| Project/Area Number |
08457630
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Human genetics
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| Research Institution | Nagasaki University |
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Principal Investigator |
JINNO Yoshihiro Nagasaki University, School of Medicine, Associate Professor., 医学部, 助教授 (20179097)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIMARU Tadayuki Nagasaki University, School of Medicine, Professor., 医学部, 教授 (20039580)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1997: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1996: ¥3,300,000 (Direct Cost: ¥3,300,000)
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| Keywords | genomic imprinting / H19 gene / cis-acting factor / repetitive sequence / PEN11B gene / atypical imprinted gene / 非定形的刷り込み遺伝子 / インプンリティング |
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| Research Abstract |
Molecular analeses of a novel gene which was isolated in the first year were a main subject in the current year.
(1) Identification of cis-acting factors
i) A repetitive sequence consisting of tandem repeats of pentanucleotides, which showed homology to the H19 downstream pentamer repeats, was found at 3.2 kb upstream of the first exon in the novel gene. Pentanucleotode of CCCAG was reiterated 30 times and CTCAG 9 times in the repetitious 375 bp sequence. It was more degenerate compared to the 280 bp H19 repeat in which pentanucleotides of CCCAG and CCCTG were repeated 24 times and 20 times, respectively.
ii) A BstUI/HhaI RFLP was identified in the 3' UTR.Allele-specificity in expression of this gene was analyzed by taking advantage of this RFLP.
iii) While it was equally expressed from both alleles in the brain, liver and kidney, two-fold allelic difference in expression was found in the placenta. Parental origin of preferrentially expressed allele could be ascertained in 5 cases among nine examined, and it was consistently maternal. Thus, we considered it to indicate an atypical imprinted gene.
iv) So far, differential methylation has not yet identified in the 5' upstream region and second intron in which another kind of repetitive sequence was present.
v) We collected 7 triplets placentae and 2 quadruplets placentae in order to identify sequences responsible for polymorphic imprinting. But, preliminary experiments suggested insuitability of using placentae for this pourpose, and therefore we abandoned this subproject.
(2) Isolation and identification of trans-acting factors
Being late in starting this subproject because of unexpectedly taking time in the above work, we are now trying to find the best condition of subtractive PCR.
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