MICRODISSECTION AND CDNA ISOLATION OF CHROMOSOME 21

该分项目的总体目标涉及使用显微解剖和 构建区域特异性微切割的微克隆技术 用于唐氏综合征高分辨分子分析的21号染色体文库 证候表型及其表达基因序列的分离 21号染色体。这些研究将提供直接的联系 细胞遗传学和分子水平的21号染色体和 应该有助于搜索与特定成分有关的基因 唐氏综合征(DS)的表型具体目标是：(1) 21号染色体亚区微切割文库的构建 区域特异性微克隆的分离和鉴定，以及(2) 区域特异性表达序列的分离与鉴定 来自21号染色体。一种显微解剖和微克隆技术 根据该计划项目开发的将用于构造库 21号染色体区域包括21ptercen、21cen-q21和21q22。 将使用特殊的细菌宿主菌株进行更完整的 文库中基因组序列的表示，特别是对于 探针缺乏的近端长臂区域21q21。独一无二 将从这些文库中分离出序列微克隆 其特征是用于分离相应的YAC克隆 镶件。这些21号染色体区域特定的文库也将被使用 对核型明显正常的DS进行鱼画分析。 来自21号染色体的唯一序列微克隆将用于 文库直接筛选法分离克隆的研究 固定化滤池杂交法。大规模分离AS 将进行尽可能多的来自21号染色体的cdna克隆和 将对分离的cdna克隆进行鉴定、区域分配和 已排序。这些区域特异性的cdna克隆将被研究 参与部分三体的DS患者以确定可能 DS特定成分表型的因果关系。

The overall aims of the subproject involve the use of microdissection and microcloning techniques to construct region-specific microdissection libraries of chromosome 21 for high resolution molecular analysis of Down syndrome phenotypes and for isolation of expressed gene sequences from chromosome 21. These studies will provide direct link between cytogenetic and molecular levels of resolution of chromosome 21 and should facilitate search for genes responsible for specific component phenotypes of Down syndrome (DS). The specific objectives are: (1) Construction of sub-region microdissection libraries of chromosome 21 for isolation and characterization of region-specific microclones, and (2) isolation and characterization of region-specific expressed sequences from chromosome 21. A microdissection and microcloning technology developed under this program project will be used to construct libraries for chromosome 21 regions including 21pter-cen, 21cen-q21 and 21q22. Special bacterial host strains will be used for a more complete representation of genomic sequences in the library, particularly for the proximal long arm region 21q21 where probes are deficient. Unique sequence microclones will be isolated from these libraries and characterized for use in isolation of corresponding YAC clones with large inserts. These chromosome 21 region-specific libraries will also be used for the FISH painting analysis of DS with apparently normal karyotype. The unique sequence microclones from chromosome 21 will be used for isolation of cDNA clones by direct screening of cDNA libraries and by the immobilized filter hybridization procedure. Large scale isolation of as many cDNA clones as possible from chromosome 21 will be carried out and the isolated cDNA clones will be characterized, regionally assigned and sequenced. These region-specific cDNA clones will be studied for involvement in DS individuals with partial trisomy to determine possible causal relationships with specific component phenotypes of DS.