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1.
Thick filament substructures in Caenorhabditis elegans: evidence for two populations of paramyosin 总被引:4,自引:2,他引:2
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《The Journal of cell biology》1993,123(2):303-311
The thick filaments of the nematode Caenorhabditis elegans contain two myosin heavy chain isoforms A and B and paramyosin, the products of the myo-3, unc-54, and unc-15 genes, respectively. Dissociation of paramyosin from native thick filaments at pH 6.36 shows a biphasic function with respect to NaCl concentration. Electron microscopy of the remaining structures shows 15-nm core structures that label with monoclonal anti-paramyosin antibody at 72.5-nm intervals. Purified core structures also show 72.5 nm repeats by negative staining. Structural analysis of native thick filaments and dissociated structures suggests that the more dissociable paramyosin is removed radially as well as processively from the filament ends. Minor proteins with masses of 20, 28, and 30 kD cosediment stoichiometrically with paramyosin in purified core structures. 相似文献
2.
In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B-dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B. 相似文献
3.
The unc-45 gene of the nematode, Caenorhabditis elegans, is essential for muscle organization and embryonic development. Genetic evidence suggests the unc-45 gene product controls muscle thick filament assembly. We report here on the determination of the gene's chromosomal location and the isolation and sequencing of its cDNA. The amino terminus of the predicted unc-45 protein contains three tandem repeats that belong in the tetratricopeptide repeat family. Tetratricopeptide motifs have been shown to be involved in protein interactions, and some of the closest homologues have chaperone-like activity. The carboxy terminus of the protein has homology with the related fungal proteins, CRO1 and She4p, which have been postulated to play a role in assembly of or interactions with a cytoplasmic myosin. We have also determined the sequence of the homologous gene from C. briggsae, which demonstrates a high level of conservation. We show that the unc-45 gene promoter can drive reporter gene expression, which is limited to muscle tissues (pharyngeal, body wall, vulval, and anal muscles), consistent with a role for the unc-45 gene in muscle development or function. 相似文献
4.
Sequence Requirements for Myosin Gene Expression and Regulation in Caenorhabditis Elegans 总被引:5,自引:1,他引:4
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Four Caenorhabditis elegans genes encode muscle-type specific myosin heavy chain isoforms: myo-1 and myo-2 are expressed in the pharyngeal muscles; unc-54 and myo-3 are expressed in body wall muscles. We have used transformation-rescue and lacZ fusion assays to determine sequence requirements for regulated myosin gene expression during development. Multiple tissue-specific activation elements are present for all four genes. For each of the four genes, sequences upstream of the coding region are tissue-specific promoters, as shown by their ability to drive expression of a reporter gene (lacZ) in the appropriate muscle type. Each gene contains at least one additional tissue-specific regulatory element, as defined by the ability to enhance expression of a heterologous promoter in the appropriate muscle type. In rescue experiments with unc-54, two further requirements apparently independent of tissue specificity were found: sequences within the 3' non-coding region are essential for activity while an intron near the 5' end augments expression levels. The general intron stimulation is apparently independent of intron sequence, indicating a mechanistic effect of splicing. To further characterize the myosin gene promoters and to examine the types of enhancer sequences in the genome, we have initiated a screen of C. elegans genomic DNA for fragments capable of enhancing the myo-2 promoter. The properties of enhancers recovered from this screen suggest that the promoter is limited to muscle cells in its ability to respond to enhancers. 相似文献
5.
The precise arrangement of molecules within the thick filament, as well as the mechanisms by which this arrangement is specified, remains unclear. In this article, we have exploited a unique genetic interaction between one isoform of myosin heavy chain (MHC) and paramyosin in Caenorhabditis elegans to probe the molecular interaction between MHC and paramyosin in vivo. Using chimeric myosin constructs, we have defined a 322-residue region of the MHC A rod critical for suppression of the structural and motility defects associated with the unc-15(e73) allele. Chimeric constructs lacking this region of MHC A either fail to suppress, or act as dominant enhancers of, the e73 phenotype. Although the 322-residue region is required for suppression activity, our data suggest that sequences along the length of the rod also play a role in the isoform-specific interaction between MHC A and paramyosin. Our genetic and cell biological analyses of construct behavior suggest that the 322-residue region of MHC A is important for thick filament stability. We present a model in which this region mediates an avid interaction between MHC A and paramyosin in parallel arrangement in formation of the filament arms. 相似文献
6.
Caenorhabditis elegans has four genes which encode skeletal myosin heavy chain isoforms. We have re-introduced clones of two of these genes, myo-3 and unc-54 at low copy number into the germline of C. elegans. The resulting loci behave as functional copies of the genes by two genetic criteria: (i) they can result in phenotypic rescue of strains carrying inactivating myo-3 or unc-54 mutations, and (ii) their presence in strains with wild-type copies of the endogenous myosin loci has genetic consequences similar to duplicating the endogenous loci. The re-introduced genes function at a level close to that of the endogenous loci. Monoclonal antibodies specific for the different isoforms have been used to localize the expressed proteins. The re-introduced genes express in precisely the same cell types as the endogenous genes, and the myosin products produced assemble into filament structures as in wild-type. Unexpectedly, we have found in the course of this work that very high copy numbers of the unc-54 gene lead to a disruption of muscle structure which may result from overexpression of the protein product. 相似文献
7.
Hydrophobicity variations along the surface of the coiled-coil rod may mediate striated muscle myosin assembly in Caenorhabditis elegans 总被引:1,自引:0,他引:1
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《The Journal of cell biology》1996,135(2):371-382
Caenorhabditis elegans body wall muscle contains two isoforms of myosin heavy chain, MHC A and MHC B, that differ in their ability to initiate thick filament assembly. Whereas mutant animals that lack the major isoform, MHC B, have fewer thick filaments, mutant animals that lack the minor isoform, MHC A, contain no normal thick filaments. MHC A, but not MHC B, is present at the center of the bipolar thick filament where initiation of assembly is thought to occur (Miller, D.M.,I. Ortiz, G.C. Berliner, and H.F. Epstein. 1983. Cell. 34:477-490). We mapped the sequences that confer A-specific function by constructing chimeric myosins and testing them in vivo. We have identified two distinct regions of the MHC A rod that are sufficient in chimeric myosins for filament initiation function. Within these regions, MHC A displays a more hydrophobic rod surface, making it more similar to paramyosin, which forms the thick filament core. We propose that these regions play an important role in filament initiation, perhaps mediating close contacts between MHC A and paramyosin in an antiparallel arrangement at the filament center. Furthermore, our analysis revealed that all striated muscle myosins show a characteristic variation in surface hydrophobicity along the length of the rod that may play an important role in driving assembly and determining the stagger at which dimers associate. 相似文献
8.
9.
Henry F. Epstein 《Molecular neurobiology》1990,4(1-2):1-25
The established observations and unresolved questions in the assembly of myosin are outlined in this article. Much of the background information has been obtained in classical experiments using the myosin and thick filaments from vertebrate skeletal muscle. Current research is concerned with problems of myosin assembly and structure in smooth muscle, a broad spectrum of invertebrate muscles, and eukaryotic cells in general. Many of the general questions concerning myosin assembly have been addressed by a combination of genetic, molecular, and structural approaches in the nematode Caenorhabditis elegans. Detailed analysis of multiple myosin isoforms has been a prominent aspect of the nematode work. The molecular cloning and determination of the complete sequences of the genes encoding the four isoforms of myosin heavy chain and of the myosin-associated protein paramyosin have been a major landmark. The sequences have permitted a theoretical analysis of myosin rod structure and the interactions of myosin in thick filaments. The development of specific monoclonal antibodies to the individual myosins has led to the delineation of the different locations of the myosins and to their special roles in thick filament structure and assembly. In nematode body-wall muscles, two isoforms, myosins A and B, are located in different regions of each thick filament. Myosin A is located in the central biopolar zones, whereas myosin B is restricted to the flanking polar regions. This specific localization directly implies differential behavior of the two myosins during assembly. Genetic and structural experiments demonstrate that paramyosin and the levels of expression of the two forms are required for the differential assembly. Additional genetic experiments indicate that several other gene products are involved in the assembly of myosin. Structural studies of mutants have uncovered two new structures. A core structure separate from myosin and paramyosin appears to be an integral part of thick filaments. Multifilament assemblages exhibit multiple nascent thick filament-like structures extending from central paramyosin regions. Dominant mutants of myosin that disrupt thick filament assembly are located in the ATP and actin binding sites of the heavy chain. A model for a cycle of reactions in the assembly of myosin into thick filaments is presented. Specific reactions of the two myosin isoforms, paramyosin, and core proteins with multifilament assemblages as possible intermediates in assembly are proposed. 相似文献
10.
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12.
Nematodes are the only group of organisms in which both cis- and trans-splicing of nuclear mRNAs are known to occur. Most Caenorhabditis elegans introns are exceptionally short, often only 50 bases long. The consensus donor and acceptor splice site sequences found in other animals are used for both cis- and trans-splicing. In order to identify the machinery required for these splicing events, we have characterized the C. elegans snRNAs. They are similar in sequence and structure to those characterized in other organisms, and several sequence variations discovered in the nematode snRNAs provide support for previously proposed structure models. The C. elegans snRNAs are encoded by gene families. We report here the sequences of many of these genes. We find a highly conserved sequence, the proximal sequence element (PSE), about 65 bp upstream of all 21 snRNA genes thus far sequenced, including the SL RNA genes, which specify the snRNAs that provide the 5' exons in trans-splicing. The sequence of the C. elegans PSE is distinct from PSE's from other organisms. 相似文献
13.
The mutation e1662 is an allele of the Caenorhabditis elegans unc-54 gene induced with the difunctional alkylating agent 1,2,7,8-diepoxyoctane. unc-54 encodes the major myosin heavy chain isozyme of body wall muscle cells. Filter-transfer hybridization and DNA sequence analysis show that e1662 is an insertion of 288 base pairs of DNA within unc-54. The inserted DNA is identical to a 288-base pair region of unc-54 located ca. 600 base pairs from the insertion site. Thus, e1662 is a displaced duplication. A 14-base pair sequence located at one end of the duplicated segment is found adjacent to the site of insertion. These homologous sequences are juxtaposed head-to-tail by the insertion event. e1662 thus contains a tandem direct repeat extending across one of its junctions. 相似文献
14.
Molecular analysis of the Drosophila nuclear lamin gene 总被引:4,自引:0,他引:4
15.
Reversion analysis of mutants of unc-22 IV, a gene affecting muscle structure and function in Caenorhabditis elegans, led to the isolation of six extragenic dominant suppressors of the “twitching” phenotype of unc-22 mutants. All six suppressors are new alleles of unc-54 I, the major body wall myosin heavy chain gene. Homozygous suppressor strains are slow, stiff and have normal muscle structure, whereas previously identified unc-54 alleles confer flaccid paralysis and drastic reduction in thick filament number and organization. Placement of the three suppressor mutations s74, s77 and s95 on the genetic fine structure map of unc-54 demonstrates that they are clustered near the right end of the map. Since this end of the gene corresponds to the 5′ end of the coding sequence, these suppressor mutations probably result in amino acid substitutions in the globular head of the myosin molecule, and should be of value in studies of myosin force generation. 相似文献
16.
Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans 总被引:32,自引:0,他引:32
N J Dibb D M Brown J Karn D G Moerman S L Bolten R H Waterston 《Journal of molecular biology》1985,183(4):543-551
We have sequenced 11 representative mutations of the unc-54 myosin heavy chain gene of Caenorhabditis elegans that affect the synthesis, assembly or enzymatic activity of the encoded myosin heavy chain. Six of the sequenced unc-54 mutations cause premature termination of protein synthesis. Four mutations (e1092, e1115, e1213, e1328) were ochre mutations, one mutation (e903) was a frameshift, which caused premature termination at a nearby UGA terminator, and one mutation (e190) was a deletion that altered the reading frame and caused termination at an ochre codon. Two mutations (e675 and s291) were inphase deletions, which resulted in a shortened myosin rod segment. These aberrant myosins fail to assemble into normal thick filaments. The sequence alterations of the missense mutations (e1152, s74, s95) indicated amino acid residues that are critical for myosin function. The mutation e1152 causes the production of a myosin heavy chain that fails to assemble into thick filaments. It had two adjacent amino acid substitutions at the extreme amino terminus of the rod, indicating a role for subfragment-2 in thick filament assembly. Mutants homozygous for s74 or s95 are very slow-moving, although they make myosin heavy chains that assemble normally. The encoded amino acid substitutions of s95 and s74 are in the 23 X 10(3) Mr and 50 X 10(3) Mr domains of the myosin head, flanking the ATP binding site. The sequenced mutations are distributed throughout the gene in the order predicted from genetic fine-structure mapping experiments. Seven of eight point mutations isolated following ethylmethane sulphonate mutagenesis were G X C to A X T transitions. A single X-ray-induced allele proved to be a deletion of two adjacent thymidine residues. The three deletion mutations were found in a region of the myosin rod with numerous direct and inverted nucleotide sequence repeats, but their origin cannot be accounted for by homologous recombination. Instead, a comparison of the deletion junctions suggests that the deletions arose by a site-specific mechanism. 相似文献
17.
Paramyosin gene (unc-15) of Caenorhabditis elegans. Molecular cloning, nucleotide sequence and models for thick filament structure 总被引:17,自引:0,他引:17
Paramyosin is a major structural component of thick filaments isolated from many invertebrate muscles. The Caenorhabditis elegans paramyosin gene (unc-15) was identified by screening with specific antibodies an "exon-expression" library containing lacZ/nematode gene fusions. Short probes recovered from the library were used to identify bacteriophage lambda and cosmid clones that encompass the entire paramyosin (unc-15) gene. From these clones, numerous subclones containing epitopes reacting with anti-paramyosin sera were obtained, providing strong evidence that the initial cloned fragment was, in fact, derived from the structural gene for paramyosin. The complete nucleotide sequence of a 12 x 10(3) base-pair region spanning the gene was obtained. The gene is composed of ten short exons encoding a protein of 866 [corrected] amino acid residues. Paramyosin is highly similar to residues 267 to 1089 of myosin heavy chain rods. For most of its length, paramyosin appears to form an alpha-helical coiled-coil and shows the expected heptad repeat of hydrophobic amino acid residues and the 28-residue repeat of charged amino acids characteristic of myosin heavy chain rods. However, paramyosin differs from myosin in having non-helical extensions at both the N and C termini and an additional "skip" residue that interrupts the 28-residue repeat. The distribution of charges along the length of the paramyosin rod is also significantly different from that of myosin heavy chain rods. Potential charge-mediated interactions between paramyosin rods and between paramyosin and myosin rods were calculated using a model successfully applied previously to the analysis of the myosin rod sequences. Myosin rods aligned in parallel show optimal charge-charge interactions at multiples of 98 residue staggers (i.e. at axial displacements of multiples of 143 A). Paramyosin rods, in contrast, appear to interact optimally at parallel staggers of 493 residues (i.e. at axial displacements of 720 A) but show only weak interaction peaks at 98 or 296 residues. Similar calculations suggest optimal interactions between paramyosin molecules and myosin rods and in their anti-parallel alignments. The implications of these results for the structure of the bare zone and the assembly of nematode thick filaments are discussed. 相似文献
18.
19.
Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail 总被引:21,自引:11,他引:10
We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity. 相似文献
20.
At least three genes encode the different isoforms of the glycolytic enzyme enolase. We have isolated the gene for the human gamma- or neuron-specific enolase and determined the nucleotide sequence from upstream to the 5' end to beyond the polyadenylation site. The gene contains 12 exons distributed over 9213 nucleotides. Introns occur at positions identical to those reported for the homologous rat gene, as well as for the human alpha- or nonneuronal enolase gene, supporting the existence of a single ancestor for the members of this gene family. Primer extension analysis indicates that the gene has multiple start sites. The putative promoter region lacks canonical TATA and CAAT boxes, is very G + C-rich, and contains several potential regulatory sequences. Furthermore, an inverted Alu sequence is present approximately 572 nucleotides upstream of the major start site. A comparison of the 5'-flanking region of the human gamma-enolase gene with the same region of the rat gene revealed a high degree of sequence conservation. 相似文献