Suppressors of daf-ll identify new daf-d genes Wendy Schackwitz and James H. Thomas Department of Genetics, University of Washington, Seattle, WA 98195 C. elegans forms a dauer larva when exposed to high levels of dauer pheromone. Several genes involved in this process have been identified. Mutations in most of these genes result in one of two phenotypes. dauer defective (Daf-d) or dauer constitutive (Daf-c). The genes have been placed in a regulatory pathway (see below) based on genetic interactions. daf-d genes are efficiently identified by looking for suppressors of daf-c genes. Although this type of screen had been performed, screens for suppressors of daf-l I had not been done, therefore there was the potential of identifying new daf-d genes by looking for suppressors of daf-l l . We have identified new daf-d genes by using EMS to generate suppressors of the daf-l l Daf-c phenotype. The screen identified 20 strong suppressor mutations (>90% suppression of the daf-l l Daf-c phenotype) that appear to identify new genes. These 20 suppressors were mapped to linkage groups and complementation tests within each group revealed that they represent 16 genes. Eight of the strongest suppressors (>97% suppression of the daf-l l Daf-c phenotype) were chosen for further analysis. These eight suppressors are being mapped in more detail. Summary of results to date:
sal43 maps to LG I between
dpy-5 and
unc-13;
sal51 maps to LG I between
aex-6 and
unc-54;
sal69 maps to LG I close to
egl-30; sal 72, sal 73, and sal 59 (allelic) map to LG I to the right or very close to
unc-13; sal 50 and
sal58 (allelic) map to LG m between
unc-93 and
unc-32; sal 71 maps to LG IV close to
dpy4; sal 76 maps to LG V close to
unc-34.; and sal 63 maps to LG X. To test the phenotype of the suppressor mutations on their own, strains that no longer carry daf-l l (
m87) have been constructed for sal 43, sal 50, sal 51, sal 69, and sal 71. To test whether the suppressor mutations conveyed a Daf-d phenotype, the ability to form dauers on starved plates at 25 C was semi-quantitatively assayed. Most plates of the wild type formed thousands of dauers under our test conditions. Suppressors sal 50 and sal 69 formed many dauers on starved plates (100-lOOOs) and thus do not confer a Daf-d phenotype. The
sal43,
sal51, and sal 71 strains formed very few dauers on starved plates (0-50 dauers). Thus these mutations confer an incompletely penetrant Daf-d phenotype. lnterestingly daf- 3 and
daf-5 mutations also confer an incompletely penetrant Daf-d phenotype (0-50 dauers), whereas
daf-12 is completely penetrant (O dauers). Limited epistasis analysis has been performned for
sal43 with several daf-c mutations. The resulting daf-d; daf-c double mutants fall into three phenotypic classes: fully suppressed, weakly suppressed, and not suppressed. The Daf-c phenotype of daf-l l (
m87), daf-l l (
m84), daf-l l (sal 95), and daf- 21
(p673) are all fully suppressed by
sal43 (0-0.6% dauer formation). The Daf-c phenotypes of
daf-7(
el372), daf- 14
(m77), and
daf4(el 364) are weakly suppressed by sal 43 (76-84% dauer formation), and daf-l 9
(m86) is not suppressed by
sal43 (100% dauer formation). This pattern of suppression can be explained by the parallel pathway model shown below. sal 43 lies downstream of the group 1 daf-c genes, and thus fully suppresses mutations in them.
sal43 only weakly suppresses the group 2 daf-c mutations because, in the double mutant, one branch of the pathway is in a dauer repressing state whereas the other branch is in a dauer stimulating state. When the two branches are summed together the result is a weak Daf-c phenotype. Further characterization of the suppressors are underway.