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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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Developmental pattern of expression of asp and  its flanking transcripts. Northern blot analysis of asp expression (asp transcript is  ∼6.5 kb). An Nco1–EcoRI  fragment from the 6.5-kb asp  cDNA clone was used as the  probe. Poly A+ is from 0–3  h-old embryos (lane 1), 3–6  h-old embryos (lane 2), first  instar larvae (lane 3), second instar larvae (lane 4),  third instar larvae (lane 5),  adult females (lane 6), and  adult males (lane 7).
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Figure 2: Developmental pattern of expression of asp and its flanking transcripts. Northern blot analysis of asp expression (asp transcript is ∼6.5 kb). An Nco1–EcoRI fragment from the 6.5-kb asp cDNA clone was used as the probe. Poly A+ is from 0–3 h-old embryos (lane 1), 3–6 h-old embryos (lane 2), first instar larvae (lane 3), second instar larvae (lane 4), third instar larvae (lane 5), adult females (lane 6), and adult males (lane 7).

Mentions: Plasmid pASP36 was constructed by inserting the NcoI–BamHI fragment from p6a (see Fig. 2) into the expression vector pET23d (Invitrogen Corp., San Diego, CA). Conditions for culture and induction were as described by the supplier. The polypeptide expressed by this clone represents the NH2-terminal portion of the Asp protein and does not include the putative actin binding domain. This polypeptide was insoluble and was purified by preparing inclusion bodies, separating the proteins by polyacrylamide gel electrophoresis, excising the appropriate band from the gel, and electroeluting the protein as described by Leppard et al. (1983). Polyclonal sera were prepared by injecting rabbits as described (Harlow and Lane, 1988).


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)

Developmental pattern of expression of asp and  its flanking transcripts. Northern blot analysis of asp expression (asp transcript is  ∼6.5 kb). An Nco1–EcoRI  fragment from the 6.5-kb asp  cDNA clone was used as the  probe. Poly A+ is from 0–3  h-old embryos (lane 1), 3–6  h-old embryos (lane 2), first  instar larvae (lane 3), second instar larvae (lane 4),  third instar larvae (lane 5),  adult females (lane 6), and  adult males (lane 7).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Developmental pattern of expression of asp and its flanking transcripts. Northern blot analysis of asp expression (asp transcript is ∼6.5 kb). An Nco1–EcoRI fragment from the 6.5-kb asp cDNA clone was used as the probe. Poly A+ is from 0–3 h-old embryos (lane 1), 3–6 h-old embryos (lane 2), first instar larvae (lane 3), second instar larvae (lane 4), third instar larvae (lane 5), adult females (lane 6), and adult males (lane 7).
Mentions: Plasmid pASP36 was constructed by inserting the NcoI–BamHI fragment from p6a (see Fig. 2) into the expression vector pET23d (Invitrogen Corp., San Diego, CA). Conditions for culture and induction were as described by the supplier. The polypeptide expressed by this clone represents the NH2-terminal portion of the Asp protein and does not include the putative actin binding domain. This polypeptide was insoluble and was purified by preparing inclusion bodies, separating the proteins by polyacrylamide gel electrophoresis, excising the appropriate band from the gel, and electroeluting the protein as described by Leppard et al. (1983). Polyclonal sera were prepared by injecting rabbits as described (Harlow and Lane, 1988).

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