Research Starter Grant: Analysis of ODS-1 in C. elegans Exposed to Anoxia

缺氧影响生物体的生长发育。自然界中的许多生物都受到氧气水平变化的影响，并适应了缺氧的生存。土壤线虫秀丽隐杆线虫（Caenorhabditis elegans）能够在很宽的氧气水平下生存，生物体的发育进程取决于氧气浓度。例如，秀丽隐杆线虫暴露在缺氧环境（0.5%至1.5%氧气）下发育缓慢，而线虫暴露在缺氧环境（0%氧气）下发育和细胞周期进程停滞。这种缺氧引起的假死，在再次接触氧气时是可逆的。除秀丽隐杆线虫以外的生物，如斑马鱼、盐水虾和果蝇，也能通过抑制发育在缺氧中存活下来。我们对缺氧对发育和细胞周期的影响知之甚少。为了充分了解缺氧诱导的假死，有必要了解各种后生动物在不同发育阶段对缺氧的遗传、生理和细胞反应。胚胎暴露在缺氧中会阻碍发育和细胞周期的进展。目前尚不清楚是否存在缺氧诱导的细胞周期阻滞的遗传和细胞基础。利用遗传模型系统（如秀丽隐杆线虫）来研究缺氧诱导的细胞周期阻滞将有助于确定是否存在对缺氧的遗传反应。秀丽隐杆线虫是一个典型的系统，其中经典的正向和反向遗传分析是可能的。线虫胚胎的大尺寸和透明度使其成为观察早期发育过程中细胞周期事件的良好系统。此外，秀丽隐杆线虫的染色体是全新中心的，具有延长染色体长度的着丝点。因此，在后期，染色体作为没有后臂的整个单位移动。此外，秀丽隐杆线虫着丝点的大尺寸（可达4微米）使研究细胞周期事件变得容易。因此，Padilla博士最近发现，线虫胚胎在缺氧条件下阻滞细胞周期进程，使线虫成为了解缺氧生存所需的细胞和遗传反应的有用系统。帕迪拉的长期研究目标是确定秀丽隐杆线虫对严重缺氧的反应机制。这个为期一年的项目的假设是，一个基因（ODS-1）是线虫胚胎纺锤体检查点的一个组成部分，该基因是通过RNA干扰（RNAi）筛选对秀丽隐杆线虫胚胎在缺氧条件下存活所必需的基因。本研究将重点研究ODS-1的特征，包括以下两个目标：目标1。测定胚胎在常氧或缺氧环境下ODS-1蛋白的亚细胞定位。目标2。目的评价ODS-1 （RNAi）胚胎的表型。这项研究将导致对线虫胚胎在缺氧条件下存活的重要基因和基因产物的表征，并将允许测试纺锤体检查点参与缺氧诱导的细胞周期停滞的假设。更广泛的影响：这是一项研究启动基金，授予在符合条件的机构接受终身职位的NSF博士后，如NSF 00-139所述。Padilla博士通过招募本科生和研究生在她的实验室从事研究项目以及在本科课堂教学，积极地将研究和教育结合起来。

Oxygen deprivation influences the growth and development of organisms. Many organisms in nature are subjected to changes in oxygen levels and have adapted to survive oxygen deprivation. The soil nematode Caenorhabditis elegans is capable of surviving a wide range of oxygen levels and the developmental progression of the organism depends on the oxygen concentration. For example, C. elegans exposed to a hypoxic environment (0.5% to 1.5% oxygen) develop slowly, but nematodes exposed to an anoxic environment (0% oxygen) arrest in their developmental and cell cycle progression. This arrest, anoxia-induced suspended animation, is reversible upon re-exposure to oxygen. Organisms other than C. elegans, such as zebrafish, brine shrimp, and fruit flies are also capable of surviving anoxia by arresting development. Little is known about the developmental and cell cycle arrest in response to anoxia. To gain a full understanding of anoxia-induced suspended animation it is necessary to understand the genetic, physiologic, and cellular responses to anoxia in a variety of metazoans at different stages of development.Embryos exposed to anoxia arrest developmental and cell cycle progression. It is not known if there is a genetic and cellular basis for anoxia induced cell cycle arrest. Using a genetic model system such as C. elegans to study anoxia induced cell cycle arrest will help determine if there is a genetic response to anoxia. C. elegans is a model system in which classical forward and reverse genetic analysis is possible. The large size and transparency of nematode embryos make them an excellent system for observing cell cycle events during early development. Additionally, C. elegans chromosomes are holocentric, with kinetochores that extend the length of the chromosomes. Thus, during anaphase chromosomes move as entire units without lagging arms. Furthermore, the large size of C. elegans kinetochores (up to 4 microns) make it easy to study cell cycle events. Thus, the recent finding by Dr. Padilla that the nematode embryo arrests cell cycle progression in anoxia, make the nematode a useful system for understanding the cellular and genetic responses required for oxygen deprivation survival.Dr. Padilla's long-term research goal is to determine the mechanisms employed by C. elegans to respond to severe oxygen deprivation. The hypothesis of this one-year project is that a gene (ODS-1), identified by an RNA interference (RNAi) screen for genes that are essential for C. elegans embryos to survive anoxia, is a component of the spindle checkpoint in the nematode embryo. The research will focus on the characterization of ODS-1, and consists of the following two aims:Aim 1. To determine the subcellular localization of ODS-1 protein in embryos exposed to a normoxic or anoxic environment.Aim 2. To evaluate the phenotype of ODS-1 (RNAi) embryos.This research will result in the characterization of a gene and gene product that appears to be important for nematode embryos to survive anoxia, and will permit the testing of the hypothesis that spindle checkpoints are involved in anoxia-induced cell cycle arrest.Broader impacts: This is a Research Starter Grant, awarded to an NSF Postdoctoral Fellow who has accepted a tenure-track position at an eligible institution, as described in NSF 00-139. Dr. Padilla actively integrates research and education by recruiting undergraduate and graduate students to work on research projects in her lab, as well as by teaching in the undergraduate classroom.