Gene, 153 (1995) 283-284 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50 GENE 08610 283 Sequence of the murine interferon-inducible RNA-dependent protein kinase (PKR) deduced from genomic clones (Translational control; protein synthesis initiation factor eIF-20~ RNA-binding protein; antiviral protein; tumor suppressor; nucleotide sequencing) Hideo Tanaka and Charles E. Samuel Department of Biological Sciences, and Interdepartmental Biochemistry and Molecular Biology Graduate Program, Universityof California, Santa Barbara, CA 93106, USA Received by R. Padmanabhan: 19 September 1994; Accepted: 17 October 1994; Received at publishers: 14 November 1994 SUMMARY The gene encoding the RNA-dependent protein kinase (PKR) was cloned from mouse genomic DNA and characterized by restriction mapping, Sc,uthern blot analysis and sequencing. The Southern analyses were consistent with the presence of a single copy of the Pk:r gene in the mouse haploid genome. The genomic nucleotide (nt) sequence, when compared to that of previously determined cDNA nt sequences, revealed 16 exons encoding the 515-amino-acid PKR. PKR is a RNA-dependent protein serine/threonine kinase inducible by interfe~7on(Samuel, 1993). In the liter- ature prior to 1993, PKR is variously known as DAI, dsI, P1 kinase, p65, p67 or TIK for the mouse enzyme; and p68 or p69 for the human enzyme (Clemens et al., 1993). The PKR kinase plays a central role in the regula- tion of protein synthesi,; in virus-infected and IFN- treated cells (Samuel, 1991; 1993) and has been implicated in the regulation of other activities including cell prolifer- ation and tumor suppression (Lengyel, 1993). Molecular cDNA clones of PKR from mouse cells have been described, although several nucleotide differences are found between the cDNA sequences (Icely et al., 1991; Feng et al., 1992). As an extension of our studies of the regulation and function of PKR, we isolated genomic clones of PKR, characterized the 5'-flanking region and determined the exon-intron sizes and junction sequences Correspondence to: Dr. C.E. Samuel, Department of Biological Sciences, University of California, Santa Barbara, CA 93106, USA. Tel. (1-805) 893-3097; Fax (1-805) 893-4724; e-mail: samuel@lifesci.lscf.ucsb.edu Abbreviations: aa, amino acid(s); bp, base pair(s); IFN, interferon; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); PKR, RNA-dependent protein kinase; Pkr, gene encoding PKR; UTR, untranslated region(s). of mouse Pkr. The mouse Pkr gene contains 16 exons and spans about 28 kb (Tanaka and Samuel, 1994). We now report the entire exon sequence of mouse Pkr determined from genomic clones. A mouse genomic library in LEMBL3 (Clontech) was screened using frag- ments of the mouse Pkr cDNA (Thomis et al, 1992) as probes. Isolated clones were characterized by restriction mapping and Southern blot analysis. The structural organization of the mouse Pkr gene determined from phage genomic clones was confirmed by Southern blot analysis of restriction digests of mouse L-cell genomic DNA; the results are consistent with the presence of a single copy of Pkr in the mouse haploid genome. Exon regions of pBluescript plasmid subclones were sequenced in both directions by the dideoxy procedure using T3 or T7 universal primers and 24 custom Pkr primers. The exon genomic nt sequence of Pkr and the deduced aa sequence are shown in Fig. 1. Mouse Pkr contains 16 exons. Exon 1 (143 bp) and part of exon 2 (16 bp) encode the 159-nt 5'-UTR of the major Pkr transcript (Tanaka and Samuel, 1994). Exon 2 includes the AUG start codon for the deduced 515-aa PKR protein; exon 16 includes the UAG stop codon and the 3'-UTR. Several differences were found between the nt sequence of the Pkr exons SSDI 0378-1119(94)00821-3