Little is known about the bacterial factors that enable pathogenic mycobacteria to survive and multiply within the macrophages of the infected host. By preparing cDNA from Mycobacterium avium bacilli grown in human-derived macrophages and in broth culture and using subtractive hybridization to remove commonly expressed genes, a procedure was developed to identify genes of M. avium that are specifically expressed when the bacilli are growing within macrophages. Total RNA was isolated from M. avium recovered 5 days after infection of human macrophages and from bacilli grown in vitro in broth. Mycobacterial mRNAs were converted to cDNA by reverse transcription. Biotin-modified cDNAs prepared from M. avium grown in broth culture were used to subtract the housekeeping genes from the cDNAs of the macrophage-derived M. avium. After each round of subtraction, a sample of the unsubtracted cDNA was amplified, labeled, and hybridized to cosmid clones of M. avium DNA. After three rounds of subtraction, the amplified DNA hybridized to approximately 1% of the cosmid clones under stringent conditions. Although the majority of the genes that are induced in phagocytized M. avium cells are expressed in the broth-grown bacilli, one DNA fragment that was identified coded for an mRNA that is highly specific for M. avium in phagosomes. This procedure will be especially useful for identifying genes that are expressed in response to growth in specific environments from organisms with genetic systems that are not well characterized
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