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. 2002 Aug;76(15):7890–7896. doi: 10.1128/JVI.76.15.7890-7896.2002

FIG. 2.

FIG. 2.

Identification of HHV8 oriLyt. (A, left side) Autoradiogram of a Southern blot of EcoRI- and DpnI-cleaved total cellular DNA from BCBL-1 cells transfected by electroporation with pDA12 with or without a plasmid expressing HHV8 ORF 50 (pEXP50). Blots were hybridized with pGEM7zf(−). The arrowhead indicates the replicated product. Lanes: 1, transfection of pDA12 plus pEXP50 (5 μg); 2, pDA12 plus pGEM7zf(−) (5 μg); 3, pDA12 plus pEXP50 (0.5 μg); 4, pDA12 plus pEXP50 (2 μg); 5, pDA12 plus pEXP50 (5 μg); 6, pDA12 plus TPA treatment for 4 days; 7, pDA12 plus TPA treatment for 2 days; 8, pDA12 plus pEXP50 (5 μg) incubated with PFA (300 μg/ml). (A, right side) Replication products are susceptible to cleavage by the restriction enzyme MboI. Cells were transfected with pDA12 and pEXP50 (5 μg), and DNA was extracted and treated with EcoRI and DpnI (lane 1) or EcoRI, DpnI, and MboI (lane 2). (B) Both the oriLyt domain and the G+C repeat regions are required for replication and identification of the OriLyt-R sequence. Transfection of HHV8 oriLyt-L and oriLyt-R subclones was done to identify essential DNA elements. All transfections contained 20 μg of oriLyt-containing plasmids plus 5 μg of pEXP50. Arrowheads indicate replicated plasmids. Refer to Table 1 and Fig. 1 for details of subclones. (B, left side) Transfection of oriLyt-L subclones. Lanes: 1, pDA13; 2, pDA14; 3, pDA15; 4, pDA16; 5, pDA18; 6, pDA12. (B, right side) Transfection of oriLyt-R subclones. Lanes: 1, pDA23; 2, pDA24; 3, pDA25.