Abstract
Toll-like receptors (TLRs) recognize various microbial components and induce immune responses. Polymorphisms in TLRs may influence their recognition of pathogen-derived molecules; swine TLRs are predicted to be associated with responses to infectious diseases such as pneumonia. In this study, we searched for single nucleotide polymorphisms (SNPs) in the coding sequences of porcine TLR1, TLR2, TLR4, TLR5, and TLR6 genes in 96 pigs from 11 breeds and elucidated 21, 11, 7, 13, and 11 SNPs, respectively, which caused amino acid substitutions in the respective TLRs. Distribution of these nonsynonymous SNPs was biased; many were located in the leucine-rich repeats, particularly in TLR1. These data demonstrated that the heterogeneity of TLR genes was preserved in various porcine breeds despite intensive breeding that was carried out for livestock improvement. It suggests that the heterogeneity in TLR genes is advantageous in increasing the possibility of survival in porcine populations.
Abbreviations
- CDS:
-
coding sequence
- LPS:
-
lipopolysaccharide
- LRR:
-
leucine-rich repeat
- RT:
-
reverse transcriptase
- PCR:
-
polymerase chain reaction
- SNP:
-
single nucleotide polymorphism
- TIR:
-
Toll/interleukin-1 receptor
- TLR:
-
Toll-like receptor
References
Akira S, Takeda K (2004) Toll-like receptor signalling. Nat Rev Immunol 4:499–511
Bell JK, Mullen GE, Leifer CA, Mazzoni A, Davies DR, Segal DM (2003) Leucine-rich repeats and pathogen recognition in Toll-like receptors. Trends Immunol 24:528–533
Beutler B (2005) The Toll-like receptors: analysis by forward genetic methods. Immunogenetics 57:385–392
Carrington M, Nelson GW, Martin MP, Kissner T, Vlahov D, Goedert JJ, Kaslow R, Buchbinder S, Hoots K, O’Brien SJ (1999) HLA and HIV-1: heterozygote advantage and B*35–Cw*04 disadvantage. Science 283:1748–1752
Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8:175–185
Excoffier L, Slatkin M (1995) Maximum-likelihood estimation of molecular haplotype frequencies in a diploid population. Mol Biol Evol 12:921–927
Fujita M, Into T, Yasuda M, Okusawa T, Hamahira S, Kuroki Y, Eto A, Nisizawa T, Morita M, Shibata K (2003) Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human Toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans. J Immunol 171:3675–3683
Hamann L, Kumpf O, Muller M, Visintin A, Eckert J, Schlag PM, Schumann RR (2004) A coding mutation within the first exon of the human MD-2 gene results in decreased lipopolysaccharide-induced signaling. Genes Immun 5:283–288
Hawn TR, Verbon A, Lettinga KD, Zhao LP, Li SS, Laws RJ, Skerrett SJ, Beutler B, Schroeder L, Nachman A, Ozinsky A, Smith KD, Aderem A (2003) A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires’ disease. J Exp Med 198:1563–1572
Jiang Q, Akashi S, Miyake K, Petty HR (2000) Lipopolysaccharide induces physical proximity between CD14 and toll-like receptor 4 (TLR4) prior to nuclear translocation of NFκB. J Immunol 165:3541–3544
Lazarus R, Vercelli D, Palmer LJ, Klimecki WJ, Silverman EK, Richter B, Riva A, Ramoni M, Martinez FD, Weiss ST, Kwiatkowski DJ (2002) Single nucleotide polymorphisms in innate immunity genes: abundant variation and potential role in complex human disease. Immunol Rev 190:9–25
Marchler-Bauer A, Bryant SH (2004) CD-Search: protein domain annotations on the fly. Nucleic Acids Res 32:W327–W331
Muneta Y, Uenishi H, Kikuma R, Yoshihara K, Shimoji Y, Yamamoto R, Hamashima N, Yokomizo Y, Mori Y (2003) Porcine TLR2 and TLR6: identification and their involvement in Mycoplasma hyopneumoniae infection. J Interferon Cytokine Res 23:583–590
Nickerson DA, Tobe VO, Taylor SL (1997) PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res 25:2745–2751
Nishimura M, Naito S (2005) Tissue-specific mRNA expression profiles of human toll-like receptors and related genes. Biol Pharm Bull 28:886–892
Schröder NW, Schumann RR (2005) Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious disease. Lancet Infect Dis 5:156–164
Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, Kimoto M (1999) MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med 189:1777–1782
Shinkai H, Muneta Y, Suzuki K, Eguchi-Ogawa T, Awata T, Uenishi H (2005) Porcine Toll-like receptor 1, 6, and 10 genes: complete sequencing of genomic region and expression analysis. Mol Immunol (in press)
Straw BE, D’Allaire S, Mengeling WL, Taylor DJ (eds) (1999) Diseases of swine, 8th edn. Blackwell, Blackwood
Suzuki K, Asakawa S, Iida M, Shimanuki S, Fujishima N, Hiraiwa H, Murakami Y, Shimizu N, Yasue H (2000) Construction and evaluation of a porcine bacterial artificial chromosome library. Anim Genet 31:8–12
Tanaka M, Ando A, Renard C, Chardon P, Domukai M, Okumura N, Awata T, Uenishi H (2005a) Development of dense microsatellite markers in the entire SLA region and evaluation of their polymorphisms in porcine breeds. Immunogenetics 57:690–696
Tanaka M, Suzuki K, Morozumi T, Kobayashi E, Matsumoto T, Domukai M, Eguchi-Ogawa T, Shinkai H, Awata T, Uenishi H (2005b) Genomic structure and gene order of the region on swine chromosome 7q1.1→q1.2. Anim Genet (in press)
Uenishi H, Eguchi T, Suzuki K, Sawazaki T, Toki D, Shinkai H, Okumura N, Hamasima N, Awata T (2004) PEDE (Pig EST Data Explorer): construction of a database for ESTs derived from porcine full-length cDNA libraries. Nucleic Acids Res 32:D484–D488
Werling D, Jungi TW (2003) TOLL-like receptors linking innate and adaptive immune response. Vet Immunol Immunopathol 91:1–12
Acknowledgements
We thank Dr. Takeshi Hayashi (NIAS) for the helpful discussions related to statistical analyses and Dr. Joan K. Lunney (APDL, ARS, USDA) for the critical reading of this manuscript. This work was supported by the Animal Genome Research Project of the Ministry of Agriculture, Forestry and Fisheries of Japan and by a Grant-in-Aid from the Japan Racing Association.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the image is a link to a high resolution version.
Supplementary Fig. 1
Comparison of the deduced amino acid sequence of porcine TLR5 gene with those of its human and mouse counterparts. The amino acid sequences encoded by human and mouse TLR5 genes are derived from Swiss-Prot accession nos. O60602 and Q9JLF7, respectively. Asterisks, colons, and periods under the aligned sequences indicate complete matches, similar amino acids among the three species, and matches in two species among three, respectively. Boxes indicate the predicted signal peptide and the transmembrane domain (GIF 789 KB)
Supplementary Fig. S2
Expression of the TLR5 gene in eight porcine tissues, as analyzed by RT-PCR. Total RNAs from the colon, kidney, liver, lung, small intestine, spleen, stomach, and thymus were purified from a 1-month-old male pig. The CDS of porcine TLR5 was encoded using a single exon. RNase-freeDNase I (Takara Bio, Otsu, Japan) was used before the samples were subjected to reverse transcription to ensure the complete degradation of genomic DNA, which contaminates the RNA samples. Primer pairs,templates treated with (+) or without (−) reverse transcriptase, and PCR cycles are shown at the top of the (GIF 130 kb)
Supplementary Fig. S3
Haplotypes of porcine TLR genes reconstructed by the expectation-maximization method (Excoffierand Slatkin 1995). Haplotypes are estimated using individuals whose genotypes of SNP loci are completely determined. Thebreeds in which the haplotypes cannot be determined are indicated as ND. Nonsynonymous SNP loci are indicated by redletters. Heterozygosity (HT) are calculated using the formula:\({\text{HT = }}{\sum {{\text{p}}^{2}_{{_{i} }} } }\) where p i is the observed frequencies of the ith haplotypes at each TLR gene. BS, Berkshire; CL, Clawn; DR, Duroc; HS,Hampshire; JH, Jinhua; LR, Landrace; LW, Large White; PB, Potbelly; MS, Meishan; MY, Middle Yorkshire; and WB,Japanese wild boar (GIF 575 kb)
Supplementary Table 1
Primers for expression analysis of porcine TLR5 (DOC 25 kb)
Rights and permissions
About this article
Cite this article
Shinkai, H., Tanaka, M., Morozumi, T. et al. Biased distribution of single nucleotide polymorphisms (SNPs) in porcine Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR5, and TLR6 genes. Immunogenetics 58, 324–330 (2006). https://doi.org/10.1007/s00251-005-0068-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00251-005-0068-z