mRNAs for α- and β-tubulin and flagellar calmodulin are among these coordinately regulated when Naegleria gruberi amebae differentiate into flagellates

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 mRNAs, which are coordinately regulated during the differentiation, were identified by in vitro translation of hybrid-selected RNA followed by two-dimensional 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 α-tubulins. The principal in vitro product, α-1, comigrated with a cytoplasmic α-tubulin, while the minor product with a more acidic pI, α-2, comigrated with flagellar α-tubulin. While Naegleria flagellar α-tubulin was found to be acetylated based on its reaction with a monoclonal antibody specific to this form, we suggest that α-2 is not likely to arise due to acetylation in vitro but probably represents the product of a second α-tubulin gene. The class III clone, also complementary to a 1.7-kb RNA, selected β-tubulin mRNA. In the course of this work it was found, using monoclonal antibodies to the α- and β-subunits of tubulin, that Naegleria α-tubulin migrated faster than β-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.