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We report the identification of several putative muscle-specific regulatory elements, and genes which are expressed preferentially in the muscle of the nematode Caenorhabditis elegans. We used computational pattern finding methods to identify cis-regulatory motifs from promoter regions of a set of genes known to express preferentially in muscle; each motif describes the potential binding sites for an unknown regulatory factor. The significance and specificity of the identified motifs were evaluated using several different control sequence sets. Using the motifs, we searched the entire C. elegans genome for genes whose promoter regions have a high probability of being bound by the putative regulatory factors. Genes that met this criterion and were not included in our initial set were predicted to be good candidates for muscle expression. Some of these candidates are additional, known muscle expressed genes and several others are shown here to be preferentially expressed in muscle cells by using GFP (green fluorescent protein) constructs. The methods described here can be used to predict the spatial expression pattern of many uncharacterized genes.

In C. elegans, assembly of hypodermal hemidesmosome-like structures called fibrous organelles is temporally and spatially coordinated with the assembly of the muscle contractile apparatus, suggesting that signals are exchanged between these cell types to position fibrous organelles correctly. Myotactin, a protein recognized by monoclonal antibody MH46, is a candidate for such a signaling molecule. The antigen, although expressed by hypodermis, first reflects the pattern of muscle elements and only later reflects the pattern of fibrous organelles. Confocal microscopy shows that in adult worms myotactin and fibrous organelles show coincident localization. Further, cell ablation studies show the bodywall muscle cells are necessary for normal myotactin distribution. To investigate myotactin's role in muscle-hypodermal signaling, we characterized the myotactin locus molecularly and genetically. Myotactin is a novel transmembrane protein of approximately 500 kd. The extracellular domain contains at least 32 fibronectin type III repeats and the cytoplasmic domain contains unique sequence. In mutants lacking myotactin, muscle cells detach when embryonic muscle contraction begins. Later in development, fibrous organelles become delocalized and are not restricted to regions of the hypodermis previously contacted by muscle. These results suggest myotactin helps maintain the association between the muscle contractile apparatus and hypodermal fibrous organelles.

Mutations in the unc-87 gene of Caenorhabditis elegans affect the structure and function of bodywall muscle, resulting in variable paralysis. We cloned the unc-87 gene by taking advantage of a transposon-induced allele of unc-87 and the correspondence of the genetic and physical maps in C. elegans. A genomic clone was isolated that alleviates the mutant phenotype when introduced into unc-87 mutants. Sequence analysis of a corresponding cDNA clone predicts a 357-amino acid, 40-kD protein that is similar to portions of the vertebrate smooth muscle proteins calponin and SM22 alpha, the Drosophila muscle protein mp20, the deduced product of the C. elegans cDNA cm7g3, and the rat neuronal protein np25. Analysis of the genomic sequence and of various transcripts represented in a cDNA library suggest that unc-87 mRNAs are subject to alternative splicing. Immunohistochemistry of wildtype and mutant animals with antibodies to an unc-87 fusion protein indicates that the gene product is localized to the I-band of bodywall muscle. Studies of the UNC-87 protein in other muscle mutants suggest that the unc-87 gene product associates with thin filaments, in a manner that does not depend on the presence of the thin filament protein tropomyosin.

Mutations in the unc-87 gene of Caenorhabditis elegans cause disorganization of the myofilament lattice in adult bodywall muscle. In order to assess the organization of specific bodywall muscle components in the absence of the unc-87 gene product, we examined the bodywall muscles of mutant animals using phalloidin and monoclonal antibodies to various muscle proteins. These studies indicated that the bodywall muscle of unc-87 embryos is initially almost wild type in its organization, but at later stages, the muscle becomes severely disorganized. To address the possibility that this disorganization is due to deterioration of the muscle as a result of contraction, we introduced into the unc-87 mutant background a mutation that decreases myosin heavy chain activity but does not substantially affect muscle structure. The improved muscle structure and motility of the double mutants are consistent with the hypothesis that at least part of the disorganization phenotype of unc-87 mutants is a consequence of the wild-type levels of force generated during muscle contraction. These results imply that the role of the unc-87 gene product is not in specifying organization but rather in serving as a structural component maintaining lattice integrity during and after contraction.

The body wall muscle cells of Caenorhabditis elegans contain an obliquely striated myofibrillar lattice that is associated with the cell membrane through two structures: an M-line analogue in the A-band and a Z-disc analogue, or dense-body, in the I-band. By using a fraction enriched in these structures as an immunogen for hybridoma production, we prepared monoclonal antibodies that identify four components of the I-band as determined by immunofluorescence and Western transfer analysis. A major constituent of the dense-body is a 107,000-D polypeptide that shares determinants with vertebrate alpha-actinin. A second dense-body constituent is a more basic and antigenically distinct 107,000-D polypeptide that is localized to a narrow domain of the dense-body at or subjacent to the plasma membrane. This basic dense-body polypeptide is also found at certain cell boundaries where thin filaments in half-bands terminate at membrane-associated structures termed attachment plaques. A third, unidentified antigen is also found closely apposed to the cell membrane in regions of not only the dense-body and attachment plaque, but also the M-line analogue. Finally, a fourth high molecular weight antigen, composed of two polypeptides of approximately 400,000-D, is localized to the I-band regions surrounding the dense-body. The attachment of the dense-body to the cell surface and the differential localization of the dense-body-associated antigens suggest a model for their organization in which the unidentified antigen is a cell surface component, and the two 107,000-D polypeptides define different cytoplasmic domains of the dense-body.

We have constructed a genomic DNA expression library and screened it with antibodies in order to clone the deb-1 gene from the nematode Caenorhabditis elegans. This gene encodes a protein found at the base of the muscle dense-bodies, structures which attach actin thin filaments to the sarcolemma. We report the complete sequence of the deb-1 gene, its localization on the C. elegans genetic map, and the finding that it encodes a protein with a sequence very similar to chicken vinculin. We also show that the difference in size between this nematode protein and chicken vinculin is due in part to the absence from the nematode sequence of one of the three internal repeats found in the chicken sequence.

The Caenorhabditis elegans gene unc-22 encodes a very large muscle protein, called twitchin, which consists of a protein kinase domain and several copies of two short motifs. The sequence of twitchin has unexpected similarities to the sequences of proteins of the immunoglobulin superfamily, cell adhesion molecules and vertebrate muscle proteins, including myosin light-chain kinase. These homologies, together with results from earlier genetic and molecular analyses, indicate that twitchin is involved in a novel mechanism of myosin regulation.

The unc-22 gene of Caenorhabolitis elegans encodes a protein which is a component of the myosin-containing A-band of the worm's striated body-wall muscle. Among 51 revertants of a transposon-induced mutant, we have identified four which retain a barely detectable mutant phenotype. Molecular analysis shows that three of these have in-frame deletions of 1.0, 1.3 and 2.0 kilobases, whereas the fourth partial revertant and two other apparently complete revertants have small insertions. All these rearrangements involve coding sequence and, in the case of the deletions, result in polypeptides that are shorter than the wild-type protein. The region of the gene containing these rearrangements contains 10 copies of a motif recognized in other regions of the gene (our unpublished data). We suggest that one explanation for the minimally mutant phenotype associated with the deletions is that the size and the repeated nature of the unc-22 protein structure make it relatively tolerant of substitutions or deletions involving one or a small number of repeated motifs. These results could explain why in some human genetic diseases, such as Duchenne's muscular dystrophy, deletions can be associated with only mild forms of the disease.

A suppressor mutation has been isolated in Caenorhabditis elegans through reversion analysis of a muscle-defective mutant. The suppressor mutation acts on specific alleles of at least six genes and in one case we have been able to show that it partially restores functional gene product to a mutant otherwise lacking that product. These and other features of the suppressor suggest that it acts at some step in information transfer, perhaps through mechanisms similar to those described previously in microorganisms.

The unc-22 gene is one of a set of genes identified using classical genetics that affect muscle structure and function in the free-living nematode Caenorhabditis elegans. Since cloning the unc-22 gene by transposon tagging, we have used conventional techniques combined with a set of Tc1 transposon insertion alleles to characterize the gene and its products. The gene extends over more than 20 kb of genomic sequence and produces a transcript of approximately 14 kb. A polyclonal antibody raised against an Escherichia coli beta-galactosidase-unc-22 fusion protein recognizes a polypeptide in nematode extracts that is between 500,000 and 600,000 daltons and labels the muscle A-band in indirect immunofluorescent microscopy. The Tc1-induced alleles have been used at every stage to verify these conclusions. The Tc1 insertions are spread over much of the region that contributes to the mature transcript; in most alleles, Tc1 sequences are incorporated into a composite unc-22-Tc1 transcript. The large protein is either absent or severely reduced in amounts in the mutants. In one case, a truncated polypeptide was also identified. The location of the protein in the A-band, along with earlier genetic data, suggests that the unc-22 product may interact with myosin to regulate its function.