Homologous recombination in the mixed homo-heteromolecular system Earlier, Nagy and Bujarski (33) demonstrated that the 66 nt portion of RAS1 did not support homologous recombination in heteromolecular system. We repeated this experiment using MH-BMV mutants. Recombinants also did not form although the entire RAS1 sequence was present in wtRNA1 and MagH-RNA3 molecules (Table 1 and Figure 3 ). To test RAS1 activity Figure 1 , white, black and gray boxes represent coding, noncoding and recombinationally active sequences, respectively, in sense (RAS1s) or antisense (RAS1as) orientation, dashed line squares encompass replaced parts of wtRNA1 and modified RNA3 molecules. Mag1-and MagH-RNA3 were created by inserting the RAS1 sequence from wtRNA1 into the RAS cloning site of PN0-RNA3 in antisense (Mag1-RNA3) or sense (MagH-RNA3) orientation. To construct MatNH-RNA3 and MatH-RNA3, the 356 nt very 3 0 end of Mag1-RNA3 or MagH-RNA3 (between KpnI and EcoRI sites) was replaced with a 379 nt portion of the wtRNA1 3 0 end (fragment containing the entire 3 0 -UTR and RAS1). Thus, both constructs contain two copies of RAS1 sequence-MatNH-RNA3 includes 5 0 RAS1as and 3 0 RAS1s, while MatH-RNA3 comprises 5 0 RAS1s and 3 0 RAS1s. Furthermore, a marker mutation DXho (marked as a white dot), removing the XhoI restriction site, was introduced into the 3 0 end of MatNH-and MatH-RNA3, to make it distinguishable from an analogous region present in wtRNA1. in the mixed homologous recombination system, a previously used, well-established procedure was applied (30, 33) . C.quinoa plants (local lesion host for BMV) were inoculated with a mixture containing in vitro transcribed wtRNA1, wtRNA2 and MatH-RNA3. After 2 weeks, when infection symptoms were well developed, the number of lesions formed on every inoculated leaf was counted to determine the infectivity of the MatH-BMV mutant. Individual local lesions were excised and total RNA was extracted separately from each of them. Then, the 3 0 -portion of RNA3 progeny accumulating in examined lesions was selectively amplified by RT-PCR involving RNA3 specific primers A and B (for their location see Figure 1 ). Reaction products were separated in a 1.5% agarose gel and their length was determined. The formation of an 800 or 400-500 nt DNA fragment indicated that the lesion contained parental or recombinant RNA3, respectively. In this way, we were able to determine the number of lesions in which a viable RNA3 recombinant was generated. The presence of recombinants in analyzed lesions was confirmed by standard northern blot hybridization. DNA fragments obtained during selective RT-PCR amplification of RNA3 were cloned and sequenced. Finally, the results obtained with our new mixed homologous recombination system were compared with analogous data previously got using the heteromolecular system (33) (see Table 1 and Figure 3 ). The data presented in Table 1 indicate that the exchange of MagH-RNA3 (carrying a single RAS1) into MatH-RNA3 (bearing two RAS1 sequences) did not affect the infectivity of the BMV mutants. The average numbers of lesions appearing on the leaves inoculated with MH-and MatH-BMV were similar: 18 and 17, respectively. Interestingly, although RAS1 did not support homologous crossovers in the heteromolecular system represented by MH-BMV, it was very active in the mixed system. About 85% of the local lesions developed during MatH-BMV infection accumulated the RNA3 recombinant instead of parental MatH-RNA3. In all of them, one RAS1 and a spacer were deleted. This indicates that crossovers occurred either within 3 0 end 5 0 RAS1 present in MatH-RNA3 (inter-or intramolecular crossovers) or within RAS1 and 5 0 RAS1 located in wtRNA1 and MatH-RNA3, respectively. Recombinant junction sites were placed in identical regions; therefore, their location could not be precisely established.