Calsequestrin, a calcium sequestering protein localized at the sarcoplasmic reticulum, is not essential for body-wall muscle function in Caenorhabditis elegans.

Calsequestrin is the major calcium-binding protein of cardiac and skeletal muscles whose function is to sequester Ca(2+ )in the lumen of the sarcoplasmic reticulum (SR). Here we describe the identification and functional characterization of a C. elegans calsequestrin gene (csq-1). CSQ-1 shows moderate similarity (50% similarity, 30% identity) to rabbit skeletal calsequestrin. Unlike mammals, which have two different genes encoding cardiac and fast-twitch skeletal muscle isoforms, csq-1 is the only calsequestrin gene in the C. elegans genome. We show that csq-1 is highly expressed in the body-wall muscles, beginning in mid-embryogenesis and maintained through the adult stage. In body-wall muscle cells, CSQ-1 is localized to sarcoplasmic membranes surrounding sarcomeric structures, in the regions where ryanodine receptors (UNC-68) are located. Mutation in UNC-68 affects CSQ-1 localization, suggesting that the two possibly interact in vivo. Genetic analyses of chromosomal deficiency mutants deleting csq-1 show that CSQ-1 is not essential for initiation of embryonic muscle formation and contraction. Furthermore, double-stranded RNA injection resulted in animals completely lacking CSQ-1 in body-wall muscles with no observable defects in locomotion. These findings suggest that although CSQ-1 is one of the major calcium-binding proteins in the body-wall muscles of C. elegans, it is not essential for body-wall muscle formation and contraction.

Muscle-specific functions of ryanodine receptor channels in Caenorhabditis elegans.

Ryanodine receptor channels regulate contraction of striated muscle by gating the release of calcium ions from the sarcoplasmic reticulum. Ryanodine receptors are expressed in excitable and non-excitable cells of numerous species, including the nematode C. elegans. Unlike vertebrates, which have at least three ryanodine receptor genes, C. elegans has a single gene encoded by the unc-68 locus. We show that unc-68 is expressed in most muscle cells, and that the phenotypic defects exhibited by unc-68 null mutants result from the loss of unc-68 function in pharyngeal and body-wall muscle cells. The loss of unc-68 function in the isthmus and terminal bulb muscles of the pharynx causes a reduction in growth rate and brood size. unc-68 null mutants exhibit defective pharyngeal pumping (feeding) and have abnormal vacuoles in the terminal bulb of the pharynx. unc-68 is required in body-wall muscle cells for normal motility. We show that UNC-68 is localized in body-wall muscle cells to flattened vesicular sacs positioned between the apical plasma membrane and the myofilament lattice. In unc-68 mutants, the vesicles are enlarged and densely stained. The flattened vesicles in body-wall muscle cells thus represent the C. elegans sarcoplasmic reticulum. Morphological and behavioral phenotypes of unc-68 mutants suggest that intracellular calcium release is not essential for excitation-contraction coupling in C. elegans.

unc-68 encodes a ryanodine receptor involved in regulating C. elegans body-wall muscle contraction.

Striated muscle contraction is elicited by the release of stored calcium ions through ryanodine receptor channels in the sarcoplasmic reticulum. ryr-1 is a C. elegans ryanodine receptor homologue that is expressed in body-wall muscle cells used for locomotion. Using genetic methods, we show that ryr-1 is the previously identified locus unc-68. First, transposon-induced deletions within ryr-1 are alleles of unc-68. Second, transformation of unc-68 mutants with ryr-1 genomic DNA results in rescue of the Unc phenotype. unc-68 mutants move poorly, exhibiting an incomplete flaccid paralysis, yet have normal muscle ultrastructure. The mutants are insensitive to the paralytic effects of ryanodine, and lack detectable ryanodine-binding activity. The Unc-68 phenotype suggests that ryanodine receptors are not essential for excitation-contraction coupling in nematodes, but act to amplify a (calcium) signal that is sufficient for contraction.

High molecular weight proteins in the nematode C. elegans bind [3H]ryanodine and form a large conductance channel.

Single-channel properties of a polypeptide fraction from the nematode Caenorhabditis elegans highly enriched in binding sites were studied in planar bilayers. [3H]Ryanodine binding sites were purified by sucrose gradient centrifugation of C. elegans microsomes solubilized in CHAPS detergent. The highest [3H]ryanodine binding activity sedimented at approximately 18% sucrose (wt/vol), and was composed of a major polypeptide with a M(r) of 360,000 and a minor polypeptide with a M(r) of 170,000. The ryanodine-binding polypeptide(s) formed a Ca(2+)-permeable channel with a permeability ratio P(divalent)/P(monovalent) = 4 and two conductance states of 215 pS and 78 pS in 0.25 M KCl. Ryanodine locked the channel in the 78 pS state and inhibited transitions between the 215 pS and 78 pS states. These data demonstrated the presence of a ryanodine receptor in C. elegans with functional properties comparable to those described in mammalian muscle.

Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes.

When DNA molecules are injected into Xenopus oocyte nuclei, they can recombine with each other. With bacteriophage lambda DNAs, it was shown that this recombination is stimulated greatly by introduction of double-strand breaks into the substrates and is dependent on homologous overlaps in the recombination interval. With plasmid DNAs it was shown that little or no recombination occurs between circular molecules but both intra- and intermolecular events take place very efficiently with linear molecules. As with the lambda substrates, homology was required to support recombination; no simple joining of ends was observed. Blockage of DNA ends with nonhomologous sequences interfered with recombination, indicating that ends are used directly to initiate homologous interactions. These observations are combined to evaluate possible models of recombination in the oocytes. Because each oocyte is capable of recombining nanogram quantities of linear DNA, this system offers exceptional opportunities for detailed molecular analysis of the recombination process in a higher organism.

Isolation and characterization of defective simian virus 40 genomes which complement for infectivity.

A new variant of simian virus 40 (EL SV40), containing the complete viral DNA separated into two molecules, was isolated. One DNA species contains nearly all of the early (E) SV40 sequences, and the other DNA contains nearly all of the late (L) viral sequences. Each genome was encircled by reiterated viral origins and termini and migrated in agarose gels as covalently closed supercoiled circles. EL SV40 or its progenitor appears to have been generated in human A172 glioblastoma cells, as defective interfering genomes during acute lytic infections, but was selected during the establishment of persistently infected (PI) green monkey cells (TC-7). PI TC-7/SV40 cells contained EL SV40 as the predominant SV40 species. EL SV40 propagated efficiently and rapidly in BSC-1, another line of green monkey cells, where it also formed plaques. EL SV40 stocks generated in BSC-1 cells were shown to be free of wild-type SV40 by a number of criteria. E and L SV40 genomes were also cloned in the bacterial plasmid pBR322. When transfected into BSC-1 cell monolayers, only the combination of E and L genomes produced a lytic infection, followed by the synthesis of EL SV40. However, transfection with E SV40 DNA alone did produce T-antigen, although at reduced frequency.

Lack of antibody in rheumatoid sera to autologous synovial macrophages and dendritic cells by antibody-dependent cellular cytotoxicity assays (ADCC).

We tested the hypothesis that antigens which stimulate the chronic immune reactivity in rheumatoid synovia might be associated with synovial macrophages and dendritic cells. ADCC assays were used to test sera from 15 RA patients and 8 non-RA controls for antibody to autologous primary and secondary cell cultures prepared by enzymatic digestion of synovial tissue. Such autoantibody was not detected in any of the 23 patients using assay conditions which minimized potential ADCC blocking effects of immune complexes and multinuclear giant cells. Collagenase producing dendritic and multinuclear giant cells. Collagenase activity and dendritic cells occurred with equal frequency in both RA and non-RA primary monolayers.