Meiotic chromosome synapsis and recombination in yeast

描述（由申请人提供）：减数分裂是一种专门的细胞程序，根据有性生殖的需要，从二倍体亲本细胞产生单倍体雄性和雌性配子。我们的研究解决了该程序的核心独特特征，即母本同源物和父本同源物之间一系列复杂的相互作用，分为三个领域：(I) 重组独立配对。这一过程也发生在某些生物体的有丝分裂细胞中（“体细胞配对”），是主要的染色体行为之一，但尚无任何基本分子机制的线索。我们将应用高通量 3C 分析来识别配对“热点”，并检查特定感兴趣变量在单个定义基因座上配对的作用。我们将通过活细胞中 GFP 标记位点的运动相关分析来进一步分析配对相互作用。如果时间允许，我们将开始研究分离（然后表征）配对分子的可能性。 (II)重组相关事件。我们将继续通过从单个重组“热点”分离和 AFM 可视化重组中间体（特别是双霍利迪连接体）来分析 DNA 水平上重组的基本性质。我们将通过寻找在过度产生时增加或减少该过程的鲁棒性的函数来启动交叉干扰的研究；此外，我们将尝试通过创建沿染色体定义的“环”并评估其影响来区分可能的干扰模型。最后，我们将通过以下方式继续探索轴成分在重组中的作用：“有丝分裂”粘连蛋白 Mcd1/Scc1/Rad21 的研究；通过关联 MNase 研究对蛋白质定位的局部变化进行高分辨率分析；并通过沿粗线期染色体的粘连蛋白和凝缩蛋白定位的高分辨率分析进一步定义染色体组织。 （三）运动与力学。我们将改进分离单个粗线期染色体作为机械研究对象的方法。我们将使用现有试剂并通过寻找“运动突变体”来追求我们最近发现的基于肌动蛋白和端粒的染色体运动。减数分裂同源间相互作用的缺陷是许多类型的遗传性不孕症以及大多数染色体非整倍性的基础，所有这些都会带来重大的社会负担（例如唐氏综合症）。对这些过程的基本了解对于这些病症的诊断和潜在改善至关重要。

DESCRIPTION (provided by applicant): Meiosis is the specialized cellular program that produces haploid male and female gametes from a diploid parental cell as required for sexual reproduction. Our research addresses the central unique feature of this program, a complex series of interactions between maternal and paternal homologs, in three areas: (I) Recombination-independent pairing. This process, which also occurs in mitotically-dividing cells of some organisms ("somatic pairing"), is one of the major chromosomal behaviors for which there is no hint of the fundamental molecular mechanism. We will apply high-throughput 3C analysis to identify pairing "hot spots" and to examine the roles of specific variables of interest to pairing at a single defined locus. We will further analyze pairing interactions by motion correlation analysis of GFP-tagged loci in living cells. As time permits, we will begin to investigate the possibility of isolating (and then characterizing) paired molecules. (II) Recombination-related events. We will continue to analyze the basic nature of recombination at the DNA level by isolation and AFM visualization of recombination intermediates (notably double Holliday junctions) from a single recombination "hot spot". We will initiate a study of crossover interference by searching for functions which, when overproduced, increase or decrease the robustness of this process; also, we will try to distinguish among possible models for interference by creating, and assessing the effects of, a defined "loop" along a chromosome. Finally, we will continue to explore the roles of axis components for recombination by: studies of the "mitotic" cohesin Mcd1/Scc1/Rad21; high resolution analysis of local variations in protein localization via linked MNase studies; and further definition of chromosome organization via high resolution analysis of cohesin and condensin localization along pachytene chromosomes. (III) Motion and mechanics. We will improve our methodology for isolation of individual pachytene chromosomes as objects for mechanical studies. We will pursue our recent discovery of actin- and telomere-based chromosomal motion using existing reagents and by searching for "motion mutants". Defects in meiotic interhomolog interactions underlie many types of hereditary infertility as well as most chromosomal aneuploidies, all of which confer significant societal burdens (e.g. Downs syndrome). Basic understanding of these processes is crucial for diagnosis and potential amelioration of these conditions.