Cloning, Sequencing and Analysis of Human Telomeric DNA

描述(由申请人提供)： 人类端粒DNA区域充满节段性复制，我们称之为亚端粒重复序列。片段重复(定义为长度大于1kb且序列一致性大于90%的重复DNA)约占人常染DNA的5%。包含人类节段性复制的染色体区域最近才进化，并且容易受到与人类疾病相关的同源驱动的染色体重排的影响。重要的是，现在已经清楚的是，功能基因和基因家族，包括通过将新的分段复制和转座的DNA片段并置而产生的嵌合转录本产生的新形成的基因，存在于分段复制的DNA区域。因此，人类基因组的5%由节段复制组成，在功能上非常重要，它与整个人类基因组中一些最新和进化最快的染色体区域有关。 亚端粒重复序列具有复杂的人端粒DNA参考序列的初始定位、测序和组装。然而，通过结合物理作图、半YAC克隆和对Half-YAC衍生材料的协作大规模测序(以及对独立衍生的重叠和相邻的BAC和粘粒进行IHGSC测序)，有可能从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年春季)。参考序列的端粒末端将延伸到末端(TTAGGG)n区(对于41个端粒中大约14个的参考等位基因)，并在一个参考等位基因的(TTAGGG)n区(大约41个端粒中的21个)相对较小的距离内延伸到亚端粒重复序列中。 然而，特定端粒上不同的亚端粒重复内容和组织有助于在人类端粒下区域看到显著的大规模变异。这些变异是可以检测到的，在给定的端粒上，染色体长度多态的范围从几个kb到超过300 kb。特定个体的亚端粒等位基因的全局互补将决定亚端粒重复序列中嵌入的功能基因的组成和剂量，以及这些基因中每个基因相对于末端(TTAGGG)n区域的位置(以及相邻的1-拷贝基因的位置)。基因剂量和基因到末端(TTAGGG)n区的距离可能对富含基因的亚端粒区域的表达有重要影响，并且取决于亚端粒基因的功能和人类潜在端粒位置效应的程度，可能对自然的人类表型变异和疾病表型都有很大贡献。大多数变异的亚端粒染色体片段还没有出现在公共序列数据库中，因此无法对这一关键染色体区域进行进一步分析。为了缩小这一差距，我们建议对人类亚端粒区域的大规模变异进行全面分析，克隆并协作测序在每个端粒携带独特亚端粒大小变异的亚端粒等位基因，并开发能够区分人类群体中单个大规模亚端粒变异的基于聚合酶链式反应的标记集。

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.