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Junctional diversification in the generation of the precursor of a discrete immune response
J George1, K M Sheehan, P H Brodeur
1Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109-0620.
Abstract:
Phosphocholine (PC)-specific antibodies that arise in the mouse in response to Proteus morganii (PM) and use V1-DFL16.1-JH1 are characterized by a number of recurring mutations. Most striking is an invariant A for G substitution in codon 95 of VH which results in an asparagine instead of aspartate at that position. Because of the apparent importance of this substitution in an anti-PC(PM) response, we wanted to determine the molecular basis for this base change. A cDNA library derived from pre-immune splenic B cells was examined for the frequency of VDJ containing the A substitution at 95 and the presence of additional point mutations in these sequences. Six different cDNA were isolated which contained an A substitution at the VD junction (frequency 0.00009); a seventh positive cDNA could not be examined. The V segments of four of these cDNA matched known germline genes and were, therefore, unmutated. Two others closely matched V in families whose members have not all been characterized, hence, it is not known whether the mutations observed are somatic or germline in origin. Sequences of 35 cDNA clones, containing the same V segment but differing in D, J and junctional nucleotides, revealed no mutations. These results indicate that the A substitution generated at codon 95 is most likely a product of V-DJ joining.
Insights
The study investigated mutations in phosphocholine (PC)-specific antibodies generated against Proteus morganii (PM). Researchers found a key mutation at codon 95, likely arising from V-DJ joining during antibody development.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Phosphocholine (PC)-specific antibodies are crucial for immune responses against pathogens like Proteus morganii (PM).
- Recurring mutations, particularly an alanine (A) for glycine (G) substitution at codon 95 of the VH gene, are observed in these antibodies.
Purpose of the Study:
- To determine the molecular basis for the invariant A for G substitution at codon 95 in anti-PC(PM) antibodies.
- To investigate the frequency and origin of this specific mutation in B cell cDNA libraries.
Main Methods:
- Construction and screening of a cDNA library from pre-immune splenic B cells.
- Sequencing of VDJ regions to identify the A substitution at codon 95 and assess other mutations.
- Analysis of V gene segments to differentiate between germline and somatic mutations.
Main Results:
- Six cDNA clones with the A substitution at the VD junction were isolated, with a frequency of 0.00009.
- Four of these clones had unmutated V segments matching known germline genes.
- No mutations were found in the D, J, and junctional nucleotides of 35 cDNA clones sharing the same V segment.
Conclusions:
- The A substitution at codon 95 is most likely a product of V-DJ joining.
- This specific mutation appears to be a significant feature of the anti-PC(PM) antibody response.