| Project/Area Number |
09671059
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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 |
内分泌・代謝学
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| Research Institution | Kobe University |
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Principal Investigator |
OKIMURA Yasuhiko Kobe University School of Medicine, Department of Medicine, Instructor., 医学部・附属病院, 助手 (30204100)
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| Co-Investigator(Kenkyū-buntansha) |
CHIHARA Kazuo Kobe University School of Medicine, Department of Medicine, Professor., 医学部, 教授 (00107955)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Growth Hormone / Growth hormon-releasing peptide receptor / Growth hormon-releasing hormone receptor / pituitary / Pit-1 / gene expression |
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| Research Abstract |
We cloned the 5'-flanking region of the human growth hormone-releasing hormone receptor (GHRH-R) gene and determined the nucleotide sequence of 2.7 kb upstream from the translation start site. RNase protection analysis showed the major transcription start site is 122bp upstream from the translation start site. The 5'-end of the longest initial 5'-RACE product was close to the site. There were no typical TATA homologies but several putative regulatory elements including Pit-i binding site-like element. Transient transfection studies using a luciferase reporter gene demonstrated 5-flanking region had promoter activity in GH3 cells (derived from rat pituitary tumor) but not in non-pituitary cells, BeWo and HeLa cells. However, co-transfection of Pit-1 expression vector increased luciferase activity in BeWo cells. Deletion study showed the regions from -310 to -130 and from -130 to -120 were important for the GHRH-R gene expression in GH3 cells, although the latter less contributied to the
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gene expression. In BeWo cells co-transfected with Pit- 1 expression vector, the region from -310 to -130 was essential for the Pit-1 - dependent expression of GHRH-R gene. The region from -310 to -120 has two putative Pit-1 binding elements, P1 and P2, located from -129 to -123 and from -171 to - 160, respectively. Both mobility shift assay and DNase-l foot print analysis showed that P2 had much higher Pit -1 binding affinity than P1. These findings were consistent with the results that the region from -310 to - 130 is an important element for Pit-i-dependent expression of GHRH-R.In addition, we cloned human growth hormone secretagogue receptor (GHS-R) gene containing the 5-flanking region of 0.6-2.9 kb. Analysis of the hGHS-R transcripts with 5'RACE suggested that putative transcription initiation site was -453 bp upstream from the translation start site. There was no TATA, CAAT, or GC box but an initiator-like sequence sequence. The 5'-flanking region was inserted into a luciferase reporter vector had promoter activity in GH3 cells. The hGHS-R promoter activity appeared to be from -734 to -608. Less
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