Epstein HF et al. (1985) J Cell Biol "Myosin and paramyosin are organized about a newly identified core structure."

Berliner GC, Miller DM, Epstein HF, Ortiz I

[J Cell Biol, 1985]

Myosin isoforms A and B are differentially localized to the central and polar regions, respectively, of thick filaments in body wall muscle cells of Caenorhabditis elegans (Miller, D.M. III, I. Ortiz, G.C. Berliner, and H.F. Epstein, 1983, Cell, 34: 477-490). Biochemical and electron microscope studies of KCl-dissociated filaments show that the myosin isoforms occupy a surface domain, paramyosin constitutes an intermediate domain, and a newly identified core structure exists. The diameters of the thick filaments vary significantly from 33.4 nm centrally to 14.0 nm near the ends. The latter value is comparable to the 15.2 nm diameter of the core structures. The internal density of the filament core appears solid medially and hollow at the poles. The differentiation of thick filament structure into supramolecular domains possessing specific substructures of characteristic stabilities suggests a sequential mode for thick filament assembly. In this model, the two myosin isoforms have distinct roles in assembly. The behavior of the myosins, including nucleation of assembly and determination of filament length, depend upon paramyosin and the core structure as well as their intrinsic molecular properties.

Epstein HF et al. (1988) J Cell Biol "Purified thick filaments from the nematode Caenorhabditis elegans: evidence for multiple proteins associated with core structures."

Epstein HF, Berliner GC, Ortiz I, Casey DL

[J Cell Biol, 1988]

The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2% with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C. elegans.

Farrer T et al. (2002) Nucleic Acids Res "Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing."

Farrer T, Kent WJ, Roller AB, Zahler AM

[Nucleic Acids Res, 2002]

GC-AG introns represent 0.7% of total human pre-mRNA introns. To study the function of GC-AG introns in splicing regulation, 196 cDNA-confirmed GC-AG introns were identified in Caenorhabditis elegans. These represent 0.6% of the cDNA- confirmed intron data set for this organism. Eleven of these GC-AG introns are involved in alternative splicing. In a comparison of the genomic sequences of homologous genes between C.elegans and Caenorhabditis briggsae for 26 GC-AG introns, the C at the +2 position is conserved in only five of these introns. A system to experimentally test the function of GC-AG introns in alternative splicing was developed. Results from these experiments indicate that the conserved C at the +2 position of the tenth intron of the let-2 gene is essential for developmentally regulated alternative splicing. This C allows the splice donor to function as a very weak splice site that works in balance with an alternative GT splice donor. A weak GT splice donor can functionally replace the GC splice donor and allow for splicing regulation. These results indicate that while the majority of GC-AG introns appear to be constitutively spliced and have no evolutionary constraints to prevent them from being GT-AG introns, a subset of GC-AG introns is involved in alternative splicing and the C at the +2 position of these introns can have an important role in splicing regulation.

Zhang J et al. (2011) BMC Genomics "GC content around splice sites affects splicing through pre-mRNA secondary structures."

Zhang J, Chen L, Kuo CC

[BMC Genomics, 2011]

BACKGROUND: Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens), mice (Mus musculus), fruit flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans) to further investigate this phenomenon. RESULTS: We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures. CONCLUSION: All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.

Epstein HF et al. (1987) J Muscle Res Cell Motil "Assemblages of multiple thick filaments in nematode mutants."

Ortiz I, Berliner GC, Epstein HF

[J Muscle Res Cell Motil, 1987]

A spectrum of thick filament-related structures exhibiting novel structural features is isolated in addition to the normal thick filaments from unc-15 and unc-82 mutants of Caenorhabditis elegans. Many assemblages have multiple myosin-coated filaments extending from both ends of central domains exhibiting paracrystalline paramyosin. The filament ends resemble the polar core structures of native thick filaments. Assemblages with filaments at only one end and short thick filaments that branch are also present. This spectrum of novel structures accumulates at high levels in specific mutants due to alterations in paramyosin or other interacting proteins. The multifilament structures are either alternative assemblages of thick filament proteins and substructures or usually transient nucleation centres active in the assembly of thick filaments which are favoured under mutant conditions.

Hamelin M et al. (1993) Journal of Neurochemistry "unc-5 and unc-6, proteins that steers migrating growth cones in C. elegans."

Hamelin M, Su M-W, Culotti JG, Zhou YW, Scott IM

[Journal of Neurochemistry, 1993]

The unc-5 and unc-6 genes of C. elegans are required to guide pioneer axon growth cone (GC) and mesodermal cell migrations in a dorsal direction on the epidermis.

Ahn IY et al. (2005) J Biochem Biophys Methods "Determination of DNA base composition by small scale acrylamide-CsCl gradient centrifugation."

Winter CE, Ahn IY

[J Biochem Biophys Methods, 2005]

In this paper we describe a simple and rapid protocol for DNA base composition determination by CsCl gradient in the presence of acrylamide. This method permits the determination of GC content in microgram amounts of DNA, and results are easily documented in photographs or graphs. The protocol was applied to the characterization of nematode DNA, but can be used for other organisms. Analyzing several experiments the mean standard deviation observed in the calculated GC content is near 1.3%.

Miller DM et al. (1983) Cell "Differential localization of two myosins within nematode thick filaments."

Miller DM, Berliner GC, Epstein HF, Ortiz I

[Cell, 1983]

The body wall muscle cells of the nematode, Caenorhabditis elegans, contain two unique types of myosin heavy chain, A and B. We have utilized an immunochemical approach to define the structural location of these myosins within body wall muscle thick filaments. By immunofluorescence microscopy, myosin B antibodies label the thick filament-containing A-bands of body wall muscle with the exception of a thin gap at the center of each A-band, and myosin A antibodies react to form a medial fluorescent stripe within each A-band. The complexes of these monoclonal antibodies with isolated thick filaments were negatively stained and studied by electron microscopy. The myosin B antibody reacts with the polar regions of all filaments but does not react with a central 0.9 um zone. The myosin A antibody reacts with a central 1.8 um zone in all filaments but does not react with the polar regions.

Rothstein N et al. (1988) Parasitology "DNA base composition of filarial nematodes."

Rothstein N, Rajan TV, Stoller TJ

[Parasitology, 1988]

We have determined the molar content of guanine + cytosine (GC content) of DNA of the filarial nematode (Brugia malayi, Brugia pahangi and Dirofilaria imitis) and of the free-living soil nematodes Caenorhabditis elegans and have analysed the DNA for the presence of methylcytosine. Two independent methods, thermal denaturation and direct analysis of base content by HPLC following enzymatic hydrolysis, reveal that the GC content of filarial nematodes is 26-28%. We have been unable to find methylcytosine in the DNA of B. malayi.

Epstein HF et al. (1985) "Nematode thick-filament structure and assembly."

Miller DM, Epstein HF, Ortiz I, Berliner GC

[1985]

Myosins from slime molds to brain cells show a remarkable commonality of general molecular properties. These characteristics include two globular domains or heads that contain ATPase and actin-binding sites and the fibrous, coiled-coil a-helical rod that interacts with other molecules in assembly. Two heavy chains (m.w. 200,000) contribute to both heads, whereas two kinds of light chains bind to each head. In this paper, we consider striated muscles and their myosins. The phylogenetically distant nematode body-wall muscles and rabbit fast skeletal muscles produce myosin heavy chains, with about 47% of the amino acid sequences in the heads and 37% of the amino acids in the rod being identical (Karn et al. 1984). Myosin heavy chains are therefore highly conserved proteins. Contrasting with the phylogenetic conservation of myosin structure and sequence is the diversity of supramolecular arrangements of myosin assemblies in striated muscles, the so-called thick filaments. The lengths of thick filaments range from 1.55 um in vertebrates, 2-4 um in insect flight muscles, 10 um in the nematode to 40 um in certain mollusks. The average diameters of these filaments range from about 15 nm in vertebrates, 20 nm in insects, 25 nm in nematodes to 50-100 nm in some molluscan muscles. The surface arrangements of the myosin heads also vary in these different species. The lattice arrangements between thick filaments and the interdigitating, actin-containing thin filaments differ in terms of symmetry and thick:thin stoichiometry between these muscles. It appears likely that other protein components of these muscles interact with the very similar myosins to produce this structural diversity. The relatively subtle differences between myosin isoforms may also be important in these interactions. We define isoform in the case of myosin, for example, as a protein that is defined as a myosin by biochemical criteria but that can be distinguished on the basis of intrinsic molecular structure from another myosin within the same organism. In this paper, we describe experiments suggesting that two genetically different isoforms of myosin play distinct roles in concert with other proteins during the assembly of thick filaments in