|Mutation Type||splice acceptor site (13 bp from exon)|
|Coordinate||52,151,225 bp (GRCm38)|
|Base Change||T ⇒ A (forward strand)|
|Gene Name||signal transducer and activator of transcription 1|
|Chromosomal Location||52,119,440-52,161,865 bp (+)|
|MGI Phenotype||Homozygotes for targeted null mutations are largely unresponsive to interferon, fail to thrive, are susceptible to viral diseases and cutaneous leishmaniasis, and show excess osteoclastogenesis leading to increased bone mass.|
|Amino Acid Change|
|Institutional Source||Beutler Lab|
Ensembl: ENSMUSP00000066743 (fasta)
Ensembl: ENSMUSP00000066743 (fasta)
|Gene Model||not available|
|Predicted Effect||probably benign|
|Phenotypic Category||decrease in NK cell response, immune system, MCMV susceptibility|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Embryos, Sperm, gDNA|
|Last Updated||08/28/2017 11:03 AM by Anne Murray|
The poison phenotype was identified among ENU-induced G3 mutant mice by screening for defects in natural killer (NK) cell function using an in vivo NK cytotoxicity assay (In vivo NK cell and CD8+ T cell cytotoxicity screen). Briefly, mice were injected with fluorescently labeled splenocytes isolated from C57BL/6J, allogeneic C3H/HeN, and TAP-deficent mice, and NK cell-specific killing was measured 48 hours later. Homozygous poison mice exhibit a reduced ability to kill TAP-deficient target cells lacking MHC class I molecules, but normal killing of C3H/HeN target cells displaying allogeneic MHC molecules. In vitro, NK cells from poison homozygotes produce normal levels of interferon (IFN)-γ and degranulate normally after stimulation with antibodies against Ly49D, NK1.1, and NKp46 receptors, or after treatment with interleukin (IL)-12, IL-2 or IL-18. However, neither IFN-γ production nor degranulation was observed when poison NK cells were exposed to YAC-1 tumor cells.
Poison homozygotes display increased susceptibility to infection by mouse cytomegalovirus (MCMV) (MCMV Susceptibility and Resistance Screen). 100% of infected mice succumb by day 5 post-infection with 105 pfu, a normally sublethal inoculum, making poison mice as susceptible to MCMV infection as BALB/c mice lacking the NK cell activating receptor Ly49H. Like poison mutants, homozygous domino mice also display MCMV susceptibility and low cytotoxic activity of NK cells, and harbor a mutation in Stat1 (1).
|Nature of Mutation|
The poison mutation was mapped to Chromosome 1, and corresponds to a T to A transversion at position 31788 in the genomic DNA sequence of the Stat1 gene (Genbank genomic region NC_000067 for linear genomic DNA sequence of Stat1). The mutation is located within intron 19, thirteen nucleotides upstream from the start of exon 20, and impairs the acceptor splice site of intron 19. cDNA sequencing demonstrates the presence of several abnormal transcripts. In one case, a sequence in the middle of exon 20 (TCCTAG, position 31850-31855) is used as an acceptor splice site, causing a deletion of 55 nucleotides from the 5’ end of exon 20 and destroying the reading frame thereafter (depicted below). A premature stop is predicted to truncate the protein after amino acid 561 (Figure 1). Other abnormal transcripts were not sequenced.
<--exon 19 <--intron 19 exon 20--> 31223 TTTTGTAAG……ATTTGTATACTTGCAG GAAAATATT……ACCATCCTAGAGCTCATTAAGAAGCACCTGCTGTGCCTCTGGAATGATGGGTAAGCGTCCCTGA 31909 542 -F--C--K- -E--N--I-……-T--I--L- -S--S--L--R--S--T--C--C--A--S--G--M--M--G--K--R--P--* 561 correct deleted aberrant
Please see the record for domino for information about Stat1.
STAT1 is required for IFN signaling. NK cell function is enhanced by IFNs, and Stat1-/- NK cells have impaired cytotoxicity relative to wild type NK cells (2). Stat1-/- mice are highly sensitive to bacterial and viral infections, and cells from these mice are unresponsive to type I IFN and IFN-γ (3;4). Stat1poison/poison mice exhibit a phenotype similar to that of knockout mice, and consistent with this, no Stat1 protein expression is detected by immunoblot in extracts of peritoneal macrophages from homozygous mutants (Figure 2).
|Primers||Primers cannot be located by automatic search.|
Poison genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.
Primers for PCR amplification
1) 94°C 2:00
2) 94°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
Poison_seq(F): 5’- TCCAGTCATCTGGGAAGTCAC-3’
Poison_seq(R): 5’- AGACTGAGTCTCACTGTCAGC-3’
The following sequence of 1084 nucleotides (from Genbank genomic region NC_000067 for linear DNA sequence of Stat1) is amplified:
31367 ggaa gcacctgcct
31381 gagtgataac actctcccac atccctcgcc ccagcctctt aatcagcagg gctctggcta
31441 aggccgtggg aaccgtgcac ggagcagttg tgcagagcta ccccagctcg agggcacctg
31501 cacagggtgg tgagaccctg cacggttcgc cagcaggagc tgagctgctc atgctggctt
31561 ctgctacagt gagcacactc ttgttttcca gtcatctggg aagtcacctt tgccgggtgc
31621 tgctcttggg aaagggagcc aaggagggag aagctctagc aagtcagaaa gaggcagggg
31681 gagccagcca ggcatttcca ctcaggaacg actgcccctc ttgcttgatc tggggtctct
31741 ttggaggggc agggcaaagc agtgctttgg ttaaccaatt gtgcatttgt atacttgcag
31801 gaaaatatta atgataaaaa tttctccttc tggccttgga ttgacaccat cctagagctc
31861 attaagaagc acctgctgtg cctctggaat gatgggtaag cgtccctgag cgctccgaag
31921 gctgggacag agtgccggcc ggccttcagg atttggcagg ggttttgctc agagatgcac
31981 acttacctgg tataaggctc cttgaaagga caccctcagg agattaagca tgtcttagga
32041 tatctatatt tagatctatc tatttaaaag ctaaattaaa aacaaaaacc tcttagtgtc
32101 attaatgtat ttattcattc tcacaaacat tgtcagtatt tactgtactg ccctaataaa
32161 gaatcagaaa ccaccagatg gggcagtgca gatctttaac ttcagctgag gctgaggctg
32221 aggcaggctg atctctgctc gtagcacagc aagctccagg cagccacggc tgacagtgag
32281 actcagtctt ttttgttaaa aggaaggaag gaaggagaga aggagaggga agggaagaga
32341 agggaagaag gaaggagaca gaaggaaaga gagaaagaga aaagtgttta gctcattcag
32401 tccagggatc aactgatatc tccggggttc ctgtcagttg accagcagtg
PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is shown in red text.
1. Crozat, K., Georgel, P., Rutschmann, S., Mann, N., Du, X., Hoebe, K., and Beutler, B. (2006) Analysis of the MCMV resistome by ENU mutagenesis, Mamm. Genome 17, 398-406.
2. Lee, C. K., Rao, D. T., Gertner, R., Gimeno, R., Frey, A. B., and Levy, D. E. (2000) Distinct requirements for IFNs and STAT1 in NK cell function, J. Immunol. 165, 3571-3577.
3. Meraz, M. A., White, J. M., Sheehan, K. C., Bach, E. A., Rodig, S. J., Dighe, A. S., Kaplan, D. H., Riley, J. K., Greenlund, A. C., Campbell, D., Carver-Moore, K., DuBois, R. N., Clark, R., Aguet, M., and Schreiber, R. D. (1996) Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway, Cell 84, 431-442.4. Durbin, J. E., Hackenmiller, R., Simon, M. C., and Levy, D. E. (1996) Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease, Cell 84, 443-450.
|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine, Peter Jurek, Katherine Timer|
|Authors||Karen Whitley, Kasper Hoebe, Bruce Beutler|