进行了测序。六个基因中的五个现已被鉴定，它们编码后期促进复合体或环体（APC/C）的亚基。该复合物充当 E3 泛素连接酶，在细胞周期中期到后期的转变过程中靶向破坏蛋白质（通过 26S 蛋白酶体）。我们将我们的突变体命名为“mat”，因为它们在减数分裂 I 期间的中期到后期转变中存在缺陷。这些 ts 突变体还表现出精母细胞减数分裂的缺陷；初级精母细胞在减数分裂 I 中期停滞，具有正常的减数分裂纺锤体，但无法分离染色体同源物。因此，这些突变体破坏了卵母细胞和精母细胞的减数分裂。为了解决 mat 基因在有丝分裂中的作用，我们在胚胎发生和幼虫发育过程中进行了上移实验。胚胎发生过程中的温度升高实验不会产生胚胎表型，然而，性腺、外阴和雄性尾部的体细胞缺陷在成人中很明显。这一观察结果表明体细胞中的有丝分裂受到 mat 突变体的影响。对于许多等位基因来说，这些上移实验也会导致不育，表明种系增殖中存在有丝分裂缺陷。 我们目前正在构建许多 APC/C 突变体的双突变体，并发现某些组合在允许的温度下具有合成致死性。我们目前正在使用一些在允许的温度下仍然存活的双突变体来重新检查 APC/C 在胚胎发生和幼虫发育期间有丝分裂细胞周期中的作用。具体来说，我们希望在种系和各种组织的发育过程中观察到更严重的有丝分裂缺陷。 为了进一步了解这些 mat 基因在发育过程中的作用，我们使用抗体染色和 GFP 转基因系来表征它们的表达模式。我们还启动了基因抑制筛选，以鉴定这些 APC/C 亚基的调节因子或底物。一种这样的抑制突变是显性的。我们期望找到新的分子，以阐明 APC/C 在多细胞生物体发育过程中不同组织和不同时间的功能和调节方式。