1992 Fiscal Year Final Research Report Summary
POSITIONAL CLONING OF CANDIDATE GENES
| Project/Area Number |
03404029
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurology
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| Research Institution | TOKYO MEDICAL AND DENTAL UNIVERSITY |
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Principal Investigator |
MIYATAKE Tadashi TOKYO MEDICAL AND DENTAL UNIVERSITY DEPARTMENT OF NEUROLOGY,PROFESSOR, 神経内科, 教授 (50048998)
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| Co-Investigator(Kenkyū-buntansha) |
UCHIHARA Toshiki TOKYO MEDICAL AND DENTAL UNIVERSITY DEPARTMENT OF NEUROLOGY,INSTRUCTOR, 神経内科, 助手 (10223570)
YANAGISAWA Katsuhiko TOKYO MEDICAL AND DENTAL UNIVERSITY DEPARTMENT OF NEUROLOGY,INSTRUCTOR, 神経内科, 助手 (10230260)
R.C.PARK MATSUMOTO TOKYO MEDICAL AND DENTAL UNIVERSITY DEPARTMENT OF NEUROLOGY,INSTRUCTOR (60165917)
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| Project Period (FY) |
1991 – 1992
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| Keywords | HEREDITARY NEURODEGENERATIVE DISEASE / REVERSE GENETICS / hn cDNA / POSITIONAL CLONING / COSMID CLONES / ADRENOLEUKODYSTROPHY / X CHROMOSOME / XQ28 |
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| Research Abstract |
By positional cloning, we attempted to identify the disease genes of hereditary neurological diseases mapped to Xq28 region including Adrenoleukodystrophy (ALD) and Emery-Dreifuss muscular dystrophy. The cosmid genomic library was constructed from a somatic cell hybrid. X3000-11.1, which carried Xq24-qter as an only human chromosome, and 1,784 cosmid clones carrying various part of human Xq24-qter region were isolated. Four hundreds of the 1,784 cosmids were identified to be Not I linking clones, and 19 independent clusters of the linking clones were regionally mapped to Xp28 region. We also identified a cDNA (QM gene) located to Xq28, which demonstrated an altered mRNA level in nontumorigenic Wilm's microcell hybrid cell line, and detalied analysis of the genomic structure of the QM gene was also performed. Southern blot analyses revealed no gross genetic alterations of the genomic DNAs from 24 ALD patients using the QM cDNA as the probe.
To isolate the transcribed sequences located in a particular chromosomal region (Xq28), the two major strategies were established. By utilizing human-specific repetitive sequences in heterogeneous nuclear RNA-complementary cDNA (hn cDNA) derived from the somatic cell hybrid,X3000-11.1, 11 transcribed sequences at Xq24-qter region were isolated Norther blot analysis revealed a hn cDNA clone was exclusively expressed in brain. The result indicates that the strategy is also useful to isolate the tissue-specific gene as well as house-keeping gene. We also tried another strategy to isolate the tissue-specific and chromosome-specific genes by utilizing the hybridization between cloned cosmid genomic DNAs and human brain cDNA clones. Subsequently, we isolated a new cDNA which was predominantly expressed in brain and skeletal muscle. The data indicates that the method was also highly useful to identify the tissue-specific genes.
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