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CODING GAP BYPASS AND ADHESION FUNCTION IN PREVOTELLA

Prevotella 中的编码间隙旁路和粘附功能

基本信息

批准号：
6355486
负责人：
Jean N Citron
金额：
$ 7.3万
依托单位：
UNIVERSITY OF MISSOURI KANSAS CITY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-04-01 至 2002-02-28
项目状态：
已结题

项目摘要

The proposed research addresses 1) the unique mechanisms used by the oral bacterium Prevotella loescheii 1295 to synthesize the SO34 adhesin protein, and 2) the mechanisms of adhesin action. The SO34 adhesin, which is encoded by the plaA gene, is a lectin- like protein that recognizes galactoside-containing receptors on Streptococcus oralis 34 cells. PlaA expression requires bypassing a 29-nucleotide (nt) gap in its coding sequence on the PlaA messenger RNA (mRN). The proposed mechanism of PlaA expression is a programmed frameshifting hop. Features of the gap region consistent with this mechanism include: 1) the presence of two (UAA) termination condons flanking the bypass region, 2) two runs of four or more identical bases (slippery sequences), 3) the ability the plaA mRNA to form a stem-loop at the beginning of the large ORF, and 4) the potential of bases in the loop to form a pseudoknot. Specific Aims of the proposed research are to: A0 Use site-directed and random mutagenesis to test whether the above features of the plaA mRNA structure are essential for efficient gap bypass. An assay that uses a beta-galactosidase reporter gence will be used to measure coding gap bypass efficiency. B) Express the SO34 adhesin and map its active site. C) Transfer and express the plaA adhesin in Prevotella and/or Bacteroides for studies of gene regulation and adhesin function. These experiments will significantly expand our understanding of genetic mechanisms in oral bacteria associated with dental plaque. Specifically, the proposed experiments will elucidate the unusual translation mechanisms used to by-pass the plaA coding gap. Expression of the plaA gene in related bacteria will facilitate testing the effect of the coding gap on plaA expression. The proposed studies will provide a solid foundation for understanding mechanisms of adhesin binding and the subsequent engineering of peptides that may prevent adhesin action. Understanding adhesin action and how to prevent adhesin binding to receptors will lay a foundation for the future development of agent that prevent plaque accumulation.

拟议的研究涉及 1）所使用的独特机制由口腔细菌 Prevotella loescheii 1295 合成 SO34 粘附素蛋白，2) 粘附素作用机制。 SO34 粘附素由 plaA 基因编码，是一种凝集素就像识别含有半乳糖苷受体的蛋白质口腔链球菌34细胞。 PlaA 表达需要绕过 PlaA 编码序列中存在 29 个核苷酸 (nt) 缺口信使RNA（mRNA）。 PlaA 的拟议机制表达是一种编程的移码跳跃。的特点符合此机制的gap区域包括：1）存在旁路区域两侧的两个 (UAA) 终止密码子，2) 两次运行四个或更多相同碱基（滑动序列），3) plaA mRNA 在开始时形成茎环的能力大 ORF 的大小，以及 4) 环中碱基形成的潜力假结。拟议研究的具体目标是： A0 使用定点突变和随机突变来测试是否 plaA mRNA结构的上述特征对于有效的间隙旁路。使用 β-半乳糖苷酶的测定记者gence将用于测量编码间隙旁路效率。 B) 表达 SO34 粘附素并绘制其活性位点图。 C) 在普雷沃氏菌和/或中转移并表达 plaA 粘附素用于研究基因调控和粘附素功能的拟杆菌。这些实验将极大地扩展我们对与牙菌斑相关的口腔细菌的遗传机制。具体来说，所提出的实验将阐明不寻常的现象用于绕过 plaA 编码间隙的翻译机制。 plaA基因在相关细菌中的表达将促进测试编码间隙对 plaA 表达的影响。这拟议的研究将为理解提供坚实的基础粘附素结合机制及其后续工程可能阻止粘附素作用的肽。了解粘附素作用以及如何防止粘附素与受体结合将奠定为今后开发牙菌斑预防剂奠定基础积累。