MULTICOPY SUPPRESSION OF AN ILV MUTATION IN E COLI K 12

肠源性大肠杆菌的重要性与日俱增 许多腹泻病的病原体。因为它被认为是一个原型 肠道细菌的生物体，继续探索了解 它的基因组成和细胞功能是至关重要的。在这 提议，PI试图解开一种乙酰羟基酸的表达 将合成酶(AHAs)基因克隆到正常拷贝的结果 质粒组。所表达的“新的”阿哈德活性是不正常的。 当克隆到高拷贝质粒pBR322中时，被缬氨酸抑制。高 在几个细菌系统中已经报道了复制现象，但没有 在K-12的AHAS系统中。具体目标包括：1)物理化学 对克隆片段所产生的酶活性进行表征， 2)确定其他ilv基因对该基因表达的影响。 活性，3)确定新表达基因的拷贝数 在染色体上，4)定位涉及的基因，5)确定和 分析该基因的DNA序列。了解基因的作用机制 在某些环境条件下的激活肯定会促进 了解一些曾经被认为具有致病性的生物 是非致病的。参与这个项目的学生将获得 在进行最先进的研究和在 在研究中收集、分析和展示科学数据 会议和国家会议。所使用的技术可应用于 双分子科学中可能感兴趣的任何领域 规划科学职业生涯的学生。

Enteropathogenic strains of Escherichia coli are increasingly important agents of many diarrheal diseases. Because it is considered a prototype organism for enteric bacteria, continuing the search for understanding of its genetic make up and cellular functions is crucial. In this proposal, the PI seeks to unravel the expression of an acetohydroxy acid synthase (AHAS) gene as a consequence of cloning it into normal copy plasmid. The "new" AHAD activity expressed is refractory to normal inhibition by valine when cloned into a high copy plasmid, pBR322. High copy phenomena have been reported in several bacterial systems, but not in the AHAS system of K-12. Specific goals involve: 1) physicochemically characterizing the resulting enzymatic activity from the cloned fragment, 2) determining the effect of other ilv genes on expression of this activity, 3) determining the number of copies of the newly expressed gene on the chromosome, 4) mapping the genes involved and 5) determining and analyzing the DNA sequence of the gene. Understanding mechanisms of gene activation under certain environmental conditions certainly facilitate understanding the recent pathogenicity of some organisms once thought to be non-pathogenic. Students involved in this project will gain an invaluable experience in conducting state-of-the-art research and in collecting, analyzing and presenting scientific data at research conferences and national meetings. Technologies used may be applied to any number of areas in the bimolecular sciences which may interest the student in planning science careers.