Ordered activation of the Ig lambda locus in Abelson B cell lines.

Abstract

During B cell development, V genes are assembled at the three different Ig gene loci (IgH, Igx, and Ig),) in a controlled and ordered fashion (1, 2). V gene rearrangements start at the IgH locus (3, 4) and continue at the Igx locus in h-producing cells (5-7), while the Igx, locus is the last to be activated (8).B cells expressing X L chains have frequently rendered one or bothJx alleles nonfunctional by the rearrangement of a recombining sequence (RS)' element (9-11) . The RS element lies 3' of the mouse Cx exon and is bordered at its 5' side by a 23-bp heptamer nonamerjoining signal (9, 10). A homologous element called the x-deleting element (xde) was also found at the human Igx locus (11) and has recently been mapped 24 kb downstream of the Cx exon (12). Rearrangements of these elements involve either an isolated heptamer in the Jx-Cx intron or a 5'-situated Vx gene segment and thus result in the deletion of part or all of the Jx locus (10, 13). Due to the lack of suitable cellular models, the order and function of RS rearrangement in developing B cells is still unclear.We have extensively subcloned and analyzed c-myc transfectants of P8, a p-producing derivative ofthe Abelson line 300-19 (6, 14, 15). Two of seven c-myc-transfected Abelson lines assemble VA genes while growing in culture. In these lines RS recombination occurred either at the same time as or before VX rearrangements, suggesting that RS recombination is functionally correlated with the activation ofthe Igx, locus. Volume 168 December 1988 2131-2137Materials and Methods Cell Lines. P8 is a derivative of the Abelson line 300-19. It carries a VDJ' and a VDJrearrangement at the IgH loci and produces intracellular u chains. While growing in culture, P8 rearranges its x loci (6). BlP8-7, B3P8-16, and B3P8-17 are c-myc-transfected derivatives ofP8 in which the expression ofthe transfected c-myc gene has been proven via Northern blot analysis (14). Subclones of these lines were isolated by limiting dilution .Southern Blot Analysis. Approximately 15 kg of genomic DNA was digested by appropriate restriction enzymes, subjected to electrophoresis through I 'Yo agarose, blotted onto nitrocellulose, and assayed for hybridization to "P-labeled probes . The Jx-specific probe (5) was the 2.7-kb Hind III fragment carrying theJx segments ; the RS-specific probe (9) was the 0.8-kb Sau 3A fragment from the RS region ; and as a Vß,1-specific probe, we used a 0.85-kb, Xba fragment (16) from the VX1 .Genomic Cloning . Genomic libraries were cloned as Hind III fragments into X Charon 28 (B allele ofBIP8-7b) and as Eco RI fragments into X Charon NM 1149 (The A allele ofBIP8-7bThis work was supported by the Bundesministerium Für Forschung und Technologie grant BCT390-2 .08 .I Abbreviation used in this paper: RS, recombining sequence.J. Exp. MED.

Murine lambda gene rearrangements: the stochastic model prevails over the ordered model.

“…Since RS recombination has only been described in a small number of cases (Durdik et al, 1984;Persiani et al, 1987;Muller and Reth, 1988), a large panel of B cell hybridomas expressing one of the four X chains were examined by Southern blot using the RS 0.8 probe (see Materials and methods). EcoRI and BglIl digested DNAs from BALB/c liver, SP2/0, and some hybridomas are shown in Figure 3.…”

“…However, this as well as the systematic deletion of a first aberrant rearrangement (that cannot be seen in hybridomas due to a complex karyotype), seems highly unlikely, since it has never been reported in transformed lines (Blomberg et al, 1981Bothwell et al, 1981Wu et al, 1982;Reilly et al, 1984;Muller et al, 1988). Moreover, in the Cx locus, when the first Vx-Jx join is nonproductive, the second Cx allele uses generally another Vx to form the second join.…”

“…Indeed, three main hypotheses have been considered (Durdik et al, 1984;Persiani et al, 1987;Muller et al, 1988): (i) there is only a chance correlation between RS expression and X locus activation without a cause-effect relation, (ii) the RS recombination allows the activation of a transpositive factor (in this case, only one RS recombination is sufficient), (iii) the RS recombination inhibits a transrepressing factor (implying the deletion of the repressor on both alleles). However, RS recombination has only been studied in a few independent murine X-expressing B cell clones (Durdik et al, 1984;Persiani et al, 1987;Muller et al, 1988 (Blackwell et al, 1989 (Reynaud et al, 1985;Thompson and Nieman, 1987;McCormack et al, 1989). Moreover, the recent description of primitive vertebrate Ig light chains, shows that the amino acid sequences are closely related to mammalian X chains (Shamblott et al, 1989).…”

“…Firstly, most of x-expressing B cells retain their X loci in the germline configuration while most of X expressing B cells have non-functional rearrangements at the x locus (x-/x-) (Alt et al, 1980;Hieter et al, 1981;Durdik et al, 1984;Siminovitch et al, 1985). Furthermore, the few Abelson murine leukemia virus (AMulv) lines that progress to X expression when propagated in culture, rearrange x first and then srart to rearrange X (Persiani et al, 1987;Muller and Reth, 1988). The RS recombination, described in the mouse by Durdik et al (1984), supports the ordered regulated model and is thought to be involved in X locus activation (Persiani et al, 1987;Muller and Reth, 1988).…”

“…Furthermore, the few Abelson murine leukemia virus (AMulv) lines that progress to X expression when propagated in culture, rearrange x first and then srart to rearrange X (Persiani et al, 1987;Muller and Reth, 1988). The RS recombination, described in the mouse by Durdik et al (1984), supports the ordered regulated model and is thought to be involved in X locus activation (Persiani et al, 1987;Muller and Reth, 1988). It consists of the deletion of part or all of the Jx -Cx locus by a recombination event between the RS sequence located downstream of the Cx segment and an heptamer nonamer signal located upstream of either Cx or Jx segments.…”