The Diversity of α-Tubulin during Artemia Prelarval Development
Artemia possess three major α-tubulins, as defined by migration patterns on two-dimensional gels, but appears to contain only one α-tubulin gene. To determine how the isotubulin diversity is generated, we have characterized Artemia tubulin mRNA and immunologically analyzed the α-tubulins during development of the organisms. Hybridization of a cloned Drosophila α-tubulin gene to northern blots reveals only one size class of tubulin mRNA in Artemia at all developmental stages examined. Quantitation on dot blots with the same tubulin gene shows that the amount of α-tubulin message remains relatively constant during development. In vitro translation of poly (A)+ mRNA yields radioactively labelled tubulin which can be recovered by taxol-driven coassembly with purified Artemia tubulin and subsequent centrifugation through sucrose. Preliminary results from two-dimensional gel electrophoresis and fluorography indicate the synthesis of two α-tubulin isoforms in vitro. Anti-tubulin antibodies, when used to stain western blots of cell-free extracts or purified tubulin from Artemia, reveal the presence of acetylated and tyrosinated α-tubulin. Acetylated tubulin has been shown by others to result from post-translational mechanisms whereas the tyrosinated tubulin may arise by post-translational modification or as a primary gene product. The results indicate that the α-tubulin diversity in Artemia is due to post-transcriptional mechanisms and support our earlier conclusion that the α-tubulin composition does not change during prelarval development.