2000 Fiscal Year Final Research Report Summary
THE STUDY OF NF1 PROMOTOR-CIS-ELEMENT
| Project/Area Number |
10670808
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Dermatology
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| Research Institution | JIKEI UNIVERSITY SCHOOL OF MEDICINE |
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Principal Investigator |
OTA Arihito JIKEI UNIVERSITY SCHOOL OF MEDICINE DERMATOLOGY FELLOW, 医学部, 助手 (20168933)
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| Co-Investigator(Kenkyū-buntansha) |
KOMATSUZAKI Makoto JIKEI UNIVERSITY SCHOOL OF MEDICINE DERMATOLOGY FELLOW, 医学部, 助手 (30266621)
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| Project Period (FY) |
1998 – 2000
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| Keywords | NEUROFIBROMATOSIS1 / NF1 / PROMOTOR / RAS |
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| Research Abstract |
A number of reporter clones have been constructed to identify cis regulatory elements in the NF1 promoter. The 5' flanking sequence of the human NF1 gene was obtained from the genomic clone cHB5. BamHI fragments from the EcoRI subclone of cHB5 yield some constructs for the initial evaluation of the NF1 promoter. Standard molecular cloning techniques were used to generate the constructs in Promega luciferase reporter vectors. Liposome mediated transfections were performed in COS7 cells and lysates were used for luciferase assays and protein determinations. All NF1 deletion constructs were transfected to COS7 cells with H-Ras(V12) mutant construct. All the luciferase expression showed 7 to 9 fold activity over the each basal activity without H-Ras(V12) mutant construct, which meant unlikely to reside Ras-responsible elements in COS7 cells. Another cell, H9c2, rat heart muscle cell was used for this study. After a series of optimization, the luciferase activity of the co-transfection showed 4.5 fold induction in the 6E construct(-143 to +473) over the transfection of 6E without H-Ras(V12) mutant construct. There are segments of NF1 promoter that appear to contain cis regulatory elements in transient liposomal transfections of H9c2 cells. We are proceeding to the next level of analysis : in vitro DNA binding studies.
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