Abstract
Zebrafish (Danio rerio) has become an increasingly important model for immunological study. Its immune system is remarkably similar to that of mammals and includes both the adaptive and innate branches. Zebrafish T cells express functional T cell receptors (TCR), and all four TCR loci are present within the genome. Using 5′-rapid amplification of cDNA ends, we cloned and sequenced zebrafish TCRβ transcripts. TCRβ VDJ coding joints demonstrate conservation of mechanisms used by other vertebrate species to increase junctional diversity. Using the sequences obtained, along with previously published data, we comprehensively annotated the zebrafish TCRβ locus. Overall, organization of the locus resembles that seen in mammals. There are 51 V segments, a single D segment, 27 Jβ1 segments, a single Jβ2 segment, and two constant regions. This description of the zebrafish TCRβ locus has the potential to enhance immunological research in zebrafish and further our understanding of mammalian TCR repertoire generation.
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Acknowledgments
NM was supported by National Institutes of Health (NIH) NICHD award 2 K12 HD001410 and an Alex’s Lemonade Stand Young Investigator award; JKF was supported by NIH NICHD award 5 K08 HD53350; JKF and NM were also supported by the Children’s Health Research Center at the University of Utah and by Primary Children’s Medical Center Foundation grants; LR was supported by NIH NIDDK T32 award DK007115; NST was supported by NIH NIAID award R21 AI079784 and the Huntsman Cancer Foundation; Huntsman Cancer Institute core facilities supported by grant P30 CA042014 also participated in this work.
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Supplemental Fig. 1
Alignment of the DJ coding joints from non-functional TCRβ cDNA transcripts. Dots indicate truncation with respect to the D or J segment used. N-nt denotes non-template nucleotide additions. J number (J#) refers to the gene identification nomenclature and is shown in bold where it occurs in more than one uniquely cloned VDJ rearrangement. Translation of highly conserved sequences is shown in the top line. The 5′-most region of each transcript is identical to genomic sequence upstream of the D cassette (GIF 83 KB)
Supplemental Fig. 2a
Zebrafish TCR Vβ segments. Alignments of 52 zebrafish TCR Vβ amino acid sequence are shown (a). Varying degrees of consensus are indicated (identical = 
, block of similar = 
, conservative = 
). Consensus sequence is listed at the bottom, and proposed FR1, FR2, and FR3 boundaries are marked by gray boxes. Arrows point to highly conserved anchor residues (GIF 436 KB)
Supplemental Fig. 2b
A neighbor-joining tree (b; Saitou and Nei 1987) based on a matrix of distances between all pairs of sequence was constructed with Vector NTI Advance (AlignX). Complete V segment amino acid sequences were included, and the analysis was performed using default settings (GIF 32.4 KB)
Supplemental Fig. 3
Vβ segment identity percentages. The amino acid percent identity of each Vβ pair is shown. Numbers greater than 75% are shaded (GIF 432 KB)
Supplemental Fig. S4
Zebrafish TCR Jβ segments. Alignments of 27 zebrafish TCR Jβ amino acid sequences are shown (a). Translations of Jβ cassettes are approximately 17 aa in length. Consensus regions are indicated (identical = 
, block of similar = 
, conservative = 
). A neighbor-joining tree (b; Saitou and Nei 1987) demonstrates seven putative Jβ families (GIF 135 KB)
Supplemental Fig. 5
Jβ segment identity percentages. The amino acid percent identity of each Jβ pair is shown (GIF 153 KB)
Supplemental Table 1
Primer sequences (DOC 56 kb)
Supplementary Material
TCR beta locus annotation (TXT 385 KB)
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Meeker, N.D., Smith, A.C.H., Frazer, J.K. et al. Characterization of the zebrafish T cell receptor β locus. Immunogenetics 62, 23–29 (2010). https://doi.org/10.1007/s00251-009-0407-6
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DOI: https://doi.org/10.1007/s00251-009-0407-6