权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Selective isolation of human promoter sequences that carry a bent DNA structure and analyzes of their structures and function

选择性分离携带弯曲DNA结构的人类启动子序列并分析其结构和功能

基本信息

批准号：
07680751
负责人：
OHYAMA Takashi
金额：
$ 1.47万
依托单位：
KONAN UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680751/
关键词：
bent DNA promoter human genome unusual DNA higher order structure chromatin ベントDNA (bent DNA)

项目摘要

Information about the localization of bent DNA around promoters and about the conformational characteristics of these curvatures is likely to help explain the role of DNA curvature in eukaryotic transcription. We developed a method that begins the first step in that process, namely selective isolation of curved eukaryotic promoters.Bent DNA fragments in a digest of human genomic DNA was separated from "normal" fragments by using a biotinylated oligonucleotide and streptavidin-coated magnetic particles. The oligonucleotide was designed to hybridize specifically to bent DNA fragments. The sequence of the oligonucleotide was 5'-(T_4N_6)_3T_4-3'(N=A,G,or C). The magnetic isolation of bent DNA fragments were successful. It took only about five hours to accomplish the isolation. In addition, about 80% of the isolated fragments were bent DNAs. Taking it into consideraton that the conventional isolation method, which employs 2D gel electrophoresis, occupies about two days, our method seemed much superior to the conventional method.Then, we tried to introduce curved fragments into a promoter trap vector pATO or into the vector pGL2-basic for luciferase assay. However, these fragments could not be introduced efficiently. It seemed that the bent DNAs captured by the above method possessed the conformations which were not favored by pATO and pGL2-basic. Therefore, we prepared bent DNA segments by the conventional method. The bent fragments thus obtained could be introduced into pGL2-basic successfully. Starting from about 700 clones, we obtained 15 bent DNA fragments that showed promoter activities and determined the sequences of two clones. Now, we are analyzing the transcription initiation site and the curved center of each clone.

关于启动子周围弯曲DNA的定位以及这些弯曲的构象特征的信息可能有助于解释DNA弯曲在真核转录中的作用。我们开发了一种方法，该方法开始了该过程的第一步，即选择性分离弯曲的真核启动子。通过使用生物素化的寡核苷酸和链霉亲和素包被的磁性颗粒，将人类基因组DNA消化物中的弯曲DNA片段与“正常”片段分离。寡核苷酸被设计为与弯曲的DNA片段特异性杂交。该寡核苷酸序列为5 '-（T_4N_6）_3T_4- 3'（N=A、G或C）。磁性分离弯曲DNA片段是成功的。只用了五个小时就完成了隔离。此外，约80%的分离片段是弯曲的DNA。考虑到传统的二维凝胶电泳分离方法需要两天左右的时间，我们的方法似乎比传统的方法上级得多。然而，这些片段不能被有效地引入。用上述方法捕获的弯曲DNA似乎具有pATO和pGL 2-basic所不喜欢的构象。因此，我们通过常规方法制备了弯曲的DNA片段。获得的弯曲片段可成功地导入pGL 2-basic中。从约700个克隆开始，我们获得了15个显示启动子活性的弯曲DNA片段，并确定了两个克隆的序列。现在，我们正在分析每个克隆的转录起始位点和弯曲中心。