A study of developmentally regulated genes in Physarum polycephalum

In the life cycle of the Protist, Physarum polycephalum, uninucleate amoebae develop into multinucleate, syncytial plasmodia via an irreversible development transition. Previously a subtracted cDNA library was screened and a novel class of genes was identified that were expressed primarily during development. These genes were called red genes (regulated in development).;My first aim was to identify and characterise more red genes. I isolated two partial cDNA clones, D11/1100 (redE) and A18/1020 (mynD). Northern blotting showed that both of the genes represented red genes; for redE this was confirmed by RT-PCR. Southern blotting showed that redE is a single-copy gene, while mynD is a single-copy gene that is a member of a gene family. A18/1020 was renamed mynD (myosin developmental) because database searches showed that the cDNA encodes part of the tail domain of a type II myosin heavy-chain protein. The complete redE coding sequence, plus some of the upstream promoter sequence was obtained by cloning a genomic restriction fragment. Database searches showed that the deduced RedE protein was not homologous to any known proteins, although it contained several potential phosphorylation and glycosylation sites. Thus, the function of RedE is unknown. As a first step towards investigating red gene function, I attempted to make a vector that would allow the manipulation of gene expression.;My second aim was to examine the replication of the red genes in the plasmodial S-phase because genes that are expressed at low levels in the plasmodium had not previously been studied. I showed that redE is replicated in early S-phase, and that two previously discovered red genes, redA and redB, are replicated in mid and early S-phase respectively