VIRULENCE DETERMINANTS OF MYCOBACTERIUM TUBERCULOSIS

结核病是人类最大的疾病之一， 在美国是一个严重的健康问题最常见的是 老年人、无家可归者、艾滋病患者和来自各国的移民 那里结核病发病率很高。 虽然结核病的病原体， 结核分枝杆菌在世纪前被发现， 基本的生理能力，遗传学，特异性毒力 决定因素，这些杆菌的致病机制几乎是 根本不存在 因此，我们建议从M. 结核病的病原体是重要的， 通过基因组DNA和重组体的分子和功能分析， 基因组文库中的分子。具体而言，我们将（i）确定 染色体DNA区域是M.结核病H37 Rv， 来自H37 Rv和H37 Ra的基因组DNA之间的消减杂交， H37 Rv和BCG之间的相互作用，（ii）制备互补DNA（cDNA）文库 从麻省从人巨噬细胞中纯化的结核H37 Rv杆菌. 感染后不同时间和肉汤中生长的H37 Rv杆菌 （iii）鉴定在不同阶段表达的基因， 巨噬细胞cDNA间消减杂交感染- 生长和肉汤生长的M.结核病H37 Rv，（iv）将基因亚克隆入 合适的载体并将它们引入H37 Ra或BCG中以确定 克隆的基因是否会增强 巨噬细胞中的无毒衍生物，和（v）表征独特的 用核苷酸序列测定和计算机确定区域和基因 分析.

Tuberculosis, one of the great scourges of mankind, is reemerging as a significant health problem in the U.S., most frequently afflicting the elderly, the homeless, individuals with AIDS, and immigrants from nations where the incidence of TB is high. Although the causative agent of TB, Mycobacterium tuberculosis, was identified a century ago, knowledge about the fundamental physiological capabilities, genetics, specific virulence determinants, and mechanism(s) of pathogenicity of these bacilli is almost nonexistent. We therefore propose to identify and characterize genes from M. tuberculosis that are important in the pathogenicity of this organism through molecular and functional analyses of genomic DNA and recombinant molecules from genomic libraries. Specifically, we will (i) identify regions of chromosomal DNA that re unique to M. tuberculosis H37Rv by subtractive hybridizations between genomic DNAs from H37Rv and H37Ra and between H37Rv and BCG, (ii) prepare complementary DNA (cDNA) libraries from M. tuberculosis H37Rv bacilli purified from human macrophages at different times after Infection and from H37Rv bacilli grown in broth culture, (iii) identify genes that are expressed at different stages of infection by subtractive hybridizations between cDNAs from macrophage- grown and broth-grown M. tuberculosis H37Rv, (iv) subclone genes into appropriate vectors and introduce them into H37Ra or BCG to determine whether or not the cloned genes will enhance survival and/or growth of the avirulent derivatives in macrophages, and (v) characterize the unique regions and genes by nucleotide sequence determination and computer analysis.