Limits...
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.

Show MeSH

Related in: MedlinePlus

The robl genomic interval. (A) A diagrammatic map of the five genes identified in the genomic region around roadblock (accession number AF141921). The entire region has been sequenced and cDNAs have been obtained for robl and genes 1 and 4. Gene 3 is a roadblock-like region, which is likely a pseudogene as it lacks any identifiable start codon. The two partially overlapping deficiencies robll(2)k10408 and roblc identify the roadblock genomic region, dotted lines correspond to regions missing in deficiencies. The EMS mutant roblz deleted a small region in one of these genes allowing us to identify it as robl. The genomic rescue region shown completely rescues roblz/robll(2)k10408. (B) The genomic sequence of the region encoding roadblock (corresponding to nucleotides 7,751–8,214 of genomic interval illustrated in A). Uppercase characters show the protein coding sequence that is translated below for each codon; lowercase characters are used to show the 5′-UTR, introns 1 and 2, and 3′-UTR. The EMS mutant roblz has a 193-bp deletion that is represented by bold characters. The deletion extends from intron 2 into the COOH terminus encoding exon 3, removing the intron's conserved branch point sequence that is underlined. Since roblz is a recessive neomorphic allele, a partially functional or aberrant protein is likely made. Reverse transcriptase–PCR analysis of roblz indicates that splicing of mutant intron 2 does not occur (data not shown). However, the mutant transcript maintains the correct reading frame through the remainder of intron 2 and exon 3. The resulting roblz protein would have an internal deletion of 54 residues (IPVKST…HEIMVA) replaced by a 12-residue insertion (GWFNCTSVCAKI) from the remainder of intron 2.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199740&req=5

Figure 1: The robl genomic interval. (A) A diagrammatic map of the five genes identified in the genomic region around roadblock (accession number AF141921). The entire region has been sequenced and cDNAs have been obtained for robl and genes 1 and 4. Gene 3 is a roadblock-like region, which is likely a pseudogene as it lacks any identifiable start codon. The two partially overlapping deficiencies robll(2)k10408 and roblc identify the roadblock genomic region, dotted lines correspond to regions missing in deficiencies. The EMS mutant roblz deleted a small region in one of these genes allowing us to identify it as robl. The genomic rescue region shown completely rescues roblz/robll(2)k10408. (B) The genomic sequence of the region encoding roadblock (corresponding to nucleotides 7,751–8,214 of genomic interval illustrated in A). Uppercase characters show the protein coding sequence that is translated below for each codon; lowercase characters are used to show the 5′-UTR, introns 1 and 2, and 3′-UTR. The EMS mutant roblz has a 193-bp deletion that is represented by bold characters. The deletion extends from intron 2 into the COOH terminus encoding exon 3, removing the intron's conserved branch point sequence that is underlined. Since roblz is a recessive neomorphic allele, a partially functional or aberrant protein is likely made. Reverse transcriptase–PCR analysis of roblz indicates that splicing of mutant intron 2 does not occur (data not shown). However, the mutant transcript maintains the correct reading frame through the remainder of intron 2 and exon 3. The resulting roblz protein would have an internal deletion of 54 residues (IPVKST…HEIMVA) replaced by a 12-residue insertion (GWFNCTSVCAKI) from the remainder of intron 2.

Mentions: Two overlapping deficiencies, robll(2)k10408 and roblc, identify the genomic interval encoding robl (Fig. 1 A). Sequencing of the entire genomic interval identified five putative gene candidates that may be affected by both deficiencies. To identify which gene encoded robl, we sequenced roblz and discovered a 193-bp deletion in the middle of a small transcription unit in the interval that we believe to be robl for several reasons. First, a 5-kb segment of this region that contains only robl, and one adjacent gene, was found to fully rescue all above-mentioned phenotypes in roblz hemizygotes. Second, this gene adjacent to robl was sequenced from roblz and found to be unaltered from the wild-type parental chromosome. In fact, this gene appears to be a robl pseudogene because it lacks any identifiable start codon. Third, robll(2)k10408 homozygotes are fully rescued by the genomic rescue construct that indicates that other genes in this interval are not essential and the observed phenotypes are robl-dependent. Finally, an NH2-terminal His-tagged robl cDNA construct under control of the hsp70Bb promoter fully rescues male roblz hemizygotes if given daily heat shock. Reducing the frequency of heat shocks results in a restoration of the described robl phenotype. This cDNA construct does not rescue an apparent female sterility seen in the rescued roblz hemizygotes, despite full rescue of all other observed robl phenotypes. Nevertheless, taken together, these data establish that the gene identified by the roblz deletion is roadblock.


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)

The robl genomic interval. (A) A diagrammatic map of the five genes identified in the genomic region around roadblock (accession number AF141921). The entire region has been sequenced and cDNAs have been obtained for robl and genes 1 and 4. Gene 3 is a roadblock-like region, which is likely a pseudogene as it lacks any identifiable start codon. The two partially overlapping deficiencies robll(2)k10408 and roblc identify the roadblock genomic region, dotted lines correspond to regions missing in deficiencies. The EMS mutant roblz deleted a small region in one of these genes allowing us to identify it as robl. The genomic rescue region shown completely rescues roblz/robll(2)k10408. (B) The genomic sequence of the region encoding roadblock (corresponding to nucleotides 7,751–8,214 of genomic interval illustrated in A). Uppercase characters show the protein coding sequence that is translated below for each codon; lowercase characters are used to show the 5′-UTR, introns 1 and 2, and 3′-UTR. The EMS mutant roblz has a 193-bp deletion that is represented by bold characters. The deletion extends from intron 2 into the COOH terminus encoding exon 3, removing the intron's conserved branch point sequence that is underlined. Since roblz is a recessive neomorphic allele, a partially functional or aberrant protein is likely made. Reverse transcriptase–PCR analysis of roblz indicates that splicing of mutant intron 2 does not occur (data not shown). However, the mutant transcript maintains the correct reading frame through the remainder of intron 2 and exon 3. The resulting roblz protein would have an internal deletion of 54 residues (IPVKST…HEIMVA) replaced by a 12-residue insertion (GWFNCTSVCAKI) from the remainder of intron 2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199740&req=5

Figure 1: The robl genomic interval. (A) A diagrammatic map of the five genes identified in the genomic region around roadblock (accession number AF141921). The entire region has been sequenced and cDNAs have been obtained for robl and genes 1 and 4. Gene 3 is a roadblock-like region, which is likely a pseudogene as it lacks any identifiable start codon. The two partially overlapping deficiencies robll(2)k10408 and roblc identify the roadblock genomic region, dotted lines correspond to regions missing in deficiencies. The EMS mutant roblz deleted a small region in one of these genes allowing us to identify it as robl. The genomic rescue region shown completely rescues roblz/robll(2)k10408. (B) The genomic sequence of the region encoding roadblock (corresponding to nucleotides 7,751–8,214 of genomic interval illustrated in A). Uppercase characters show the protein coding sequence that is translated below for each codon; lowercase characters are used to show the 5′-UTR, introns 1 and 2, and 3′-UTR. The EMS mutant roblz has a 193-bp deletion that is represented by bold characters. The deletion extends from intron 2 into the COOH terminus encoding exon 3, removing the intron's conserved branch point sequence that is underlined. Since roblz is a recessive neomorphic allele, a partially functional or aberrant protein is likely made. Reverse transcriptase–PCR analysis of roblz indicates that splicing of mutant intron 2 does not occur (data not shown). However, the mutant transcript maintains the correct reading frame through the remainder of intron 2 and exon 3. The resulting roblz protein would have an internal deletion of 54 residues (IPVKST…HEIMVA) replaced by a 12-residue insertion (GWFNCTSVCAKI) from the remainder of intron 2.
Mentions: Two overlapping deficiencies, robll(2)k10408 and roblc, identify the genomic interval encoding robl (Fig. 1 A). Sequencing of the entire genomic interval identified five putative gene candidates that may be affected by both deficiencies. To identify which gene encoded robl, we sequenced roblz and discovered a 193-bp deletion in the middle of a small transcription unit in the interval that we believe to be robl for several reasons. First, a 5-kb segment of this region that contains only robl, and one adjacent gene, was found to fully rescue all above-mentioned phenotypes in roblz hemizygotes. Second, this gene adjacent to robl was sequenced from roblz and found to be unaltered from the wild-type parental chromosome. In fact, this gene appears to be a robl pseudogene because it lacks any identifiable start codon. Third, robll(2)k10408 homozygotes are fully rescued by the genomic rescue construct that indicates that other genes in this interval are not essential and the observed phenotypes are robl-dependent. Finally, an NH2-terminal His-tagged robl cDNA construct under control of the hsp70Bb promoter fully rescues male roblz hemizygotes if given daily heat shock. Reducing the frequency of heat shocks results in a restoration of the described robl phenotype. This cDNA construct does not rescue an apparent female sterility seen in the rescued roblz hemizygotes, despite full rescue of all other observed robl phenotypes. Nevertheless, taken together, these data establish that the gene identified by the roblz deletion is roadblock.

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