| Project/Area Number |
01570131
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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 |
General medical chemistry
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| Research Institution | Osaka University |
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Principal Investigator |
NOGUCHI Tamio Osaka Univ. Faculty of Med. Assist. Prof., 医学部, 講師 (70135721)
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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: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1989: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Pyruvate kinase genes / Enhancer / Transcription factor / Tissue-specific expression / Alternative splicing / cis-Acting element / CATアッセイ / 肝特異的転写因子 |
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| Research Abstract |
Cis-Acting elements responsible for tissue- (cell-) specific expression of the pyruvate kinase L gene was studied by transient DNA transfer experiments with chloramphenicol acetyltransferase fusion and by transgenic mice carrying the fusion gene. We found three positive regulatory regions required for expression of the L-type isozyme in adult rat hepatocytes. These regions were designated as PKL-I, PKL-II, and PKL-III. PKL-I showed enhancer-like activity alone, whereas PKL-II PKL-III did not have any independent effect. The inclusion of all three elements oriented in the same direction had the maximum synergistic effect, indicating that these elements function as a unit. This unit enhanced expression from heterologous as well as homologous promoters in a manner that was independent of its orientation and position relative to the cap site. The activity of the unit was not detected in HeL a cells. The 5' flanking region of the L gene containing the unit also directed tissue-specific expr
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ession of transgene in a manner similar to that of the endogeneous L-type isozyme. Thus, this unit is responsible for tissue (cell)-specific expression of the L-type isozyme. Gel retardation assay indicated that the transcription factor called LF-B1 bound to PKL-I and that different unknown nuclear proteins bound to PKL-II and PKL-III. Genomic clones containing the human pyruvate kinase M gene were isolated and their sequences were determined. The gene is approximately 32 kb long and consists of 12 exons and 11 introns. The 5' flanking region of the gene showed high sequence similarity with that of the rat M gene. Bacterial chloramphenicol acetyl-transferase assay revealed that the upstream region from -493 to - 51 contained a cis-acting element (s) that was essential for expression of the M gene in Hela calls. Long stretches of conserved regions were found in the introns around the M_1- and M_2-specific exons, suggesting that these regions may be involved in the alternative splicing machinery. Less
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