Loss-of-function analyses of zebrafish PERK. (A) A phylogenetic tree constructed by the neighbor-joining method for the full-length amino acid sequences of PERK proteins. c, chicken; ce, C. elegans; d, D. melanogaster; h, human; m, mouse; xt, Xenopus tropicalis; z, zebrafish. (B) The expression of perk, chop, and bip analyzed by RT-PCR using total RNA from 8-h–postfertilization embryos injected or not with 1 pmol perkMO. s, spliced form; us, unspliced form. The amount of cDNA used for RT-PCR was standardized by the ef1α expression. (C) Induced expression of gstp1 after 12-h treatment of 5 μg/mL tunicamycin (TM), 2 μM thapsigargin (TG), or 100 μM diethyl maleate (DEM) in WT AB larvae injected or not with 1 pmol perkMO. The numbers in each picture indicate the positive larvae/tested larvae. (D) The deletion in exon 13 of perk in the it312-mutant genome and schematic representations of the WT and mutant proteins. A dotted bar indicates an artificial 66-aa C-terminal region generated by the frame shift mutation. IRE1-like, IRE1-like domain; Kinase, kinase domain; SP, signal peptide region; TM, transmembrane domain.
|
