Poster abstracts

Poster number 38 submitted by Benjamin Linger

Isolation of the tetrahymena telomeric protien complex

Benjamin R. Linger (Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati), Carolyn M. Price (Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati)

Abstract:
In order to maintain genome integrity, the telomeric DNA from cells with linear chromosomes is packaged into a protective nucleoprotein complex. In the absence of such a complex, the telomeres are recognized as DNA damage and subject to repair by non-homologous end joining (NHEJ). The resulting chromosome fusions lead to profound genome instability. Although telomeric nucleoprotein complexes are known to contain a variety of unique telomere proteins plus general DNA repair factors, the full protein complement of a telomere has not been determined for any organism. Our goal is to isolate a native telosome with all its component proteins. We are using the ciliate Tetrahymena thermophila due to the abundance of genetic tools available and its atypical biology that generates thousands of telomeres per cell. To isolate a native telosome, we have engineered the rDNA chromosome so that the entire telosome can be released by restriction digestion. This chromosome is an excellent source for telosome isolation because of its high copy number of ~10,000 molecules per cell. We have shown that the telosome can be released from the engineered rDNA by digestion with PvuII. We have also verified that the telomere protein POT1a associates with the releasable telomeres. To promote affinity purification of released telomeres, we subsequently added a TAP-tag to the N-terminus of the POT1a gene in the cell line with the engineered rDNA chromosome. Initial immunoprecipitation of soluble TAP-POT1a and subsequent mass spectrometry has identified two potential POT1a binding partners for which we are now pursuing functional analysis. The homology observed between telomere proteins identified to date suggests that characterization of the Tetrahymena telosome components will facilitate identification of key components of human telomeres and provide insight into how these components maintain functional telomeres that are essential for genome stability.

Keywords: telomerase, telomere