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Somatic hypermutation introduces insertions and deletions into immunoglobulin V genes.

Wilson PC, de Bouteiller O, Liu YJ, Potter K, Banchereau J, Capra JD, Pascual V - J. Exp. Med. (1998)

Bottom Line: Antigen-directed selection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response.No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic VH4 repertoire of the tonsil donor.The lack of similar instances in unmutated IgD+CD38- follicular mantle cDNA clones statistically associates these events to the somatic hypermutation process (P = 0.014).

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology Center, Department of Microbiology, University of Texas Southwestern Medical Center at Dallas, Texas 75235-9140, USA.

ABSTRACT
During a germinal center reaction, random mutations are introduced into immunoglobulin V genes to increase the affinity of antibody molecules and to further diversify the B cell repertoire. Antigen-directed selection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response. The mutations of Ig genes are typically basepair substitutions, although DNA insertions and deletions have been reported to occur at a low frequency. In this study, we describe five insertion and four deletion events in otherwise somatically mutated VH gene cDNA molecules. Two of these insertions and all four deletions were obtained through the sequencing of 395 cDNA clones (approximately 110,000 nucleotides) from CD38+IgD- germinal center, and CD38-IgD- memory B cell populations from a single human tonsil. No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic VH4 repertoire of the tonsil donor. These six insertions or deletions and three additional insertion events isolated from other sources occurred as triplets or multiples thereof, leaving the transcripts in frame. Additionally, 8 of 9 of these events occurred in the CDR1 or CDR2, following a pattern consistent with selection, and making it unlikely that these events were artifacts of the experimental system. The lack of similar instances in unmutated IgD+CD38- follicular mantle cDNA clones statistically associates these events to the somatic hypermutation process (P = 0.014). Close scrutiny of the 9 insertion/deletion events reported here, and of 25 additional insertions or deletions collected from the literature, suggest that secondary structural elements in the DNA sequences capable of producing loop intermediates may be a prerequisite in most instances. Furthermore, these events most frequently involve sequence motifs resembling known intrinsic hotspots of somatic hypermutation. These insertion/deletion events are consistent with models of somatic hypermutation involving an unstable polymerase enzyme complex lacking proofreading capabilities, and suggest a downregulation or alteration of DNA repair at the V locus during the hypermutation process.

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Predicted amino  acid sequences of nine cDNA  clones with insertions or deletions. Note that all of these clones  are extensively mutated. In all but  clone tm121, the insertions or deletions occur in CDR1 or CDR2.  (A and B) Two clones with insertions (A) and four clones with deletions (B) from a single tonsil. (C)  Three additional cDNA clones  with insertions isolated from various sources. Sequence data available from GenBank/DDBJ under  accession numbers AF013615  through AF013626.
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Figure 1: Predicted amino acid sequences of nine cDNA clones with insertions or deletions. Note that all of these clones are extensively mutated. In all but clone tm121, the insertions or deletions occur in CDR1 or CDR2. (A and B) Two clones with insertions (A) and four clones with deletions (B) from a single tonsil. (C) Three additional cDNA clones with insertions isolated from various sources. Sequence data available from GenBank/DDBJ under accession numbers AF013615 through AF013626.

Mentions: In a large scale analysis of VH genes from both the IgM and IgG compartments of B cell subpopulations separated from a single human tonsil, six clones that contained DNA insertions or deletions were isolated. These insertions and deletions were apparently selected in that they involved nucleotide triplets or multiples of nucleotide triplets, leaving the cDNAs (transcripts) in frame, and they were localized to the CDR1 and CDR2 (Fig. 1, A and B). The six clones with insertions or deletions were identified from the sequencing of 395 cDNA clones (∼110,000 nucleotides) from GC and memory B cell subpopulations, resulting in a frequency of <2% of clones analyzed (∼1 event/18,000 nucleotides). All six events were in IgG transcripts. Two events were obtained from IgD−CD38+ GC and four events from IgD−CD38− memory cell populations. None of the IgM VH cDNAs analyzed from this tonsil had insertions or deletions, although we have observed such events in IgM transcripts in the past and in subsequent analyses, as described below.


Somatic hypermutation introduces insertions and deletions into immunoglobulin V genes.

Wilson PC, de Bouteiller O, Liu YJ, Potter K, Banchereau J, Capra JD, Pascual V - J. Exp. Med. (1998)

Predicted amino  acid sequences of nine cDNA  clones with insertions or deletions. Note that all of these clones  are extensively mutated. In all but  clone tm121, the insertions or deletions occur in CDR1 or CDR2.  (A and B) Two clones with insertions (A) and four clones with deletions (B) from a single tonsil. (C)  Three additional cDNA clones  with insertions isolated from various sources. Sequence data available from GenBank/DDBJ under  accession numbers AF013615  through AF013626.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199186&req=5

Figure 1: Predicted amino acid sequences of nine cDNA clones with insertions or deletions. Note that all of these clones are extensively mutated. In all but clone tm121, the insertions or deletions occur in CDR1 or CDR2. (A and B) Two clones with insertions (A) and four clones with deletions (B) from a single tonsil. (C) Three additional cDNA clones with insertions isolated from various sources. Sequence data available from GenBank/DDBJ under accession numbers AF013615 through AF013626.
Mentions: In a large scale analysis of VH genes from both the IgM and IgG compartments of B cell subpopulations separated from a single human tonsil, six clones that contained DNA insertions or deletions were isolated. These insertions and deletions were apparently selected in that they involved nucleotide triplets or multiples of nucleotide triplets, leaving the cDNAs (transcripts) in frame, and they were localized to the CDR1 and CDR2 (Fig. 1, A and B). The six clones with insertions or deletions were identified from the sequencing of 395 cDNA clones (∼110,000 nucleotides) from GC and memory B cell subpopulations, resulting in a frequency of <2% of clones analyzed (∼1 event/18,000 nucleotides). All six events were in IgG transcripts. Two events were obtained from IgD−CD38+ GC and four events from IgD−CD38− memory cell populations. None of the IgM VH cDNAs analyzed from this tonsil had insertions or deletions, although we have observed such events in IgM transcripts in the past and in subsequent analyses, as described below.

Bottom Line: Antigen-directed selection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response.No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic VH4 repertoire of the tonsil donor.The lack of similar instances in unmutated IgD+CD38- follicular mantle cDNA clones statistically associates these events to the somatic hypermutation process (P = 0.014).

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology Center, Department of Microbiology, University of Texas Southwestern Medical Center at Dallas, Texas 75235-9140, USA.

ABSTRACT
During a germinal center reaction, random mutations are introduced into immunoglobulin V genes to increase the affinity of antibody molecules and to further diversify the B cell repertoire. Antigen-directed selection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response. The mutations of Ig genes are typically basepair substitutions, although DNA insertions and deletions have been reported to occur at a low frequency. In this study, we describe five insertion and four deletion events in otherwise somatically mutated VH gene cDNA molecules. Two of these insertions and all four deletions were obtained through the sequencing of 395 cDNA clones (approximately 110,000 nucleotides) from CD38+IgD- germinal center, and CD38-IgD- memory B cell populations from a single human tonsil. No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic VH4 repertoire of the tonsil donor. These six insertions or deletions and three additional insertion events isolated from other sources occurred as triplets or multiples thereof, leaving the transcripts in frame. Additionally, 8 of 9 of these events occurred in the CDR1 or CDR2, following a pattern consistent with selection, and making it unlikely that these events were artifacts of the experimental system. The lack of similar instances in unmutated IgD+CD38- follicular mantle cDNA clones statistically associates these events to the somatic hypermutation process (P = 0.014). Close scrutiny of the 9 insertion/deletion events reported here, and of 25 additional insertions or deletions collected from the literature, suggest that secondary structural elements in the DNA sequences capable of producing loop intermediates may be a prerequisite in most instances. Furthermore, these events most frequently involve sequence motifs resembling known intrinsic hotspots of somatic hypermutation. These insertion/deletion events are consistent with models of somatic hypermutation involving an unstable polymerase enzyme complex lacking proofreading capabilities, and suggest a downregulation or alteration of DNA repair at the V locus during the hypermutation process.

Show MeSH
Related in: MedlinePlus