Analysis of the antifreeze glycoprotein containing genomic locus in the Antarctic notothenioid fish dissostichus mawsoni

Development of the Antarctic Circumpolar Current (ACC) circa 25 mya resulted in cooling of the high latitude waters of the Southern Ocean to a chilly -1.86 ??C (near the freezing point of seawater) and extinction of most of the late Eocene temperate fish fauna. A notothenioid ancestral stock survived and went through an adaptive radiation that gave rise to a variety of ecotypes that filled the empty niches. The notothenioid fishes now account for 95% of the fish biomass that inhabits the continental shelf of Antarctica and islands of the Scotia Arc. The adaptive radiation was linked to the evolution of antifreeze glycoproteins (AFGPs). High blood levels of AFGPs (25 to 35 mg/ml) lower their freezing point a few tenths of a degree below that of seawater (-1.86oC) and are a vital part of their freeze avoidance strategy. The AFGP gene evolved from a trypsinogen-like protease (TLP) gene, and presumably through an ancestral intermediate, a chimeric AFGP/TLP gene. All three types of genes (TLP, AFGP, and chimeric AFGP/TLP) are found in Antarctic notothenioid genomes, but it is not known whether the chimeric gene is transcribed and translated into a protein that would provide both AFGP and TLP molecules. The AFGP/TLP genomic locus of an Antarctic notothenioid, Dissostichus mawsoni was characterized in order to determine the mechanism of gene family expansion that would provide the high blood AFGP concentrations. The AFGP/TLP locus was isolated by screening a bacterial artificial chromosome (BAC) library for AFGP/TLP positive clones. Seven BAC clones representing two haplotypes encompassed the AFGP/TLP locus. Assembly of the AFGP/TLP locus was complicated by its highly repetitive nature. Thus, an assembly protocol was developed which entailed construction of subclone libraries of two insert size ranges (1-5 kbp and 5-30 kbp) for some of the positive BAC clones. BAC clone shotgun subclone libraries were then sequenced and subjected to automated and manual sequence reconstruction. Matching of paired-end sequences of some of the 1-5 kbp and all of the 5-30 kbp shotgun subclones to the locus sequence assembly was carried out to establish the linear order of genes. The AFGP/TLP locus assembly and analysis showed a high AFGP gene dosage (14 AFGP polyprotein genes in haplotype 1 and 8 AFGP polyprotein genes in haplotype 2) that very likely resulted from segmental duplications of the AFGP gene and its flanking regions, as seen in the >95 % sequence identity between AFGP gene modules. Thus it is clear that extensive AFGP gene duplication resulting in high gene dosage is the molecular basis for the high serum AFGP concentrations observed in the Antarctic notothenioids. Besides the AFGP genes, the locus contains three AFGP/TLP chimeric genes and two TLP genes. Bayesian and Maximum Likelihood phylogenetic reconstructions of the AFGP, TLP, and AFGP/TLP chimeric coding regions indicated that the AFGP gene family arose from an ancestral chimeric gene related to a specific chimeric gene in the locus. Analysis of this extant paralog of the chimeric gene ancestor of AFGP gene revealed that the first stand-alone AFGP gene was most likely formed by slipped misalignment on the template strand during DNA replication in the chimeric ancestor, resulting in the removal of the bulk of the TLP coding regions. We hypothesize that extensive AFGP gene duplication may have been propagated by a recombination hotspot located downstream of all AFGP genes. Double stranded DNA breakage at this recombination hotspot may have resulted in AFGP gene duplication via non-homologous segmental duplication by unequal crossing-over. Increased AFGP gene dosage conceivably was selected for upon advent of icy Antarctic marine conditions, increasing survival fitness in Antarctic notothenioids in the form of increased serum AFGP concentrations. Examination of the AFGP polypeptide coding regions of AFGP and AFGP/TLP chimeric genes (exon 2) showed that the AFGP genes encode predominantly the smaller AFGP molecules, consistent with their high abundance observed in the serum. The larger AFGP molecules are predominantly encoded in the AFGP/TLP chimeric genes. The chimeric genes are transcribed, and the tissue distribution of the chimeric gene transcript expression is similar to that of AFGP genes, suggesting that the chimeric gene may be functional in present day Antarctic notothenioids as an AFGP, providing the larger serum AFGP molecules. Three types of trypsinogen genes are also associated with the AFGP/TLP genomic locus. Bayesian and Maximum Likelihood phylogenetic reconstructions using spliced D. mawsoni trypsinogen coding sequences, and a large sampling of vertebrate trypsinogen sequences from the NCBI EST and nucleotide databases, indicate D. mawsoni trypsinogens belonged to two of the three previously classified teleost trypsinogen gene types, group I (digestive) trypsinogen and group III (cold active) trypsinogen. Group I and group III trypsinogens are located within clade I and clade III respectively identified in our analysis. Phylogenetic analysis and intron-exon structure mapping revealed that clade III trypsinogens consists of two distinct subclades (clade IIIA and IIIB), encompassing the previously classified cold-active group III trypsinogens. BLAST searches of NCBI ESTs from different teleosts revealed clade III trypsinogens to be present in more basal warm-water teleosts (catfish, Siluriformes), suggesting they evolved in a time of warm climate, and thus were not of cold adaptive origin thus the cold active capability of some extant paralogs may be a subsequent evolutionary acquisition. Antarctic notothenioid clade I and III trypsinogen transcript abundance were determined by relative qPCR. Both clade III trypsinogens transcripts were higher than that of clade I in the Antarctic notothenioid D. mawsoni. Tissue expression distributions of Antarctic notothenioid clade III trypsinogens, determined by PCR, were also broader than those of temperate O. mykiss clade III paralogs. Absolute qPCR quantification of transcript expression showed that in a warm-acclimated Antarctic notothenioid fish, clade III trypsinogen transcripts decreased while clade I trypsinogen transcripts increased. These analyses suggest clade III trypsins, which are expressed at very low levels in warm water teleosts, were recruited in Antarctic notothenioids for potential cold-active capabilities, and evolved into the primarily expressed trypsinogen type in these fishes. The clade III trypsinogens that persist at low transcript levels in warm-water teleosts may perform other proteolytic functions unrelated to digestion or their potential cold-active capabilities