Cytogenetics of Meiosis of Maize

描述（由申请人提供）：减数分裂对于所有有性生殖生物体都是必不可少的，本文所述的研究将进一步加深我们对玉米和所有生物体中这一过程的理解。减数分裂的机制是一个主要的医学兴趣的主题，因为减数分裂期间不准确的染色体分离（非整倍性）是导致几种先天性畸形的原因，这是人类过早终止妊娠和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，并且有可能进行极好的细胞学研究。进入减数分裂前期是在细胞周期依赖性开关的控制下，无减数分裂1（am 1），其功能是将有丝分裂转换为减数分裂细胞周期所必需的。将从分子、生物化学和细胞学水平研究急性髓细胞白血病的功能。我们将使用表达微阵列来确定是否减数分裂基因的表达改变aml突变等位基因。将在不存在第一次分裂1（afd 1）细胞核的情况下研究染色体结构，其中RAD 51安装非常严重减少。对于各种afdl等位基因，我们将把RAD 51的安装程度与细线期/偶线期染色体重塑的程度相关联。我们将制备抗AFD 1抗体，用于免疫纯化AFD 1相互作用蛋白，并将其定位于减数分裂染色体上。我们建议，RAD 51复合物的同源性搜索和重组所需的。为了分析我们的20个去突触突变体中的潜在重组缺陷，我们将使用针对关键重组途径组分如SPO 11、RAD 51、BLM或MSH 4和MLH 1的抗体，基于蛋白质复合物分布的缺陷对它们在配对/重组途径中的位置进行分类。我们将在不同种类的RAD 51基因座缺陷突变体的成员之间产生双突变体，以分析潜在的上位相互作用。我们将进一步细胞学和分子特征的三个突变体差同源突触1（phs 1），desynapticCS（dsyCS），和隔离II（seglI）是严重缺乏的RAD 51灶，并确定他们是否是缺乏在一个步骤需要加载到染色体上的RAD 51复合物。我们将克隆dsyCS和seglI，使用转座子标记策略，表征其功能，如果时间允许，克隆其他基因的配对/重组途径的后期阶段有缺陷。

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.