Genomic cloning and genetic mapping of the rat Nramp1 (Bcg) gene on chromosome 9.

Natural resistance in mice to infection with unrelated intracellular pathogens such as several species of mycobacterium (Goto et al. 1989), Salmonella typhimurium (Plant and Glynn 1976), and Leishmania donovani (Bradley 1977) is controlled in part by the phenotypic loci on mouse Chromosome (Chr) 1, Bcg, Ity, and Lsh respectively. Nramp1 (natural resistance-associated macrophage protein) has been identified by positional cloning (Vidal et al. 1993) as the gene for the Bcg/Ity/Lsh loci, which are now considered alleles of Nramp1. Human, mouse, and chicken homologs of the Nramp1 gene have been cloned and their chromosomal locations mapped (Cellier et al. 1994; Vidal et al. 1993; Hu et al. 1995). Their protein sequences are all highly conserved. Nramp1 is expressed primarily in macrophages and polymorphonuclear leukocytes, encoding a novel integral membrane protein (Vidal et al. 1993; Cellier et al. 1994) with similarities to several eukaryotic membrane proteins, including the CruA nitrite/ nitrate concentrator of Aspergillus nidulans. These observations suggest that Nramp1 may be involved in the transport or metabolism of nitric oxide, which is a key effector produced by activated macrophages. Analysis of the Nramp1 gene sequence from inbred mouse strains has indicated that disease susceptibility is associated with a nonconservative glycine to aspartic acid substitution within the fourth transmembrane domain of the Nramp1 protein (Malo et al. 1994). The rat counterpart of the Nramp1 gene has not been isolated. Inbred LEW/N rats are known to be extraordinarily susceptible to many experimental autoimmune, inflammatory, and infectious diseases (Wilder 1993) including endotoxin-induced uveitis, bacterial cell wall-induced arthritis, adjuvant arthritis, Yersenia-induced arthritis, etc., while F344/N rats are relatively resistant. To facilitate studies of Nramp1 in rats similar to those carried out in mice and humans and to evaluate the role of Nramp1 in experimental arthritis in the rat, we have genomically cloned and chromosomally mapped the rat Nramp1 gene. To clone the rat Nramp1 gene, we first designed a pair of PCR primers, forward primer 58-GCACCTATGCAGGCAGTTCGTGA-38 and reverse primer 58-TAAGCAGTCAGGCCCAAGTAACCT-38, from the published mouse Nramp1 gene coding sequence (Vidal et al. 1993). These primers were used to amplify a rat genomic DNA fragment. The PCR reaction was set up in a total volume of 50 ml containing 0.5 mg of rat genomic DNA, 4 mM of each primer, 2.5 U of Taq polymerase, and 2.5 mM of each dNTP in 1× PCR reaction buffer (Perkin Elmer, Branchburg, N.J.). Thirty-five cycles of amplification were performed at 94°C for 1 min, 65°C for 1 min, and 72°C for 3 min. A 0.4-kb DNA product, as expected, was observed after electrophoresis in a 1.2% agarose gel. The PCR product was purified with a QIAEX DNA Gel Extraction Kit (QIAGEN Inc., Chatsworth, Calif.) and was subcloned into a PCR TAII vector (Invitrogen, San Diego, Calif.). To confirm the identity of the PCR product, we sequenced both strands of the insert (GenBank Accession No. U53822). The nucleotide sequence was 82% and 93% identical, respectively, with published human and mouse Nramp1 cDNA sequences. No intron sequences were found in this fragment. A rat Nramp1 P1 genomic DNA clone was obtained from Genome Systems Inc. (St. Louis, Mo.) by PCR screening with the Nramp1 primers described above. To verify the authenticity of the Nramp1 P1 clone, we used two approaches: (1) the P1 clone DNA and genomic DNA were amplified with the primers described above, and identical size PCR products were found (0.4 kb). (2) Both the P1 and genomic DNAs were digested with the restriction enzymes NotI and BglII, separated by agarose gel electrophoresis, and transferred to a Gene Screen Plus hybridization transfer membrane (NEN Research Products, Boston, Mass.). Southern hybridization with the Nramp1 PCR fragment described above produced identical banding patterns for both the rat P1 and genomic DNA (data not shown). For generation of a polymorphic dinucleotide repeat marker associated with the Nramp1 gene for genotyping and mapping analyses, the P1 DNA was digested with NotI and BglII for Southern blot analysis with a P end-labeled (CA)15 oligonucleotide, as described previously (Zha et al. 1993). A 1.8-kb fragment from the P1 digest hybridized with the (CA)15 probe, which was then purified from an agarose gel and subcloned into PGEM-3Z vector (Promega, Madison, Wis.). The fragment was sequenced in both directions (GenBank Accession No. U53568), and PCR primers of D9Arb3 flanking the CA repeats (forward primer 58-ACCTCCAAACACATGTACACCTGG-38 and reverse primer 58CACACCACGCGTGTCTTTAGATGA-38) were designed for analysis of length variation of the dinucleotide repeats in various inbred rat strains. The following inbred rat strains were analyzed, LEW/N, F344/N, LOU/MN, WBB1/N, WBB2/N, MR/N, MNR/N, ACI/N, SHR/N, WKY/N, BN/SsN, BUF/N, and LER/N (Genetic Resource Section of the Veterinary Resources Program of the National Center for Research Resources at the National Institutes of Health) and DA/Bkl (Marie Griffiths, Salt Lake City, Utah). The P-labeled PCR reaction in 25 ml was performed as previously reported (Zha et al. 1993). Denatured PCR products were run on 8% polyacrylamide, 8 M urea sequencing gels and imaged by autoradiography. A polymorphism was detected between LEW/N and F344/N rats with this primer set. The allele sizes in all 16 inbred rat strains examined, including F344/N and LEW/N, are listed in Table 1. The Nramp1 gene (D9Arb3) was mapped to rat Chr 9, between Cryg and D9Arb4 (Fig. 1), by genotyping 40 (F344/N × LEW/N) F2 progeny. This intercross mapping panel has been used extensively by our group for linkage mapping (Zha et al. 1993). The segregation of the markers in the F2 generation was analyzed with MAPMAKER, version 3.0 (Lincoln et al. 1992), which constructs linkage maps based on maximum likelihood calculations. Correspondence to: L. Ge Mammalian Genome 7, 856–857 (1996).