Studies of genes showing differential expression during plasmodium development in physarum polycephalum

Uninucleate amoebae of the protist Physarum polycephalum develop into multinucleate, syncytial plasmodia following a developmental transition that is initiated by the multiallelic mating-type locus, matA. As a step towards understanding the processes involved in plasmodium development, genes that are expressed primarily during the amoebal-plasmodial developmental transition were previously identified from a subtracted cDNA library. The initial aim of the research described in this thesis was to perform sequence analysis of four such cDNA clones. This analysis revealed that the coding sequences of the four genes were incomplete. The N-terminus of the deduced P4/10P transcript contains homology to Rhizobium nodulation N proteins, redB encodes a calcium-binding protein with homology to sarcoplasmic proteins from invertebrates while both redA and D6/18P are novel genes. Northern blotting analysis was then performed to examine the unique expression pattern of redA and redB in more detail and confirmed that these genes are expressed primarily during the developmental transition. Southern blotting analysis indicated that redA and redB do not belong to gene families. The complete coding sequence for redA was deduced from a genomic clone obtained by inverse PCR and two introns were identified in the 5' region of the gene; it is possible that the 3' region of redA contains a third intron. To investigate the role of redA, gene disruption studies were initiated; however, in P. polycephalum such studies are limited due to considerably low transformation efficiency. A further limitation to transformation studies using P. polycephalum is the availability of only one selectable marker.