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: 3753544
• 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/3753544
• 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.

这项研究旨在确定，在遗传和结构 水平，普氏菌菌毛相关粘附素和 它们的菌毛在克隆和测序后， 半乳糖苷特异性菌毛相关粘附素基因 在成熟蛋白质中的中断的翻译区域开始 氨基酸29的密码子（实际上是赭石终止子）是 表征了 存在一段138个核苷酸 含有两个赭石终止子，polyA滑动区，茎 在基因组DNA和粘附素中发现了环和假结 mRNA确定核糖体翻译需要旁路或 位于两个赭石密码子之间的至少29个核苷酸的“hop”。 通过PCR复制整个区域，并将其框内融合到 5'末端的lacZ序列，证明E.大肠杆菌能够 有效地通过旁路区域进行阅读; 基因的表达水平为对照的4 - 25%。 基于内部10个氨基酸序列的DNA探针 从纯P. loescheii消化物中分离的酸性肽 放线菌特异性粘附素与限制性基因组DNA反应 从口腔细菌中分离出来 该探针随后被用于 筛选GT-11 λ噬菌体基因组罗氏毕赤酵母文库。 目前，含有噬菌体阳性插入物的DNA正在被 分离出来进行克隆。 类似地，罗氏疟原虫菌毛的N-末端序列 从结构的纯化制备物确定。 一 放射性寡核苷酸探针制备如下： 翻译，并发现与消化的基因组DNA反应。 的 上述P. loescheii lambda GT-11文库目前是 用寡核苷酸筛选。 核糖醇-5-P脱氢酶基因存在于核糖醇中， 操纵子已经被克隆，并且大约50%被测序。 的核糖醇 目前正在寻求临时秘书处的运输， 脱氢酶基因