Mycobacterium aviumGenes Expressed during Growth in Human Macrophages Detected by Selective Capture of Transcribed Sequences (SCOTS)

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Abstract
Selective capture of transcribed sequences (SCOTS) has been employed to identify 54 cDNA molecules that represent 46 genes that are expressed byMycobacterium aviumduring growth in human macrophages. Some cDNA molecules correspond to genes that are apparently expressed 48 h after infection of macrophages, while others correspond to genes expressed 110 h after infection, and still others correspond to genes expressed throughout the course of infection in our model system. Genes expressed byM. aviumduring growth in macrophages include genes encoding enzymes of several biosynthetic pathways (pyrimidines, mycobactin, and polyketides); genes that encode enzymes involved in intermediary metabolism, energy metabolism (tricarboxylic acid cycle, glyoxalate shunt), and nitrogen metabolism; and genes that encode regulatory proteins. A number of genes of unknown function were also identified, including genes that code for proteins similar to members of the PPE family of proteins ofMycobacterium tuberculosisand proteins similar to those encoded by theM. tuberculosis mcegenes, which have been previously associated with mycobacterial virulence. The SCOTS technique, followed by enrichment for cDNA molecules that are up-regulated or are uniquely expressed byM. aviumduring growth in human macrophages (compared to growth in laboratory broth culture), allows recovery and identification of a greater diversity of cDNA molecules than does subtractive hybridization between cDNA mixtures from macrophage-grown and broth-grownM. avium.Data are presented demonstrating the reproducibility of recovery of a subset of cDNA molecules from cDNA mixtures purified by SCOTS on several different occasions. These results further demonstrate the beneficial utility of the SCOTS technique for identifying genes whose products are needed for successful survival and growth by an organism in a specific environment.