Cell Cycle Regulation in C elegans

线虫的细胞周期调控

• 批准号: 6420985
• 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/6420985
• 关键词: Caenorhabditis elegans; cell cycle; developmental genetics; embryogenesis; fertilization; meiosis; temperature sensitive mutant

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 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. In collaboration with Dr. Diane Shakes (College of William and Mary, Williamsburg, VA) and Dr. Jill Schumacher (University of Houston), we have determined that 4 of the 6 genes encode subunits of the Anaphase Promoting Complex (APC). This complex serves as an E3 ubiquitin ligase that target proteins for destruction 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 examined germline proliferation and have observed cell cycle delays. Germ cells appear to spend a greater time in M-phase. We believe this delay is due to the cells having difficulty progressing through anaphase, and not due to an absolute arrest. Temperature shift-up experiments during embryogenesis do not result in embryonic phenotypes, however, somatic defects in the gonad, vulva, and male tail are apparent. This observation further 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. To further understand the role of these genes during development, we are characterizing their expression patterns. Genetic suppression screens are also planned in order to identify regulators or substrates of these APC subunits.

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，卵母细胞染色体在减数分裂 I 中期停滞，并且永远不会分离，也不会挤出极体。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。我们与 Diane Shakes 博士（弗吉尼亚州威廉斯堡威廉玛丽学院）和 Jill Schumacher 博士（休斯顿大学）合作，确定了 6 个基因中的 4 个编码后期促进复合体 (APC) 的亚基。该复合物充当 E3 泛素连接酶，在细胞周期的中期到后期转变期间靶向破坏蛋白质。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，我们检查了种系增殖并观察到了细胞周期延迟。生殖细胞似乎在 M 期花费更长的时间。我们认为这种延迟是由于细胞难以通过后期，而不是由于绝对停滞。胚胎发生过程中的温度升高实验不会产生胚胎表型，但性腺、外阴和雄性尾部的体细胞缺陷是明显的。这一观察结果进一步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，这些上移实验也 导致不育。为了进一步了解这些基因在发育过程中的作用，我们正在表征它们的表达模式。还计划进行基因抑制筛选，以确定这些 APC 亚基的调节因子或底物。