1. Immunology and Inflammation
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Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus

  1. Jeannine A Ott
  2. Caitlin D Castro
  3. Thaddeus C Deiss
  4. Yuko Ohta
  5. Martin F Flajnik
  6. Michael F Criscitiello  Is a corresponding author
  1. College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, United States
  2. University of Maryland at Baltimore, United States
  3. Texas A&M Health Science Center, Texas A&M University, United States
Research Article
Cite this article as: eLife 2018;7:e28477 doi: 10.7554/eLife.28477
16 figures, 7 tables and 1 additional file

Figures

Alignment of Gamma V clones suggests minimal somatic hypermutation.

Thymocyte clones for nine γV groups from three different predicted V genes. CDR regions are marked above the scale for each γV alignment. Amino acids are shown under the nucleotide consensus sequence, and dots represent identity to this sequence. We highlighted nonsynonymous changes in black; synonymous changes are underlined. Gaps are used for alignment purposes and indicate a shortened sequence (at the beginning or end). Sequences are identified by a single clone number or a group of identical clones condensed to a single line (the number of clones are indicated). Clone numbers that contain ‘THY’ are from thymus, ‘PBL’ are from peripheral blood leukocytes, and ‘SPV’ are from spiral valve (intestine). We deposited all 69 sequences into GenBank under accession numbers KY351639 – KY351707.

https://doi.org/10.7554/eLife.28477.002
Alignment of Delta V clones suggests somatic hypermutation.

Thymocyte clones for 12 δV groups from seven different predicted V genes. CDR regions are marked above the scale for each δV alignment. Amino acids are shown under the nucleotide consensus sequence, and dots represent identity to this sequence. We highlighted nonsynonymous changes in black; synonymous changes are underlined. Gaps are used for alignment purposes and indicate a shortened sequence (at the beginning or end). Sequences are identified by a single clone number or a group of identical clones condensed to a single line (the number of clones are indicated). Clone numbers that contain ‘THY’ are from thymus, ‘PBL’ are from peripheral blood leukocytes, and ‘SPV’ are from spiral valve (intestine). We deposited all 112 sequences into GenBank under accession numbers KY346705 – KY346816.

https://doi.org/10.7554/eLife.28477.003
Alignment of Beta V clones illustrates a lack of somatic hypermutation.

Thymocyte clones for βV groups from six different predicted V genes. CDR regions are marked above the scale for each βV alignment. Amino acids are shown under the nucleotide consensus sequence, and dots represent identity to this sequence. We observed three nonsynonymous changes within a single sequence (highlighted in black). Sequences are identified by a single clone number or a group of identical clones condensed to a single line (the number of clones are indicated). Clone numbers that contain ‘PBL’ are from peripheral blood leukocytes and ‘THY’ are from thymus. We deposited all 57 sequences into GenBank under accession numbers KY351708 – KY351764.

https://doi.org/10.7554/eLife.28477.004
CDR3s of TcR Alpha chain are diverse.

Amino acid (aa) alignment of TcR αV1 thymocyte clones illustrating diversity of the third complementarity-determining region (CDR3). All clones contain identical variable (V) region sequence (aa 1–61). We grouped clones by shared, identical joining (J) regions (purple boxes) and highlight the differences in the V-J join (CDR3 region) in red boxes.

https://doi.org/10.7554/eLife.28477.005
Figure 4—source data 1

CDR3 regions diversified by exonuclease activity and addition of N and P nucleotides.

Alignment of nucleotides belonging to the join between variable (V) and joining (J) segments within TcRα thymocyte clones. We determined the putative ends of each V segment and putative beginning of each J segment by comparing alignments between different sharks, assuming that identical nucleotides between sharks were germline. The last number of each sequence name indicates the number of clones containing that nucleotide sequence between the V and J segments.

https://doi.org/10.7554/eLife.28477.006
Alignment of Alpha V cDNA clones suggest somatic hypermutation at shark TCRα.

Thymocyte clones for all 11 αV groups with the same CDR3 from six different predicted V genes. Locations of framework regions (FR), complementarity determining regions (CDR), joining regions (J), and constant (C) regions are marked above the scale for each αV. In absence of germline sequence information, we used a Geneious-derived nucleotide consensus sequence for analysis of nucleotide changes in thymocyte clones. Amino acids are shown under the consensus sequence, and dots represent identity to this sequence. Nonsynonymous changes are highlighted in black; synonymous changes are underlined. Gaps are used for alignment purposes and indicate either a shortened sequence (at the beginning or end) or insertions or deletions within the sequence. Sequences are identified by a single clone number or a group of identical clones condensed to a single line (the number of clones are indicated). Clone numbers that contain ‘THY’ are from thymus, ‘PBL’ are from peripheral blood leukocytes, and ‘SPV’ are from spiral valve (intestine). We did not use clones from αV1.3 and αV7.1 because they did not contain mutations in FR or CDR regions. We deposited all 42 sequences into GenBank under accession numbers KY189332 – KY189354 or KY366469 – KY366487.

https://doi.org/10.7554/eLife.28477.009
Mutation frequencies differed within TcR V regions.

Mutability of complementarity determining regions (CDR), especially CDR1, exceeded that of framework regions (FR) for all mutations together (black bars) and for nonsynonymous mutations alone (NSYN, hatched bars). We found no statistical difference in synonymous mutations (SYN, white bars) between CDRs and FRs.

https://doi.org/10.7554/eLife.28477.012
Figure 6—source data 1

Frequency of mutation in TcR alpha V framework and complementarity determining regions for all mutation, nonsynonymous (NSYN) mutation only, or synonymous (SYN) mutation only.

https://doi.org/10.7554/eLife.28477.013
Mutation and motif locations within individual domains of TcR αV sequences.

(A) Number of mutations (both single and tandem, 283 total) to A:T nucleotides (black bars, 122 mutations) or G:C nucleotides (blue bars, 161 mutations) observed at each position along the sequence length of TcR αV sequences. We counted mutations to Geneious-derived consensus sequences within framework and complementarity-determining region (CDR) domains (41 sequences within 11 αV groups). (B) Number of WA/TW motifs (black bars, indicating possible polymerase η action) or DGYW/WRCH motifs (blue bars, indicating hotspots for AID activity, and thus possible mutation) at each position along the sequence length. Position indicates the forward (3’ to 5’) location of the mutable base of each motif within a Geneious-derived consensus sequence for each TcR αV group (eleven groups). Red boxes indicate the location of CDR1 (positions 76–99), CDR2 (positions 151–171), and CDR3 (positions 300–328) within each panel. [WA/TW: A:T is the mutable position; DGYW/WRCH: G:C is the mutable position; W = A/T, D = A/G/T, Y = C/T, R = A/G, and H = T/C/A].

https://doi.org/10.7554/eLife.28477.014
Shark thymus expresses AID.

(A) Expression of AID in shark spleen (black bar), thymus (hashed bar), and forebrain (gray bar) using real-time quantitative PCR. Expression was measured using the delta delta Cq method and are normalized against shark muscle tissue using β2M as a reference gene. Data represent expression fold-change differences at four cDNA concentrations. (B) In situ hybridization of mRNA using ribo-probes on adult shark thymus sections. 1,2: Two different fields probing for TcRβ antisense. 3,4: AID antisense. 5,6: TcRβ sense. 7,8: AID sense. All micrographs at 10X magnification; black scale bar in lower right of each panel is 100 uM. Anatomical structures are designated on the top panels [c: cortex; m: medulla; sc: subcapsular region; cmj: corticomedullary junction].

https://doi.org/10.7554/eLife.28477.018
Figure 9 with 2 supplements
AID expression localized to inner cortex and cortico-medullary junction.

(a) H and E staining of fixed shark thymus tissue illustrating thymic architecture (10x). The densely packed cells at the margins of the image comprise the cortex (cor), while the less densely packed cells in the center constitute the medulla (med). The region at the junction between cortex and medulla incorporates the corticomedullary junction (CMJ), delineated generally by a hashed white circle. [b-z] Single molecule RNA fluorescence in situ hybridization (FISH) probing fixed thymus sections simultaneously for AID (probes labeled with Quasar 670; pseudo colored red) and TcRα (probes labeled with CalFluor Red 610; pseudo colored green) and counterstained with DAPI (blue). (b) Composite of seven Z-stacked images (10x) depicting overall thymic architecture and the localization of AID expression to the inner cortex and cortico-medullary junction regions of shark thymus. We superimposed (and minimally adjusted) the outlined CMJ boundaries from (a) onto (b) to elucidate the junction between cortex and medulla. [c-z] We obtained images of each fluorophore using 10x, 20x, and 63x magnification and merged Z-stacked images together. Individual (10x) fluorophore images of DAPI [c-f], TcRα [g-j], and AID [k-n] and Z-stacked merged images [o-r] illustrate AID and TcRα expression in four locations of shark thymus. White boxes indicate the magnified regions of the 10x and 20x images shown in the 20x [s-v] and 63x [w-z] images, respectively. Scale bars [a,b,c,g,k,o] 150 μm, [s] 75 μm, and [w] 30 μm. [cor: cortex; med: medulla; CMJ: corticomedullary junction].

https://doi.org/10.7554/eLife.28477.019
Figure 9—figure supplement 1
Localization of AID and TcRα probes is independent.

Single molecule RNA fluorescence in situ hybridization (FISH) showing three separate regions of fixed thymus sections demonstrating the independent localization of our probes. We probed individually for TcRα (a-c) (probes labeled with CalFluor Red 610; pseudo colored green), simultaneously for both TcRα and AID (d-f), or individually for AID (probes labeled with Quasar 670; pseudo colored red) and counterstained with DAPI (blue). There were two tissue sections in between imaged regions of TcRα section (a-c) and combined AID/TcRα section (d-f). The combined AID/TcRα section (d-f) was consecutive with the AID only section (g-h). We obtained images of each fluorophore using 10x magnification and merged Z-stacked images together. Scale bar 100 μm.

https://doi.org/10.7554/eLife.28477.020
Figure 9—figure supplement 2
Lack of AID and TcRα probe hybridization in shark brain.

Single molecule RNA fluorescence in situ hybridization (FISH) showing fixed brain sections on four separate slides demonstrating the lack of probe localization in non-immune tissue. We probed simultaneously for both TcRα and AID (a-c), individually for AID (probes labeled with Quasar 670 and imaged with Cy5 filter; pseudo colored red) (d-f), individually for TcRα (probes labeled with CalFluor Red 610 and imaged with Cy3 filter; pseudo colored green) (g-i), or for neither probe (negative control) (j-l). We show probe staining in shark thymus tissue as a positive control [m-o]. We counterstained all slides with DAPI (blue). We imaged fluorescence detected using each filter (DAPI, Cy3/Cy5) regardless of the probe used to illustrate that background fluorescence is not dependent on probe application. We obtained images of each fluorophore using 10x magnification and merged Z-stacked images together (DAPI fluorescence [a,d,g,j,m]; Cy3 (green) and Cy5 (red) fluorescence [b,e,h,k,n]; merged [c,f,I,l,o]). Scale bar 150 μm.

https://doi.org/10.7554/eLife.28477.021
Model predicting how AID acts on T cells in the thymus.

CD4/CD8 double negative (DN) thymocytes in the subcapsular region (SC) and cortex rearrange the β chain, using a surrogate pTα receptor to test for expression signaling. Cells with productive β arrangements then proliferate and express both CD4 and CD8, becoming double positive thymocytes (DPs). As DPs move toward the inner cortex and cortico-medullary junction (CMJ) where α chain rearranges, cells may begin to express AID. Non-productive rearrangements can be rescued from apoptosis by receptor editing or by receptor salvaging, in which AID catalyzes SHM to produce cells with improved affinity to MHC:Ag complexes (to pass positive selection). Salvaged thymocytes then proliferate and express either CD4 or CD8 on their surface as single-positive (SP) cells. AID-mediated receptor salvaging may also reduce recognition of self-peptide, rescuing self-reactive thymocytes from apoptosis (to pass negative selection). [sc: subcapsular region; cmj: corticomedullary junction; green shading indicates region of AID expression].

https://doi.org/10.7554/eLife.28477.022
Observed TcR Alpha/Delta germline Vs exhibit high sequence identity.

Nucleotide (A) and amino acid (B) alignments of 17 germline variable (V) region gene segments. Two V groups contained three identical germline gene segments each (highlighted in gray), leaving only 13 unique V gene segments. Boxes surround conserved amino acids. Numbers at the ends of sequences indicate percent identity to the first germline sequence (α/δ V5) within the alignment.

https://doi.org/10.7554/eLife.28477.024
Observed germline sequences align only to TcR αV4 clones.

Nucleotide alignments of TcR αV4 thymocyte clones to known germline V segments. Highlighted and underlined bases denote nonsynonymous and synonymous differences (respectively) to the germline V segment. Boxed regions represent nucleotides of the third complementarity-determining region (CDR3) according to IMGT guidelines, accounting for differences between clone and germline sequences. We highlight the single nucleotide/amino acid change between α/δ V7 and α/δ V10 germline segments in red.

https://doi.org/10.7554/eLife.28477.025

Tables

Table 1
Summary of sequence data used in this paper.

Putative subfamilies within each TcR alpha V family share at least 85% nucleotide identity using nearest-neighbor consensus trees of V segments. Number of TcR alpha nucleotide (NUC), amino acid (AA) sequences or sequence groups within each category. Highlighted columns specifically refer to data used in this study. (See results for detailed descriptions of sequences included within each column.)

https://doi.org/10.7554/eLife.28477.007
Alpha V segmentPutative # Sub-familiesAll cloned sequencesComplete CDR3-J junction*Unique V Region Unique V segmentUnique CDR3-J§Groups with identical CDR3-J#CDR3-J groups in Study**Sequences in each dataset††
NUCAANUCAANUCAA
TRA V114040353424163432742, 3, 3, 5
TRA V231818131313101212315
TRA V33217194555235345150514
TRA V436028222221212121622, 2
TRA V5435341513981313312
TRA V6299775677100
TRA V759660494839384848922, 2
TRA V931919141412111313400
TRA V10245452926211927261024, 9
539447239229179163226224481345
  1. *A full list of these sequences can be found in Table 1—source data 1.

    V Region includes all bases between the 1 st predicted nucleotide of the V segment to the last predicted nucleotide of the J segment (V and J).

  2. ‡V Segment includes all bases between the 1 st predicted nucleotide of the V segment to the last predicted nucleotide of the V segment (V only).

    §CDR3-J includes all bases after the last predicted nucleotide of the V segment to the last predicted nucleotide of the J segment.

  3. #Number of groups with identical CDR3-J sequences, which we used to determine sequence relatedness (see text for details).

    **Number of groups with identical CDR3-J sequences used in this study. (Those not used contained no mutation with V segments.)

  4. ††Total number of sequences for each alpha V used to assess somatic hypermutation within this study (e.g., for aV1, 4 different clonal groups contained 2, 3, 3, and 5 identical CDR3-J regions, respectively).

Table 1–source data 1

Tab-delimited text file containing sequences used in this paper for analysis of somatic hypermutation of TcR alpha chain.

https://doi.org/10.7554/eLife.28477.008
Table 2
Frequencies of somatic hypermutation in nurse shark alpha V groups (αV G) containing the same CDR3.

Mutation frequency is the total number of nucleotide changes to a Geneious-derived consensus sequence divided by the total number of nucleotides. We counted synonymous (S) and nonsynymous (N) mutations separately for each FR and CDR for 11 different CDR3 groups in seven predicted alpha V genes. [FR: framework region; CDR: complementarity-determining region; Seqs: sequences; Nuc: nucleotides; Freq: frequency]

https://doi.org/10.7554/eLife.28477.010
FR1FR2FR3FR Means
αV Group# Seqs# CodonsTotalMutationsTotalMutation# CodonsTotalMutationsTotalMutation# CodonsTotalMutationsTotalMutation
NucSNMutationFreq (%)NucSNMutationFreq (%)NucSNMutationFreq (%)SNALL
αV1.1325225212146.222171530221.307413694482.16861824
αV1.2525375108184.800172551893.5294161555101.626162137
αV1.43252251010.444171531342.614413694592.4396814
αV2.15253750000.000172551010.392426300000.000101
αV3.14253000441.333172040000.000404800110.208055
αV4.12251501232.000171020221.96143258117186.977121123
αV4.22251500000.000171021121.961432582793.4883811
αV5.12251500664.000171022354.902422521231.19031114
αV7.222515047117.333171021343.922412461672.84661622
αV10.14253000000.000172041120.980414924040.813516
αV10.2925675211131.926174594481.743401080311141.29692635
Sum4127530752050701872091122739455504935488367125192
Mean Mutation Freq (%)0.651.632.280.571.291.870.690.951.640.661.221.88
Standard Deviation2.9944.5476.5311.1362.2522.8413.1253.4145.4294.7428.20111.861
CDR1CDR2CDR3CDR Means
AlphaV Group# Seqs# CodonsTotalMutationsTotalMutation# CodonsTotalMutationsTotalMutation# CodonsTotalMutationsTotalMutation
NucSNMutationFreq (%)NucSNMutationFreq (%)NucSNMutationFreq (%)SNALL
αV1.138722245.5565451012.22210901677.7784812
αV1.2581205497.5005751234.00081201454.16771017
αV1.438720111.3895450000.0005450224.444033
αV2.1571050110.9525750000.0003450000.000011
αV3.146720334.1676720000.0001120000.000033
αV4.127423149.5246360112.7786362025.556527
αV4.227421124.7626360000.0006360000.000112
αV5.127420337.14363607719.4447420000.00001010
αV7.225300000.0007421124.7626360225.556134
αV10.147843033.5716720000.0007840333.571336
αV10.2971891894.76261622242.46971893031.58761016
Sum417787015243963696513186673571724275481
Mean Mutation Freq (%)1.722.764.480.721.872.590.952.313.271.172.353.52
Standard Deviation1.6902.3162.9790.6882.0892.2481.0272.0672.2722.6593.7545.626
Table 3
Number and frequency of DNA mutations that occur in tandem within framework regions (FR) and complementarity determining regions (CDR) in nurse shark alpha V (αV) groups.

All mutations include both tandem and point mutations within a region. [Seqs: sequences]

https://doi.org/10.7554/eLife.28477.011
FRCDR
# Nucleotides Tandemly MutatedAll mutationFrequency of tandem mutation# Nucleotides Tandemly MutatedAll mutationFrequency of tandem mutation
αV Group# Seqs234Sum234Sum
αV1.1310162425.030061250.0
αV1.25410113729.7510131776.5
αV1.4330061442.9000030.0
αV25000010.0000010.0
αV342004580.01002366.7
αV4.12410112347.80103742.9
αV4.2211051145.501032150.0
αV5210021414.311051050.0
αV7.22410112250.01002450.0
αV10.1430066100.0000060.0
αV10.29410113531.421071643.8
Total4127517319238.01350418150.6
Table 4
Target nucleotide mutation frequency in DGYW/WRCH or WA/TW mutation hotspots within framework regions (FR) and complementarity determining regions (CDR).

[DGYW/WRCH (G:C is the mutable position; D = A/G/T, Y = C/T, W = A/T, R = A/G, and H = T/C/A); WA/TW (A:T is the mutable position; W = A/T); ‘ALL’ refers to nucleotides within a hotspot motif; ‘All other’ refers to nucleotides outside a hotspot motif]

https://doi.org/10.7554/eLife.28477.015
Mutated baseHotspot motifRegionTotal nucleotidesObserved # MutationsExpected # MutationsMutation freq (%)χ2 pT-test P
G/CDGYW/WRCHFR9275618.166.040.00000.0267
CDR4003526.228.75
ALL13279137.526.860.0000
Outside Motif440271124.481.61
A/TWA/TWFR25315436.072.130.00150.2248
CDR4002621.124.03
ALL29318055.972.520.0000
Outside Motif36904165.031.11
  1. *T-test analysis was used to compare mutations within hotspot motifs to those outside hotspot motifs. Mutations to G and C nucleotides occurred significantly more often within DGYW/WRCH motifs than outside these motifs, while mutations to A and T nucleotides showed no preference for WA/TW motifs.

    †χ2 analysis was used to compare observed and expected numbers of mutations between FR and CDR regions and between mutations inside and outside hotspot motifs. More mutations to all nucleotides occurred within hotspots than outside hotspots, and significantly more mutations occurred to nucleotides within CDRs than FRs.

Table 5
Bias in base substitution during somatic hypermutation of TcR alpha V genes within all sequence regions (ALL), framework regions (FR), or complementarity determining regions (CDR).

Probability of occurrence is the proportion of that base out of the total nucleotides. [Nuc: nucleotides; OBS: Observed; EXP: expected; MI: mutability index; ChiSq: Chi squared]

https://doi.org/10.7554/eLife.28477.016
aALLBaseOccurrenceProbability of occurrenceOBSEXPMI*ChiSq
G28950.2307765.051.180.0022
C28340.2258563.681.33
A34980.2786478.600.810.0068
T33680.2675775.680.75
Total125951.00283283
GC Mutation: 57.0%; Transitions: 42.8%; Transversions: 25.1%
bFRBaseOccurrenceProbability of OccurrenceOBSEXPMI*ChiSq
G23780.235844.501.300.0037
C22680.225642.441.32
A27790.273852.000.730.0082
T28350.284053.050.75
Total102601.00192192
GC Mutation: 59.0%; Transitions: 41.7%; Transversions: 24.0%
cCDRBaseOccurrenceProbability of OccurrenceOBSEXPMIChiSq
G5170.221920.150.940.1336
C5660.242922.061.31
A7190.312628.020.930.3620
T5330.231720.770.82
Total23351.009191
GC Mutation: 52.9%; Transitions: 45.1%; Transversions: 27.5%
  1. *Mutability Index, as first defined in Chen et al., 2012. χ2 analysis was used to compare observed and expected numbers of mutations. G and C mutated significantly more often than expected, while A and T mutated significantly less often than expected. Base composition: 23.0% G, 22.5% C 27.8% A, 26.7% T.

Table 6
Frequencies of somatic hypermutation in nurse shark thymus and peripheral lymphoid tissue (blood and spiral valve).

Mutations were analyzed only in alpha V groups containing the same third complementarity-determining region (CDR). Mutation frequency was measured as the total number of nucleotide changes to a Geneious-derived consensus sequence divided by the total number of nucleotides in all sequences. Nonsynymous (N) and synonymous (S) mutations (mut) were counted separately for each framework (FR) and CDR for two predicted alpha V genes. [FR1, FR2, FR3, CDR1, CDR2, and CDR3 refer to the first, second, or third FR or CDR region, respectively.]

https://doi.org/10.7554/eLife.28477.017
Tissue typeMut typeFR Mutations (#)CDR Mutations (#)FR mutation frequencyCDR mutation frequency
 FR1FR2FR3All FRCDR1CDR2CDR3All CDRFR1FR2FR3CDR1CDR2CDR3
Thymus
 (6 sequences)
N8872313370.5700.8710.3170.0710.3270.136
S8261610010.5700.2180.2720.0710.0000.000
ALL1610133923381.1401.0890.5900.5291.0751.235
Total Nucleotides140491822054527378279243900
Periphery
 (2 sequences)
N7461702241.4961.3070.8330.0001.8521.587
S401500000.8550.0000.1390.0000.0000.000
ALL11472202242.3501.3070.9720.0001.8521.587
Total Nucleotides4683067201494126108126360
Table 7
List of forward (F) and reverse (R) primers used to generate T cell receptor (TcR) sequences and expression data.

[AID: Activation induced cytidine deaminase; B2M: beta-2 microglobulin; α: alpha; β: beta; γ: gamma; δ: delta; V: variable region; C: constant region].

https://doi.org/10.7554/eLife.28477.023
PrimerF/RIDLocationNucleotide sequence (5' to 3')Amino acidTm
TcR αV1FMFC370leader region of αV1ATG TTG CCT GAA GCT CMLPEA55
RMFC191alpha C regionCAT TGG TGG ATA GCA AGC CCT TCG ATSKGLLSTN76
TcR αV4FMFC122beginning of αV4GTC TCC TCA GTT GTT CGT ACVSSVVR58
RMFC123end of αV4CAG TAA TAC ACA GCA GCG TCDAAVYY58
FMFC374leader region of αV4TGG ATT GTG TGG GCA GTAWIVWAV54
RMFC191alpha C regionCAT TGG TGG ATA GCA AGC CCT TCG ATSKGLLSTN76
TcR αV5FMFC124beginning of αV5CTC AGG AAG GAG AGA TTA TCA CQEGEII60
RMFC125end of αV5CAA TGA TAC ACG GCG GAG TCDSAVYH60
FMFC124beginning of αV5CTC AGG AAG GAG AGA TTA TCA CQEGEII60
RMFC191alpha C regionCAT TGG TGG ATA GCA AGC CCT TCG ATSKGLLSTN76
TcR αV7FMFC376end of leader αV7AGC GAT GGA GTT TCT GTG ATTSDGVSVI58
RMFC191alpha C regionCAT TGG TGG ATA GCA AGC CCT TCG ATSKGLLSTN76
TcR αV10FMFC378leader region of αV10CTA TTT CTT CAC TAC CGC AGYFFTTA56
RMFC191alpha C regionCAT TGG TGG ATA GCA AGC CCT TCG ATSKGLLSTN76
TcR α 5'FGeneRacer 5' Nestedhomologous to RNA oligoGGA CAC TGA CAT GGA CTG AAG GAG TA--78
TcR α 3'RMFC191alpha C regionCAT TGG TGG ATA GCA AGC CCT TCG ATSKGLLSTN76
TcR βV1FMFC126beginning of bV1CTC CGT ACA TCG TCT CTA TTGPYIVSI60
RMFC127end of βV1CAC GCA CAG AAA TAG ACA GCAVYFCA58
TcR βV2FMFC128beginning of βV2CTA CGT GGA GCA GTC TCC ATCYVEQSP63
RMFC129end of βV2GCA CGC ACA ATA ATA GAC AGC CAVYYCAC62
TcR βV3FMFC130beginning of βV3CTA CGT GGA ACA GTC TCC TTCYVEQSP61
RMFC131end of βV3CAC GCG CAG AAA TAG ACA GVYFCA57
TcR βV5FMFCb50beginning of βV5GTT CGG TGC TCT TTC TCT GCMFGALSLH60
RMFCb54end of βV5GAC TGC AGT ATC AGT CGG CAC CLVPTDTAV66
TcR γV1FMFCg56beginning of γV1GTC GCT GTA TTA CTG GCT CAT TGMSLYYWL63
RMFCg59end of γV1GAG CGC ACA GTA ATA GGT GGC AGTATYYCAL67
TcR γV3FMFCg58beginning of γV3GAA GGG TCA CGT CCT TGC GMKGHVLA62
RMFCg61end of γV3GAT CCC AGA GTC ATC CTCEDDSGI56
FMFC170beginning of γV3CAA TAA CCA GAG CAC CGG GITRAP56
RMFC171end of γV3AGA TCC CAG AGT CGT CCT CEDDSGI56
TcR δV3FMFCd62beginning of δV3GAT TCC CCG TCC CTG GTG TCDSPSLVS65
RMFCd66end of δV3CAG TGC ACA GTG ATA CAC AGCAVYHCAL61
TcR δV5FMFCd63beginning of δV5GCA GCT ACT CAG TAT CTG GMQLLSIW57
RMFCd67end of δV5GAA AGC ACA GTA ATA CAG AGALYYCAF54
TcR δV7FMFC172beginning of δV7CTG TCA CTC AGT TAT TCT CCT CVTQLFS60
RMFC173end of δV7GCA GCC CAG TTA TAG TCA AACLTITGL60
TcR δV12FMFC174beginning of δV12CAG AGC CCA CCT CAG TTA CQSPPQ;60
RMFC175end of δV12GAG CGC AGT AAT AGA TGG CAIYYCA57
TcR δV16RMFC176end of δV16GCA GCT CCG AGA TAG ACA ACLSISEL60
FMFC177beginning of δV16GAG TCC TGG CTC ACG CAA TCESWLTQ63
TcR δV17FMFC178beginning of δV17CAG TCT TGG TCA GAA ATA ACCQSWSEIT57
RMFC179end of δV17CAA CTG AAG ATA AGT GAT CGITYLQL54
AIDFMFC342beginning of AID exon 1AGG CAC GAG ACC TAC ATG TTGRHETYML61
RMFC347end of AID exon 2TGA ACC AGG TGA GGC GGT AYRLTWF60
B2MFMFC211first cysteineAAC GTG TTG CTC TGT CAT GCNVLLCHA58
RMFC212before second cysteineGGG GTG AAC TCC ACA TAA CGRYVEFTP60

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