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.

我们一直在研究来自秀丽隐杆线虫的一类温度敏感（TS）胚胎致死突变体，这些突变体在减数分裂的中期中停滞。在野生型动物中，减数分裂的预言中的卵母细胞被精子施肥。受精后，卵母细胞染色体经历了两种减数分裂师，从而丢弃了极性体内的额外染色体。这些第一个减数分裂分裂很重要，因为此阶段染色体隔离的任何错误都可能导致胚胎异常数量的染色体导致胚胎，这可能会导致致死性。在我们的突变体中，卵母细胞染色体在减数分裂I的中期中停滞，从不将它们的染色体同源物分开，从不挤压极性体。为了分子识别第一个减数分裂分裂所需的基因，我们绘制了突变体和测序的候选基因。现在已经鉴定出六个基因中的五个，它们编码了促进复合物或循环体（APC/C）的后期亚基。该复合物用作靶向蛋白质的E3泛素连接酶，以破坏（由26S蛋白体）在中期为细胞周期的后期转变。我们将突变体命名为“ MAT”，因为它们在减数分裂过程中的中期向后期转变中的缺陷。原发性精子细胞在减数分裂I的比例中停滞，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，这些突变体在卵母细胞和精子细胞中都破坏了减数分裂。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转移实验。胚胎发生过程中的温度转移实验不会导致胚胎表型，但是，成年人显然是性腺，外阴和雄性尾巴的体细胞缺陷。该观察结果表明，躯体中的有丝分裂分裂受MAT突变体的影响。对于许多等位基因，这些转移实验也导致不育，表明种系增殖中有丝分裂缺陷。 我们目前正在构建许多此类APC/C突变体的双重突变体，并发现某些组合在允许温度下是合成的致命。目前，我们正在使用一些在允许温度下仍然可行的双突变体，以重新检查APC/C在胚胎生成和幼虫发育过程中有丝分裂细胞周期期间的作用。具体而言，我们希望在种系和各种组织的发展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基因在发育中的作用，我们正在使用抗体染色和GFP转基因线来表征它们的表达模式。我们还启动了遗传抑制筛查，以识别这些APC/C亚基的调节剂或底物。一种这样的抑制突变是主要的。我们预计找到新的分子，这些分子阐明了APC/C在多细胞生物体开发过程中在不同组织和不同时间的调节。