Cytogenetics of Meiosis of Maize

描述(申请人提供)：减数分裂对所有有性繁殖生物体都是必不可少的，这里描述的研究将进一步加深我们对这一过程的理解，不仅在玉米中，而且在所有生物体中。减数分裂的机制是一个重要的医学课题，因为减数分裂过程中不准确的染色体分离(非整倍体)是几种先天性畸形的原因，这是提前终止妊娠和人类配子产生不良的主要原因。我们的目标是了解减数分裂过程中染色体分离的机制，特别是同源染色体如何配对和突触。玉米是唯一一种存在大量影响减数分裂的突变体的生物，因此有可能进行极好的细胞学研究。进入减数分裂前期受控于细胞周期依赖的开关，无融合生殖1(AM1)，它的功能是将有丝分裂细胞周期转换为减数分裂细胞周期。AML的功能将在分子、生化和细胞学水平上进行研究。我们将使用表达微阵列来确定AML突变等位基因中减数分裂基因的表达是否发生了变化。染色体结构将在没有第一分裂1(Afd1)核的情况下进行研究，在那里RAD51安装非常严重地减少。对于不同的AFDL等位基因，我们将RAD51的安装程度与细线/合线染色体重塑的程度相关联。我们将制备抗AFD1抗体，以免疫纯化AFD1相互作用蛋白，并对FD1在减数分裂染色体上进行定位。我们认为RAD51复合体是进行同源搜索和重组所必需的。为了分析我们的20个去突触突变体中潜在的重组缺陷，我们将使用针对关键重组途径组件的抗体，如SPO 11、RAD51、BLM或MSH4和MLH1，根据蛋白质复合体分布的缺陷来分类它们在配对/重组途径中的位置。我们将在不同类别的RAD51基因座缺陷突变体的成员之间进行双突变，以分析潜在的上位性相互作用。我们将进一步从细胞学和分子学的角度对RAD51位点严重缺陷的三个突变体进行鉴定，包括同源突触1(Phs1)、结突触CS(DsyCS)和分离II(SeglI)，并确定它们是否缺乏将RAD51复合体加载到染色体上所需的步骤。我们将使用转座子标签策略克隆dsyCS和seglI，鉴定它们的功能，并在时间允许的情况下，克隆在配对/重组途径的后期阶段有缺陷的其他基因。

DESCRIPTION (provided by applicant): Meiosis is essential for all sexually reproducing organisms and the studies described here will further our understanding of this process not only in maize but in all organisms. The mechanism of meiosis is a topic of major medical interest since inaccurate chromosome segregation (aneuploidy) during meiosis is causal in several congenital malformations, a major cause of premature termination of pregnancy, and of poor gamete production in humans. Our goal is to understand the mechanism of chromosome segregation during meiosis, particularly how homologous chromosomes pair and synapse. Maize is the only organism where there is a large collection of mutants that affect meiosis, and it is possible to do superb cytology. Entrance into meiotic prophase is under control of a cell cycle dependent switch, ameiotic 1 (am1) whose function is required to convert a mitotic to a meiotic cell cycle. The function of aml will be studied at a molecular, biochemical and cytological level. We will use expression microarrays to determine whether meiotic gene expression is altered in aml mutant alleles. Chromosome structure will be studied in absence of first division 1 (afd1) nuclei where RAD51 installation is very severely reduced. For various afdl alleles, we will correlate extent of RAD51 installation with extent of leptotene/zygotene chromosome remodeling. We will make anti-AFD1 antibody to immuno-purify AFD1 interacting proteins, and for localization of FD1 on meiotic chromosomes. We propose that RAD51 complexes are required for both the homology search and recombination. To analyze potential recombination defects in our 20-desynaptic mutants, we will use antibodies against key recombination pathway components such as SPO 11, RAD51, BLM or MSH4 and MLH1 to classify their position in the pairing/recombination pathway based on deficiencies in protein complex distribution. We will make double mutants between members of various classes of RAD51 loci deficient mutants to analyze potential epistatic interactions. We will further cytologically and molecularly characterize the three mutants poor homologous synapsis1 (phs1), desynapticCS (dsyCS), and segregation II (seglI) that are severely deficient in RAD51 foci and determine whether they are deficient in a step required to load RAD51 complexes onto chromosomes. We will clone dsyCS and seglI, using transposon-tagging strategies, characterize their function, and if time permits, clone other genes defective in later stages of the pairing/recombination pathway.