The Cytogenetics of Meiosis of Maize

描述（由申请人提供）：减数分裂基因功能的失败对人类有深远的影响，导致非整倍体、自然流产、出生缺陷和不育。玉米染色体的极好的细胞学，其发达的遗传和基因组资源，大量的减数分裂突变体收集，以及获得大量高度同步化的性母细胞的能力，使其成为一种整合细胞学，分子，基因组学和蛋白质组学方法来回答减数分裂中的关键问题的优秀生物体。进行减数分裂的能力取决于减数分裂细胞周期的转换，以及独特的细线期染色体结构的形成。我们假设在玉米中，这种转换是由蛋白质AMEIOTIC 1（AM 1）介导的，并且成功转换的指标是细线染色体的形成。其次，细线期染色体结构的变化调节下游减数分裂过程。为了验证这些假设，我们将阐明AM 1功能的机制，使用等位基因系列来定义功能域，蛋白质组学来识别相互作用的蛋白质，反向遗传学来解决相互作用的蛋白质的功能。为了鉴定受AM 1调控的基因，我们将使用来自野生型和ami突变体的减数分裂RNA的微阵列和定量RT-PCR。为了确定细线期染色体如何调节下游过程，我们将使用超高分辨率结构照明（SI）光学显微镜分析细线期染色体的结构，并将结构与功能联系起来。我们将发展一个综合模型的染色粒和轴向元件组织使用SI和阐明的变化，染色体对和突触。我们将在平铺阵列上使用ChIP来识别组蛋白修饰和确定的染色体区域中的RAD 21/REC 8结合位点。基因内重组或染色体结构缺陷的突变体将用于确定染色粒结构中的空间约束如何影响重组。我们将确定的定义功能的染色质重塑发生在细线偶线过渡在野生型，并在am 1-pra 1细胞在这个阶段被捕，并确定是否小RNA代谢是负责这些变化。最后，我们将描述其他三个RAD 21/REC 8 cohesin复合物家族成员在建立细线染色体结构和姐妹染色单体凝聚力的作用。由于减数分裂是一个进化上保守的过程，我们对玉米减数分裂的了解与人类有关。

DESCRIPTION (provided by applicant): Failure of meiotic gene function has profound consequences in humans, leading to aneuploidy, spontaneous abortions, birth defects and infertility. The superb cytology of the maize chromosome, its well developed genetic and genomic resources, the large meiotic mutant collection, and the ability to obtain large amounts of highly synchronized meiocytes make it an excellent organism for integrating cytological, molecular, genomics and proteomic approaches to answer key questions in meiosis. The ability to perform meiosis depends on a switch to the meiotic cell cycle, and the formation of unique leptotene chromosome architecture. We hypothesize that in maize this switch is mediated by the protein AMEIOTIC1 (AM1), and that the indicator of a successful switch is the formation of the leptotene chromosome. Secondly, changes in leptotene chromosome architecture regulate downstream meiotic processes. To test these hypotheses, we will elucidate the mechanism of AM1 function using an allelic series to define functional domains, proteomics to identify interacting proteins, and reverse genetics to resolve the functions of interacting proteins. To identify genes that are regulated by AM1, we will use microarrays and quantitative RT-PCR using meiotic RNA from wild type and ami mutants. To determine how leptotene chromosomes regulate downstream processes, we will analyze the architecture of the leptotene chromosome using ultra high resolution structured illumination (SI) light microscopy and relate structure to function. We will develop an integrated model of chromomere and axial element organization using SI and elucidate changes as chromosomes pair and synapse. We will use ChIP on tiling arrays to identify histone modifications and RAD21/REC8 binding sites in a defined chromosome region. Mutants deficient in intragenic recombination or chromosome architecture will be used to determine how spatial constraints in chromomere architecture affect recombination. We will identify the defining features of the chromatin remodeling that occurs at the leptotene zygotene transition in wild type, and in am1-pra1 cells arrested at this stage, and determine whether small RNA metabolism is responsible for these changes. Finally, we will characterize the role of the three other RAD21/REC8 cohesin complex family members in establishing leptotene chromosome structure and sister chromatid cohesion. Since meiosis is an evolutionarily conserved process what we learn about meiosis in maize is relevant to humans.