Figure 1.
Sequence alignments of zebrafish IFIT proteins with Clustal W (1.8) program.
Zebrafish IFIT family genes were identified by BLAST search of zebrafish genome. The putative TPR motifs were indicated by boxes, according to the rule that each motif should contain more than 4 conserved residues of the 8 key sites within the consensus sequences of TPR motifs summarized by NCBI. Identical (*) and similar (. and :) residues are indicated.
Table 1.
Statistics for IFIT homologous gene search from databases.
Figure 2.
Gene organization of IFIT loci from mammals, birds and zebrafish.
The exon/intron structures of eutherian mammals (A), monotreme platypus (B), birds (C) and zebrafish (D) were compared. Chimpanzee and rhesus monkey IFIT1P genes that are located away from the main IFIT loci, and Zebra Finch IFITA residing in other scaffold, were identified by GENSCAN. The exons are indicated by black boxes, and the introns by grey lines. The orientation of the open reading frames is indicated by arrowheads.
Figure 3.
Comparative analyses of gene synteny of vertebrate IFIT loci.
Approximately 10-million-bp DNA sequences flanking IFIT loci of the indicated species were analyzed for gene colinearity. For dog Chromosome 4, Zebra Finch LEG22, western clawed frog GL180711.1, GL181739.1, and torafugu Scaffold 622, only 1170 kb, 195 kb, 25 kb, 12 kb and 7 kb were analyzed due to the incomplete genome information. The surrounding genes, if needed, were predicated by GENSCAN and compared by local blast software. Only the conserved flanking genes were shown. The red surrounding genes are conserved in all vertebrate lineages. The black is seen in birds, amphibians, fish and dog. The grey is conserved in mammals, birds and amphibians. The blue is in birds and fish, the yellow in fish and amphibians, the purple exclusively in fish lineages, and the green only in mammalian lineages.
Figure 4.
Phylogenetic analyses of IFIT family proteins identified in this study.
A neighbor-joining tree was constructed based on analysis of 107 IFIT family protein sequences using Geneious Pro 5.4.6 program, with bootstrap values for 1000 replicates. Human FKBP4 and zebrafish FKBP4 as well as the TPR-containing proteins identified from acorn worm, lancelet, hagfish and sea lamprey were included, indicating that these proteins are not real IFIT homologues (indicated by black). There are long C-terminus of zebrafish IFIT5A (from position 509 to 773 amino acid) and N-terminus of dog IFIT5L (from 1 to 245 amino acid), which were not conserved and then were deleted to exclude disturbance for multiple alignments. The blue represented IFIT genes from fishes, the green from birds and reptiles, the yellow from amphibians, and the red from mammals.
Figure 5.
RT-PCR detection of zebrafish IFIT genes in response to poly(I:C) and IFN.
ZFL cells seeded in 6-well plates were transfected with poly(I:C) (4 µg/well, final concentration 4 µg/mL) or with PBS as control (A), and ZF4 cells seeded in 6-well plates were transfected with zebrafish IFN1 construct (4 µg/well, final concentration 4 µg/mL) or with pcDNA3.1 as control (B). At the indicated time points, the treated cells were extracted for detection of IFIT mRNAs.
Figure 6.
Comparative analyses of ISRE-containing promoters of IFIT family genes.
(A) More than 1 kb of 5′ flanking sequences upstream of transcription start sites of IFIT genes from human, chimpanzee, rhesus monkey, mouse, rat, horse, pig, cattle, dog, chicken, Zebra Finch, and zebrafish were searched for ISRE motifs by Geneious and TFSEARCH combined with manual inspections. ISRE motifs are indicated by black rectangles and the numbers above show the sequence distances from the transcription start sites. (B) Schematic representations of six zebrafish IFIT gene promoter-driven luciferase constructs.
Figure 7.
Activation of zebrafish IFIT promoters by recombinant IFN, Poly I:C and IRF3/IRF7.
(A) EPC or CAB cells seeded in 24-well plates were cotransfected with 0.25 µg of zebrafish IFIT promoter-driven luciferase constructs and 0.025 µg of pRL-TK. 24 h post transfection, cells were treated by recombinant IFN (20 ng/ml) for another 24 h, and then harvested for detection of luciferase activity. The fold induction was normalized by the luciferase activity from control cells. (B) EPC or CAB cells seeded in 24-well plates were cotransfected as in (A). 24 h post transfection, cells were transfected again with 4 µg/ml poly(I:C) for another 24 h, then harvested for detection of luciferase activity. (C and D) EPC (C) and CAB cells (D) seeded in 24-well plates were cotransfected as in (A) with the indicated plasmids. 48 h post transfection, cells were harvested for detection of luciferase activity. Luciferase activities were presented as relative light units (RLU) normalized to the amount of Renilla luciferase activities.