,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.
