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Identification of Replication Units in Mammalian Genome by Fiber-FISH

通过 Fiber-FISH 鉴定哺乳动物基因组中的复制单位

基本信息

批准号：
09660080
负责人：
OKUMURA Katsuzumi
金额：
$ 2.18万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660080/
关键词：
DNA replication FISH DNA fiber Replicon Chromatin structure Bromodeoxyuridine PWS AS replication timing intranuclear structures

项目摘要

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.

荧光原位杂交(FISH)是基因定位和生物学分析的有力工具。本工作将FISH技术应用于动物细胞基因组DNA复制过程的分析。Fibre-FISH使我们能够在几个kb的水平上分析基因组距离。新合成的DNA可以通过检测掺入的胸苷类似物溴脱氧尿苷(BrdU)在DNA纤维上追逐。本研究的主要目的是检验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，对掺入BrdU的检测方法进行了改进。DNA碱性变性可有效、均匀地检测到复制DNA纤维。DNA纤维上DNA复制的进程是以时间依赖的方式进行的。其次，在这种条件下，可以用FISH显示基因组DNA，也可以同时检测新合成的DNA和特定的基因组序列。此外，还建立了HL60细胞的高度同步化方法。这些结果表明，应用这些精细技术可以确定复制子和复制子簇结构域。作为相关研究，发展了一种分析特定基因组序列包装状态的方法。这对于理解包括DNA复制域在内的核内基因组组织是很重要的。基因组克隆的核内包装状态可以在核晕上可视化。这项技术被应用于印记基因Snrpn，表明父亲等位基因上的Snrpn基因比母亲等位基因上的Snrpn基因更紧密。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。