srf-4,
srf-8 and
srf-9 (surface) mutants were isolated by Chris Link based on their ectopic surface binding to the lectin wheat germ agglutinin (WGA), which binds to N-acetylglucosamine. In contrast to
srf-2,
srf-3,
srf-5 and
srf-6 mutants, which have no gross morphological defects,
srf-4,
srf-8 and
srf-9 mutants have multiple defects, including uncoordinated movement, protruding vulva, abnormal egg laying, and abnormal copulatory bursae and gonad morphology, and therefore have been categorized as pleiotropic mutants (1). Each of these pleiotropic mutations enhances the defects associated with elevated
lin-12 activity (1, 2). A broad underlying defect in the pleiotropic mutants, which would interfere with the proper expression of one or more cuticle component, may also affect other processes required for normal development. Glycosylation, protein targeting, sorting, secretion or assembly of extracellular matrices are among the potentially affected processes.Prior genetic mapping located
srf-4,
srf-8 and
srf-9 to specific intervals of LGV (1). In order to identify these loci, we are using a candidate gene approach: we have searched the physical intervals that contain each of the pleiotropic srf genes for ORFs encoding products with homology to known components of glycosylation and secretion pathways in other organisms. These candidates were tested by dsRNAi to determine if their loss of function results in the characteristic phenotypes of the pleiotropic srf mutants. Using this approach we have identified two genes one in the physical interval containing
srf-8 and another in the interval containing
srf-4- whose loss of function results in ectopic WGA surface binding in the
rrf-3 mutant strain. In addition, RNAi silencing of these genes in
lin-12(
n302) mutants results in a significant increase of Muv progeny, indicating that their loss of function increases
lin-12 activity. We are currently sequencing these candidates in
srf-8 and
srf-4 background and will report our progress.(1)Link C. et al (1992) Genetics 131: 867-881 (2)Sundaram and Greenwald (1993) Genetics 135: 755-763.