Telomere Formation, Sites and Intermediates, During Macronuclear Development of Oxytricha Trifallax

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A new technique allows rapid and precise mapping of telomere addition sites (TASs) at chromosome ends of the hypotrichous ciliate macronucleus. The macronucleus is carved developmentally from a copy of the cell's gremlin nucleus, involving the generation of 40,000 new telomeres, following the breakage of chromatids of polytenized germline chromosomes, creating chromosomes with just one or a few genes each. Some chromosomes are created using exactly the same TAS pair (called "fixed TASs"), but others are generated by alternative processing. In this case, some chromatids escape breakage altogether and use a heterogeneous set of TASs in a region dictated by cis-acting sequences (called "mixed" TASs). One hypothesis of how this may work is the "hesitant cutter" hypothesis, whereby the TAS spectrum results from the action of a loose complex of the chromatid cutter and enzyme, telomerase. This model suggests that the region is anchored by a binding site ("CBS") for the complex and a relatively poor cut site, such that the cutter is slow to cut; meanwhile the telomerase dissociates, the cut is made, the end left unprotected to nuclease erosion. The nuclease could then proceed through the 3' strand until it pauses under the influence of a cis-acting pause site, often at dT in a string of dTs. This could allow telomerase to act when the erosion is paused. Mapping TASs in mixed regions will allow the uncovering of three types of cis-acting sequences: the CBS, cut, and pause sites. Numerous allelic variants of TAS regions will be so mapped, refining the effective site sequences. In addition, fixed TASs will be mapped to allow the refinement of a consensus site already identified, 3' TAY (Y=purine). This project will help illuminate the processes of chromosome breakage, telomere formation and the action of telomerase. Basic understanding of these processes will be key to understanding cellular senescence.