1998 Fiscal Year Final Research Report Summary
Identification and cDNA cloning of the transcriptional factors regulating the tissue-specific expression of human alpha-folate receptor gene
| Project/Area Number |
09670793
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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 |
Pediatrics
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| Research Institution | Kanazawa University |
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Principal Investigator |
SAIKAWA Yutaka University Hospital, Kanazawa University Assistant Professor, 医学部・附属病院, 講師 (60283107)
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| Project Period (FY) |
1997 – 1998
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| Keywords | alpha human folate receptor gene / tissue specific expression of the gene / drug resistance / transcriptional factor |
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| Research Abstract |
1. I-dentification of the nuclear DNA-binding proteins involving the specific expression of alpha human folate receptor (hFR) gene : we have identified two promoters which are independently active in a tissue-specific manner in regulation of the alpha hFR gene expression. To further characterize the regulation of these promoters, the transcriptional elements involved in the promoter located upstream of exon 1 were investigated in the transport-defective methotrexate-resistant KB cells (02) by sequence analysis of the promoter, gel shift assays, nuclear run-on assays, and RNase protection assays. Compared to wtKB cells grown in physiologic concentrations of folate, we demonstrate that (1) the 02 cells expressed 2% of a hFR protein and its mRNA ; (2) the transcription rate of a hFR gene was reduced 7 fold in the 02 cells relative to wtKB cells ; (3) the nuclear protein(s) that forms a complex with a -352/-461 (109 bp) DNA fragment located approximately 400 bp upstream of the transcription start site was demonstrated in wtKB cells and was significantly reduced in the C2 cells ; (4) based on competitive gel shift assays using several synthetic oligonucleotides corresponding - 426/-461 DNA sequences, a -4391-452 DNA fragment contained the specific binding sites of this nuclear protein(s). This sequence contains potential binding sites of CCAAT-enhancer binding protein beta, GATA-2, and Ets-1. The supershift assays using antibodies against these proteins will be performed to characterize the factor(s) to this region.
2. Tissue specific expression of the nuclear factor(s) : the binding of nuclear protein(s) to this DNAfragment was also decreased in MCF-7, MDCK, ACHN, SN12C, and TK- 10 celllines in which expression of the alpha hFR is not detectable. These results suggest that this factor may be involved in the tissue-specific expression of alpha hFR gene and in the modulation of receptor expression in antifolate resistant KB cells.
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Research Products
(18 results)
