 | Fig. 1. (a) Southern blot analysis of virus genomes. Viruses were purified by sucrose density gradient centrifugation and the DNA was extracted as described (Esposito et al., 1981). These DNA samples were digested with either PstI/XbaI or HpaI and the resultant DNA fragments were separated by electrophoresis on a 1 % agarose gel, transferred to nitrocellulose filters and probed with a 600 bp DNA fragment that had been excised from plasmid p B8R by digestion with HindIII/EcoRI. Following PstI/XbaI digestion, a band of just under 4 kb was detected with all viruses. In addition, a fragment of 2.6 kb was found with WR and vB8R-rev, whereas this was reduced to 1.8 kb in vB8R and vB8R-R. This fragment was slightly smaller in the vsmIFN- R genome due to the replacement of the WT B8R gene with the mouse IFN-R gene. Following HpaI digestion, a band of 2.2 kb was seen with WR and vB8R-R and this was slightly smaller with vsmIFN-R and reduced to 1.5 kb with vB8R and vB8R-R. (b) Schematic representation of the B8R gene locus and flanking regions. Open boxes represent ORFs to the right and left of B8R. Stippled box indicates the mouse IFN-R gene expressed from vsmIFN-R. Hatched boxes indicate the DNA used as probe in (a). Horizontal line with arrowheads indicates the region of vB8R genome deleted in vB8R. Vertical lines indicate the positions of restriction endonuclease sites and horizontal lines indicate the fragments detected in (a) and their predicted sizes. |
