Cloning, Sequencing and Analysis of Human Telomeric DNA

描述（由申请人提供）： 人类端粒DNA区域充满了我们称之为亚端粒重复序列的片段重复。片段重复（定义为长度大于1 kb且序列同一性大于90%的重复DNA）约占人类常染色体DNA的5%。含有人类片段重复的染色体区域最近已经进化，并且容易受到与人类疾病相关的同源驱动的染色体重排。重要的是，现在清楚的是，功能基因和基因家族，包括新形成的基因，其来源于通过新的片段重复和转座的DNA片段并置产生的嵌合转录物，填充片段重复的DNA区域。因此，5%的人类基因组由片段性重复组成，在功能上非常重要，并且与整个人类基因组中一些最新和最快速进化的染色体区域相关。 亚端粒重复序列具有复杂的初始定位、测序和人端粒DNA参考序列的组装。然而，通过结合物理作图、半YAC克隆和半YAC衍生材料的协作性大规模测序（以及独立衍生的重叠和相邻BAC和cosmetic的IHGSC测序），可以从41个常染色质端粒区域中的每一个获得并验证完成的“参考”序列（到2003年春天）。参考序列的端粒末端将延伸到末端（TTAGGG）n区（对于41个端粒中的大约14个的参考等位基因）中，并延伸到一个参考等位基因（41个端粒中的大约21个）的（TTAGGG）n区的相对小的距离内的亚端粒重复序列中。 然而，差异subtelomeric重复内容和组织在特定的端粒有助于显着的大规模变化，在人类subtelomeric区域。这些变化是可检测的染色体长度多态性范围从几kb到大于300 kb在一个给定的端粒。给定个体中亚端粒等位基因的全局互补将决定嵌入亚端粒重复序列中的功能基因的组成和剂量以及这些基因中的每一个的位置（和相邻1拷贝基因的位置）相对于末端（TTAGGG）n段。基因剂量和基因距离终端（TTAGGG）n道可能有重要的后果，在基因丰富的subtelomeric区域的表达，并根据subtelomeric基因的功能和潜在的端粒位置在人类的影响程度，可以大大有助于自然人类表型变异和疾病表型。大多数变异亚端粒染色体片段尚未在公共序列数据库中表示，因此无法进一步分析这一关键染色体区域。为了缩小这一差距，我们建议进行一个全面的分析，在人类的subtelomeric区域的大规模变化，克隆和协作序列的subtelomeric等位基因携带独特的subtelomeric大小变异在每个端粒，并开发基于PCR的标记集能够区分个人的大规模subtelomeric变异在人群中。

DESCRIPTION (provided by applicant): Human telomeric DNA regions are filled with segmental duplications we refer to as subtelomeric repeats. Segmental duplications (defined as duplicated DNA greater than 1 kb in length and greater than 90% sequence identity) comprise approximately 5% of euchromatic human DNA. Chromosome regions containing human segmental duplications have evolved very recently, and are susceptible to homology-driven chromosomal rearrangements associated with human disease. Importantly, it is now clear that functional genes and gene families, including newly-formed genes derived from chimeric transcripts generated by juxtaposition of new segmentally duplicated and transposed DNA segments, populate segmentally duplicated DNA regions. The 5% of the human genome comprised of segmental duplications is therefore very important functionally, and is associated with some of the newest and most rapidly-evolving chromosome regions in the entire human genome. Subtelomeric repeat sequences have complicated initial mapping, sequencing, and assembly of reference sequences for human telomeric DNA. However by combining physical mapping, half-YAC cloning, and collaborative large-scale sequencing of half-YAC derived materials (as well as IHGSC sequencing of independently-derived overlapping and adjacent BACs and cosmids) it was possible to acquire and validate a finished "reference" sequence from each of the 41 euchromatic telomere regions (by the spring of 2003). The telomeric end of the reference sequence will extend into the terminal (TTAGGG)n tract (for reference alleles of approximately 14 of 41 telomeres) and into subtelomeric repeat sequences within a relatively small distance of the (TTAGGG)n tracts of one reference allele (approximately 21 of 41 telomeres). However, differential subtelomeric repeat content and organization at specific telomeres contribute to remarkable large-scale variations seen in human subtelomeric regions. These variations are detectable as chromosome length polymorphisms ranging from a few kb to greater than 300 kb at a given telomere. The global complement of subtelomeric alleles in a given individual will determine the composition and dosage of functional genes embedded in the subtelomeric repeats as well as the positions of each of these genes (and the positions of adjacent 1-copy genes) relative to terminal (TTAGGG)n tracts. Both gene dosage and gene distance from terminal (TTAGGG)n tracts may have important consequences for expression in gene-rich subtelomeric regions, and depending upon subtelomeric gene functions and the extent of potential telomere position effects in humans, could contribute substantially to both natural human phenotypic variation and to disease phenotypes. Most variant subtelomeric chromosome segments are not yet represented in the public sequence databases, and are therefore inaccessible for further analysis of this key chromosome region. In order to close this gap, we propose to carry out a comprehensive analysis of large-scale variations in human subtelomeric regions, to clone and collaboratively sequence subtelomeric alleles carrying unique subtelomeric size variants at each telomere, and to develop PCR-based marker sets capable of distinguishing individual large-scale subtelomeric variants in the human population.