In order to demonstrate that Mat0-RNA3 can be used as an effective tool in recombination studies, we applied it to examine the recombination activity of two specific sequences derived from the HCV genome. The first, 101 nt sequence is placed within the 5 0 -portion of the HCV genome (within the fragment encoding E2 protein) and is named HVR (40, 42) . The second, called the sequence X (X) constitutes a 98 nt 3 0 end of HCV genomic RNA. It has been shown that X represents the most conservative fragment of HCV genome (43) . Both sequences were obtained by a standard RT-PCR method involving viral RNA isolated from the blood of infected patients as a template (40, 41) . Amplified fragments were inserted into the 5 0 -cloning site of Mat0-RNA3 in two different orientations (sense and antisense), then only in sense orientation into the 3 0 -cloning site (for details see Materials and Methods). As a result, four different Mat0-RNA3 derivatives were generated: (i) MatH-HVR-RNA3, possessing two copies of HVR in sense orientation (3 0 and 5 0 HVRs); (ii) MatH-X-RNA3, with two copies of X in sense orientation (3 0 and 5 0 Xs); (iii) MatNH-HVR-RNA3, with two copies of HVR, the 3 0 -copy in sense and the 5 0 in antisense orientation (3 0 HVRs and 5 0 HVRas); (iv) MatNH-X-RNA3, with two copies of X located in different orientation (3 0 Xs and 5 0 Xas) ( Figure 5B ). The former two were applied to test HVR's and X's ability to support homologous crossovers while the latter two to examine Xs/Xas' and HVRs/HVRas' capacity to induce nonhomologous, heteroduplex-mediated recombination. Unlike previously tested mutants (MatNH-and MatH-BMV), in MatH-HVR-, MatNH-HVR-, MatH-X-and MatNH-X-BMV, the examined sequences were present only in the recombination vector. They were absent in the two other genomic RNAs, so that recombination crossovers could involve only RNA3 molecules.