A previous study on cloning and sequencing of the sheep TRB locus revealed that the D-J-C region is organized in three independent clusters tandem aligned, with D-J-C cluster 3 additional with respect to the other mammalian TRB loci 12. D-J-C cluster 1 contains one TRBD, six TRBJ and one TRBC gene. D-J-C cluster 3, located at 2.4 Kb downstream cluster 1, includes one TRBD, five TRBJ and one TRBC gene. Finally, D-J-C cluster 2 is positioned at 2.6 Kb downstream cluster 3 with one TRBD, seven TRBJ and one TRBC gene (fig. 1).

To evaluate the contribution of each gene cluster in the formation of the β-chain repertoire, a total of 72 clones containing rearranged V-D-J-C transcripts with a correct open reading frame were analyzed. All cDNA clones were registered in EMBL database with the Accession numbers from FM993913 to FM993984. 21 of these clones were derived from perinatal thymus (pSTMOS series) of a Moscia Leccese breed sheep, 15 from adult thymus (pSTA series) and 19 from spleen (pSMA series) of a Gentile di Puglia breed sheep, 17 from peripheral blood (pSSAR series) of a Sarda Ionica breed sheep. The clones were obtained by RT-PCR. The 5' primer was chosen on the YLCASS amino acid motif of the TRBV genes as members of the TRBV subgroups with this motif which seem to be the most frequently used 16 while the 3'-primer was designed on a conserved region of the three TRBC genes 12.

The deduced amino acid sequences of the V-D-J regions of all 72 cDNA clones are reported in the Table 1 together with the corresponding TRBC genes, according to the tissue of origin. Among the clones only one sequence is shared between blood (pSSAR25) and adult thymus (pSTA03). No tissue-specific expression of the genes was found. A total of 16 TRBJ genes were recovered within the different cDNAs. Thus, only one out of 17 functional TRBJ genes present in the genomic sequence was completely absent (TRBJ2.6). Besides, all TRBJ sequences match well with the corresponding genomic ones, and the high level of sequence similarity observed among the different animals is consistent with a close phylogeny of sheep breeds. The TRBJ2 cluster seems to be preferentially used (38/72 = 52.7%) and, although the numbers are too low to be statistically relevant, a slight increase in the use of TRBJ2.3 (14/38 = 36.8%) and TRBJ2.7 (10/38 = 26.3%) genes can be observed. Moreover, 20 clones retain a member of the TRBJ3 cluster, with the TRBJ3.4 gene (9/20 = 45%) more frequently used, while 14 clones retain the TRBJ1 gene set, without any preferential usage.

Three nucleotide differences at the N-terminus allow to distinguish the three TRBC gene isotypes: TRBC1 differs with respect to TRBC2 and TRBC3 genes for two nucleotide substitutions in the third and fourth codons; TRBC3 (as well as TRBC1 gene) is distinguishable from TRBC2 because of a silent nucleotide substitution at the third position of the first codon 12. On the basis of these criteria, the N-terminus of the TRBC portions within the cDNA sequences was analyzed and a significant group of cDNAs with the TRBC3 gene (35/72 = 48.6%) identified. Moreover, 25 clones retain the TRBC2 (34.7%) and 12 clones are with the TRBC1 (16.6%) gene (Table 1).

More complex is the determination and the contribution of the genes involved in the CDR3 formation. The CDR3 β region is defined as a stretch of nucleotides running after the codon encoding the cystein in position 104 of the TRBV gene to the codon before that which encodes the phenylalanine of the FGXG motif of the TRBJ gene http://imgt.cines.fr/ 17. The corresponding amino acid sequence of the CDR3 loop deduced from the nucleotide sequences reveals that it is heterogeneous for amino acid composition (Table 1). The mean length of the CDR3 loop was approximately the same in spleen (mean 12.3 aa, range 10-16 aa) and adult thymus (mean 12.6 aa, range 9-16 aa), but larger in blood (mean 13.9 aa, range 10-15 aa) and young thymus (mean 13.7 aa, range 10-20 aa). For comparison, human peripheral blood CDR3β loop is about 12.7 residue long 18 and mouse is 11.9 residue long 19. A similar CDR3 length and size range was reported in thymus and peripheral blood lymphocytes of piglets (mean 13.1 aa, range 10-17 aa) 20.

For a close inspection of the CDR3 s, the nucleotide sequences have been excised from each cDNA sequence and analyzed in detail. In the absence of the TRBV germline sequences, the deletions at the 3'-end of the TRBV and the nucleotide addition at the V-D junctions cannot be accurately estimated. However, the comparison of the 72 V-D-J junctions after the ASS motif allowed the determination of the probable 3'-end of the TRBV gene that has not been trimmed by exonuclease during rearrangement in a significant proportion of sequences (Table 1). By the comparison of the TRBD genomic sequences, the nucleotides located in the CDR3 regions were considered to belong to a TRBD gene if they constituted a stretch of at least four consecutive residues corresponding to the TRBD1, TRBD3 or TRBD2 germline sequences. In this way the 72 sequences were grouped according to the TRBD1 (fig. 2a, 36 sequences), TRBD3 (fig. 2b, 16 sequences) or TRBD2 (fig. 2c, 8 sequences) gene usage. 12 sequences with no recognizable TRBD genes were grouped separately (fig. 2d). These last sequences could be interpreted as direct V-J junctions. However, it is also possible that nucleotide trimming masked the initial participation of D gene during the rearrangement. In the other cases the degree of germline nucleotide trimming in the 3'-V and 5'-J as well as the 5' and 3' D region is similar in all groups (fig. 2). Nucleotides that could not be attributed to any template sequence are considered N-elements. The mean length for N-D-N addition, including D region, is 15 nt (range 6-23 bases) for the first group (fig. 2a), 13.8 nt (range 4-22 bases) for the second group (fig. 2b) and 16 nt (range 6-33 bases) for the group with TRBD2 participation (fig. 2c). The mean of N addition in the clones without TRBD sequence (fig. 2d) is 8.3 nt (range 2-16 bases). Particular features of the CDR3 region of the clones are the presence within the D region of nucleotide substitutions as well as the presence of insertion (psTMos 13 in fig. 2b) and deletion (psTA12 in fig. 2a) with respect to the germline sequences. Although the numbers are too low to be statistically relevant, a trend towards longer CDR3 length in TRBD2 (mean 42.3 bp, range 27-60) with respect to TRBD1 (mean 40.3 bp range 33-54) and TRBD3 (mean 38.5 bp, range 30-48), or with no apparent TRBD (mean 36.2 bp, range 30-42) transcripts was evident.

These data together suggest that all three TRB D-J-C clusters are used to generate in sheep functional TR β-chain with no specific influence of any clusters.

Since the genomic organization of the 3' region of the sheep TRB locus is known (fig. 1) 12, the formal interpretation of the D-J-C arrangements is possible. The intra-cluster rearrangements represent a consistent portion of the repertoire (41.6%), with 10 TRBD1-TRBJ1, 9 TRBD3-TRBJ3 and 6 TRBD2-TRBJ2 rearrangements (Table 1). A similar number of rearrangements (53.3%) can be interpreted by direct 5'- to- 3' joining across the clusters (inter-cluster rearrangements) with 20 TRBD1-TRBJ2, 6 TRBD1-TRBJ3 and 6 TRBD3-TRBJ2 rearrangements (Table 1). Interestingly, we also observed two TRBD2-TRBJ3 (psTMOs23 and psTA09, italics in Table 1) and one TRBD3-TRBJ1 (psSAR08, italics in Table 1) joining. Since the D- J-C cluster 2 is located downstream D- J-C cluster 3 as well as D- J-C cluster 3 is downstream D- J-C cluster 1 within the TRB locus, both these junctions may only be explained by chromosomal inversion, or with more probability, by trans-rearrangement occurring during TRB locus recombination.

A systematic analysis of the constant region of the transcripts also revealed that multiple splice variants are present. In fact, the canonical splicing is present in 49/72 (68%) clones with 10 TRBJ1-TRBC1, 17 TRBJ3-TRBC3 and 22 TRBJ2-TRBC2 transcripts (Table 1). A group of 7 clones (4 TRBJ1-TRBC3 and 3 TRBJ3-TRBC2) comes from an alternative or cis-splicing mechanism (9.7%). Finally, it is noteworthy that 16 clones (22.2%, bold in Table 1) with TRBJ2 genes showed TRBC3 or TRBC1 instead of the expected TRBC2 gene. Since TRBC3 as well as TRBC1 genes are located upstream TRBJ2 cluster in the germline DNA, TRBJ2 joined to TRBC1 or TRBC3 sequences cannot be a cis-spliced product of a single precursor RNA. Consequently, they must be the product of a trans-splicing between a transcript with TRBJ2-TRBC2 genes and a transcript containing TRBC1 or TRBC3 genes.

We excluded that all these non canonical sequences may be the result of PCR artifacts since the crossover points have not as expected a random distribution, but they always lie at the D-J or/and J-C junction, giving rise to products of the appropriate length and sequence.

The presence of splice variants may suggest the involvement of the TRBC gene in generating the TR β-chain functional diversity.

To complete the analysis of the TRBC domain in the cDNA clones, the whole constant portion of the transcripts was retrieved from the sequences and aligned according to the three TRBC isotypes for each animal in the different tissues.

The comparison of the 72 cDNAs showed the presence of different sequences that can be identified for the nucleotide variability in 14 different positions, 12 located in the first and two in the third exon, resulting in six amino acid substitutions all grouped in the first exon, and as a consequence, in the extracellular domain of the chain (fig. 3). By means of these variations, we observed a number of different sequences in excess. For example, five different groups of sequences were assigned to the TRBC3 gene in the young thymus of the Moscia Leccese breed individual. This number is certainly higher than the expected two allelic forms, at the most, of the gene. In order to understand the origin of the additional sequences, we have isolated by PCR the allelic variants of all three TRBC genes from the young thymus genomic DNA of the Moscia Leccese individual, used as a reference model with respect to the others. The specificity of the PCR reactions was achieved by using a reverse primer which binds to either TRBC1 and TRBC3 (B40) or TRBC2 3'-UTR (B42) sequences, and completely TRBC specific forward primers complementary to a specific region upstream the TRBC1 (CC1), TRBC3 (CC3) and TRBC2 (CC2) coding regions (see Methods). The three different PCR products were sequenced, and in every case, two allelic forms for each TRBC gene were obtained (data not shown). The comparison of the genomic with the corresponding sequences within the young thymus cDNAs allows us to establish that the first two more abundant groups of TRBC3 sequences represent the two allelic forms of the TRBC3 genes (pink and lilac in fig. 3), while alternative splicing of the third exon and DNA recombinational diversification process with the TRBC2 gene can have generated the other three groups of TRBC3 sequences (mixed color in fig. 3). Moreover, the two groups of TRBC2 cDNA sequences (green and yellow in fig. 3) perfectly matched with the two allelic forms (data not shown). Only one allelic form was recovered for the TRBC1 gene (italics in fig. 3), while the other TRBC1 sequence can have been generated by a mechanism of DNA recombinational diversification with the allele of TRBC3 gene (mixed color).

After deducing the allelic variants of the three constant genes in the other tissues, alternative splicing and recombinational diversification can explain the excess of the sequences also in those cases.

Thymus, spleen and blood were obtained from animals of three different autochthonous breeds. One thymus was collected from one neonatal Moscia Leccese sheep; spleen and the other thymus from one adult Gentile di Puglia; and blood from one young Sarda Ionica sheep. All animals were conventionally reared outbred sheep and were healthy at the time of sample collection. All animal manipulations were carried out with the approval of the Bari Animal Ethics Commitee and in compliance with Institutional Animal Care and Use Comittee (IACUC) requirements.

The different organs were removed from the animals, immediately frozen in liquid nitrogen and stored at -70°C until preparation of RNA. In the case of blood, RNA was prepared before freezing.

Total RNA was extracted from tissues under the protocol approved by the manufacturer (Trizol reagent, Invitrogen). First-strand cDNA synthesis was performed by reverse transcription of 5 μgr of total RNA primed with 2,5 μl of oligodT (0,5 μg/μl) using 2 μl dNTP (10 mM), 2 μl DTT (100 mM) and 1 μl PowerScript™ReverseTrancriptase (Clontech) in the recommended buffer in a total of 20 μl. The genes of interest were amplified from 10% of cDNA preparations using a sense V primer (VB3; 5'-TATCTCTGTGCCAGCAGC-3') complementary to a conserved region in the 3'-end of sheep TRBV genes 16 and an antisense CB3 primer (5'-CACCAGGGCGCTGACCAG-3'; AM420900; 8,222-8,239 positions) (5'-CACCAGGGCGCTGACCAG-3'; AM420900; 17,442-17,459 positions) (5'-CACCAGGGCGCTGACCAG-3'; AM420900; 26,702-26,719 positions) located in the third exon of the sheep TRBC genes. All the PCR were performed in a 50 μl volume with 5 μl 10× buffer, 2 μl MgCl2 (50 mM), 1 μl dNTP (10 mM), 0,5 μl of Taq Platinuum 5 U/μl (Invitrogen) and 2 μl of the sense and antisense primers (10 mM). After 2 min of initial denaturation at 94°C, the samples were subjected to 35 cycles of amplification (30 s at 94°C, 30 s at 58°C, 30 s at 72°C). The final cycle was extended for 10 min at 72°C. Amplified cDNA fragments were purified by using the PureLink PCR Purification Kit (Invitrogen-Life Technologies), ligated into StrataClone PCR Cloning Vector and transformed into StrataClone Competent Cells (Stratagene).

Genomic DNA was isolated from the young Moscia Leccese thymus by standard techniques. For the DNA amplifications, 50-200 μgr of thymus DNA was used with the TaKaRa LA Taq in 50 μl reactions, according to the recommendations (TAKARA BIO INC.). The cycling conditions were as follows: 94°C for 1 min; 35 cycles of 30 s denaturation at 95°C, 1 min annealing at 58°C, 2 min polymerization at 68°C; and 68°C for 10 min. The primer combinations used were CC1 and B40 for the TRBC1 gene, CC3 and B40 for the TRBC3 gene and CC2 and B42 for the TRBC2 gene. The CC1 (5'-CTGTGGCCCCTTTCCTTGTT-3'; AM420900, 6,805-6,824 positions), CC3 (5'-ACACACACAGCCCCTACCA-3', AM420900, 16,324-16,342 positions) and CC2 (5'-AGAGATGGGTTGTCGTAGG-3', AM420900, 25,117-25,136 positions) are designed on the 5'- end specific of the TRBC1, TRBC3 and TRBC2 genes respectively. B40 (5'-TCAGGGCAGTAACAGGCT-3'; AM420900, 8587-8569 positions) (5'-TCAGGGCAGTAACAGGCT-3'; AM420900; 17832-17815 positions) is complementary to the 3'UTR of TRBC1 as well as TRBC3 genes, while B42 (5'-ATGACTCGGGACGCACTT-3', AM420900, 27,040-27,057 positions) is complementary to the 3'UTR of the TRBC2 genes. Amplified DNA fragments were purified by PureLink PCR Purification Kit (Invitrogen-Life Technologies) and used directly for DNA sequencing.

The CDR3 size was calculated by the number of amino acids between the amino acid after the conserved 2^nd cysteine in the V gene (pos.104), and the amino acid before the phenylalanina of the FGXG motif in the J gene http://imgt.cines.fr/ 17. This method gives the CDR3 length with three amino acids more than that done in Kabat et al 36.

Nucleotide sequences were determined by a commercial service. DNA sequence data were processed and analyzed using the blasta program http://www.ncbi.nlm.nih.gov/BLAST, Clustal W http://www.ebi.ac.uk/clustalw/index.html 37 and IMGT database http://imgt.cines.fr/) 17.