mRNAs for alpha- and beta-tubulin and flagellar calmodulin are among those coordinately regulated when Naegleria gruberi amebae differentiate into flagellates.

Abstract

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...

Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amebae into flagellates.

“…They show a coordinate and transient 20-fold increase after induction of the differentiation of amebae into flagellates (30). Three of the DS RNAs code for proteins found in the flagellar axoneme: a-and P-tubulin and a flagellum-specific form of calmodulin (CaM-1) (35). The fourth DS RNA, which is unidentified, is regulated in concert with the axonemal protein RNAs.…”

“…2A). Detailed descriptions of the cDNA plasmids used in this work have been reported previously (30,35 The two NS cDNA plasmids pcNg 2 and pcNg 3-28 contained sequences of unidentified poly(A)+ RNAs that are present in both amebae and differentiating cells ( Fig. 2A, rows 6 and 7).…”

“…2A, row 5). The cDNA inserts in these plasmids show no cross hybridization, each insert hybridizes to a single band on Northern (RNA) blots (30), and they each select specific mRNAs in hybrid selection-in vitro translation experiments (35). Therefore, we assayed the concentration of all six RNAs in the products of a run-on transcription using hybridization to a single filter containing all six plasmids plus pBR322.…”

“…We tested this possibility by comparing the size of the a-and ,-tubulin primary transcripts synthesized in 40-min nuclei. Tubulin RNAs were isolated by hybrid selection (35) and fractionated by agarose gel electrophoresis. Both the a-and ,-tubulin RNAs were heterogeneous, but they extended over the same size range (Fig.…”

“…Furthermore, the P-tubulin plasmid shows the same degree of specificity as the a-tubulin plasmids when it is used to hybrid select RNA from either total VOL. 8,1988 on May 10, 2018 by guest http://mcb.asm.org/ Downloaded from poly(A)+ RNA (35) or from in vitro transcription reactions (Fig. 4).…”