Mouse interferon-inducible RNA-dependent protein kinase Pkr gene: cloning and sequence of the 5'-flanking region and functional identification of the minimal inducible promoter.
The RNA-dependent protein kinase (PKR) is implicated in the antiviral and antiproliferative actions of interferon (IFN). As an extension of our structural characterization of the exon-intron organization of the mouse Pkr gene, we now have isolated and characterized the mouse Pkr promoter region required for IFN-inducible transcription. Transient transfection analyses, using reporter constructs possessing various 5'-flanking fragments of the Pkr gene, led to the identification of a functional IFN-inducible promoter. A single IFN-stimulated response element (ISRE) was present in a minimal 44-nt TATA-less promoter identified by deletion analysis; the 13-nt ISRE differed from previously described ISRE elements in that the 3'-nt was a purine instead of a pyrimidine. The sequence immediately upstream of the ISRE possessed the 15-nt KCS element that was exactly conserved in sequence and position between the mouse and human Pkr promoters. A single gamma IFN-activated sequence (GAS)-like element and multiple recognition sites for factors including NF-kappaB and NF-IL6 involved in responses to various cytokine and hormone signals in inflammatory responses were also present in the 5'-flanking region. Northern blot analysis showed efficient IFN-alpha induced accumulation of 2.4kb, 4.5kb and approx. 6kb Pkr transcripts, but neither IFN-gamma nor IL-6 induced detectable Pkr mRNA accumulation in L cells.
Tanaka
H
H
Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, USA.
Samuel
C E
CE
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D007372:Interferons / Q000494:pharmacology*; D007739:L Cells; D051379:Mice; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D019892:eIF-2 Kinase / Q000235:genetics*
D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma; D004247:DNA; D007372:Interferons; D015500:Chloramphenicol O-Acetyltransferase; D019892:eIF-2 Kinase