Telomeres, the physical ends of eukaryotic chromosomes, are required for the stable maintenance and complete duplication of linear chromosomes. The special structure of telomeres and the variety of functions ascribed to them suggest that multiple gene products are involved in their replication and function. Genetic and biochemical approaches are described to identify and characterize proteins that interact with the telomeres of Saccharomyces cerevisiae. Yalpha26 and RAP1, two candidates for genes encoding telomere binding cross reacts with antiserum raised to a protein known to bind specifically to telomeres in the ciliated protozoan Oxytricha nova. Moreover, excess copies of Yalpha 26 affect telomere length in yeast. DNA sequencing will reveal if Yalpha26 is indeed related to the Oxytricha gene. The phenotype of null and overexpressing alleles on viability, telomere lengths, and chromosome stability will also be determined. RAP1, an essential gene, has high affinity in vitro for telomeric sequences as well as for the transcription control regions of a variety of genes. To determine if the RAP1 protein interacts with telomeres in vivo, the phenotype (telomere lengths, chromosome stability, transcription levels) of strains overexpressing or deficient in RAP1 will be determined. Biochemical methods will also be used to identify proteins that interact with telomeres. The chromatin structure of a chromosomal telomere will be determined in normal cells and in cell grown under conditions shown previously to affect telomere structure. Methods for affinity purification of telomeric chromatin will be developed that will enable us to ask directly if Yalpha26 (or RAP1) is found on telomeres, and, ultimately, to identify other telomere associated proteins. Finally, six lps (linear plasmid stability) mutants that lose linear artificial chromosomes at elevated frequency but lose circular chromosomes at wild-type rates will be analyzed by molecular biological and genetic methods to determine if and how their products interact with yeast telomeres.
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