Margaret A. Shears

Liver-specific and seasonal expression of transgenic Atlantic salmon harboring the winter flounder antifreeze protein gene.

We have analyzed the inheritance and expression of a line of transgenic salmon harboring the antifreeze protein gene from the winter flounder. The genomic clone 2A-7 coding for a major liver-type antifreeze protein gene (wflAFP-6) was integrated into the salmon genome. From a transgenic founder (# 1469), an F3 generation was produced. In this study, southern blot analysis showed that only one copy of the antifreeze protein transgene was integrated into a unique site in F3 transgenic fish. The integration site was cloned and characterized. Northern analysis indicated that the antifreeze protein mRNA was only expressed in the liver and showed seasonal variation. All of the F3 offspring contained similar levels of the antifreeze protein precursor protein in the sera and the sera of these offspring showed a characteristic hexagonal ice crystal pattern indicating the presence of antifreeze activity. In addition, the antifreeze protein precursor protein level was found to vary with the season, being highest in the month of November and lowest in May. This study had demonstrated a tissue-specific and stable expression of the antifreeze protein transgene in the F3 generation of the transgenic salmon 1469 line.

Isolation and characterization of type I antifreeze proteins from cunner, Tautogolabrus adspersus, order Perciformes.

Antifreeze proteins (AFPs) are produced by many species of teleost fish that inhabit potentially lethal ice‐laden seawater and afford them protection from freezing. To date type I AFPs have been fully characterized in two teleost orders: Pleuronectiformes and Scorpaeniformes. In this study, we report the isolation and complete characterization of a type I AFP present in fish from a third order: cunner (Tautogolabrus adspersus), order Perciformes (family Labridae). This protein was purified from blood plasma and found to belong to what is now known as classical type I AFP with their small size (mass 4095.16 Da), alanine richness (> 57 mol%), high α‐helicity (> 99%) with the ability to undergo reversible thermal denaturation, 11 amino acid (ThrX10) repeat regions within the primary structure, the capacity to impart a hexagonal bipyramidal shaping to ice crystals and the conservation of an ice‐binding site found in many of the other type I AFPs. Partial de novo sequencing of the plasma AFP accounted for approximately half of the peptide mass. Sequencing of a combined liver and skin cDNA library indicated that the protein is produced without a signal sequence. In addition the translated product of the AFP cDNA suggests that it codes for the AFP isolated from plasma. These results further solidify the hypothesis that type I AFPs are multiphyletic in origin and suggest that they represent remarkable examples of convergent evolution within three orders of teleost fish.

Antifreeze proteins in the grubby sculpin, Myoxocephalus aenaeus and the tomcod, Microgadus tomcod: comparisons of seasonal cycles

SynopsisAntifreeze protein levels in the plasma of the grubby sculpin, Myoxocephalus aenaeus and the tomcod, Microgadus tomcod of Long Island coastal waters start to increase by November in anticipation of midwinter freezing conditions. Peak levels of antifreeze, as measured by the difference in plasma melting and freezing points, were detected in January for both species. The thermal hysteresis values reached 0.459°C in sculpin and 0.51°C in tomcod. Antifreeze peptides and glycopeptides start to disappear when water temperatures begin to rise and are at insignificant levels by late spring. Aspects of the seasonal cycle and the level of antifreeze activity were compared in three sympatric species (sculpin, tomcod, flounder); in two closely related but ecologically distinct gadids (tomcod, Atlantic cod); and within the genus Myoxocephalus.

Antifreeze proteins in the urine of marine fish.

Several species of marine teleosts have evolved blood plasma antifreeze polypeptides which enable them to survive in ice-laden seawater. Four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence and secondary structure are currently known. Although all of these antifreezes are relatively small (2.6–33 kd) it was generally thought that they were excluded from the urine by a variety of glomerular mechanisms. In the present study antifreeze polypeptides were found in the bladder urine of winter flounder (Pseudopleuronectes americanus), sea raven (Hemitripterus americanus), ocean pout (Macrozoarces americanus) and Atlantic cod (Gadus morhua). Since the plasma of each of these fish contains a different antifreeze class it would appear that all four classes of antifreeze can enter the urine. The major antifreeze components in the urine of winter flounder were found to be identical to the major plasma components in terms of high performance liquid chromatography retention times and amino acid composition. It is concluded that plasma antifreeze peptides need not be chemically modified before they can enter the urine.