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

四膜虫肌球蛋白重链基因 TETMYO-1 的表征

• 批准号: 9604137
• 负责人: Ray Gavin
• 金额: $ 5万
• 依托单位: CUNY Brooklyn College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604137&HistoricalAwards=false
• 关键词: Characterization; TETMYO; Myosin; Heavy; Chain

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.

9604137 Gavin Technical：基础体相关肌动蛋白和肌球蛋白可能构成一个收缩系统，可能在纤毛运动中起作用，因此在纤毛原生动物的运动中具有重要意义。纤毛基底体与相关的纤维复合物存在于许多脊椎动物组织中，如气管上皮和纤毛运动，这是这些组织生理学的一个组成部分。一个非常规的肌球蛋白重链基因（TETMYO-1）的头部结构域先前被克隆和测序。免疫荧光显微镜初步结果表明，TETMYO-1基因编码的肌球蛋白定位于四膜虫细胞骨架的基底。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，并准备一个基因组敲除该基因的结构体。将从基因的156个核苷酸片段中制备特异性抗体，将其克隆到载体中并在大肠杆菌中表达。表达的蛋白将被纯化并作为免疫原引发多克隆抗体。该抗体将用于TETMYO-1在四膜虫细胞中的免疫金定位。超微结构定位将集中在皮质结构、大核和细胞质上，以证明TETMYO-1的定位。我们将特别关注与基体相关的微丝，以确定肌动蛋白丝是否与肌动蛋白丝共定位，肌动蛋白丝已被证明在四膜虫的每个细胞骨架基体周围形成笼状结构。为了准备基因组敲除TETMYO-1在四膜虫中的功能，将开发一个破坏结构。一个包含抗生素抗性编码序列的卡带，其两侧是启动子和终止子序列，将与克隆的TETMYO-1片段连接。非技术：纤毛的跳动为许多单细胞生物的运动提供动力，并使一些动物器官表面的液体流动。在单细胞生物和脊椎动物组织中，纤毛运动的一个显著特征是，纤毛的跳动和方向与相邻和远端纤毛的跳动和方向是协调的。这种协调是如何实现的是细胞生物学中的一个重要问题，对于理解单细胞生物的生理过程和一些脊椎动物器官的功能至关重要。本项目将研究单细胞生物四膜虫中可能在协调纤毛跳动方向中起重要作用的收缩蛋白的功能。阐明这种蛋白的功能将为了解协调运动在某些细胞和组织中如何发生提供重要的见解。