Host Resistance Locus Research Articles

Balancing Selection for Pathogen Resistance Reveals an Intercontinental Signature of Red Queen Coevolution.

The link between long-term host–parasite coevolution and genetic diversity is key to understanding genetic epidemiology and the evolution of resistance. The model of Red Queen host–parasite coevolution posits that high genetic diversity is maintained when rare host resistance variants have a selective advantage, which is believed to be the mechanistic basis for the extraordinarily high levels of diversity at disease-related genes such as the major histocompatibility complex in jawed vertebrates and R-genes in plants. The parasites that drive long-term coevolution are, however, often elusive. Here we present evidence for long-term balancing selection at the phenotypic (variation in resistance) and genomic (resistance locus) level in a particular host–parasite system: the planktonic crustacean Daphnia magna and the bacterium Pasteuria ramosa. The host shows widespread polymorphisms for pathogen resistance regardless of geographic distance, even though there is a clear genome-wide pattern of isolation by distance at other sites. In the genomic region of a previously identified resistance supergene, we observed consistent molecular signals of balancing selection, including higher genetic diversity, older coalescence times, and lower differentiation between populations, which set this region apart from the rest of the genome. We propose that specific long-term coevolution by negative-frequency-dependent selection drives this elevated diversity at the host's resistance loci on an intercontinental scale and provide an example of a direct link between the host’s resistance to a virulent pathogen and the large-scale diversity of its underlying genes.

An alternative route of bacterial infection associated with a novel resistance locus in the Daphnia\u2013Pasteuria host\u2013parasite system

To understand the mechanisms of antagonistic coevolution, it is crucial to identify the genetics of parasite resistance. In the Daphnia magna–Pasteuria ramosa host–parasite system, the most important step of the infection process is the one in which P. ramosa spores attach to the host’s foregut. A matching-allele model (MAM) describes the host–parasite genetic interactions underlying attachment success. Here we describe a new P. ramosa genotype, P15, which, unlike previously studied genotypes, attaches to the host’s hindgut, not to its foregut. Host resistance to P15 attachment shows great diversity across natural populations. In contrast to P. ramosa genotypes that use foregut attachment, P15 shows some quantitative variation in attachment success and does not always lead to successful infections, suggesting that hindgut attachment represents a less-efficient infection mechanism than foregut attachment. Using a Quantitative Trait Locus (QTL) approach, we detect two significant QTLs in the host genome: one that co-localizes with the previously described D. magna PR locus of resistance to foregut attachment, and a second, major QTL located in an unlinked genomic region. We find no evidence of epistasis. Fine mapping reveals a genomic region, the D locus, of ~13 kb. The discovery of a second P. ramosa attachment site and of a novel host-resistance locus increases the complexity of this system, with implications for both for the coevolutionary dynamics (e.g., Red Queen and the role of recombination), and for the evolution and epidemiology of the infection process.

The genetic basis of resistance and matching-allele interactions of a host-parasite system: The Daphnia magna-Pasteuria ramosa model.

Negative frequency-dependent selection (NFDS) is an evolutionary mechanism suggested to govern host-parasite coevolution and the maintenance of genetic diversity at host resistance loci, such as the vertebrate MHC and R-genes in plants. Matching-allele interactions of hosts and parasites that prevent the emergence of host and parasite genotypes that are universally resistant and infective are a genetic mechanism predicted to underpin NFDS. The underlying genetics of matching-allele interactions are unknown even in host-parasite systems with empirical support for coevolution by NFDS, as is the case for the planktonic crustacean Daphnia magna and the bacterial pathogen Pasteuria ramosa. We fine-map one locus associated with D. magna resistance to P. ramosa and genetically characterize two haplotypes of the Pasteuria resistance (PR-) locus using de novo genome and transcriptome sequencing. Sequence comparison of PR-locus haplotypes finds dramatic structural polymorphisms between PR-locus haplotypes including a large portion of each haplotype being composed of non-homologous sequences resulting in haplotypes differing in size by 66 kb. The high divergence of PR-locus haplotypes suggest a history of multiple, diverse and repeated instances of structural mutation events and restricted recombination. Annotation of the haplotypes reveals striking differences in gene content. In particular, a group of glycosyltransferase genes that is present in the susceptible but absent in the resistant haplotype. Moreover, in natural populations, we find that the PR-locus polymorphism is associated with variation in resistance to different P. ramosa genotypes, pointing to the PR-locus polymorphism as being responsible for the matching-allele interactions that have been previously described for this system. Our results conclusively identify a genetic basis for the matching-allele interaction observed in a coevolving host-parasite system and provide a first insight into its molecular basis.

Genome-Wide Association Study of Spot Form of Net Blotch Resistance in the Upper Midwest Barley Breeding Programs.

Pyrenophora teres f. maculata, the causal agent of spot form of net blotch (SFNB), is an emerging pathogen of barley in the United States and Australia. Compared with net form of net blotch (NFNB), less is known in the U.S. Upper Midwest barley breeding programs about host resistance and quantitative trait loci (QTL) associated with SFNB in breeding lines. The main objective of this study was to identify QTL associated with SFNB resistance in the Upper Midwest two-rowed and six-rowed barley breeding programs using a genome-wide association study approach. A total of 376 breeding lines of barley were evaluated for SFNB resistance at the seedling stage in the greenhouse in Fargo in 2009. The lines were genotyped with 3,072 single nucleotide polymorphism (SNP) markers. Phenotypic evaluation showed a wide range of variability among populations from the four breeding programs and the two barley-row types. The two-rowed barley lines were more susceptible to SFNB than the six-rowed lines. Continuous distributions of SFNB severity indicate the quantitative nature of SFNB resistance. The mixed linear model (MLM) analysis, which included both population structure and kinship matrices, was used to identify significant SNP-SFNB associations. Principal component analysis was used to control false marker-trait association. The linkage disequilibrium (LD) estimates varied among chromosomes (10 to 20 cM). The MLM analysis identified 10 potential QTL in barley: SFNB-2H-8-10, SFNB-2H-38.03, SFNB-3H-58.64, SFNB-3H-78.53, SFNB-3H-91.88, SFNB-3H-117.1, SFNB-5H-155.3, SFNB-6H-5.4, SFNB-6H-33.74, and SFNB-7H-34.82. Among them, four QTL (SFNB-2H-8-10, SFNB-2H-38.03 SFNB-3H-78.53, and SFNB-3H-117.1) have not previously been published. Identification of SFNB resistant lines and QTL associated with SFNB resistance in this study will be useful in the development of barley genotypes with better SFNB resistance.

Identification of Loci Controlling Restriction of Parasite Growth in Experimental Taenia crassiceps Cysticercosis

Human neurocysticercosis (NC) caused by Taenia solium is a parasitic disease of the central nervous system that is endemic in many developing countries. In this study, a genetic approach using the murine intraperitoneal cysticercosis caused by the related cestode Taenia crassiceps was employed to identify host factors that regulate the establishment and proliferation of the parasite. A/J mice are permissive to T. crassiceps infection while C57BL/6J mice (B6) are comparatively restrictive, with a 10-fold difference in numbers of peritoneal cysticerci recovered 30 days after infection. The genetic basis of this inter-strain difference was explored using 34 AcB/BcA recombinant congenic strains derived from A/J and B6 progenitors, that were phenotyped for T. crassiceps replication. In agreement with their genetic background, most AcB strains (A/J-derived) were found to be permissive to infection while most BcA strains (B6-derived) were restrictive with the exception of a few discordant strains, together suggesting a possible simple genetic control. Initial haplotype association mapping using >1200 informative SNPs pointed to linkages on chromosomes 2 (proximal) and 6 as controlling parasite replication in the AcB/BcA panel. Additional linkage analysis by genome scan in informative [AcB55xDBA/2]F1 and F2 mice (derived from the discordant AcB55 strain), confirmed the effect of chromosome 2 on parasite replication, and further delineated a major locus (LOD = 4.76, p<0.01; peak marker D2Mit295, 29.7 Mb) that we designate Tccr1 (T. crassiceps cysticercosis restrictive locus 1). Resistance alleles at Tccr1 are derived from AcB55 and are inherited in a dominant fashion. Scrutiny of the minimal genetic interval reveals overlap of Tccr1 with other host resistance loci mapped to this region, most notably the defective Hc/C5 allele which segregates both in the AcB/BcA set and in the AcB55xDBA/2 cross. These results strongly suggest that the complement component 5 (C5) plays a critical role in early protective inflammatory response to infection with T. crassiceps.

Molecular Mapping of Hypersensitive Resistance from Tomato ‘Hawaii 7981’ toXanthomonas perforansRace T3

Bacterial spot of tomato (Solanum lycopersicum) is caused by four species of Xanthomonas. The disease causes significant yield losses and a reduction in fruit quality. Physiological races have been described with tomato race 3 (T3) corresponding to strains of Xanthomonas perforans. The breeding line Hawaii 7981 (hereafter H7981) shows a hypersensitive reaction (HR) to race T3 strains conditioned by the interaction of the host resistance locus Xv3 and the bacterial effector avrXv3. The Xv3 gene is required for H7981-derived resistance to be effective under field conditions, though its expression is subject to genetic background. The segregation of HR in F(2) populations derived from H7981 crossed to processing tomato parents OH88119 and OH7870 was studied in 331 progeny, with the two independent crosses providing validation. We screened 453 simple-sequence repeat, insertion/deletion, and single-nucleotide polymorphism markers and identified 44 polymorphic markers each for the OH88119 and OH7870 populations covering 84.6 and 73.3% of the genome, respectively, within 20 centimorgans (cM). Marker-trait analysis using all polymorphic markers demonstrated that Xv3-mediated resistance maps to chromosome 11 in the two independent crosses. Allelism tests were conducted in crosses between lines carrying Xv3 derived from H7981, Rx-4 derived from plant introduction (PI) 128216, and resistance derived from PI 126932. These allelism tests suggested that the loci conditioning HR to race T3 strains are linked within 0.1 cM, are allelic, or are the same gene.

Identification and characterization of Cri1, a locus controlling mortality during Citrobacter rodentium infection in mice

Infection of inbred mouse strains with Citrobacter rodentium represents an ideal model to reveal the genetic factors controlling host resistance to noninvasive enteric bacterial pathogens. We have chosen a positional cloning approach to identify putative gene(s) that control the known difference in survival between resistant C57BL/6J and susceptible C3H/HeJ and C3H/HeOuJ mice. Our work has identified one major locus within proximal chromosome 15 that is responsible for the marked susceptibility of both C3H strains, and we formally exclude Tlr4 from control of survival to this pathogen. We have named this new host resistance locus Cri1 (Citrobacter rodentium infection 1). The Cri1 genetic interval currently spans ∼16 Mb and it confers survival to the infection in a recessive manner. Transfer of the Cri1 locus from the surviving B6 mice into a congenic mouse with a C3Ou genetic background confirms its overall chromosomal localization and its highly significant effect on host survival. The C3Ou.B6-Cri1 mice thus produced have also enabled us to dissociate the control of mouse survival from the control of bacterial load early in the infection as well as from control of colonic hyperplasia.

Fast, adaptive evolution at a bacterial host-resistance locus: The PFam54 gene array in Borrelia burgdorferi

Microbial pathogens have evolved sophisticated mechanisms for evasion of host innate and adaptive immunities. PFam54 is the largest paralogous gene family in the genomes of Borrelia burgdorferi, the Lyme disease bacterium. One member of PFam54, the complement-regulator acquiring surface proteins 1 (Bb Crasp-1), is able to abort the alternative pathway of complement activation via binding human complement-regulator factor H (FH). The gene coding for BbCRASP-1 exists in a tandem array of PFam54 genes in the B. burgdorferi genome, a result apparently of repeated gene duplications. To help elucidate the functions of the large number of PFam54 genes, we performed phylogenomic and structural analyses of the PFam54 gene array from ten B. burgdorferi genomes. Analyses based on gene tree, genome synteny, and structural models revealed rapid adaptive evolution of this array through gene duplication, gene loss, and functional diversification. Individual PFam54 genes, however, do not show high intra-population sequence polymorphisms as genes providing evasion from adaptive immunity generally do. PFam54 members able to bind human FH are not monophyletic, suggesting that human FH affinity, however strong, is an incidental rather than main function of these PFam54 proteins. The large number of PFam54 genes existing in any single B. burgdorferi genome may target different innate-immunity proteins of a single host species or the same immune protein of a variety of host species. Genetic variability of the PFam54 gene array suggests that universally present PFam54 lineages such as BBA64, BBA65, BBA66, and BBA73 may be better candidates for the development of broad-spectrum vaccines or drugs than strain-restricted lineages such as BbCRASP-1.

Dominant Role of the sst1 Locus in Pathogenesis of Necrotizing Lung Granulomas during Chronic Tuberculosis Infection and Reactivation in Genetically Resistant Hosts

Significant host heterogeneity in susceptibility to tuberculosis exists both between and within mammalian species. Using a mouse model of infection with virulent Mycobacterium tuberculosis (Mtb), we identified the genetic locus sst1 that controls the progression of pulmonary tuberculosis in immunocompetent hosts. In this study, we demonstrate that within the complex, multigenic architecture of tuberculosis susceptibility, sst1 functions to control necrosis within tuberculosis lesions in the lungs; this lung-specific sst1 effect is independent of both the route of infection and genetic background of the host. Moreover, sst1-dependent necrosis was observed at low bacterial loads in the lungs during reactivation of the disease after termination of anti-tuberculosis drug therapy. We demonstrate that in sst1-susceptible hosts, nonlinked host resistance loci control both lung inflammation and production of inflammatory mediators by Mtb-infected macrophages. Although interactions of the sst1-susceptible allele with genetic modifiers determine the type of the pulmonary disease progression, other resistance loci do not abolish lung necrosis, which is, therefore, the core sst1-dependent phenotype. Sst1-susceptible mice from tuberculosis-resistant and -susceptible genetic backgrounds reproduce a clinical spectrum of pulmonary tuberculosis and may be used to more accurately predict the efficacy of anti-tuberculosis interventions in genetically heterogeneous human populations.

The Relationship of Host-Mediated Induced Resistance to Polymorphism in Gene-for-Gene Relationships

Gene-for-gene relationships are a common feature of plant-parasite interactions. Polymorphism at host resistance and parasite avirulence loci is maintained if there is negative, direct frequency-dependent selection on alleles of either gene. More specifically, selection of this kind is generated when the disease is polycyclic with frequent auto-infection. When an incompatible interaction occurs between a resistant host and an avirulent parasite, systemic defenses are triggered, rendering the plant more resistant to a later attack by another parasite. However, induced resistance (IR) incurs a fitness cost to the plant. Here, the effect of IR on polymorphism in gene-for-gene interactions is investigated. First, in an infinite population model in which parasites have two generations per host generation, increasing the fitness cost of IR increases selection for susceptible plants at low disease severity, while increasing the effectiveness of IR against further parasite attacks enhances selection for resistant plants at high disease severity. This reduces the possibility of polymorphism being maintained in host and parasite populations. In finite population models, the number of plants varies over time as a function of the disease burden of the population. Polymorphism in gene-for-gene relationships is then more stable at high disease prevalence and severity if IR reactions are more costly when there is competition for resources between plants.

Chromosomal mapping of host resistance loci to Trichinella spiralis nematode infection in rats

The differences in host response among strains of rats to intestinal nematode parasite Trichinella spiralis infection could provide a powerful benefit for further elucidation of molecular interactions between the host and the parasite. Using several strains of rats, we previously observed that DA strain is a strong responder and F344 strain is a weak responder with respect to expulsion of the adult worm. To identify the host resistance loci, quantitative trait loci (QTLs) analysis in F2 population from crosses between DA and F344 strains was performed. One significant QTL (designated as Tspe) was mapped to the middle region of chromosome 9. In addition, the effect of DA allele at Tspe locus could act recessively and lead to the rejection of more adult worms from the gut. The results from the present study provide more insights on host-parasite interactions, which may be useful in facilitating the development of novel approaches for treatment and control of intestinal parasites in human and domestic livestock.

Host-parasite interactions and the evolution of gene expression.

Interactions between hosts and parasites provide an ongoing source of selection that promotes the evolution of a variety of features in the interacting species. Here, we use a genetically explicit mathematical model to explore how patterns of gene expression evolve at genetic loci responsible for host resistance and parasite infection. Our results reveal the striking yet intuitive conclusion that gene expression should evolve along very different trajectories in the two interacting species. Specifically, host resistance loci should frequently evolve to co-express alleles, whereas parasite infection loci should evolve to express only a single allele. This result arises because hosts that co-express resistance alleles are able to recognize and clear a greater diversity of parasite genotypes. By the same token, parasites that co-express antigen or elicitor alleles are more likely to be recognized and cleared by the host, and this favours the expression of only a single allele. Our model provides testable predictions that can help interpret accumulating data on expression levels for genes relevant to host−parasite interactions.

NK gene complex haplotype variability and host resistance alleles to murine cytomegalovirus in wild mouse populations

The NK gene complex (NKC) on mouse chromosome 6 encodes receptors that are expressed on NK cells, such as Ly49H, and is involved in regulating NK cell control of virus infections, such as murine cytomegalovirus (MCMV). In the present study, we investigated the level of allelic heterogeneity in NKC loci in populations of outbred wild mice. This work revealed extensive levels of heterogeneity within two wild mouse populations. Analysis of MCMV replication in a population of specific pathogen-free outbred wild mice revealed that low viral titres, which are normally associated with the Cmv1(r) allele of the Cmv1 host resistance locus, were not prevalent in the mice tested. Hence, NKC-mediated resistance associated with Cmv1(r)/Ly49H-like effects was rare in this population. Overall, these data indicate that the NKC region is highly polymorphic and thus it is very likely that it confers on mice sufficient variability to cope with infection by a range of pathogens.

Host–pathogen interactions: Host resistance factor Nramp1 up-regulates the expression of Salmonella pathogenicity island-2 virulence genes

Nramp1 (Natural resistance-associated macrophage protein-1; also known as Slc11a1) is a host resistance gene that provides protection against several intracellular pathogens, including Salmonella enterica serovar Typhimurium. Little is known about the dynamic interplay that occurs between mammalian host resistance determinants such as Nramp1 and pathogens during infection. To explore these interactions, we examined the effect of Nramp1 on expression of Salmonella typhimurium (STM) virulence factors. We demonstrate that Salmonella pathogenicity island 2 (SPI2) is essential for replication of STM in spleens of infected Nramp1(+/+) mice. Furthermore, the presence of Nramp1 in transfected cell lines and congenic knockout mice resulted in the up-regulation of STM SPI2-associated virulence genes critical for intramacrophage survival. This Nramp1-dependent up-regulation of SPI2 was mimicked in vitro by chelation of iron, demonstrating the iron-responsive nature of expression of STM SPI2-associated virulence genes. We propose that acquisition of SPI2 by S. enterica not only enabled this bacterium to become an effective intracellular pathogen but also allowed the bacterium to withstand the effects of macrophage defense mechanisms such as Nramp1 early in the evolution of its pathogenic character. These dynamic Nramp1-pathogen interactions may be essential for regulating the course of an infection. This study demonstrates the presence of a previously undescribed direct influence of a mammalian innate host resistance locus on a pathogen at the genetic level.

Evolution of gene‐for‐gene systems in metapopulations: the effect of spatial scale of host and pathogen dispersal

The concept of gene‐for‐gene coevolution is a major model for research on disease resistance in crop plants. However, few theoretical or empirical studies have examined such systems in natural situations, and as a consequence, there is little knowledge of how spatial effects are likely to influence the evolution of host resistance and pathogen virulence in gene‐for‐gene interactions. In this work, a simulation approach was used to investigate the epidemiological and genetic consequences of varying host and pathogen dispersal in metapopulation situations. The results demonstrate clear impacts of dispersal distance on the total number of host and pathogen genotypes that are maintained, as well as on genetic variation at individual host resistance and pathogen virulence loci. Several other important results also emerged from this study. In contrast to the predictions of many earlier nonspatial models, so‐called ‘super‐races’ of pathogens do not always evolve and dominate, indicating that it is not necessary to assume costs of resistance or virulence to maintain high levels of polymorphism in biologically realistic situations. The rate of evolution of both resistance and virulence depend on the scale of dispersal, with greater mixing (as a function of dispersal scale) resulting in a faster approach to a dynamic endpoint. The model in this paper also predicts that, despite the greater total genotypic diversity of pathogens across the metapopulation, variation in host resistance will generally be greater than variation in pathogen virulence within local populations.

Chromosomal mapping of the host resistance locus to rodent malaria (Plasmodium yoelii) infection in mice.

The disease outcome in malaria caused by the protozoan parasite Plasmodium is influenced by host genetic factors. To identify host genes conferring resistance to infection with the malaria parasite, we undertook chromosomal mapping using a whole-genome scanning approach in cross-bred mice. NC/Jic mice all died with high parasitemia within 8 days of infection with 1 x 10(5) parasitized erythrocytes. In contrast, 129/SvJ mice all completely excluded malaria parasites from the circulation and remained alive 21 days after infection. We performed linkage analysis in backcross [(NC/Jic x 129/SvJ)xNC/Jic] mice. The Pymr ( Plasmodium yoelii malaria resistance) locus was mapped to the telomeric portion of mouse Chromosome (Chr) 9. This locus controls host survival and parasitemia after infection. The Char1 locus ( P. chabaudi resistance locus 1), controlling host survival and peak parasitemia in P. chabaudi infection, was previously mapped to the same region. This host resistance locus mapping to Chr 9 may represent a ubiquitous locus controlling susceptibility to rodent malaria. Elucidation of the function of this gene will provide valuable insights into the mechanism of host defense against malaria parasite infection.

Sequence-Ready BAC Contig, Physical, and Transcriptional Map of a 2-Mb Region Overlapping the Mouse Chromosome 6 Host-Resistance Locus Cmv1

The host-resistance locus Cmv1 controls viral replication of mouse cytomegalovirus (MCMV) in the spleen of infected mice. Cmv1 maps on distal chromosome 6, very tightly linked to the Ly49 gene family within a 0.35-cM interval defined proximally by Cd94/Nkg2d and distally by D6Mit13/D6Mit111/D6Mit219/Prp/Kap. To facilitate the cloning of the gene, we have created a high-resolution physical map of the Cmv1 genetic interval that is based on long-range restriction mapping by pulsed-field gel electrophoresis, fluorescence in situ hybridization analysis of interphase nuclei, and the assembly of a cloned contig. A contig of BAC and YAC clones was assembled using probes derived from the minimal genetic interval. Individual clones from the region were validated by (1) restriction digest fingerprinting, (2) STS content mapping, (3) Southern hybridizations, and (4) sequencing and mapping of clone ends. This contig contains 25 YACs anchored by 71 STSs and 73 BACs anchored by 40 STSs. We also report the cloning of 31 new STSs and 18 new polymorphic markers. A minimum tiling path was defined that consists of either 4 YACs or 13 BACs covering 1.82 Mb between D6Ott8, the closest proximal marker, and D6Ott115, the closest distal marker. Gene distribution in the region includes 14 Ly49 genes as well as 3 new additional transcripts. This high-resolution, sequence-ready BAC contig provides a backbone for the identification of Cmv1 and its relationship with genes involved in innate immunity.

The Nramp1 protein and its role in resistance to infection and macrophage function.

Susceptibility to infectious diseases is under genetic control in humans. Animal models provide an ideal tool to study the genetic component of susceptibility and to identify candidate genes that can then be tested for association or linkage studies in human populations from endemic areas of disease. The Nramp1 gene was isolated by positional cloning the host resistance locus Bcg/Ity/Lsh, and mutations at this locus impair the resistance of mice to infections with intracellular parasites, such as Salmonella, Leishmania, and Mycobacterium. Allelic variants at the human Nramp1 homologue have recently been found to be associated with susceptibility to tuberculosis and leprosy in humans. The Nramp1 protein is an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. After phagocytosis, Nramp1 is targeted to the membrane of the microbe-containing phagosome, where it may modify the intraphagosomal milieu to affect microbial replication. Although the biochemical mechanism of action of Nramp1 at that site remains unknown, Nramp homologues have been identified in many other animal species and actually define a protein family conserved from bacteria to humans. Some of these homologues have been shown to be divalent cation transporters. Recently, a second member of the mammalian Nramp family, Nramp2, was discovered and shown to be mutated in animal models of iron deficiency. The Nramp2 protein was subsequently shown to be the major transferrin-independent iron uptake system of the intestine. Together, these results suggest that Nramp1 may control intracellular microbial replication by actively removing iron or other divalent cations from the phagosomal space.

Genetic and physical mapping of the mouse host resistance locus Lgn1.

Genetic and physical mapping of the mouse host resistance locus Lgn1.

Genomic Structure, Promoter Sequence, and Induction of Expression of the Mouse Nramp1 Gene in Macrophages

A candidate gene for the mouse chromosome 1 host resistance locus Bcg/Ity/Lsh was recently cloned and designated Nramp (natural resistance-associated macrophage protein). Nramp is part of a small family of at least two genes, Nramp1 and Nramp2. Primer extension and cDNA cloning were used to isolate the complete 5' end of the Nramp1 mRNA. Analysis of genomic cosmid and bacteriophage clones overlapping the complete Nramp1 gene revealed that the gene was composed of 15 exons and spanned 11.5 kb of genomic DNA. Positioning of introns on the coding portion of the mRNA revealed a modular relationship between coding exons and predicted structural domains of the protein, with 8 of the 12 transmembrane (TM) domains encoded by individual exons. Northern blotting analysis indicated that Nramp1 expression was restricted to J774A.1 and RAW 264.7 macrophage lines and was dramatically increased by treatment with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Primer extension and S1 nuclease mapping experiments were used to locate the transcription initiation site of Nramp1 and revealed the presence of one major and several minor initiation sites. Nucleotide sequencing of the corresponding region failed to detect classical TATA and CAAT elements, but identified two putative initiator sequences located near the major initiation site. Consensus sequences for binding of the macrophage and B-cell-specific transcription factor PU.1, as well as several LPS (NF-IL6) and IFN-gamma response elements, were also identified.