Analysis of Chromosome Condensation and Cohesion in Arabidopsis

为了更好地理解减数分裂过程中发生的分子和生化过程，研究人员对拟南芥的T-DNA标记突触突变体syn1进行了表征。syn1突变破坏了减数分裂I轻体体前的染色体凝聚和配对，导致染色体断裂。拟南芥SYN1蛋白与S. pombe RAD21和RAD21样蛋白相似，它们在有丝分裂过程中是染色体凝聚和姐妹染色单体内聚所必需的。因此，拟南芥SYN1是减数分裂过程中染色体凝聚和姐妹染色单体凝聚所必需的rad21样蛋白。基于与SYN1的相似性，两个额外的拟南芥RAD21同源物SYN2和SYN3最近被分离出来并正在被表征。本文提出的实验旨在进一步表征拟南芥SYN1、SYN2和SYN3，并了解它们如何参与染色体凝聚和姐妹染色单体内聚。研究表明，syn1植物在减数分裂过程中存在染色体凝聚和姐妹染色单体凝聚缺陷；然而，目前尚不清楚SYN1在这些过程中扮演什么角色，也不清楚这两个过程是如何联系在一起的。荧光原位杂交（FISH）实验将用于检测syn1植物减数分裂过程中的染色体配对和姐妹染色单体内聚。此外，将研究SYN1在染色体上的分布，以确定其定位的时间和位置，以及它是否参与沿染色体臂、着丝粒或两者的内聚。最后，SYN1的改变形式将被引入SYN1植物中，以开始识别功能所必需的蛋白质区域。根据它们的序列，SYN2和SYN3很可能在染色体凝聚和/或姐妹染色单体内聚中起重要作用；然而，它们是否在减数分裂、有丝分裂或两者都起作用尚不清楚。此外，rad21样蛋白在有丝分裂和减数分裂中是否以相同的方式起作用尚不清楚。这些问题将通过分离和鉴定SYN2和SYN3缺陷系来解决。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。提出的研究将对染色体凝聚和姐妹染色单体内聚的过程产生新的见解，并更好地理解这两个重要事件是如何联系在一起的。减数分裂是导致单倍体配子产生的一系列高度有序的事件，对真核生物有性生殖至关重要。单轮DNA复制，随后同源染色体配对和重组启动了这一过程，最终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的分离。姐妹染色单体内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，它们保证了细胞分裂过程中遗传信息的适当分离。虽然对有丝分裂过程中染色体凝聚的理解已经取得了相当大的进展，但真核生物染色体动力学中最不为人所知的方面之一可能是姐妹染色单体内聚的机制。

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.