|Coordinate||106,224,586 bp (GRCm38)|
|Base Change||T ⇒ C (forward strand)|
|Gene Name||toll-like receptor 9|
|Chromosomal Location||106,222,598-106,226,883 bp (+)|
FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The protein encoded by this gene is a member of the Toll-like receptor (TLR) family which plays a fundamental role in pathogen recognition and activation of innate immunity. TLRs are highly conserved from Drosophila to humans and share structural and functional similarities. They recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, and mediate the production of cytokines necessary for the development of effective immunity. The various TLRs exhibit different patterns of expression. This gene is preferentially expressed in immune cell rich tissues, such as spleen, lymph node, bone marrow and peripheral blood leukocytes. Studies in mice and human indicate that this receptor mediates cellular response to unmethylated CpG dinucleotides in bacterial DNA to mount an innate immune response. [provided by RefSeq, Jul 2008]
PHENOTYPE: Nullizygous mice exhibit impaired immune responses to CpG DNA and altered susceptibility to EAE and parasitic infection. ENU-induced mutants may exhibit altered susceptibility to viral infection or induced colitis and impaired immune response to unmethylated CpG oligonucleotides. [provided by MGI curators]
|Amino Acid Change||Serine changed to Proline|
|Institutional Source||Beutler Lab|
S359P in Ensembl: ENSMUSP00000082207 (fasta)
|Gene Model||not available|
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Phenotypic Category||Autosomal Semidominant|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Semidominant|
|Local Stock||Sperm, gDNA|
|Last Updated||2016-05-13 3:09 PM by Anne Murray|
|Record Created||2010-04-20 11:48 AM by Hua Huang|
The CpG11 phenotype was identified in a screen for ENU-induced homozygous mutants with impaired responses to Toll-like receptor (TLR) ligands (TLR Signaling Screen). Peritoneal macrophages from the index CpG11 mouse (G4562) produced normal amounts of tumor necrosis factor (TNF)-α in response to all TLR ligands tested, except oligodeoxynucleotides containing CpG motifs (CpG ODNs). CpG11 heterozygotes produced intermediate levels of TNF-α relative to wild type and CpG11 homozygotes, demonstrating that the mutation is semidominant (Figure 1).
|Nature of Mutation|
The candidate gene Tlr9 was sequenced directly, and a T to C transition was identified at position 1181, in exon 2 of 2 total exons.
1166 CTCCACCTGGCAAGTTCCTTTAAGAACCTGGTG 353 -L--H--L--A--S--S--F--K--N--L--V-
The mutated nucleotide is indicated in red lettering, and results in a serine to proline substitution at amino acid 359 of the TLR9 protein.
Please see the record for CpG1 for more information about Tlr9.
The extracellular domains of TLRs contain multiple leucine rich repeats (LRRs) that mediate ligand recognition by TLRs. TLR9 has 25 LRRs in its ectodomain. The CpG11 mutation is located in the predicted eleventh leucine rich repeat (LRR) of the TLR9 ectodomain (Figure 2).
Based on the 3D structures of TLR1, TLR2 (1), TLR3 (2-4), and other LRR-containing proteins (5), the serine mutated in CpG11 is predicted to lie in the surface-exposed α-helical second leucine-rich sequence of the affected LRR, at a position thought to permit occupancy by any amino acid, but adjacent to an invariant phenylalanine. The CpG11 phenotype indicates that a proline at this position fails to support normal function of TLR9. The mutation may disrupt ligand binding, receptor dimerization, or destroy proper folding or localization of the receptor.
|Primers||Primers cannot be located by automatic search.|
CpG11 genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide insertion.
CpG11 (F): 5’- CATGGACGGGAACTGCTACTACAAG -3’
CpG11(R): 5’-ATGAAGTTCTTAGAAGCAGGGGTGC -3’
1) 95°C 2:00
2) 95°C 0:30
3) 56°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 29X
6) 72°C 7:00
7) 4°C ∞
Primers for sequencing
CpG11_seq(F): 5’- TGAGCAATCTCACCCATCTG -3’
CpG11_seq(R): 5’- GACAACAGCTCCTCCTGCTC -3’
The following sequence of 885 nucleotides (from Genbank genomic region NC_000075 for linear genomic sequence of Tlr9, sense strand) is amplified:
1441 ggaactgcta ctacaagaac ccctgcacag gagcggtgaa ggtgacccca ggcgccctcc
1501 tgggcctgag caatctcacc catctgtctc tgaagtataa caacctcaca aaggtgcccc
1561 gccaactgcc ccccagcctg gagtacctcc tggtgtccta taacctcatt gtcaagctgg
1621 ggcctgaaga cctggccaat ctgacctccc ttcgagtact tgatgtgggt gggaattgcc
1681 gtcgctgtga ccatgccccc aatccctgta tagaatgtgg ccaaaagtcc ctccacctgc
1741 accctgagac cttccatcac ctgagccatc tggaaggcct ggtgctgaag gacagctctc
1801 tccatacact gaactcttcc tggttccaag gtctggtcaa cctctcggtg ctggacctaa
1861 gcgagaactt tctctatgaa agcatcaccc acaccaatgc ctttcagaac ctaacccgcc
1921 tgcgcaagct caacctgtcc ttcaattacc gcaagaaggt atcctttgcc cgcctccacc
1981 tggcaagttc ctttaagaac ctggtgtcac tgcaggagct gaacatgaac ggcatcttct
2041 tccgcttgct caacaagtac acgctcagat ggctggccga tctgcccaaa ctccacactc
2101 tgcatcttca aatgaacttc atcaaccagg cacagctcag catctttggt accttccgag
2161 cccttcgctt tgtggacttg tcagacaatc gcatcagtgg gccttcaacg ctgtcagaag
2221 ccacccctga agaggcagat gatgcagagc aggaggagct gttgtctgcg gatcctcacc
2281 cagctccgct gagcacccct gcttctaaga acttcat
Primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is indicated in red.
1. Jin, M. S., Kim, S. E., Heo, J. Y., Lee, M. E., Kim, H. M., Paik, S. G., Lee, H., and Lee, J. O. (2007) Crystal Structure of the TLR1-TLR2 Heterodimer Induced by Binding of a Tri-Acylated Lipopeptide. Cell. 130, 1071-1082.
2. Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) The Molecular Structure of the Toll-Like Receptor 3 Ligand-Binding Domain. Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980.
3. Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Crystal Structure of Human Toll-Like Receptor 3 (TLR3) Ectodomain. Science. 309, 581-585.
4. Liu, L., Botos, I., Wang, Y., Leonard, J. N., Shiloach, J., Segal, D. M., and Davies, D. R. (2008) Structural Basis of Toll-Like Receptor 3 Signaling with Double-Stranded RNA. Science. 320, 379-381.
|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine|
|Authors||Hua Huang, Bruce Beutler|