ORGANIZATION AND EXPRESSION OF MYCOPLASMA GENES

这项提议的总体目标是(通过DNA序列)确定 分析)选定的膜表面蛋白的一级结构 致关节炎病原体猪鼻支原体，并定义一个 重申可能有助于基因组多样性的基因组元素 并反映了遗传物质在该物种之间的传播 支原体。Hyorhinis是该属70多种植物之一。 支原体，一群多样性的、快速进化的无壁小细胞 原核生物(750 kb基因组)，与革兰氏阳性有亲缘关系 真细菌。它会引起猪的慢性退行性关节炎。M. 鼻咽癌通过结构与宿主细胞表面相互作用 暴露在它的单一限制膜上。这些也是 免疫反应有助于疾病进程，包括一个主要的 最近定义的一组膜蛋白，共价修饰 脂类。不同菌株间存在重要的表型差异。 鼻息肉，包括致关节炎潜能、细胞吸附程度和 表膜抗原的变异。膜的独特之处 蛋白质的结构和加工不被理解，基因也不被理解 猪鼻支原体或其他已知支原体表型变异的基础。 拟议研究的第一个目标是获得初级dna序列。 以及最近鉴定的编码积分的克隆基因的表达 猪鼻分枝杆菌的膜蛋白具有潜在的新的脂类部位 酰化作用，或编码ATPase的膜亚单位。的关键功能 支原体膜蛋白的加工、锚定和表面抗原性 结构将被确定，并高度保守的比较 功能膜蛋白(ATPase)将由类似的 优生菌对应物。第二个目标是定义结构和 最近发现的重复基因组的可能传播 元素，在不同的菌株之间高度重复。 猪鼻分枝杆菌，并在其他支原体的选择菌株中分布 物种。这作为基因组多样性的潜在创造者是很重要的 在猪鼻分枝杆菌中的传播，可能有助于理解 支原体中的遗传物质。标准的限制技术 作图和桑格双脱氧测序将表征现有的 从猪鼻分枝杆菌重组基因组文库中克隆的DNA片段。 支原体蛋白的克隆表达及鉴定 片段将在大肠杆菌中利用噬菌体或质粒定向合成 微细胞或高表达宿主载体系统，缺失分析， 重组蛋白产品的抗原性分析和胰酶图谱。 互补分析将用于支原体基因功能的评估 在大肠杆菌中。

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.