Rothenburg S , Deigendesch N , Dey M , Dever TE , Tazi L .

Intramural NIH HHS

	Figure 1. PCR for cloning of T. nigroviridis PKR genes. (A) Results for 5' RACE PCRs with T. nigroviridis cDNA are shown for primers specific for PKR1 (lane 1) and PKR2 (lane 2). M denotes the 1 kb marker. Fragments are labeled in kb. (B) shows the results of 3' RACE PCR using four different forward primers used in primary and nested PCRs for PKR1 (lanes 1–4) and three different forward primers combinations for PKR2 and PKR3 (lanes 5–7). The smaller fragment represents PKR2 and the larger one represents PKR3. (C) PCR products are shown using primers covering the complete open reading frames of PKR1 (lane 1), PKR2 (lane 2) and PKR3 (lane 3). (D) PCR reactions of overlapping regions were performed with genomic DNA to elucidate the genomic organization of PKR1. Lanes 1 and 2 show the PCR products obtained with primers spanning the region between the 5' untranslated region of PKR1 and exon 15 and exon 14 and the 3' untranslated region of exon 19, respectively. PCR product shown in lane 3 was obtained with primers covering exon 17 of PKR1 and exon 7 of PKR2. PCR products were cloned and completely sequenced.
	Figure 2. Schematic presentation of the domain organization of PKR and PKZ genes. The domain organization of PKR and PKZ genes from (A) H. sapiens (Hs), (B) X. laevis (Xl), (C)-(E) T. nigroviridis (Tn), (F) G. aculeatus (Ga) and (G), (H) D. rerio (Dr) are shown in the following colors: dsRNA binding domains (dsR), dark blue; Z-DNA binding domains (Zα), light blue; kinase domains (KD), red; kinase inserts (KI), yellow. Total length of deduced open reading frames in amino acids (aa) is indicated above the schematics.
	Figure 3. Comparison of expression patterns of zebrafish PKR and PKZ after induction with poly(I:C). PCRs were performed with cDNA prepared from the indicated tissues with primers covering the complete ORFs of zebrafish PKR (upper panel), PKZ (middle panel) or RACK1, the latter of which is constitutively expressed and served as control (lower panel). Zebrafish were either treated with poly(I:C) (indicated by plus) or with PBS (minus).
	Figure 4. Genomic arrangement of tandemly arranged PKR and PKZ genes in T. nigroviridis, X. tropicalis and D. rerio. The genomic arrangement and relative orientation of three PKR genes in T. nigroviridis and X. tropicalis and PKR and PKZ in D. rerio are shown. Arrows indicate 5' to 3' orientation of genes. The distance or approximate sizes of intergenic regions are indicated.
	Figure 5. Phylogenetic relationship of PKR and PKZ genes. The phylogenetic tree was constructed from the kinase domains of PKR (blue branches) and PKZ (red branches), omitting the KI, using maximum likelihood and BMCMC approaches. Both analyses resulted in the same tree topology. Significant bootstrap values above 70 (for maximum likelihood analysis) and significant posterior probabilities converted to percentages above 95 (for Bayesian analysis) are shown above and below the branches, respectively. Asterisks denote evident duplication events. Human and zebrafish PERK were used as outgroups for rooting the phylogenetic tree. The following abbreviations were used: Bt, Bos taurus (cattle); Ss, Sus scrofa (pig); Cf, Canis familiaris (dog); Oc, Oryctolagus cuniculus (European rabbit); Ec, Equus caballus (horse); Mam, Macaca mulatta (Rhesus macaque); Cae, Chlorocebus aethiops (African green monkey); Pt, Pan troglodytes (chimpanzee); Hs, Homo sapiens (human); Mm, Mus musculus (house mouse); Rn, Rattus norvegicus (Norway rat); Ma, Mesocricetus auratus (golden hamster); Oa, Ornithorhynchus anatinus (platypus); Md, Monodelphis domestica (gray short-tailed opossum); Gg, Gallus gallus (chicken); Xt, Xenopus tropicalis (western clawed frog); Xl, Xenopus laevis (African clawed frog); Tn, Tetraodon nigroviridis (green spotted pufferfish); Tr, Takifugu rubripes (torafugu); Ga, Gasterosteus aculeatus (three spined stickleback); Ol, Oryzias latipes (Japanese medaka); Dr, Danio rerio (zebrafish); Pp, Pimephales promelas (fathead minnow); Ca, Carassius auratus (goldfish); Ssa, Salmo salar (Atlantic salmon).
	Figure 6. Multiple sequence alignment of the kinase domains of PKR and PKZ genes from various species. Multiple sequence alignment of the kinase domains PKR and PKZ from the indicated species (abbreviations are explained in Figure 5 legend) was performed using MUSCLE [64]. Secondary structure elements as reported for human PKR [10] as well as numbering of residues relative to human PKR are shown above the sequences. Residues involved in PKR inter-dimer contacts (pluses) and eIF2α recognition (asterisks) are marked above the sequences. Residues or deletions that show evidence for convergent evolution (blue) or are lineage specific (red) are colored. The backgrounds of residues that are highly conserved are colored as follows: 100% conservation, dark green; 90–99% conservation, light green; 80–89% conservation, yellow; conservation of functionally conserved residues, salmon pink.
	Figure 7. Exon/intron organization of PKR and PKZ genes. The exon/intron structure of T. nigroviridis PKR1, D. rerio PKR and PKZ, G. aculeatus PKR and H. sapiens PKR (as reported [47]). Untranslated regions of exons are represented as white boxes, while colored and gray boxes denote exons of ORFs. Exonic parts encoding dsRBDs (dark blue), Z-DNA binding domains (Zα; light blue), kinase domains (red) and KIs (yellow) are colored. Exons are drawn to scale. Lengths (in bp) of exonic parts contributing to the ORFs are shown below the exons. Lengths (in bp) of introns are shown in between exons. Question marks indicate intron sizes of unknown length.
	Figure 8. Both zebrafish PKR and PKZ phosphorylate yeast eIF2α. (A) Plasmids expressing full-length human (hs) PKR or zebrafish (dr) PKZ or two different alleles of PKR, under the control of a galactose-inducible promoter, were introduced into S. cerevisiae strains H2557 (wild-type eIF2α, upper plates) and J223 (eIF2α-S51A, lower plates) as indicated. After two rounds of single colony purification, the transformants were streaked out simultaneously on SD-ura (non-inducing conditions, left) or SGal-ura (inducing conditions, right) medium and grown for three days (SD-ura) or four days (SGal-ura). (B) Western blot analyses of extracts from strain H2557 transformed with vector, HsPKR, DrPKR or DrPKZ. SDS-PAGE was used to separate 4 μg of WCEs which were blotted onto nitrocellulose membranes. Tagged PKR and PKZ were detected using anti-Flag-tag antibody (upper panel). eIF2α phosphorylated on Ser51 and total eIF2α are shown in middle and bottom panels, respectively.

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