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Genetic Diversity and mRNA Expression of Porcine MHC Class I Chain-Related 2 (SLA-MIC2) Gene and Development of a High-Resolution Typing Method.

Dadi H, Le M, Dinka H, Nguyen D, Choi H, Cho H, Choi M, Kim JH, Park JK, Soundrarajan N, Park C - PLoS ONE (2015)

Bottom Line: The number of alleles of the SLA-MIC2 gene in pigs is somewhat lower compared to the number of alleles of the porcine MHC class I and II genes; however, the level of heterozygosity was similar.Our results indicate the comprehensiveness of using genomic DNA-based typing for the systemic study of the SLA-MIC2 gene.The method developed for this study, as well as the detailed information that was obtained, could serve as fundamental tools for understanding the influence of the SLA-MIC2 gene on porcine immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biotechnology, Konkuk University, Seoul, 143-701, Korea.

ABSTRACT
The genetic structure and function of MHC class I chain-related (MIC) genes in the pig genome have not been well characterized, and show discordance in available data. Therefore, we have experimentally characterized the exon-intron structure and functional copy expression pattern of the pig MIC gene, SLA-MIC2. We have also studied the genetic diversity of SLA-MIC2 from seven different breeds using a high-resolution genomic sequence-based typing (GSBT) method. Our results showed that the SLA-MIC2 gene has a similar molecular organization as the human and cattle orthologs, and is expressed in only a few tissues including the small intestine, lung, and heart. A total of fifteen SLA-MIC2 alleles were identified from typing 145 animals, ten of which were previously unreported. Our analysis showed that the previously reported and tentatively named SLA-MIC2*05, 07, and 01 alleles occurred most frequently. The observed heterozygosity varied from 0.26 to 0.73 among breeds. The number of alleles of the SLA-MIC2 gene in pigs is somewhat lower compared to the number of alleles of the porcine MHC class I and II genes; however, the level of heterozygosity was similar. Our results indicate the comprehensiveness of using genomic DNA-based typing for the systemic study of the SLA-MIC2 gene. The method developed for this study, as well as the detailed information that was obtained, could serve as fundamental tools for understanding the influence of the SLA-MIC2 gene on porcine immune responses.

No MeSH data available.


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.
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pone.0135922.g001: 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.

Mentions: We designed sets of PCR primers against the SLA-MIC2 sequence contained within a bacterial artificial chromosome (BAC) sequence, AJ251914, and attempted to amplify the region between introns 1 and 4, or the full-length SLA-MIC2 cDNA, according to the exon-intron information of the pig SLA-MIC2 gene from a previous report [5]. Following the results of MICA and MICB expression from humans [34], we selected the lung and small intestine as initial RNA sources [13,21,34]. However, we were unable to obtain amplicons from either genomic DNA PCR or RT-PCR. Through additional database searches, we identified another BAC sequence (CT737281) that contained the SLA-MIC2 gene, but with a sequence discrepancy from the end of exon 4 to exon 6 of SLA-MIC2, as compared to AJ251914. We also realized that the exon-intron organization from AJ251914 was different from the current in silico annotation (NM_001114274) of the SLA-MIC2 gene according to the Ensemble genome browser (http://asia.ensembl.org/index.html). To resolve the discrepancy, we performed RT-PCR with SLA-MIC2-specific forward primers and a poly A specific oligo (dT)17 reverse primer. Among the several primer combinations used, a SLA-MIC2 exon 3-specific forward primer (MIC2-cDNA-sF) produced a 670-bp cDNA product. From this result, we were able to identify the differences in both the nucleotide sequence and the position of exon-intron boundaries for SLA-MIC2 exons 5 and 6 between the reported information and our findings, resulting in the precise characterization of the exon-intron structures of full-length SLA-MIC2 cDNA (Fig 1 and S1 Fig). The porcine MIC2 gene encodes a polypeptide of 374 amino acids consisting of a leader sequence (exon 1), three extracellular domains α1–3 (exons 2, 3 and 4), a transmembrane domain (exon 5), and a cytoplasmic domain (exon 6) (Fig 2 and S1 Fig), which were defined by comparative analysis with human MICA.


Genetic Diversity and mRNA Expression of Porcine MHC Class I Chain-Related 2 (SLA-MIC2) Gene and Development of a High-Resolution Typing Method.

Dadi H, Le M, Dinka H, Nguyen D, Choi H, Cho H, Choi M, Kim JH, Park JK, Soundrarajan N, Park C - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549063&req=5

pone.0135922.g001: 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.
Mentions: We designed sets of PCR primers against the SLA-MIC2 sequence contained within a bacterial artificial chromosome (BAC) sequence, AJ251914, and attempted to amplify the region between introns 1 and 4, or the full-length SLA-MIC2 cDNA, according to the exon-intron information of the pig SLA-MIC2 gene from a previous report [5]. Following the results of MICA and MICB expression from humans [34], we selected the lung and small intestine as initial RNA sources [13,21,34]. However, we were unable to obtain amplicons from either genomic DNA PCR or RT-PCR. Through additional database searches, we identified another BAC sequence (CT737281) that contained the SLA-MIC2 gene, but with a sequence discrepancy from the end of exon 4 to exon 6 of SLA-MIC2, as compared to AJ251914. We also realized that the exon-intron organization from AJ251914 was different from the current in silico annotation (NM_001114274) of the SLA-MIC2 gene according to the Ensemble genome browser (http://asia.ensembl.org/index.html). To resolve the discrepancy, we performed RT-PCR with SLA-MIC2-specific forward primers and a poly A specific oligo (dT)17 reverse primer. Among the several primer combinations used, a SLA-MIC2 exon 3-specific forward primer (MIC2-cDNA-sF) produced a 670-bp cDNA product. From this result, we were able to identify the differences in both the nucleotide sequence and the position of exon-intron boundaries for SLA-MIC2 exons 5 and 6 between the reported information and our findings, resulting in the precise characterization of the exon-intron structures of full-length SLA-MIC2 cDNA (Fig 1 and S1 Fig). The porcine MIC2 gene encodes a polypeptide of 374 amino acids consisting of a leader sequence (exon 1), three extracellular domains α1–3 (exons 2, 3 and 4), a transmembrane domain (exon 5), and a cytoplasmic domain (exon 6) (Fig 2 and S1 Fig), which were defined by comparative analysis with human MICA.

Bottom Line: The number of alleles of the SLA-MIC2 gene in pigs is somewhat lower compared to the number of alleles of the porcine MHC class I and II genes; however, the level of heterozygosity was similar.Our results indicate the comprehensiveness of using genomic DNA-based typing for the systemic study of the SLA-MIC2 gene.The method developed for this study, as well as the detailed information that was obtained, could serve as fundamental tools for understanding the influence of the SLA-MIC2 gene on porcine immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biotechnology, Konkuk University, Seoul, 143-701, Korea.

ABSTRACT
The genetic structure and function of MHC class I chain-related (MIC) genes in the pig genome have not been well characterized, and show discordance in available data. Therefore, we have experimentally characterized the exon-intron structure and functional copy expression pattern of the pig MIC gene, SLA-MIC2. We have also studied the genetic diversity of SLA-MIC2 from seven different breeds using a high-resolution genomic sequence-based typing (GSBT) method. Our results showed that the SLA-MIC2 gene has a similar molecular organization as the human and cattle orthologs, and is expressed in only a few tissues including the small intestine, lung, and heart. A total of fifteen SLA-MIC2 alleles were identified from typing 145 animals, ten of which were previously unreported. Our analysis showed that the previously reported and tentatively named SLA-MIC2*05, 07, and 01 alleles occurred most frequently. The observed heterozygosity varied from 0.26 to 0.73 among breeds. The number of alleles of the SLA-MIC2 gene in pigs is somewhat lower compared to the number of alleles of the porcine MHC class I and II genes; however, the level of heterozygosity was similar. Our results indicate the comprehensiveness of using genomic DNA-based typing for the systemic study of the SLA-MIC2 gene. The method developed for this study, as well as the detailed information that was obtained, could serve as fundamental tools for understanding the influence of the SLA-MIC2 gene on porcine immune responses.

No MeSH data available.