ANALYSIS OF MEIOTIC CHROMOSOME SYNAPSIS IN YEAST

减数分裂是有性生殖生物为了使生殖细胞中的染色体数目减少一半而进行的一个基本过程。当减数分裂失败时，染色体不平衡的配子产生。在哺乳动物中，这种染色体不平衡的配子受精产生的受精卵是不能存活的，导致了大量的自然流产。在产生可存活后代的情况下，观察到21三体或特纳综合征（XO）所见的精神和形态缺陷。了解减数分裂过程是如何准确分离同源染色体的，可能最终提供监测和防止这一过程失败所需的知识。在减数分裂I中同源物的适当分离要求它们首先通过称为突触复合体（SC）的多蛋白结构的形成而形成物理关联。SC是由沿称为轴向元件（ae）的蛋白质核心复制的姐妹染色单体对凝聚形成的，然后通过插入一个中心区域形成突触。酵母的遗传研究表明，ae对于产生和包装交叉非常重要，从而确保正确的分离。酵母ae的三个关键减数分裂特异性成分是HOP1、RED1和MEK1。遗传实验表明，Hop1p/Red1p复合物和Red1p同质寡聚物之间的平衡对AE功能很重要，并且这种化学计量是由Mek1p激酶调节的。这项资助的重点是通过定义HOP1、RED1和MEK1在减数分裂中的特定作用来了解ae在酵母中的功能。为此，开发了一种新的筛选方法来分离RED1中的功能突变体。这种筛选已经成功地发现了RED1的一个等位基因，该等位基因在与Hop1p结合时存在特异性缺陷。该突变体为确定哪些减数分裂过程需要Red1p/Hop1异聚物提供了有用的工具。将在HOP1中寻求类似的功能突变体分离，并进行补充实验。假设Hop1p同源寡聚物在减数分裂双链断裂末端的结合中具有与red1无关的功能，将使用染色质免疫沉淀技术进行测试。为了了解AE组装是如何被调节的，将使用遗传和生化方法来鉴定通过磷酸化保守的苏氨酸来激活Mek1p的激酶。

Meiosis is a fundamental process that sexually reproducing organisms undergo in order to reduce by half the chromosome number in germ cells. When meiosis fails, chromosomally imbalanced gametes result. In mammals the zygotes generated by fertilization of such chromosomally imbalanced gametes are inviable and account for a large number of spontaneous abortions. In cases where viable offspring are produced, mental and morphological defects such as those seen for Trisomy 21 or Turner syndrome (XO) are observed. Understanding how the meiotic process works to accurately segregate homologous chromosomes may ultimately provide the knowledge needed to monitor and prevent failures of this process. Proper segregation of homologs at Meiosis I requires that they first become physically associated by formation of a multi-protein structure called the synaptonemal complex (SC). The SC is formed by condensation of replicated pairs of sister chromatids along protein cores called axial elements (AEs) that are then synapsed by the insertion of a central region. Genetic studies in yeast have demonstrated that AEs are important for generating and packaging crossovers so that they ensure proper disjunction. Three key meiosis-specific components of yeast AEs are HOP1, RED1 and MEK1. Genetic experiments suggest that a balance between Hop1p/Red1p complexes and Red1p homo-oligomers is important for AE function and that this stoichiometry is regulated by the Mek1p kinase. The focus of this grant is to understand how AEs function in yeast by defining the specific roles of HOP1, RED1, and MEK1 during meiosis. Towards this end, a novel screen designed to isolate separation of function mutants in RED1 has been developed. This screen has already been successful in discovering an allele of RED1 that is specifically defective in binding to Hop1p. This mutant provides a useful tool to determine which meiotic processes require Red1p/Hop1 heterooligomers. Similar separation of function mutants will be sought in HOP1 and complementary experiments performed. The hypothesis that Hop1p homo-oligomers have a RED1-independent function in binding near the ends of meiotic double strand breaks will be tested using the chromatin immunoprecipitation technique. To understand how AE assembly is regulated, genetic and biochemical approaches will be used to identify the kinase responsible for activating Mek1p by phosphorylation of a conserved threonine.