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.

有关弯曲DNA在启动子周围的定位以及这些曲率的构象特性的信息可能有助于解释DNA曲率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，即弯曲的真核启动子的选择性隔离。通过使用生物素化的寡核苷酸和链甲蛋白链球菌蛋白涂的磁性颗粒，将人类基因组DNA的摘要中的eNT DNA片段与“正常”片段分离出来。寡核苷酸的设计旨在专门杂交弯曲的DNA片段。寡核苷酸的序列为5' - （T_4N_6）_3T_4-3'（n = a，g或c）。弯曲DNA片段的磁隔离成功。完成隔离只花了大约五个小时。另外，约80％的孤立片段是弯曲的DNA。考虑到采用2D凝胶电泳的常规分离方法占据了大约两天的时间，我们的方法似乎优于常规方法。然后，我们试图将弯曲的片段引入启动子陷阱矢量PATO或载体PGL2-Basic for Luciferase分析中。但是，这些碎片无法有效地引入。看来，以上方法捕获的弯曲DNA具有Pato和PGL2-Basic不支持的构象。因此，我们通过常规方法制备了DNA片段。因此获得的弯曲片段可以成功地引入PGL2基础。从约700个克隆开始，我们获得了15个弯曲的DNA片段，这些碎片片段显示出启动子活性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。