Telomeres are specialized DNA-protein structures found at the ends of eukaryotic chromosomes which are necessary for the stable maintenance and normal replication of chromosomes. The presence of telomeric sequences at the ends of linear chromosomes is a universal property of all eukaryotic cells. There is a direct correlation between the gradual shortening of telomere length in human somatic cells in vivo and in vitro with the ability of cells to divide. This implies a direct link between telomere length and the aging process. It has been suggested that activation of telomere synthesis may be essential for the growth of immortalized human cells. There are also several lines of evidence linking abnormalities at the telomere with cancer cells. Telomeric DNA is maintained by a ribonucleoprotein enzyme (telomerase) whose RNA moiety serves as a template, dictating the species-specific telomeric DNA repeat synthesized. Thus, telomerase represents an unusual reverse transcriptase that is template-independent because it contains its own template in the form of an RNA. The experimental system that has been the most amenable to the study of telomeres and telomere synthesis is that of the ciliated protozoans.
The aim of the proposed research is to investigate the possible involvement of telomerase RNA (ThR) in the enzymatic activity of telomerase beyond that of a templating function, using the cilate Tetrahymena thermophila as the experimental system. The research plan is logically divided into two objectives. The fist will be to identify the primary and secondary structural features of TER that are invariant throughout evolution, and therefore essential to telomerase activity. This phylogenetic approach will center on ciliate species that are closely allied with T. thermophila, i.e. species that synthesize (GGGGTT) telomeric repeats and are in the suborder Tetrahymenina. Cross- hybridization and PCR will be used to identify and clone additional TER genes. Inclusion of additional TER gene sequences with the fourteen known sequences (seven published) in a phylogenetic analysis will help to more completely define conserved nucleotides that may be of functional importance. This information will help narrow our selection of nucleotides and structural element targeted for mutagenesis to those that are conserved in all tetrahymenine species. The second objective of the proposed research will be to experimentally test the refined TER secondary structure model. This will be accomplished by site-directed mutagenesis of absolutely conserved primary and secondary structural elements, followed by introduction of the mutated TER gene into Tetrahymena thermophila by DNA-mediated transformation. Mutated TER genes will be cloned into a selectable, Tetrahymena-specific transformation vector and introduced into cells by electroporation. Transformants will be fully characterized for assembly of the plasmid- borne TER transcripts into telomerase and for their ability to synthesize telomeric DNA in vivo. The effect of mutant telomerase RNAs on telomerase activity will also be quantitated in a series of in vitro assays. Telomerase will be partially purified from transformants expressing mutant TER and characterized more completely with respect to number of kinetic parameters (substrate and dNTP binding, Vmax, etc.)
