| Project/Area Number |
02680216
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
分子遺伝学・分子生理学
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| Research Institution | National Institute of Genetics |
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Principal Investigator |
KANEDA Sumiko National Institute of Genetics, Department of Molecular Genetics, Assistant, 分子遺伝研究系, 助手 (60152815)
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| Project Period (FY) |
1990 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1992: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1991: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1990: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | thymidylate synthase / cell cycle / intron / enhancer / チミジル酸合成酵 / 細胞周期 / プロセシング / 形質転換 |
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| Research Abstract |
Thymidylate synthase(TS) plays an essential role in regulating the balanced supply of the four DNA precursors for normal DNA replication. We isolated lambda phage clones containing the functionally active human TS gene and determined the complete nucleotide sequence and the major transcriptional start sites. The biologically active unit spans about 16 kilo bases and is composed of seven exons and six introns. There is no typical promoter sequence. A human TS minigene containing 5'- and 3'-flanking sequences, all the exons and only intron 1 showed a normal frequency of stable transformation when transfected into TS-negative mutant cells, whereas minigenes in which intron 1 was replaced by intron 2 or deleted in the above construct showed only a few percent of the above frequency. Introduction of intron 1 into the above intronless or intron 2-minigene restored the transforming activities regardless of its position and orientation. Deletion analysis revealed two positive and one negative regulatory sequences in the 5'-end of intron 1, each of which seemed to bind specific proteins as shown by gel shift analysis. Intron 1 also stimulated expression of a TS promoter-CAT gene construct but not that of an SV40 promoter-CAT gene construct. These results clearly indicates that the intron 1 regulatory sequence cluster is a promoter specific enhancer which is basically different from known transcriptional enhancers. Intron 1 was also shown to be a major determinant of the cell cycle-dependent fluctuation of TS mRNA at posttranscriptional steps. Therefore, we postulated that there is a link between transcription and processing of the precursor mRNA in the expression of human TS minigenes.
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