REPLICATION ASYNCHRONY AND IMPRINTING

复制异步和印记

• 批准号: 2191498
• 负责人: RALPH SCOTT HANSEN
• 金额: $ 20.63万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2191498
• 关键词: DNA methylation; DNA replication; flow cytometry; genetic library; genetic polymorphism; genetic transcription; genomic imprinting; human genetic material tag; human tissue; in situ hybridization; nucleic acid repetitive sequence; oligonucleotides; plasmids

Genomic imprinting refers to the process of epigenetic change that occurs during germ cell development that results in either maternal- or paternal- specific gene expression. Imprinting appears to be involved in the development of several inherited disorders in humans as well as in the ontogeny of several forms of cancer. Because only a few imprinted genes have been discovered, identification of other imprinted genes is of primary importance to the understanding of imprinting as well as to the isolation of candidate disease genes. Most regions of the genome replicate synchronously with respect to their chromosomal homologue. However, it has been established that chromosomal regions currently known to contain imprinted genes replicate asynchronously. This proposal describes a general method for scanning the human genome far imprinted genes based on the replication asynchrony of their chromosomal alleles. The method involved the separation of dividing cells into different fractions of S- phase by flow cytometry and isolating newly replicated DNA from each of these fractions. The late-replicating DNA is predicted to be enriched in asynchronously-replicating inter-Alu sequences and will be used as a template for Alu PCR amplification; a plasmid library will be prepared from these products and replication profiles of inter-Alu products from different fractions of S-phase will be used to analyze the clones; the inter-Alu clones will also be screened for their presence on single human chromosomes by hybridization to a monochromosomal hybrid panel; sequences that exhibit biphasic or broad replication profiles and that are localized to a single chromosome will be sequenced; locus-specific PCR primers will be developed to verify replication asynchrony, determine allele-specific replication timing and test for gene expression; positive inter-Alu clones will be used for the isolation of cosmid clones; cosmids will be examined for chromosomal location and replication asynchrony by in situ hybridization, for methylation imprinting in genomic DNA, and for transcriptional expression. Monoallelic expression of candidate genes will be examined after obtaining expressed polymorphisms. Preliminary data indicate that the scanning method is likely to identify several replication-imprinted regions. Initial screening resulted in 3/123 inter- Alu clones that replicated asynchronously and localized to single chromosomes. A chromosome 15 clone was further characterized because of its possible localization to a known region of replication-imprinting, the Prader-Willi/Angelman locus. This sequence was localized outside the region usually deleted in Prader-Willi patients and was expressed in human cell lines. Other candidates were localized to chromosomes 17 and 19, each of which may have imprinted regions based on synteny with mouse chromosomes. Further characterization of these, as well as future clones, may help to identify imprinted genes that have major roles in human disease.

基因组印记是指发生的表观遗传变化的过程 在生殖细胞发育过程中，导致母体或父亲 特定基因表达。印迹似乎参与了 在人类以及人类中的几种遗传疾病的发展 几种形式的癌症的个体发育。因为只有几个印迹基因 已经发现了其他印迹基因的识别是 对理解印迹以及对 分离候选疾病基因。基因组复制的大多数区域 就其染色体同源物同步。但是，它有 已经确定染色体区域目前已知包含 印迹基因不同步复制。该提议描述了 扫描人基因组的一般方法远基于印迹基因 其染色体等位基因的复制异步。该方法 涉及将细胞分离为不同的s-的不同部分 通过流式细胞仪进行阶段，并从每个 这些分数。预计将延迟重复的DNA富含 异步复制的alu间序列，将用作 ALU PCR扩增的模板；将准备一个质粒库 从这些产品和alu Inter-Alu产品的复制曲线中 S期的不同部分将用于分析克隆。这 Alu Inter-Alu克隆也将被筛选以使其在单人类中的存在 通过杂交与单色杂种杂交材料染色体；序列 展示了双相或广泛的复制曲线，并且本地化 将对单个染色体进行测序；基因座特异性PCR引物将 开发以验证复制异步，确定特定等位基因 复制时间和基因表达的测试；阳性的alu间克隆 将用于隔离宇宙克隆；将检查宇宙 用于染色体位置和复制异步 杂交，用于基因组DNA中的甲基化，用于 转录表达。候选基因的单相表达将 在获得表达的多态性后进行检查。初步数据 表明扫描方法可能可以识别几个 复制刻有区域。初始筛选导致3/123 Alu克隆，将异步复制并本地化为单个 染色体。由于 它可能定位到已知的复制刻印区域， Prader-Willi/Angelman Locus。这个序列本地化在 通常在Prader-Willi患者中删除的区域，并在人类中表达 细胞系。其他候选人本地化为17和19染色体，每个候选人 其中可能具有基于与小鼠同步的印迹区域 染色体。这些以及未来克隆的进一步表征 可能有助于确定在人类中具有重要作用的印迹基因 疾病。