Cloning and characterization of the altA  -tubulin gene of Physarum

Abstract

A cDNA clone derived from the altA locus, encoding one of several a-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, alA-tubulin, is 92% identical to the other known Physarum a-tubulins, alB and a2B, which are products of two tightly linked genes at the altB locus. The nucleotide sequence of the altA coding region is 82 YO 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 a-tubulin it encodes suggest that alA is the substrate for post-translational acetylation, giving rise to the a3-tubulin isoform found specifically in amoebae and flagellates. Northern blot analysis of plasmodia1 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.

Two alleles of a developmentally regulated alpha-tubulin locus in Physarum polycephalum replicate on different schedules.

“…The levels of expression of both the altA and the altB genes reach a peak near mitosis just before the onset of S phase, though apparently at substantially different levels (12). Is there a relationship between the expression pattern and the replication schedule of each locus?…”

“…The partial cDNA clone Ppc-cx125 is derived from the altB locus (40,59). The partial cDNA clone a501 is derived from the 3' region of the altA gene (12 A double digestion using Dral and MluI was found to satisfy both criteria (Fig. 1).…”

Microtubules in Microorganisms: How Tubulin Isotypes Contribute to Diverse Cytoskeletal Functions

“…Chlamydomonas reinhardtii tua1, tua2 tub1, tub2 each isoform: differential anti-tubulin drug resistance Kato- Minoura et al (2020). Physarum polycephalum altA, altB(N), altB(E), betA, betB, betC altA, altB(N), altB(E): MT assembly in plasmodia and myxamoeba Burland et al (1983), Roobol et al (1984), Cunningham et al (1993);Gerber et al (2022), altA builds flagellar MTs via PTM in amoeba and flagellates Green and Dove (1984), Blindt et al (1986), Diggins and Dove (1987), Sasse et al (1987); betA: MT structure assembly during myxamoeba stage Burland et al (1988), Diggins-Gilicinski et al (1989), Gerber et al (2022; betB: constitutively expressed in plasmodia and myxamoeba Werenskiold et al (1988), Paul et al (1989), benzimidazole resistance to both plasmodia and myxamoeba Burland et al (1984); betC: anastral mitotic spindle assembly in plasmodia Burland et al (1983), Roobol et al (1984), Gerber et al (2022), MT assembly in developing cells during amoeboid-plasmodial transition Solnica-Krezel et al (1988). *In Table 2, gene names are presented using organism-specific syntax.…”

“…Although the α-tubulin population contains two isoelectric variants, one was found to originate via acetylation of AltA ( Green and Dove, 1984 ; Blindt et al, 1986 ; Diggins and Dove, 1987 ; Sasse et al, 1987 ), leaving only one major α-isotype expressed in the myxamoeba phase ( Singhofer-Wowra and Little, 1987 ). While ALTB(E) encodes one of the two major α-tubulin isoelectric variants in plasmodia, the remaining four α-tubulin genes, and particularly ALTC and ALTD , potentially encode the other isoelectric variants ( Cunningham et al, 1993 ).The single α-tubulin expressed in myxamoeba was also found to be less prone to mutation ( Singhofer-Wowra et al, 1986 ) than its more divergent and multiple plasmodial counterparts ( Krämmer et al, 1985 ). This suggests that the tubulin mutational rate may be limited by the structural constraints of highly conserved MT structures like the axoneme, which is present in myxamoeba and not the plasmodial phase, but also supports the idea that utilizing multiple tubulin isotypes allows individual isotypes to more readily coevolve with specific MT-dependent processes.…”