The ends of eukaryotic chromosomes are protected by nucleoprotein structures called telomeres consisting of GC-rich repeat sequences. Studies have shown that mutated telomeres are detrimental to the cell, causing chromosomal fusions and increased genomic instability (Dandjinou et. al., 1999). Previous research in our lab on Tetrahymena thermophila (T. thermophila) has shown a severe anaphase arrest (Kirk et al., 1997) and extensive loss of telomere sequences in response to telomeric DNA mutations. In other eukaryotes, telomere loss results in telomeric fusions (Tong et. al., 2001). Therefore, in our telomeric mutants I hypothesize the existence of telomeric fusions. In order to test our hypothesis, I developed a polymerase chain reaction (PCR) assay that can detect telomere fusions. Primers were designed from the sequences just adjacent to the telomere called telomere-associated sequences (TAS). I designed a positive control to ensure that our assay could overcome the typical difficulties encountered in amplifying long GC-rich PCRproducts. Clones of T. thermophila TAS sequences were cut and ligated to mimic potential in vivo fusion events. PCR on my positive control demonstrated that our assay can amplify telomeric fusions up to 841 bp. Preliminary assays of the mutant DNA did not amplify any fusion products. However, reamplification of the products from PCR on digested DNA yielded products. Southern blot analysis of these products using a telomeric probe showed signals that indicate fusions. However, sequence analysis showed the signals to be artifacts produced by the reamplification. The study suggests that micronuclear fusions up to 841 bp may not exit in our mutants and if they do, the number of such fusion events is beyond the amplification limit of my assay.
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