Cloning and characterization of the 5'-flanking region of the rat P4Halpha gene encoding the prolyl 4-hydroxylase alpha(I) subunit.
Prolyl 4-hydroxylase (P4H), an alpha(2)beta(2) tetramer, plays a crucial role in collagen synthesis. It catalyzes the hydroxylation of proline residues in X-Pro-Gly sequences to form 4-hydroxyproline. We isolated a genomic clone of the rat P4Halpha(I) gene. Approx. 0.6 kb of the fragment, which contained the 5'-flanking region, exon 1, and intron 1, was sequenced. Computer analysis revealed several motifs that may act as binding sites for basal transcription factors. To elucidate the regulation of the rat P4Halpha(I) gene expression, we assessed the 0.6 kb 5'-flanking region of the P4Halpha(I) gene for basal promoter activity. A series of deletion mutants of the 5'-flanking region linked to the luciferase gene was constructed. The basal expression level of these constructs was determined in fetal rat lung fibroblasts and Hepa-1 hepatoma cells. By measuring the luciferase activity, we found a positive-regulatory region at positions -246 to -165 bp.
Takahashi
Shigeru
S
Laboratory of Environmental Molecular Physiology, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, 192-0392, Tokyo, Japan.
Dohi
Naoki
N
Takahashi
Yuji
Y
Miura
Takashi
T
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D005809:Genes, Regulator; D015698:Genomic Library; D051379:Mice; D008969:Molecular Sequence Data; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000737:chemistry; D051381:Rats; D015183:Restriction Mapping; D014157:Transcription Factors / Q000737:chemistry; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D012333:RNA, Messenger; D014157:Transcription Factors; D011393:Procollagen-Proline Dioxygenase