An extrachromosomal switch recombination substrate reveals kinetics and substrate requirements of switch recombination in primary murine B cells

Abstract

Ig class switch recombination occurs in B lymphocytes upon activation, and is targeted to distinct switch (S) regions by cytokine-mediated induction of switch transcripts spanning the entire S region and the adjacent constant region gene segments. Using a novel type of switch recombination substrate, constructed according to the intron-exon structure of the IgH locus, but with heterologous elements, we here have tested the structural requirements for targeting and the kinetics of switch recombination in activated primary murine B cells. When transfected at various times after activation, up to 10% of the transfected B cells perform recombination of the substrate within 12 h. Switch recombination in primary B cells is restricted to the first 72 h after onset of activation, then rapidly decreases to background levels, as obtained in plasmacytoma cells or with substrates carrying no S region sequences. In terms of structural requirements, switch recombination is targeted to any transcription unit that contains an intronic S region and depends on processing of the primary transcript by splicing.

Class switch recombination was specifically targeted to immunoglobulin (Ig)G4 or IgA in Hodgkin's disease‐derived cell lines

“…The preferential targeting of constant region genes indicates that class switch recombination occurs in a highly regulated process in HD‐derived cell lines. It has been shown for both murine and human B cells that class switch recombination is specifically directed not only with respect to onset and duration (Petry et al , 1999), but also with respect to the targeting of switch regions involved (Winter et al , 1987; Athan et al , 1989; Irsch et al , 1993, 1994). Particular switch regions are selected for recombination by cytokine‐induced transcription (Stavnezer‐Nordgren & Sirlin, 1986; Lutzker & Alt, 1988; for review see Lorenz & Radbruch, 1996; Hein et al , 1998).…”

“…‘Instability’ of the IgH loci of switching cells has been reported for endogenous IgH loci as well as for recombination substrates that had been introduced into cells activated for switch recombination (Ott & Marcu, 1989; Petry et al , 1999). DNA strand breaks are also introduced in Ig gene loci during other stages of B‐cell differentiation, e.g.…”

A Pak- and Pix-dependent branch of the SDF-1α signalling pathway mediates T cell chemotaxis across restrictive barriers

“…A Kozak sequence, initiation codon, FLAG tag and two unique restriction sites (AscI and ClaI) were inserted into the polylinker of pBI-EGFP, using the appropriate annealed oligonucleotides (details available upon request from D. Yablonski), to create pBI-FLAG. pBI-H-2K k was derived from pBI-FLAG, by replacing the EGFP coding sequence with a truncated form of the allelic mouse major histocompatibility complex antigen, H-2K k [37], isolated from the plasmid pMACS K k .II (Miltenyi Biotec GmbH, Germany). The Pak inhibitory fragments used in this paper are depicted in Figure 1.…”

Germline Transcription and Switch Recombination of a Transgene Containing the Entire H Chain Constant Region Locus: Effect of a Mutation in a STAT6 Binding Site in the γ1 Promoter

“…Toward this end, many substrates have been developed for S recombination. Substrate studies have demonstrated the ability of S regions to stimulate recombination, and, like recombination of H chain genes, recombination of the substrates is usually increased by transcription (17)(18)(19)(20)(21). Recombination of substrates is usually better in B cell lines than in non-B cell lines (17-19, 22, 23).…”