Table 1.
Primer sequences and amplification conditions used for MIC2 analysis.
Fig 1.
General strategy of genomic sequence-based genotyping for pig SLA-MIC2.
The diagram shows the location of each primer for PCR and sequencing. The sizes (bp) of introns and exons are indicated.
Fig 2.
Comparison of amino acid sequences of MIC genes among pigs, humans, and cattle.
A representative sequence of each functional MIC gene from each species was selected, and amino acid sequences were compared throughout the entire coding region to evaluate sequence conservation. The accession numbers for the sequences are BoLA-MIC1 (BK006541), BoLA-MIC2 (BK006542), and BoLA-MIC3 (BK006543) for cattle, and MICA (NM_000247) and MICB- (NM_005931) for humans. Potential sites for N-linked glycosylation are underlined, and cysteine residues are indicated in squares for SLA-MIC2. Gaps are indicated by dashes and identical residues are indicated by dots. Stars above the sequences indicate conserved N-linked glycosylation sites, and plus signs above the sequences indicate a cysteine residue that is conserved across species. The starting points of protein domains are indicated above the annotated sequence, and the numbers above the sequence indicate the number of amino acids starting from the α1 domain excluding the leader peptide.
Fig 3.
Comparison of the mRNA expression levels of SLA-MIC2 in various pig tissues.
a) The RT-PCR products (1080 bp) of SLA-MIC2 exons 2 to 6 in different tissues, amplified from RNA isolated from nine-week-old male pigs. b) Standard GAPDH gene expression levels in different tissues, as visualized by the intensity of RT-PCR product staining on an agarose gel. c) The photodensity ratios between the amplified SLA-MIC2 and GAPDH products.
Table 2.
Comparison of the allele frequency of porcine MIC2 among seven pig breeds using high-resolution genomic sequence-based typing.
Fig 4.
Amino acid sequence similarities between swine leukocyte antigen SLA-MIC2 and MIC orthologs of humans and cattle.
The different protein domains are indicated below the x-axis. MICA and MICB are from humans; BoLA-MIC1, 2, and 3 are from cattle.
Table 3.
Differences in porcine MIC2 heterozygosity among seven breeds of pigs.
Fig 5.
A phylogenetic tree showing the relationships of MIC orthologous genes in different mammals including pigs (MIC2), cattle (BoLA-MIC1, 2, and 3), humans (MICA and B), chimpanzees (Patr-MICA/B), rhesus macaques (Mamu-MIC1 and 2), and mice and rats (Mr1).
A phylogenetic tree was constructed using the sequences corresponding to MIC exons 2, 3, and 4 using the neighbor joining method. The numbers on the nodes indicate the bootstrap values above 50% (n = 1000). The accession numbers of sequences are indicated in parentheses. SLA-1*0401(AF464016), one of the most common SLA molecules of swine, was used as an out-group. Bar below the tree indicates distance scale.