Assembly and Stability of Microtubules from Antarctic Fish at Low Temperatures

拟议研究的长期目标是在分子水平上确定使南极鱼类的微管蛋白能够在这些适应寒冷的脊椎动物经历的-2至2摄氏度的低温下聚合的结构适应，并检查鱼类微管的动力学。这个项目的具体目标有四个：1)克隆和测序与编码南极鱼类α和β微管蛋白的信使RNA(CDNA)互补的DNA；2)确定南极鱼类基因组中微管蛋白基因的结构和数量；3)确定南极鱼类微管蛋白结构域的组织和功能；4)研究南极鱼类微管在低温下的动态行为。实验将在微管蛋白的cDNA和基因上进行，这些微管蛋白是从三种南极鱼种中提纯的微管蛋白进行的。将通过标准方法建立忽略隐翅虫的α和β微管蛋白cDNA和基因的核苷酸序列。微管组装所需的微管蛋白结构域将通过对蛋白质的有限消化和微管组装分析来确定。最后，将用电子显微镜和视频增强光学显微镜方法研究南极鱼类微管在低温下的动力学。将南极鱼类微管蛋白的一级序列和结构域与温带和温血脊椎动物微管蛋白的一级序列和结构域进行比较，可以揭示低温下微管组装所必需的结构特征。这项工作还将首次提供有关南极极端变温动物微管蛋白基因结构的有价值的信息。

The long-range goals of the proposed research are to determine, at the molecular level, the structural adaptations that enable the tubulins of Antarctic fishes to polymerize at the low body temperatures of -2 to +2 C experienced by these cold-adapted vertebrates, and to examine the dynamics of the fish microtubule. The specific objectives of this project are four in number: 1) to clone and to sequence DNAs complementary to messenger RNAs (cDNAs) that encode the alpha and beta tubulins of an Antarctic fish; 2) to determine the structure and number of the tubulin genes within the genome of an Antarctic fish; 3) to determine the organization and functions of the structural domains of tubulins from Antarctic fishes; and 4) to characterize the dynamic behaviors of Antarctic fish microtubule at low temperatures. Experiments will be performed on tubulin cDNAs and genes derived from the nucleic acids of Notothenia coriiceps neglecta and on microtubule proteins purified from three Antarctic fish species: N. coriiceps neglecta, N. gibberifrons, and Chaenocephalus aceratus. The nucleotide sequences of the alpha and beta tubulin cDNAs and genes from N. coriiceps neglecta will be established by standard methods. The structural domains of tubulins that are necessary for the assembly of cold-stable microtubule will be defined by limited proteolytic digestion of the proteins followed by microtubule assembly assays. Finally, the dynamics of Antarctic fish microtubule at low temperatures will be examined by electron-microscopic and video-enhanced light- microscopic methods. Comparison of the primary sequences and structural domains of tubulins from Antarctic fishes with those of tubulins from temperate and warm-blooded vertebrates should reveal the structural features necessary for microtubule assembly at low temperatures. This work will also provide, for the first time, valuable information concerning the structure of tubulin genes from an extreme Antarctic poikilotherms.