scholarly journals Gene Knockouts Reveal Separate Functions for Two Cytoplasmic Dyneins in Tetrahymena thermophila

1999 ◽  
Vol 10 (3) ◽  
pp. 771-784 ◽  
Author(s):  
Seungwon Lee ◽  
Julie C. Wisniewski ◽  
William L. Dentler ◽  
David J. Asai
Keyword(s):  
Division Of Labor ◽  
Heavy Chain ◽  
Phagocytic Activity ◽  
Cell Shape ◽  
Tetrahymena Thermophila ◽  
Cytoplasmic Dynein ◽  
Targeted Disruption ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Gene Knockouts

In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophilapresents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of twoTetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, andDYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.

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.

scholarly journals Molecular analysis of the gamma heavy chain of Chlamydomonas flagellar outer-arm dynein

1994 ◽  
Vol 107 (3) ◽  
pp. 497-506 ◽  
Author(s):  
C.G. Wilkerson ◽  
S.M. King ◽  
G.B. Witman
Keyword(s):  
Heavy Chain ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Complete Sequence ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Microtubule Binding Domain ◽  
Helical Content ◽  
Axonemal Dynein ◽  
Dynein Heavy Chains

We report here the complete sequence of the gamma dynein heavy chain of the outer arm of the Chlamydomonas flagellum, and partial sequences for six other dynein heavy chains. The gamma dynein heavy chain sequence contains four P-loop motifs, one of which is the likely hydrolytic site based on its position relative to a previously mapped epitope. Comparison with available cytoplasmic and flagellar dynein heavy chain sequences reveals regions that are highly conserved in all dynein heavy chains sequenced to date, regions that are conserved only among axonemal dynein heavy chains, and regions that are unique to individual dynein heavy chains. The presumed hydrolytic site is absolutely conserved among dyneins, two other P loops are highly conserved among cytoplasmic dynein heavy chains but not in axonemal dynein heavy chains, and the fourth P loop is invariant in axonemal dynein heavy chains but not in cytoplasmic dynein. One region that is very highly conserved in all dynein heavy chains is similar to a portion of the ATP-sensitive microtubule-binding domain of kinesin. Two other regions present in all dynein heavy chains are predicted to have high alpha-helical content and have a high probability of forming coiled-coil structures. Overall, the central one-third of the gamma dynein heavy chain is most conserved whereas the N-terminal one-third is least conserved; the fact that the latter region is divergent between the cytoplasmic dynein heavy chain and two different axonemal dynein heavy chains suggests that it is involved in chain-specific functions.

Identification and molecular evolution of new dynein-like protein sequences in rat brain

1995 ◽  
Vol 108 (5) ◽  
pp. 1883-1893 ◽  
Author(s):  
Y. Tanaka ◽  
Z. Zhang ◽  
N. Hirokawa
Keyword(s):  
Rat Brain ◽  
Heavy Chain ◽  
Phylogenetic Trees ◽  
Cytoplasmic Dynein ◽  
Amino Acid Sequences ◽  
Adult Brain ◽  
Chain Gene ◽  
Evolutionary Analysis ◽  
Degenerate Primers ◽  
Dynein Heavy Chain

RT-PCR cloning was performed to find unknown members of the dynein superfamily expressed in rat brain. Six kinds of degenerate primers designed for the dynein catalytic domain consensuses were used for extensive PCR amplifications. We have sequenced 550 plasmid clones which turned out to include 13 kinds of new dynein-like sequences (DLP1-8, 9A/B, 10–12) and cytoplasmic dynein heavy chain. In these clones, alternative splicing was detected for a 105 nt-domain containing the CFDEFNRI consensus just downstream of the most N-terminal P-loop (DLP9A and 9B). By using these obtained sequences, initial hybridization studies were performed. Genomic Southern blotting showed each sequence corresponds to a single copy of the gene, while northern blotting of adult brain presented more than one band for some subtypes. We further accomplished molecular evolutionary analysis to recognize their phylogenetic origins for the axonemal and non-axonemal (cytoplasmic) functions. Different methods (UPGMA, NJ and MP) presented well coincident phylogenetic trees from 44 partial amino acid sequences of dynein heavy chain from various eukaryotes. The trunk for all the cytoplasmic dynein heavy chain homologues diverged directly from the root of the phylogenetic tree, suggesting that the first dynein gene duplication defined two distinct functions as respective subfamilies. Of particular interest, we found a duplication event of the cytoplasmic dynein heavy chain gene giving rise to another subtype, DLP4, located between the divergence of yeast and that of Dictyostelium. Such evolutionary topology builds up an inceptive hypothesis that there are at least two non-axonemal dynein heavy chains in mammals.

scholarly journals 2M1448 Properties of human cytoplasmic dynein heavy chain 1 and 2(Molecular motor 3,The 48th Annual Meeting of the Biophysical Society of Japan)

2011 ◽  
Vol 51 (supplement) ◽  
pp. S97
Author(s):  
Ichikawa Muneyoshi ◽  
Yuta Watanabe ◽  
Takashi Murayama ◽  
Yoko Yano Toyoshima
Keyword(s):  
Annual Meeting ◽  
Heavy Chain ◽  
Molecular Motor ◽  
Cytoplasmic Dynein ◽  
Dynein Heavy Chain ◽  
Biophysical Society

scholarly journals Cytoplasmic Dynein, the Dynactin Complex, and Kinesin Are Interdependent and Essential for Fast Axonal Transport

1999 ◽  
Vol 10 (11) ◽  
pp. 3717-3728 ◽  
Author(s):  
MaryAnn Martin ◽  
Stanley J. Iyadurai ◽  
Andrew Gassman ◽  
Joseph G. Gindhart ◽  
Thomas S. Hays ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Heavy Chain ◽  
Cytoplasmic Dynein ◽  
Genetic Interactions ◽  
Organelle Transport ◽  
Fast Axonal Transport ◽  
Anterograde Transport ◽  
Dynein Heavy Chain ◽  
Conventional Kinesin ◽  
Dynactin Complex

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150Glued(Glued) component of the dynactin complex with the use of genetic techniques in Drosophila.cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150Glued were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued orcDhc64C mutations were stronger than those betweenGlued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.

scholarly journals A fertility region on the Y chromosome of Drosophila melanogaster encodes a dynein microtubule motor

1993 ◽  
Vol 90 (23) ◽  
pp. 11132-11136 ◽  
Author(s):  
J Gepner ◽  
T S Hays
Keyword(s):  
Drosophila Melanogaster ◽  
Y Chromosome ◽  
Heavy Chain ◽  
Male Fertility ◽  
Chromosome Region ◽  
Cytoplasmic Dynein ◽  
Dynein Heavy Chain ◽  
Microtubule Motor ◽  
Hydrolysis Of

A clone encoding a portion of the highly conserved ATP-binding domain of a dynein heavy-chain polypeptide was mapped to a region of the Drosophila melanogaster Y chromosome. Dyneins are large multisubunit enzymes that utilize the hydrolysis of ATP to move along microtubules. They were first identified as the motors that provide the force for flagellar and ciliary bending. Seven different dynein heavy-chain genes have been identified in D. melanogaster by PCR. In the present study, we demonstrate that one of the dynein genes, Dhc-Yh3, is located in Y chromosome region h3, which is contained within kl-5, a locus required for male fertility. The PCR clone derived from Dhc-Yh3 is 85% identical to the corresponding region of the beta heavy chain of sea urchin flagellar dynein but only 53% identical to a cytoplasmic dynein heavy chain from Drosophila. In situ hybridization to Drosophila testes shows Dhc-Yh3 is expressed in wild-type males but not in males missing the kl-5 region. These results are consistent with the hypothesis that the Y chromosome is needed for male fertility because it contains conventional genes that function during spermiogenesis.

scholarly journals The primary structure of rat brain (cytoplasmic) dynein heavy chain, a cytoplasmic motor enzyme.

1993 ◽  
Vol 90 (17) ◽  
pp. 7928-7932 ◽  
Author(s):  
Z. Zhang ◽  
Y. Tanaka ◽  
S. Nonaka ◽  
H. Aizawa ◽  
H. Kawasaki ◽  
...  
Keyword(s):  
Rat Brain ◽  
Heavy Chain ◽  
Primary Structure ◽  
Cytoplasmic Dynein ◽  
Dynein Heavy Chain
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