scholarly journals The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

1997 ◽  
Vol 8 (4) ◽  
pp. 607-620 ◽  
Author(s):  
S H Myster ◽  
J A Knott ◽  
E O'Toole ◽  
M E Porter
Keyword(s):  
Heavy Chain ◽  
Insertional Mutagenesis ◽  
Northern Blot Analysis ◽  
Immunoblot Analysis ◽  
Transcription Unit ◽  
Peptide Sequence ◽  
Dynein Heavy Chain ◽  
The Individual ◽  
Intermediate Chains ◽  
Specific Peptide

Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex.

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.

scholarly journals Insights into the Structural Organization of the I1 Inner Arm Dynein from a Domain Analysis of the 1β Dynein Heavy Chain

2000 ◽  
Vol 11 (7) ◽  
pp. 2297-2313 ◽  
Author(s):  
Catherine A. Perrone ◽  
Steven H. Myster ◽  
Raqual Bower ◽  
Eileen T. O'Toole ◽  
Mary E. Porter
Keyword(s):  
Heavy Chain ◽  
Structural Organization ◽  
Microscopic Analysis ◽  
Transcription Unit ◽  
Light Chains ◽  
Flagellar Motility ◽  
Electron Microscopic ◽  
Dynein Heavy Chain ◽  
Complex Transformation ◽  
Motility Mutant

To identify domains in the dynein heavy chain (Dhc) required for the assembly of an inner arm dynein, we characterized a new motility mutant (ida2-6) obtained by insertional mutagenesis.ida2-6 axonemes lack the polypeptides associated with the I1 inner arm complex. Recovery of genomic DNA flanking the mutation indicates that the defects are caused by plasmid insertion into theDhc10 transcription unit, which encodes the 1β Dhc of the I1 complex. Transformation with Dhc10 constructs encoding <20% of the Dhc can partially rescue the motility defects by reassembly of an I1 complex containing an N-terminal 1β Dhc fragment and a full-length 1α Dhc. Electron microscopic analysis reveals the location of the missing 1β Dhc motor domain within the axoneme structure. These observations, together with recent studies on the 1α Dhc, identify a Dhc domain required for complex assembly and further demonstrate that the intermediate and light chains are associated with the stem regions of the Dhcs in a distinct structural location. The positioning of these subunits within the I1 structure has significant implications for the pathways that target the assembly of the I1 complex into the axoneme and modify the activity of the I1 dynein during flagellar motility.

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.

Mice with a mutation in the dynein heavy chain 1 gene display sensory neuropathy but lack motor neuron disease

2009 ◽  
Vol 215 (1) ◽  
pp. 146-152 ◽  
Author(s):  
Luc Dupuis ◽  
Anissa Fergani ◽  
Kerstin E. Braunstein ◽  
Judith Eschbach ◽  
Nathalie Holl ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Motor Neuron Disease ◽  
Heavy Chain ◽  
Sensory Neuropathy ◽  
Dynein Heavy Chain

Assignment1 of the human dynein heavy chain gene DNAH17L to human chromosome 17p12 by in situ hybridization and radiation hybrid mapping

1999 ◽  
Vol 84 (3-4) ◽  
pp. 188-189 ◽  
Author(s):  
L. Bartoloni ◽  
J.-L.C. Blouin ◽  
A.J. Sainsbury ◽  
A. Gos ◽  
M.A. Morris ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Human Chromosome ◽  
Heavy Chain ◽  
Radiation Hybrid ◽  
Radiation Hybrid Mapping ◽  
Chain Gene ◽  
Hybrid Mapping ◽  
Heavy Chain Gene ◽  
Dynein Heavy Chain

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.

Immunoglobulin heavy-chain enhancer is required to maintain transfected gamma 2A gene expression in a pre-B-cell line

1990 ◽  
Vol 10 (3) ◽  
pp. 1076-1083
Author(s):  
B Porton ◽  
D M Zaller ◽  
R Lieberson ◽  
L A Eckhardt
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Heavy Chain ◽  
Gene Activation ◽  
Immunoglobulin Heavy Chain ◽  
Transcription Unit ◽  
Enhancer Activity ◽  
Igh Genes ◽  
High Level

The immunoglobulin heavy-chain (IgH) enhancer serves to activate efficient and accurate transcription of cloned IgH genes when introduced into B lymphomas or myelomas. The role of this enhancer after gene activation, however, is unclear. The endogenous IgH genes in several cell lines, for example, have lost the IgH enhancer by deletion and yet continue to be expressed. This might be explained if the role of the enhancer were to establish high-level gene transcription but not to maintain it. Alternatively, other enhancers might lie adjacent to endogenous IgH genes, substituting their activity for that of the lost IgH enhancer. To address both of these alternatives, we searched for enhancer activity within the flanking regions of one of these IgH enhancer-independent genes and designed an experiment that allowed us to consider separately the establishment and maintenance of expression of a transfected gene. For the latter experiment we generated numerous pre-B cell lines stably transformed with a gamma 2a gene. In this gene, the IgH enhancer lay at a site outside the heavy-chain transcription unit, between DH and JH gene segments. After expression of the transfected gene was established, selective conditions were chosen for the outgrowth of subclones that had undergone D-J joining and thus IgH enhancer deletion. Measurements of gamma 2a expression before and after enhancer deletion revealed that the enhancer was required for maintenance of expression of the transfected gene. The implication of this finding for models of enhancer function in endogenous genes is discussed.

Using dynein heavy chain 5 and creatine kinase levels in cervical fluid and blood for early diagnosing of ectopic pregnancy

2020 ◽  
Author(s):  
Cagdas Sahin ◽  
Zihni Onur Uygun ◽  
Ismet Hortu ◽  
Ali Akdemir ◽  
Meltem Kocamanoglu ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Ectopic Pregnancy ◽  
Heavy Chain ◽  
Dynein Heavy Chain

scholarly journals Isolated beta-heavy chain subunit of dynein translocates microtubules in vitro.

1988 ◽  
Vol 107 (5) ◽  
pp. 1793-1797 ◽  
Author(s):  
W S Sale ◽  
L A Fox
Keyword(s):  
Ionic Strength ◽  
Sea Urchin ◽  
Heavy Chain ◽  
Glass Surface ◽  
Functional Assay ◽  
Atpase Subunits ◽  
Low Ionic Strength ◽  
Intermediate Chains ◽  
Sea Urchin Sperm

Our goal was to assess the microtubule translocating ability of individual ATPase subunits of outer arm dynein. Solubilized outer arm dynein from sea urchin sperm (Stronglocentrotus purpuratus) was dissociated into subunits by low ionic strength buffer and fractionated by zonal centrifugation. Fractions were assessed by an in vitro functional assay wherein microtubules move across a glass surface to which isolated dynein fractions had been absorbed. Microtubule gliding activity was coincident with the 12-S beta-heavy chain-intermediate chain 1 ATPase fractions (beta/IC1). Neither the alpha-heavy chain nor the intermediate chains 2 and 3 fractions coincided with microtubule gliding activity. The beta/IC1 ATPase induced very rapid gliding velocities (9.7 +/- 0.88 micron/s, range 7-11.5 micron/s) in 1 mM ATP-containing motility buffers. In direct comparison, isolated intact 21-S outer arm dynein, from which the beta/IC1 fraction was derived, induced slower microtubule gliding rates (21-S dynein, 5.6 +/- 0.7 micron/s; beta/IC1, 8.7 +/- 1.2 micron/s). These results demonstrate that a single subdomain in dynein, the beta/IC1 ATPase, is sufficient for microtubule sliding activity.

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