Identifying and characterizing genes required for the meiosis-to-mitosis transition

在受精过程中，精子发出一个信号，指示卵子完成雌性减数分裂，并过渡到有丝分裂细胞分裂，从而形成新的有机体。然而，目前尚不清楚哪些分子成分参与其中，以及它们如何指定正确的细胞分裂。这一建议的重点是我们发现的一种分子途径，这是秀丽隐杆线虫从减数分裂到有丝分裂转变所必需的。我们的方法确定了卵母细胞和精子特异性成分，我们现在准备利用我们的系统进一步了解这一关键途径。我们可以在秀丽隐杆线虫上进行实验，这在人类或其他哺乳动物身上是不可能做到的。受精、减数分裂和第一次有丝分裂都在一小时内发生，所有的过程都可以在子宫内用高分辨率活细胞显微镜观察到。每个阶段的具体参数都可以精确量化。此外，个体基因的活性可以通过突变或RNA干扰（RNAi）有效地改变，以测定发育中的生物体内的影响。******利用NSERC-DG基金，我们发现了3个高度相似的基因，memi-1， memi-2和memi-3，它们编码减数分裂II （MII）所需的卵母细胞因子。MEMI蛋白在减数分裂早期存在，但在MII开始后不久就被降解，并且在有丝分裂胚胎中未被检测到。这三个基因的缺失导致受精卵在减数分裂I期流产并过早进入有丝分裂（即跳过MII期）。这与不能运动的精子不能使卵母细胞受精的情况非常相似。因此，我们假设MEMI“感知”精子的进入。对我们有利的是，我们有一个超形态突变，MEMI -1(sb41)，它导致“过量”的MEMI活性。sb41干扰MEMI-1降解，导致胚胎不能正确地从MII过渡到有丝分裂。利用基于rnai的memi-1抑制因子筛选（sb41），我们发现了一种精子PP1磷酸酶，它是memi的遗传激活因子，也是触发MII的难以捉摸的精子信号的理想候选者。******在最近使用ems诱变技术筛选memi-1（sb41）抑制因子的试验中，我们恢复了相同的PP1磷酸酶，以及许多新基因。为了进一步了解MEMI通路，我们将：*** 1)鉴定和表征新的ems诱导的MEMI -1抑制因子（sb41）。*** 2)鉴定MEMI蛋白的翻译后修饰，这些修饰可能发生在减数分裂到有丝分裂的转变过程中。*** 3)利用接近结扎和酵母2杂交分析MEMI物理相互作用。******虽然对协调受精胚胎复杂细胞分裂特征的机制了解有限，但我们提出的目标有可能揭示核心成分及其分子作用。发现的每一个新基因都将为研究生和本科生的独立研究项目提供理想的训练机会。

During fertilization, sperm deliver a signal that instructs the egg to complete female meiosis and transition to mitotic cell divisions that build the new organism. However, it is unclear which molecular components are involved and how they specify the correct cell divisions. This proposal focuses on a molecular pathway that we discovered, which is required for the meiosis-to-mitosis transition in the nematode C. elegans. Our approach identified oocyte- and sperm-specific components, and we are now poised to exploit our system to further understand this critical pathway. We can perform experiments on C. elegans that would be impossible to do in humans or other mammals. Fertilization, the meiotic divisions, and the first mitotic division all occur within an hour, and all processes can be viewed in utero with high-resolution live-cell microscopy. Specific parameters of each stage can be quantified with precision. Furthermore, the activity of individual genes can be efficiently altered by mutation or RNA interference (RNAi) to assay the effects within a developing organism.******Using NSERC-DG funds, we discovered 3 highly-similar genes, memi-1, memi-2, and memi-3, which encode oocyte factors required for meiosis II (MII). MEMI proteins are present during early meiosis, but they are degraded shortly after MII begins, and are undetected in mitotic embryos. Loss of all three genes results in fertilized embryos that abort meiosis I and enter mitosis prematurely (i.e., they skip MII). This is very similar to what happens when immotile sperm cannot fertilize the oocyte. Thus, we hypothesize that MEMI “senses” sperm entry. To our advantage, we have a hypermorphic mutation, memi-1(sb41), which results in “excess” MEMI activity. sb41 interferes with MEMI-1 degradation, resulting in embryos that fail to transition from MII to mitosis properly. Using an RNAi-based suppressor screen of memi-1(sb41), we discovered a sperm PP1 phosphatase, which is a genetic activator of memi and ideal candidate for the elusive sperm signal that triggers MII.******In a recent pilot screen for memi-1(sb41) suppressors using EMS-mutagenesis, we recovered the same PP1 phosphatase, as well as many new genes. To further understand the MEMI pathway, we will:*** 1) identify and characterize the new EMS-induced suppressors of memi-1(sb41).*** 2) identify post-translational modifications in MEMI proteins that are hypothesized to occur during the meiosis-to-mitosis transition.*** 3) analyze MEMI physical interactions using proximity ligation and yeast 2-hybrid assays.******Although there is limited knowledge of the mechanisms that coordinate the complex cell divisions characteristic of fertilized embryos, our proposed aims have a potential to reveal core components and their molecular roles. Each of the new genes identified will provide ideal training opportunities for independent research projects, at both the graduate and undergraduate level.