Activation of a  -Tubulin Gene during Early Development of the Plasmodium in Physarum polycephalum

Abstract

Uninucleate amoebae of Physarum polycephalum strain CL undergo apogamic development to form multinucleate plasmodia via an intermediate stage of large, uninucleate cells irreversibly committed to plasmodial development. This amoebal-plasmodia1 transition involves major changes in tubulin gene expression and the organization of microtubular structures. We analysed the expression of the betC locus, which encodes the plasmodial-specific P2-tubulin, during plasmodial development. A key question addressed was the timing of expression of betC in relation to the last open mitosis of the amoeba and the first closed mitosis of the plasmodium during the transition. Culture conditions were improved to yield partly synchronous differentiating cultures containing 50-60 % committed cells, in order to facilitate biochemical analysis of development. Northern blotting indicated that betC RNA was virtually absent from amoebae and from early differentiating cultures. However, betC transcripts could already be detected in differentiating cultures containing only 0.1 % of committed cells; the relative amount of betC transcripts increased as the percentage of committed cells in differentiating cultures increased. In fully developed plasmodia, there was at least a 330-fold increase in the betC transcript level compared to that in amoebae. We conclude that betC is activated during the amoebal-plasmodia1 transition immediately before or during the commitment event. Small amounts of P2-tubulin polypeptide could first be detected by Western blotting around the stage of the first closed mitosis. Thus P2-tubulin may participate in the first closed mitosis that committed cells undergo during their development into plasmodia.

Identification of three genes expressed primarily during development in Physarum polycephalum

“…1990), we could not detect proP mRNA in RNA from amoebae. The expression pattern of proP is similar to that previously observed for two other plasmodium-specific genes, β2-tubulin and fragminP (Solnica-Krezel et al 1988;T'Jampens et al 1997).…”

Characterization of npf mutants identifying developmental genes in Physarum

“…( 1 989), with modifications for the 6-11B-1 antibody described by Solnica-Krezel et al (1990). Cells were examined using a Zeiss Axiophot microscope.…”

“…This transition involves many changes in cellular organization and gene expression (Bailey e t a/., 1987;Sweeney e t al., 1987;Solnica-Krezel et al, 1988). Genetic analysis of wild-type isolates of P. pobcephaltlm revealed that plasmodium formation is a sexual process and identified three naturally polymorphic loci that are involved in its control : matB and matC, which influence the frequency of fusion between haploid amoebae ; and matA, which controls development of matAheteroallelic fusion cells into diploid plasmodia (Dee, 1987;Kawano e t al., 1987).…”

Variable pathways for developmental changes of mitosis and cytokinesis in Physarum polycephalum.

“…Amebae were cultured on liver infusion agar plates at 30°C, a temperature restrictive to plasmodial development, as described by Blindt et al (1986). CL amebae were induced to develop into plasmodia as described by Solnica-Krezel et al (1988). The proportion of cells committed to plasmodium formation was determined as in Youngman et al (1977) and Solnica-Krezel et al (1990).…”

“…The reorganization of microtubules during this amebal-plasmodial transition (APT) is accompanied by changes in the repertoire of expressed tubulin isotypes. At least two tubulin isotypes,/31A and ct3, disappear (Burland et al, 1984;Diggins and Dove, 1987;Sasse et al, 1987), whereas at least three, Ncd, Etx2, and ~2, appear (Monteiro et al, 1987;Solnica-Krezel et al, 1988.…”