MAMMALIAN GENOME MAPPING OF TISSUE-SPECIFIC CDNAS

组织特异性 CDNAS 的哺乳动物基因组图谱

• 批准号: 2208994
• 负责人: Michael F. Seldin
• 金额: $ 37.45万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-05 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2208994
• 关键词: animal genetic material tag; animal tissue; artificial chromosomes; complementary DNA; genetic mapping; genetic markers; genetic polymorphism; genome; human genetic material tag; in situ hybridization; laboratory mouse; linkage mapping; nucleic acid sequence; polymerase chain reaction; sequence tagged sites; southern blotting

Tissue specific cDNAs will be rapidly mapped to mouse chromosomal segments in order to facilitate the definition of both mouse and human genomic organization. An interspecific mouse cross between Mus spretus and a laboratory inbred strain will allow definition of cDNA RFLV markers and mapping by haplotype analysis of most if not all cDNA clones. Equalized neonatal thymus specific cDNA libraries will be obtained by the application of a novel solid phase system in which cDNAs will be bound and eluted from immobilized genomic sequences. This technique should allow the generation of a library of tissue specific cDNAs that cross hybridize between mammalian species by the selection mouse cDNAs that bind to human genomic sequences. Unique cDNAs (approximately 1000) will then be mapped after PCR amplification of sequences. A panel of 38 interspecific backcross mouse DNAs that has been characterized for over 45O markers throughout the mouse genome will be used for rapid mapping. This panel divides the mouse genome into over 470 different segments. A stratified mapping approach will be subsequently utilized to divide the genome into 1000 different segments. In addition, reference markers at 5 cM intervals that have been analyzed in 100 to 400 meiotic events will further define the mapped location of new cDNA markers. The results will provide a putative human regional localization by the application of comparative mapping knowledge. Selected cDNAs, a total of 200, will be used to precisely define the borders of conserved linkage groups by in situ hybridization on human chromosomes. The cDNA clones will also be partially sequenced to provide sequenced tagged sites, allow PCR screening of YAC libraries, and enable the mapping of single stranded conformational polymorphisms by interested investigators. Since 5' as well as 3' sequence information will be obtained, these transcripts will be compared with previously characterized genes. Novel transcripts will provide a resource for molecular genetic studies of thymocyte maturation. In addition, the mapping of over 1000 unique neonatal thymus transcripts may determine whether there are regions of the genome that are transcriptionally active in a coordinate fashion. The mapped cDNA clones will be made easily available to other investigators and should dramatically facilitate closure of long range restriction maps as well as provide additional tools for contig construction of gene rich regions of both the mouse and human genome. The cDNA map will also be integrated with the developing microsatellite STS maps in other laboratories. This will allow integration of efforts to define a contig of the mouse genome.

组织特异性 cDNA 将快速定位到小鼠染色体片段 为了促进小鼠和人类基因组的定义 组织。斯普雷图斯 (Mus spretus) 和 实验室近交系将允许定义 cDNA RFLV 标记和 通过对大多数（如果不是全部）cDNA 克隆进行单倍型分析进行作图。均衡化 新生儿胸腺特异性 cDNA 文库将通过 应用一种新颖的固相系统，其中 cDNA 将被结合并 从固定的基因组序列中洗脱。该技术应该允许 产生交叉杂交的组织特异性 cDNA 文库 通过选择与人类结合的小鼠 cDNA 来区分哺乳动物物种 基因组序列。然后将绘制独特的 cDNA（大约 1000 个） PCR扩增序列后。由 38 个种间组成的小组 回交小鼠 DNA 已被鉴定超过 45O 个标记 整个小鼠基因组将用于快速作图。这个面板 将小鼠基因组分为 470 多个不同的片段。分层的 随后将利用作图方法将基因组分为 1000 个不同的部分。此外，参考标记以 5 cM 间隔 在 100 到 400 个减数分裂事件中进行的分析将进一步定义 新 cDNA 标记的映射位置。结果将提供一个 通过应用比较法推定人类区域定位 测绘知识。选定的 cDNA（总共 200 个）将用于 通过原位精确定义保守连锁群的边界 人类染色体上的杂交。 cDNA 克隆也将部分被 测序以提供测序标记位点，允许对 YAC 进行 PCR 筛选 文库，并实现单链构象的映射 感兴趣的研究人员进行多态性研究。由于 5' 和 3' 序列 将获得信息，这些成绩单将与 先前表征的基因。新颖的成绩单将提供资源 用于胸腺细胞成熟的分子遗传学研究。此外， 超过 1000 个独特的新生儿胸腺转录本的图谱可能会确定 基因组中是否存在转录活跃的区域 以协调的方式。绘制的 cDNA 克隆将很容易制作 可供其他研究人员使用，并应极大地促进 关闭长范围限制图并提供额外的工具 用于小鼠和人类基因丰富区域的重叠群构建 基因组。 cDNA 图谱也将与正在开发的 其他实验室的微卫星 STS 地图。这将允许集成 定义小鼠基因组重叠群的努力。