| Research Abstract |
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.
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