STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE SFID REGION OF ESCHERICHIA COLI

本课题的总体目标是探索细胞的调控机制。 在大肠埃希氏菌中的分离。拟议的工作重点是蜂窝 SfiD基因的作用。这个基因的突变阻止了两个基因的作用 分裂抑制物，Su1a和SfiC，其合成作为部分刺激 对DNA损伤的依赖于recA的反应。Su1a防止“FtsZ环” 形成，即FtsZ细胞分裂启动蛋白的聚集 在细胞分裂前的信封上。SfiD突变允许FtsZ 在Su1a存在的情况下形成环。SfiD基因被定位 在大肠杆菌物理图谱上的千碱基对3,102和3,110之间，或 在基因图谱上大约需要63.7分钟。与第二种表型相关的 有sfid突变的人在低于29℃的温度下生长极差。 学位。这种“冷敏感性”可能归因于细胞的部分溶解。 在低温下。在互补性研究中，突变的sfiD等位基因 对sfid plus是隐性的。一些拟议的实验是 针对SFID区域的结构分析。这些措施包括 全长和部分sfiD基因片段亚克隆入噬菌体 载体和DNA序列测定。的核苷酸位置 突变的sfiD25和sfiD27等位基因将通过组合建立 聚合酶链式反应、基因克隆和测序。在其他实验中， 将检测sfiD在细胞壁完整性上的突变， 直接或通过对肽聚糖的抗生素抑制剂的反应 综合。作为一种识别其产物可能相互作用的基因的手段 在sfid中，第二个位置抑制突变的映射位置 反转的冷敏表型将被确定。最后， 为探索SfiD的生理作用而设计的额外实验 将包括监测SFID生产过剩对部门的影响 SfiD plus与sfiD突变体的频率和Su1a稳定性测量 菌株。

The overall aim of this project is to probe the mechanisms regulating cell division in Escherichia coli. The proposed work focuses on the cellular role of the sfiD gene. Mutations at this locus prevent the action of two division inhibitors, Su1A and SfiC, whose synthesis is stimulated as part of the recA-dependent response to DNA damage. Su1A prevents "FtsZ ring formation," i.e. aggregation of the FtsZ cell division initiator protein on the cell envelope just prior to division. An sfiD mutation allows FtsZ ring formation in the presence of Su1A. The sfiD gene is located between kilobase pair 3,102 and 3,110 on the E. coli physical map, or roughly 63.7 minutes on the genetic map. A second phenotype associated with the sfiD mutations is extremely poor growth at temperatures below 29 degree. This "cold sensitivity" may be attributable to partial cell lysis at low temperature. In complementation studies, the mutant sfiD alleles are recessive to sfid plus. A number of the proposed experiments are directed toward structural analysis of the sfiD region. These include subcloning of complete and partial sfiD gene segments into phagemid vectors and DNA sequence determination. The nucleotide positions of mutant sfiD25 and sfiD27 alleles will be established using a combination of PCR, gene cloning and sequencing. In other experiments the effect of mutations in sfiD on the integrity of the cell wall will be examined, either directly or by response to antibiotic inhibitors of peptidoglycan synthesis. As a means of identifying genes whose products may interact with sfiD, the map positions of second site suppressor mutations that reverse the cold sensitivity phenotype will be determined. Finally, additional experiments designed to explore the physiological role of SfiD will include monitoring the effect of SfiD overproduction on division frequency and Su1A stability measurements in sfiD plus versus sfiD mutant strains.