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 determine its role in man.


METHODS
A yeast artificial chromosome carrying NRAMP was subcloned and positive clones sequenced. The transcriptional start site was mapped using 5' RACE PCR. Polymorphic variants were amplified by PCR. Linkage analysis was used to map NRAMP.


RESULTS
NRAMP spans 12kb and has 15 exons encoding a 550 amino acid protein showing 85% identity (92% similarity) with Nramp. Two conserved PKC sites occur in exon 2 encoding the Pro/Ser rich SH3 binding domain, 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 transmembrane spanning domain, site of the murine susceptibility mutation; and exon 11 encoding a conserved transport motif. No mutations comparable to the murine susceptibility mutation were found. The transcriptional initiation site mapped 148 bp 5' of the translational initiation codon. 440bp of 5' flanking sequence contained putative promoter region elements: 6 interferon-gamma response elements, 3 W-elements, 3 NF kappa B binding sites and 1 AP-1 site. Nine purine-rich GGAA core motifs for the myeloid-specific PU.1 transcription factor were identified, two combining with imperfect AP1-like sites to create PEA3 motifs. TATA, GC and CCAAT boxes were absent. A possible enhancer element containing the Z-DNA forming dinucleotide repeat t(gt),ac(gt),ac(gt),g was polymorphic (4 alleles; n = 4,9,10,11), and was used to map NRAMP to 2q35.


CONCLUSIONS
This analysis provides important resources to study the role of NRAMP in human disease.
University of Cambridge Clinical School, Department of Medicine, Addenbrooke's Hospital, United Kingdom.
J M
JM
Blackwell
C H
CH
Barton
J K
JK
White
S
S
Searle
A M
AM
Baker
H
H
Williams
M A
MA
Shaw
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics*; D027682:Cation Transport Proteins; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast; D002889:Chromosomes, Human, Pair 2; D003001:Cloning, Molecular; D017931:DNA Primers; D005091:Exons; D005260:Female; D008040:Genetic Linkage; D006801:Humans; D007438:Introns; D033862:Iron-Binding Proteins; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010375:Pedigree; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D000099173:Natural Resistance-associated Macrophage Protein 1; D000099236:Solute Carrier Family 11, Member 2
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D017931:DNA Primers; D033862:Iron-Binding Proteins; D008565:Membrane Proteins; D000099173:Natural Resistance-associated Macrophage Protein 1; D000099236:Solute Carrier Family 11, Member 2