| Project/Area Number |
01571221
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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 |
Biological pharmacy
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| Research Institution | University of Shizuoka |
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Principal Investigator |
TAKEISHI Keiichi University of Shizuoka, School of Food & Nutritional Sciences, Professor, 食品栄養科学部, 教授 (90012608)
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| Co-Investigator(Kenkyū-buntansha) |
HORIE Nobuyuki University of Shizuoka, School of Food & Nutritional Sciences, Research Associat, 食品栄養科学部, 助手 (70209287)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Thymidylate synthase / Human genomic DNA / Promotor / CAT vector / Regulatory factor / Differentiation / DNA binding protein / 転写制御因子 / ミニ遺伝子 |
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| Research Abstract |
In this research project we studied factors involved in the control of expression of human thymidylate synthase (TS) gene.
First, we attempted to identify an unusual promotor sequence of human TS gene utilizing two kinds of CAT (chloramphenicol acetyltransferase) vectors with an enhancer sequence. Using these vectors the promoter activity was first successfully detected and it was concluded that the promotor existed within about 400 base pairs upstream from the transcriptional initiation site. This success opened the way to identify the promotor sequence at the level of a base.
Secondly, we searched for nuclear protein factors that bind to several DNA fragments derived from the 5'-terminal region of human TS gene. For this purpose, we examined nuclear extracts from human cultured cells by the gel mobility shift assay method. As the result, three kinds of nuclear factors were found. One of these factors was confirmed to be a factor which binds to an octamer sequence. The other two factors showed the change in the quantity which depended on the stage of differentiation of human promyelocytic leukemia HL-60 cells, i. e., one decreased during the cellular differentiation, whereas the other increased. More interestingly, it was revealed that both the factors bind to the same DNA region around the ATG initiation codon of human TS gene.
We are planning to purify the nuclear factors for the preparation of antibodies against them and for the determination of their partial amino acid sequences. Utilizing these materials or data, we are going to clone the genes for the factors and to elucidate their role in the regulation of the expression of human TS gene.
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