| Project/Area Number |
10670132
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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 |
Pathological medical chemistry
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| Research Institution | Chiba University |
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Principal Investigator |
HATANO Masahiko Chiba University, Graduate Shool of Medicine, Associate Professor, 大学院・医学研究科, 助教授 (20208523)
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| Co-Investigator(Kenkyū-buntansha) |
OKADA Seiji Chiba University, Graduate Shool of Medicine, Assistant Professor, 大学院・医学研究科, 助手 (50282455)
TOKUHISA Takeshi Chiba University, Graduate Shool of Medicine, Professor, 大学院・医学研究科, 教授 (20134364)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1999: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1998: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | NCX / Hirschsprung-related disease / transcription factor / homebox / DNA binding / SSCP / RDA / 遺伝子発現調節 / 神経堤細胞 / 疾患モデル動物 |
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| Research Abstract |
1. The murine Ncx (Enx,Hox11L1) gene is specifically expressed in a neuronal subset of neural crest derived tissues. In attempts to elucidate the regulatory DNA element of the tissue-specific expression, we sequenced the 5'-flanking region of the Ncx gene. The transcriptional initiation site was determined at the 297 nucleotides (-297) upstram from the ATG start codon (+1). A retinoic acid response element was located on the region between -1163 and -1150. Transient transfection assays with the 5'-flanking sequences fused to the luciferase gene showed that the region between -1387 and -1368 was crucial for the tissue-specific enhancer activity. Furthermore, nuclear proteins extracted from neural crest derived cells such as murine and human neuroblastoma cells bind to the DNA region between -1387 and -1368. This DNA element was also conserved in the 5'-flanking region of the human NCX gene. Our observations strongly suggest that the DNA element (-1387 and -1368) contributes to tissue-sp
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ecific expression of the Ncx gene in murine and human species.
2. We determined specific Ncx protein binding consensus DNA sequences by PCR based random oligonucleotide selection. Optimal Ncx binding sequences were 5'-CGGTAATTGG-3' (a TAAT core) and 5'-CGGTAAGTGG-3' (a TAAG core), which coincided with the Hox11 binding sequence. Both Ncx and Hox11 could specifically bind to the TAAT and the TAAG core oligonucleotide in vitro by electrophoretic mobility shift assay. However, they could efficiently transactivate the luciferase reporter plasmid linked to the TAAT core sequence but not to the TAAG core sequence. Thus, Ncx and Hox11 act as a transcriptional activator via their target sequence, 5'-CGGTAATTGG-3'. We are now trying to identify downstream of Ncx gene by representational difference analysis (RDA).
3. We cloned human NCX gene and determined its structure. Human NCX gene consists of 3 exons and NCX cDNA sequence was highly homologous to mirine Ncx. We established PCR-SSCP method by desinging several sets of primers which can amplify each exons and exon-intron boundaries. Genomic DNA was isolated from peripheral blood of 33 patients with Hirschsprung-related disease. We could identify some silent mutations in coding region or some nucleotides changes in introns. However we could not find any significant mutations which cause amino acid substitution or stop codon. Less
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