Comparison of the immunoglobulin heavy‐chain complementarity determining region‐3 structure among the DNA sequences and the μ‐ and γ‐transcripts in human B‐lineage cells

Abstract

SUMMARYTo study the recombinational significance of the (immunoglobulin heavy) IgH chain gene in human Bcell development, we compared the complementarity determining region (CDR)-3 sequences of the DNA and the m -transcripts from human normal pre-B cells and mature B cells, and the g -transcripts from bone marrow cells. The CDR-3 sequences were longer in the DNA than in the m -and g -transcripts, and this was independent of whether or not the rearrangement was productive. The DLR family genes were less frequently used in the m -and g -transcripts. When translated into amino acids, all CDR-3 sequences from the m -and g -transcripts were productive, although 26 . 2% of the DNA sequences had stop codons in the D element and/or frameshifts of the JH gene segments. The CDR-3 of the productive DNA sequences in pre-B cells frequently (26 . 6%) contained at least three continuous hydrophobic amino acids, which were mainly coded by the DLR and DXP family genes at the third reading frame. However, such motifs were rare in the m -transcripts of pre-B (7 . 7%) and mature B cells (3 . 9%), and in the g -transcripts of bone marrow cells (1 . 1%) as well as in the DNA of mature B cells (10 . 4%). These findings suggested that the length and/or hydrophobicity of the IgH CDR-3 might play a role in the selection mechanisms of B-cell development.

Utilization of Ig heavy chain variable, diversity, and joining gene segments in children with B-lineage acute lymphoblastic leukemia: implications for the mechanisms of VDJ recombination and for pathogenesis

“…Similar to the findings previously reported for immature B cells, 66 and in keeping with a previous study in ALL, 33 only 21% of V H -J H rearrangements detected were potentially productive. Unlike normal B cells, this does not lead to loss of leukemic clone, demonstrating that ALL does not require signals mediated by Ig signaling for survival.…”

B‐cell precursors differentiated from cord blood CD34+ cells are more immature than those derived from granulocyte colony‐stimulating factor‐mobilized peripheral blood CD34+ cells

“…High molecular weight DNA was extracted from cultured cells by a standard method. The IgH CDR‐3 sequences were amplified by polymerase chain reaction (PCR) using a set of primers corresponding to the consensus sequences of the 3′ ends of the V H and J H genes (consensus V H and consensus J H , Table 1) as previously reported 23,26 . Amplified products were separated on 8% polyacrylamide gels, and fragments spanning the 50–250 bp region were isolated.…”

“…The IgH CDR-3 sequences were ampli®ed by polymerase chain reaction (PCR) using a set of primers corresponding to the consensus sequences of the 3k ends of the V H and J H genes (consensus V H and consensus J H , Table 1) as previously reported. 23,26 Ampli®ed products were separated on 8% polyacrylamide gels, and fragments spanning the 50±250 bp region were isolated. The fragments were cloned into pT7Blue T-vector (Novagen, Madison, WI), then transfected into Escherichia coli strain DH5a.…”

“…Late pre‐B cells have rearranged IgL genes, and production of IgL causes the expression of the surface µ‐chain (sµ), which is characteristic of B cells 15 . The development of rapid cloning and sequencing techniques has resulted in a substantial accumulation of immunoglobulin variable region gene sequences at various stages of B‐cell development, and has revealed stage‐specific trends in the usage of V, D and J genes, the degree of N nucleotide addition, and the rate of somatic mutations 16–27 …”

Expressed Murine and Human CDR-H3 Intervals of Equal Length Exhibit Distinct Repertoires that Differ in their Amino Acid Composition and Predicted Range of Structures

“…Sequences obtained from genomic DNA were excluded because they are less likely to be expressed as a protein than cDNA/ RNA-derived sequences. 73 The selected sequences from both databases were joined into one table with human sequences and one table with murine sequences. The sequences were sorted alphabetically to identify and remove duplicate sequences, which could originate from submission of one sequence to both databases, or from repeated submissions to the same database.…”