DIAGRAMA DE PROCESO DE FLUJO

overexpression of the gene. The m utants were transform ed into w ildtype cells and the phenotypes w ere exam ined by m icroscopy and FACS analysis 20 hours after induction of the prom oter at 32°C. O verproduction of the w ildtype cdcISp or any of the m utants caused the cells to elongate, unlike cells containing the vector alone (Fig. 2.2 A). These results indicate that all of the cdcISp m utants are capable of blocking mitosis.

N ext the ability of the cdcISp m u tan ts to in d u ce D N A re-replication was exam ined. FACS analysis show ed an increase in DNA content of cells over­ expressing cdclS-150-577p and cdclS-150-577 (T374A)p. Over 40% of the cells had a DNA content of greater than 4C (Fig. 2.2 B d and e). These m utants, like the w ildtype protein, were therefore able to induce re-replication. The C-term inus of cdcISp m ay even induce re-replication to a greater extent th an the w ildtype protein. Only a proportion of cells are able to induce re-replication as can be seen from the continued presence of a 2C peak (Fig. 2.2 B b, d and e). This is because cells contain a variable plasm id copy num ber and a m inim um num ber of plasmids will be required in a cell before re-replication can be induced. Cells overproducing cdclS-l-141p an d cdclS-N TPp still elongated to a sim ilar extent as the other m utants, as dem onstrated by the forw ard scatter dot plot. H ow ever only a small apparent increase in DNA content was generated (Fig. 2.2 B c and f), and less than 10% of cells had a DNA content of greater than 4C. To verify that cdclS-l-141p and cdclS -N T P p w ere n o t cau sin g a low level of re-re p lic atio n lead in g to diploidization, cells were plated onto m edia containing Phloxin B (PB) at the end of the tim ecourse. Phloxin B is a red dye th at allow s differentiation betw een haploid and diploid cells. Only living cells are able to pum p out Phloxin B and as there is a greater proportion of dead or dying cells in a diploid colony compared to a haploid colony as diploid cells are more sick, the diploid colonies stain a darker pink. Only pale pink colonies, indicative of haploid cells, grew on the PB plates w hen cdcl8-l-141p and cdcl8-NTPp had been expressed indicating that there were no diploid colonies present. The increase in DNA content observed is m ost likely due to either an increase in background staining or m itochondrial DNA (Sazer and Sherwood, 1990) in the subset of cells expressing the constructs.

C h a p te r 2 C h a racterizatio n of c d c t 8 do m ain s

W estern blots w ere perform ed to verify th at the cdcl8 m u tan t proteins were expressed in all cases. Antibodies against the C-term inus of cdcl8p indicated that cdcl8-w tp (66kDa), cdcl8-150-577p (48kDa), cdcl8-150-577 (T374A)p (48kDa) and cdcl8-N TPp (66kDa) w ere all expressed to equal levels (Fig. 2.2 C, lanes 1-4). Several bands of lower m olecular w eight are also seen and these are m ost likely degradation products as cdcl8 is a highly unstable protein (Muzi-Falconi et al., 1996). It is surprising that m ore cdcl8p products of low er m olecular w eight are present w hen the C-term inus of cdcl8p is expressed, as it is m ore stable than the w ildtype protein (Baum et al., 1998).

To detect the N -term inus of cdcl8p (cdcl8-l-141p), antibodies against the entire p rotein w ere used. No band w as seen in the vector alone control. H ow ever, a cdcl8p band of about 16kDa was present in the cdcl8-l-141p transform ant lane. Endogenous cdcl8p was not detected as it is expressed at a m uch lower level than that induced by the n m tl prom oter 20 hours after derepression (Fig. 2.2 D lanes 1 and 2).

These results indicate that all of the m utants w ere able to m aintain a block over mitosis, while those expressing the C-term inus of the protein, cdcl8-150-577p and cdcl8-150-577 (T374A)p, could also efficiently prom ote DNA re-replication.

2.2 CdclSp induced re-replication is not caused by

inhibition of the cdc2p/cdcl3p mitotic kinase

It has been suggested that DNA re-replication induced by high levels of cdcl8p occurs indirectly due to the inhibition of the cd c2 p /cd cl3 p mitotic kinase (Wolf et al., 1996). There are two possibilities for how this inhibition could be achieved. Either cdcl8p could directly bind and inhibit cdc2p, or overproduction of cdcl8p could saturate the proteolytic m achinery that d egrades both itself and ru m lp (Kominami and Toda, 1997). The latter could subsequently lead to an accumulation of the CDK in h ib ito r an d a decrease in the m itotic kinase activity. These possibilities w ere investigated in several ways. Firstly, the mitotic kinase activity w as assayed in cdcl3p im m unoprecipitates derived from cells overproducing cdcl8-w tp and cdcl8-150-577p. The cdc2p kinase activity was followed during a 20