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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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Specificity of affinity-purified anti-Drosophila Bub1 antibodies. Identical amounts of Drosophila third instar larval brain extracts, from either wild-type (Oregon R) or bub1 mutant homozygotes, were loaded onto the indicated lanes of a Western blot probed with affinity-purified anti-Drosophila Bub1 antibodies (see Materials and Methods). A doublet of ∼165 kD (the predicted size for Drosophila Bub1) is recognized in wild-type extracts. These two bands are almost completely absent in brain extracts made from bub1 mutant brains. The antibody also recognizes a band of ∼100 kD that is not Bub1-specific and that is also recognized by pre-immune IgY. This cross-reacting band serves as an internal loading control.
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Figure 1: Specificity of affinity-purified anti-Drosophila Bub1 antibodies. Identical amounts of Drosophila third instar larval brain extracts, from either wild-type (Oregon R) or bub1 mutant homozygotes, were loaded onto the indicated lanes of a Western blot probed with affinity-purified anti-Drosophila Bub1 antibodies (see Materials and Methods). A doublet of ∼165 kD (the predicted size for Drosophila Bub1) is recognized in wild-type extracts. These two bands are almost completely absent in brain extracts made from bub1 mutant brains. The antibody also recognizes a band of ∼100 kD that is not Bub1-specific and that is also recognized by pre-immune IgY. This cross-reacting band serves as an internal loading control.

Mentions: In order to examine Drosophila Bub1 distribution during the cell cycle, affinity-purified antibodies were generated against a LacZ/Bub1 fusion protein as described in Materials and Methods. Affinity-purified IgY identifies two bands of ∼165 kD (the predicted molecular mass for Drosophila Bub1) on Western blots of larval brain extracts. These bands disappear almost completely in brain extracts made from bub1 mutants to levels <2–3% of wild-type (Fig. 1), and are completely absent in identical blots probed with preimmune IgY made from the same chickens (not shown). The antibody preparations also recognize a 100-kD band unaffected by bub1 mutations (Fig. 1); this band is also seen when blots are probed with preimmune IgY (not shown). The two Bub1-specific bands probably represent alternatively spliced or phosphorylated forms of Bub1 as shown previously by Roberts et al. 1994. The near complete absence of these bands in extracts from l(2)K06109 and l(2)K03113 homozygotes indicate that these mutations represent strongly hypomorphic, near alleles of Drosophila bub1. It is possible that the residual low levels (seen at longer exposures) represent the perdurance of maternally supplied product contributed by the heterozygous mothers of these mutant animals.


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)

Specificity of affinity-purified anti-Drosophila Bub1 antibodies. Identical amounts of Drosophila third instar larval brain extracts, from either wild-type (Oregon R) or bub1 mutant homozygotes, were loaded onto the indicated lanes of a Western blot probed with affinity-purified anti-Drosophila Bub1 antibodies (see Materials and Methods). A doublet of ∼165 kD (the predicted size for Drosophila Bub1) is recognized in wild-type extracts. These two bands are almost completely absent in brain extracts made from bub1 mutant brains. The antibody also recognizes a band of ∼100 kD that is not Bub1-specific and that is also recognized by pre-immune IgY. This cross-reacting band serves as an internal loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199734&req=5

Figure 1: Specificity of affinity-purified anti-Drosophila Bub1 antibodies. Identical amounts of Drosophila third instar larval brain extracts, from either wild-type (Oregon R) or bub1 mutant homozygotes, were loaded onto the indicated lanes of a Western blot probed with affinity-purified anti-Drosophila Bub1 antibodies (see Materials and Methods). A doublet of ∼165 kD (the predicted size for Drosophila Bub1) is recognized in wild-type extracts. These two bands are almost completely absent in brain extracts made from bub1 mutant brains. The antibody also recognizes a band of ∼100 kD that is not Bub1-specific and that is also recognized by pre-immune IgY. This cross-reacting band serves as an internal loading control.
Mentions: In order to examine Drosophila Bub1 distribution during the cell cycle, affinity-purified antibodies were generated against a LacZ/Bub1 fusion protein as described in Materials and Methods. Affinity-purified IgY identifies two bands of ∼165 kD (the predicted molecular mass for Drosophila Bub1) on Western blots of larval brain extracts. These bands disappear almost completely in brain extracts made from bub1 mutants to levels <2–3% of wild-type (Fig. 1), and are completely absent in identical blots probed with preimmune IgY made from the same chickens (not shown). The antibody preparations also recognize a 100-kD band unaffected by bub1 mutations (Fig. 1); this band is also seen when blots are probed with preimmune IgY (not shown). The two Bub1-specific bands probably represent alternatively spliced or phosphorylated forms of Bub1 as shown previously by Roberts et al. 1994. The near complete absence of these bands in extracts from l(2)K06109 and l(2)K03113 homozygotes indicate that these mutations represent strongly hypomorphic, near alleles of Drosophila bub1. It is possible that the residual low levels (seen at longer exposures) represent the perdurance of maternally supplied product contributed by the heterozygous mothers of these mutant animals.

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