| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1995: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
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.
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