ESTABLISHMENT OF CHROMOSOME MAPPING OF WHOLE BAC CLONES IN SCHISTOSOMA MANSONI.

本项目作为曼氏血吸虫基因组计划的一部分，促进了BAC克隆染色体定位技术的发展，建立了所有克隆的地址。在项目的最后四年中，我们完成了以下项目。(1)完善了染色体制备、BAC克隆DNA标记、染色体DNA变性、染色体上信号位置测定等技术。这些改进使我们能够建立FISH（荧光原位杂交）染色体定位。该技术也适用于丝胞染色体。(2)由于曼氏血吸虫的5、6、7号染色体体积小，形状相似，如果不熟悉识别，仅凭形状很难识别。因此，为了提高染色体鉴定的客观技术，我们对每个染色体制作了特异性的染色探针。(3) BAC克隆染色体图谱的建立使我们能够定位染色体上的一些特定基因，如腺苷琥珀酸裂解酶和HOX基因。(4)为补充FISH技术，将引物原位标记技术（primed in situ labeling technology， PRIS）应用于曼氏血吸虫染色体，对染色体上的DNA进行PCR扩增。(5)本项目建立的技术也应用于灵长类动物的基因定位和染色体分化实验。该项目的第一个目标是对20000个BAC克隆进行染色体定位，但尚未实现。然而，如果世界上其他机构的合作，这将是可能的。我们的技术将移交给其他寄生虫基因组计划项目组，使项目成果为全球寄生虫基因组计划的发展做出贡献。

This project has promoted objectives that develops techniques of chromosome mapping of BAC clones and establishes address of all the clones, as a part of genome project of Schistosoma mansoni. We have done following items during the last four years of the project.(1)We improved adequate techniques for chromosome preparation, labeling of BAC clone DNA, denaturation of chromosome DNA, and measuring location of signal on chromosome. These improvements allowed us to establish FISH (fluorescence in situ hybridization) for chromosome mapping. The techniques were available for filarial chromosomes as well.(2)Because chromosomes 5,6, and 7 of Schistosoma mansoni are little in size and similar in shape, they are very difficult to be identified by only their shape if you are not familiarized to identifying them. Therefore, we produced specific painting probes to each chromosome in order to improve the objective technique of chromosome identification.(3)Establishment of chromosome mapping of BAC clones allowed us to localize some specific genes on chromosomes, for instance adenylosuccinate lyase and HOX genes.(4)To supplement for FISH technique, PRIS(primed in situ) labeling technique, that makes PCR amplification of DNA on chromosomes, was applied to chromosomes of Schistosoma mansoni.(5)Techniques that have been established in the project were also applied to experiments of gene mapping and chromosome differentiation in primates.The first aim of the project that makes chromosome mapping of 20000 BAC clones was not able to attain yet. However, it will be possible according to collaboration of other institutions in the world. Our techniques are going to handover to other project teams of parasite genome project, so that outcomes produced in the project will contribute for development of the parasite genome project on the move in the world.