To validate the real participation of the third additional D-J-C cluster and compare its usage with respect to the others in the formation of the TR β-chain repertoire, we analyzed transcripts of 72 unique D-J-C rearrangements recovered from four different tissues of four different animals, belonging to three different ovine breeds. Although the analyzed sequences lacked the TRBV genes, the presence of the CDR3 β region, the TRBJ gene as well as most of the TRBC gene sequence was sufficient to permit a comprehensive analysis of the expressed TR β chain. Data presented here show that the mechanisms for generating diversity in sheep β chain polypeptides appear to adhere to the paradigms established through the study of humans and rodents. However, the diversity is enhanced by somatic rearrangement of 3 TRBD and 17 TRBJ genes that, by virtue of the expected recombination imprecision and N-region addition, maximizes diversity in the CDR3 region, thus expanding the potential repertoire of antigen specificities (Table 1). In spite of the presence of a longer coding nucleotide sequence in TRBD genes if compared with the human and mouse counterpart , the overall size of the CDR3 region is conserved in all tissues among the different mammalian species (Table 1). This conservation was archived by a greater deletion at the 5'end of TRBJ genes and a concomitant increase in N-nucleotide addition at the V-D-J junction during rearrangement (fig. 2). This suggests that the length of CDR3 in TR β chain is essential for T-cell function.
While there is not a specific influence of any cluster in the formation of the sheep β-chain in the different tissues, a dissimilar usage of the genes can be identified and it could depend on the sheep TRB genomic organization. Consistent with a promoter-enhancer facilitated recombination model, in human and mouse, assembly of the DJβ1 cassette is dependent on the interaction of the enhancer with the PDβ1 promoter positioned immediately 5' of the TRBD1 gene. Assembly of DJβ2 proceeds independent from that of DJβ1, albeit with less efficiency. Also in this case, an undefined PDβ2 region continues to associate with the enhancer . Our analyses suggest that also in sheep the mechanisms selectively alter D usage, so that the "privileged" TRBD1 gene can account for the 60% of the total clones with respect to 26.6% of TRBD3 and 13.3% of TRBD2. This may reside in the greater efficiency of the PDβ1 promoter activity with respect to the PDβ3 or PDβ2. A striking conservation of the PDβ1 and PDβ2 (as well as PDβ3) regions among sheep, human and mouse  can support this observation, whereas the activity of the two similar PDβ3 and PDβ2 promoters could be correlated with their position from 5' to 3' within the locus.
The prominent utilization of the members of the TRBJ2 with respect to the TRBJ3 and TRBJ1 sets, as deducted from our cDNA collection, results from inter cluster or trans-rearrangements. It is possible that the preferential usage of the TRBJ2 set could depend on the number of genes that lie in the genomic region, if multiple Jβ 12-RSs are important for increasing the local concentration of the RAG proteins that first bind a 12-RS and then capture a 23-RS to form a synaptic complex . In this regard, it is notable that the six sheep TRBJ1 genes lie in about 2.1 Kb, the five TRBJ3 genes in about 900 bp, while the seven TRBJ2 genes are grouped in about 1 Kb. Recently, Franchini et al.  have demonstrated, by means of an in vitro RAG1/2 mediated DNA coupled cleavage assay using various pair-wise RS combinations, that in mouse, the coupled cleavage of Dβ1-Jβ1 and Dβ2-Jβ2 substrates are similar and are both weak if compared to Dβ1-Jβ2 substrates, suggesting that Jβ2 RSs are better partners than Jβ1 RSs. In the same way, in sheep there could be the presence of a hierarchy efficiency of coupled cleavage with the Dβ1-Jβ2 > Dβ1-Jβ3 > Dβ1-Jβ1.
As the increment of the number of TRBD and TRBJ genes produces larger variation in TR β chain, particularly in CDR3 region as expected, similarly, the presence of an additional TRBC gene seems to affect the variety of the β chain repertoire. In fact careful analysis of the cDNA constant regions obtained from the different animals showed a level of unexpected variability in the first exon of the TRBC genes (fig. 3) if compared with that established in the genomic sequence . By using the single nucleotide variations present in the first and third exon of the TRBC genes as hallmarks, we demonstrated that alternative splicing concerning the first and/or the third exon and/or somatic recombinatiorial processes are involved in the diversification of the constant region of the sheep β-chain. The alternative splicing can occur either in cis or in trans. The presence of a cis-splicing mechanism comes from the analysis of six clones with TRBJ1 - TRBC3 and TRBJ3 - TRBC2 arrangement, while the presence of a trans-splicing process derives from the analysis of 16 clones with TRBJ2 spliced to TRBC3 or TRBC1 instead of the expected TRBC2 gene (Table 1). TRBJ2 to TRBC1 or TRBC3 splicing could be possible only when TRBV-TRBD-TRBJ transcripts are spliced with a transcript of the other allele. As a consequence, trans-splicing of two RNA separate precursors is the only logical possibility. The involvement of interallelic trans-splicing has already been documented in IgH chains . Beyond this case the presence of interallelic trans-splicing in vertebrates is problematical to demonstrate. It has been documented to be an essential process for the expression of the lola Drosophila gene. Lola encodes 20 protein isoforms belonging to a family of BTB zinc-finger transcriptional factor . Genetic tests have demonstrated that some isoforms were generating thought intrallelic trans-splicing . No particular sequences for trans-splicing have been identified around the exon-intron boundary in the lola gene; therefore, the basic mechanism of trans-splicing is likely to be shared with those of cis-splicing and occur co-transcriptionally where nascent pre mRNA are produced in close proximity, as is the case for cis-splicing . It is possible that also in sheep TRB locus, the cis and trans-splicing shared the same mechanism.
Investigation of the constant domain of the sheep cDNAs led us to deduce that a minimal set of sequences are also generated by a somatic recombinatorial process (fig. 3). Somatic recombinatorial diversification occurs in vertebrates, yeast and plants [27–29], and such a modification of germline sequences can generate individuals with different starting gene repertoires in different tissues.
The precise effect and significance of the variability in the constant region of TR β-chain remain to be determined. It might create diversity in the T cell function. The extracellular domain of the TRBC molecule consists of well-defined regions . The pattern of amino acid replacements in the sheep cDNA was located, beyond the N- terminus, one both in the TRBC E β-strand and in the DE loop and two in the FG loop. This last is TR β-chain specific loop in all mammalian species and contains 12 residues that are conserved between the two TRBC isotypes in human and mouse. In sheep sequences, the FG loop is one amino acid longer and underwent replacement among the three TRBC genes. So the Gln in position 106 in the first half part of the loop can be replaced by Glu; while the Asp in position 115 of the second part of the loop can be substituted by Ala (fig. 3). Three-dimensional structures of the TR  have shown that the FG loop of the TR β chain exists as an elongated, rigid element forming a sidewall of a cavity created by the asymmetric disposition of Cα and Cβ domains that receive the ε subunit of the CD3 complex . Therefore a primary function of the Cβ FG loop in the thymus is to facilitate negative selection, while following maturation, αβ T cells are dependent on the Cβ FG loop to their activation . Our hypothesis is that amino acid replacement in the FG loop of the sheep TRBC genes can be modified by the sensitivity of αβ T cell for cognate peptide recognition, and this can be correlated with the function of the αβ T cell in sheep.