Positive and negative regulation of the transcription of the human protein kinase C beta gene.
To analyze the mechanism of the cell type-specific expression of protein kinase C beta (PKC beta), we isolated the 5'-portion of the human gene for PKC beta and identified multiple positive and negative regulatory sequences that regulate its transcription. S1 nuclease mapping as well as primer extension analysis of the 5'-end of the PKC beta mRNA identified a putative transcriptional initiation site (position +1) 484 base pairs (bp) upstream of the first ATG codon. The 5'-upstream sequence contains a CCAAT sequence at position -110, but no TATA box. The transcriptional activities of various 5'-deletion mutants of the PKC beta gene upstream region, fused to the chloramphenicol acetyltransferase structural gene, were examined in terms of chloramphenicol acetyltransferase expression after transfection into three kinds of rodent cell lines: P19 and GH4C1, which are positive for the expression of PKC beta mRNA; and 3Y1, which is negative. Mutants containing a 5'-flanking sequence longer than 1.9 kilobases (kb) showed chloramphenicol acetyltransferase activities of the same order as the expression of the endogenous gene. This indicates that this region contains sequences regulating the cell-type specificity of PKC beta gene expression and that the specificity is determined at least partially at the level of transcription. The 1.9-kb sequence contains at least three positive elements: P1 (-56 to -234 bp), P2 (-234 to -411 bp), and PN (-1.4 to -1.9 kb). PN is active only in P19 cells, P1 in GH4C1 and P19 cells, and P2 in all three cell lines. In addition to these positive elements, there are negative elements: N1 (-411 to -674 bp), which is active in all three cell lines; and PN, which is active only in GH4C1 cells. These results suggest the presence of multiple trans-acting factors that act on these positive and negative cis-acting elements and regulate the cell type-specific expression of the PKC beta gene.
Niino
Y S
YS
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
Ohno
S
S
Suzuki
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D015971:Gene Expression Regulation, Enzymologic; D015698:Genomic Library; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D010957:Plasmids; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D011493:Protein Kinase C