ROLE OF SURFACE MOLECULES IN COLONIZATION AND BIOLOGICAL MIMICRY

表面分子在定植和生物拟态中的作用

• 批准号: 3775675
• 负责人: J LONDON
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3775675
• 关键词: adhesin; bacterial genetics; ecology; gene expression; infection; molecular cloning; nucleic acid probes; nucleic acid sequence; oral bacteria; pilus

This study seeks to define, at the genetic and structural level, the Prevotella loescheii fimbrial-associated adhesins and the fimbriae bearing them. After cloning and sequencing the galactoside-specific fimbrial-associated adhesin gene, the interrupted translation region within the mature protein beginning at the codon for amino acid 29 (actually an ochre terminator) was characterized. Existence of a stretch of 138 nucleotides containing the two ochre terminators, a polyA slip region, stem loop and pseudoknot was demonstrated in genomic DNA and adhesin mRNA establishing that ribosomal translation requires a bypass or "hop" of at least 29 nucleotides located between two ochre codons. The entire region was replicated by PCR and fused in frame into the 5' terminus of lacZ to demonstrate that E. coli is capable of reading through the bypass region efficiently; expression of the gene occurred at levels varying between 4 and 25% of the control. A DNA probe based on the sequence of an internal 10 amino acid peptide isolated from a digest of pure P. loescheii actinomyces-specific adhesin reacted with restricted genomic DNA from this oral bacterium. The probe was subsequently used to screen a GT-11 lambda phage genomic P. loescheii library. Currently, DNA containing the phage-positive insert is being isolated for cloning. Similarly, the N-terminal sequence of P. loescheii fimbriae was determined from a purified preparation of the structures. A radioactive oligonucleotide probe was prepared following reverse translation and was found to react with digested genomic DNA. The above mentioned P. loescheii lambda GT-11 library is currently being screened with the oligonucleotide. The ribitol-5-P dehydrogenase gene found in the ribitol operon has been cloned and is roughly 50% sequenced. The ribitol PTS transport is currently being sought in the flanking regions of the dehydrogenase gene.

这项研究试图在基因和结构上定义 水平，罗氏普氏杆菌菌毛相关粘附素和 承载它们的菌毛。在克隆和测序之后 半乳糖苷特异性菌毛相关粘附素基因 成熟蛋白开始时的中断翻译区域 在氨基酸29的密码子(实际上是赭石终止子)是 特色化的。存在138个核苷酸的延伸 含有两个赭石终止子，Polya滑移区，茎 基因组DNA和粘附素中存在环和伪结 确定核糖体翻译需要旁路或 位于两个赭色密码子之间的至少29个核苷酸组成的“HOP”。 整个区域通过聚合酶链式反应进行复制，并在框架内融合到 LacZ的5‘末端，以证明大肠杆菌能够 有效地读取旁路区域；表达 基因发生的水平在对照的4%到25%之间。 一种基于内部10个氨基酸序列的DNA探针 从罗氏拟青霉消化液中分离得到的酸性多肽 放线菌特异性粘附素与限制性基因组DNA的反应 从这种口腔细菌中。该探头随后被用于 GT-11噬菌体基因组文库的筛选 目前，含有噬菌体阳性插入片段的DNA正在 被隔离以进行克隆。 类似地，罗氏平菇菌毛的N-端序列 是从结构的纯化制剂中确定的。一个 用反相法制备放射性寡核苷酸探针 翻译，并被发现与消化的基因组DNA反应。这个 以上提到的P.loescheii lambda GT-11文库目前 用寡核苷酸进行筛查。 核糖醇中发现的核糖醇-5-P脱氢酶基因 操纵子已经被克隆，大约50%被测序。核糖醇 目前正在寻求PTS运输的侧翼地区 脱氢酶基因。