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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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Asp expression constructs. The upper portion of the  figure shows the asp cDNA indicating restriction cleavage sites  and localization of the putative actin and calmodulin binding  sites. Segments of the protein expressed in E. coli are indicated  by shaded bars. The truncated protein expressed in flies transformed by pMBO1366 is indicated by a solid line.
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Figure 4: Asp expression constructs. The upper portion of the figure shows the asp cDNA indicating restriction cleavage sites and localization of the putative actin and calmodulin binding sites. Segments of the protein expressed in E. coli are indicated by shaded bars. The truncated protein expressed in flies transformed by pMBO1366 is indicated by a solid line.

Mentions: To further characterize the asp gene product, we raised rabbit polyclonal antibodies to a truncated Asp protein expressed in E. coli. The NcoI–BamHI fragment of cDNA 6a (Fig. 4) was subcloned into the expression vector pET23d. The resulting construct, pASP36, expresses a polypeptide corresponding to amino acid residues 1–512, with a predicted molecular weight of 55.9 kD. This polypeptide does not contain the putative actin binding domain 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)

Asp expression constructs. The upper portion of the  figure shows the asp cDNA indicating restriction cleavage sites  and localization of the putative actin and calmodulin binding  sites. Segments of the protein expressed in E. coli are indicated  by shaded bars. The truncated protein expressed in flies transformed by pMBO1366 is indicated by a solid line.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Asp expression constructs. The upper portion of the figure shows the asp cDNA indicating restriction cleavage sites and localization of the putative actin and calmodulin binding sites. Segments of the protein expressed in E. coli are indicated by shaded bars. The truncated protein expressed in flies transformed by pMBO1366 is indicated by a solid line.
Mentions: To further characterize the asp gene product, we raised rabbit polyclonal antibodies to a truncated Asp protein expressed in E. coli. The NcoI–BamHI fragment of cDNA 6a (Fig. 4) was subcloned into the expression vector pET23d. The resulting construct, pASP36, expresses a polypeptide corresponding to amino acid residues 1–512, with a predicted molecular weight of 55.9 kD. This polypeptide does not contain the putative actin binding domain 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