Characterization of mua-3 phenotypes

Temperature-dependent lethality:
Eggs were isolated via hypochlorite treatment (Sulston and Hodgkin 1988) and then harvested in M9 buffer to obtain a synchronous population. Synchronized populations of YJ35 mua-3(uy19) mutants and wild-type were transferred to E. coli HB101 seeded NGM plates at the L1 stage and cultivated at 15°, 20°, or 25° in triplicate. Synchronized populations of approximately 100 mua-3 and wild-type L1s were placed onto each plate. At 25°, approximately 30 hr after L1s were given food, L4s started to molt. At 20°, L4s started to molt after approximately 40 hr, and at 15° L4s started to molt after approximately 60 hr. The majority of death occurred during the molt; therefore, timeframes for the experiments were designated accordingly. The number of dead animals and the number of total animals were counted to determine percent survival of mua-3 and wild-type at each hour from 50 hr at 15°, 37 hr at 20°, and 30 hr at 25°.

Sterility:
To determine the number of offspring produced by individual animals of OT136 mua-3(rh195) mutants and wild-type, five L4s of each strain were transferred singly to E. coli-seeded NGM plates and their progeny were counted. Each worm was transferred to a new plate every day to avoid crowding and to visualize all the progeny easily. Progeny were counted 3 d later.