GENETIC AND MOLECULAR ANALYSIS OF IMPRINTED GENES

基因组印记导致亲本等位基因的特异性表达 对于在生长调节中起重要作用的内源性基因， 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有关， 发育综合征五个印记基因已被表征， 但小鼠的印记基因的总数和它们的潜力 在癌症和其他疾病中的作用是未知的。一种筛选抗肿瘤药物的方法， 新的印记基因的小鼠基因组已经开发出来，其基于 CpG岛的差异甲基化。 该提案描述了 限制性标志基因组扫描（RLGS），其使用高分辨率 双向凝胶电泳鉴定末端标记的稀有位点 使用Not I和类似的内切核酸酶切割基因组DNA。两 数千个RLGS地标可以被识别为单个两个中的点， 可以容易地 用不同的限制酶组合产生。遗传变异 因为这些标志已经在常见的近交系之间被确定， 实验室小鼠和其他小鼠物种之间的差异。我们已经筛选了超过 小鼠基因组中的2000个菌株特异性位点， 使用一种标志性的酶，即甲基化， 敏感（NotI）。亲本等位基因特异性NotI消化通过 分析不同小鼠基因型之间的相互F1组合。 8个基因座被鉴定出在不同的基因型中， 亲本基因小鼠11号染色体上的一个位点已经被鉴定为 其显示与U2小分子衍生的氨基酸序列同源性 核蛋白辅助因子小亚基-U2 AF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。 印迹基因的研究进展该提案概述了 实验计划，将表征五个额外的印迹RLGS 已被确定为确定的第一步的基因座（Irlgs基因座） 印记的分子基础和进化的原因 维持不同基因的单等位基因表达。我们亦建议 在小鼠基因组中筛选另外3500 - 4000个地标， Irlgs位点作为确定内源基因总数的手段 被植入了印记可以克隆印迹小鼠基因的序列， 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， 确定哺乳动物基因组中印记基因的数量及其 可能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.