The dense body and M-lines of body wall muscle cells are related integrin-mediated attachment complexes providing a transmembrane link from the myofilments within the sarcomeres to the basal lamina between muscle and hypodermis. This arrangement helps transmit contractile force from the myofilament lattice to the worms surface during locomotion. Because dense bodies and M-lines are homologous to the integrin-mediated attachments made by vertebrate cells, they offer an opportunity for genetic investigation of an important class of conserved adhesion complexes.A subset of genes identified in a previous genome-wide screen for Pat mutants (Paralyzed, Arrested elongation at Two-fold) have been shown by us and others to encode components of dense bodies and M-lines. Mutations that block the expression of several membrane and membrane-proximal components, including PAT-3/integrin, UNC-112, PAT-4/integrin-linked kinase and PAT-6/actopaxin, block the proper recruitment of membrane-distal attachment components and the attachment of myofilaments.Here we report progress on the molecular isolation
pat-9.
pat-9 mutant embryos have defects in sarcomere assembly very similar those in
unc-112,
pat-4 and
pat-6 loss-of-function mutants. We have used standard three-factor and single nucleotide polymorphism mapping to position
pat-9 to an interval of ~1 mb near the right end of the X chromosome. There are ~50 annotated genes in this region, but none are predicted to encode proteins previously associated with integrin adhesion complexes. We used SAGE (Serial Analysis of Gene Expression) data generated by the Canadian Genome Project from sorted C. elegans muscle cells
(http://elegans.bcgsc.ca/), in combination with a computational algorithm designed to identify cis-regulatory motifs from the promoter regions of genes expressed preferentially in muscle (Larry Schriefer, personal communication), to narrow down this list to 10 candidates likely to be expressed in body wall muscle cells. Standard RNAi experiments for two of these genes produced Pat progeny. DNA sequence analysis of T27B1.2 isolated from
pat-9 embryos and N2 adults revealed a single base pair difference. The
pat-9 sequence has a G to A change disrupting the 5 splice site at the start of the fourth intron. Transformation rescue experiments are underway to determine whether T27B1.2 corresponds to
pat-9.