Characterization of TETMYO-1, A Myosin Heavy Chain Gene in Tetrahymena

9604137 Gavin技术：基底相关肌动蛋白和肌球蛋白可以构成一个收缩系统，该系统可能在纤毛运动中发挥作用，因此在纤毛虫原生动物中具有超越运动的意义。许多脊椎动物组织中都存在纤毛基底和纤维复合体，如气管上皮和纤毛运动，它们是这些组织生理的组成部分。一个非常规肌球蛋白重链基因(TETMYO-1)的头部结构域已被克隆和测序。初步的免疫荧光显微镜结果表明，TETMYO-1基因编码的肌球蛋白定位于四膜虫细胞骨架的基底层。这个为期一年的项目的目标是确定TETMYO-1编码的肌球蛋白的超微结构定位，并准备构建该基因的基因组敲除结构。将从该基因的156个核苷酸片段中制备特异性抗体，并将其克隆到载体上并在大肠杆菌中表达。表达的蛋白将被纯化并用作免疫原以诱导多克隆抗体。该抗体将用于TETMYO-1在四膜虫细胞中的免疫金定位。超微结构的定位将集中在皮质结构、大核和细胞质上，以寻找TETMYO-1定位的证据。为了确定肌球蛋白是否与肌动蛋白细丝共定位，以确定肌球蛋白是否与肌动蛋白细丝共定位，从而在四膜虫每个细胞骨架基体周围形成笼状结构，人们将特别关注这些微丝。为了准备基因组敲除四膜虫的TETMYO-1功能，将开发一种干扰结构。含有抗生素耐药性编码序列和终止子序列的盒将连接到克隆的TETMYO-1片段。非技术：纤毛的敲打为许多单细胞生物体的移动提供了动力，并在一些动物器官的表面上移动液体。在单细胞生物和脊椎动物组织中纤毛运动的一个显着特征是纤毛的拍打和方向与相邻纤毛和远缘纤毛的拍打和方向相协调。如何实现这种协调是细胞生物学中的一个重要问题，对于了解单细胞生物体的生理过程和一些脊椎动物器官的功能至关重要。该项目将研究一种收缩蛋白的功能，该蛋白可能在协调单细胞生物体四膜虫纤毛跳动方向方面发挥重要作用。阐明这种蛋白质的功能将为一些细胞和组织中如何发生协调运动提供重要的见解。

9604137 Gavin Technical: Basal body-associated actin and myosin could constitute a contractile system that might have a role in ciliary motion and theefore have significance beyond motility in ciliated protozoa. Ciliated basal bodies with associated fibrillar complexes are present in many vertebrate tissues, such as, tracheal epithelium and ciliary motion, that are an integral part of the physiology of these tissues. The head domain of an unconventional myosin heavy chain gene (TETMYO-1) was previously cloned and sequenced. Preliminary immunofluorescence microscopy results indicated that the myosin encoded by the TETMYO-1 gene is localized in the basal bodies of the Tetrahymena cytoskeleton. The objectives of this one-year project are to determine the ultrastructural localization of the myosin encoded by TETMYO-1 and to prepare a construct for a genomic knockout of this gene in Tetramymena. Specific antibodies will be prepared from 156-nucleotide fragment of the gene that will be cloned intoa vector and expressed in E. Coli. The expressed protein will be ourified and used as an immunogen to elicit polyclonal antibodies. The antibodieq will be used for immunogold localization of the TETMYO-1 in Tetrahymena cells. The ultrastructural localization will focus on cortical structures, the macronucleus, and the cytoplasm for evidence of TETMYO-1 localization. Particular attention will be focused on basal body-associated microfilaments in order to determine whether the mysosin colocalizes with actin filaments that have been shown to form a cage-like structure around each cytoskeletal basal body in Tetrahymena. To prepare for a genomic knockout of TETMYO-1 function in Tetrahymena, a disruption construct will be devleoped. A cassette that contains an antibiotic resistance coding sequence flanked by promoter and a terminator sequence will be ligated to a cloned TETMYO-1 fragment. Non Technical: The beating of cilia provides the motive force for the movement of many unicellular organisms and to mo ve fluids over the surface of some animal organs. A remarkable feature of the motion of cilia in both unicellular organisms and in vertebrate tissues is that the beating and direction of the cilia is coordinated with that of both adjacent and distal cilia. How this coordination is achieved is an important question in cell biology that is essential to understand physiological processes of unicellular organisms and the function of some vertebrate organs. This project will investigate the function of a contractile protein that may have an essential role in coordinating the direction of cilia beating in the unicellular organism Tetrahymena. Elucidating the function of this protein will provide important insights into how coordinated movement occurs in some cells and tissues.