The long term goal of this research is to explain how telomeres perform their various roles in cells. At present we know that telomeres are essential for proper cell growth and development. However, we only have limited information about telomere structure and we do not understand the relationship between their structure and their function. Hypotrichous ciliates (Euplotes and Oxytricha) will be used as the experimental system because these organisms have sufficient telomeric material to perform structural studies; their macronuclei contain literally millions of telomeres. Three complimentary approaches will be used to examine the importance of specific features of telomere structure during certain cellular events. Firstly, the structure of the telomeric complex from Euplotes crassus will be studied. The DNA-protein interactions will be analyzed by chemical and nuclease footprinting. The protein components of the complex will be identified and their DNA-binding properties studied. The structural features of the Euplotes and the Oxytricha nova telomeric complexes will be compared so that conserved features of possible functional importance can be identified. Secondly, de novo telomere formation will be studied during macronuclear development. As Euplotes crassus will mate synchronously, one can obtain large numbers of macronuclei at specific stages in development. By studying telomere structure during the various stages in development, it should be possible to identify the steps that lead to formation of the mature telomeric complex. These studies should clarify which structural features are required in certain cellular events, and should elucidate how the various components of a mature telomere interact with each other to form a functional structure. Lastly, changes in telomere structure associated with DNA replication will be studied. These studies should clarify how telomeres function during replication. Pre-replicating DNA molecules will be obtained from isolated replication bands. Newly- replicated molecules will be obtained by pulse labeling with biotinylated dUTP and affinity purifying the labeled molecules. Differences in DNA sequence and structure. DNA-protein interactions, and protein composition will be sought.
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