[Show abstract][Hide abstract] ABSTRACT: To better understand the function and diversity of gammadelta T cells, we determined the genomic sequence encoding diversity (D) segments of the porcine TCR delta chain and its upstream regions, because pigs and other artiodactyls have relatively high proportions of gammadelta T cells. The revealed sequence contained 28 variable (V) alpha/delta segments, including 4 TRDV1 and at least 6 Ddelta segments, a much higher number than in humans and mice. All 6 of the Ddelta segments that had canonical recombination signal sequences were functionally utilized in expressed TCR delta chain genes. The multiplicity of Ddelta segments enabled the use of more than 3 Ddelta segments in a single functional TCR delta chain. The increased number of TCR delta segments was acquired by the duplication of the germline sequence, which occurred after the divergence of artiodactyls from primates and rodents. These data demonstrate that the pig is able to generate a highly diversified repertoire of TCR delta chain molecules.
[Show abstract][Hide abstract] ABSTRACT: A complete genomic nucleotide sequence for porcine pTalpha gene was obtained from a BAC clone, which revealed a novel exon 2 missing in human and murine counterparts. Cattle and dog genomic sequences showed the counterparts corresponding to porcine exon 2. Using thymocyte RNA and RT-PCR, three types of porcine pTalpha-chain cDNA sequences, pTalpha1, pTalpha2 and pTalpha3, were obtained. These three different cDNA sequences were alternatively spliced products with pTalpha1 consisting of exons 1, 2, 3, 4, and 5, pTalpha2 consisting of exons 1, 2, 4, and 5, and pTalpha3 consisting of exons 1, 2, 3 and the intron down stream of exon 3. pTalpha1 and pTalpha2 correspond to previously reported pTalphaa, and pTalphab, respectively, and pTalpha3 is reported for the first time. Using RT-PCR, pTalpha3 appeared expressed predominantly in the thymocyte RNA. The chromosome location of pTalpha was investigated using Radiation Hybrid Map and FISH, both of which revealed the location at SSC7q11-q12.
[Show abstract][Hide abstract] ABSTRACT: Complete porcine CD3zeta-chain cDNA sequence was obtained for the first time, and its genomic nucleotide sequence was investigated from exon 2 down to CD3eta-chain exon 8. The sequence of porcine CD3zeta-chain showed homologous amino acid sequence with human and murine counterparts, in contrast to CD3eta-chain exon 8 with diversity among animals previously investigated. CD3eta-chain peptide is an alternative splice form of CD3zeta-chain exon 7 splicing to CD3eta-chain exon 8 instead of CD3zeta-chain exon 8. The genomic sequences revealed that the splice acceptor sequences of CD3eta-chain exon 8 of all animals investigated to be completely uniform. Further, CD3eta-chain exon 8 amino acid sequences retained the unique characters of having high proline (Pro) and positively charged amino acid content except for rats and mice. Although the biological role of CD3eta-chain remains to be enigmatic, these evidences suggests the evolutional pressure to maintain its sequence.
[Show abstract][Hide abstract] ABSTRACT: Porcine T-cell receptor alpha (TCRalpha)-chain cDNA clones were isolated from libraries made from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes (PBL) of a 5-month-old Clawn strain pig. Among 109 cDNA clones with the Jalpha-gene segment, 44 different Jalpha-gene segments were found out of the 61 Jalpha-gene segments previously identified in the porcine germline sequence. Among the 103 complete TCRalpha-chain cDNA clones with the rearranged Valpha- and Jalpha-gene segments, 33 different Valpha-gene segments were identified, which randomly rearranged to Jalpha-gene segments indicating lack of any specific combinations between Valpha- and Jalpha-gene segments with only one exception of the same set of Jalpha-gene segments in duplicate clones. Among the cDNA clones from PBL of an individual 5-month-old Clawn strain pig, a broad distribution of the Jalpha-gene segment usage was observed over the entire Jalpha-gene cluster. The Jalpha-gene segment usage in an individual 1-month-old thymus from a LW strain pig also gave a pattern consistent with the 5-month-old pig. These distributions of the Jalpha-gene segment usage were similar to the previously reported patterns for human T-cells and those of adult murine T-cells. Among the porcine cDNA clones isolated, TCRalpha-chain CDR3 length ranged from 4 to 14 amino acids with the average being 9.35 amino acids. Present report provides groundwork for further studies on porcine TCRalpha-chain expression.
[Show abstract][Hide abstract] ABSTRACT: Pig (Sus scrofa) TRA clones were isolated from cDNA libraries of total RNA from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes of a 5-month-old Clawn strain pig. Among 103 complete TRA cDNA clones from both sources, 33 different TRAV genes were identified. By comparing their sequence identities against one another, these pig TRAV genes were grouped into 20 subgroups, including 13 subgroups, each containing only a single member. All of these pig subgroups gave corresponding human and mouse functional counterparts, suggesting their functional commonality. An exception was the Va01 gene segment, which lacked a functional human counterpart. The present report provides groundwork for studies on pig TRA expression.
[Show abstract][Hide abstract] ABSTRACT: We successfully cloned and sequenced porcine toll-like receptor (TLR2) and TLR6 cDNA from porcine alveolar macrophages stimulated with 10 microg/ml lipopolysaccharide (LPS). The open reading frames (ORFs) of the porcine TLR2 and TLR6 cDNA were shown to be 2358 and 2391 bp in length and to encode 785 and 796 amino acids, respectively. The predicted amino acid sequence of porcine TLR2 was 72.3% homologous to human TLR2 and 61.0% homologous to murine TLR2. That of porcine TLR6 was 74.4% homologous to human TLR6 and 66.1% homologous to murine TLR6. Porcine TLR2 and TLR6 genes were both mapped to porcine chromosome 8 (TLR2: SSC8q21.1 --> 21.5; TLR6: SSC8p11.1 --> p21.1) by fluorescence in situ hybridization (FISH) and radiation hybrid mapping. Western blot analysis confirmed that TLR2 and TLR6 proteins were both expressed in porcine alveolar macrophages. Further, antiporcine TLR2 and TLR6 antibodies synergistically blocked tumor necrosis factor-alpha (TNF-alpha) production by porcine alveolar macrophages stimulated with Mycoplasma hyopneumoniae. These results indicated that both TLR2 and TLR6 are important in the recognition of M. hyopneumoniae in porcine alveolar macrophages and will be useful in understanding innate immunity against M. hyopneumoniae.
Journal of Interferon & Cytokine Research 11/2003; 23(10):583-90. · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A complete genomic region of 131.2 kb including the swine T-cell receptor alpha/delta constant region (TRAC/TRDC) and joining segments (TRAJ/TRDJ) was sequenced. The structure of this region was strikingly conserved in comparison to that of human or mouse. All of the 61 TRAJ segments detected in the human genomic sequence were detected in the swine sequence and the sequence of the protein binding site of T early alpha, the sequence of the alpha enhancer element and the conserved sequence block between TRAJ3 and TRAJ4 are highly conserved. Insertion of the repetitive sequences that interspersed after the differentiation of the species in mammals such as short interspersed nucleotide elements is markedly suppressed in comparison to other genomic regions, while the composition of the mammalian-wide interspersed sequences is relatively conserved in human and swine. This observation indicates the existence of a highly selective pressure to conserve this genomic region around TRAJ throughout the evolution of mammals.
[Show abstract][Hide abstract] ABSTRACT: We cloned and sequenced a cDNA that contains the coding sequence of the porcine interleukin-18 receptor alpha chain (PoIL-18Ralpha). Based on the conserved nucleotide sequences between human (HuIL-18Ralpha) and murine IL-18Ralpha (MuIL-18Ralpha), we performed reverse transcription-polymerase chain reaction (RT-PCR) with total RNA prepared from porcine peripheral blood lymphocytes (PBLs) stimulated with PoIL-12 to clone the cDNA of PoIL-18Ralpha. The open reading frame (ORF) of the PoIL-18Ralpha cDNA is 1620 base pairs (bp) in length and encodes 539 amino acids. The predicted amino acid sequence showed 68.2% and 50.2% identity to the human and murine amino acid sequences, respectively. Stimulation with concanavalin A (ConA) and IL-12, but not with IL-4, was shown to upregulate the expression of IL-18Ralpha mRNA in pig PBLs by RT-PCR analysis. Flow cytometric analysis also demonstrated that IL-18Ralpha was constitutively expressed on PoPBLs, and this expression was augmented by ConA stimulation. Furthermore, the PoIL-18Ralpha gene was mapped by fluorescence in situ hybridization (FISH) to porcine chromosome 3 (3q13-q14), near the location at which the IL-1beta gene had already been mapped. The present results will be helpful for understanding PoIL-18 and interferon gamma (IFN-gamma)-mediated T helper 1 (Th1) cell development.
Journal of Interferon & Cytokine Research 10/2002; 22(9):995-1002. · 3.30 Impact Factor