GENETIC AND MOLECULAR ANALYSIS OF IMPRINTED GENES

基因组印记导致亲代等位基因的特异性表达 内源基因在生长调节中发挥重要作用 哺乳动物胚胎。这些基因的失调似乎与 与人类肿瘤和新生儿癌症以及许多 发育综合症。已鉴定出五个印记基因 小鼠但印记基因的总数及其潜力 在癌症和其他疾病中的作用尚不清楚。一种筛选方法 已开发出新印记基因的小鼠基因组 CpG 岛的差异甲基化。 该提案描述了 使用高分辨率的限制性标志基因组扫描 (RLGS) 二维凝胶电泳识别末端标记的稀有位点 使用 Not I 和类似的核酸内切酶切割基因组 DNA。两个 数千个 RLGS 地标可以被识别为单个两个中的点 三维凝胶和基因组的附加标志可以很容易地 用不同的限制性内切酶组合产生。遗传变异 对于这些地标，已在常见的近交系和 实验室小鼠和其他小鼠物种之间的差异。我们筛选了超过 小鼠基因组中 2000 个品系特异性位点用于差异化 使用甲基化这一标志性酶对亲本基因进行甲基化 敏感（NotI）。通过检测亲本等位基因特异性 NotI 消化 分析不同小鼠基因型之间的相互 F1 组合。 确定了 8 个位点，显示出差异甲基化 父母的基因。小鼠 11 号染色体上的一个基因座已被鉴定 这显示了与 U2 小的衍生氨基酸序列同源性 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 印记基因表征的进展。该提案概述了 实验程序将表征另外五个印记 RLGS 位点（Irlgs 位点）已被确定为确定的第一步 印记的分子基础和进化原因 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另外 3500 - 4000 个标志物以寻找新的 Irlgs 位点作为确定内源基因总数的手段 那些被印记的。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。此信息将提供更完整的机会 确定哺乳动物基因组中印记基因的数量及其 潜在参与癌症和其他发育障碍 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.