scholarly journals Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins.

1994 ◽  
Vol 5 (1) ◽  
pp. 57-70 ◽  
Author(s):  
B H Gibbons ◽  
D J Asai ◽  
W J Tang ◽  
T S Hays ◽  
I R Gibbons
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Sea Urchins ◽  
Early Stage ◽  
Single Gene ◽  
Cytoplasmic Dynein ◽  
Phosphate Binding ◽  
Atp Binding Site ◽  
Heavy Chains ◽  
Amino Acid Region

Transcripts approximately 14.5 kilobases in length from 14 different genes that encode for dynein heavy chains have been identified in poly(A)+ RNA from sea urchin embryos. Analysis of the changes in level of these dynein transcripts in response to deciliation, together with their sequence relatedness, suggests that 11 or more of these genes encode dynein isoforms that participate in regeneration of external cilia on the embryo, whereas the single gene whose deduced sequence closely resembles that of cytoplasmic dynein in other organisms appears not to be involved in this regeneration. The four consensus motifs for phosphate binding found previously in the beta heavy chain of sea urchin dynein are present in all five additional isoforms for which extended sequences have been obtained, suggesting that these sites play a significant role in dynein function. Sequence analysis of a approximately 400 amino acid region encompassing the putative hydrolytic ATP-binding site shows that the dynein genes fall into at least six distinct classes. Most of these classes in sea urchin have a high degree of sequence identity with one of the dynein heavy chain genes identified in Drosophila, indicating that the radiation of the dynein gene family into the present classes occurred at an early stage in the evolution of eukaryotes. Evolutionary changes in cytoplasmic dynein have been more constrained than those in the axonemal dyneins.

scholarly journals A map of photolytic and tryptic cleavage sites on the beta heavy chain of dynein ATPase from sea urchin sperm flagella.

1988 ◽  
Vol 106 (5) ◽  
pp. 1607-1614 ◽  
Author(s):  
G Mocz ◽  
W J Tang ◽  
I R Gibbons
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Terminal Region ◽  
Beta Chain ◽  
Phosphate Binding ◽  
Atp Binding Site ◽  
Heavy Chains ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Sea Urchin Sperm

NH2-terminal analysis of the alpha and beta heavy chain polypeptides (Mr greater than 400,000) from the outer arm dynein of sea urchin sperm flagella, compared with that of the 230,000- and 200,000-Mr peptides formed upon photocleavage of dynein by irradiation at 365 nm in the presence of vanadate and ATP, shows that the NH2 termini of the intact chains are acetylated and that the 230,000- and 200,000 Mr peptides constitute the amino- and carboxy-terminal portions of the heavy chains, respectively. Tryptic digestion of the beta heavy chain is known to separate it into two particles, termed fragments A and B, that sediment at 12S and 6S (Ow, R. A., W.-J. Y. Tang, G. Mocz, and I. R. Gibbons, 1987. J. Biol. Chem. 262:3409-3414). Immunoblots against monoclonal antibodies specific for epitopes on the beta heavy chain, used in conjunction with photoaffinity labeling, show that the ATPase-containing fragment A is derived from the amino-terminal region of the beta chain, with the two photolytic sites thought to be associated with the purine-binding and the gamma-phosphate-binding areas of the ATP-binding site spanning an approximately 100,000 Mr region near the middle of the intact beta chain. Fragment B is derived from the complementary carboxy-terminal region of the beta chain.

scholarly journals A family of dynein genes in Drosophila melanogaster.

1994 ◽  
Vol 5 (1) ◽  
pp. 45-55 ◽  
Author(s):  
K Rasmusson ◽  
M Serr ◽  
J Gepner ◽  
I Gibbons ◽  
T S Hays
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Amino Acid Level ◽  
Single Copy ◽  
Cytoplasmic Dynein ◽  
Amino Acid Identity ◽  
Chain Gene ◽  
Phosphate Binding ◽  
Atp Binding Site ◽  
Dynein Heavy Chain

We report the identification and initial characterization of seven Drosophila dynein heavy chain genes. Each gene is single copy and maps to a unique genomic location. Sequence analysis of partial clones reveals that each encodes a highly conserved portion of the putative dynein hydrolytic ATP-binding site in dyneins that includes a consensus phosphate-binding (P-loop) motif. One of the clones is derived from a Drosophila cytoplasmic dynein heavy chain gene, Dhc64C, that shows extensive amino acid identity to cytoplasmic dynein isoforms from other organisms. Two other Drosophila dynein clones are 85 and 90% identical at the amino acid level to the corresponding region of the beta heavy chain of sea urchin axonemal dynein. Probes for all seven of the dynein-related sequences hybridize to transcripts that are of the appropriate size, approximately 14 kilobases, to encode the characteristic high molecular weight dynein heavy chain polypeptides. The Dhc64C transcript is readily detected in RNA from ovaries, embryos, and testes. Transcripts from five of the six remaining genes are also detected in much lesser amounts in tissues other than testes. All but one of the dynein transcripts are expressed at comparable levels in testes suggesting their participation in flagellar axoneme assembly and motility.

Sequence analysis of the Chlamydomonas alpha and beta dynein heavy chain genes

1994 ◽  
Vol 107 (3) ◽  
pp. 635-644 ◽  
Author(s):  
D.R. Mitchell ◽  
K.S. Brown
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Catalytic Domain ◽  
Nucleotide Binding ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Coding Region ◽  
Gamma Chain ◽  
Heavy Chains ◽  
Dynein Heavy Chains

We have sequenced genomic clones spanning the complete coding region of one heavy chain (beta) and the catalytic domain of a second (alpha) of the Chlamydomonas reinhardtii flagellar outer arm dynein ATPase. The beta heavy chain gene (ODA-4 locus) spans 20 kb, is divided into at least 30 exons, and encodes a predicted 520 kDa protein. Comparison with sea urchin beta dynein sequences reveals homology that extends throughout both proteins. Over the most conserved central catalytic region, the Chlamydomonas alpha and beta chains are equally divergent from the sea urchin beta chain (64% and 65% similarity, respectively), whereas the Chlamydomonas gamma chain is more divergent from urchin beta (54% similarity). The four glycine-rich loops identified as potential nucleotide-binding sites in other dynein heavy chains are also present in Chlamydomonas alpha and beta dyneins. Two of these four nucleotide-binding motifs are highly conserved among flagellar dyneins, but only the motif previously identified as the catalytic site in sea urchin dynein is highly conserved between flagellar and cytoplasmic dynein heavy chains. Predictions of secondary structure suggest that all dynein heavy chains possess three large domains, with the four nucleotide-binding consensus sequences located in a central 185 kDa domain that is bounded on both sides by regions that form multiple, short alpha-helical coiled-coils.

scholarly journals Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains.

1988 ◽  
Vol 107 (6) ◽  
pp. 2657-2667 ◽  
Author(s):  
A L Ingold ◽  
S A Cohn ◽  
J M Scholey
Keyword(s):  
Monoclonal Antibodies ◽  
Sea Urchin ◽  
Heavy Chain ◽  
Bovine Brain ◽  
Drosophila Embryo ◽  
Detectable Effect ◽  
Heavy Chains ◽  
Sea Urchin Egg ◽  
Dependent Inhibition ◽  
Mgatpase Activity

We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo 116-kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesin-induced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP-sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility.

scholarly journals Cytoplasmic Dynein Heavy Chain 1b Is Required for Flagellar Assembly in Chlamydomonas

1999 ◽  
Vol 10 (3) ◽  
pp. 693-712 ◽  
Author(s):  
Mary E. Porter ◽  
Raqual Bower ◽  
Julie A. Knott ◽  
Pamela Byrd ◽  
William Dentler
Keyword(s):  
Caenorhabditis Elegans ◽  
Expression Pattern ◽  
Heavy Chain ◽  
Insertional Mutagenesis ◽  
Sea Urchins ◽  
Amorphous Material ◽  
Cytoplasmic Dynein ◽  
Genetic Approach ◽  
Dynein Heavy Chain ◽  
Flagellar Assembly

A second cytoplasmic dynein heavy chain (cDhc) has recently been identified in several organisms, and its expression pattern is consistent with a possible role in axoneme assembly. We have used a genetic approach to ask whether cDhc1b is involved in flagellar assembly in Chlamydomonas. Using a modified PCR protocol, we recovered two cDhc sequences distinct from the axonemal Dhc sequences identified previously. cDhc1a is closely related to the major cytoplasmic Dhc, whereas cDhc1b is closely related to the minor cDhc isoform identified in sea urchins, Caenorhabditis elegans, and Tetrahymena. TheChlamydomonas cDhc1b transcript is a low-abundance mRNA whose expression is enhanced by deflagellation. To determine its role in flagellar assembly, we screened a collection of stumpy flagellar (stf) mutants generated by insertional mutagenesis and identified two strains in which portions of the cDhc1bgene have been deleted. The two mutants assemble short flagellar stumps (<1–2 μm) filled with aberrant microtubules, raft-like particles, and other amorphous material. The results indicate that cDhc1b is involved in the transport of components required for flagellar assembly in Chlamydomonas.

Isolation and characterization of a novel dynein that contains C and A heavy chains from sea urchin sperm flagellar axonemes

1994 ◽  
Vol 107 (2) ◽  
pp. 345-351 ◽  
Author(s):  
E. Yokota ◽  
I. Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Heavy Chain ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Heavy Chains ◽  
Isolation And Characterization ◽  
Sea Urchin Sperm

A novel dynein (C/A dynein), which is composed of C and A heavy chains, two intermediate chains and several light chains, was isolated from sea urchin sperm flagella. The C/A dynein was released by the treatment with 0.7 M NaCl plus 5 mM ATP from the axonemes depleted of outer arm 21 S dynein. Sedimentation coefficient of this dynein was estimated by sucrose density gradient centrifugation to be 22–23 S. The C/A dynein particle appeared to be composed of three distinct domains; two globular head domains and one rod domain as seen by negative staining electron microscopy. The mobility of ‘A’ heavy chain of C/A dynein on SDS-gel electrophoresis was similar to that of A heavy chains (A alpha and A beta) of 21 S dynein. However, UV-cleavage patterns of C and A heavy chains of C/A dynein were different from those of A heavy chains of 21 S dynein. Furthermore, an antiserum raised against A heavy chain of C/A dynein did not crossreact with A heavy chains of 21 S dynein. Under the conditions in which the C/A dynein was released, some of inner arms were removed concomitantly from axonemes as observed by electron microscopy. These results suggested that C/A dynein is a component of the inner arms.

A comparative study of repeated sequences in the SM50 gene of some sea urchins

Zygote ◽  
1999 ◽  
Vol 8 (S1) ◽  
pp. S75-S75
Author(s):  
Masayuki Goto ◽  
Masahiro Matsumoto ◽  
Takashi Kitajima ◽  
Akiya Hino
Keyword(s):  
Amino Acid ◽  
Sea Urchin ◽  
Sequence Data ◽  
Sea Urchins ◽  
Rabbit Antiserum ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Immunofluorescent Staining ◽  
Upstream Region ◽  
Amino Acid Region

Spicule matrix proteins of sea urchin embryo are the specific products of the micromere / primary mesenchyme cell (PMC) lineage, and are considered to be involved in spicule formation (Wilt, 1999). One of these proteins, SM50, has been described for three species: Strongylocentrotus purpuratus (SP), Lytechinus pictus (Lp) and Hemicentrotus pulcherrimus (Hp) (for references see Wilt, 1999). The nucleotide and amino acid sequences are well conserved in these species. SM50 proteins of these species have repetitive amino acid sequences in the carboxyl-terminal half of the proteins. Therefore, examination of SM50 sequences, especially the repetitive sequence region, in various species will help an understanding of the process of sea urchin ontogeny and evolution. In this study we tried to amplify, by PCR, the SM50 sequences of species for which no sequence data are reported.Total DNA was extracted from the sperm of sea urchins by standard procedures. The purified DNA was subjected to PCR to amplify the repetitive amino acid region and its upstream region. The primers were designed based on the highly conserved sequences in the reported SM50 as Consensus-Degenerate Hybrid Oligonucleotide Primers (Rose et al., 1997). The amplified products were gel-purified, and sequenced using ABI PRISM 310 Genetic Analyzer using PCR primers. The determined nucleotide sequences were translated into amino acid sequences and compared among species with a phylogenetic tree constructed by the neighbour-joining method. For indirect immunofluorescent staining, embryos were fixed with 70% methanol and reacted with rabbit antiserum against recombinant SM50 protein.

scholarly journals The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella.

1985 ◽  
Vol 101 (4) ◽  
pp. 1400-1412 ◽  
Author(s):  
W S Sale ◽  
U W Goodenough ◽  
J E Heuser
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Heavy Chains ◽  
Low Salt ◽  
Spherical Head ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Globular Head

Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.

scholarly journals Dynein from Dictyostelium: primary structure comparisons between a cytoplasmic motor enzyme and flagellar dynein.

1992 ◽  
Vol 119 (6) ◽  
pp. 1597-1604 ◽  
Author(s):  
M P Koonce ◽  
P M Grissom ◽  
J R McIntosh
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Cellular Slime Mold ◽  
Reading Frame ◽  
Dynein Heavy Chain ◽  
Binding Domains

We report here the cloning and sequencing of a cytoplasmic dynein heavy chain gene from the cellular slime mold Dictyostelium discoideum. Using a combination of approaches, we have isolated 14,318 bp of DNA sequence which contains an open-reading frame of 4,725 amino acids. The deduced molecular weight of the polypeptide predicted by this reading frame is 538,482 D. Overall, the polypeptide sequence is 51% similar and 28% identical to the recently published sequences of the beta-dynein heavy chain from sea urchin flagella (Gibbons, I. R., B. H. Gibbons, G. Mocz, and D. J. Asai. 1991. Nature (Lond.). 352: 640-643; Ogawa, K. 1991. Nature (Lond.). 352:643-645). It contains four GXXXXGKT/S motifs that form part of a consensus sequence for ATP-binding domains; these motifs are clustered near the middle of the polypeptide. The distribution of the regions sharing sequence similarity between the Dictyostelium and sea urchin heavy chain polypeptides suggests that the amino termini of dyneins may contain domains that specify axonemal or cytoplasmic functions.

scholarly journals Evidence for Four Cytoplasmic Dynein Heavy Chain Isoforms in Rat Testis

1998 ◽  
Vol 9 (2) ◽  
pp. 237-247 ◽  
Author(s):  
Peggy S. Criswell ◽  
David J. Asai
Keyword(s):  
Heavy Chain ◽  
Broad Band ◽  
Cytoplasmic Dynein ◽  
Peptide Sequence ◽  
Gene Encoding ◽  
Rat Testis ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Low Ionic Strength ◽  
Dynein Heavy Chains

Recent studies have revealed the expression of multiple putative cytoplasmic dynein heavy chain (DHC) genes in several organisms, with each gene encoding a separate protein isoform. This finding is consistent with the hypothesis that different isoforms do different things, as is the case for the axonemal dyneins. Furthermore, the large number of tasks ascribed to cytoplasmic dynein suggests that there may be additional isoforms not yet identified. Two of the mammalian cytoplasmic dynein heavy chains are DHC1a and DHC1b. DHC1a is conventional cytoplasmic dynein and is found in all organisms examined. DHC1b is expressed in organisms that have multiple dyneins, and has been implicated in the intracellular trafficking of molecules in unciliated and ciliated cells. In the present study, we examined the DHC1b protein from rat testis. Testis cytoplasmic dynein contains a large amount of dynein heavy chain reactive with an antibody raised against a peptide sequence of rat DHC1b. The testis anti-DHC1b immunoreactive protein is slightly smaller than testis DHC1a, as assessed by SDS-PAGE. In Northern blots, the DHC1b mRNA is smaller than the DHC1a mRNA. In sucrose gradients made in low ionic strength, DHC1a sedimented at approximately 20S, and the anti-1b immunoreactive heavy chains sedimented in a broad band centered at approximately 14S. The V1-photolysis reaction of individual sucrose gradient fractions revealed three distinct patterns of photolysis, suggesting that there are at least three separate 1b-like heavy chain isoforms in testis. Using a high-stringency Western blotting protocol, the anti-1b antibody and the anti-DHC2 antibody recognized the same heavy chain and specifically bound to one of the three 1b-like heavy chains. We conclude that rat testis contains three 1b-like dynein heavy chains, and one of these is the product of the DHC1b/DHC2 gene previously identified.

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