COMPLETE SEQUENCING AND GENE EXPRESSION IN M AVIUM

描述：(改编自申请者S摘要)本项目目标 是为了确定禽分枝杆菌的完整DNA序列 染色体和检测基因表达的全局变化 体外环境条件的变化。可行性和可操作性 微生物基因组测序的成本效益已经得到证明 最近被私家侦探和他的研究所。将使用的方法将是 以前与流感嗜血杆菌等一起使用的那些，尽管更大的基因组 其大小(4.7 MBP对1.8 MBP)将需要将近3倍 扩大规模。将对序列进行标准分析。检视 基因表达的变异，估计4500个基因的PCR产物将 排列在滤膜上，与从RNA制备的标记基因杂交 据估计在27种不同的条件下生长 (包括生长阶段、营养和氧气消耗、温度、pH、 自由基、抗菌剂和巨噬细胞内的生长)。这个 申请人认为，完整的禽类分枝杆菌序列，结合 目前正在确定结核分枝杆菌和麻风杆菌的完整序列， 应该通过以下方式在基因组和基因上进行高度信息量的比较 基因基础。该序列将在疫苗方面有潜在应用 毒力机制的发展、鉴定和抗菌剂 治疗和抵抗。

DESCRIPTION: (Adapted from Applicant,s Abstract) The goals of this project are to determine the complete DNA sequence of the Mycobacterium avium chromosome and examine global changes in gene expression with various changes in environmental conditions in vitro. The feasability and cost-effectiveness of microbial genome sequencing has been demonstrated recently by the P.I. and his institute. The approaches to be used will be those used previously with H. influenzae, etc., although the larger genome size (4.7 Mbp versus 1.8 for H. influenzae) would require a nearly 3 fold scaleup. Standard analyses of the sequence will be conducted. To examine variation in gene expression, PCR products of the estimated 4500 genes will be arrayed on filters for hybridization to labelled cDNA prepared from RNA of M. avium grown under an estimated 27 different sets of conditions (involving growth phase, nutrient and oxygen depletion, temperature, pH, radicals, antimicrobial agents, and growth within macrophages). The applicant believes that complete M. avium sequence, in conjuction with the complete M. tuberculosis and M. leprae sequences currently being determined, should lead to highly informative comparisons on both a genome and gene by gene basis. The sequence will have potential applications to vaccine development, identification of virulence mechanisms, and antimicrobial therapy and resistance.