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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，产生具有不同特征的子细胞命运对于胚胎时期细胞多样性的产生非常重要发育，并可能调节许多组织中的干细胞功能（例如，表皮、肠道、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，人们对如何以及如何分裂知之甚少哺乳动物中发生不对称分裂。我们的长期目标是鉴定果蝇中控制不对称细胞分裂的基因，以及确定同源鼠基因是否控制不对称细胞胚胎发生期间或成体干细胞群中的分裂。这拟议的研究可能在哺乳动物干细胞中具有临床应用永生化、基因治疗和干细胞肿瘤的治疗。我们将重点关注果蝇中枢神经系统干细胞（神经母细胞），它们分裂不对称地产生新的神经母细胞和更加分化的神经母细胞子细胞（GMC）。至少有 4 个蛋白质被分成子代 GMC，包括 Prospero 转录因子，从神经母细胞特异性向 GMC 特异性转变所必需的基因表达。本提案的具体目标是： (1) 识别调节神经母细胞不对称细胞分裂的新基因。我们将筛选 Prospero 蛋白的错误定位，并表征通过标准分子遗传学方法产生突变体。 (2) 表征 miranda，一种编码一种新型卷曲螺旋蛋白的基因，该蛋白是 Prospero 在神经母细胞中的不对称定位。我们将排序影响米兰达定位的蛋白质阳性米兰达 EMS 等位基因（3 个等位基因）或 Prospero 结合（2 个等位基因）来映射每个功能领域;使用酵母两种杂交筛选来特异性鉴定蛋白质绑定 Miranda 本地化域；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域（与丘吉尔博士）。 (3) 进一步表征繁荣功能。我们将确定 prospero RNA 是否翻译神经母细胞（但不是 GMC）的抑制对于建立不同的兄弟姐妹命运；测定 Prospero 中磷酸化的作用本土化;并测定 Prospero 在成人中的表达和功能感觉神经系统。 (4) 分离米兰达的鼠同源物（和具体目标 1) 中确定的基因，并表征它们在调节胚胎发生和成体茎中的不对称分裂细胞。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。