Antisense transcripts of V(D)J rearrangements; artifacts caused by false priming?
Somatic hypermutation (SH) of V(D)J rearrangements at the IGH and IGL loci diversifies the IG repertoire during the germinal center response. SH is absolutely dependent on the enzyme activation induced cytidine deaminase (AID) that initiates the SH process by deaminating C nucleotides in ssDNA. Mutations from G and C are thought to occur as a result of strand symmetrical deamination of C by AID on the coding and non-coding strands respectively. Mutations from A and T are introduced in a strand biased way during error prone repair of the AID induced lesion. SH is linked to transcription and it has been proposed that bidirectional transcription across V(D)J rearrangements occurs in activated and quiescent B cells and that it is co-opted to facilitate the accessibility of the two DNA strands by regulating accessibility of single stranded DNA to AID. We have developed a quantitative method to study directional transcription. Our method controls for differences in efficiency and specificity of reverse transcription that are known to be able to generate false positive data. This method does not detect antisense transcripts in exonic or intronic regions within the hypermutation domain of the spontaneously hypermutating cell line Ramos, or in human blood B cells or tonsil cells, providing convincing evidence that antisense transcripts are rare or absent in human B cells.
Zhao
Yuan
Y
Department of Immunobiology, King's College London, Guy's Hospital Campus, St. Thomas' St, London SE1 9RT, UK.
Dunn-Walters
Deborah K
DK
Barone
Francesca
F
Spencer
Jo
J
D016477:Artifacts; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D005091:Exons; D005189:False Positive Reactions; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D015328:Gene Rearrangement, B-Lymphocyte, Light Chain; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D014066:Palatine Tonsil / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012326:RNA Splicing; D016372:RNA, Antisense / Q000235:genetics*; D027041:Somatic Hypermutation, Immunoglobulin; D014158:Transcription, Genetic
D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D016372:RNA, Antisense