|Coordinate||124,867,746 bp (GRCm38)|
|Base Change||G ⇒ A (forward strand)|
|Gene Name||CD4 antigen|
|Chromosomal Location||124,864,692-124,888,221 bp (-)|
FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a membrane glycoprotein of T lymphocytes that interacts with major histocompatibility complex class II antigenes and is also a receptor for the human immunodeficiency virus. This gene is expressed not only in T lymphocytes, but also in B cells, macrophages, and granulocytes. It is also expressed in specific regions of the brain. The protein functions to initiate or augment the early phase of T-cell activation, and may function as an important mediator of indirect neuronal damage in infectious and immune-mediated diseases of the central nervous system. Multiple alternatively spliced transcript variants encoding different isoforms have been identified in this gene. [provided by RefSeq, Aug 2010]
PHENOTYPE: Mice homozygous for knock-out alleles exhibit abnormal immune system morphology and physiology. [provided by MGI curators]
|Amino Acid Change||Glutamine changed to Stop codon|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000024044] [ENSMUSP00000038536] [ENSMUSP00000145267]|
AA Change: Q359*
|Predicted Effect||probably null|
|Phenotypic Category||Autosomal Recessive|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice|
|Last Updated||2016-05-13 3:09 PM by Bruce Beutler|
|Record Created||2013-06-27 11:47 PM by Kuan-Wen Wang|
The craw phenotype was discovered while screening N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice for T-dependent (TD) humoral responses. Craw mice lack T-dependent IgG responses to both ovalbumin administered with aluminum hydroxide (OVA-Alum; Figure 1) and recombinant Semliki Forest virus (rSFV)-encoded β-galactosidase (rSFV-β-gal; Figure 2); craw mice exhibit normal T-independent IgM responses to 4-hydroxy-3-nitrophenylacetyl-Ficoll (NP-Ficoll) (not shown). Flow cytometric analysis of peripheral blood from craw mice determined that the craw mice do not have detectable amounts CD4+ T cells, but exhibit a strong increase in the frequency of CD8+ T cells (Figure 3).
|Nature of Mutation|
The Cd4 gene was directly sequenced as a candidate gene due to the lack of CD4+ T cells in the craw mice. The craw mutation is a C to T transition at base pair 124,867,746 (v38) on chromosome 6, or base pair 20,465 in the GenBank genomic region NC_000072 encoding Cd4. The mutation corresponds to residue 1,245 in the NM_013488 mRNA sequence in exon 7 of 10 total exons.
The mutated nucleotide is indicated in red. The mutation results in substitution of a premature stop codon (*) for glutamine (Q) at amino acid 359 in the CD4 protein.
Cd4 encodes CD4, a member of the immunoglobin superfamily that is a co-receptor for the T cell receptor (TCR). Together with the TCR, CD4 engages major histocompatibility complex (MHC) class II molecules in antigen presenting cells (1-4). Additional data suggests that CD4 may function to initiate, or augment, the early stages of T cell activation rather than stabilizing TCR-MHC interaction (5). The craw mutation is a nonsense mutation within the extracellular immunoglobulin (Ig)-like C2-type 3 domain (i.e., D4; Figure 4).
Please see the record thoth for information about Cd4.
Signaling via the CD4-TCR complex is essential at multiple stages of thymocyte differentiation, T-cell activation, and homeostasis. Coding of the premature stop codon in craw results in loss of peripheral CD4+ T cells as well as the loss of T-dependent humoral responses, indicating that any protein product expressed in craw is non-functional in generating a TCR-mediated signaling response.
craw(F):5'- AGCTCTTAGCCAGGAAGACCTCAC -3'
craw(R):5'- TCCACGCTTACAGCTTGAACCC -3'
craw_seq(F):5'- CTCACTCCTAAGCTGTGTGGAAG -3'
craw_seq(R):5'- TTGAACCCCGAGCAGCAG -3'
Craw 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.
Craw(F): 5’- AGCTCTTAGCCAGGAAGACCTCAC-3’
Craw(R): 5’- TCCACGCTTACAGCTTGAACCC-3’
Craw_seq(F): 5’- CTCACTCCTAAGCTGTGTGGAAG-3’
Craw_seq(R): 5’- TTGAACCCCGAGCAGCAG-3’
1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40X
6) 72°C 10:00
7) 4°C ∞
The following sequence of 509 nucleotides is amplified (Chr.6: 124867421-124867929, GRCm38; NC_000072):
1 agctcttagc caggaagacc tcactcctaa gctgtgtgga agtgggaagt aactggagga
61 tacaagtggg aacctagagc ccaggacccc tctctctcct cacttctatc tccaacctct
121 gtacaaaatg cctccgtgag gagctaggga actgagactc taggtcggca tagcttgtcc
181 ctggggcttt ccacaggtga agagtccgag gcttagggtg aggccttaga aactggatcc
241 ttaccctgga tcctggagtc catcttgacc ttatcacctt cactcagtag acactgccac
301 agccctgtct caggggccac cacttgaact actttctgct cctcagagac cctggcctcc
361 tggttctcct gcttcagggt cagtctcatc ttgggagagg taggtcccat cacctcacag
421 gtcaaagtat tgttgagctg agccactgca gaggaaggag aggcagagag ctggatcctg
481 ctgctcgggg ttcaagctgt aagcgtgga
Primer binding sites are underlined and the sequencing primer is highlighted; the mutated nucleotide is shown in red text (C>T, Chr. (+) strand; G>A, sense strand).
1. Maddon, P. J., Littman, D. R., Godfrey, M., Maddon, D. E., Chess, L., and Axel, R. (1985) The Isolation and Nucleotide Sequence of a cDNA Encoding the T Cell Surface Protein T4: A New Member of the Immunoglobulin Gene Family. Cell. 42, 93-104.
2. Maddon, P. J., Molineaux, S. M., Maddon, D. E., Zimmerman, K. A., Godfrey, M., Alt, F. W., Chess, L., and Axel, R. (1987) Structure and Expression of the Human and Mouse T4 Genes. Proc Natl Acad Sci U S A. 84, 9155-9159.
3. Gorman, S. D., Tourvieille, B., and Parnes, J. R. (1987) Structure of the Mouse Gene Encoding CD4 and an Unusual Transcript in Brain. Proc Natl Acad Sci U S A. 84, 7644-7648.
4. Doyle, C., and Strominger, J. L. (1987) Interaction between CD4 and Class II MHC Molecules Mediates Cell Adhesion. Nature. 330, 256-259.
|Science Writers||Anne Murray|
|Authors||Kuan-Wen Wang, Jin Huk Choi, Ming Zeng, Bruce Beutler|