GENOMIC ORGANIZATION AND SEQUENCE OF THE HUMAN NRAMP GENE - IDENTIFICATION AND MAPPING OF A PROMOTER REGION POLYMORPHISM

Background: Murine Nramp is a candidate for the macrophage resistance gene Ity/Lsh/Bcg. Sequence analysis of human NRAMP was undertaken to d etermine its role in man. Materials and Methods: A yeast artificial ch romosome carrying NRAMP was subcloned and positive clones sequenced. T he transcriptional start site was mapped using 5' RACE PCR. Polymorphi c variants were amplified by PCR. Linkage analysis was used to map NRA MP. Results: NRAMP spans 12kb and has 15 exons encoding a 550 amino ac id protein showing 85% identity (92% similarity) with Nramp. Two conse rved PKC sites occur in exon 2 encoding the Pro/Ser rich SH3 binding d omain, and in exon 3. Striking sequence similarities (57 and 53%) were observed with yeast mitochondrial proteins, SMF1 and SMF2, especially within putative functional domains: exon 6 encoding the second transm embrane spanning domain, site of the murine susceptibility mutation; a nd exon 11 encoding a conserved transport motif. No mutations comparab le to the murine susceptibility mutation were found. The transcription al initiation site mapped 148 bp 5' of the translational initiation co don. 440bp of 5' flanking sequence contained putative promoter region elements: 6 interferon-gamma response elements, 3 W-elements, 3 NF kap pa B binding sites and 1 AP-1 site. Nine purine-rich GGAA core motifs for the myeloid-specific PU.1 transcription factor were identified, tw o combining with imperfect AP1-like sites to create PEA3 motifs. TATA, GC and CCAAT boxes were absent. A possible enhancer element containin g the Z-DNA forming dinucleotide repeat t(gt),ac(gt),ac(gt),g was poly morphic (4 alleles; n=4,9,10,11), and was used to map NRAMP to 2q35. C onclusions: This analysis provides important resources to study the ro le of NRAMP in human disease.