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Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis.

Bowman AB, Patel-King RS, Benashski SE, McCaffery JM, Goldstein LS, King SM - J. Cell Biol. (1999)

Bottom Line: The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here.Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein.We propose that roadblock/LC7 family members may modulate specific dynein functions.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, Department of Pharmacology, University of California San Diego, La Jolla, California 92093-0683, USA.

ABSTRACT
Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.

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Molecular analysis of LC7 from the Chlamydomonas outer dynein arm. (A) Two tryptic peptides from outer arm dynein LC7 were completely sequenced, yielding a total of 26 residue assignments. The actual mass of each peptide is in agreement with the calculated mass once methionine oxidation of the upper peptide is incorporated. (B) DNA and predicted protein sequence for the Chlamydomonas LC7 cDNA clone. Both peptide sequences are found in the coding region (26/26 residues correct). These sequences are indicated in bold type and are contiguous in the primary structure. The polyadenylation signal is underlined. This sequence is available in the NCBI GenBank (accession number AF140239). (C) Southern blot of genomic DNA from Chlamydomonas strain S1D2 digested with BamHI, PstI, PvuII, and SmaI and probed with the full-length LC7 cDNA. The data suggest that there is a single gene for LC7 in Chlamydomonas. (D) Northern blot analysis of RNA from nondeflagellated cells and from those actively regenerating flagella (30′ postDF). A single message of ∼0.95 kb that is induced in regenerating cells is evident.
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Figure 2: Molecular analysis of LC7 from the Chlamydomonas outer dynein arm. (A) Two tryptic peptides from outer arm dynein LC7 were completely sequenced, yielding a total of 26 residue assignments. The actual mass of each peptide is in agreement with the calculated mass once methionine oxidation of the upper peptide is incorporated. (B) DNA and predicted protein sequence for the Chlamydomonas LC7 cDNA clone. Both peptide sequences are found in the coding region (26/26 residues correct). These sequences are indicated in bold type and are contiguous in the primary structure. The polyadenylation signal is underlined. This sequence is available in the NCBI GenBank (accession number AF140239). (C) Southern blot of genomic DNA from Chlamydomonas strain S1D2 digested with BamHI, PstI, PvuII, and SmaI and probed with the full-length LC7 cDNA. The data suggest that there is a single gene for LC7 in Chlamydomonas. (D) Northern blot analysis of RNA from nondeflagellated cells and from those actively regenerating flagella (30′ postDF). A single message of ∼0.95 kb that is induced in regenerating cells is evident.

Mentions: The Chlamydomonas outer dynein arm contains eight distinct light chain components (Piperno and Luck 1979; Pfister et al. 1982). Previously, we cloned and described all of these proteins except for LC7. To clone LC7, we purified and sequenced two tryptic LC7 peptides isolated from outer arm dynein (Fig. 2 A). Based upon this sequence, PCR primers were designed and an LC7 cDNA clone isolated. The largest cDNA clone was 864 bp in length (Fig. 2 B) and contained a single open reading frame of 105 residues with a predicted mass of 11,928 D and a calculated pI of 7.85. Both peptide sequences obtained from purified LC7 were found in this clone (26/26 residues correct) and were both preceded by the predicted basic residue. Three in frame stop codons were present upstream of the first Met residue and a 489-bp 3′ untranslated region, including a perfect copy of the Chlamydomonas polyadenylation signal, followed the stop codon.


Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis.

Bowman AB, Patel-King RS, Benashski SE, McCaffery JM, Goldstein LS, King SM - J. Cell Biol. (1999)

Molecular analysis of LC7 from the Chlamydomonas outer dynein arm. (A) Two tryptic peptides from outer arm dynein LC7 were completely sequenced, yielding a total of 26 residue assignments. The actual mass of each peptide is in agreement with the calculated mass once methionine oxidation of the upper peptide is incorporated. (B) DNA and predicted protein sequence for the Chlamydomonas LC7 cDNA clone. Both peptide sequences are found in the coding region (26/26 residues correct). These sequences are indicated in bold type and are contiguous in the primary structure. The polyadenylation signal is underlined. This sequence is available in the NCBI GenBank (accession number AF140239). (C) Southern blot of genomic DNA from Chlamydomonas strain S1D2 digested with BamHI, PstI, PvuII, and SmaI and probed with the full-length LC7 cDNA. The data suggest that there is a single gene for LC7 in Chlamydomonas. (D) Northern blot analysis of RNA from nondeflagellated cells and from those actively regenerating flagella (30′ postDF). A single message of ∼0.95 kb that is induced in regenerating cells is evident.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199740&req=5

Figure 2: Molecular analysis of LC7 from the Chlamydomonas outer dynein arm. (A) Two tryptic peptides from outer arm dynein LC7 were completely sequenced, yielding a total of 26 residue assignments. The actual mass of each peptide is in agreement with the calculated mass once methionine oxidation of the upper peptide is incorporated. (B) DNA and predicted protein sequence for the Chlamydomonas LC7 cDNA clone. Both peptide sequences are found in the coding region (26/26 residues correct). These sequences are indicated in bold type and are contiguous in the primary structure. The polyadenylation signal is underlined. This sequence is available in the NCBI GenBank (accession number AF140239). (C) Southern blot of genomic DNA from Chlamydomonas strain S1D2 digested with BamHI, PstI, PvuII, and SmaI and probed with the full-length LC7 cDNA. The data suggest that there is a single gene for LC7 in Chlamydomonas. (D) Northern blot analysis of RNA from nondeflagellated cells and from those actively regenerating flagella (30′ postDF). A single message of ∼0.95 kb that is induced in regenerating cells is evident.
Mentions: The Chlamydomonas outer dynein arm contains eight distinct light chain components (Piperno and Luck 1979; Pfister et al. 1982). Previously, we cloned and described all of these proteins except for LC7. To clone LC7, we purified and sequenced two tryptic LC7 peptides isolated from outer arm dynein (Fig. 2 A). Based upon this sequence, PCR primers were designed and an LC7 cDNA clone isolated. The largest cDNA clone was 864 bp in length (Fig. 2 B) and contained a single open reading frame of 105 residues with a predicted mass of 11,928 D and a calculated pI of 7.85. Both peptide sequences obtained from purified LC7 were found in this clone (26/26 residues correct) and were both preceded by the predicted basic residue. Three in frame stop codons were present upstream of the first Met residue and a 489-bp 3′ untranslated region, including a perfect copy of the Chlamydomonas polyadenylation signal, followed the stop codon.

Bottom Line: The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here.Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein.We propose that roadblock/LC7 family members may modulate specific dynein functions.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, Department of Pharmacology, University of California San Diego, La Jolla, California 92093-0683, USA.

ABSTRACT
Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.

Show MeSH
Related in: MedlinePlus