MAPPING AND MICRODISSECTION OF CHROMSOME 21

这个实验室的长期目标是使用蜂窝和 研究组织和表达的分子方法学 人类染色体及其组成基因和调控 正常和疾病状态下的序列。这样做的具体目的是 成分建议是利用体细胞遗传学、分子生物学 FINE的探针、显微解剖和微克隆技术 人类染色体的结构作图、分离和表达 21.这些研究将为细胞遗传学提供直接链接桥接 和21号染色体的分子分辨率，可以促进我们的 寻找病因学背后的基因和调控序列 唐氏综合症。建议的研究包括(1)测绘 21号染色体使用一系列定义明确的末端缺失， (2)从染色体的特定区域微克隆DNA序列 21；(3)显微解剖和显微注射联合应用治疗 大片段DNA在培养细胞中的表达研究 动物。对于终端删除映射，我们将建立大的 人类/中国人21号染色体末端缺失的数目 含有21q+易位染色体的细胞杂交 T(8；21)(q22；q22.3)AML-M2白血病细胞。终端删除 将由X射线产生，并由BrdU+可见光选择 方法利用易位的营养缺陷性标记GlyB 从21q+易位染色体的8号染色体到21号染色体。 将使用大量的DNA探针来确定缺失 线性和亚显微分辨率。这些删除将是 用于所有基因和DNA探针的精细区域定位 被分配给21号染色体，并用于解析许多 该染色体上紧密相连的基因和DNA标记。为 显微解剖21号染色体，我们将使用新开发的 显微解剖和微克隆技术。21号染色体可以是 细分为五个地区和地区特定的图书馆将 构建用于提供来自定义区域的DNA探针 用于可能识别相关DNA序列的染色体 患有唐氏综合症。最后，我们将介绍大块的 21号染色体解剖区域的连续DNA序列 通过显微注射进入细胞，研究它们的整合和 表情。这项工作将扩展到转基因小鼠。这 研究语言表达的方法显得尤为重要 像唐氏综合症这样的复杂疾病。

The long-term goal of this laboratory is to use cellular and molecular methodologies to study the organization and expression of human chromosomes and their constituent genes and regulatory sequences in normal and disease states. The specific aim of this component proposal is to use somatic cell genetics, molecular probes, microdissection and microcloning techniques for fine structure mapping, dissection and expression of human chromosome 21. These studies will provide direct link bridging cytogenetic and molecular resolution of chromosome 21 and can facilitate our search for genes and regulatory sequences underlying the etiology of Down syndrome. The proposed studies include (1) mapping of chromosome 21 using a series of well-defined terminal deletions, (2) microcloning of DNA sequences from defined areas of chromosome 21, and (3) combined use of microdissection and microinjection for studying expression of large blocks of DNA in cultured cells and animals. For terminal deletion mapping, we will establish large numbers of terminal deletions of chromosome 21 by using a human/CHO cell hybrid containing a 21q+ translocation chromosome derived from AML-M2 leukemia cells of t(8;21)(q22;q22.3). Terminal deletions will be produced by X-ray and selected by BrdU + visible light method using the auxotrophic marker GlyB which was translocated from chromosome 8 to 21 of the 21q+ translocation chromosome. Large numbers of DNA probes will be used to define the deletions in linear and submicroscopic resolution. These deletions will be used for refined regional mapping of all genes and DNA probes assigned to chromosome 21, and for resolving the order of many closely linked genes and DNA markers on this chromosome. For microdissecting chromosome 21, we will use the newly developed microdissection and microcloning techniques. Chromosome 21 can be subdivided into five regions and region-specific libraries will be constructed for providing DNA probes from defined areas of the chromosome for possible identification of DNA sequences associated with Down syndrome. Finally we will introduce large blocks of contiguous DNA sequences from dissected regions of chromosome 21 into cells by microinjection for studying their integration and expression. This work will be extended to transgenic mice. This approach appears particularly important for studying expression of complex disorders like Down syndrome.