Fig. 4

CYCLIN A2 and CYCLIN B1 expression partially inhibits rereplication in emi1-deficient cells.

(A) Amino acid alignment of the dead box domain of wild-type human CYCLIN A2 and CYCLIN B1 and dead box (DB) mutant proteins. (B) Expression of CYCLIN A-DB and CYCLIN B-DB GFP fusion constructs at 5 somites, as visualized by fluorescence microscopy. Embryos injected with either DNA construct showed significant and pervasive mosaic expression at this stage and the level of expression was not altered by co-injection of emi1 MO. (C) Representative example of FACS scanning of propidium iodide (PI)-stained total cells from embryos injected with the indicated morpholinos and DNA constructs. Note the partial decrease in >G2 population in embryos injected with emi1 MO and CYCLIN A-DB as compared to emi1 MO only (quantitation in panel D). Also, embryos injected with emi1 MO and CYCLIN B-DB showed a partially decreased >G2 population and a partially increased G1 population as compared to emi1 MO only (quantitation in panel D). (D) Quantitation of the cell cycle distribution in embryos injected with the indicated morpholinos and DNA constructs. There was a significant difference between the percentage of cells with >G2 DNA content in embryos injected with emi1 MO and CYCLIN A-DB as compared to emi1 MO only. Also, there was a significant difference between the percentage of cells with >G2 DNA content and in G1 phase in embryos injected with emi1 MO and CYCLIN B-DB as compared to emi1 MO only. The results shown are average of 3 independent experiments and error bars indicate standard deviation. (E) Normalized average cell size in embryos injected with the indicated morpholinos and DNA constructs, as indicated by FSC of total cells in the FACS analysis. There was no significant decrease in the size of cells in embryos injected with emi1 MO and CYCLIN A-DB or CYCLIN B-DB as compared to emi1 MO only. If anything, there was a slight but significant increase in cell size of embryos injected with emi1 MO and CYCLIN A-DB as compared to emi1 MO only.