FIG.4.
Analysis of Vλ sequences cloned from sIgM loss variants. (A) Fluctuation analysis of the generation frequency of sIgM loss variants. The abundance of sIgM loss variants was determined in several parallel cultures derived from sIgM-positive single cells after clonal expansion (3 weeks); median percentages are noted above each data set and are indicated by the dashed lines. WT, wild type. (B) Comparison of Vλ sequences from sIgM loss cells sorted from parental sIgM-positive clones of wild-type or brca2tr cells. Each horizontal line represents the rearranged Vλ1/Jλ (402 bp). Point mutations (lollipop shape) and gene conversion tracts (horizontal bars above lines) are indicated. (C) Proportions of Vλ sequences carrying different number of point mutations (PM), gene conversions (GC), or mutations of ambiguous origin (Amb) among sorted sIgM-negative populations. Segment sizes are proportional to the number of sequences carrying the number of mutations indicated around the peripheries of the pie charts. The total number of Vλ sequences analyzed is indicated in the centers of the charts, with the data compiled from analysis of three brca2tr cells. (D) Nucleotide substitution preferences deduced from point mutations in sequences from brca2tr cells. All of the V sequences having more than two point mutations showed different patterns, while 12 of the 27 analyzed V sequences having a single-base substitution showed identical events. Thus, most of the mutations may represent independent events rather than clonal expansion. (E) Ig diversification patterns in wild-type cells and rad51c, rad51d, and brca2tr mutants. The frequencies of gene conversion tracts and nontemplated point mutations per mutated sequence in sorted sIgM-negative cells are plotted. The ratio of point mutations per gene conversion is shown to the right of each bar. The mutation data are derived from 105 WT sequences from 5 subclones, 38 each of rad51c and rad51d sequences from two subclones, and 40 brca2tr sequences from three subclones. Ambiguous mutations (44) were excluded from this analysis. Data indicated by asterisks are from reference 44.