| Project/Area Number |
12670128
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Pathological medical chemistry
|
|---|
| Research Institution | TOHOKU University |
|---|
Principal Investigator |
NATA Koji Tohoku University, Graduate School of Medicine, Research Associate, 大学院・医学系研究科, 助手 (90202233)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KATO Ichiro Toyama Medical & Pharmaceutical University, School of Medicine, Associate Professor, 医学部, 助教授 (50250741)
TAKASAWA Shin Tohoku University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (50187944)
|
|---|
| Project Period (FY) |
2000 – 2001
|
| Project Status |
Completed (Fiscal Year 2001)
|
| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
|
|---|
| Keywords | FK506 / cyclic ADP-ribose / FK506 binding protein / ryanodine receptor / calcium channel / gene family / chromosomal localization / luciferase assay / FISH法 |
|---|
| Research Abstract |
Cyclic ADP-ribose (cADPR) induces the release of Ca^<2+> from microsomes of pancreatic islets for insulin secretion. It has been demonstrated that cADPR binds to FK506-binding protein (FKBP) on rat islet ryanodine receptor and that the binding of cADPR to FKBP frees the ryanodine receptor from FKBP, causing it to release Ca^<2+> (J. Biol. Chem. 272, 3133, 1997). In this study, we have isolated human FKBP12.6 and FKBP 12 gene and determined their structure and chromosomal localization.
1. Human FKBP12.6 gene spanned about 16 kbp in length. The FKBP12.6 gene consisted of four exons and three introns. Human FKBP 12 gene spanned about 20 kbp in length. The FKBP 12 gene consited of five exons and four introns. The positions of exon-intron junction of the FKBP12.6 and FKBP12 genes were perfectly matched except that FKBP12 has an additional exon 5, to code exclusively for 3'-untranslated region.
2. Fluorescence in situ hybridization revealed that the FKBP12.6 gene was located on chromosome 2p21
… More
-23 and the FKBP12 gene was located on chromosome 20p13.
Next, we analyzed the promoter regions of human FKBP12.6 and FKBP 12 genes with luciferase assay.
3. A series of 5'-deletions from -1691 of human FKBP12.6 gene and a series of 5'-deletions from -1706 of FKBP 12 gene were transfected into NB-1 cells for luciferase assay.
4. Deletion of the sequence from -1691 to +8 caused no significant changes of the expression of luciferase. Further deletion to +74 caused a marked loss of the expression. These results suggest the importance of +8〜+74 sequence for the expression of the human FKBP 12.6 gene.
5. Deletion of the sequence from -1706 to -179 caused no significant changes of the expression of luciferase. Further deletion to -74 caused a marked loss of the expression. Insertion of -179〜-74 sequence into -179〜-74 deleted reporter plasmid with reverse orientation and insertion into 3'-portion of the luciferase gene in -179〜-74 deleted reporter plasmid recovered the expression. These results suggest that -179〜-74 sequence acts as an enhancer for the expression of the human FKBP 12 gene. Less
|
