Analysis of Chromosome Condensation and Cohesion in Arabidopsis

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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.