The parent NIH grant has supported the development of methods for examining DNA-protein complexes by electron microscopy (EM), and transferring these methods to other laboratories. One research focus involves telomere structure. Here, a pressing need is to develop yeast systems which will allow EM to be used to probe telomere structure. Dr. Tomaska at Comenius University is highly experienced in yeast biochemistry and genetics. Two short collaborative visits he made to the US laboratory resulted in two papers demonstrating the feasibility and productivity of this international collaboration. Funding is now required to establish and foster further work. Dr. Tomaska will carry out the yeast developmental work in his laboratory and then obtain training in the EM preparative methods in the US laboratory with the goal of establishing these methods at Comenius University.
The first Aim i s the development of a S. cerevisiae minichromosome system for isolation and visualization of yeast telomeres. The minichromosome system developed by Runge and Zakian will be optimized for the isolation of multicopy linear minichromosomes on sucrose gradients with replication and telomere-specific proteins bound. Using EM and electrophoretic methods, the structure of the yeast telomeres will be examined with and without specific telomere-binding proteins.
The second aim i s to study the replication of yeast linear mitochrondrial genomes as a model system for elucidation of alternative, telomerase-independent, pathways of telomere maintenance. Proteins that specifically interact with the terminal structures of linear mtDNA molecules will be characterized further and examined by EM. The nature of the nuclear control over the replication of mitochondrial telomeres will be studied to analyze the specific coordination between the nuclear and mitochondrial genomes. This research will be primarily done in The Slovak Republic as an extension of NIH grant R01-GM31819-17.
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