Primary structure of an alpha-tubulin gene of Physarum polycephalum.

An alpha-tubulin gene of Physarum was isolated as a phage-lambda NM1149 recombinant (designated phage-lambda N alpha Tu). Phage-lambda N alpha Tu contained a 4700 base-pair HindIII nuclear DNA fragment of an allele of the altB locus of Physarum (one of four unlinked alpha-tubulin gene loci). Subfragments of the 4700 base-pair insert of phage-lambda N alpha Tu were cloned into phage M13 and the nucleotide sequence was determined by the dideoxy chain termination method. The start point of transcription was identified by primer extension and a putative polyadenylation site was located by S1 nuclease analysis. The 4650 base-pair HindIII insert into phage-lambda N alpha Tu spans the complete gene; sequences upstream from the 5' end contain the RNA transcription promoter elements (the TATA and CCAAT boxes). The nucleotide sequence encoding alpha-tubulin contains seven intervening sequences, ranging from 63 to 222 nucleotides in size. The exons have a sequence that is identical with a Physarum alpha-tubulin cDNA clone, except for three base changes, one leading to a Val codon in place of a Met codon, another leading to a Glu codon in place of an Asp codon, and the third change is silent. The genomic clone provides the nucleotide sequence coding for the last 26 amino acid residues missing from the cDNA clone. The new sequence data indicate that the alpha-tubulin gene has a C-terminal methionine codon and not a tyrosine codon, which has been found in all alpha-tubulin genes sequenced to date.