Comparative Feline Genomics: High-Throughput BAC/PAC Contig Mapping

比较猫科动物基因组学：高通量 BAC/PAC 重叠群作图

• 批准号: 6433111
• 负责人: STEPHEN J O'BRIEN
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6433111
• 关键词: cats; genetic library; genetic mapping; major histocompatibility complex; molecular cloning

The genome organization of the recently sequenced 3.6 Mb human Major Histocompatibility Complex (MHC) will be best understood in a comparative evolutionary context. To develop a physical map of the domestic cat major histocompatibility complex (MHC) for nucleotide sequencing, we have constructed two large insert domestic cat genomic DNA libraries, one using the P1 artificial chromosome (PAC) system with an average insert size of 80 kb and a second using the bacterial artificial chromosome (BAC) system with an average insert size of 137 kb which has been arrayed in 384-well plates. State-of-the-art, high-throughput analysis technologies are being applied to BAC/PAC contig mapping of the feline MHC. Using feline and/or human MHC DNA probes, we have thus far identified 452 BAC and 66 PAC DNA clones containing the feline MHC. Putative MHC positive BAC/PAC clones are arranged in a 96-well plate format and rescreened by colony hybridization and BAC/PAC DNAs are purified for fingerprint analysis and Southern hybridization. To generate contig maps of large insert clones, complete Hind III digested BAC/PAC DNAs are fingerprinted by electrophoresis through agarose, stained, imaged, and the data analyzed using the Fingerprinting Contigs (FPC) software developed by C. Soderlund which assembles a minimal tiling path of clones using an algorithm to cluster clones into contigs based on their probability of coincidence scores. Using these appoaches, we have constructed a sequence ready 660 kbp BAC/PAC contig map of the domestic cat MHC class II region consisting of 24 BACs and 12 PACs with a gene order similar to, but distinct from human and mice: DPB/DPA, Ring3, DMB, TAP1, DOB, DRB2, DRA3, DRB2, DRA2 and DRA1. FISH analyses of selected class I and II PAC clones confirmed that the domestic cat MHC lies in the pericentromeric region of chromosome B2. However, apparently unlike the human and mouse MHC, both the domestic cat DRA and DRB genes have undergone multiple duplications, functional DPB/DPA genes have been deleted as well as the entire DQB/DQA region.

最近测序的 3.6 Mb 人类主要组织相容性复合物 (MHC) 的基因组组织将在比较进化背景下得到最好的理解。 为了开发用于核苷酸测序的家猫主要组织相容性复合物（MHC）的物理图谱，我们构建了两个大型插入家猫基因组 DNA 文库，一个使用平均插入大小为 80 kb 的 P1 人工染色体（PAC）系统，另一个使用 P1 人工染色体（PAC）系统，平均插入大小为 80 kb。第二个使用细菌人工染色体 (BAC) 系统，平均插入片段大小为 137 kb，已排列在 384 孔板中。 最先进的高通量分析技术正应用于猫科动物 MHC 的 BAC/PAC 重叠群作图。 使用猫科动物和/或人类 MHC DNA 探针，我们迄今已鉴定出 452 个含有猫科动物 MHC 的 BAC 和 66 个 PAC DNA 克隆。将推定的 MHC 阳性 BAC/PAC 克隆排列在 96 孔板中，通过菌落杂交进行重新筛选，并纯化 BAC/PAC DNA 用于指纹分析和 Southern 杂交。 为了生成大插入克隆的重叠群图谱，通过琼脂糖电泳对完整的 Hind III 消化的 BAC/PAC DNA 进行指纹识别、染色、成像，并使用 C. Soderlund 开发的指纹识别重叠群 (FPC) 软件对数据进行分析，该软件可组装最小平铺使用算法根据克隆的重合分数概率将克隆聚类为重叠群的克隆路径。 使用这些方法，我们构建了家猫 MHC II 类区域的序列就绪 660 kbp BAC/PAC 重叠群图，由 24 个 BAC 和 12 个 PAC 组成，其基因顺序与人类和小鼠相似但不同：DPB/DPA， Ring3、DMB、TAP1、DOB、DRB2、DRA3、DRB2、DRA2 和 DRA1。 对选定的 I 类和 II 类 PAC 克隆的 FISH 分析证实，家猫 MHC 位于 B2 染色体的着丝粒周围区域。 然而，与人类和小鼠MHC明显不同的是，家猫DRA和DRB基因都经历了多次重复，功能性DPB/DPA基因以及整个DQB/DQA区域都被删除。