Figure 2
Generation of pam−/− zebrafish. (A) Schematic depicting the domains in zebrafish Pam protein. The signal sequence (orange), PHM domain (purple) with two copper-binding sites, linker region (Exon A; black), PAL catalytic domain (red), transmembrane domain (TMD; yellow) and cytosolic domain (CD; green) are shown; residue numbers indicate the boundaries corresponding to each domain. (B) Predicted protein sequences for three pam mutant lines generated through CRISPR-Cas9 genome editing; frame-shift mutations (in red) result in truncation of all three proteins before the beginning of the PHM domain. The colored lines indicate the signal sequence (orange) and beginning of the PHM domain (blue). (C) PHM enzyme assays of embryos collected at the indicated developmental stages from wildtype siblings (pam+/+) and both heterozygous (pammbg5+/−) and homozygous (pammbg5−/−) pammbg5 animals. At all stages, the homozygous mutant had no detectable PHM activity. Data plotted as mean ± SD (n = 3). (D) PAL enzyme activity in pam+/+, pammbg5+/− and pammbg5−/− 7 dpf zebrafish embryos; no PAL activity was detected in the homozygous pammbg5−/− embryos. Data plotted as mean ± SD (n = 3).
