Phenotypic Mutation 'huckle' (pdf version)
Allelehuckle
Mutation Type intron
Chromosome2
Coordinate101,471,568 bp (GRCm39)
Base Change A ⇒ T (forward strand)
Gene Rag1
Gene Name recombination activating 1
Synonym(s) Rag-1
Chromosomal Location 101,468,627-101,479,846 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The protein encoded by this gene is involved in activation of immunoglobulin V-D-J recombination. The encoded protein is involved in recognition of the DNA substrate, but stable binding and cleavage activity also requires RAG2. Defects in this gene can be the cause of several diseases. [provided by RefSeq, Jul 2008]
PHENOTYPE: Homozygotes for targeted null mutations exhibit arrested development of T and B cell maturation at the CD4-8- thymocyte or B220+/CD43+pro-B cell stage due to inability to undergo V(D)J recombination. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_009019; MGI: 97848

MappedYes 
Amino Acid Change
Institutional SourceBeutler Lab
Gene Model not available
AlphaFold P15919
PDB Structure RAG1 DIMERIZATION DOMAIN [X-RAY DIFFRACTION]
Crystal structure of the RAG1 nonamer-binding domain with DNA [X-RAY DIFFRACTION]
Crystal structure of the RAG1 nonamer-binding domain with DNA [X-RAY DIFFRACTION]
Crystal structure of the core RAG1/2 recombinase [X-RAY DIFFRACTION]
SMART Domains Protein: ENSMUSP00000077584
Gene: ENSMUSG00000061311

DomainStartEndE-ValueType
Pfam:RAG1_imp_bd 11 288 5.7e-120 PFAM
RING 290 328 1.39e-3 SMART
ZnF_C2H2 353 376 2.61e1 SMART
PDB:3GNB|A 389 464 3e-44 PDB
ZnF_C2H2 725 750 7e1 SMART
Predicted Effect probably benign
Meta Mutation Damage Score Not available question?
Is this an essential gene? Possibly nonessential (E-score: 0.391) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance 100% 
Alleles Listed at MGI

All alleles(12) : Targeted, knock-out(2) Targeted, other(8) Chemically induced(2)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00940:Rag1 APN 2 101472733 missense probably damaging 1.00
IGL01125:Rag1 APN 2 101472346 missense probably damaging 0.99
IGL01836:Rag1 APN 2 101472239 missense probably damaging 1.00
IGL02216:Rag1 APN 2 101473726 missense possibly damaging 0.91
IGL02271:Rag1 APN 2 101473733 missense probably damaging 0.99
IGL02293:Rag1 APN 2 101473391 missense probably benign 0.39
IGL02601:Rag1 APN 2 101473018 missense probably damaging 1.00
Anne UTSW 2 101473861 missense probably damaging 0.99
busted UTSW 2 101472292 missense probably damaging 1.00
cloth UTSW 2 101473009 missense probably damaging 1.00
defective UTSW 2 101473055 missense probably damaging 1.00
doll UTSW 2 101472415 missense probably damaging 1.00
dysfunctional UTSW 2 101474629 missense probably damaging 1.00
furchte UTSW 2 101474852 missense probably benign 0.05
horrorshow UTSW 2 101472968 missense probably damaging 1.00
maladaptive UTSW 2 101645647 intron probably benign
scarecrow UTSW 2 101472852 missense probably damaging 1.00
R0658:Rag1 UTSW 2 101473028 missense probably damaging 0.99
R1126:Rag1 UTSW 2 101473034 missense probably damaging 1.00
R1177:Rag1 UTSW 2 101472623 missense probably benign 0.10
R1319:Rag1 UTSW 2 101473537 missense probably damaging 1.00
R1513:Rag1 UTSW 2 101473336 missense possibly damaging 0.95
R1859:Rag1 UTSW 2 101474407 missense probably benign 0.03
R2218:Rag1 UTSW 2 101474491 missense probably benign
R3932:Rag1 UTSW 2 101473384 missense probably damaging 1.00
R4127:Rag1 UTSW 2 101472416 missense probably damaging 1.00
R4365:Rag1 UTSW 2 101473288 missense probably damaging 1.00
R4620:Rag1 UTSW 2 101474025 missense probably damaging 1.00
R4815:Rag1 UTSW 2 101473861 missense probably damaging 0.99
R5070:Rag1 UTSW 2 101472656 missense probably damaging 1.00
R5209:Rag1 UTSW 2 101474560 missense probably benign 0.01
R5239:Rag1 UTSW 2 101473300 missense possibly damaging 0.91
R5390:Rag1 UTSW 2 101473079 missense probably benign
R5607:Rag1 UTSW 2 101474137 missense probably damaging 1.00
R6259:Rag1 UTSW 2 101474797 missense possibly damaging 0.83
R6412:Rag1 UTSW 2 101472865 missense probably damaging 0.99
R6633:Rag1 UTSW 2 101473055 missense probably damaging 1.00
R6679:Rag1 UTSW 2 101474629 missense probably damaging 1.00
R6723:Rag1 UTSW 2 101473990 missense probably damaging 0.99
R6853:Rag1 UTSW 2 101472566 missense probably damaging 0.99
R6867:Rag1 UTSW 2 101472292 missense probably damaging 1.00
R6974:Rag1 UTSW 2 101472137 missense probably damaging 0.99
R7071:Rag1 UTSW 2 101473807 missense probably damaging 0.99
R7124:Rag1 UTSW 2 101474128 missense probably damaging 0.99
R7248:Rag1 UTSW 2 101472123 missense probably damaging 0.99
R7256:Rag1 UTSW 2 101472415 missense probably damaging 1.00
R7567:Rag1 UTSW 2 101474006 missense probably damaging 0.98
R7581:Rag1 UTSW 2 101473649 missense possibly damaging 0.95
R7830:Rag1 UTSW 2 101472404 missense probably damaging 1.00
R7941:Rag1 UTSW 2 101472691 missense probably benign 0.24
R8024:Rag1 UTSW 2 101472852 missense probably damaging 1.00
R8434:Rag1 UTSW 2 101473009 missense probably damaging 1.00
R8688:Rag1 UTSW 2 101472968 missense probably damaging 1.00
R8918:Rag1 UTSW 2 101472098 missense probably benign
R9116:Rag1 UTSW 2 101475137 missense probably benign 0.38
R9116:Rag1 UTSW 2 101472820 missense probably damaging 1.00
R9210:Rag1 UTSW 2 101474852 missense probably benign 0.05
R9409:Rag1 UTSW 2 101473192 missense probably damaging 1.00
R9562:Rag1 UTSW 2 101473327 missense probably damaging 1.00
R9565:Rag1 UTSW 2 101473327 missense probably damaging 1.00
R9594:Rag1 UTSW 2 101474701 missense probably benign
R9658:Rag1 UTSW 2 101473229 missense possibly damaging 0.83
R9779:Rag1 UTSW 2 101474153 missense probably damaging 1.00
X0018:Rag1 UTSW 2 101474892 missense probably damaging 0.99
X0018:Rag1 UTSW 2 101473942 missense probably damaging 1.00
Z1176:Rag1 UTSW 2 101473604 missense probably damaging 1.00
Mode of Inheritance Autosomal Recessive
Local Stock Sperm
Repository
Last Updated 2019-03-21 1:17 PM by Diantha La Vine
Record Created 2010-08-30 7:16 PM by Carrie N. Arnold
Record Posted 2011-01-05
Phenotypic Description

The huckle phenotype was identified among ENU-mutagenized G3 mice and is characterized by a complete deficiency of CD8+ and CD4+ T cells in the blood.  B cells are almost completely absent.  The NK cell population is expanded.

Nature of Mutation

The candidate genes Rag1, Rag2, and Prkdc were sequenced.  A T to A transversion was identified at position 1232 of the Rag1 transcript, within exon 2 of 2 total exons.

1215 GTGAGTCTGGAAAAATATAACCACCATGTGTCA

364  -V--S--L--E--K--Y--N--H--H--V--S-

The mutated nucleotide is indicated in red lettering, and converts codon 369 (tyrosine) to a stop codon.

Illustration of Mutations in
Gene & Protein
Putative Mechanism
Figure 1. Domain structure of RAG1. The positions of catalytic triad residues are shown above the diagram. The central domain contains binding sites for the RSS heptamer and for RAG2, as well as a zinc finger motif (ZFB, hatched box). The C-terminal domain (C-term) binds DNA in a non-sequence-specific manner, and may mediate dimerization. The huckle mutation converts codon 369 (tyrosine) to a stop codon. ZDD, zinc-binding dimerization domain; RING, RING finger domain; ZFA, C2H2 zinc finger motif A; NBR, nonamer binding region; ZFB, zinc finger motif B. This image is interactive. Other mutations found in RAG1 are noted in red. Click on the mutations for more specific information.  

The huckle mutation creates a premature stop codon that would truncate the protein after amino acid 368, which lies within the zinc-binding dimerization domain (ZDD) that encompasses a zinc RING finger motif (amino acids 288-339) and a C2H2 zinc finger domain (amino acids 349-378; designated ZFA) (Figure 1) (1).  The ZDD forms part of the non-core regions of RAG1 (amino acids 1-383 and 1009-1040) that when deleted still yield an active protein capable of recombining a plasmid substrate (2;3).  The phenotype of huckle mice closely resembles that of Rag1 null mice, consistent with a complete lack of RAG1 protein expression resulting from the premature stop codon.

Please see the record for maladaptive for information about Rag1.

Primers Primers cannot be located by automatic search.
Genotyping

Huckle 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

huckle (F): 5’- AAGTTCAGCAGTTCCCACAGTCAG-3’

huckle (R): 5’- GCCTCCCAGTGATAGCTTTCACAG -3’

PCR program

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

huckle_seq(F): 5’- AGAGCCCAGTGAAGTCCTTTC -3’

huckle_seq(R): 5’- GCATGATGGCCTCTAATTCATCAG -3’

The following sequence of 931 nucleotides (from Genbank genomic region NC_000068 for linear genomic sequence of Rag1, sense strand) is amplified:

              a agttcagcag ttcccacagt caggtctact tcccaaggaa agtgaccgtg

5341 gagtggcacc cccacacacc gtcctgtgac atctgtttta ctgcccatcg gggactcaag

5401 aggaagagac atcagcccaa tgtgcagctc agcaagaaac taaaaactgt gctcaaccac

5461 gcgagacggg accgtcgcaa gagaactcag gctagggtca gcagcaagga agtcctgaag

5521 aagatctcca actgcagtaa gattcatctc agtaccaagc ttcttgccgt ggacttccca

5581 gcacactttg tgaaatccat ctcctgccag atatgcgaac acattctggc tgatcccgtg

5641 gagaccagct gcaagcatct attctgtagg atctgcattc tcagatgtct caaagtcatg

5701 ggcagctatt gtccctcttg ccgatatccg tgcttcccta ctgacctgga gagcccagtg

5761 aagtcctttc tgaacatctt gaattctctc atggtcaagt gtcccgcgca agattgcaat

5821 gaggaagtga gtctggaaaa atataaccac catgtgtcaa gccacaaaga atctaaagag

5881 actttggtgc atatcaataa agggggacgg cctcgccagc atctcctgtc actgacgaga

5941 agggcgcaga aacatcggct gagggagctc aagattcaag tcaaagaatt tgctgacaaa

6001 gaagaaggtg gagatgtgaa ggctgtctgc ttgacattgt ttctcctggc actgagggcg

6061 aggaatgagc acaggcaagc tgatgaatta gaggccatca tgcaaggcag gggctccggg

6121 cttcaaccag ctgtttgctt ggccatccgt gtcaatacct tcctcagctg tagccaatac

6181 cataagatgt acaggactgt gaaagctatc actgggaggc

Primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is indicated in red.

References
Science Writers Eva Marie Y. Moresco
AuthorsCarrie N. Arnold, Elaine Pirie, Bruce Beutler
Edit History
2011-08-12 3:47 PM (current)
2011-01-20 11:39 AM
2011-01-06 11:18 AM
2011-01-06 11:18 AM
2011-01-05 11:16 AM