Analysis of Chromosome Condensation and Cohesion in Arabidopsis

作为研究的一部分，以更好地了解减数分裂过程中发生的分子和生物化学过程，T-DNA标记的，突触突变的拟南芥，syn 1，已被确定。syn 1突变破坏了减数分裂I的细丝体之前的染色体凝聚和配对，并导致染色体片段化。拟南芥SYN 1已被分离并发现表现出与S.粟酒酵母RAD 21和RAD 21样蛋白，其是有丝分裂期间染色体凝聚和姐妹染色单体凝聚所需的。因此，拟南芥SYN 1是一个类似RAD 21的蛋白质，在减数分裂过程中染色体凝聚和姐妹染色单体凝聚所需。基于与SYN 1的相似性，最近分离出另外两个拟南芥RAD 21同源物SYN 2和SYN 3，并正在对其进行表征。本实验旨在进一步研究拟南芥SYN 1、SYN 2和SYN 3在染色体凝聚和姐妹染色单体凝聚中的作用。已经证明，SYN 1植物在减数分裂过程中染色体凝聚和姐妹染色单体凝聚方面存在缺陷;然而，目前尚不清楚SYN 1在这些过程中扮演什么角色，以及这两个过程是如何联系起来的。荧光原位杂交（FISH）实验将进行检查染色体配对和姐妹染色单体的凝聚力在syn 1植物减数分裂。此外，将研究SYN 1在染色体上的分布，以确定其定位的时间和位置，以及其是否参与沿着染色体臂、着丝粒或两者的内聚力。最后，将SYN 1的改变形式引入syn 1植物中，开始鉴定蛋白质的功能所必需的区域。基于它们的序列，SYN 2和SYN 3很可能在染色体凝聚和/或姐妹染色单体凝聚中起重要作用;然而，尚不清楚它们是否在减数分裂、有丝分裂或两者中起作用。此外，目前还不清楚是否RAD 21样蛋白在有丝分裂和减数分裂中以相同的方式发挥作用。这些问题将通过隔离和表征SYN 2和SYN 3中有缺陷的线路来解决。将进行表达研究，以检查基因的组织特异性和表达时间，以及免疫定位实验，以检查蛋白质在染色体上的分布。拟议的研究将产生新的见解染色体凝聚和姐妹染色单体凝聚的过程，并更好地了解这两个重要的事件是如何联系在一起的。减数分裂是真核生物有性生殖的关键过程，是一系列高度有序的事件，最终导致单倍体配子的产生。单轮DNA复制，随后同源染色体配对和重组启动该过程，其在两个分裂阶段达到高潮，第一个是分离同源染色体的还原分裂，第二个是姐妹染色单体分离。姐妹染色单体凝聚和染色体凝聚的建立是减数分裂和有丝分裂过程中的中心事件，它们确保了细胞分裂过程中遗传信息的正确分离。虽然已经取得了相当大的进展，对有丝分裂过程中染色体凝聚的理解，也许真核细胞染色体动力学的最不了解的方面之一是姐妹染色单体凝聚的机制。

As part of studies to better understand the molecular and biochemical processes that occur during meiosis, a T-DNA tagged, synaptic mutant of Arabidopsis, syn1, has been characterized. The syn1 mutation disrupts chromosome condensation and pairing prior to leptonema of meiosis I and results in fragmentation of the chromosomes. Arabidopsis SYN1 has been isolated and found to exhibit similarity to S. pombe RAD21 and RAD21-like proteins, which are required for chromosome condensation and sister chromatid cohesion during mitosis. Therefore, Arabidopsis SYN1 is a RAD21-like protein that is required for chromosome condensation and sister chromatid cohesion during meiosis. Based on similarity to SYN1, two additional Arabidopsis RAD21 homologues, SYN2 and SYN3 have recently been isolated and are being characterized. Experiments proposed here are designed to further characterize Arabidopsis SYN1, SYN2 and SYN3 and understand how they participate in chromosome condensation and sister chromatid cohesion. It has been demonstrated that syn1 plants are defective in chromosome condensation and sister-chromatid cohesion during meiosis; however it is not clear what role SYN1 plays in these processes or how the two processes are linked. Fluorescence in situ hybridization (FISH) experiments will be conducted to examine chromosome pairing and sister-chromatid cohesion during meiosis in syn1 plants. In addition, the distribution of SYN1 on chromosomes will be investigated to determine when and where it is localized and if it is involved in cohesion along the chromosome arms, the centromeres or both. Finally altered forms of SYN1 will be introduced into syn1 plants to begin to identify regions of the protein that are necessary for function . Based on their sequence, it is very likely that SYN2 and SYN3 play important roles in chromosome condensation and/or sister chromatid cohesion; however it is not clear if they function during meiosis, mitosis or both. Also, it is not clear if RAD21-like proteins function in a same way during mitosis and meiosis. These questions will be addressed through the isolation and characterization of lines defective in SYN2 and SYN3. Expression studies to examine the tissue-specificity and timing of expression of the genes as well as immunolocalization experiments to examine the distribution of the proteins on chromosomes, will be conducted. The proposed studies will yield new insights into the processes of chromosome condensation and sister chromatid cohesion and a better understanding of how these two important events are linked. Meiosis, a highly ordered series of events that results in the production of haploid gametes, is essential for eukaryotic sexual reproduction. A single round of DNA replication, followed by homologous chromosomes pairing and recombination initiates the process, which culminates in two division phases, the first a reductive division that segregates homologous chromosomes and the second in which sister chromatids are separated. The establishment of sister chromatid cohesion and chromosome condensation are central events during both meiosis, and mitosis, which ensure the proper segregation of genetic information during cellular division. While considerable progress has been made towards understanding chromosome condensation during mitosis, perhaps one of the least understood aspects of eukaryotic chromosome dynamics is the mechanism of sister-chromatid cohesion.