Genomic basis for antifreeze peptide heterogeneity and abundance in an Antarctic eel pout: gene structures and...

Type III antifreeze protein (AFP) in the Antarctic eel pout (Lycodichthys dearborni) occurs as a heterogeneous family of three major and at least five minor variants collectively maintained at high year-round blood levels (> 20 mg/ml). Two major AFPs (RD1, RD2) are 7 kD in size, and the third (RD3) is 14 kD and is composed of two 7-kD AFP domains linked by a 9-residue connector. The genomic basis for the heterogeneity and abundance of these AFPs was investigated in this study. Genomic library screening statistics and restriction mapping analyses of 16 genomic clones together indicate an AFP gene family of over 40 genes, which would provide a sizable gene dosage for high AFP output. Two genomic clones, each containing 2 AFP genes, were characterized in detail. Three of the genes, were characterized in detail. Three of the genes encode the 7-kD AFP RD2, and are arrayed in direct 8.3-kb tandem repeats. The three gene sequences are nearly 100% identical for a distance of over 3 kb (inclusive of 1.7-kb 5' and 1-kb 3' flanking sequences), indicating strong selective pressure from the freezing Antarctic waters on maintaining functionality of AFP genes for producing adequate levels of AFPs for survival. The fourth AFP gene has multiple exons and translates into a multimeric AFP composed of at least six 7-kD AFP domains successively linked a 9-residue connector sequence similar to that of the dimeric 14-kD AFP, RD3. The presence of an unusually large (2.7-kb) AFP messenger RNA beside two small ones (0.9 kb and 0.7 kb) in Northern blot of liver RNA is consistent with the presence of a large functional multiexon AFP gene. Substantial sequence identity (83%) between the 1.2-kb introns of the multiexon AFP gene and the intron and 5' flanking sequences of RD2 genes suggests that the former could arise from recombinant events that linked two adjacent 7-kD AFP genes in the 8.3-kb tandem repeats followed by duplication events to produce the multiple exons.