| Project/Area Number |
62480260
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Hematology
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
AOKI Nobuo Department of Medicine, Tokyo Medical and Dental University, 医学部, 教授 (20048937)
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| Co-Investigator(Kenkyū-buntansha) |
MIURA Osamu Department of Medicine, Tokyo Medical and Dental University, 医学部, 助手 (10209710)
KATO Atsushi Department of Medicine, Tokyo Medical and Dental University, 医学部, 助手 (50183265)
HIROSAWA Shinsaku Department of Medicine, Tokyo Medical and Dental University, 医学部, 助手 (50143574)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 1988: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1987: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Keywords | Alpha 2 plasmin inhibitor / Chromosome / Gene / Congenital deficiency of alpha 2 plasmin inhibitor / 染色体 / 遺伝子発現 / アルファ2プラスミンインヒビター欠損症 / 線維素溶解 / 転写調節 / 血栓溶解 / 遺伝子ファミリー / cDNA |
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| Research Abstract |
1. We have isolated overlapping phage genomic clones covering an area of 26 kilobases that encodes the human alpha_2-plasmin inhibitor (alpha_2PI). The alpha_2PI gene contains 10 exons and 9 introns distributed over 16 kilobases of DNA. To our knowledge, the number of introns is the highest yet reported for a member of the serine protease inhibitor (serpin) superfamily. All introns are located in the 5'-half of the corresponding mRNA. The 5'-untranslated region and the leader sequence are interrupted by 3 introns totaling 6 kilobases. A "TATA box" sequence is located 17 nucleotides upstream from the proposed transcription initiation site. Multiple "GC box" sequences, G+C-rich sequences, and "CCAAT box"-like sequence, the hepatitis B virus enhancer element-like sequence and the human immunodeficiency virus enhancer-like sequence appear in the 5'-flanking region.
2. The human alpha_2PI gene (PLI) was mapped by in situ hybridization using a genomic DNA probe which contained exons coding for the signal peptide and a portion of the mature protein. The results allowed the chromosome localization of the gene to 18q11.1 q11.2.
3. The molecular genetic basis of a familial deficiency of alpha_2PI was studied. Southern blot hybridization analysis with human alpha_2PI cDNA and genomic DNA probes demonstrated no gross deletion or rearrangement of the gene. BY sequencing all the coding exons and exonintron boundaries of the gene of a homozygote, we identified a single cytidine nucleotide insertion in the exon coding for the carboxyl-terminal reqion. This frameshift mutation leads to an alteration and elongation of the carboxyl-terminal portion of the deduced amino acid sequence. In a transient expression assay, the alpha_2PI level in the culture medium of the cells transfected with the mutated alpha_2PI expression vector was very low and only 4% of that of the cells transfected with the normal vector.
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