Cloning and characterization of the altA alpha-tubulin gene of Physarum

Abstract

A cDNA clone derived from the altA locus, encoding one of several alpha-tubulins in Physarum, was sequenced and used to determine the developmental and cell cycle expression patterns of its corresponding gene. The predicted amino acid sequence of the altA gene product, alpha 1A-tubulin, is 92% identical to the other known Physarum alpha-tubulins, alpha 1B and alpha 2B, which are products of two tightly linked genes at the altB locus. The nucleotide sequence of the altA coding region is 82% identical to the two altB genes. Expression of the altA gene was found in all three cell types examined - amoeba, flagellate and plasmodium - but at substantially different levels in each. The peak level of altA message detected in flagellates was 14-fold higher than in amoebae, while the peak level in plasmodia was 5-fold lower than in amoebae. The expression pattern of altA and the predicted amino acid sequence of the alpha-tubulin it encodes suggest that alpha 1A is the substrate for post-translational acetylation, giving rise to the alpha 3-tubulin isoform found specifically in amoebae and flagellates. Northern blot analysis of plasmodial RNA samples from specific times in the cell cycle showed that the level of altA message varies over the cell cycle in a pattern similar to transcripts from other tubulin genes, with a peak at mitosis and little or no message detected during most of interphase.