tailieunhanh - Báo cáo khoa học: A designed curved DNA segment that is a remarkable activator of eukaryotic transcription

To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase (tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. | ỊFEBS Journal A designed curved DNA segment that is a remarkable activator of eukaryotic transcription Noriyuki Sumida1 Jun-ichi Nishikawa1 Haruka Kishi1 Miho Amano1 Takayo Furuya1 Haruyuki Sonobe1 and Takashi Ohyama2 3 1 Department of Biology Faculty of Science and Engineering Konan University Kobe Japan 2 Department of Biology Schoolof Education Waseda University Tokyo Japan 3 Graduate Schoolof Science and Engineering Waseda University Tokyo Japan Keywords chromatin chromatin engineering curved DNA supercoil transcription activator Correspondence T. Ohyama Department of Biology School of Education Waseda University 1-6-1 Nishi-Waseda Shinjuku-ku Tokyo 169-8050 Japan Fax 81 3 3207 9694 Tel 81 3 5286 1520 E-mail ohyama@ Received 11 September 2006 revised 19 October 2006 accepted 25 October 2006 doi To identify artificial DNA segments that can stably express transgenes in the genome of host cells we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp T32 and 180 bp T20 were able to activate transcription from the herpes simplex virus thymidine kinase tk promoter by approximately 150-fold and 70-fold respectively compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. Interestingly T20 was found to activate transcription in all the stable transformants irrespective of the locus. This suggests that the T20 segment may allow stable expression of transgenes which is of importance in many fields and may also be useful for the construction of nonviral .