| Summary: | 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 ...
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