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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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ELISA assays showing the expression of clone pg86  with a six amino acid insertion at  the FW1/CDR1 junction.  Clone pg86 (IgG heavy chain)  was coexpressed with the κ light  chain FS6κ in insect cells using  the baculovirus expression system. Expression of pg86 and its  ability to pair with the FS6κ light  chain was tested using capture  ELISAs. Wells were coated with  goat anti–human IgG. Supernatants containing recombinant antibodies were added in serial  twofold dilutions. Bound antibody  was detected with phosphatase-conjugated goat anti-human IgG  (A), and goat anti–human Cκ (B).
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Figure 5: ELISA assays showing the expression of clone pg86 with a six amino acid insertion at the FW1/CDR1 junction. Clone pg86 (IgG heavy chain) was coexpressed with the κ light chain FS6κ in insect cells using the baculovirus expression system. Expression of pg86 and its ability to pair with the FS6κ light chain was tested using capture ELISAs. Wells were coated with goat anti–human IgG. Supernatants containing recombinant antibodies were added in serial twofold dilutions. Bound antibody was detected with phosphatase-conjugated goat anti-human IgG (A), and goat anti–human Cκ (B).

Mentions: To facilitate the analysis of large numbers of VH gene transcripts for the presence of insertions or deletions, first strand cDNA produced as described above was PCR amplified using Expand high fidelity polymerase (Boehringer Mannheim) to reduce errors resulting from Taq polymerase alone. The products of this PCR amplification were cloned as described above and screened using 32P-labeled, gene-specific oligonucleotides (VH4-39:5′-ATTGGGAGTATCTATTATAGT-3′; L-6 as above). Positive colonies were picked and used to inoculate overnight cultures. A 1 μl aliquot from each 24-h culture was used to directly inoculate 25-μl PCR amplification mixtures in 96-well–format PCRs. The internal PCR reactions used 32P-labeled, gene-specific oligonucleotides to amplify a 230-base fragment including the VH4-39 CDR1 (L-4, as above, and VH4-39-3′: 5′-GCTCCCACTATAATAGATACT-3′) or for analysis of VH6 genes a 166-nucleotide fragment including the CDR1 and CDR2 of VH6 (VH6FW1: 5′-TGCCATCTCCGGGGACAGTGT-3′, VH6FW3: 5′-TGTGTCTGGGTTGATGGTTAT-3′). Aliquots of each clone were also used to inoculate amplifications of CDR3 regions using FW3-specific (ssFW3: 5′-CTGAA[C/G]CTGAGCTCTGTGAC[T/C]) and Cμ- or Cγ-specific oligonucleotides (CμD: 5′-GGAATTCTCACAGGAGACGA-3′, Cγ-140 as above) to analyze the diversity of the populations under study; the distribution of CDR3 size variations of several hundred VH sequences cloned in this analysis were used to produce an expected distribution of CDR3 sizes for comparison (see Fig. 5 B). The amplification products were electrophoresed on 0.6X-TBE, 5% urea-acrylamide sequencing gels (Long Ranger; J.T. Baker, Phillipsburg, NJ) and analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA.) using the Image Quant software supplied by the manufacturer. Clones that differed from the expected size and those clones in lanes adjacent to aberrantly migrated bands were used to produce plasmid preparations from which the inserts were sequenced in either direction.


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)

ELISA assays showing the expression of clone pg86  with a six amino acid insertion at  the FW1/CDR1 junction.  Clone pg86 (IgG heavy chain)  was coexpressed with the κ light  chain FS6κ in insect cells using  the baculovirus expression system. Expression of pg86 and its  ability to pair with the FS6κ light  chain was tested using capture  ELISAs. Wells were coated with  goat anti–human IgG. Supernatants containing recombinant antibodies were added in serial  twofold dilutions. Bound antibody  was detected with phosphatase-conjugated goat anti-human IgG  (A), and goat anti–human Cκ (B).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: ELISA assays showing the expression of clone pg86 with a six amino acid insertion at the FW1/CDR1 junction. Clone pg86 (IgG heavy chain) was coexpressed with the κ light chain FS6κ in insect cells using the baculovirus expression system. Expression of pg86 and its ability to pair with the FS6κ light chain was tested using capture ELISAs. Wells were coated with goat anti–human IgG. Supernatants containing recombinant antibodies were added in serial twofold dilutions. Bound antibody was detected with phosphatase-conjugated goat anti-human IgG (A), and goat anti–human Cκ (B).
Mentions: To facilitate the analysis of large numbers of VH gene transcripts for the presence of insertions or deletions, first strand cDNA produced as described above was PCR amplified using Expand high fidelity polymerase (Boehringer Mannheim) to reduce errors resulting from Taq polymerase alone. The products of this PCR amplification were cloned as described above and screened using 32P-labeled, gene-specific oligonucleotides (VH4-39:5′-ATTGGGAGTATCTATTATAGT-3′; L-6 as above). Positive colonies were picked and used to inoculate overnight cultures. A 1 μl aliquot from each 24-h culture was used to directly inoculate 25-μl PCR amplification mixtures in 96-well–format PCRs. The internal PCR reactions used 32P-labeled, gene-specific oligonucleotides to amplify a 230-base fragment including the VH4-39 CDR1 (L-4, as above, and VH4-39-3′: 5′-GCTCCCACTATAATAGATACT-3′) or for analysis of VH6 genes a 166-nucleotide fragment including the CDR1 and CDR2 of VH6 (VH6FW1: 5′-TGCCATCTCCGGGGACAGTGT-3′, VH6FW3: 5′-TGTGTCTGGGTTGATGGTTAT-3′). Aliquots of each clone were also used to inoculate amplifications of CDR3 regions using FW3-specific (ssFW3: 5′-CTGAA[C/G]CTGAGCTCTGTGAC[T/C]) and Cμ- or Cγ-specific oligonucleotides (CμD: 5′-GGAATTCTCACAGGAGACGA-3′, Cγ-140 as above) to analyze the diversity of the populations under study; the distribution of CDR3 size variations of several hundred VH sequences cloned in this analysis were used to produce an expected distribution of CDR3 sizes for comparison (see Fig. 5 B). The amplification products were electrophoresed on 0.6X-TBE, 5% urea-acrylamide sequencing gels (Long Ranger; J.T. Baker, Phillipsburg, NJ) and analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA.) using the Image Quant software supplied by the manufacturer. Clones that differed from the expected size and those clones in lanes adjacent to aberrantly migrated bands were used to produce plasmid preparations from which the inserts were sequenced in either direction.

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