Research Objectives:
As the Southern Ocean cooled during the past 25 million years, the fishes of antarctic coastal waters evolved biochemical and physiological adaptations that maintain their essential cellular processes. The long-range goals of this research are to determine, at the molecular level, the adaptations that enhance the assembly of microtubules, the function of kinesin motors, and the expression of globin and tubulin genes. Specific objectives are
• Determine the primary sequence changes and posttranslational modifications that contribute to the efficient polymerization of antarctic fish tubulins at low temperatures
• Evaluate the biochemical adaptations required for efficient function of their brain kinesin motor at low temperatures
• Characterize the structure, organization, and promoter-driven expression of globin and tubulin genes from an antarctic rockcod (Notothenia coriiceps) and a temperate congener (N. angustata).
Brain tubulins from antarctic fishes differ from those of temperate and warm-blooded vertebrates both in unusual primary sequence substitutions and in posttranslational C-terminal glutamylation. Potential adaptations of antarctic fish tubulins will be tested directly by production of wild-type and site-directed tubulin mutants for functional laboratory analysis. Researchers will determine the capacity of fish tubulins to form “cold-stable” microtubules, and test the role of the carboxy-terminal charge status of tubulin in cold adaptation of microtubule assembly after enzymatic manipulation.
Three unusual substitutions in the kinesin motor domain of Chionodraco rastrospinosus may enhance mechanochemical activity at low temperatures. To test the functional significance of these changes, fish residues will be converted to those found in mammalian brain kinesin. Reciprocal substitutions will be introduced into the framework of the mammalian kinesin motor domain. After production in Escherichia coli and purification, the functional performance of the mutant motor domains will be evaluated.
Molecular adaptation of gene expression in N. coriiceps will also be analyzed. Structural features that support efficient expression will be assessed. Comparison with N. angustata should help delineate elements of the regions that are important for high-level expression at low temperatures. The functions of possible regulatory elements will be tested by deletion analysis and by specific mutagenesis. Together, these studies should reveal the molecular adaptations of antarctic fishes that maintain efficient cytoskeletal assembly, mechanochemical motor function, and gene expression at low temperatures and advance the molecular understanding of the poikilothermic mode of life.
