|Coordinate||36,770,983 bp (GRCm38)|
|Base Change||A ⇒ G (forward strand)|
|Gene Name||zeta-chain (TCR) associated protein kinase|
|Synonym(s)||ZAP-70, TZK, Srk|
|Chromosomal Location||36,761,798-36,782,818 bp (+)|
FUNCTION: This gene encodes a member of the protein tyrosine kinase family. The encoded protein is essential for development of T lymphocytes and thymocytes, and functions in the initial step of T lymphocyte receptor-mediated signal transduction. A mutation in this gene causes chronic autoimmune arthritis, similar to rheumatoid arthritis in humans. Mice lacking this gene are deficient in alpha-beta T lymphocytes in the thymus. In humans, mutations in this gene cause selective T-cell defect, a severe combined immunodeficiency disease characterized by a selective absence of CD8-positive T lymphocytes. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2014]
PHENOTYPE: Mutant mice show T cell defects. Null mutants lack alpha-beta T cells in the thymus and have fewer T cells in dendritic and intestinal epithelium. Spontaneous and knock-in missense mutations affect T cell receptor signaling, one of the former resulting in severe chronic arthritis. [provided by MGI curators]
|Amino Acid Change||Histidine changed to Arginine|
|Institutional Source||Beutler Lab|
H58R in Ensembl: ENSMUSP00000027291 (fasta)
|Gene Model||not available|
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Last Updated||2017-08-08 8:01 PM by Diantha La Vine|
|Record Created||2010-06-02 4:59 PM by Carrie N. Arnold|
The wanna mutation was discovered while screening N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice for aberrant T-dependent and T-independent B cell responses. Wanna mice lack a T-dependent immuoglobin G (IgG) response to model antigens encoded by a recombinant suicide vector based on the Semliki Forest Virus (rSFV), but display normal T-independent IgM responses to haptenated ficoll and serum levels of IgA. Flow cytometric analysis of peripheral blood lymphocytes from the four original index mice showed a lack of peripheral CD4+ and CD8+ T cells (lower panels in CD4 vs CD8 plots), but had normal frequencies of natural killer (NK) cells (lower panels in CD8 vs NK1.1 plots) and mature B cells (not shown).
|Nature of Mutation|
The wanna mutation was mapped to Chromosome 1 by outcrossing homozygous animals to the closely related C57BL/10J strain and backcrossing F1 animals back to wanna mice. The DNA samples from phenotypically wild type and mutant animals were pooled separately and analyzed by bulk segregation analysis (BSA) using a panel of 124 single nucleotide polymorphisms (SNPs) (1). Sequencing of the candidate gene Zap70 on Chromosome 1 found an A to G transition at position 264 of the Zap70 transcript using Genbank record NM_009539, in exon 2 of 13 total exons.
248 GACGTGCGCTTCCACCATTTCCCCATCGAGCGC 53 -D--V--R--F--H--H--F--P--I--E--R-
The mutated nucleotide is indicated in red lettering and causes a histidine to arginine change at amino acid 58 of the encoded protein.
The wanna mutation results in a histidine to arginine substitution of a conserved residue in the N-terminal SH2 domain (Figure 5). Along with the C-terminal SH2 domain, this domain is required to bind to the phosphorylated tyrosines of immunoreceptor tyrosine-based activation motifs (ITAMS) of the ζ and CD3ε chains of the T cell receptor (TCR; see the record for tumormouse). The N-terminal SH2 domain makes van der Waals contacts with the second phosphorylated tyrosine of an ITAM [(D/E)xxYxxI/Lx(6-8)YxxI/L] according to crystal structure studies (FIgure 6) (2). The phenotypes of wanna mice provide in vivo confirmation that His 58 is important for ZAP70 function.
Please see the record for murdock for more information about Zap70.
|Primers||Primers cannot be located by automatic search.|
Wanna genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transition.
Wanna(F): 5’- ACTGACTCCCGTGCAAAGATGAGG -3’
Wanna(R): 5’- TCAAGCATAGCACATGCTGAGGAC -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 8
Primers for sequencing
Wanna_seq(F): 5'- ATGATGGCCCTGAAACGC -3'
Wanna_seq(R): 5'- GAGGACTACAGCCTATAGTCTCTG -3'
The following sequence of 777 nucleotides (NCBI Mouse Genome Build 37.1, Chromosome 1, bases 36,827,587 to 36,828,363) is amplified:
actgactccc gtgcaaagat gaggcaccat gatggccctg aaacgccttt gcacccacag ggtccagcga tgcccgatcc cgcggcgcac ctgccattct tctatggcag catctcgcgg gctgaggccg aggagcacct gaagctggca ggcatggccg acgggctgtt cctcctgcgc cagtgtttgc gctccctggg cggctacgtg ctgtcgttgg tgcacgacgt gcgcttccac catttcccca tcgagcgcca actcaacggc acgtacgcca tcgcgggcgg gaaggcgcac tgcggcccgg ccgagctctg ccagttctac tctcaggacc ccgacgggct gccctgcaac ctgcgtaagc cgtgtaaccg gccgcccgga ctggagccac agcccggggt cttcgactgc ctgcgtgatg ctatggtgcg cgactacgtg cgccagacct ggaagctgga ggtgagggcg tggtcagagg gggagtgggc gaggcatgga ggctgatgct gtgtcttcat tggcttgcaa gatgctctga ggcctgggat ttggaagtga cagaatgctg aggccctagt tgagcccatg acctggaagg aggacttgca ggtccaggct tgcgcagcct gaatgatgct gtggtgcttc accatctatg atgcgcctga aggtgccaga gaggaccatg aagggcaggc ctgtggccta cagaaggatg gcagacagag actataggct gtagtcctca gcatgtgcta tgcttga
Primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is indicated in red.
1. Xia, Y., Won, S., Du, X., Lin, P., Ross, C., La Vine, D., Wiltshire, S., Leiva, G., Vidal, S. M., Whittle, B., Goodnow, C. C., Koziol, J., Moresco, E. M., and Beutler, B. (2010) Bulk Segregation Mapping of Mutations in Closely Related Strains of Mice. Genetics 186, 1139-1146.
|Science Writers||Nora G. Smart|
|Illustrators||Diantha La Vine|
|Authors||Carrie Arnold, Elaine Pirie, and Bruce Beutler|