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Fig. 2. Molecular and phenotypic characterisation of bubR1^Rev1. (A) Genomic organisation of bubR1¹, bubR1^Rev1 and bubR1^Rev74 alleles. bubR1^Rev1 and bubR1^Rev74 were isolated from a genetic screen designed to identify imprecise excisions of the P element inserted in bubR1¹ (l(2)K03110). Arrows indicate pairs of primers used for PCR to determine whether the ends of the P elements were intact. (B) Southern blot showing the difference between wild-type and bubR1 alleles. Although bubR1^Rev74 and wild-type strains are identical, indicating that a perfect P element excision had taken place, bubR1^Rev1 genomic organisation shows a smaller band at 4.3 kb (asterisk), indicating a 2 kb deletion within the P element. The genomic DNA was digested with HindIII and EcoRI. (C) Western blot analysis of BubR1 protein levels in wild-type and bubR1 alleles. Tubulin was used as a loading control. Total proteins from wild-type third instar larval brains and female ovaries shows high levels of BubR1. BubR1 could only be detected from a minimum of two wild-type embryo extract at 30-90 minutes AEL. Homozygous bubR1¹ and bubR1^Rev1 third instar larval brains show a significant reduction in BubR1 protein levels. BubR1 protein level is significantly decreased in homozygous bubR1^Rev1 ovaries and undetectable from protein extract of two bubR1^Rev1 embryos. (D) Quantification of the mitotic phenotype in third instar larval brains from wild-type and bubR1 mutant allele combinations. In mutant genetic background, we observed a decrease in prometaphase-metaphase mitotic figures and an increase in the level of Sister Chromatid Separation (SCS). (E,F) Third instar larval neuroblasts incubated in 10 µM colchicine for 30 minutes induces a prometaphase-like arrest in wild-type cells (E), whereas most bubR1^Rev1 mutant cells (F) undergo SCS. Scale bar: 5 µm.

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