POLYMORPHIC OLFR-CONTAINING DUPLICATIONS NEAR TELOMERES

描述：本提案的长期目标是调查 人类染色体亚端粒区域的组织，以及它们的 与嗅觉受体（OLFR）家族的进化有关。 来自多个来源的证据表明，这些地区是由 大的序列块以各种组合存在于 多条染色体的端粒。 这些区域的存在和数量 在染色体和个体之间有差异。 申请人假设 这些亚端粒区域是通过复制、缺失和 同源和非同源染色体之间的交换， 对于基因家族多样性的产生很重要， OLFR，它们优先与这些区域相关，并且它们 可能形成临床上重要的染色体重排的来源。 该项目的具体目标是描述一个这样的亚端粒 区域，即3q上的区域，通过（1）构建该区域的物理地图（2） 识别各种重复元素在其他元素上的分布， 染色体，并定位差异分布的边界 （3）数据库 200 kb的3q，以确定该地区的基因，并寻找基序， （4）在细胞遗传学上测定正常变异 和序列水平，并用它来描述这些最近的演变， 区域和它们所包含的基因，以及（5）测试基因是否在附近复制 端粒的表达，并映射额外的OLFR基因，以确定是否 与亚端粒区域的关联在该家族中是常见的。 3q的参考物理地图将通过使用 染色体3粘粒文库排列在高密度过滤器上。 目的是步行到 端粒重复序列的一端，和独特的染色体DNA上 另一 将通过键入已定义STS的克隆来确认Walks 从该区域，通过FISH和限制性片段作图。 10粘粒 将使用分布在参考地图切片路径上的克隆 对于代表10个不同人群的50个个体的FISH， 还有6种不同的灵长类动物狗和老鼠 这将定义 不同染色体上每个区块的相对频率 人口，并将用于构建系统发育树的基础上， 等位基因频率 25个来自其他种群的无关个体将 还使用一种粘粒进行了研究， 染色体分布 此外，流上的STS映射已排序 染色体和动物园印迹上的Southern印迹将用于研究 FISH检测到或未检测到的序列同源性程度。 亚端粒区域的进化也将通过测序进行研究 OLFRA和OLFRB基因来自3个不同的染色体， 最大限度地扩大种群间的差异。 其他OLFR基因在人类基因组中的染色体分布将是 使用FISH和通过PCR获得的OLFR序列库进行评估， 简并引物和已知的含OLFR的克隆。 表达测定 利用RT-PCR技术对嗅神经元中的亚端粒OLFR基因进行研究， 神经上皮mRNA和嗅觉cDNA文库的杂交或PCR， 以及序列分析来揭示假基因。

DESCRIPTION: The long-term goal of this proposal is to investigate the organization of the subtelomeric regions of human chromosomes, and their relationship to the evolution of the olfactory receptor (OLFR) family. Evidence from several sources suggests that these regions are made up of large blocks of sequence which are present in various combinations at the telomeres of multiple chromosomes. The presence and number of these regions varies among chromosome and among individuals. The applicant hypothesizes that these subtelomeric regions have evolved by duplications, deletions and exchanges among homologous and nonhomologous chromosomes, that this process has been important for the generation of diversity in gene families as the OLFR, which are preferentially associated with these regions, and that they may form a source of clinically important chromosomal rearrangements. The specific aims of the project are to characterize one such subtelomeric region, that on 3q, by (1) constructing a physical map of the region (2) identifying the distribution of the various repetitive elements on other chromosomes, and locating the boundaries of differentially distributed segments (3) sequencing 200 kb of 3q to identify genes in the region, and look for motifs that may predispose to recombination (4) assay normal variation at the cytogenetic and sequence levels and use this to describe the recent evolution of these regions and the genes they contain and (5) to test if genes duplicated near telomeres are expressed, and mapping additional OLFR genes to determine if association with subtelomeric regions is common in this family. The reference physical map of 3q will be constructed by walking using a chromosome 3 cosmid library arrayed on dense filters. The aim is to walk to the telomeric repeat sequence at one end, and to unique chromosomal DNA on the other. Walks will be confirmed by typing the clones for STS defined from the region, by FISH and by restriction fragment mapping. 10 cosmid clones distributed across the tiling path in the reference map will by used for FISH in 50 individuals representing 10 different human populations, as well as in 6 different primate species, dog and mouse. This will define the relative frequency of each block on each chromosome in different populations, and will be used to construct phylogenetic trees based on allele frequencies. 25 unrelated individuals from other populations will also studied using one cosmid showing particularly striking variation in chromosomal distribution. In addition STS mapping on flow sorted chromosomes and southern blotting on zoo-blots will be used to study the extent of sequence homology detected or undetected by FISH. The evolution of the subtelomeric regions will also be studied by sequencing the OLFRA and OLFRB genes from multiple chromosomes in 3 different populations chosen to maximize interpopulational differences. The chromosomal distribution of other OLFR genes in the human genome will be assessed using FISH with a pool of OLFR sequences obtained by PCR using degenerate primers, and known OLFR-containing clones. Assay for expression of subtelomeric OLFR genes will be attempted using RT-PCR on olfactory neuroepithelium mRNA, and hybridization or PCR on olfactory cDNA libraries, as well as sequence analysis to reveal pseudogenes.