Identification of Replication Units in Mammalian Genome by Fiber-FISH

荧光原位杂交（FISH）是基因定位和生物学分析的有力工具。本研究将FISH技术应用于动物细胞基因组DNA复制过程的分析。Fiber-FISH可以在几个kb的水平上分析基因组间的距离。新合成的DNA可以通过检测掺入的溴脱氧尿苷（BrdU）（一种胸苷类似物）在DNA纤维上追踪。本研究的主要目的是探讨这两种精细技术的结合方法及其在DNA复制过程分析中的可行性。首先，改进了掺入BrdU的检测方法。DNA的碱性变性被发现均匀和有效地检测复制的DNA纤维。以时间依赖的方式追踪DNA纤维上的DNA复制的进展。第二，在此条件下，FISH技术可以实现基因组DNA的可视化，并可以同时检测新合成的DNA和特定的基因组序列。建立了HL 60细胞的高同步化方法。这些结果表明，复制子和复制子簇结构域可以通过应用这些精细技术来确定。作为相关研究，开发了用于分析特定基因组序列的包装状态的方法。这对于理解包括DNA复制结构域在内的核内基因组结构具有重要意义。基因组克隆的核内包装状态可以在核晕上可视化。将该技术应用于印迹基因SNRPN，表明父本等位基因上的SNRPN基因比母本等位基因上的SNRPN基因更紧凑。这些信息对于阐明DNA复制机制与基因表达和染色质结构的关系是非常重要的。

Florescence in situ hybridization (FISH) is a powerful tool for gene mapping as well as biological analyses. In this work, the FISH technique was applied to the analysis of DNA replication process in the animal cell genome. Fiber-FISH allows us to analyze the genomic distances in several kb level. Newly synthesized DNAs can be chased on a DNA fiber by detecting incorporated bromodeoxyuridine (BrdU), a thymidine analog. The primary purpose in this study is to examine the combined method of these two fine techniques and its feasibility for the analysis of DNA replication process. Firstly, the detection method of the incorporated BrdU was improved. Alkaline danaturation of DNA was found to detect the replicating DNA fiber uniformly and efficiently. The progress of the DNA replication on a DNA fiber was chased in a time dependent manner. Secondly, in this condition, the genomic DNA could be visualized by FISH.The simultaneous detection of newly synthesized DNA and specific genome sequences was also possible. A highly synchronizing method of HL60 cells was also developed. These results indicate that replicons and replicon cluster domains can be determined by the application of these finetechniques. As a related studies, a method for analysis of a packaging status of the specific genome sequences was developed. This is an important for the understanding of the intranuclear genome organization including DNA replication domains. Intranuclear packaging status of the genomic clones could be visualized on a nuclear halo. This technique was applied to the imprinted gene, SNRPN, suggesting that a SNRPN gene on the paternal allele is more compacted than that on the maternal allele. These information is very important for clarifying the DNA replication mechanismin relation to the gene expression and chromatin structures.