GENETIC AND MOLECULAR ANALYSIS OF IMPRINTED GENES

基因组印迹导致亲本等位基因的特异性表达 对于在植物生长调节中起重要作用的内源基因 哺乳动物的胚胎。这些基因的失调似乎与 与人类的肿瘤和新生儿癌症以及一些 发育综合症。五个印记基因已经在 而是小鼠的印记基因总数及其潜力 在癌症和其他疾病中的作用尚不清楚。一种筛选的方法 小鼠的新印记基因的基因组已经开发出来，这是基于 CpG岛的差异甲基化。这份提案描述了 使用高分辨率的限制性里程碑基因组扫描(RLGS) 双向凝胶电泳法鉴定末端标记的稀有位点 使用Not I和类似的内切酶切割基因组DNA。二 数以千计的RLGS地标可以识别为一个单独的两个点 基因组的维度凝胶和额外的标志物可以很容易地 用不同的限制性内切酶组合生产。遗传变异 这些标志已经在常见的近交系和 在实验室小鼠和其他小鼠物种之间。我们已经放映了不止 小鼠基因组中2000个菌株特异性位点的差异 使用一种标志性的酶，即甲基化，对父母基因进行甲基化 敏感(Noti)。亲本等位基因特异性NotI酶切检测 分析不同小鼠基因型间的正反交F1组合。 共鉴定出8个基因座，表现出不同的甲基化 父母的基因。小鼠11号染色体上的一个基因座已被鉴定 它显示了一个衍生的氨基酸序列与U2小分子同源性 核蛋白辅助因子小亚基-U2AF结合蛋白。 第二个基因座的分子克隆已经确定，工作正在进行中 印记基因特征的研究进展。这项提案概述了 实验计划将表征另外五个印记的RLG 基因座(Irlgs基因座)已被确定为确定 印记的分子基础及其进化原因 维持不同基因的单等位基因表达。我们还建议 在小鼠基因组中筛选新的3500-4000个标志性基因 IRGS基因座作为确定内源基因总数的一种手段 都是有印记的。印记小鼠基因的克隆序列可以是 用于鉴定人类同源基因及其在人类中的连锁 基因组。这些信息将提供一个更完整的机会来 确定哺乳动物基因组中印记基因的数量和它们的 可能参与癌症和其他发育障碍的研究 人类。

Genomic imprinting results in the specific expression of parental alleles for endogenous genes that play significant roles in growth regulation of mammalian embryos. Disregulation of these genes appears to be associated with neoplasias and neonatal cancers in humans and a number of developmental syndromes. Five imprinted genes have been characterized in the mouse but the total number of imprinted genes and their potential roles in cancer and other diseases is unknown. A method of screening the mouse genome for new imprinted genes has been developed that is based upon differential methylation of CpG islands. This proposal describes restriction landmark genomic scanning (RLGS) which uses high resolution two-dimensional gel electrophoresis to identify end-labeled, rare-site cleavages of genomic DNA using Not I and similar endonucleases. Two thousand RLGS landmarks can be identified as spots in a single two dimensional gel and additional landmarks for a genome can be readily produced with different restriction enzyme combinations. Genetic variation for these landmarks has been identified between common inbred strains and between laboratory mice and other Mus species. We have screened more than 2000 strain-specific sites in the mouse genome for differential methylation of parental genes using a landmark enzyme that is methylation sensitive (NotI). Parent allele-specific NotI digestion was detected by analyzing reciprocal F1 combinations between various mouse genotypes. Eight loci were identified that showed differential methylation of parental genes. One locus on mouse chromosome 11 has been characterized which shows a derived amino acid sequence homology with the U2 small nucleoprotein auxiliary factor small subunit -U2AF binding protein. Molecular clones for a second locus have been identified and work is in progress to characterize the imprinted gene. This proposal outlines an experimental program that will characterize five additional imprinted RLGS loci (Irlgs loci) that have been identified as a first step in determining the molecular basis for imprinting and the evolutionary reasons for sustaining monoallelic expression of different genes. We also propose to screen an additional 3500 - 4000 landmarks in the mouse genome for new Irlgs loci as a means of determining the total number of endogenous genes that are imprinted. The cloned sequences for imprinted mouse genes can be used to identify the homologous human genes and their linkage in the human genome. This information will provide a more complete opportunity to establish the number of imprinted genes in the mammalian genome and their potential involvement in cancer and other developmental disorders in humans.