Bejsovec AM et al. (1984) "Genetic techniques for analysis of nematode muscle."

Anderson P, Eide DJ, Bejsovec AM

[1984]

Current knowledge concerning the protein components of muscle is based largely on biochemical analysis of myofibrillar preparations. Such in vitro studies are limited because direct evidence for the in vivo function of isolated proteins is difficult to obtain. In vitro techniques, futhermore, are restricted often to the study of abundant proteins. Very little is known about minor sarcomeric components or how the sarcomere is assembled overall. Certainly, the assembly and function of a structure as complex as the sarcomere require many more than the dozen or so proteins commonly studied. Genetic techniques provide an alternative approach to the study of muscle. Mutations that cause muscle disfunction define genes required to construct a normal muscle. cell. The nature of mutant defects provides insights into the functions of the wild-type gene products. Genetic analysis is not restricted to the study of abundant proteins. The genes for even low-abundance proteins are subject to mutation, and if a gene product is required for muscle assembly or function, such mutants will be muscle-defective. Macromolecular complexes provide special opportunities for genetic intervention, because gene products that directly interact in the structure can often be identified. Mutational defects that affect one member of an interacting pair of proteins can be compensated by mutations affecting its interacting partner. Not all genes, hwoever, are directly accessible to genetic analysis. For example, genes whose products are essential for cell or organism viability and genes having more than one functional copy present special problems.....

Bejsovec AM et al. (1983) Worm Breeder's Gazette "muscle defective revertants of unc-105(n490)."

Bejsovec AM, Anderson P

[Worm Breeder's Gazette, 1983]

We have isolated a variety of muscle defective mutants by reverting the paralyzed mutant unc-105(n490). n490 is severely paralyzed, apparently due to hyper-contraction of body-wall muscle cells. Revertants having improved motility are easily visualized among their paralyzed siblings. Wild-type revertants have been described by Joan Park (see Newsletter 7:49) as null alleles of unc-105. We find that partial revertants of n490 are often muscle defective. Being muscle defective, such mutants are unable to generate the force required for the n490 mutant phenotype. Thus, they are isolated as suppressors of hyper-contraction. Although these revertant strains have an uncoordinated phenotype, they are usually more motile than n490, and this serves as the basis for their selection. We used this reversion to isolate spontaneous unc-54 and unc-22 mutations (see 1983 Meeting Abstracts), and have now begun to analyze other classes of n490 revertants. We hope that among these revertants will be novel muscle defective mutants. We mutagenized cultures of n490 and picked partial revertants among the F2 progeny. As expected, many Twitcher revertants (presumably unc- 22) were observed; we have not analyzed this class. The remaining partial revertants were screened for abnormal muscle using polarized light microscopy. We are in the process of analyzing 117 partial revertants of n4900 74 of these revertants are muscle defective. Thus far, we have identified twenty additional alleles of unc-54 and one allele each of unc-52, utations complement alleles of all the known genes having a similar muscle defective mutant phenotype. These mutations may define new muscle genes. Interestingly, another 21 of the muscle defective revertants appear to contain dominant Unc's that have a recessive lethal phenotype. Several of these mutations have been segregated away from n490; they are muscle defective as heterozygotes and are inviable as homozygotes. Finally, two additional alleles of unc-15 have been identified among our collection of spontaneous revertants. We hope that these revertants will define new loci involved in development and function of the body-wall muscle and provide additional alleles of the already identified genes.

Bejsovec AM et al. (1987) International C. elegans Meeting "molecular and ultrastructural analysis of dominant unc-54 mutations."

Anderson P, Bejsovec AM

[International C. elegans Meeting, 1987]

Bejsovec AM et al. (1985) International C. elegans Meeting "new types of muscle-defective mutants."

Anderson P, Bejsovec AM

[International C. elegans Meeting, 1985]

Bejsovec AM et al. (1988) Worm Breeder's Gazette "antimorphic mutations in the head region of the unc-54 myosin heavy chain gene."

Bejsovec AM, Anderson P

[Worm Breeder's Gazette, 1988]

We are investigating the molecular basis of dominant antimorphic mutations at the unc-54 locus. These dominant-uncoordinated, recessive lethal mutations disrupt the assembly of thick filaments in the body wall muscle. By genetic and ultrastructural criteria, the mutant gene product interacts with both wild-type myosin heavy chain B, the unc-54 gene product, and myosin heavy chain A, the minor heavy chain class found in body wall muscle thick filaments. The strong disruptive effect is mediated by very low levels of full length protein - depending on the allele the amount ranges from less than 2% to approximately 20% of wild-type levels. Following EMS mutagenesis, these antimorphic missense alleles arise at a frequency equal to that of recessive null alleles at the unc-54 locus. To determine what portions of the myosin heavy chain are affected by these mutations, we have employed a technique developed by Myers et al. (Science 230: 1242, 1985) for detecting single base changes in genomic DNA. Base changes have been localized for 10 independently isolated EMS-induced alleles; all 10 show changes in the region of the gene encoding the globular head. [See Figure 1] One allele, unc-54(r342), carries 2 separate point mutations - one in the head and one in the rod portion of the gene. Both mutations have been sequenced and only the one located in the head creates an amino acid substitution. We have used the polymerase chain reaction ( PCR) technique (Saiki et al., Science 239: 487, 1988) to amplify and then sequence without cloning the affected region from mutant genomic DNA. Mutant sequences confirm the accuracy of mismatch positions estimated by the Myers technique. The head mutations sequenced so far create non-conservative substitutions for amino acids that are highly conserved among many myosin heavy chain sequences (including the yeast heavy chain). Thus we have mutationally identified parts of the globular head that are important in thick filament assembly. [See Figure 1]

Bejsovec AM et al. (1989) International C. elegans Meeting "myosin-heavy-chain mutations that disrupt thick-filament assembly."

Anderson P, Bejsovec AM

[International C. elegans Meeting, 1989]

Bejsovec AM et al. (1988) Genes Dev "Myosin heavy-chain mutations that disrupt Caenorhabditis elegans thick filament assembly."

Anderson P, Bejsovec AM

[Genes Dev, 1988]

We have investigated Caenorhabditis elegans mutants in which altered unc-54 myosin heavy-chain protein interferes with assembly of thick myofilaments. These mutants have a dominant, muscle-defective phenotype, because altered myosin heavy-chain B (MHC B), the product of the unc-54 gene, disrupts assembly of wild-type MHC B. The mutant MHC B also interferes with assembly of wild-type myosin heavy-chain A (MHC A), the product of another MHC gene expressed in body-wall muscle cells. Because of disrupted MHC A assembly, dominant unc-54 mutants also exhibit a recessive-lethal phenotype. Dominant unc-54 mutations are missense alleles, and the defects in thick filament assembly result from mutant protein that is of normal molecular weight. Accumulation of mutant MHC B in amounts as little as 2% of wild-type levels is sufficient to disrupt assembly of both wild-type MHC A and MHC B. Dominant unc-54 mutations occur at remarkably high frequency following ethylmethane sulfonate (EMS) mutagenesis; their frequency is approximately equal to that of recessive, loss-of- function mutations. This unusually high gain-of-function frequency implies that many different amino acid substitutions in the myosin heavy-chain B protein can disrupt thick filament assembly.

Bejsovec AM et al. (1990) Cell "Functions of the myosin ATP and actin binding sites are required for C. elegans thick filament assembly."

Anderson P, Bejsovec AM

[Cell, 1990]

We have determined the positions and sequences of 31 dominant mutations affecting a C. elegans muscle myosin heavy chain gene. These mutations alter thick filament structure in heterozygotes by interfering with the ability of wild-type myosin to assemble into stable thick filaments. These assembly-disruptive mutations are missense alleles affecting the globular head of myosin. The most strongly dominant alleles alter highly conserved residues of the myosin ATP binding site, indicating that functions of the myosin ATPase are important for thick filament assembly. Other alleles alter the site at which myosin binds actin.

Dierick H et al. (1999) Curr Top Dev Biol "Cellular mechanisms of wingless/Wnt signal transduction."

Dierick H, Bejsovec AM

[Curr Top Dev Biol, 1999]

Wg/Wnt signaling regulates cell proliferation and differentiation in species as divergent as nematodes, flies, frogs, and humans. Many components of this highly conserved process have been characterized and work from a number of laboratories is beginning to elucidate the mechanism by which this class of secreted growth factor triggers cellular decisions. The Wg/Wnt ligand apparently binds to Frizzled family receptor molecules to initiate a signal transduction cascade involving the novel cytosolic protein Dishevelled and the serine/threonine kinase Zeste-white 3/GSK3. Antagonism of Zw3 activity leads to stabilization of Armadillo/beta-catenin, which provides a transactivation domain when complexed with the HMG box transcription factor dTCF/LEF-1 and thereby activates expression of Wg/Wnt-responsive genes. The Wg/Wnt ligands pass through the secretory pathway and associate with extracellular matrix components; recent work shows that sulfated glycosaminoglycans are essential for proper transduction of the signal. Mutant forms of Wg in Drosophila reveal separable aspects of Wg function and suggest that proper transport of the protein across cells is essential for cell fate specification. Complex interactions with the Notch and EGF/Ras signaling pathways also play a role in cell fate decisions during different phases of Drosophila development. These many facets of Wg/Wnt signaling have been elucidated through studies in a variety of species, each with powerful and unique experimental approaches. The remarkable conservation of this pathway suggests that Wg/Wnt signal transduction represents a fundamental mechanism for the generation of diverse cell fates in animal embryos.

Saenz-Narciso, Beatriz et al. (2011) International Worm Meeting "Characterizing the adrenomedullin homologue in C. elegans."

Martinez, Alfredo, Cabello, Juan, Gomez-Orte, Eva, Saenz-Narciso, Beatriz

[International Worm Meeting, 2011]

Adrenomedullin (AM) is a peptide hormone that shows multiple physiological functions in vertebrates, such as bronchodilation, neurotransmission, hormone regulation, antimicrobial activity or growth regulation. Deregulation of AM has been shown in different human pathologies such as diabetes, cancer, hypertension, heart failure, and sepsis. Research on molecules capable of modulating AM and its effects may lead to the discovery of new pharmacological agents to treat these important human diseases. Highly interesting is the role of AM in cancer. It has been proven that AM acts as an autocrine/paracrine tumor cell survival factor and its elevated expression in cancer cells can promote angiogenesis leading to tumor progression. While AM has been well characterized in other research models, little is known about this molecule in C. elegans. We have found and characterized a mutant in the AM homologue of C.elegans, here called wam-1. 4D microscopy analysis of wam-1 mutants shows defects in the migration of the hermaphrodite gonad. There are also several defects in embryonic development. Some embryos have cells that are excluded out of the worm during development while fate specification, as well as proliferation is normal. This suggests a function of wam-1 in cell adhesion. To localize the expression of the worm AM homologue protein, we performed inmunostaining using mouse anti-AM antibodies. The AM protein was localized in the hypodermis. In contrast, we also generated transgenic lines expressing GFP under the control of the wam-1 promoter. GFP expression was detected in the mouth of the worm, although due to its homology with secreted hormones, the protein could play a role far away from the secretory cells. At the moment, we are performing a detailed molecular and genetic characterization, in order to understand the function of this molecule.