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

Figure 2.

Figure 2

Construction of MatNH- and MatH-RNA3 mutants. BMV genomic molecules are schematically presented as in 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′ end of Mag1-RNA3 or MagH-RNA3 (between KpnI and EcoRI sites) was replaced with a 379 nt portion of the wtRNA1 3′ end (fragment containing the entire 3′-UTR and RAS1). Thus, both constructs contain two copies of RAS1 sequence—MatNH-RNA3 includes 5′RAS1as and 3′RAS1s, while MatH-RNA3 comprises 5′RAS1s and 3′RAS1s. Furthermore, a marker mutation ΔXho (marked as a white dot), removing the XhoI restriction site, was introduced into the 3′ end of MatNH- and MatH-RNA3, to make it distinguishable from an analogous region present in wtRNA1.