MEIOTIC CHROMOSOME SYNAPSIS AND RECOMBINATION IN YEAST

这项资助解决了酵母减数分裂过程中同源染色体之间的相互作用。第一部分同源并列。(A)Pre-DSB同源基因配对的机制：我们建议使用我们的Cre-loxP重组实验来鉴定(1)配对-缺陷突变体，(2)使用我们的高分辨率FISH方法来配对“热点”。我们认为，同源连接的工作从一个分子过程顺序地转移到另一个分子过程；这将通过FISH和相关突变体的免疫细胞学进行研究。第二部分重组及其控制。(A)我们将继续对交叉控制进行理论分析，这将分离出该控制“特别稳健”的突变体，并进一步分析我们最近发现的染色单体干扰。(B)我们将进一步分析一种新的重组中间体，单端入侵，关于详细的结构和功能依赖；异源双链DNA形成的时间将进一步研究；我们将利用凝胶和EM方法对来自单个重组热点、野生型和Zip1突变株的双Holliday连接进行详细的分析、纯化和分析。(C)我们将利用我们新发现的依赖于Zip1的、依赖于温度的停滞条件来研究关键的转换点，从而研究联会复合体成分Zip1的作用(S)；我们还将分离温度敏感的Zip1等位基因，并利用它们通过温度漂移实验来确定Zip1的作用时间(S)；还将分离某些特殊类型的Zip1等位基因；以及将对细线体Zip1斑块相对于其他蛋白质的定位进行细胞学评估。(D)我们将通过(1)对相关突变体中重组的遗传和物理分析，以及(2)旨在鉴定和表征含有MLHL的蛋白质/蛋白质和蛋白质/DNA复合体的生化分析，来研究MLHL和可能定义新功能的相关蛋白质的作用。第三部分：染色体/染色质结构。(A)我们将利用我们新开发的方法C-SCAN，该方法在单个实验中确定感兴趣的染色体蛋白质在整个第三染色体长度上的分布。我们将继续分析Red1(及其相关的Hop1)，还将分析Zip1、选定的其他减数分裂特异蛋白质和染色体结构的几个基本成分。(B)我们认为减数分裂前期涉及染色质压缩和解压的多个周期，其中某些周期对重组至关重要。我们将通过遗传学、C扫描等方法进一步研究染色体蛋白Bdf1和组蛋白状态的变化。可以解决线圈的超卷状态。

This grant addresses the interactions between homologous chromosomes during meiosis in yeast. Part I. Homolog Juxtaposition. (A) Mechanism of pre-DSB homolog pairing: We propose to identify (1) pairing-defective mutants using our Cre-loxP recombination assay and (2) pairing "hot spots" using our high resolution FISH method. We think that the job of inter-homolog connection passes sequentially from one molecular process to another; this will be investigated by FISH and immunocytology of relevant mutants. Part II. Recombination and its Control. (A) We will continue theoretical analysis of crossover control, which will isolate mutants in which that control is "extra robust," and further analyze the chromatid interference we recently discovered. (B) We will further analyze a new recombination intermediate, single end invasions, with regard to detailed structured and functional dependencies; the timing of heteroduplex DNA formation will be investigated further; and we will analyze purify and analyze in detail, by gel and EM methods, double Holliday junctions from a single recombination hot spot, from wild type and Zipl mutant strains. (C) We will investigate the role(s) of synaptonemal complex component Zipl by exploiting our newly-discovered Zip1-dependent, temperature-dependent arrest condition to study a critical transition point; we will also isolate thermosensitive Zip1 alleles and use them to define time(s) of Zipl action by temperature shift experiments; certain special types of zipl alleles will also be isolated; and the localization of leptotene Zip1 patches relative to other proteins will be assessed cytologically. (D) We will investigate the roles of Mlhl and related proteins, which likely define new functions, by (1) genetic and physical analysis of recombination in relevant mutants and (2) biochemical analysis aimed at identifying and characterizing Mlhl-containing protein/protein and protein/DNA complexes. Part III. Chromosome/Chromatin Structure. (A) We will exploit our newly developed method, C-SCAN, which determines, in a single experiment, the distribution of a chromosomal protein of interest all along the length of Chromosome III. We will continue analysis of Red1 (and its relative Hop1) and will also analyze Zipl, selected other meiosis-specific proteins and several basic components of chromosome structure. (B) We think that meiotic prophase involve multiple cycles of chromatin compaction and decompaction, certain of which are critical for recombination. We will further investigate chromosomal protein Bdf1 and variations in histone status in this regard by genetic, C-SCAN and other methods. Supercoiling status of loops may be addressed.