A shuttle vector system for the investigation of immunoglobulin gene hypermutation: absence of enhanced mutability in intermediate B cell lines.
Somatic hypermutation focused to the rearranged V(D)J segment of the immunoglobulin (Ig) loci contributes substantially to antibody gene diversification. However, neither the precise B cell subset subject to hypermutation nor the molecular mechanism(s) involved is known. One model proposes that Ig segments may be uniquely susceptible to DNA nicking and subsequent error-prone repair during a specific B cell developmental stage. We describe an SV40-based shuttle vector system for testing such a model. Plasmids containing two distinct Ig segments juxtaposed to the supF marker gene have been passaged through cell lines representing intermediate stages of B cell development, rescued and screened for marker gene mutations. To date we have not demonstrated enhanced supF mutation in any cell line examined, irrespective of the adjacent Ig segment. Thus, these cell lines exhibit normal DNA repair mechanisms and no evidence of increased endonuclease activity on the Ig segments tested. The feasibility of this system will allow similar experiments using other Ig target sequences exposed to a broader range of B cells.
Johnston
J M
JM
Program in Human Molecular Biology and Genetics, University of Utah School of Medicine, Salt Lake City 84112.
Yu
M T
MT
Carroll
W L
WL
D000916:Antibody Diversity; D001402:B-Lymphocytes / Q000166:cytology / Q000502:physiology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D004260:DNA Repair; D005803:Genes, Immunoglobulin; D016153:Genes, Suppressor; D005822:Genetic Vectors; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D066298:In Vitro Techniques; D009154:Mutation; D012365:RNA, Transfer, Tyr; D014162:Transfection
D007143:Immunoglobulin Heavy Chains; D012365:RNA, Transfer, Tyr