Fig. 4. PIM^kenadba does not rescue sister chromatid separation in pim mutants. (A-P) Embryos were labeled with a DNA stain (A,D,E,H,I,L,M,P; DNA) and anti-Cyclin B (B,F,J,N; CYCB) at the stage of mitosis 15 (A-C,E-G,I-K, M-O) or after mitosis 15 (D,H,L,P). High-magnification views from epidermal regions, including merged views (C,G,K,O; DNA in red and anti-CycB in green), are shown. Mitosis 15 proceeds normally in pim⁺ sibling embryos (A-D; pim⁺) as well as in pim-mutant embryos with a recombined g>stop>pim transgene lacking the stop cassette (E-H; pim^- g>pim), as evidenced by normal telophase figures (arrows) during mitosis 15 and normal nuclear counts after mitosis 15 (white numbers in D,H,L,P). By contrast, sister chromatid separation does not occur during mitosis 15 in pim-mutant embryos with either an unrecombined g>stop>pim^kenadba transgene (I-K; pim^- g>s>pim^kenadba) or the recombined transgene lacking the stop cassette (M-O; pim^- g>pim^kenadba). Instead of normal late mitotic figures, these embryos contained decondensing metaphase plates (arrowheads) during mitosis 15 and a twofold lower nuclear count after mitosis. (Q-T) Expression of g>pim^kenadba in pim⁺ embryos allows normal proliferation during the early mitotic divisions but not during the late divisions in the CNS. DNA staining at stage 14 reveals the presence of many large polyploid abnormal nuclei (arrowheads) in the CNS of g>pim^kenadba embryos (R,T; g>pim^kenadba), which are absent in control siblings (Q,S; pim⁺). S and T show high-magnification views with the CNS from the embryos displayed in Q and R, respectively. (U) Co-immunoprecipitation experiments show that PIM^kenadba-myc associates normally with SSE and THR. Anti-myc immunoprecipitates (IP anti-myc) isolated from extracts (extract) of embryos expressing Cdk1-myc (Cdk1-myc), PIM^kenadba-myc (pim^kenadba-myc) or PIM-myc (pim-myc) were probed by immunoblotting for the presence of SSE (SSE), THR (THR), Cyclin B (CYCB) and tubulin (TUB).

Return to article