Abstract Microtubules, which are subcellular "pipe-like" filaments composed of the protein tubulin, and their associated motors, dynein and kinesin, participate in many fundamental cellular processes, including cell division, nerve growth and regeneration, cell motility, and the organization and transport of organelles within cells. In these processes, microtubules serve as rigid "railroad tracks" for the movement of motors and their cargoes ("trains"), and the motors themselves are efficient, high-velocity, regulative "locomotives." The microtubles and microtubule motors of cold-adapted Antarctic fishes are unique in their capacity to assemble and to function at body temperatures (-2 to +2 oC) well below those of warm- blooded and temperate organisms. The long-range goals of the proposed research are to determine, at the molecular level, the adaptations that enhance the assembly of microtubules, the expression of tubulin genes, and the activity of microtubule motors from Antarctic fishes in this extreme thermal regime. The objectives of this project are: 1) to determine the structural features (e.g., changes in their amino acid sequences) that enable the tubulins of Antarctic fishes to polymerize efficiently at low temperatures; 2) to characterize the structure, organization, and expression of an alpha-tubulin gene cluster from an Antarctic rockcod, Notothenia coriiceps; and 3) to examine the biochemical adaptations required for efficient function of Antarctic fish flagellar dynein motors at low temperatures. Molecular adaptations of the Antarctic fish tubulins will be determined by comparing the amino acid sequences of alpha- and beta-tubulins from Notothenia coriiceps, to those from its temperate relative, the New Zealand black cod, Notothenia angustata. Gene expression in N. coriiceps will be analyzed by defining the organization and transcription- regulating elements of an alpha-tubulin gene cluster. The structure of the cluster will be characterized b y sequence analysis using standard DNA sequencing methods. Features of N. coriicepsalpha-tubulin transcription-regulating elements (promoters) that support efficient gene expression at cold temperatures will be assessed by use of reporter gene constructs in primary cell cultures derived from N. coriiceps tissues. Comparison to results obtained with N. angustata promoters should help to delineate elements of alpha-tubulin promoter regions that are important for high-level expression at low temperatures. Finally, the biochemical adaptations required for efficient function of N. coriiceps flagellar dyneins at low temperatures will be assayed by measurement of the temperature dependence of the motor activities of purified inner- and outer-arm dyneins. For comparison, flagellar dyneins from the temperate trout Salmo gairdneri (and possibly from N. angustata) will also be analyzed. Taken together, these studies should reveal the molecular adaptations of Antarctic fishes that maintain efficient microtubule assembly, mechano-chemical motor function, and gene expression at low temperatures. In the broadest sense, this research program should advance the molecular understanding of the cold-adapted mode of life,
