Mammalian Genome 5, 390-392 (1994). e 9 Springer-VerIag New YorkInc. [994 TNP1 gene: a sheep polymorphic marker for a conserved region among mammals F. Pitel, 1 I. Lantier, 2 K. Tabet-Aoul, 3 N. Saidi-Mehtar, 3 J.-M. Elsen, 4 F. Lantier, 2 J. Gellin 1 1Laboratoire de G6n6tique Cellulaire, INRA, Centre de recherches de Toulouse, Castanet-Tolosan 31326, France 2Laboratoire de Pathologie Infectieuse et Immunologie, INRA, Centre de recherches de Tours, Nouzilly 37380, France 3Laboratoire de Biologie mol6culaire et G6n6tique, Universit60ran Es-Senia, Oran, Algeria 4Station d'Am61ioration G6n6tique des Animaux, INRA, Centre de recherches de Toulouse, Castanet-Tolosan 31326, France Received: 24 September 1993 /Accepted: 31 January 1994 One of the conserved regions between human and mouse genomes concerns the long arm of human Chromosome (Chr) 2 and the proximal region of mouse Chr 1. A num- ber of loci have been mapped to this homologous segment including the Ity/Lsh/Bcg locus (immunity to Salmonella typhimurium, Leishmania donovani and Mycobacterium boris). In mouse, this locus controls the resistance to sev- eral infections from microorganisms, including Salmonel- la typhimurium (Plant and Glynn 1976). The Ity gene al- so controls the murine resistance to Salmonella abortus ovis, a serotype specific of sheep and goat (Oswald et al. 1992). In sheep, a genetic component of the susceptibili- ty to this infection has been evidenced (Lantier et al. 1990). This susceptibility could be controlled by an ovine equiv- alent of the Ity gene. A first step towards testing this hy- pothesis was to prove the conservation of the Ity cluster in sheep. This has been partly checked previously with the synteny of three genes from this region: villin (VIL), fibronectin (FN-1), and cholinergic receptor gamma (CHNRG) genes (Tabet-Aoul et al. 1992). Our objective was to obtain a PCR-usable polymorphic marker of this re- gion, expecting it to constitute a marker for the suspected major gene controlling the trait of resistance to this abortive disease. The conserved region corresponding to the Ity/Lsh/Bcg locus in mouse includes the transition protein 1 gene (Hei- daran et al. 1989; Luerssen et al. 1990). This gene encodes a spermatid-specific basic nuclear protein found in haploid spermatogenic cells during spermiogenesis. TNP 1 is high- ly conserved among mammals (Kremling et al. 1989). We cloned a sheep fragment of this gene after PCR amplifi- cation, which enabled us to assign this locus to a sheep chromosome and to prove its synteny with other known markers of the Ity cluster. In order to amplify an ovine TNP1 gene fragment specifically, a primer pair was chosen after the alignment of five TNP1 Genbank sequences: the human genomic se- Correspondence to: F. Pitel quence (accession number M59924; Luerssen et al. 1990) and four cDNA sequences from mouse (X12521; Kleene et al. 1988), rat (M17096; Heidaran and Kistler 1987), pig and cattle (X16170 and X16171; Kremling et al. 1989). The sequences were compared by using Multalin software (Corpet 1988), which allows the alignment of several se- quences simultaneously. This comparison was carried out on a 230-bp-long cDNA sequence, corresponding to the portion shared by all the published sequences. The se- quence of each TNP1 primer was then chosen among the most homologous segments observed: (+) 5'-GGCATGA- GGAGGGGCAAGAAC-3', (-) 5'-TCACAAGTGGGAG- CGCAAATTG-3'. These sequences are not the "consen- sus" sequences given by the software, but the bovine se- quences found in the selected segments. We assumed that the ovine sequence would be more similar to its bovine ho- molog than to the other species. The lower primer ended with the bovine stop codon TGA. Sheep genomic DNA was subjected to PCR amplifi- cation (PHC-3, Techne) with the following cycling condi- tions: denaturation at 94~ for 1 rain (first cycle, 5 min); annealing at 65~ for 1 min; elongation at 72~ for 2 rain (last cycle, 5 min). Thirty cycles were performed in the presence of 1 unit of Taq polymerase (Promega Ltd), 10 ng DNA, 25 gM of each TNP1 primer, 2 mu MgC12, 0.2 mM of each deoxynucleotide, 50 mM KC1, 10 mM Tris-HC1 (pH 9.0 at 25~ 0.1% Triton X-100, in a final volume of 25 ~tl. The PCR result was a clear and intense band, which suggests the high degree of conservation of the primer se- quences between sheep and other mammals. The obtained fragment was cloned and sequenced (Sequenase, USB Se- quencing kit): it was a 358-bp-long insert; 315 bp were ovine-specific, the other 43 corresponded to the consensus primers. The nucleic acid sequence of this fragment is shown in Fig. 1. The comparison with TNP1 cDNA se- quences from different species ensured the identity of this sheep TNP1 gene portion. It identified a probable 220-bp- long intron, also found in the human genomic sequence (Luerssen et al. 1990), and defined the end of the fragment as the presumed 3' end of the coding region. If one ex- cludes the intron present in this fragment and the primers,