Nramp2 Gene Research Articles

Determination of cadmium and/or drought stress effects on some plant phytohormone contents and hormone gene expressions in bean (Phaseolus vulgaris L)

Plants can sometimes be under the influence of more than one stress factor. In this case, combined stress factors can cause different responses in plants. In this study, the phytohormonal activity and hormone gene expressions of beans (Phaseolus vulgaris L.) were investigated in different drought levels and soils with different levels of cadmium accumulation. Plants were grown at 4 different cadmium (Cd) levels (Cd: 0, Cd1: 100, Cd2: 150, and Cd3: 200 mg kg$^{-1}$) and 3 irrigation levels (D0: 100%, D1: 75%, and D2: 50% of field capacity). Abscisic acid (ABA), indole acetic acid (IAA), gibberellic acid (GA), salicylic acid (SA), cytokinin, zeatin, and jasmonic acid contents and gene expressions related to them were investigated. As a result of the study, we determined that there was a significant decrease in the content of IAA, GA, SA, cytokinin, zeatin, and jasmonic acid with increasing doses of drought and Cd. Especially with the coexistence of both stress factors, this reducing effect (the reductions in these hormones were 86.6%, 64.1%, 71.9%, 66.7%, 45.5%, and 73.9% as compared to D0, respectively) on the measured parameters was greater. However, ABA content increased significantly with drought and/or Cd stresses, especially with Cd3D2, about a thousand times higher than the control. On the other hand, mRNA transcript levels of some hormone genes (ABA 8'-hydroxylase, adenylate isopentenyltransferase (AIPT3), auxin-related protein (AUX/ IAA), MeJA biosynthesis and gibberellin 20-oxidase and NRAMP2 gene known to be involved in Cd transport, were detected. According to the results, while ABA 8'-hydroxylase, gibberellin 20-oxidase, and NRAMP2 genes were overexpressed in all stress treatments, AIPT3 and AUX/IAA genes were down-regulated and also, MeJA biosynthesis gene varied depending on dose and stress.

Genetic and transcriptional variations in NRAMP-2 and OPAQUE1 genes are associated with salt stress response in wheat

Key messageSNP alleles on chromosomes 4BL and 6AL are associated with sensitivity to salt tolerance in wheat and upon validation can be exploited in the development of salt-tolerant wheat varieties.The dissection of the genetic and molecular components of salt stress response offers strong opportunities toward understanding and improving salt tolerance in crops. In this study, GWAS was employed to identify a total of 106 SNP loci (R2 = 0.12–63.44%) linked to salt stress response in wheat using leaf chlorophyll fluorescence, grain quality and shoot ionic (Na+ and K+ ions) attributes. Among them, 14 SNP loci individually conferred pleiotropic effects on multiple independent salinity tolerance traits including loci at 99.04 cM (R2 ≥ 14.7%) and 68.45 cM (R2 ≥ 4.10%) on chromosomes 6AL and 4BL, respectively, that influenced shoot Na+-uptake, shoot K+/Na+ ratio, and specific energy fluxes for absorption (ABS/RC) and dissipation (DIo/RC). Analysis of the open reading frame (ORF) containing the SNP markers revealed that they are orthologous to genes involved in photosynthesis and plant stress (salt) response. Further transcript abundance and qRT-PCR analyses indicated that the genes are mostly up-regulated in salt-tolerant and down-regulated in salt-sensitive wheat genotypes including NRAMP-2 and OPAQUE1 genes on 4BL and 6AL, respectively. Both genes showed highest differential expression between contrasting genotypes when expressions of all the genes within their genetic intervals were analyzed. Possible cis-acting regulatory elements and coding sequence variation that may be involved in salt stress response were also identified in both genes. This study identified genetic and molecular components of salt stress response that are associated with Na+-uptake, shoot Na+/K+ ratio, ABS/RC, DIo/RC, and grain quality traits and upon functional validation would facilitate the development of gene-specific markers that could be deployed to improve salinity tolerance in wheat.

Expression kinetics of natural resistance associated macrophage protein (NRAMP) genes in Salmonella Typhimurium-infected chicken

BackgroundSalmonella enterica serovar Typhimurium (Salmonella Typhimurium) is a zoonotic pathogen responsible for severe intestinal pathology in young chickens. Natural resistance-associated macrophage protein (NRAMP) family has been shown to be associated with resistance to intracellular pathogens, including Salmonella Typhimurium. The role of NRAMP proteins in macrophage defence against microbial infection has been ascribed to changes in the metal-ion concentrations inside the bacteria-containing phagosomes. The present study was conducted to investigate tissue-specific (liver, spleen and caecum) expression kinetics of NRAMP gene family (NRAMP1 and NRAMP2) in broilers from day 0 to day 15 after Salmonella Typhimurium challenge concomitant to clinical, blood biochemical and immunological parameters survey.ResultsClinical symptoms appeared 4 days post-infection (dpi) in infected birds. Symptoms like progressive weakness, anorexia, diarrhoea and lowering of the head were seen in infected birds one-week post-infection. On postmortem examination, liver showed congestion, haemorrhage and necrotic foci on the surface, while as the spleen, lungs and intestines revealed congestion and haemorrhages. Histopathological alterations were principally found in liver comprising of necrosis, reticular endothelial hyperplasia along with mononuclear cell and heterophilic infiltration. Red Blood Cell (RBC) count, Haemoglobin (Hb) and Packed Cell Volume (PCV) decreased significantly (P < 0.05) in blood while heterophil counts increased up to 7 days post-infection. Serum glucose, aspartate transaminase (AST) and alanine transaminase (ALT) enzymes concentrations increased significantly throughout the study. A gradual increase of specific humoral IgG response confirmed Salmonella infection. Meanwhile, expression of NRAMP1 and NRAMP2 genes was differentially regulated after infection in tissues such as liver, spleen and caecum known to be the target of Salmonella Typhimurium replication in the chicken.ConclusionThus the specific roles of NRAMP1 and NRAMP2 genes in Salmonella Typhimurium induced disease may be supposed from their differential expression according to tissues and timing after per os infection. However, these roles remain to be analyzed related to the severity of the disease which can be estimated by blood biochemistry and immunological parameters.

Exploring genetic polymorphism in innate immune genes in Indian cattle (Bos indicus) and buffalo (Bubalus bubalis) using next generation sequencing technology

Activation of innate immunity initiates various cascades of reactions that largely contribute to defense against physical, microbial or chemical damage, prompt for damage repair and removal of causative organisms as well as restoration of tissue homeostasis. Genetic polymorphism in innate immune genes plays prominent role in disease resistance capabilities in various breeds of cattle and buffalo. Here we studied single nucleotide variations (SNP/SNV) and haplotype structure in innate immune genes viz CHGA, CHGB, CHGC, NRAMP1, NRAMP2, DEFB1, BNBD4, BNBD5, TAP and LAP in Gir cattle and Murrah buffalo. Targeted sequencing of exonic regions of these genes was performed by Ion Torrent PGM sequencing platform. The sequence reads obtained corresponding to coding regions of these genes were mapped to reference genome of cattle BosTau7 by BWA program using genome analysis tool kit (GATK). Further variant analysis by Unified Genotyper revealed 54 and 224 SNPs in Gir and Murrah respectively and also 32 SNVs was identified. Among these SNPs 43, 36, 11,32,81,21 and 22 variations were in CHGA, CHGB, CHGC, NRAMP1, NRAMP2, DEFB1 and TAP genes respectively. Among these identified 278 SNPs, 24 were found to be reported in the dbSNP database. Variant analysis was followed by structure formation of haplotypes based on multiple SNPs using SAS software revealed a large number of haplotypes. The SNP discovery in innate immune genes in cattle and buffalo breeds of India would advance our understanding of role of these genes in determining the disease resistance/susceptibility in Indian breeds. The identified SNPs and haplotype data would also provide a wealth of sequence information for conservation studies, selective breeding and designing future strategies for identifying disease associations involving samples from distinct populations.

Characterization of chicken natural resistance-associated macrophage protein encoding genes (Nramp1 and Nramp2) and association with salmonellosis resistance

Natural resistance-associated macrophage protein 1 and 2 encoding genes (Nramp1 and Nramp2) are related to many diseases. We cloned the cDNA of chicken Nramp1 and Nramp2 genes, characterized their expression and polymorphisms, and investigated the association of some SNPs with resistance to salmonellosis. The Nramp1 cDNA was 1746 bp long and the Nramp2 cDNA was 1938 bp long. These cDNAs are similar to previously reported cDNAs, varying by two and one amino acids, respectively. The chicken Nramp1 gene expressed predominantly in liver, thymus and spleen in both females and males. The Nramp2 gene expressed in almost all tissues, but predominantly in breast muscle, leg muscle, cerebrum, cerebellum, lung, kidney, and heart in both females and males. We identified 45 SNPs and 2 indels in the chicken Nramp1 gene; three of 13 SNPs in the exons were missense mutations (Arg223Gln, Ala273Glu and Arg497Gln). Association analysis indicated that A24101991G is significantly associated with chicken salmonellosis resistance. These results will be useful for functional investigation of chicken Nramp1 and Nramp2 genes.

The Nramp (Slc11) proteins regulate development, resistance to pathogenic bacteria and iron homeostasis in Dictyostelium discoideum

The Dictyostelium discoideum genome harbors two genes encoding members of the Nramp superfamily, which is conserved from bacteria (MntH proteins) to humans (Slc11 proteins). Nramps are proton-driven metal ion transporters with a preference for iron and manganese. Acquisition of these metal cations is vital for all cells, as they act as redox cofactors and regulate key cellular processes, such as DNA synthesis, electron transport, energy metabolism and oxidative stress. Dictyostelium Nramp1 (Slc11a1), like its mammalian ortholog, mediates resistance to infection by invasive bacteria. We have extended the analysis to the nramp2 gene, by generating single and double nramp1/nramp2 knockout mutants and cells expressing GFP fusion proteins. In contrast to Nramp1, which is recruited to phagosomes and macropinosomes, the Nramp2 protein is localized exclusively in the membrane of the contractile vacuole, a vesicular tubular network regulating cellular osmolarity. Both proteins colocalize with the V-H(+)-ATPase, which can provide the electrogenic force for vectorial transport. Like nramp1, nramp2 gene disruption affects resistance to Legionella pneumophila. Disrupting both genes additionally leads to defects in development, with strong delay in cell aggregation, formation of large streams and multi-tipped aggregates. Single and double mutants display differential sensitivity to cell growth under conditions of iron overload or depletion. The data favor the hypothesis that Nramp1 and Nramp2, under control of the V-H(+)-ATPase, synergistically regulate iron homeostasis, with the contractile vacuole possibly acting as a store for metal cations.

SLC11A1 (formerly NRAMP1) and disease resistance.

Slc11a1 (formerly Nramp1) has many pleiotropic effects on macrophage (m) activation, including regulation of the CXC chemokine KC, interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), major histocompatibility complex (MHC) class II molecules, tumour necrosis factor α (TNFα), nitric oxide (NO) release, l-arginine flux, oxidative burst and tumoricidal as well as antimicrobial activity (reviewed by Blackwell and Searle, 1999; Blackwell et al., 2000). A naturally occurring Gly→Asp mutation at amino acid 169 of Slc11a1 makes mice as susceptible to Leishmania donovani, Salmonella typhimurium and Mycobacterium bovis as gene-disrupted mice (Vidal et al., 1995). Hence, the mutation is a functional null. This mutation also confers susceptibility to a range of other pathogens in mice, including Mycobacterium lepraemurium (Brown et al., 1982; Skamene et al., 1984), Mycobacterium intracellulare (Goto et al., 1989), Toxoplasma gondii (Blackwell et al., 1994), Candida albicans (Puliti et al., 1995) and Leishmania infantum (Leclercq et al., 1996). In man, SLC11A1 is linked or associated with multiple infectious (Shaw et al., 1997; Abel et al., 1998; Bellamy et al., 1998; Marquet et al., 1999; Cervino et al., 2000; Gao et al., 2000; Greenwood et al., 2000; Ryu et al., 2000; Mohamed et al., 2001) and autoimmune (Shaw et al., 1996; Hofmeister et al., 1997; Esposito et al., 1998; Maliarik et al., 2000; Sanjeevi et al., 2000; Singal et al., 2000; Yang et al., 2000) diseases. The infectious diseases include viral (HIV), bacterial (tuberculosis, leprosy, meningococcal meningitis) and protozoan (visceral leishmaniasis) pathogens. The autoimmune diseases include rheumatoid arthritis, juvenile rheumatoid arthritis, diabetes, sarcoidosis and Crohn's disease. Mutation in the closely related Slc11a2 (Nramp2) gene causes microcytic anaemia in mice (Fleming et al., 1997), but disease association in man has not been reported. Slc11a1 and Slc11a2 are polytopic integral membrane proteins with 10–12 putative membrane-spanning domains (Vidal et al., 1993; Gunshin et al., 1997). In both, the natural functional null mutation occurs in transmembrane domain 4 (Vidal et al., 1993; Fleming et al., 1997). Both Slc11a1 and Slc11a2 have protein kinase C (PKC) binding sites (Vidal et al., 1993; Barton et al., 1994; Gruenheid et al., 1995), but only Slc11a1 has a Pro–Ser-rich N-terminus (Barton et al., 1994). Here, we review current knowledge on the evolution, function and roles of Slc11a1/SLC11A1 in disease.

Nramp2 Analysis in Hemochromatosis Probands

ABSTRACTThe mechanism that leads to iron overload in hereditary hemochromatosis is not yet fully understood and genes other than HFE may be involved. Nramp2 is an intestinal iron transporter, upregulated by dietary iron deficiency, which also colocalizes with transferrin in recycling endosomes. The purpose of the present study was to analyze the coding region of the Nramp2 gene in 14 hemochromatosis probands which did not carry any HFE mutations on both chromosomes. We confirmed the existence of a polymorphism (1254 T → C), which presumably is not associated with hereditary hemochromatosis, but we did not find any mutation. On the other hand, we identified 17 splice variants of the Nramp2 mRNA. Eight corresponded to activation of cryptic splicing sequences between exons 3 and 4. They were observed in a majority of hemochromatosis probands and control subjects. This indicates the existence of an important splicing instability in this region. At this stage, the biological significance of these variants is unclear. Our study did not find evidence for the involvement of the Nramp2 gene in hereditary hemochromatosis. The remaining question is whether hemochromatosis probands in our study have iron overload because of environmental factors or due to mutation in gene(s) other than HFE and Nramp2.

Heterologous expression, functional characterization and localization of two isoforms of the monkey iron transporter Nramp2

Natural resistance-associated macrophage protein 2 (Nramp2) has been suggested to be involved in transferrin-independent iron uptake. Two isoforms of the Nramp2 gene generated by alternative splicing of the 3ʹ exons were identified in mouse, rat and human, but it is unclear if they perform distinct functions. To rationalize our previous work, which indicated an increase in iron deposition in a Parkinsonian monkey brain, two monkey Nramp2 isoforms were isolated for a comparative study to assess their relative iron-uptake abilities, tissue distribution and subcellular localization. The monkey Nramp2 isoforms, 2a and 2b, exhibit approx. 98% identity at the amino acid level when compared with the human homologues. The Nramp2a transcript contains a canonical iron-responsive element (IRE), whereas that of Nramp2b lacks the IRE motif in the 3ʹ untranslated region. By reverse transcriptase (RT)-PCR, the mRNAs of both isoforms were detected in all tissues examined. The amino acid differences at the C-terminus neither affected the protein expression levels in HEK-293T and COS-7 cells nor altered the subcellular localization and tissue distribution of the isoforms. Similar levels of iron uptake were detected in the HEK-293T cells transfected with either the Nramp2a or 2b gene, and a reduction of iron from the ferric (Fe3+) to the ferrous (Fe2+) state is necessary before transport can take place. However, this transferrin-independent uptake of iron into the cells is not a Ca2+-dependent process.

Heterologous expression, functional characterization and localization of two isoforms of the monkey iron transporter Nramp2.

Natural resistance-associated macrophage protein 2 (Nramp2) has been suggested to be involved in transferrin-independent iron uptake. Two isoforms of the Nramp2 gene generated by alternative splicing of the 3' exons were identified in mouse, rat and human, but it is unclear if they perform distinct functions. To rationalize our previous work, which indicated an increase in iron deposition in a Parkinsonian monkey brain, two monkey Nramp2 isoforms were isolated for a comparative study to assess their relative iron-uptake abilities, tissue distribution and subcellular localization. The monkey Nramp2 isoforms, 2a and 2b, exhibit approx. 98% identity at the amino acid level when compared with the human homologues. The Nramp2a transcript contains a canonical iron-responsive element (IRE), whereas that of Nramp2b lacks the IRE motif in the 3' untranslated region. By reverse transcriptase (RT)-PCR, the mRNAs of both isoforms were detected in all tissues examined. The amino acid differences at the C-terminus neither affected the protein expression levels in HEK-293T and COS-7 cells nor altered the subcellular localization and tissue distribution of the isoforms. Similar levels of iron uptake were detected in the HEK-293T cells transfected with either the Nramp2a or 2b gene, and a reduction of iron from the ferric (Fe(3+)) to the ferrous (Fe(2+)) state is necessary before transport can take place. However, this transferrin-independent uptake of iron into the cells is not a Ca(2+)-dependent process.

Analysis of single nucleotide polymorphisms in the NRAMP2 gene, for association with inflammatory bowel disease

Analysis of single nucleotide polymorphisms in the NRAMP2 gene, for association with inflammatory bowel disease

Nutrient absorption

Some key advances occurred last year in understanding mechanisms involved in nutrient absorption. A novel "prechylomicron transport vesicle" was identified; its movement to the Golgi is the rate-limiting step for triacylglycerol absorption. A scavenger receptor (type BI) in the brush border membrane appears to facilitate cholesterol uptake. Several studies define mechanisms for gastrointestinal peptide hormone stimulation of glucose uptake. An oligopeptide transporter, PepT1, is transcriptionally upregulated by certain dietary amino acids and dipeptides. Surprisingly, both insulin and fasting double the maximum velocity for dipeptide uptake (via PepT1), but they act by different mechanisms. Three transporters, SMVT (sodium-dependent multivitamin transporter for biotin and pantothenate), SVCT (for vitamin C), and CaT1 (for Ca uptake from the lumen) have been cloned and are active when expressed in various cells. Additional studies provide insights on Ca absorption and vitamin D action in aging, estrogen deficiency, and adaptation to a low Ca diet. Nramp2, also called DMT1 (divalent metal ion transporter), seems to be a major regulator of transferrin-independent, nonheme iron uptake. Finally, the protein HFE associates with the transferrin receptor and is part of an iron-sensing mechanism that regulates iron absorption. It is defective in hereditary hemochromatosis. HFE and Nramp2 (DMT1) genes are reciprocally regulated.

Dinucleotide repeat polymorphism in the third intron of the NRAMP2/DMT1 gene.

Nramp1 (natural resistance-associated macrophage protein 1), encoding a polytropic integral membrane protein, was isolated as a candidate of the mouse Lsh/Ity/Bcg locus, which regulates macrophage activation for anti-microbial activity against intracellular pathogens. The NRAMP2 gene was cloned from human genome as a homologue of NRAMP1. We found a polymorphic dinucleotide repeat in the third intron of the NRAMP2 gene. This polymorphism will be a useful genetic marker to study disease associated with susceptibility to infection with intracellular pathogens.

Role of iron in Nramp1-mediated inhibition of mycobacterial growth.

Innate resistance to mycobacterial growth is mediated by a gene, Nramp1. We have previously reported that Nramp1 mRNA from macrophages of Mycobacterium bovis BCG-resistant (Bcgr) mice is more stable than Nramp1 mRNA from macrophages of BCG-susceptible (Bcgs) mice. Based on these observations and on reports that show that the closely related Nramp2 gene is a metal ion transporter, we evaluated the effect of iron on the growth of Mycobacterium avium within macrophages as well as on the stability of Nramp1 mRNA. The addition of iron to macrophages from Bcgs mice resulted in a stimulation of mycobacterial growth. In contrast, iron increased the capacity of macrophages from Bcgr mice to control the growth of M. avium. When we treated recombinant gamma interferon (IFN-gamma)-activated macrophages with iron, we found that iron abrogated the growth inhibitory effect of IFN-gamma-activated macrophages from Bcgs mice but that it did not affect the capacity of macrophages from Bcgr mice to control microbial growth. A more detailed examination of the effect of iron on microbial growth showed that the addition of small quantities of iron to resident macrophages from Bcgr mice stimulated antimicrobial activity within a very narrow dose range. The effect of iron on the growth inhibitory activity of macrophages from Bcgr mice was abrogated by the addition of catalase or mannitol to the culture medium. These results are consistent with an Fe(II)-mediated stimulation of the Fenton/Haber-Weiss reaction and hydroxyl radical-mediated inhibition of mycobacterial growth.

Reciprocal Regulation of HFE and Nramp2 Gene Expression by Iron in Human Intestinal Cells

The newly identified hemochromatosis gene, HFE, and a candidate iron transporter gene, Nramp2, have been proposed as key factors responsible for the regulation of intestinal iron absorption. Although the exact functions of these proteins in intestinal iron absorption are unknown, HFE may be required for the down-regulation of iron absorption that occurs with increasing iron status, and Nramp2 may up-regulate iron absorption when iron status is low. Thus, we examined whether the expression of the HFE and Nramp2 genes are regulated by iron status in the human intestinal cell line Caco-2. HFE mRNA and HFE protein were increased and Nramp2 mRNA was decreased by increasing cellular iron status in Caco-2 cells. This iron-mediated modulation of mRNA levels was specific to iron. Moreover, super-induction of HFE mRNA in the presence of cycloheximide suggests that HFE gene expression may be controlled by a short-lived repressor protein. HFE and Nramp2 mRNA levels also changed in opposite directions during cellular differentiation. This reciprocal modification of the HFE and Nramp2 gene expression during both iron treatment and cell differentiation in Caco-2 cells is consistent with an opposing role for these proteins in homeostatic regulation of human intestinal iron absorption.

Recently Identified Molecular Aspects of Intestinal Iron Absorption

Gene mapping techniques to identify gene mutations in humans and animals with phenotypic abnormalities in iron metabolism are providing important insights into the probable molecular mediators of intestinal iron absorption. Positional gene cloning in humans with hereditary hemochromatosis has identified a mutation in a novel major histocompatibility complex (MHC) gene called HFE that is likely to be involved in regulating intestinal iron absorption. In addition, recent observations based on positional cloning strategies in the mk/mk mouse and the Belgrade (b/b) rat rodent models of hypochromic, microcytic anemia have shown that the phenotypic abnormality in iron metabolism is associated with a mutation in the Nramp2 gene. Functional cloning studies in Xenopus oocytes have characterized DCT1 (Nramp2) as an iron-regulated proton-coupled divalent cation transporter. Nramp2 is likely to be the membrane transporter that functions in controlling iron entry across the apical membrane and in the export of iron out of endosomal vesicles. The observation that the expression of both HFE and Nramp2 mRNAs are reciprocally regulated by cellular iron status in Caco-2 cells, a human intestinal cell line, lends additional credence to the notion that these proteins may work in concert to regulate intestinal iron absorption.

The Human Nramp2 Gene: Characterization of the Gene Structure, Alternative Splicing, Promoter Region and Polymorphisms

ABSTRACT: Nramp2 is a gene encoding a transmembrane protein that is important in metal transport, in particular iron. Mutations in nramp2 have been shown to be associated with microcytic anemia in mk/mk mice and defective iron transport in Belgrade rats. Nramp2 contains a classical iron responsive element in the 3′ untranslated region that confers iron dependent mRNA stabilization. In this report, we describe a splice variant form of human nramp2 that has the carboxyl terminal 18 amino acids substituted with 25 novel amino acids and has a new 3′ untranslated region lacking a classical iron-responsive element. This splice form of nramp2, nramp2 non-IRE, was found to be derived from splicing of an additional exon into the terminal coding exon.The nramp2 gene is comprised of 17 exons and spans more than 36 kb. It contains an additional 5′ exon and intron (exon and intron 1) and an additional 3′ exon (exon 17) and intron (intron 16) as compared to nramp1, a homologous gene. The additional exons and introns account for much of the difference in length between nramp2 (>36 kb) and nramp1 (12 kb). The exon-intron borders of nramp2 exons 3-15 are homologous to nramp1 exons 2-14. The nramp2 5′ regulatory region contains two CCAAT boxes but lacks a TATA box. The 5′ regulatory region of nramp2 also contains five potential metal response elements (MRE's) that are similar to the MRE's found in the metallothionein-IIAgene, three potential SP1 binding sites and a single γ-interferon regulatory element.Five single nucleotide mutations or polymorphisms were identified within the nramp2 gene. One of these, 1303C→A, occurs in the coding region of nramp2 and results in an amino acid change from leucine to isolecine. A polymorphism, 1254T/C, also occurs in the coding region of nramp2 but does not cause an amino acid change. The other 3 polymorphisms are within introns (IVS2+11A/G, IVS4+44C/A, and IVS6+538G/Gdel). In addition, a polymorphic microsatellite TATATCTATATATC (TA)6-7(CA)10-11CCCCCTATA (TATC)3(TCTG)5TCCG (TCTA)6was identified in intron 3.Analysis of cDNA derived by direct amplification of reversed transcribed RNA or cDNA clones isolated from a library provide evidence of skipping of exons 10 and 12 of nramp2. Deletion of either of these exons would result in a sequence that remains in frame yet would generate a protein that would lack transmembrane spanning region 7 or 8 respectively. The deletion of a single transmembrane domain would have severe topological consequences.The coding region of the nramp2 gene of hemochromatosis patients with or without mutations in the hemochromatosis gene,HFE, were examined and found to be normal. One hemochromatosis patient, with a normalHFEgenotype, was heterozygous for the 1303C→A mutation. Furthermore, in an examination of hemochromatosis patients with mutantHFEand normalHFEgenes, we did not observe a linkage disequilibrium of either group with a particular nramp2 haplotype. These data suggest that mutations in nramp2 are not commonly associated with hemochromatosis.

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.