Figure 6.
Figure 6. Somatic hypermutation of transgenic VDJ exons and flanking sequences. Part of the transgenic VDJ exon and 3′ flanking sequences (from germinal center B cells) were amplified, cloned, and sequenced. Mutations in the 3′ 171 bp of VDJH2 exon and 329 bp of 3′ flanking sequences are presented as pie charts, in which the sectors of the pie are proportional to the number of sequences with a given number of mutations. The number of sequences is shown in the center of the pie chart, and the number of mutations in some sectors is noted. For lines 820, 820Δ, 774, and 774Δ, the data represent a compilation of sequences from two sorted cell samples derived from independent mice. For lines 336 and 336Δ, the data are derived from one sorted cell sample each; a second set of sequences from the same cell sample yielded 0.21% mutations in line 336 DNA and 0.02% mutations in line 336Δ DNA. For all three pairs of transgenic lines tested, the mutation frequency is greater in intact versus the enhancer-deleted line, P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). Mutation frequencies in the intronic 329-bp were: line 820, 0.36% (27 mutations in 7557 nt); line 820Δ, 0.03% (4 mutations in 13,818 nt); line 774, 0.40% (43 mutations in 10,857 nt); line 774Δ, 0.11% (14 mutations in 12,831 nt); line 336, 0.16% (44 mutations in 27,636 nt); and 336Δ, 0.03% (9 mutations in 30,268 nt). For each set of paired transgenic lines, the difference in intronic mutations was significant at P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). In separate experiments, transgenic V region mutations were also determined in cloned mRNA (VDJCγ1, VDJCγ2a, and VDJCγ2b pooled sequences) from spleens of immunized line 820 mice (see text). The 820 VDJ exon had 1.67% mutations (128 mutations in 7680 nt sequenced from 30 clones). The 820 Cγ region had 0.1% mutations (10 mutations in 10,041 nt sequenced in the same 30 clones). By a two-tailed χ2 test, with Yate’s correction and one degree of freedom, the difference in mutation frequency in V and C is significant at P < 0.0001.

Somatic hypermutation of transgenic VDJ exons and flanking sequences. Part of the transgenic VDJ exon and 3′ flanking sequences (from germinal center B cells) were amplified, cloned, and sequenced. Mutations in the 3′ 171 bp of VDJH2 exon and 329 bp of 3′ flanking sequences are presented as pie charts, in which the sectors of the pie are proportional to the number of sequences with a given number of mutations. The number of sequences is shown in the center of the pie chart, and the number of mutations in some sectors is noted. For lines 820, 820Δ, 774, and 774Δ, the data represent a compilation of sequences from two sorted cell samples derived from independent mice. For lines 336 and 336Δ, the data are derived from one sorted cell sample each; a second set of sequences from the same cell sample yielded 0.21% mutations in line 336 DNA and 0.02% mutations in line 336Δ DNA. For all three pairs of transgenic lines tested, the mutation frequency is greater in intact versus the enhancer-deleted line, P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). Mutation frequencies in the intronic 329-bp were: line 820, 0.36% (27 mutations in 7557 nt); line 820Δ, 0.03% (4 mutations in 13,818 nt); line 774, 0.40% (43 mutations in 10,857 nt); line 774Δ, 0.11% (14 mutations in 12,831 nt); line 336, 0.16% (44 mutations in 27,636 nt); and 336Δ, 0.03% (9 mutations in 30,268 nt). For each set of paired transgenic lines, the difference in intronic mutations was significant at P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). In separate experiments, transgenic V region mutations were also determined in cloned mRNA (VDJCγ1, VDJCγ2a, and VDJCγ2b pooled sequences) from spleens of immunized line 820 mice (see text). The 820 VDJ exon had 1.67% mutations (128 mutations in 7680 nt sequenced from 30 clones). The 820 Cγ region had 0.1% mutations (10 mutations in 10,041 nt sequenced in the same 30 clones). By a two-tailed χ2 test, with Yate’s correction and one degree of freedom, the difference in mutation frequency in V and C is significant at P < 0.0001.

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