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Figure 4

Figure 4. From: Drosophila roadblock and Chlamydomonas Lc7.

A large family of robl/LC7-like proteins. BLAST analysis has identified several mammalian ESTs, Drosophila genes, and a gene from C. elegans that are highly homologous to robl/LC7. (A) A dendrogram of the robl/LC7-like family members identified is shown. This dendrogram was generated using the GCG pileup command. We identified at least five Drosophila roadblock-like genes by searching the BDGP-derived ESTs and genomic sequences. Previously unidentified genes have been designated by their cytological location determined by BDGP. Also, two classes of robl/LC7-like genes have been identified as mammalian ESTs (identified by a representative EST accession number). (B) An alignment of the protein family is shown. The alignment was generated by the same GCG pileup command as an MSF file. Boxshade was used to illustrate aligned amino acid identity (dark shaded residues) and similarity (light shaded residues).

Aaron B. Bowman, et al. J Cell Biol. 1999 July 12;146(1):165-180.
Figure 5

Figure 5. From: Drosophila roadblock and Chlamydomonas Lc7.

Coimmunostaining of larval segmental nerves for (left column) SYT and (right column) ChAT revealed distal axonal accumulations of synaptic cargo in robl mutants. (A and B) In wild type, there is only a low background staining observed. (C and D) Segmental nerves from roblz hemizygotes have accumulations of SYT and ChAT. (E and F) In roblz hemizygotes the segmental nerves at the anterior, coming off of the ventral ganglion (located at the bottom left quadrant in images E and F), show a decreased frequency of SYT accumulations but an increased frequency of ChAT accumulations when compared with more posterior regions. (G and H) The segmental nerves located at the posterior show an increased frequency of SYT accumulations with a corresponding decreased frequency of ChAT accumulations. (I and J) Segmental nerves from robl null homozygotes (roblk/roblk) show abundant accumulations of SYT and ChAT.

Aaron B. Bowman, et al. J Cell Biol. 1999 July 12;146(1):165-180.
Figure 6

Figure 6. From: Drosophila roadblock and Chlamydomonas Lc7.

Transmission EM cross-sections of robl mutant third instar larval segmental nerves revealed two classes of axonal cargo accumulations and severe axonal loss and nerve degeneration. (A) Nerves from roblz hemizygous larvae had axons that swelled with transported material (dashed circles) and showed a loss of axons and nerve degeneration (area designated within arrows). (B) Axons from wild-type nerves do not show this swelled dense membranous axonal morphology. Two classes of axonal accumulations were observed in roblz hemizygotes: (C) small single component (small clear vesicles) axonal accumulations and (D) larger multi-component axonal accumulations. (E) Severely sluggish roblz hemizygous larvae showed increased axonal loss and degeneration. (F) All roblz homozygous larvae consistently showed a high degree of axonal loss and nerve degeneration. Bars: (A, B, E, and F) 500 nm; (C and D) 200 nm.

Aaron B. Bowman, et al. J Cell Biol. 1999 July 12;146(1):165-180.
Figure 3

Figure 3. From: Drosophila roadblock and Chlamydomonas Lc7.

LC7 is a component of the outer dynein arm. (A) Flagellar axonemes were prepared from wild-type Chlamydomonas (WT) and from mutants lacking the outer arm (oda9), inner arm I1 (ida1, ida2, and ida3), and inner arms I2/3 (ida4). Samples were electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie blue (upper panel) or blotted to nitrocellulose and probed with the R7178 antibody to detect LC7. The LC7 protein is highly reduced only in the strain lacking outer dynein arms. (B) A high salt extract of wild-type axonemes was sedimented through a 5–20% sucrose gradient and fractions electrophoresed in 5–15% acrylamide gels. Fraction 1 is the bottom of the gradient. The upper panel shows the gel stained with Coomassie blue, the lower panel is an immunoblot probed with R7178 to detect the LC7 protein. All the salt extractable LC7 protein precisely comigrates at ∼18 S with components of the outer dynein arm (e.g., IC1 and IC2).

Aaron B. Bowman, et al. J Cell Biol. 1999 July 12;146(1):165-180.
Figure 8

Figure 8. From: Drosophila roadblock and Chlamydomonas Lc7.

A robl/LC7-like protein is present in cytoplasmic dynein. (A) Western blot analysis was performed on samples from the fractionation of a rat brain homogenate. Blots were probed with mAb 74-1 and the R7178 rabbit polyclonal to detect IC74 of cytoplasmic dynein and the Mr ∼12,000 robl/LC7-like protein, respectively. (B) Rat brain proteins eluted from microtubules with ATP were sedimented in a 5–20% sucrose gradient. Samples were probed with the 74-1 and R7178 antibodies. The robl/LC7-like protein precisely comigrates with IC74 of cytoplasmic dynein. (C) Rat brain homogenate immunoprecipitates of cytoplasmic dynein (antibody 74-1), kinesin (antibody H-2), dynactin (antibody 50-1), and a bead control were probed with the R7178 antibody. The robl/LC7-like protein is detectable only in the cytoplasmic dynein (IC74) sample. (D) Drosophila embryo immunoprecipitates of cytoplasmic dynein (antibody 74-1) or the bead control were probed with the 6883 anti-robl antibody and 74-1 anti-IC74 antibody. The roadblock protein was only precipitated in the cytoplasmic dynein (IC74) sample. (E) Equally loaded Drosophila robl null and wild-type larval homogenates were probed with the 6883 anti-robl antibody and with the 3A5 anti-tubulin antibody. The roadblock protein is undetectable in the robl null larvae, whereas tubulin is detected at about equal levels in null and wild-type larvae.

Aaron B. Bowman, et al. J Cell Biol. 1999 July 12;146(1):165-180.
Figure 7

Figure 7. From: Drosophila roadblock and Chlamydomonas Lc7.

Severe mitotic defects were revealed in robl mutants examined by third instar larval brain squash analysis. Examples of typical wild-type mitotic figures are shown (designated by arrows): (A) a normal prometaphase figure, (B) a normal metaphase figure, and (C) a normal anaphase figure. Multiple abnormal mitotic figures are observed in roblz hemizygotes, including (designated by arrows): (D and E) aneuploid figures, (F) aneuploid figures with hypercondensed chromosomes, and (G) mutant anaphase figures with hypercondensed chromosomes disorganized around the presumptive poles. Mitotic figures are infrequently found in roblz homozygotes such as (designated by arrows): (H) a mutant anaphase structure with severe chromosome bridging, (I) another anaphase structure with a lagging chromosome and a single chromosome bridge, and (J) apparent telophase bridging. (K) The mitotic index of roblz hemizygotes is about five times that of wild type, the error bars indicate the SEM. roblz hemizygotes are late pupal lethal, when dissected from their pupal cases they show (L) rough pupal eyes. (M) A wild-type pupal eye is shown for comparison.

Aaron B. Bowman, et al. J Cell Biol. 1999 July 12;146(1):165-180.
Figure 9

Figure 9. From: Drosophila roadblock and Chlamydomonas Lc7.

Summary of dynein-associated proteins. (A) A complete table of outer dynein arm–associated light chains is shown. The nominal mass refers to the Mr determined by SDS-PAGE analysis; actual predicted mass of the proteins is given parenthetically. Previous work has elucidated biochemical interactions amongst these proteins. (B) A model of cytoplasmic dynein organization is shown. The cytoplasmic dynein particle is built around two heavy chains that form the stems and globular heads of the complex. Associated with the stems are a series of accessory proteins including: two IC74 intermediate chains that mediate dynein–dynactin interactions, two copies of the Tctex1 light chain (or of the related rp3 protein), one dimer of the highly conserved 10 kD/LC8 DLC (LC8 dimer), and a 22-kD polypeptide (the location of which is speculative). The present study indicates that cytoplasmic dynein also contains a robl/LC7-like protein that, by analogy with flagellar outer arm dynein, is located at the base of the dynein particle. A table of known proteins associated with cytoplasmic dynein is given; the majority are conserved in axonemal dyneins.

Aaron B. Bowman, et al. J Cell Biol. 1999 July 12;146(1):165-180.
Figure 2

Figure 2. From: Drosophila roadblock and Chlamydomonas Lc7.

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.

Aaron B. Bowman, et al. J Cell Biol. 1999 July 12;146(1):165-180.
Figure 1

Figure 1. From: Drosophila roadblock and Chlamydomonas Lc7.

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.

Aaron B. Bowman, et al. J Cell Biol. 1999 July 12;146(1):165-180.

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