STREPTOCOCCUS PLASMIDS--MOLECULAR GENETIC ANALYSIS

链球菌质粒--分子遗传学分析

基本信息

批准号：
2060261
负责人：
VICKERS BURDETT
金额：
$ 22.25万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1979
资助国家：
美国
起止时间：
1979-01-01 至 1997-11-30
项目状态：
已结题

项目摘要

The only approach to the control of bacterial infections is the use of antibacterial drugs. However, antibiotic resistance can compromise treatment. One of the most frequently used antibiotics is tetracycline. The Tet M resistance determinant, which we discovered during the course of this project, has proven to have an extremely broad host range among bacterial pathogens. We have shown that Tet(M) protein protects the protein biosynthetic machinery of the cell from the inhibitory action of antibiotic at the level of the ribosome. The goal of this project is to understand the molecular basis for this protection mechanism with the hope that such information may be useful in design of new tetracycline derivatives which may be useful in treating infections with a possible secondary outcome being a better understand the mechanism of tetracycline action. To this end we plan to study the certain steps during protein synthesis for their susceptibility to tetracycline and the consequence of the presence of Tet(M) protein. In particular preliminary experiments suggest that Tet(M) protein may function as a homolog of the normal elongation factor G (which functions during ribosome translocation). some extent the approaches outlined below will be guided by this observation. Two general approaches will be employed: (1) A biochemical approach will be used to evaluate the physical interaction of Tet(M) with protein biosynthetic components. For example, we have shown that the protein has high affinity for the ribosome, although it may also interact with elongation factors. The nature of such interactions will be examined by use of protein-protein and protein-nucleic acid crosslinking reagents to deduce interaction of Tet(M) with these components, and by application of alkylation protection methods to assess rRNA interactions with tetracycline in the presence and absence Tet(M). (2) These biochemical experiments will be complemented by genetic analysis of Escherichia coli chromosomal mutations that were isolated during the previous grant period which confer tetracycline sensitivity on the cell even in the presence of functional Tet(M). Our current hypothesis is that such mutations alter the gene(s) that encode tetracycline binding activities and/or components which interact directly with Tet(M). Preliminary mapping of these mutations place them within the major cluster of ribosomal protein genes (73 min). We hope to clone and identify the corresponding genes and characterize the nature of the mutations conferring this phenotype. In a related approach, we plan attempted isolation of mutations in the rRNA coding sequences to see if we can identify alterations in these molecules that result in tetracycline resistance.

控制细菌感染的唯一方法是使用抗菌药物。但是，抗生素耐药性会损害治疗。最常用的抗生素之一是四环素。我们在课程中发现的TET M阻力决定因素在这个项目中，事实证明，在细菌病原体。我们已经表明TET（M）蛋白保护细胞的蛋白质生物合成机械从抑制作用的抑制作用核糖体水平的抗生素。这个项目的目标是了解这种保护机制的分子基础希望此类信息对新的四环素的设计可能有用可能在治疗感染可能有用的衍生物次要结果是更好地了解四环素的机制行动。为此，我们计划研究蛋白质期间的某些步骤综合其对四环素的敏感性和 TET（M）蛋白的存在。特别是初步实验表明TET（M）蛋白可以用作正常的同源物伸长因子G（核糖体易位在功能）。一些以下概述的方法将以这一观察为指导。将采用两种一般方法：（1）生化方法将用于评估TET（M）与蛋白质的物理相互作用生物合成成分。例如，我们已经表明蛋白质具有核糖体的高亲和力，尽管它也可能与伸长因素。这种互动的性质将由使用蛋白质 - 蛋白质和蛋白核酸交联试剂推断Tet（M）与这些组件的相互作用，并通过应用评估与RRNA相互作用的烷基化保护方法四环素在存在和不存在TET（M）的情况下。（2）这些生化实验将通过大肠杆菌的遗传分析来补充在上一个赠款期间分离的染色体突变即使存在功能性TET（M）。我们目前的假设是这种突变改变了编码四环素结合活动和/或组件的基因与TET（M）直接相互作用。这些初步映射突变将它们置于主要的核糖体蛋白基因簇中（73分钟）。我们希望克隆并确定相应的基因和表征赋予这种表型的突变的性质。在相关方法，我们计划在rRNA中尝试隔离突变编码序列，以查看我们是否可以识别这些分子的变化这导致四环素抗性。