MOLECULAR GENETIC ANALYSIS OF ASYMMETRIC CELL DIVISIONS

不对称细胞分裂的分子遗传学分析

基本信息

批准号：
6490243
负责人：
Chris Q Doe
金额：
$ 14.19万
依托单位：
UNIVERSITY OF OREGON
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-01-01 至 2003-12-31
项目状态：
已结题

项目摘要

Asymmetric cell divisions, which produce daughter cells with different fates, are important for generating cell diversity during embryonic development, and may regulate stem cell function in many tissues (e.g. epidermis, intestine, blood, liver, germ cells, and the nervous system). Despite the clinical importance of understanding the regulation of asymmetric cell divisions, remarkably little is known about how and where asymmetric divisions occur in mammals. Our long-term goal is to identify genes controlling asymmetric cell divisions in Drosophila, and to determine if homologous murine genes control asymmetric cell divisions during embryogenesis or in adult stem cell populations. The proposed research may have clinical applications in mammalian stem cell immortalization, gene therapy, and treatment of stem cell neoplasms. We will focus on Drosophila CNS stem cells (neuroblasts), which divide asymmetrically to produce a new neuroblast and a more differentiated daughter cell (GMC). At least 4 proteins are partitioned into the daughter GMC, including the Prospero transcription factor, which is necessary for the transition from neuroblast-specific to GMC-specific gene expression. The specific aims of this proposal are: (1) To identify new genes regulating neuroblast asymmetric cell divisions. We will screen for mis-localization of Prospero protein, and characterize mutants by standard molecular genetic methods. (2) To characterize miranda, a gene encoding a novel coiled-coil protein required for the asymmetric localization of Prospero in neuroblasts. We will sequence protein-positive miranda EMS alleles that affect Miranda localization (3 alleles) or Prospero binding (2 alleles) to map each functional domain; use a yeast two hybrid screen to identify proteins specifically binding the Miranda localization domain; screen for novel Miranda- binding "cargo" proteins; and determine the structure of Miranda functional domains (with Dr. M. Churchill). (3) To further characterize prospero function. We will determine if prospero RNA translational repression in neuroblasts (but not GMCs) is necessary to establish distinct sibling fates; assay the role of phosphorylation in Prospero localization; and assay prospero expression and function in the adult sensory nervous system. (4) To isolate murine homologues of miranda (and genes identified in Specific Aim 1), and to characterize their role in regulating asymmetric divisions during embryogenesis and in adult stem cells. We will do RNA and protein localization studies; our collaborator, Dr. G. Oliver will generate and assay the gene knock-out mice.

不对称细胞分裂，产生具有不同的子细胞命运对于在胚胎期间产生细胞多样性很重要发育，并可能调节许多组织中的干细胞功能（例如表皮，肠，血液，肝脏，生殖细胞和神经系统）。尽管了解调节的临床重要性不对称的细胞分裂，关于如何和在哺乳动物中发生不对称分裂的地方。我们的长期目标是确定控制果蝇中不对称细胞分裂的基因，确定同源鼠基因是否控制不对称细胞胚胎发生或成年干细胞种群中的分裂。这拟议的研究可能在哺乳动物干细胞中应用永生化，基因治疗和干细胞肿瘤的治疗。我们将专注于果蝇中枢神经系统干细胞（神经细胞），该干细胞分裂不对称产生新的神经细胞和更分化的女儿细胞（GMC）。至少有4种蛋白分配到女儿GMC，包括Prospero转录因子，从神经细胞特异性到GMC特异性的过渡所必需的基因表达。该提议的具体目的是：（1）确定调节神经细胞不对称细胞分裂的新基因。我们将筛选出普遍蛋白的错误定位，并表征通过标准分子遗传学方法突变体。（2）表征米兰达（Miranda Prospero在神经细胞中的不对称定位。我们将序列蛋白质阳性的miranda EMS等位基因会影响米兰达定位（3个等位基因）或Prospero绑定（2个等位基因）以绘制每个功能领域;使用酵母两个混合屏幕专门识别蛋白质约束米兰达定位领域；小说米兰达的屏幕结合“货物”蛋白；并确定米兰达的结构功能领域（M. Churchill博士）。（3）进一步表征 Prospero功能。我们将确定Prospero RNA转化是否建立神经细胞中的抑制（但不是GMC）是必要的独特的兄弟姐妹命运；测定磷酸化在Prospero中的作用本土化;并在成年人中测定Prospero表达和功能感觉神经系统。（4）隔离米兰达的鼠类同源物（和特定目标中鉴定出的基因1），并表征其在调节胚胎发生过程中的不对称分裂和成年茎细胞。我们将进行RNA和蛋白质定位研究；我们的合作者G. Oliver博士将生成并分析基因敲除老鼠。