Proc. Natl. Acad. Sci. USA Vol. 88, pp. 7699-7703, September 1991 Immunology Chicken T-cell receptor a8-chain diversity: An evolutionarily conserved D encoded glycine turn within the hypervariabfe CDR3 domain (N region/junctional diversity/immunologic repertoire/diversity gene segment/complementarity-determining region) WAYNE T. MCCORMACK, LARRY W. TJOELKER, GREGORY STELLA, CHRISTINA E. POSTEMA, AND CRAIG B. THOMPSON* Howard Hughes Medical Institute, Departments of Internal Medicine and Microbiology/Immunology, University of Michigan Medical School, Ann Arbor, MI 48109 Communicated by J. Herbert Taylor, June 10, 1991 ABSTRACT Unlike mammals, chickens generate an im- species (11). The germ-line repertoire of chicken Vat genes munoglobulin (Ig) repertoire by a developmentally regulated consists of only two Va families, Vat and V,2 (12, 13), which process of intrachromosomal gene conversion, which results in bear structural similarities to the mammalian VpI and VP1I nucleotide substitutions throughout the variable regions of the subgroups (14). The chicken Van family consists of six nearly Ig heavy- and light-chain genes. In contrast to chicken Ig genes, identical Vp genes (average amino acid identity, 97%), and the we show in this report that diversity of the rearranged chicken V2 family consists ofthree to five genes (average amino acid T-cell receptor (TCR) a-chain gene is generated by junctional identity, 95%), depending on the chicken strain examined. heterogeneity, as observed in rearranged mammalian TCR Unlike chicken Ig V segments, all chicken Va segments that genes. This junctional diversity increases during chicken de- have been sequenced contain heptamer and nonamer ele- velopment as a result of an increasing base-pair addition at the ments. Multiple distinct rearrangements of the TCR,8 locus Vp-Dp and Dp-Jp joints (where V, D, and J are the variable, are detected when Southern blots of chicken thymic and diversity, and joining gene segments). Despite the junctional T-cell-line DNA are probed with Va1 and V2 probes (11-13), hypervariability, however, almost all functional Vp-Dp-Jp suggesting that most or all chicken Va genes are functional. junctions appear to encode a glycine-containing #-turn. Such Comparison of cDNA sequences revealed three unique Jo a turn may serve to position the amino acid side chains of a sequences (11), and genomic screening with an oligonucleo- hypervariable TCR a-chain loop with respect to the antigen- tide probe identified a fourth Jo gene segment (13). Sequence binding groove of the major histocompatibility complex mol- alignments of TCR3 cDNA and genomic clones revealed ecule. Consistent with this hypothesis, the germ-line Dp nucle- 12-25 nucleotides at the V9-Je junction that were not en- otide sequences of chickens, mice, rabbits, and humans have coded by the germ-line Va and Jp segments, and suggested the been highly conserved and encode a glycine in all three reading presence of Do segments (11). frames. We have examined the molecular mechanisms for the generation of diversity in the chicken TCRE locus by iden- Immunoglobulins (Ig) and T-cell receptors (TCR) are the tifying the germ-line chicken Do gene segment and by ana- antigen-recognition molecules on the surfaces of B and T lyzing rearranged chicken TCRJ3 genes isolated from the cells, respectively (1-3). Whereas B cells recognize soluble developing thymus and mature spleen.t We observe that antigens, T cells recognize antigenic peptides associated with chicken TCR,8 diversity is generated by junctional variabil- cell surface molecules encoded by the major histocompati- ity, rather than by gene conversion, as in chicken Ig genes. bility complex (MHC) (4, 5). Genetic analyses of the mam- Further, the junctional diversity increases during develop- malian Ig and TCR gene families have shown that somatic ment due to increased random base-pair (N-nucleotide) ad- diversity is generated by junctional and combinatorial diver- dition. Within the junctional variability, however, most re- sity during the assembly offunctional Ig and TCR genes from arranged chicken TCR,8 genes have at least one glycine variable (V), diversity (D, for Ig heavy, TCRf3, and TCRS residue, which is encoded in the germ-line by an evolution- chains), and joining (J) gene segments (1-3). In contrast, arily conserved Do segment. The implications of a conserved birds use distinct molecular mechanisms to generate somatic glycine for TCR structure and antigen recognition are dis- diversity in their Ig loci. Instead of using junctional and cussed. combinatorial variation to create somatic Ig gene diversity, chickens rearrange only single V and J segments for both the heavy (H)- and the light (L)-chain Ig lpci (6, 7). After MATERIALS AND METHODS rearrangement, the VH and VL segments undergo sequence Isolation of Rearranged Chicken TCRE Genes. Polymerase diversification by intrachromosomal gene conversion using chain reaction (PCR; ref. 15) amplification, cloning, and families of V pseudogene segments as sequence donors sequencing of rearranged VP1.1-Dp-JP3 genes from 4-week (6-10). Given these differences between mammalian and thymus DNA were performed as described (16). Thirty cycles avian Ig gene diversification, it was of interest to determine of amplification (1.5 min at 940C, 3 min at 720C) were the molecular mechanisms for the diversification of chicken performed in a Coy thermal cycler. PCR primers included a TCRf3 genes. The recent description of chicken TCR8 cDNA and ge- Abbreviations: CDR, complementarity-determining region; MHC, nomic sequences revealed evolutionarily conserved struc- major histocompatibility complex; TCR, T-cell antigen receptor; tural features of the TCR,8 chains in avian and mammalian TdT, terminal deoxynucleotidyltransferase. *To whom reprint requests should be addressed at: Howard Hughes Medical Institute, 1150 West Medical Center Drive, MSRB-I, Room The publication costs of this article were defrayed in part by page charge 3510, Ann Arbor, MI 48109. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M73064). 7699 Downloaded by guest on September 28, 2021 7700 Immunology: McCormack et al. Proc. Natl. Acad Sci. USA 88 (1991) sense primer at the 5' end of V91.1 (5'-CCCCGTCGACGT- probe spanning this core sequence hybridized to a 1.8-kb TCAGACAAAGAGAGTGTAATCCA-3') and an antisense HindIII fragment isolated from a genomic clone encompass- primer in the 3' flanking region of J3 (5'-GGAAGQCGjjC- ing the 20-kb region 5' of the J4 cluster. Nucleotide sequenc- CGAGGTGAGGGAGATGACAAGCACAGAGCAGG- ing of this genomic fragment revealed a single germ-line Do 3'). Sal I and Not I restriction sites (underlined) at the 5' ends segment (Fig. 1B). Rearrangement of only one Dp segment of the primers facilitated cloning into the pBluescript SK(-) with the four J4 segments was detected when Southern blots plasmid vector (Stratagene). ofthymic DNA were hybridized with probes located 5' ofD's Identification of the Chicken Germ-Line Dp Gene Segment. (data not shown). Comparison ofthe chicken, mouse (21, 22), An oligonucleotide probe (5'-GGGACAGGGGGATC-3') rabbit (20), and human (18, 19) germ-line Do segments spanning the putative chicken Do gene segment (11) was revealed 100% nucleotide sequence identity for the first 11 bp end-labeled with [y-32P]ATP by T4 polynucleotide kinase ofchicken Dp and the human and mouse D,91.1 gene segments, (17). Southern blots containing restriction digests of cloned one nucleotide substitution in mouse Dp2.1 and rabbit D>, genomic fragments ofthe germ-line chicken TCRF3 locus (11) and a 3-bp insertion in human D2.1 (Fig. 18). There is some were prehybridized in lOx Denhardt's solution/0.9 M variation in the sequence and length at the 3' ends of the NaCl/90 mM Tris-HCl, pH 8/0.6 mM EDTA/0.1% SDS at germ-line D,9 segments. Except for the conserved heptamer 420C for several hours. Blots were hybridized at 420C over- and nonamer elements ofthe recombination signal sequence, night in the same solution after addition of labeled probe (106 there is no significant sequence homology in the 5' or 3' dpm/ml). Blots were washed twice at room temperature and flanking regions ofthe genomic D,9 gene ofchicken compared twice at 420C in 0.9 M NaCl/0.09 M trisodium citrate, pH with mouse, rabbit, and human (Fig. 1B). 7/0.1% SDS. A 1.8-kilobase (kb) HindIII genomic fragment Non-Germ-Line-Encoded Nuceotide Additons to the Vp positive for hybridization with the Dp oligonucleotide probe and Dp-Jp Juncos. Alignment of the germ-line Dp segment was subcloned into pGEM-7Zf(+) (Promega) and sequenced with our initial rearranged TCRJ3 clones (Fig. UA) revealed a using primers specific for the SP6 and T7 sites of the vector number of non-germ-line base pairs at the Vp-Ds and Ds-J and internal sequences. junctions, suggesting that N-nucleotide addition may account for a large part of the TCR,3 junctional diversity. N nucleo- tides are template-independent G+C-rich polynucleotide ad- RESULTS ditions to V(D)J joints of mammalian Ig and TCR genes, Chicken TCRN Diversity Is Focused at the Vp-j Junction. believed to be mediated by terminal deoxynucleotidyltrans- To assess the diversity ofthe chicken TCRj3 repertoire, gene ferase (TdT) (23-25). This is in contrast to rearranged chicken rearrangements involving the V91.1 and 4J,3 genes were cloned Ig genes, which lack N nucleotides at V(D)Jjoints (6-10). To from the thymus of a 4-week-old chicken after PCR ampli- assess the contribution of N nucleotides to TCR8 diversity, fication (15, 16). Comparison ofthese clones to the germ-line additional rearranged Vpl.1-DP-J3 clones were isolated from Vp and J4 sequences reveals no sequence variation within the thymocyte DNA prepared from 18-day embryos (early) and Vp or J4 regions, but extensive variation is observed at the from a chicken 4 weeks after hatching (late).