These relatively minor and slow to accumulate effects on cell survival are consistent with a previous description of growth kinetics in PCF T. brucei following TbORC1/CDC6 RNAi. Analysis of DNA content by fixing and DAPI-staining the cells, and then counting the ratio of nuclear and kinetoplast DNA visible in individual cells, revealed potential replication-associated defects. As has been described for TbORC1/CDC6,Bortezomib structure and confirmed here, RNAi of each gene resulted in the accumulation of aberrant cells that do not conform to the 1N1K, 1N2K or 2N2K DNA configurations that mark the normal course of cell division in T. brucei. In all cases, these aberrant cells were 0N1K ‘zoids’, indicating that they had lost nuclear DNA, and their accumulation was mirrored by similar reduction in 1N1K cell numbers, suggesting they arise from cytokinesis of 1N2K cells that have undergone kinetoplast replication and division but have failed to complete nuclear DNA replication. The numbers of these zoids, which never amounted to more than,5% of the uninduced cells,BYL719 appeared to reflect the extent of mRNA loss following RNAi: TbORC1/CDC6 and TbORC4 mRNA levels were relatively strongly reduced and zoids made up 31% and 28%, respectively, of the population 6 days after RNAi; Tb7980 and Tb3120 RNAi appeared less effective and zoids accumulated to a lesser extent. Despite this pronounced accumulation of zoids, we have not to date been able to demonstrate directly a role for the putative ORC-like factors in nuclear DNA replication. To do this, we attempted to measure the extent of 59 bromodeoxyuridine incorporation after RNAi by dot-blotting genomic DNA and probing with anti-BrdU monoclonal antibody. For each of TbORC4, Tb7980 and Tb3120, we have been unable to detect any reduction in BrdU signal four, six or even 10 days post-RNAi induction. However, it appears that this assay is relatively insensitive unless RNAi is strongly penetrative. When we analysed an RNAi clone that resulted in,90% loss of TbORC1/ CDC6 mRNA, BrdU incorporation was detectably reduced after four days, and this was concomitant with loss of 4C DNA and increased amounts of S-phase and sub-2C DNA in FACS analysis of propidium iodide -stained DNA, very similar to that described by Godoy et al. In contrast, in another clone in which only,75% loss of TbORC/CDC6 mRNA could be seen, no effect on BrdU incorporation could be detected after 6 days and the perturbation of DNA content by FACS was noticeably less severe. As the maximum extent of mRNA loss we have seen post-RNAi is,75% for ORC4, it seems likely that the extent of RNAi is below a threshold needed to see an effect on nuclear DNA replication by the BrdU dot blot assay adopted.