A complex pattern of microsatellite polymorphism within the bovine NRAMP1 gene
Authors: Hor˘ín P. , Rychlík I. , Templeton J. W. , Adams L. G. 
Published: March 2003

Nucleotide sequence polymorphism due to a variation in the number of GT dinucleotide repeats was found in the 3’ untranslated region (nucleotide positions 1781–1804) of the bovine natural resistance‐associated macrophage protein (NRAMP1) gene. The total variation in the number of GT repeats resulted not only from changes in the number of GT repeats but also from variation in the number of 5’ adjacent Gs. Two types of event may explain the polymorphism recently termed ‘allelic homoplasy’ (Grimaldi & Crouau‐Roy, 1997). Besides addition and/or deletion of GT repeats, a T vs. G transversion at position 1782 at the 5’ end of the GT repeat array generated variability in 3’ UTR of the bovine NRAMP1 gene. Another substitution site (G→A), interrupting the GT repeat array at position 1805, previously reported in cattle and not found in related species, was not found to show within‐species polymorphism. Although the functional significance of the polymorphism reported remains unknown, its detection allows investigation of associations with resistance and/or susceptibility to important intracellular pathogens in cattle.Microsatellites, short repetitive sequences of usually two to six nucleotides, represent an important part of DNA in multicellular organisms (Ramel, 1997). Due to their extensive polymorphism, they are used in gene mapping, parentage testing, and evolutionary biology. Despite their widespread use, mechanisms generating the variability of microsatellites still have not been completely clarified. The major mechanism leading to microsatellite allelic diversity is addition or deletion of small numbers of repeats, mostly of a single repeat unit, due to strand slippage during DNA replication (Goldstein & Pollock, 1997). Detailed analyses of sequence variability at individual microsatellite loci may reveal complex mutation patterns (Macaubas et al., 1997).In mice, the locus Bcg/Lsh/Ity, controlling natural resistance to intracellular parasites, and its candidate gene, NRAMP1 (for natural resistance‐associated macrophage protein), have been identified. Homologous sequences in humans and other species were detected subsequently (Vidal et al., 1993; Skamene et al., 1998). Here, we describe a complex pattern of microsatellite polymorphism revealed during our search for polymorphisms within the bovine homologue of the mouse NRAMP1 (bovine NRAMP1) gene (Feng et al., 1996).Fifty ng of genomic DNA, isolated from blood of Czech Red Pied and Czech Black Pied cattle, was amplified using primers specific for the 3’ untranslated region (nucleotide positions 1745–1955). The forward primer NRAMPF (5’‐GTGGAATGAGTGGGCACAGT‐3’) and the reverse primer NRAMPR (5’‐CTCTCCGTCTTGCTGTGCAT‐3’) were designed based on the sequence published previously (Feng et al., 1996). PCR was performed in a volume of 25 μL, containing 1 × reaction buffer (50 mM KCl, 10 mM Tris‐HCl, pH 9.0, and 0.1% Triton X‐100), 0.2 mM of each deoxyribonucleotide, 1.0 mM MgCl2, 0.5 μM of each of the primers, and 1 U Taq DNA polymerase (Promega, Madison, USA). After an initial denaturation of 2 min at 94 °C, 30 cycles at 94 °C for 30 s, 62 °C for 30 s and 72 °C for 30 s were performed, followed by the final extension at 72 °C for 5 min. Expected restriction patterns with HaeIII confirmed the identity of the PCR product of the predicted size (211 bp). DNAs from five cows were subcloned and sequenced. The PCR products, purified by the QIA quick gel extraction kit (Qiagen, Hilden, Germany), were ligated into a T‐vector using the Pin Point™ Xa1 T‐Vector System (Promega, Madison, USA), and Escherichia coli DH5α cells were transformed. The double‐stranded recombinant DNA was purified using the QIAprep minispin kit (Qiagen, Hilden, Germany). Clones were sequenced in both directions by the automatic ALF DNA Sequencer (Pharmacia, Uppsala, Sweden).The detected sequences were identical to the published bovine Nramp1 sequence (Feng et al., 1996), except for a polymorphism within the 3’ untranslated region of the gene (nucleotide positions 1781–1804) due to a variation in the number of GT dinucleotide repeats and a T → G substitution. Different sequences with variable numbers of GT repeats were detected. The variation in the number of GT repeats was related to a variation in the number of 5’ adjacent Gs (Fig. 1).. Homologies of the 3’ untranslated region NRAMP1 sequences in cattle and related species (see Table 1 for references).imageThe sequence with (GT)12 reported by Feng et al. (1996) was detected in seven of our 11 clones, and in all five cattle analysed. The number of clones from each animal differed between one and three. The sequence containing (GT)10 was identified in two different clones, each of them belonging to a different animal. Thus, two individuals were heterozygotes for the 3’ UTR NRAMP1 sequence. Two other clones with different total numbers of repeats (GT)9 and (GT)11 were rejected because the sequence was not found in other clones.The (GT)12 and (GT)10 sequences were confirmed in at least two different clones from at least two different cattle. Sequences detected in other clones, like 1782T + (GT)10 = (GT)11 and 1782G + (GT)9 = (GT)9, followed the same complex pattern. However, they were found in single clones and not confirmed in other cattle. The G → T transversion, along with the length GT repeat polymorphism, leading to the same (GT)10 length variants, were also defined independently in other breeds in the Texas laboratory: in outbred Bos indicus (Brahman), Bos taurus (Angus), and Brahman × Angus crossbreds. At least five clones (up to 10 in the case of the Angus herd sire) were sequenced from each of the animals in this study. The variants from the Texas study were shown to be transmitted to progeny in embryo transfer sibships (Adams et al., submitted). Taken together, the data support the view that the variants reported here are not PCR derived.Two types of event may explain the patterns observed. Besides addition and/or deletion of the GT repeats, a G vs. T transversion at the position 1782 at the 5’ end of the GT array generated an additional polymorphism in 3’ UTR.Consequently, in the case of 1782G, the total number of GT is not increased by an additional GT. In the case of 1782T, the existing number of GT repeats increases by one due to an additional GT unit generated by the substitution within the flanking polyG array.Therefore, the polymorphic patterns detected may be described as 1782T + (GT)11 = (GT)12 for the originally published sequence, and 1782G + (GT)10 = (GT)10 for the new allele detected. We propose the designation bovine NRAMP1.1 for the originally reported sequence, and bovine NRAMP1.2 for the 1782G + (GT)10 = (GT)10 allelic sequence.More than one mutational event thus may explain the polymorphism observed. In other multicellular organisms, point mutations have been shown to accompany repeat arrays (Ramel, 1997). In a (CA)n microsatellite located in the HLA‐DQB1 region, three types of mutation have been shown to generate new alleles (Macaubas et al., 1997). Ellegren et al. (1993) identified an intronic microsatellite polymorphism associated with the bovine MHC which they explained as a combination of length mutations and point mutations, termed ‘allelic homoplasy’. Our results can be explained by the same combination of two mutation mechanisms. Such mechanism versatility is particularly important in estimating genetic distances (Goldstein & Pollock, 1997).An AT dinucleotide, interrupting the GT repeat array at position 1805, suggests another G → A substitution in this region. This sequence, however, was identical in all 11 clones reported here. A homologous NRAMP1 gene 3’ untranslated region containing a GT repeat array was described in sheep (Pitel et al., 1994; Matthews & Crawford, 1998), red deer (Matthews & Crawford, 1998), American bison (Feng et al., Texas A&M University, College Station, TX, USA, unpublished), and water buffalo (Hashad et al., Texas A&M University, College Station, TX, USA, unpublished) (Table 1). The GT repeat array was not interrupted by an AT dinucleotide in any of these studies, and the number of GT repeats ranged from 13 (American bison) to 22 (sheep). Without the 1805G → A transition, the total number of GT repeats in cattle would be longer by four repeat units. Interruptions of repeat arrays occur in other organisms (Macaubas et al., 1996), and have been reported in other bovine genes as well (Ellegren et al., 1993).

3’ untranslated region sequences in cattle and related species
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In all the above‐mentioned species, the nucleotide position 1782 is occupied by T; in cattle, it can also be occupied by G (see Fig. 1). Therefore, it seems that a 1782T → G transversion occurred exclusively in cattle and contributed to the within‐species polymorphism. The functional significance of the polymorphism reported is not known. However, associations of a 3’ UTR mutation with susceptibility to tuberculosis in humans (Bellamy et al., 1998) and with susceptibility to brucellosis in cattle (Adams, L.G. et al., Texas A&M University, College Station, TX, USA, submitted) have been defined statistically. Another association of the NRAMP1 gene with susceptibility to intracellular pathogens has been described for the G → A transition at position 783 in mice (Vidal et al., 1993) but not for its analogue in cattle (Feng et al., 1996). These results justify a search for suitable association markers for important cattle intracellular pathogens, like Mycobacterium spp. or Salmonella spp. Finding such polymorphisms could be useful both theoretically and practically.