INNATE RESISTANCE TO MYCOBACTERIA--ROLE OF NRAMP GENE

结核病是北美的一个主要问题， 艾滋病中这种感染的流行率和破坏性影响的增加 患者，以及出现高毒力和多药耐药 菌株M.结核 宿主的防御机制 感染，以及长期生存的细菌机制 对宿主吞噬细胞内的作用仍知之甚少 阐明和 了解自然界中遗传差异的分子基础 在人类和哺乳动物中观察到的对分枝杆菌感染的易感性 实验动物，应该提供新的见解的机制， 宿主防御这些感染。 在老鼠体内， 结核分枝杆菌感染是由Bcg/Ity/Lsh基因控制的。 我们已经克隆了一个Bcg的候选基因，命名为nramp（天然的 抵抗相关的巨噬细胞蛋白），其编码一种新的 巨噬细胞特异性膜转运蛋白。 两大目标，如果 这个建议是正式的证明，nramp和Bcg是 相同的基因，以及对这些基因的综合遗传和生化分析， Nramp蛋白，目的是了解其生物学作用， 巨噬细胞 一种携带无效等位基因的突变小鼠品系的产生 在胚胎中同源重组产生的nramp基因座上， 干细胞将用于确定1）nramp和Bcg是否相同 进一步分析nramp在巨噬细胞中的作用 功能 作为一种替代方法，我们将使用转基因动物， 引入nramp的Bcg'等位基因的克隆拷贝（129/sv，显性） Bcgs小鼠背景（C57 BL/6 J，隐性），并确定它是否 可以逆转C57 BL/6 J小鼠对感染的易感性。 结构 并提出了nramp基因的功能特征， 特别强调顺式作用序列和反式作用序列的鉴定， 组成型或诱导型巨噬细胞的作用因子 该基因的特异性表达。 我们还将确定是否新颖 与疾病易感性相关的NRAMP突变， 在其他近交系小鼠品系中鉴定，但也在人类中鉴定， NRAMP同源物在来自家族谱系的个体中的分离， 易患结核病。 Nramp的生化分析 蛋白质，包括产生特异性抗体， 鉴定其细胞和亚细胞定位，假定 翻译后修饰，膜相关转运 官能团，特别是关于活性氮中间体， 也提出了。 总之，这些研究应该有助于理解 nramp在巨噬细胞介导抗分枝杆菌中的作用 感染，可能为治疗干预提供新的靶点 调节宿主对结核病的防御。

Tuberculosis is a major source of concern in North America due to the increased prevalence and devastating effect of this infection in AIDS patients, and the emergence of highly virulent and multidrug resistant strains of M. tuberculosis. The host mechanisms of defense against this infection, and the bacterial mechanisms underlying long-term survival within the host phagocytes remain poorly understood. Elucidating and understanding the molecular basis of genetic differences in natural susceptibility to mycobacterial infections observed in humans and in experimental animals, should provide new insight into the mechanisms of host defense against these infections. In the mouse, natural resistance to infection with mycobacteria is controlled by the Bcg/Ity/Lsh gene. We have cloned a candidate gene for Bcg designated nramp (natural resistance associated macrophage protein) which codes for a novel macrophage specific membrane transport protein. The two major goals if this proposal are the formal demonstration that nramp and Bcg are the same gene, and the comprehensive genetic and biochemical analysis of the Nramp protein, with the aim of understanding its biological role in the macrophage. The creation of a mutant mouse strain carrying a null allele at the nramp locus generated by homologous recombination in embryonal stem cells will be used to determine 1) if nramp and Bcg are the same gene, and 2) to further analyze in vivo the role of nramp in macrophage function. As an alternative approach, we will use transgenic animals to introduce a cloned copy of the Bcg' allele of nramp (129/sv, dominant) onto a Bcgs mouse background (C57BL/6J, recessive), and determine if it can reverse susceptibility to infection in C57BL/6J mice. A structural and functional characterization of the nramp gene is proposed, with special emphasis on the identification of cis-acting sequences and trans- acting factors responsible for constitutive or inducible macrophage specific expression of this gene. We will also determine if novel mutations in nramp and associated with disease susceptibility can be identified in additional inbred mouse strains, but also in the human NRAMP homolog in individuals from familial pedigrees segregating for susceptibility to tuberculosis. A biochemical analysis of the Nramp protein, including the production of specific antibodies, and the identification of its cellular and subcellular localization, putative post-translational modifications, membrane associated transport functions, in particular with respect to reactive nitrogen intermediates, are also proposed. Together, these studies should help understand the role of nramp in macrophage mediated resistance to mycobacterial infections, perhaps providing a new target for therapeutic intervention in the modulation of host defenses against tuberculosis.