Gap junctions (connexons) are important for the coupling of cell populations in early development of C. elegans and are responsible for about one-third of the connections in its nervous system as represented by electrical junctions. A classical gap junction is made up of a hexameric array of subunits called connexins that span the plasma membrane with four transmembrane regions, two extracellular loop regions, one cytoplasmic loop and both cytoplasmic N and C terminals. We have used a battery of polyclonal antibodies obtained from immunisation to small peptides with sequences derived from both the cytoplasmic and extracellular domains of known homologous vertebrate connexins. A few of these antibodies have produced interesting staining patterns in the nervous system and the gut of hatched animals. Permeation and fixation solutions are being tested to try to obtain staining in early embryos. These positive antibodies have been used with Western immunoblots and have banding patterns with characteristic molecular weights that correspond to vertebrate connexins. Further hybridisations have been completed from both native and denaturing gels to observe the change from multimeric arrays to monomeric bands. Recent computer searches of both ACeDB and the Kohara cDNA sequence databases have not produced any genes with similar DNA homologies to vertebrate connexin genes. Oligonucleotides that we have used to these conserved peptide domains are beginning to yield some positive results that shall be tested by sequencing.

Nawrocki LW et al. (1993) International C. elegans Meeting "The cloning and molecular characterization of C. elegans GABA receptors subunits."

Fleming JT, Nawrocki LW, Miller KG

[

International C. elegans Meeting,

1993]

Clein M et al. (1991) International C. elegans Meeting "SELECTING FOR EXTRAGENIC SUPPRESSORS OP unc-55"

Walthall WW, Clein M

[

International C. elegans Meeting,

1991]

The embryonic DD and postembryonic VD motoneurons innervate different sets of muscles. Inspite of differences in lineal origin and target specificity the two classes of D motoneurons exhibit a number of biochemical and morphological similarities. Our goal is to understand the genetic basis for these similarities and differences. A survey of the neural Unc's reveals mutations in a number of genes that cause identical changes in both classes. Currently only one gene, unc- 55, a ventral coiler, has been identified that specifically affects one of the two classes. Reconstructions by Nawrocki et al., have shown that the Unc-55 allele e402 respecifies the VD motoneurons so that they innervate the same targets as the DD motoneurons. Our working hypothesis is that initially the genetic programs for both dasses of D motoneurons is the same and that unc-55 is necessary and sufficient to create the different specificities. A maze was designed to aid in the search for suppressors of unc-55. The maze, whose structure was inspired by the benzene ring, is effective in enriching for noncoiler phenotypes. Typically 95 to 100X of N2 animals navigate the maze and find the food in a 15 to 20 hour period. However, due to a number of blind alleys, <SX of unc-ff animals are successful. unc-ff strains CB 1170 and ER14 have been mutagenized with EMS following Brenner's procedure and F2 animals placed on the maze. The equivalent of 400,000 mutagenized chromosomes from CBl 170 and 100,000 chromosomes from ER14 have been screened with the maze. Presently we are characterizing two extragenic suppressors that are not coilers. Both are derived from ER14, each is X-linked and recessive. When separated from unc-55 they exhibit difficulties in moving backward but they are not coilers. Further characterization is underway.