Fig. 4

Sequences, models, and codon usage of cunner AFPs and Ala-rich C-terminal regions of GIMAP genes. A) AFP isoforms aligned with Ala highlighted in yellow, acidic and basic residues in red and blue font, respectively; Gly and Pro highlighted in pink, polar residues other than Thr highlighted in green, and aliphatic residues highlighted in gray, with the spacing of Thr residues (black highlighting) indicated above and asterisks indicating 100% conservation below. The last two residues (faded gray) are naturally removed when the C-terminus is amidated [29]. The AFPs are numbered sequentially as they appear in JAJGRF010000003.1, bases 6,121,000 to 6,371,000. B) Models of the long (AFP-1) and short (AFP-2) cunner AFPs generated using AlphaFold2-Colab [88] and rendered using PyMOL [87]. Residues are colored as above but with backbone atoms in light gray, Thr in green and other polar non-charged residues in dark green. Two 180°-degree rotations are displayed with their termini marked N and C. C) Ala-rich C-terminal regions of Cunner-A5 and Ballan-A1 GIMAPs relative to the shorter Cunner A isoforms. The Ala and Thr residues within the extension are colored as in A above, with the end of the GTPase domain in italics. Conservation between the unambiguously aligned residues of the four isoforms with extensions is indicated below the alignment by asterisks (all four sequences identical) or dots (three of four sequences identical). The beginnings of the two segments used to derive Ala codon usage are indicated with arrows. Accession numbers and reannotated sequences are given in Supplementary Table 1. D) Ala-codon usage of cunner AFPs (all isoforms, 322 codons) compared to the Ala-rich extensions of Cunner-A5 (26 codons), Ballan-A1 (75 codons), and Ala codons sampled from more than 5 million coding sequences from teleost fishes