Assembly and Stability of Microtubules from Antarctic Fish at Low Temperatures

这项研究的长期目标是在分子水平上确定使南极鱼类的微管能够在这些适应寒冷的脊椎动物所经历的-2至+2摄氏度的低温下聚合的结构适应性，并检查鱼类微管的动力学。该项目的具体目标有四个：1)克隆和测序南极鱼类编码α和β微管的信使rna （cdna）的互补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)确定南极鱼类微管结构域的组织和功能；4)低温条件下南极鱼类微管的动态行为特征。实验将进行 在微管蛋白互补dna和基因的核酸中提取出来的 Notothenia coriiceps neglecta和微管蛋白 纯化从三个南极鱼类:n . coriiceps neglecta: gibberifrons, Chaenocephalus aceratus。用标准的方法建立了猪胸草的α和β微管蛋白cdna和基因的核苷酸序列。冷稳定微管组装所必需的微管结构域将通过有限的蛋白质水解消化和微管组装分析来确定。最后，将采用电子显微镜和视频增强光显微方法研究南极鱼类微管在低温下的动力学。将南极鱼类微管的初级序列和结构域与温带和温血脊椎动物的微管进行比较，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宝贵信息。

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.