This paper presents evidence that the phenotypic transformation of Naegleria gruberi from amebae to flagellates that occurs when cells are placed in a nutrient-free aqueous environment is dependent on transcription and translation. RNA and protein are synthesized during the hour-long differentiation. Actinomycin D and daunomycin selectively inhibit RNA synthesis, and cycloheximide selectively inhibits protein synthesis, throughout the time required for differentiation. These inhibitors prevent differentiation if added soon after the cells are transferred to nonnutrient buffer but cease to block specific differentiation events at subsequent, reproducible times, the transition points. After each transition point, morphogenesis can occur in the presence of the inhibitor and in the virtual absence of transcription or translation. A map of the transition points indicates that RNA synthesis is required until halfway through the temporal process from initiation to flagellum assembly, and that protein synthesis is required until three-fourths of the way through. Even when flagellum outgrowth can occur in the presence of cycloheximide, the length of the flagella formed is determined by the extent of synthesis of an unknown "limiting precursor." The transition points for formation of flagella and for formation of the streamlined flagellate body shape are temporally separate. These results indicate that differentiation in Naegleria involves a redirection of cell metabolism to produce new RNA and protein molecules that are essential for morphogenesis.

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