Figure 4.

 κ chain demethylation and rearrangement. (a) B220⁺ spleen cell DNA from wild-type (wt) mice (lanes 1,2), from mice carrying a neomycin resistance gene replacing the κ intronic enhancer [iEκT^−/−] (Takeda et al. 1993) (lanes 3,4), and from mice carrying a prerearranged κ-transgene [Lκ] (Sharpe et al. 1991; Betz et al. 1994) (lanes 5,6) was digested by HindIII–BglII with (+) or without (−) HhaI. Blot hybridization was performed with probe 4 (see legend to Fig. 1) to visualize the endogenous κ genes exclusively. Both the endogenous κ alleles in iEκT^−/− and Lκ mice were found to be fully methylated in non-B cells (data not shown). (b) B220⁺ spleen cell DNA taken from iEκT^−/− mice was digested by HindIII (lane 1) with (+) or without (−) SacII (lane 2) or SacII–HhaI (lane 3) and analyzed by blot hybridization using probe 1. Note that although <50% of the κ alleles have the SacII site unmethylated (2.5 kb), all of these molecules are also unmethylated at the HhaI sites, suggesting that the demethylation is of an allelic nature.