Primary structure of an α-tubulin gene of Physarum polycephalum

Abstract

An α-tubulin gene of Physarum was isolated as a phage-λ NM1149 recombinant (designated phage-λ NαTu). Phage-λ NαTu contained a 4700 base-pair HindIII nuclear DNA fragment of an allele of the altB locus of Physarum (one of four unlinked α-tubulin gene loci). Subfragments of the 4700 base-pair insert of phage-λ NαTu 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 S 1 nuclease analysis. The 4650 base-pair HindIII insert into phage-λ NαTu 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 α-tubulin contains seven intervening sequences, ranging from 63 to 222 nucleotides in size. The exons have a sequence that is identical with a Physarum α-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 α-tubulin gene has a C-terminal methionine codon and not a tyrosine codon, which has been found in all α-tubulin genes sequenced to date.