Figure 3. Inhibition of UNG2 in G1 phase... | Rockefeller University Press

Figure 3.

$Figure 3. Inhibition of UNG2 in G1 phase inhibits transversion mutation and increases transition mutation at IgV G:C base pairs. SWHEL splenocytes were cultured overnight with recombinant CD40L and transduced to express GFP fusion proteins. 2 d later, ≤104 sorted GFP+ cells were injected into primed male congenic hosts, along with 108 HEL-SRBC. Individual GFP+ cells that bound red fluorescent HEL were recovered from host spleens 6 d after adoptive transfer by flow cytometric sorting into 96-well plates. (A) Map of the SWHEL VDJH allele. The dashed box shows the 523-bp window sequenced. VDJ-coding regions are indicated by the gray boxes, with CDRs indicated in darker gray. Nested PCR primers and the sequencing primer (453) are indicated by arrows. The position of a 562bp germline deletion (relative to the wild-type C57BL/6 IgH allele) is indicated by “Δ.” (B) The distribution of DNA content among GFP+ cells from hosts that received SWHEL cells transduced to express the proteins indicated. Vertical scales are arbitrarily adjusted to mimic Fig. 1 C. (C) Expression of ugi-GFP increases the fraction of cells in post–G1 phase in vivo (ipso facto, it also decreases the fraction of cells in G1 phase). SWHEL splenocytes were transduced with GFP (circles) or ugi-GFP (triangles) and adoptively transferred. 5 d later, the distribution of GFP+ve splenocytes between G1 (2n DNA) and post-G1 (>2n DNA) phases was determined by DAPI fluorescence. The fractions of GFP+ve cells with >2n DNA content from three independent experiments (black, gray and white symbols) are plotted. *, P < 0.05, two-tailed paired Student’s t test. (D–F) The mean number per sequence (±SEM) of transversion mutations at G:C base pairs (D), transition mutations at G:C base pairs (E), or mutations at A:T base pairs (F) detected in single GFP+HEL+ splenocytes transduced to express the proteins indicated by x-axis labels. The total number of hosts, sequences, and mutations contributing to each column are given in Table 1. The multiplier placed above the brackets indicates the change in mutation in ugi+ cells relative to appropriate control cells, accompanied by a significance indicator (according to one-way nonparametric ANOVA combined with Dunn’s multiple comparisons) ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05, respectively. None of the A:T mutation means (in F) were significantly different from any other.$

Inhibition of UNG2 in G1 phase inhibits transversion mutation and increases transition mutation at IgV G:C base pairs. SW_HEL splenocytes were cultured overnight with recombinant CD40L and transduced to express GFP fusion proteins. 2 d later, ≤10⁴ sorted GFP⁺ cells were injected into primed male congenic hosts, along with 10⁸ HEL-SRBC. Individual GFP⁺ cells that bound red fluorescent HEL were recovered from host spleens 6 d after adoptive transfer by flow cytometric sorting into 96-well plates. (A) Map of the SW_HEL VDJ_H allele. The dashed box shows the 523-bp window sequenced. VDJ-coding regions are indicated by the gray boxes, with CDRs indicated in darker gray. Nested PCR primers and the sequencing primer (453) are indicated by arrows. The position of a 562bp germline deletion (relative to the wild-type C57BL/6 IgH allele) is indicated by “Δ.” (B) The distribution of DNA content among GFP⁺ cells from hosts that received SW_HEL cells transduced to express the proteins indicated. Vertical scales are arbitrarily adjusted to mimic Fig. 1 C. (C) Expression of ugi-GFP increases the fraction of cells in post–G1 phase in vivo (ipso facto, it also decreases the fraction of cells in G1 phase). SW_HEL splenocytes were transduced with GFP (circles) or ugi-GFP (triangles) and adoptively transferred. 5 d later, the distribution of GFP^+ve splenocytes between G1 (2n DNA) and post-G1 (>2n DNA) phases was determined by DAPI fluorescence. The fractions of GFP^+ve cells with >2n DNA content from three independent experiments (black, gray and white symbols) are plotted. *, P < 0.05, two-tailed paired Student’s t test. (D–F) The mean number per sequence (±SEM) of transversion mutations at G:C base pairs (D), transition mutations at G:C base pairs (E), or mutations at A:T base pairs (F) detected in single GFP⁺HEL⁺ splenocytes transduced to express the proteins indicated by x-axis labels. The total number of hosts, sequences, and mutations contributing to each column are given in Table 1. The multiplier placed above the brackets indicates the change in mutation in ugi⁺ cells relative to appropriate control cells, accompanied by a significance indicator (according to one-way nonparametric ANOVA combined with Dunn’s multiple comparisons) ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05, respectively. None of the A:T mutation means (in F) were significantly different from any other.