Abstract. We have examined the nature of the requirement for RNA synthesis during the differentiation of Naegleria gruberi amebae into flagellates (Fulton, C., and C. Walsh, 1980, J. CellBioL, 85:346-360) by looking for poly(A)+RNAs that are specific to differentiating cells. A cDNA library prepared from poly(A)+RNA extracted from cells 40 min after initiation of the differentiation (40-min RNA), the time when formation of flagella becomes insensitive to inhibitors of RNA synthesis, was cloned into pBR322. Recombinant clones were screened for sequences that were complementary to 40-min RNA but not to RNA from amebae (0-min RNA). Ten of these diffentiation-specific (DS) plasmids were identified. The DS plasmids were found to represent at least four different poly(A)+RNAs based on cross-hybridization, restriction mapping, and Northern blot analysis.Dot blot analysis was used to quantify changes in DS RNA concentration. The four DS RNAs appeared coordinately during the differentiation. They were first detectable at 10-15 min after initiation, reached a peak at 70 min as flagella formed, and then declined to low levels by 120 min when flagella reached full length. The concentration of the DS RNAs was found to be at least 20-fold higher in cells at 70 min than in amebae. The changes in DS RNA concentration closely parallel changes in tubulin mRNA as measured by in vitro translation

Abstract. Three of four mRNAs that are specific to the differentiation of Naegleria gruberi amebae into flagellates (Mar, J., J. H. Lee, D. Shea, and C. J. Walsh, 1986, J. Cell Biol., 102:353-361)have been identified as coding for flagellar proteins. The products of these rnRNAs, which are coordinately regulated during the differentiation, were identified by in vitro translation of hybrid-selected RNA followed by twodimensional gel electrophoresis and antibody binding. Six cross-hybridizing clones complementary to a 1.7-kb RNA (class II) all selected mRNA that was translated into two rThe principal in vitro product, r comigrated with a cytoplasmic ~t-tubulin, while the minor product with a more acidic pI, a-2, comigrated with flagellar tx-tubulin. While Naegleria flagellar ~t-tubulin was found to be acetylated based on its reaction with a monoclonal antibody specific to this form, we suggest that ct-2 is not likely to arise due to acetylation in vitro but probably represents the product of a second tt-tubulin gene. The class III clone, also complementary to a 1.7-kb RNA, selected I~-tubulin mRNA. In the course of this work it was found, using monoclonal antibodies to the ct-and 13-subunits of tubulin, that Naegleria ~t-tubulin migrated faster than 13-tubulin on SDS-PAGE. The class IV clone, which hybridizes with a 0.5-kb RNA, selected an mRNA that was translated into a heat stable calcium-binding protein, flagellar calmodulin.T I~ rapid and synchronous differentiation of Naegleria amebae into flagellates is easily induced by washing amebae free of the bacteria or axenic medium that serves as a food source (reviewed by Fulton in references 7 and 8). When amebae differentiate into flagellates they form basal bodies (11), flagellar rootlets (24), and flagellar axonemes in just over 1 h (5, 37). By 120 min after initiation, 95-100% of the cells have full length flagella (10) and most have developed a microtubule-based cytoskeleton (42). In concert with the formation of cytoplasmic microtubules, the cells assume an asymmetric oval form, the flagellate shape (8). Neither tubulin (12, 13, 19) nor translatable tubulin mRNA (23) appear to be present in amebae in appreciable amounts. Yet within 10-15 rain after the initiation of the differentiation, functional tubulin mRNA appears (23).The formation of flagella by Naegleria amebae is not an obligate part of the life cycle (7). Apparently, amebae can grow and divide for an unlimited number of generations without ever forming flagella. This fact, combined with the complexity of the flagellar apparatus (the flagellar axoneme alone contains nearly 200 different proteins [29]), raises intriguing questions about the regulatory mechanisms involved in the differentiation. Based on studies using inhibitors of RNA and protein synthesis, the formation of flagella in Naegleria requires the synthesis of new RNA and protein (14). At least 70% (19) and probably >90% (12) of the tubulin in the flagellar axoneme is included among the proteins synthesized de novo during the differentiati...