Nramp: Deprive and conquer?

Solute carriers 11 (Slc11) evolved from bacterial permease (MntH) to eukaryotic antibacterial defense (Nramp) while continuously mediating proton (H+)-dependent manganese (Mn2+) import. Also, Nramp horizontal gene transfer (HGT) toward bacteria led to mntH polyphyly. Prior demonstration that evolutionary rate-shifts distinguishing Slc11 from outgroup carriers dictate catalytic specificity suggested that resolving Slc11 family tree may provide a function-aware phylogenetic framework. Hence, MntH C (MC) subgroups resulted from HGTs of prototype Nramp (pNs) parologs while archetype Nramp (aNs) correlated with phagocytosis. PHI-Blast based taxonomic profiling confirmed MntH B phylogroup is confined to anaerobic bacteria vs. MntH A (MA)'s broad distribution; suggested niche-related spread of MC subgroups; established that MA-variant MH, which carries 'eukaryotic signature' marks, predominates in archaea. Slc11 phylogeny shows MH is sister to Nramp. Site-specific analysis of Slc11 charge network known to interact with the protonmotive force demonstrates sequential rate-shifts that recapitulate Slc11 evolution. 3D mapping of similarly coevolved sites across Slc11 hydrophobic core revealed successive targeting of discrete areas. The data imply that pN HGT could advantage recipient bacteria for H+-dependent Mn2+ acquisition and Alphafold 3D models suggest conformational divergence among MC subgroups. It is proposed that Slc11 originated as a bacterial stress resistance function allowing Mn2+-dependent persistence in conditions adverse for growth, and that archaeal MH could contribute to eukaryogenesis as a Mn2+ sequestering defense perhaps favoring intracellular growth-competent bacteria.

Determination of Glyphosate and AMPA in Blood Can Predict the Severity of Acute Glyphosate Herbicide Poisoning.

OBJECTIVE: To evaluate a potential association between blood and urine concentration of glyphosate and its metabolite, aminomethylphosphonic acid (AMPA), with severity of acute glyphosate (herbicide) poisoning. METHODS: In our retrospective study of acute glyphosate poisoning, we examined records from the French National Database of Poisonings, dated between January 1, 2004, and December 31, 2016. We compared the severity of poisoning among case individuals using the Fisher exact test and Wilcoxon test. Also, we calculated ROC curves to determine the cutoff for blood and urine concentration. RESULTS: A total of 17 plasma glyphosate, 11 urine glyphosate, 13 plasma AMPA, and 10 urine AMPA specimens were included in our study, with collection dates ranging from January 1, 2004, through December 31, 2016. CONCLUSION: The optimal cutoff we discovered for blood concentration of AMPA was 0.88 mg/L; for glyphosate, it was 600 mg/L. The cutoff plasma concentration of AMPA has never been described in the literature, to our knowledge.

Dairy goats adjust their meal patterns to the fibre content of the diet.

Few studies have investigated how meal patterns of ruminants are affected by diet fibre content. Dairy goats (N = 32) in late lactation and early gestation were housed in eight groups of four goats, with all combinations of breed (Alpine and Saanen) and lactation number (1 and 2) represented in each group. Each goat had access to its own individual feed trough placed on a weigh scale with data logged automatically. All goats were fed the same total mixed ration (TMR; 30% concentrate and 44.6% NDF in DM) ad libitum for a control period of 22 days. Using the same feed ingredients, half of the groups were then offered a High fibre diet (20% concentrate; 47.3% NDF), and the other half a Low fibre diet (40% concentrate; 41.5% NDF) for a treatment period of 16 days. Daily meal patterns (meal frequency, duration and size, feeding rate, daily feed intake and daily feeding time) were computed for each animal using a meal criterion of 8 min. The last 10 days for each period (control and treatment) were used to calculate individual period means and individual differences between the two periods. During the control period, the goats ate on average 12.1 ± 0.49 meals/day, consuming 4.2 ± 0.10 kg fresh TMR daily. When the ration changed, all measures of feeding behaviour except meal size changed asymmetrically for the goats on the two diets. Goats fed the High fibre diet reduced their meal frequency by 10%, and the first meal after feed distribution lasted 11% longer, leading to a 9% reduction in feeding rate and no significant changes in daily feed intake and daily feeding time. Goats on the Low fibre diet did not significantly change their meal frequency or meal size, but the combined changes nevertheless led to a 9% increase in daily feed intake. On the Low fibre diet, goats were able to increase their feeding rate by a third, leading to a reduction in meal durations, thus reducing daily feeding time by 13%. Goats adapt their feeding behaviour to the fibre proportion of the offered diet, with more changes when fibre content is lowered, which needs to be taken into account when comparing phenotypes and adaptability of small ruminants to different diets.

CSACI guidelines for the ethical, evidence-based and patient-oriented clinical practice of oral immunotherapy in IgE-mediated food allergy.

BACKGROUND: Oral immunotherapy (OIT) is an emerging approach to the treatment of patients with IgE-mediated food allergy and is in the process of transitioning to clinical practice. OBJECTIVE: To develop patient-oriented clinical practice guidelines on oral immunotherapy based on evidence and ethical imperatives for the provision of safe and efficient food allergy management. MATERIALS AND METHODS: Recommendations were developed using a reflective patient-centered multicriteria approach including 22 criteria organized in five dimensions (clinical, populational, economic, organizational and sociopolitical). Data was obtained from: (1) a review of scientific and ethic literature; (2) consultations of allergists, other healthcare professionals (pediatricians, family physicians, nurses, registered dieticians, psychologists, peer supporters), patients and caregivers; and patient associations through structured consultative panels, interviews and on-line questionnaire; and (3) organizational and economic data from the milieu of care. All data was synthesized by criteria in a multicriteria deliberative guide that served as a platform for structured discussion and development of recommendations for each dimension, based on evidence, ethical imperatives and other considerations. RESULTS: The deliberative grid included 162 articles from the literature and media reviews and data from consultations involving 85 individuals. Thirty-eight (38) recommendations were made for the practice of oral immunotherapy for the treatment of IgE mediated food allergy, based on evidence and a diversity of ethical imperatives. All recommendations were aimed at fostering a context conducive to achieving objectives identified by patients and caregivers with food allergy. Notably, specific recommendations were developed to promote a culture of shared responsibility between patients and healthcare system, equity in access, patient empowerment, shared decision making and personalization of OIT protocols to reflect patients' needs. It also provides recommendations to optimize organization of care to generate capacity to meet demand according to patient choice, e.g. OIT or avoidance. These recommendations were made acknowledging the necessity of ensuring sustainability of the clinical offer in light of various economic considerations. CONCLUSIONS: This innovative CPG methodology was guided by patients' perspectives, clinical evidence as well as ethical and other rationales. This allowed for the creation of a broad set of recommendations that chart optimal clinical practice and define the conditions required to bring about changes to food allergy care that will be sustainable, equitable and conducive to the well-being of all patients in need.

Reduced in vitro functional activity of human NRAMP1 (SLC11A1) allele that predisposes to increased risk of pediatric tuberculosis disease.

Polymorphic variants within the human natural resistance-associated macrophage protein-1 (NRAMP1, also known as SLC11A1) gene have been shown to impact on susceptibility to tuberculosis in different human populations. In the mouse, Nramp1 is expressed at the macrophage phagosomal membrane and its activity can be assayed by the relative acquisition of mannose 6-phosphate receptor (M6PR) in Salmonella-containing vacuoles. Based on this M6PR recruitment assay, we have now developed an assay in primary human macrophages to test the function of human NRAMP1 gene variants. First, we established that M6PR acquisition was significantly higher (P = 0.002) in human U-937 monocytic cell lines transfected with NRAMP1 as compared to untransfected U-937 cells. Second, the M6PR assay was shown to be highly reproducible for NRAMP1 activity in monocyte-derived macrophages (MDM) from healthy volunteers. Finally, the assay was investigated in MDM from pediatric tuberculosis patients and significantly lower NRAMP1 activity was detected in MDM from individuals homozygous for the NRAMP1-274 high-risk allele (CC genotype) in comparison to heterozygous individuals (CT genotype; P=0.013). The present study describes both an assay for human NRAMP1 functional activity and concomitant evidence for reduced NRAMP1 function in the common genetic variant shown to be associated with tuberculosis susceptibility in pediatric patients.

Recent progress in structure-function analyses of Nramp proton-dependent metal-ion transporters.

The natural resistance-associated macrophage protein (Nramp) homologs form a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions (Me2+, including Mn2+, Fe2+, Co2+, and Cd2+). The Nramp, or solute carrier 11 (SLC11), family is conserved in eukaryotes and bacteria. Humans and rodents express 2 parologous genes that are associated with iron disorders and immune diseases. The NRAMP1 (SLC11A1) protein is specific to professional phagocytes and extrudes Me2+ from the phagosome to defend against ingested microbes; polymorphisms in the NRAMP1 gene are associated with various immune diseases. Several isoforms of NRAMP2 (SLC11A2, DMT1, DCT1) are expressed ubiquitously in recycling endosomes or specifically at the apical membrane of epithelial cells in intestine and kidneys, and can contribute to iron overload, whereas mutations impairing NRAMP2 function cause a form of congenital microcytic hypochromic anemia. Structure-function studies, using various experimental models, and mutagenesis approaches have begun to reveal the overall transmembrane organization of Nramp, some of the transmembrane segments (TMS) that are functionally important, and an unusual mechanism coupling Me2+ and proton H+ transport. The approaches used include functional complementation of yeast knockout strains, electrophysiology analyses in Xenopus oocytes, and transport assays that use mammalian and bacterial cells and direct and indirect measurements of SLC11 transporter properties. These complementary studies enabled the identification of TMS1 and 6 as crucial structural segments for Me2+ and H+ symport, and will help develop a deeper understanding of the Nramp transport mechanism and its contribution to Me2+ homeostasis in human health and diseases.

Acquisition of Mn(II) in addition to Fe(II) is required for full virulence of Salmonella enterica serovar Typhimurium.

The roles of the genes feoB (ABC ferrous iron transporter), mntH (proton-dependent manganese transporter), and sitABCD (putative ABC iron and/or manganese transporter) in Salmonella pathogenicity were investigated by using mutant strains deficient in one, two, or three transporters. Our results indicated that sitABCD encodes an important transporter of Mn(II) and Fe(II) which is required for full virulence in susceptible animals (Nramp1(-/-)) and for replication inside Nramp1(-/-) macrophages in vitro. The mntH sitABCD double mutant (mutant MS) showed minimal Mn(II) uptake and increased sensitivity to H(2)O(2) and to the divalent metal chelator 2,2'-dipyridyl (DP) and was defective for replication in macrophages. In vivo MS appeared to be as virulent as the sitABCD mutant in Nramp1(-/-) animals. The ferrous iron transporter Feo was required for full virulence in 129/Sv Nramp1(-/-) mice, and infection with multiple mutants lacking FeoB was not fatal. The sitABCD feoB mutant (mutant SF) and the mntH sitABCD feoB mutant (mutant MSF) showed minimal Fe(II) uptake and were slightly impaired for replication in susceptible macrophages. MSF showed reduced growth in minimal medium deficient in divalent cations. The role of the mntH gene, which is homologous to mammalian Nramp genes, was also investigated after overexpression in the double mutant MS. MntH preferred Mn(II) over Fe(II) and could suppress MS sensitivity to H(2)O(2) and to DP, and it also improved the intracellular survival in Nramp1(-/-) macrophages. This study indicates that acquisition of Mn(II), in addition to Fe(II), is required for intracellular survival and replication of Salmonella enterica serovar Typhimurium in macrophages in vitro and for virulence in vivo.

Regulation of NRAMP1 gene expression by 1alpha,25-dihydroxy-vitamin D(3) in HL-60 phagocytes.

The natural resistance-associated macrophage protein 1 (Nramp1) is a proton-dependent transporter of divalent metals. We studied NRAMP1 expression during HL-60 differentiation induced by VD and VD agonists. NRAMP1 and CD14 gene expression differed in kinetics of induction, mRNA levels and stability, and response to VD combined with PMA, whereas a combination of VD and IFN-gamma induced similar up-regulation. NRAMP1 protein expression paralleled the accumulation of mRNA and was localized in the phagosomal membrane after phagocytosis. A promoter construct extending 647 bp upstream of NRAMP1 ATG showed myeloid-specific transcription in transient transfection assays, which was up-regulated by VD in HL-60. In HL-60 clones stably transfected with this construct, transcription was apparently induced through indirect VD genomic effects, and there was accordance between the levels of reporter transcription and endogenous NRAMP1 mRNA in response to VD but not to IFN-gamma. Thus, VD genomic effects stimulate NRAMP1 transcription and protein expression in maturing phagocytes.

Inactivation of the Mycobacterium tuberculosis Nramp orthologue (mntH) does not affect virulence in a mouse model of tuberculosis.

Mycobacterium tuberculosis is an intracellular pathogen which can survive and multiply within the phagosomal compartment of the macrophage, and in doing so has to withstand the various macrophage defense mechanisms, which include limitation of iron and other metals. Analysis of the complete genome sequence of M. tuberculosis revealed an extensive array of cation transporters, including mntH, an orthologue of the eukaryotic Nramp (natural resistance-associated macrophage protein) gene, that encodes a proton-dependent divalent metal transporter. To assess the effect of this transporter on intracellular survival and pathogenesis, an mntH knock-out mutant of M. tuberculosis H37Rv was created and assayed in bone marrow-derived macrophages and in a murine model of tuberculosis. In neither of these systems was any loss of fitness associated with inactivation of mntH, demonstrating that Nramp orthologues are not important determinants of mycobacterial virulence.

Polyphyletic origins of bacterial Nramp transporters.

The redox-active metals iron and manganese are required for energy metabolism, protection against oxidative stress and defense against infections. In eukaryotes, both divalent metals are transported by Nramp transporters. The sequence of these transporters was remarkably conserved during evolution. Several bacterial Nramp homologs (MntH) are also proton-dependent manganese transporters. Here, we present phylogenetic evidence for the polyphyletic origins of three groups of MntH proteins and for possible Nramp horizontal gene transfer with eukaryotes. We propose that the evolution of the MntH/Nramp family is related to adaptation to oxidative environments, including those arising during infection of animals and plants.

Identification of the Escherichia coli K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter.

The Escherichia coli mntH (formerly yfeP) gene encodes a putative membrane protein (MntH) highly similar to members of the eukaryotic Nramp family of divalent metal ion transporters. To determine the function of E. coli MntH, a null mutant was created and MntH was overexpressed both in wild-type E. coli and in the metal-dependent mutant hflB1(Ts). At the restrictive temperature 42 degrees C, the mntH null mutation reduces the suppression of hflB1(Ts) thermosensitivity by exogenous divalent metals. Conversely, overexpression of MntH restores growth at 42 degrees C, increases suppression of the ts phenotype by Fe(II) and Ni(II) and renders hflB1(Ts) cells hypersensitive to Mn(II). Transport studies in intact cells show that MntH selectively facilitates uptake of 54Mn(II) and 55Fe(II) in a temperature-, time- and proton-dependent manner. Competition studies in uptake assays and growth inhibition experiments in hflB1(Ts) mutants together indicate that MntH is a divalent metal cation transporter of broad substrate specificity. The functional characteristics of MntH suggest that it corresponds to the previously described manganese transporter of E. coli. This study indicates that proton-dependent divalent metal ion uptake has been preserved in the Nramp family from bacteria to humans.

Expression of the human NRAMP1 gene in professional primary phagocytes: studies in blood cells and in HL-60 promyelocytic leukemia.

In the mouse, mutations at the natural resistance-associated macrophage protein 1 (Nramp1) gene abrogate resistance to infection with antigenically unrelated intracellular parasites such as Mycobacterium, Salmonella, and Leishmania. Nramp1 expression is restricted to reticuloendothelial organs and peripheral blood leukocytes, where the protein may function as a membrane transporter of an as yet to be identified substrate. To identify the human blood cell type(s) expressing NRAMP1 mRNA and determine how Nramp1 expression is regulated in these cells, we have examined separated populations of peripheral blood leukocytes and in vitro cell lines. We observed that polymorphonuclear leukocytes (PMN) are the major site of NRAMP1 expression, followed to a lesser degree by monocytes (MN). Migration of MN to tissues (alveolar macrophages) or maturation in vitro (long-term culture) was associated with a higher level of NRAMP1 expression compared with blood MN. Northern analyses of RNA from model cultured cells showed absence of NRAMP1 expression in transformed cell lines from either erythroid or lymphoid T or B lineages as well as progenitors of the monocyte/macrophage pathway (KG1, U937, THP1), and the HL-60 promyelocytic leukemia. Induction of differentiation of HL-60 cells toward either the monocyte/macrophage (vitamin D3, phorbol ester) or the granulocyte pathways (DMF, DMSO), as measured by induction of IL8-Rb, c-FMS, and CD14 marker gene expression, was concomitant with a strong induction of NRAMP1 expression. These results suggest that NRAMP1 expression is specific to the myeloid lineage and is acquired during the maturation of PMN and MN. The possibility that NRAMP1 may be a component of the phagosomal/endosomal apparatus common to PMN and MN is discussed.

The macrophage-specific membrane protein Nramp controlling natural resistance to infections in mice has homologues expressed in the root system of plants.

In mice, natural resistance or susceptibility to infection with Mycobacteria, Salmonella, and Leishmania is controlled by a gene named Bcg. Bcg regulates the capacity of macrophages to limit intracellular replication of the ingested parasites, and is believed to regulate a key bactericidal mechanism of this cell. Recently, we have cloned the Bcg gene and shown that it encodes a novel macrophage-specific membrane protein designated Nramp. A routine search of the public databases for sequences homologous to Nramp identified 3 expressed sequence tags (EST) that show strong similarities to the mammalian protein. We report the identification and cloning of a full-length cDNA clone corresponding to a plant homologue (OsNramp1) of mammalian Nramp. Predicted amino acid sequence of the plant protein indicates a remarkable degree of similarity (60% homology) with its mammalian counterpart, including identical number, position, and composition of transmembrane domains, glycosylation signals, and consensus transport motif, suggesting an identical overall secondary structure and membrane organization for the two proteins. This high degree of structural similarity indicates that the two proteins may be functionally related, possibly through a common mechanism of transport. RNA hybridization studies and RT-PCR analyses indicate that OsNramp1 mRNA is expressed primarily in roots and only at very low levels in leaves/stem. DNA hybridization studies indicate that OsNramp1 is not a single gene, but rather forms part of a novel gene family which has several members in all plants tested including cereals such as rice, wheat, and corn, and also in common weed species. The striking degree of conservation between the macrophage-specific mammalian Nramp and its OsNramp1 plant homologue is discussed with respect to possible implications in the metabolism of nitrate in both organisms.

Nramp defines a family of membrane proteins.

Nramp (natural resistance-associated macrophage protein) is a newly identified family of integral membrane proteins whose biochemical function is unknown. We report on the identification of Nramp homologs from the fly Drosophila melanogaster, the plant Oryza sativa, and the yeast Saccharomyces cerevisiae. Optimal alignment of protein sequences required insertion of very few gaps and revealed remarkable sequence identity of 28% (yeast), 40% (plant), and 55% (fly) with the mammalian proteins (46%, 58%, and 73% similarity), as well as a common predicted transmembrane topology. This family is defined by a highly conserved hydrophobic core encoding 10 transmembrane segments. Other features of this hydrophobic core include several invariant charged residues, helical periodicity of sequence conservation suggesting conserved and nonconserved faces for several transmembrane helices, a consensus transport signature on the intracytoplasmic face of the membrane, and structural determinants previously described in ion channels. These characteristics suggest that the Nramp polypeptides form part of a group of transporters or channels that act on as yet unidentified substrates.

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.

Identification of polymorphisms and sequence variants in the human homologue of the mouse natural resistance-associated macrophage protein gene.

The most common mycobacterial disease in humans is tuberculosis, and there is evidence for genetic factors in susceptibility to tuberculosis. In the mouse, the Bcg gene controls macrophage priming for activation and is a major gene for susceptibility to infection with mycobacteria. A candidate gene for Bcg was identified by positional cloning and was designated "natural resistance-associated macrophage protein gene" (Nramp1), and the human homologue (NRAMP1) has recently been cloned. Here we report on (1) the physical mapping of NRAMP1 close to VIL in chromosome region 2q35 by PCR analysis of somatic cell hybrids and YAC cloning and (2) the identification of nine sequence variants in NRAMP1. Of the four variants in the coding region, there were two missense mutations and two silent substitutions. The missense mutations were a conservative alanine-to-valine substitution at codon 318 in exon 9 and an aspartic acid-to-asparagine substitution at codon 543 in the predicted cytoplasmic tail of the NRAMP1 protein. A microsatellite was located in the immediate 5' region of the gene, three variants were in introns, and one variant was located in the 3' UTR. The allele frequencies of each of the nine variants were determined in DNA samples of 60 Caucasians and 20 Asians. In addition, we have physically linked two highly polymorphic microsatellite markers, D2S104 and D2S173, to NRAMP1 on a 1.5-Mb YAC contig. These molecular markers will be useful to assess the role of NRAMP1 is susceptibility to tuberculosis and other macrophage-mediated diseases.

Cloning and characterization of a second human NRAMP gene on chromosome 12q13.

The mouse Nramp1 gene was initially identified by positional cloning as a candidate for the host resistance locus Bcg on mouse Chromosome (Chr) 1. Subsequent studies have shown that Nramp is a small gene family of at least three members in the mouse genome. We report the isolation and characterization of a cDNA clone corresponding to the second member of the human NRAMP family, NRAMP2. Predicted amino acid sequence analysis of the NRAMP2 polypeptide identifies a polytopic membrane protein highly homologous to NRAMP1, with 66% identical residues (80% overall homology), resulting in identical predicted secondary structures for the two proteins. Sequence conservation is particularly high in the predicted transmembrane domains (90%), suggesting that these regions play a key role in the structural and functional aspects common to both proteins. As opposed to its NRAMP1 counterpart, whose expression is restricted to phagocytic cells, Northern blotting analyses indicate that NRAMP2 mRNA transcripts are expressed at low levels in all tissues analyzed. Fluorescence in situ hybridization has identified 12q13 as the chromosomal location for human NRAMP2.

Identification and characterization of a second mouse Nramp gene.

The Nramp gene was isolated as a candidate for the host resistance locus Bcg/Ity/Lsh, which controls natural resistance of mice to several types of infections. We have isolated by cross-hybridization cDNA clones corresponding to a second mouse Nramp gene, which we designate Nramp2. Nucleotide and predicted amino acid sequence analyses of full-length cDNA clones for Nramp2 indicate that this novel Nramp protein is closely homologous to the previously described Nramp and that the two genes form part of a small gene family. The two Nramp proteins encode integral membrane proteins that share 63% identical residues and an overall homology of 78%. They share very similar secondary structure, including identical hydropathy profiles and predicted membrane organization, with a minimum of 10 and most probably 12 transmembrane domains, a cluster of predicted N-linked glycosylation sites, and a consensus transport motif. Analysis of the distribution of Nramp2 mRNA transcripts in normal mouse tissues by Northern blotting revealed that the Nramp2 gene produces several mRNAs, including prominent 3.3- and 2.3-kb species generated by the use of alternative polyadenylation signals. In contrast to the previously described macrophage-specific Nramp gene, Nramp2 mRNAs were found to be expressed at low levels in all tissues tested. Using a polymorphic (GT)26 dinucleotide repeat identified in the 3' untranslated region of the mRNA, we have mapped the Nramp2 gene to the distal part of mouse chromosome 15 between markers D15Mit41 and D15Mit15, with the gene order and intergene distance (in cM): centromere-56.1-D15Mit41-(1 +/- 1)-Nramp2-(5 +/- 2)-D15Mit15.