The distribution of telomerase was not affected (Figs. 4K,L) but importantly, G-quadruplex structures were still visible in the replication band showing that this helicase is required for resolving G-quadruplex structure during replication (Figs. 4G,H).
When the expression of telomerase was silenced, G-quadruplex structures were still visible in the replication band (Figs. 4M,N) but also no staining with the anti-helicase antibody was observed in the replication band (Figs. 4O,P).
Since flow cytometrical observations and pfu measurements show that about 35% of the cells have adhered viruses at this stage, we conclude that this level is below the level of detection by PFGE, and that viral genomes become visible after their replication inside host cells.
The percentage of GFP positive cells visible (indicating active virus replication) following 120 h incubation was subsequently assessed from fluorescence microscopy images quantified using image analysis software.
In contrast, sites of HSV-2 MS-GFP replication were not visible in the eyes or facial epithelium of 0ΔNLS-immunized mice at any time beyond Day 1 post-challenge (Fig. S6).
However, incubation with sera resulted in virus replication which was visible by the presence of CPE (cytopathic effect) and was demonstrated by a PRRSV-specific staining.
Therefore, the time between ori1 replication and its visible separation into two foci is in the range 11.5 16.5 min. The analysis also showed that 50% of ter3 loci had separated by 8 min after the last time at which a replisome was present.
At the 45-min time point, clear PCNA peaks were no longer visible, probably due to asynchrony of replication forks.
Fragile sites are therefore believed to represent unreplicated regions of the genome that have escaped the replication checkpoint, and are visible as gaps and breaks on metaphase chromosomes [ 25].
In the infected, DNaseI-treated cells, the replication body was no longer visible.
