Complete nucleotide sequence and genomic structure of the human
NRAMP1 gene region on Chromosome region 2q35
Thomas J. Hudson,
James M. Musser,
McGill Centre for the Study of Host Resistance, McGill University Health Centre, Montreal, Quebec, Canada
Department of Human Genetics, McGill University, Montreal, Quebec, Canada
Montreal Genome Centre, McGill University Health Centre, Montreal, Quebec, Canada
Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes
of Health, Hamilton, Montana 59840, USA
Received: 18 February 2000 / Accepted: 19 April 2000
Abstract. Several lines of independent evidence suggest that hu-
man Natural Resistance Associated Macrophage Protein 1 gene
(NRAMP1) is an important regulator of susceptibility to infectious
diseases caused by certain intracellular pathogens. Here, we report
the nucleotide sequence of 32198 bp of genomic DNA overlapping
NRAMP1 on chromosomal region 2q35. The NRAMP1 gene
spans 13604 bp. The gene and its 5Ј genomic region are highly
enriched for DNA repeat sequences. A second gene was identified
in the immediate vicinity of NRAMP1 and was tentatively named
Nuclear LIM Interactor-Interacting Factor (NLI-IF) by analogy to
its closest ortholog. The human NLI-IF gene begins 4721 bp
downstream of the NRAMP1 stop codon and is composed of seven
exons varying in size from 57 bp to 1644 bp. The gene gives rise
to a 2655-bp mRNA transcript that contains a 783-bp open reading
frame. The predicted molecular weight of the 261-amino acid NLI-
IF protein is 29.2 kDa. Several putative gene regulatory elements
were identified in the 5Ј upstream region of NLI-IF, including
consensus binding sequences for Sp1, AP-2, NF-kappa B, and PU
1. The NLI-IF amino acid sequence has homology to proteins that
have a high degree of homology with the NLI-interacting factor
from Gallus gallus and are found in divergent species ranging
from yeast to plants. NLI-IF is part of a human gene family en-
coding four related proteins of unknown function. Northern blot
analysis of 15 different human tissues revealed a 2.6-kb NLI-IF
mRNA that was ubiquitously expressed, but at varying levels. A
second transcript with estimated size of 7 kb was expressed only in
the placenta. Our data provide new sequence information about the
NRAMP1 gene region that will be useful in the search for genetic
variants causally involved in altered susceptibility to infectious
There is growing evidence that genetic factors of the host play a
decisive role in several infectious diseases. For example, it has
now been clearly established that the murine Nramp1 gene con-
trols innate resistance/susceptibility to a wide range of intracellular
macrophages parasites (Vidal et al. 1993, 1996; Malo et al. 1994).
The human Nramp1 homologue, NRAMP1, has been cloned and
mapped to Chromosome (Chr) region 2q35 (Liu et al. 1995). Sev-
eral NRAMP1 polymorphisms have been identified (Liu et al.
1995; Buu et al. 1995) and used for linkage and association analy-
sis with infectious disease. A large sib-pair study in South Vietnam
indicated significant linkage between leprosy and NRAMP1 hap-
lotypes (Abel et al. 1998). A case-control study in West Africa
found that risk of tuberculosis is associated with specific
NRAMP1 alleles (Bellamy et al. 1998), and recent findings indi-
cated that variants of the human NRAMP1 gene are associated
with susceptibility to HIV infection (Marquet et al. 1999).
Despite these advances, it is not clear how NRAMP1 contrib-
utes to human disease susceptibility. The only known amino acid
polymorphism that one could speculate to affect NRAMP1 func-
tion is a N543D polymorphism in the carboxy-terminal region of
the protein (Liu et al. 1995). Likewise, a possible regulatory poly-
morphism has been located in the 5Ј promoter region of NRAMP1
(Searle and Blackwell 1999). However, genetic analysis clearly
shows that these polymorphisms cannot fully explain NRAMP1
association or linkage with infectious disease phenotypes (Abel et
al. 1998). This observation suggests that at least some biologically
significant NRAMP1 polymorphisms have not yet been detected.
To facilitate the identification of additional polymorphisms, we
have sequenced 32 kb of genomic DNA surrounding NRAMP1.
We found that NRAMP1 is located in a genome region highly
enriched for genomic repeat elements and detected a second ubiq-
uitously expressed gene in the immediate vicinity of NRAMP1.
Materials and methods
Generation of M13 sequencing library.
An NRAMP1-positive P1
artificial chromosome (PAC) clone (P9I18) was selected from a Chr 2-
specific PAC library (Gingrich et al. 1996). Two liters of PAC culture were
grown overnight in LB medium supplemented with 25 mg/ml kanamycin.
PAC DNA was recovered from the culture with the QIAGEN plasmid kit.
To confirm that the entire NRAMP1 gene was included in this PAC clone,
a series of PCR reactions was performed on clone P9I18 DNA by using
specific primers from each region of the gene. Next, DNA from clone
P9I18 was ultrasonicated, size selected on agarose gels, and the 2-kb band
fraction was subcloned into M13 bacteriophages at the Whitehead Institute
Center for Genome Research (Cambridge, Mass.).
Automated sequencing and sequence analysis.
M13 subclones were
sequenced by using big-dye-labeled standard M13 primers from Applied
Biosystems (ABI; Foster City, Calif.) under conditions recommended by
the supplier. The reactions were run on a PERKIN ELMER ABI PRISM
377. Assembly of DNA sequences was done with the Staden Package
(version 1998.0). Initially, M13 subclones were sequenced randomly. Gaps
between contigs were closed by using specific primers from the contig
extremities. Sequencing with specific primers was done with ABI big-dye
terminators using dye-labeled dideoxynucleotides as chain terminators.
Correspondence to: E. Schurr, Montreal General Hospital Research Insti-
tute, Rm L11-521, 1650 Cedar Avenue, Montreal, Que, H3G 1A4 Canada;
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned the accession numbers AF229162 and
Mammalian Genome 11, 755–762 (2000).
© Springer-Verlag New York Inc. 2000