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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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Polyacrylamide gel assay to identify insertions or deletions  into VH genes. (A) Phosphorimage of a polyacrylamide gel: each lane contains the hot-PCR products (32P-labeled) of the VH gene and the CDR3  of an individual clone. (B) A comparison of the distribution of CDR3  sizes of the 485 CDR3s assayed to the distribution of 500 CDR3s observed in sequences from this report indicates that the clones assayed by  electrophoresis were a polyclonal population. CDR3 sizes were measured  from the most 3′ Tyr residue (common to all V genes analyzed) to the  most 5′ Cμ or Cγ residue. CDR3 lengths for those assayed by electrophoresis were extrapolated based on sequencing of 75 out of the 485  clones assayed. The x-axis is the number of amino acids greater than the  shortest CDR3 found.
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Figure 3: Polyacrylamide gel assay to identify insertions or deletions into VH genes. (A) Phosphorimage of a polyacrylamide gel: each lane contains the hot-PCR products (32P-labeled) of the VH gene and the CDR3 of an individual clone. (B) A comparison of the distribution of CDR3 sizes of the 485 CDR3s assayed to the distribution of 500 CDR3s observed in sequences from this report indicates that the clones assayed by electrophoresis were a polyclonal population. CDR3 sizes were measured from the most 3′ Tyr residue (common to all V genes analyzed) to the most 5′ Cμ or Cγ residue. CDR3 lengths for those assayed by electrophoresis were extrapolated based on sequencing of 75 out of the 485 clones assayed. The x-axis is the number of amino acids greater than the shortest CDR3 found.

Mentions: To determine whether or not these insertion/deletion events were associated with somatic hypermutation, we analyzed their occurrence in unmutated FM transcripts. This was done using either direct sequencing or PCR amplification of portions of the VH genes spanning the CDRs, followed by size comparisons on polyacrylamide gels (Fig. 3). Any clones that ran aberrantly, and the clones in adjacent lanes, were sequenced (75 out of the 485 clones). None of these 75 clones were related based on CDR3 homology. To ensure that the remaining 410 FM clones were polyclonal, the CDR3s were PCR amplified and loaded on the sequencing gels simultaneously to the VH gene amplification products for size comparisons (Fig. 3 A). The size distribution of these CDR3s was similar to that of ∼500 VH gene sequences analyzed in this study (Fig. 3 B), providing evidence that our FM sample is polyclonal.


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)

Polyacrylamide gel assay to identify insertions or deletions  into VH genes. (A) Phosphorimage of a polyacrylamide gel: each lane contains the hot-PCR products (32P-labeled) of the VH gene and the CDR3  of an individual clone. (B) A comparison of the distribution of CDR3  sizes of the 485 CDR3s assayed to the distribution of 500 CDR3s observed in sequences from this report indicates that the clones assayed by  electrophoresis were a polyclonal population. CDR3 sizes were measured  from the most 3′ Tyr residue (common to all V genes analyzed) to the  most 5′ Cμ or Cγ residue. CDR3 lengths for those assayed by electrophoresis were extrapolated based on sequencing of 75 out of the 485  clones assayed. The x-axis is the number of amino acids greater than the  shortest CDR3 found.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Polyacrylamide gel assay to identify insertions or deletions into VH genes. (A) Phosphorimage of a polyacrylamide gel: each lane contains the hot-PCR products (32P-labeled) of the VH gene and the CDR3 of an individual clone. (B) A comparison of the distribution of CDR3 sizes of the 485 CDR3s assayed to the distribution of 500 CDR3s observed in sequences from this report indicates that the clones assayed by electrophoresis were a polyclonal population. CDR3 sizes were measured from the most 3′ Tyr residue (common to all V genes analyzed) to the most 5′ Cμ or Cγ residue. CDR3 lengths for those assayed by electrophoresis were extrapolated based on sequencing of 75 out of the 485 clones assayed. The x-axis is the number of amino acids greater than the shortest CDR3 found.
Mentions: To determine whether or not these insertion/deletion events were associated with somatic hypermutation, we analyzed their occurrence in unmutated FM transcripts. This was done using either direct sequencing or PCR amplification of portions of the VH genes spanning the CDRs, followed by size comparisons on polyacrylamide gels (Fig. 3). Any clones that ran aberrantly, and the clones in adjacent lanes, were sequenced (75 out of the 485 clones). None of these 75 clones were related based on CDR3 homology. To ensure that the remaining 410 FM clones were polyclonal, the CDR3s were PCR amplified and loaded on the sequencing gels simultaneously to the VH gene amplification products for size comparisons (Fig. 3 A). The size distribution of these CDR3s was similar to that of ∼500 VH gene sequences analyzed in this study (Fig. 3 B), providing evidence that our FM sample is polyclonal.

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