Structure and expression of an α‐tubulin gene of Physarum polycephalum

Fragments of Physarum polycephalum DNA generated by partial digestion with Sau 3A wer cloned into phage‐λ EMBL4. A recombinant (phage‐λ EαTu) containing an α‐tubulin (Eα‐tubulin) gene was isolated. The Eα‐tubulin gene is part of the altB locus. The gene was sequenced and was found to contain seven intervening sequences. the α‐tubulin isotype (Eα‐tubulin) encoded by the gene has a methionine residue at the C‐terminus. The Eα‐tubulin gene has much in common with the Nα‐tubulin gene cloned into phage‐λ NM1149 [M. J. Monteiro & R. A. Cox (1987) J. Mol. Biol. 193 , 427–438]. However, the gene products Eα‐tubulin and Nα‐tubulin differ in amino acid sequence near to the C‐terminus. E‐peptide (corresponding to amino acids 440–448 of Eα‐tubulin) and N‐peptide (corresponding to amino acids 437–445 of Nα‐tubulin) were synthesised and used to raise antibodies (E‐antibodies and N‐antibodies). The antibodies were used to show that on two‐dimensional gel elctrophoresis Nα‐tubulin travels to the α1 position and Eα‐tubulin moves to the α2 position. Gene‐specific DNA probes were used to show that transcripts of the Eα‐tubulin gene were present in the plasmodial but not in the amoebal phase of the life cycle. The E‐ and N‐antibodies detected Eα‐ and Nα‐tubulins in plasmodia but not in amoebae, confirming that the expression of the Eα‐ and Nα‐tubulin genes is regulated during development. Eα‐ and Nα‐tubulin were shown to be components of spindle microtubules by indirect immunofluorescence microscopy.