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. 2005 Jul 7;33(12):e105. doi: 10.1093/nar/gni106

Figure 4.

Figure 4

Non-homologous recombination in the heteromolecular and mixed homo-heteromolecular systems. As in Figure 3, light and thicker lines represent viral genomic RNAs and nascent recombinant RNA, respectively. WtRNA1 is red (the recombinationally active sequence RAS1s which it contains is additionally boxed), wtRNA2 is black and modified RNA3 is blue. When inserted into RNA3 the wtRNA1-derived RAS1s sequence is also shown as a red box, whereas the complementary RAS1as sequence is shown as a green box. The portion of the RNA3 recombinant synthesized on wtRNA1 is red, the portion synthesized on the RAS1as sequence is green and the fragment synthesized on RNA3 is blue. The black dot symbolizes the ΔXho mutation present in MatNH-RNA3. RF, recombination frequency. Dashed line squares encompass the region identical in both systems [the region where crossovers occur, shown in detail in (E)]. (A) Heteromolecular system (M1-BMV). A detailed description of the M1-BMV genome is presented in Figure 1. All nascent RNA3 molecules accumulating in M1-BMV infected plants were recombinants (RF = 100%). (B) Mixed system (MatNH-BMV). Three copies of RAS1 are located in the MatNH-BMV genome, two in MatNH-RNA3 (3′RAS1s and 5′RAS1as) and one in wtRNA1 (RAS1s). The recombination frequency observed during infection with MatNH-BMV was 95%. Of the identified recombinants, 10% were without the ΔXho marker, and 90% with the ΔXho marker. (C) Putative scenario of RAS1s/RAS1as-mediated non-homologous recombination in the heteromolecular system. Owing to the presence of RAS1s and RAS1as sequences in wtRNA1 and Mag1-RNA3, respectively, they are capable of forming a local double-stranded structure supporting non-homologous crossovers (for details see Figure 1). (D) Putative scenarios of RAS1s/RAS1as-mediated non-homologous recombination in the mixed system. The presence of 3′RAS1s and 5′RAS1as sequences in MatH-RNA3 and RAS1s in wtRNA1 creates several opportunities of heteroduplex formation: between wtRNA1 RAS1s and MatNH-RNA3 5′RAS1as (intermolecular), between two pairs of RAS1s/RAS1as sequences of two different MatNH-RNA3 molecules (intermolecular) and between 5′RAS1as and 3′RAS1s of the same MatNH-RNA3 molecule (intramolecular). Recombinants are generated if BMV replicase initiates nascent strand synthesis at the 3′ end of wtRNA1 or MatNH-RNA3 and then switches to MatNH-RNA3 within the local double-stranded region. (E) Recombinants identified during M1- and MatNH-BMV infection. Boxed fragments of recombining wtRNA1/Mag1-RNA3, wtRNA1/MatNH-RNA3 and MatNH-RNA3/MatNH-RNA3 molecules are practically identical in both systems (except for the ΔXho mutation present in MatNH-RNA3). The locations of the junction sites are marked with arrows and letters. The numbers indicate how many recombinants of the same type were isolated. Upper case letters refer to M1-BMV, lower case letters refer to MatNH-BMV.