Worm Breeder's Gazette 16(1): 40 (October 1, 1999)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
CIML, Case 906, 13288 Marseille Cedex 9, France
We are interested in investigating the function of nematode homologues of proteins known to be important for innate immunity in other organisms. Genetic studies with the mouse have shown that a single locus is involved in innate resistance against a range of pathogens including Mycobacterium bovis (BCG), M. intracellulare, M. lepraemurium, Salmonella typhirium and Leishmania. The corresponding gene codes for a protein Nramp1 (Natural resistance-associated macrophage protein; Vidal et al., 1993, Cell, 73, 469-485) that is specifically expressed in macrophages. A highly similar protein, Nramp2, is expressed ubiquitously and appears not to have a role in defense against infection. The Nramp proteins are members of a family of metal ion transporters, that includes the yeast Smf proteins.
C. elegans possesses three genes similar in sequence to genes of the Nramp/smf family, the neighbours, K11G12.4 (smf-1) and K11G12.3 (smf-2) and a third gene on Y69A2 (smf-3). They each encode predicted proteins that are between 40% and 55% identical to the human Nramp1 protein. Phylogenetic analysis gives no clue as to whether any of these three nematode proteins is orthologous to Nramp1 (see http://pbil.univ-lyon1.fr/members/duret/nrm_tot.html ; seqcel is SMF-3). On the otherhand, while murine Nramp2 is capable of abolishing the hypersensitivity to EGTA of a smf1/smf2 double mutant, Nramp1 is unable to do so (Pinner et al., 1997, J. Biol. Chem., 272, 28933-28938), suggesting a relatively simple approach to resolving this question.
Two full-length cDNAs were available in Yuji Kohara's collection : yk452d4 for smf-1 and yk357h3 for smf-3.We obtained the cDNA corresponding to smf-2 by reverse transcription. The three cDNAs were cloned into a yeast expression vector (with a myc tag at their C-terminus to confirm expression) and, in collaboration with the laboratory of Philippe Gros, will be used to transform a yeast smf1/smf2 double mutant. If a given gene complements the yeast mutant phenotype, it would considered as Nramp2, rather than Nramp1, orthologue.
In parallel we are currently studying the expression patterns and sub-cellular localisation of the three C. elegans SMF proteins, as well as the phenotype, in terms of metal ion/EGTA sensitivity and susceptibility to fungal and bacterial infection of a putative smf-1 null mutant.
Thanks to Laurent Duret for the phylogenetic analysis, Yuji Kohara
for cDNAs and David Hughes and the Sanger Centre for the smf-1
knockout.