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Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila.

Basu J, Bousbaa H, Logarinho E, Li Z, Williams BC, Lopes C, Sunkel CE, Goldberg ML - J. Cell Biol. (1999)

Bottom Line: We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase.Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry.Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

View Article: PubMed Central - PubMed

Affiliation: Section of Genetics and Development, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
We have characterized the Drosophila mitotic checkpoint control protein Bub1 and obtained mutations in the bub1 gene. Drosophila Bub1 localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element-induced, near- mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants. We have also investigated the relationship between Bub1 and other kinetochore components. We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

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Expression of reaper in bub1 mutants. Imaginal discs were stained with X-gal to follow expression of a reaper-lacZ reporter construct (rpr-lacZ) in larvae of various genotypes. (A) rpr-lacZ/rpr-lacZ. (B) bub1/bub1 without the rpr reporter gene. (This control was necessitated by the fact that the P elements causing the bub1 mutations contain a lacZ gene.) (C and D) bub1/bub1; rpr-lacZ/rpr-lacZ imaginal discs showing very high levels of β-galactosidase expression, indicating that the cell death program is activated in many cells. Similar results were observed in the brains of these same animals (not shown). Bar, 100 μm.
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Figure 6: Expression of reaper in bub1 mutants. Imaginal discs were stained with X-gal to follow expression of a reaper-lacZ reporter construct (rpr-lacZ) in larvae of various genotypes. (A) rpr-lacZ/rpr-lacZ. (B) bub1/bub1 without the rpr reporter gene. (This control was necessitated by the fact that the P elements causing the bub1 mutations contain a lacZ gene.) (C and D) bub1/bub1; rpr-lacZ/rpr-lacZ imaginal discs showing very high levels of β-galactosidase expression, indicating that the cell death program is activated in many cells. Similar results were observed in the brains of these same animals (not shown). Bar, 100 μm.

Mentions: A striking feature of Drosophila bub1 mutants is the occurrence of significantly elevated frequencies of apoptotic nuclei in larval brains (Fig. 5 and Fig. 6). This result was unexpected, as expression of a dominant negative form of mouse Bub1 has been reported to reduce the frequency of apoptotic nuclei in nocodazole-treated cells (Taylor and McKeon 1997), implying that loss of checkpoint function prevents the apoptotic response. The reasons for the apparent dichotomy between our results in Drosophila and those from mouse tissue culture cells are not clear. These effects could be organism or cell type–specific, or the differences could reflect unusual consequences of the dominant negative forms of Bub1 utilized in the mouse study.


Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila.

Basu J, Bousbaa H, Logarinho E, Li Z, Williams BC, Lopes C, Sunkel CE, Goldberg ML - J. Cell Biol. (1999)

Expression of reaper in bub1 mutants. Imaginal discs were stained with X-gal to follow expression of a reaper-lacZ reporter construct (rpr-lacZ) in larvae of various genotypes. (A) rpr-lacZ/rpr-lacZ. (B) bub1/bub1 without the rpr reporter gene. (This control was necessitated by the fact that the P elements causing the bub1 mutations contain a lacZ gene.) (C and D) bub1/bub1; rpr-lacZ/rpr-lacZ imaginal discs showing very high levels of β-galactosidase expression, indicating that the cell death program is activated in many cells. Similar results were observed in the brains of these same animals (not shown). Bar, 100 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199734&req=5

Figure 6: Expression of reaper in bub1 mutants. Imaginal discs were stained with X-gal to follow expression of a reaper-lacZ reporter construct (rpr-lacZ) in larvae of various genotypes. (A) rpr-lacZ/rpr-lacZ. (B) bub1/bub1 without the rpr reporter gene. (This control was necessitated by the fact that the P elements causing the bub1 mutations contain a lacZ gene.) (C and D) bub1/bub1; rpr-lacZ/rpr-lacZ imaginal discs showing very high levels of β-galactosidase expression, indicating that the cell death program is activated in many cells. Similar results were observed in the brains of these same animals (not shown). Bar, 100 μm.
Mentions: A striking feature of Drosophila bub1 mutants is the occurrence of significantly elevated frequencies of apoptotic nuclei in larval brains (Fig. 5 and Fig. 6). This result was unexpected, as expression of a dominant negative form of mouse Bub1 has been reported to reduce the frequency of apoptotic nuclei in nocodazole-treated cells (Taylor and McKeon 1997), implying that loss of checkpoint function prevents the apoptotic response. The reasons for the apparent dichotomy between our results in Drosophila and those from mouse tissue culture cells are not clear. These effects could be organism or cell type–specific, or the differences could reflect unusual consequences of the dominant negative forms of Bub1 utilized in the mouse study.

Bottom Line: We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase.Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry.Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

View Article: PubMed Central - PubMed

Affiliation: Section of Genetics and Development, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
We have characterized the Drosophila mitotic checkpoint control protein Bub1 and obtained mutations in the bub1 gene. Drosophila Bub1 localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element-induced, near- mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants. We have also investigated the relationship between Bub1 and other kinetochore components. We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

Show MeSH
Related in: MedlinePlus