Comparative analysis of two Nramp loci from rainbow trout.

Abstract

Innate resistance to intracellular parasites is controlled in part by Nramp1 (Natural resistance-associated macrophage protein 1) in mammals and birds. To isolate Nramp homologs from rainbow trout, a combination of library screening and rapid amplification of cDNA ends was performed. Two closely related Nramp loci, designated OmNramp alpha and OmNramp beta, were cloned and characterized. OmNramp alpha and OmNramp beta encode two highly conserved proteins of 585 and 558 amino acids, respectively. Deduced amino acid seqences showed that the OmNramp alpha and OmNramp beta proteins share 90% of their residues and contain all of the signature features of the Nramp family of proteins: 12 transmembrane domains, two N-linked glycosylation sites, and a conserved transport motif. Phylogenetic analysis supported a close relation to Nramp2 proteins, a related member of the Nramp family. Despite this relation, juvenile trout expressed OmNramp alpha in a manner consistent with an Nramp1 homolog and OmNramp beta similar to an Nramp2 locus. Both trout loci were expressed at relatively high amounts in the ovaries of juveniles, a finding not reported in the investigations of previously characterized mammalian and avian homologs. These results suggest a role for Nramp loci in the follicular development of teleost fishes, as well as in mammals. Because salmonid fishes are ancestral tetraploids, fragments of OmNramp alpha and OmNramp beta were isolated from smelt, a diploid relative, to determine whether the trout loci represent duplicates of a single gene. Homologous sequences for both loci were found in smelt, supporting the hypothesis that OmNramp alpha and OmNramp beta are indeed independent loci that were present before the chromosomal duplication of salmonids. The isolation of Nramp loci from rainbow trout may eventually produce a genetic tool for the control of disease in aquaculture operations. Determining the involvement of trout homologs in innate immunity may also provide insight regarding the evolution of host resistance to pathogens.