The prevalence in fraction B progenitor B cells of inverted sequences was equivalent to fraction F mature, recirculating B cells (4 out of 71 vs. Among the 326 iD-containing CDR-H3 sequences, four contained iD sequences in i-RF1 and seven used i-RF2 (Tables S1 and S2). Although D H inversions were more frequent in ΔD-iD B cells than in the wild-type or depleted D H locus with single DFL16.1 gene segment ( ΔD-DFL) controls, their prevalence did not increase with development even though iD i-RF1 recapitulates the normally preferred tyrosine-enriched sequence of DSP2.2 RF1. In fraction B CD19 +CD43 +BP-1 −IgM − progenitor B cells, 74% of the sequences used RF1 and, in fraction F CD19 +CD43 −IgM +IgD + mature, recirculating B cells, 80% used the mutant RF1 (Fig. We found no evidence of selection during development for use of D H RFs that lacked arginine, histidine, and asparagine. Amino acid utilization in ΔD-iD/ΔD-iD mature B cells reflects dominant use of iD RF1 We used gene targeting via homologous recombination in a BALB/c embryonic stem cell line ( 12) to create an IgH allele (depleted D H locus with a single, mutated DFL16.1 gene segment containing inverted DSP 2.2 sequence ) limited to this single, modified D H. iD RF2 and RF3 by deletion and RF1 and RF3 generated by inversion (i-RF2 and i-RF3) maintain a preference for hydrophobic amino acids, and RF3 and i-RF3 continue to incorporate termination codons. iD RF1 generated by inversion (i-RF1) encompasses the original tyrosine enriched sequence of DSP2.2 RF1. DFL16.1 sequence encoding tyrosine and serine at the 5′ and 3′ termini of the D H gene segment was retained to preserve microhomology between the 3′ end of the D H and the 5′ end of J H ( 9, 10). We termed this hybrid DFL16.1-inverted DSP2.2 D element iD for inverted D. This allowed us to replace central RF1 codons for tyrosine and glycine with those for arginine, histidine, and asparagine. We replaced the central portion of DFL16.1, the most V H-proximal D H ( 11), with the complete inverted coding sequence of DSP2.2 ( Fig.
![charged amino acids charged amino acids](https://image.slidesharecdn.com/classif-191003202156/95/classif-amino-acids-proteins-5-638.jpg)
The extensive range of diversity available to CDR-H3 has functional consequences because its location at the center of the antigen-binding site, as classically defined, permits this interval to often play a significant role in antigen recognition and binding ( 4– 6). Together, these mechanisms create a CDR-H3 repertoire that ranges from unmodified and intact germline-encoded sequence to rearrangements where extensive nibbling and N addition no longer permit identification of the original D H. The terminal deoxynucleotidyl transferase (TdT) catalyzed insertion of N nucleotides at the sites of joining permits the inclusion of nongermline sequence into CDR-H3 ( 1, 2).
![charged amino acids charged amino acids](https://openoregon.pressbooks.pub/app/uploads/sites/220/2021/02/2.3.png)
Imprecision in joining these gene segments permits exonucleolytic loss as well as palindromic (P junction) gain of terminal V H, D H, and J H sequence.
![charged amino acids charged amino acids](http://guweb2.gonzaga.edu/faculty/cronk/CHEM240/images/diprotic_system_charges.gif)
Unlike H chain complementarity determining regions 1 and 2, which are entirely encoded by the V H gene segment, CDR-H3 is created de novo by the VDJ rearrangement process ( 1– 3).