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First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02361

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The Drosophila Cdc6/18 protein has functions in both early and late S phase in S2 cells

¹ Department Basic Medical Sciences, St Georges Hospital Medical School, London, SW17 0RE, UK
² Genetics and Molecular Biology Research Group, Institute of Biology and Environmental Sciences, University College of Nyiregyhaza, Sostoi ut 31/B, Nyiregyhaza 4400, Hungary

View larger version (109K): [in a new window]   Fig. 1. Sequence comparison of the cdc6 proteins from Drosophila, human, S. cerevisiae and S. pombe. Black shading represents identity and grey shading represents similarity.

View larger version (21K): [in a new window]   Fig. 2. Cellular distribution of DmCdc6. (A) Characteristics of the Dmcdc6 antibody used for this study. Crude extracts of Drosophila S2 cells were loaded and analysed by western blotting with Dmcdc6 antibody. The sizes of molecular weight markers are indicated at the right of the figure. Text indicates the position of the Dmcdc6 and a band that is apparently unrelated (as determined by the use of affinity purified antibodies). Those bands marked with an asterisk are degradation products of Dmcdc6 that increase in intensity as S2 cell fractions are aged. (B) S2 cells in mid-log phase were fractionated and analysed by western blotting. 500,000 equivalent cells were loaded per lane. C, cytoplasmic extract; Chr, chromatin-bound extract; Np, nucleoplasmic extract; T, total cell extract. (C) FACS analysis of exponential phase S2 cells. (D) Proliferating S2 cells were treated with aphidicolin (as described in Materials and Methods) and fractionated as in Fig. 2A. The top panel shows the distribution of DmCdc6 in cellular fractions of untreated cells (Ctrl) and cells treated with aphidicolin (Aph). The bottom panel shows a Coomassie Blue-stained gel of the region of gel between 14 and 21 kDa to show that the distribution of histones is as would be expected for an efficient fractionation. (E) FACS profile of control and aphidicolin-treated cells on days 1 and 2 of the experiment. The fractionation above corresponds to the day 1 sample, although no significant difference was seen between day 1 and day 2. (F) Total cell extract from 500,000 cells exponentially growing (Exp.) or quiescent (Q.) were analysed for the presence of Cdc6 by western blot. (G) FACS profile of nondividing S2 cells prepared as described in Materials and Methods. (H) Proliferating S2 cells were treated with ecdysone as described in Materials and Methods, and the total amount of DmCdc6 in untreated (–) and treated (+) was analysed by western blotting. (I) FACS profile of untreated cells (left) compared with cells that had been treated with ecdysone as described in Materials and Methods (right).

View larger version (18K): [in a new window]   Fig. 3. DmCdc6 RNA interference. (A) The regions of the DmCdc6 gene used for RNAi and RT-PCR in all experiments presented in the paper. DmCdc6 RNAi 1 and 2 were used in the standard Dmcdc6 knockouts presented in Fig. 3, and RT-PCR 1 is used in both this Figure and Fig. 7 to check for the presence of bulk DmCdc6. UTR, RNAi and RTPCR2 are used to knockout and check for, respectively, the presence of the endogenous DmCdc6 in the experiments discussed in Fig. 7. (B) Western blot analysis of Cdc6 depletion by RNAi: total cell extracts of 500,000 cells untreated (–) or treated with DmCdc6 double-stranded RNA (+) at day 3 and day 5 of the experiment were loaded in each well. (C) Depletion of DmCdc6 mRNA detected by RT-PCR at day 3 and day 5. –, no treatment; +, treated with Dm Cdc6 double-stranded RNA and m, treated with unrelated double-stranded RNA. The Df31 gene amplification serves as a nonspecific control and the ORC1 gene amplification serves as a specific control to test for cross reaction of the probes with the DmORC1 gene. (D) Comparison of the growth of untreated S2 cells, cells treated with DmCdc6 double-stranded RNA and cells treated with an unrelated double-stranded RNA. (E) FACS analysis of S2 cells treated with DmCdc6 RNAi compared with mock treated cells and cells treated with RNAi against cyclin E.

View larger version (29K): [in a new window]   Fig. 4. S2 cells treated with DmCdc6 double-stranded RNA show abnormalities. Cells treated with DmCdc6 double-stranded RNA (Cdc6 RNAi) or untreated (Ctrl) were stained for tubulin (red) and DNA (blue) (A), or for lamin (green) and DNA (blue) (B).

View larger version (68K): [in a new window]   Fig. 5. Treatment of S2 cells DmCdc6 double-stranded RNA causes a large number of cells to stain with antiphospho H3 antibody. Cells either treated with Cdc6 double-stranded RNA (Cdc6 RNAi) or untreated (Ctrl)were stained for tubulin (green) phospho H3 (red) and DNA (blue). The DNA and phospho H3 costaining appears purple.

View larger version (21K): [in a new window]   Fig. 6. Overexpression of DmCdc6 in S2 cells. Two independent transformed S2 cell lines (S2 Cdc6-1 and S2 Cdc6-2) were analysed for expression of DmCdc6 either with or without induction by copper. The protein levels were visualised by western blotting with antibody against either the DmCdc6 protein itself or the His tag present in the overexpressed protein. In each case total cell extract from 500,000 cells was loaded. The western was also developed with anti tubulin antibody as a loading control.

View larger version (33K): [in a new window]   Fig. 7. Overexpression of DmCdc6 in S2 cells has only a small effect on the cells. (A) FACS profile of cells in which DmCdc6 has been overexpressed. (B) Overexpressed Dm Cdc6 is nuclear: S2 Cdc6-1 and S2 Cdc6-2 cells were fractionated and analysed by western blotting. 500,000 equivalent cells were loaded per lane. T, total cell extract; C, cytoplasmic extract; Np, nucleoplasmic extract; Chr, chromatin-bound extract. (C) Immunofluorescent staining of S2 Cdc6-1 and S2 Cdc6-2 cell lines using the antibody directed against the His tag on the protein. The His staining is shown in green and the DNA, which is stained with DAPI, is shown in blue. (D) RTPCR to show the effects of the depletion of the endogenous DmCdc6 on S2 cells overexpressing DmCdc6. Normal S2, S2 Cdc6-1 and S2 Cdc6-2 cells were treated with UTR RNAi (Fig. 3A) and then subject to RTPCR using either RTPCR1 or RTPCR2 to detect total and endogenous DmCdc6, respectively. ORC1 in this case serves as the negative control. + indicates RNAi treatment and – no RNAi treatment. (E) Growth analysis of normal S2, S2 Cdc6-1 and S2 Cdc6-2 cells with and without Cdc6 5' UTR RNAi treatment.