ORGANIZATION AND EXPRESSION OF MYCOPLASMA GENES

本提案的总体目标是确定（通过DNA序列 分析）的膜表面蛋白的选择的一级结构 致关节炎病原体猪鼻支原体，并定义一种 重复的基因组元件可能有助于基因组多样性 在这个物种中，并反映了遗传物质的传播， 支原体 M.猪鼻鱼是猪鼻鱼属70多个物种之一， 支原体，一组多样的，快速进化的小无壁 原核生物（750 kb基因组），与革兰氏阳性菌遗传相关 真细菌 它会导致猪的慢性退行性关节炎。 M. 猪鼻通过结构与宿主细胞表面相互作用 暴露在其单一的限制膜上。 这些也是 免疫反应有助于疾病过程，包括一个主要的 一组最近定义的膜蛋白，被共价修饰， 脂质。 重要的表型差异存在于M. 猪鼻，包括关节炎的可能性，细胞吸附程度和 表面膜抗原的变异。 膜的独特功能 蛋白质的结构和加工过程还不清楚，基因也不清楚。 表型变异的基础。猪鼻支原体或其他已知支原体。 本研究的第一个目的是获得原始DNA序列 和表达最近鉴定的克隆基因编码的整合 M.具有潜在新脂质位点的猪鼻 酰化或编码ATP酶的膜亚基。 的关键特征 支原体膜蛋白加工、锚定和表面抗原 结构将被确定，并比较高度保守的 功能性膜蛋白（ATP酶）将用类似物制成 真细菌对应物。 第二个目标是确定结构， 可能是最近发现的重复基因组 元素，这是高度重复的可变模式之间的菌株 M.猪鼻支原体，并分布于其它支原体的筛选菌株中 物种 这作为基因组多样性的潜在发生器是重要的 在M。猪鼻，并可能有助于了解传播 支原体中的遗传物质。 限制的标准技术 作图和桑格双脱氧测序将是表征现有的 从M.猪鼻肌 克隆的支原体蛋白的表达和鉴定 片段将在E.杆菌 微细胞或高表达宿主-载体系统，缺失分析， 重组蛋白产物的抗原性分析和胰蛋白酶作图。 互补分析将用于评估支原体基因功能 在大肠杆菌

The overall objectives of this proposal are to determine (by DNA sequence analysis) the primary structure of selected membrane surface proteins of the arthritogenic pathogen Mycoplasma hyorhinis, and to define a reiterated genomic element that may contribute to genomic diversity within this species and reflect transmission of genetic material among mycoplasmas. M. hyorhinis is one of over 70 species in the genus Mycoplasma, a group of diverse, rapidly evolving small wall-less procaryotes (750 kb genomes), phylogenetically related to gram positive eubacteria. It causes chronic degenerative arthritis in swine. M. hyorhinis interacts with the surface of host cells through structures exposed on its single limiting membrane. These are also targets of immune responses contributing to disease processes, and include a major group of recently defined membrane proteins covalently modified by lipids. Important phenotypic differences occur among strains of M. hyorhinis, including arthritogenic potential, degree of cytadsorption and variation of surface membrane antigens. Unique features of membrane protein structure and processing are not understood, nor is the genetic basis of phenotypic variation in M. hyorhinis or other mycoplasma known. The first aim of the proposed research is to obtain primary DNA sequence and expression of recently identified cloned genes encoding integral membrane proteins of M. hyorhinis with potentially novel sites of lipid acylation, or encoding membrane subunits of ATPase. Critical features of mycoplasma membrane protein processing, anchorage and surface antigenic structure will be determined, and comparison of highly conserved functional membrane proteins (ATPase) will be made with analogous eubacterial counterparts. The second aim is to define the structure and possible transmission of a recently identified repetitive genomic element, that is highly reiterated in variable patterns among strains of M. hyorhinis, and is distributed in selecting strains of other mycoplasma species. This is important as a potential generator of genomic diversity within M. hyorhinis, and may be useful in understanding transmission of genetic material among mycoplasmas. Standard techniques of restriction mapping and Sanger dideoxy sequencing will be to characterize existing DNA fragments cloned from recombinant genomic libraries of M. hyorhinis. Expression and characterization of mycoplasma proteins from cloned fragments will utilize phage- or plasmid-directed synthesis in E. coli minicells or high expression host-vector systems, deletion analysis, antigenic analysis and tryptic mapping of recombinant protein products. Complementation analysis will be used to assess mycoplasma gene function in E. coli.