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

一种新的技术允许快速和精确地定位染色体末端的端粒添加位点（TASs）。巨核是在发育过程中从细胞的小颗粒核的一个拷贝中切割出来的，涉及到40,000个新的端粒的产生，随着多殖生殖系染色体染色单体的断裂，产生了每条只有一个或几个基因的染色体。一些染色体是由完全相同的TAS对（称为“固定TAS”）产生的，但其他染色体是通过替代处理产生的。在这种情况下，一些染色单体完全避免断裂，并在由顺式作用序列决定的区域使用一组异构的TASs（称为“混合”TASs）。其中一种假说是“犹豫切割器”假说，即TAS谱是染色单体切割器和端粒酶的松散复合物作用的结果。该模型表明，该区域由复合体的结合位点（“CBS”）和相对较差的切割位点锚定，因此切割机切割缓慢；与此同时端粒酶解离，切割完成，端粒不受核酸酶侵蚀。然后，核酸酶可以通过3'链，直到在顺式作用暂停位点的影响下暂停，通常在一系列dT中的dT处。这可以让端粒酶在侵蚀停止时发挥作用。在混合区域中映射TASs将允许发现三种类型的顺式作用序列：CBS、cut和pause位点。许多TAS区域的等位基因变异将被绘制出来，完善有效位点序列。此外，将绘制固定的TASs，以允许改进已经确定的共识位点，3' TAY （Y=嘌呤）。该项目将有助于阐明染色体断裂、端粒形成和端粒酶的作用过程。对这些过程的基本了解将是理解细胞衰老的关键。

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.