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

一种新的技术可以快速和精确的定位端粒添加位点（TAS）在染色体末端的hypotrichous纤毛虫大核。 大核是从细胞的小精灵核的拷贝发育而来的，涉及40，000个新端粒的产生，在多张力生殖系染色体的染色单体断裂之后，产生每个只有一个或几个基因的染色体。 一些染色体是使用完全相同的TAS对（称为"固定TAS"）创建的，但其他染色体是通过替代处理生成的。 在这种情况下，一些染色单体完全逃脱断裂，并在由顺式作用序列决定的区域中使用一组异质的TAS（称为"混合" TAS）。 关于这可能如何起作用的一个假说是"犹豫切割器"假说，其中TAS谱是由染色单体切割器和酶端粒酶的松散复合物的作用引起的。 该模型表明，该区域由复合物的结合位点（"CBS"）和相对差的切割位点锚定，使得切割物切割缓慢;同时端粒酶解离，进行切割，末端不受核酸酶侵蚀保护。 然后核酸酶可以继续通过3 '链，直到它在顺式作用暂停位点的影响下暂停，通常在一串dT中的dT处。 这可以让端粒酶在腐蚀停止时发挥作用。 在混合区域中映射TAS将允许发现三种类型的顺式作用序列：CBS、切割和暂停位点。 TAS区域的许多等位基因变体将被如此定位，从而细化有效位点序列。 此外，将定位固定的TAS以允许对已经鉴定的共有位点3 'TAY（Y =嘌呤）进行细化。 该项目将有助于阐明染色体断裂、端粒形成和端粒酶作用的过程。 对这些过程的基本理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.