| Co-Investigator(Kenkyū-buntansha) |
MATSUO Noritaka Oita Medical University, Biochemistry, Assistant Professor, 医学部, 助手 (10284788)
SHIRABE Koumei Oita Medical University, Biochemistry, Associate Professor, 医学部, 助教授 (50179058)
MOMOTA Ryusuke Okayama University Medical School, Molecular Biology, Assistant Professor, 医学部, 助手 (80263557)
OOHASHI Toshitaka Okayama University Medical School, Molecular Biology, Assistant Professor, 医学部, 助手 (50194262)
NINOMIYA Yoshifumi Okayama University Medical School, Molecular Biology, Professor, 医学部, 教授 (70126241)
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| Research Abstract |
We isolated the 5' flanking region of mouse alphal(XI) collagen gene. As seen in human gene, this promoter has several transcription start sites. Comparison of the sequence of the promoter with consensus regulatory elements showed that there was no TATA or CCAAT box, and that there were several potential Sp1 binding sites.
Two promoter fragments , 617 bp (short promoter) and 5 kb (long promoter) in size, sharing the same 3 end were subcloned in the upstream of luciferase. And three Sma fragments, 3.5 kb, 3.0 kb and 7.0 kb in the first intron were subeloned in the downstream of the these constructs. We transfected the DNAs into bovine aorta smooth muscle cells ad human chondrosarcoma cells. The luciferase activity of short promoter fragment was higher than that of long promoter in both cells. Three fragments of first intron seemed to depress the activity of promoter.
To analyze alternative splicing in the acidic region, we made mini genes for the deletion of exon 6A, 6B, 7, 8 and 6A-8, respectively. We transfected these genes in 204 (rhabdomyosarcoma cell), RCS (rat chondrosarcoma) and 293 (kidney cell). In 293 cell, the exon 6A-7-8 is main splice form, and exon 6B is not expressed. In 204 cell, exon 6A-7-8 is the abundant splice form while 7-8 is also present. In the RCS cell, five splice forms are present. In order to analyze the function of the acidic domain in vivo, we have also begun engineering the gene targeting constructs for the deletion of exon 6A and 6B.
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