Preferential expression of one  -tubulin gene during flagellate development in Physarum

Abstract

The microbial eukaryote Physarum pofycephahm displays several distinct cell types in its life cycle, including amoebae, flagellates and plasmodia. Despite its relative simplicity, Physarum has a tubulin gene family of complexity comparable to that of Drosophifa. We have identified P-tubulin cDNAs from Physarum that are derived from the betA P-tubulin locus and encode P1A tubulin. We have also identified a partial cDNA for the unlinked betB P-tubulin gene, which encodes P1B tubulin. The polypeptide sequences encoded by betA and betB show 99 % identity, but the nucleotide sequences show only 85 % identity, consistent with an ancient duplication of these genes. The betB gene is expressed in amoebae, flagellates and plasmodia, whereas betA is expressed only in amoebae and flagellates. During the amoeba-flagellate transition the level of betA transcript increases over 100-fold, while the level of betB transcript changes very little. Thus Physarum has a mechanism for regulating the level of discrete P-tubulin transcripts differentially during flagellate development. A need for this differential regulation could account for the maintenance of the virtually isocoding betA and betB P-tubulin genes.

Cloning and characterization of the altA -tubulin gene of Physarum

“…Two P-tubulin genes, betA and k t B , are expressed in amoebae (Burland et al, 1984). During flagellate induction, however, only betA message shows an increased abundance (Paul et al, 1992). Remarkably, the magnitude of betA induction is much greater than altA induction over the same time course: while altA message increased by about 14-fold from its steady-state level in amoebae (Fig.…”

“…The construction and screening of two Physarum cDNA libraries, ML5 and ML6, are described by Paul et al (1992). The source of RNA for ML6 cDNA synthesis was strain LU352 (Dee et al, 1989), grown axenically (McCullough et al, 1978).…”

“…1986). About 300 ng of a606 transcript was translated iu vifro a s described (Paul et al, 1992). A mixture of the radiolabelled product and unlabelled plasmodial lysate was resolved by two-dimensional PAGE and blotted onto a nitrocellulose membrane as described (Burland et al, 1988).…”

“…6) is accompanied by a coordinate induction of a specific j3-tubulin message (Paul et al, 1992). Two P-tubulin genes, betA and k t B , are expressed in amoebae (Burland et al, 1984).…”

“…This clone included a 500 bp insert, designated a501, that hybridized preferen tially to polymorphic restriction fragments characteristic of the altA locus on Southern blots of Physarurzz genomic DNA (data not shown). In order to obtained a full-length cDNA from this locus, the a501 fragment was used to probe the amoebal/flagellate cDNA library ML6 (Paul et a/., 1992), which includes size-selected cDNA inserts of greater than 1 kb. From among the positive plaques detected, one containing a 1450 bp cDNA insert, designated a606, was characterized further.…”