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The Drosophila gene abnormal spindle encodes a novel microtubule-associated protein that associates with the polar regions of the mitotic spindle.

Saunders RD, Avides MC, Howard T, Gonzalez C, Glover DM - J. Cell Biol. (1997)

Bottom Line: Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins.The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins.Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Campaign, Cell Cycle Genetics Group, Department of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, Scotland.

ABSTRACT
abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp1 directs the synthesis of a COOH terminally truncated or internally deleted peptide of approximately 124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH2-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34(cdc2) and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.

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Shortened proteins are produced by the asp1 mutant  and by pMBO1366 transformants. Western blot analysis of asp  expression using the antibody Rb3133. (Lane 1) Proteins from 8  wild-type larval brains; (lane 2) proteins from 8 brains of larvae  transformed by pMBO1366; (lane 3) proteins from 12 asp1/asp1  larval brains.
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Figure 5: Shortened proteins are produced by the asp1 mutant and by pMBO1366 transformants. Western blot analysis of asp expression using the antibody Rb3133. (Lane 1) Proteins from 8 wild-type larval brains; (lane 2) proteins from 8 brains of larvae transformed by pMBO1366; (lane 3) proteins from 12 asp1/asp1 larval brains.

Mentions: The resulting serum Rb3133 recognizes one polypeptide of ∼220 kD in immunoblots of third instar larval brains (Fig. 5). To confirm that Rb3133 reacts specifically with the Asp protein, brains from larvae of the transgenic strain 1366 (see Materials and Methods) were used in Western blot analysis (Fig. 5, lane 2). Flies transformed with pMBO1366 are expected to synthesize two forms of the Asp protein: full length protein of 220 kD, derived from the endogenous copy of asp at 96A and a truncated form derived from the transgene. As expected, the serum detects the 220-kD wild-type asp protein plus a 124-kD polypeptide corresponding to the COOH-terminal truncated form of the asp protein encoded by plasmid pMBO1366. When extracts from brains of asp1 homozygous larvae were analyzed (Fig. 5, lane 3), the 220-kD protein is no longer observed, but the serum labels a polypeptide of ∼130 kD. As the polyclonal antibodies were raised against the NH2 terminus of the Asp protein, it seems that the asp1 mutation results in a COOH terminus truncation or in an internal deletion of the Asp protein.


The Drosophila gene abnormal spindle encodes a novel microtubule-associated protein that associates with the polar regions of the mitotic spindle.

Saunders RD, Avides MC, Howard T, Gonzalez C, Glover DM - J. Cell Biol. (1997)

Shortened proteins are produced by the asp1 mutant  and by pMBO1366 transformants. Western blot analysis of asp  expression using the antibody Rb3133. (Lane 1) Proteins from 8  wild-type larval brains; (lane 2) proteins from 8 brains of larvae  transformed by pMBO1366; (lane 3) proteins from 12 asp1/asp1  larval brains.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139842&req=5

Figure 5: Shortened proteins are produced by the asp1 mutant and by pMBO1366 transformants. Western blot analysis of asp expression using the antibody Rb3133. (Lane 1) Proteins from 8 wild-type larval brains; (lane 2) proteins from 8 brains of larvae transformed by pMBO1366; (lane 3) proteins from 12 asp1/asp1 larval brains.
Mentions: The resulting serum Rb3133 recognizes one polypeptide of ∼220 kD in immunoblots of third instar larval brains (Fig. 5). To confirm that Rb3133 reacts specifically with the Asp protein, brains from larvae of the transgenic strain 1366 (see Materials and Methods) were used in Western blot analysis (Fig. 5, lane 2). Flies transformed with pMBO1366 are expected to synthesize two forms of the Asp protein: full length protein of 220 kD, derived from the endogenous copy of asp at 96A and a truncated form derived from the transgene. As expected, the serum detects the 220-kD wild-type asp protein plus a 124-kD polypeptide corresponding to the COOH-terminal truncated form of the asp protein encoded by plasmid pMBO1366. When extracts from brains of asp1 homozygous larvae were analyzed (Fig. 5, lane 3), the 220-kD protein is no longer observed, but the serum labels a polypeptide of ∼130 kD. As the polyclonal antibodies were raised against the NH2 terminus of the Asp protein, it seems that the asp1 mutation results in a COOH terminus truncation or in an internal deletion of the Asp protein.

Bottom Line: Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins.The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins.Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Campaign, Cell Cycle Genetics Group, Department of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, Scotland.

ABSTRACT
abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp1 directs the synthesis of a COOH terminally truncated or internally deleted peptide of approximately 124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH2-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34(cdc2) and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.

Show MeSH
Related in: MedlinePlus