The study of expression of a plasmodial-specific gene in physarum polycephalum.

The aim of the project was to investigate expression of the plasmodial-specific gene, P46 (Sweeney et al., 1987), during the apogamic amoebal-plasmodial transition of Physarum polycephalum. The intention was to establish the onset of transcription of the P46 gene in relation to the extended cell cycle that occurs during development (Bailey et al., 1987), and also in relation to commitment to plasmodial development. Initially, it was intended to use in situ hybridization to analyse expression of the P46 gene in individual developing cells. This method proved to be insufficiently sensitive for this purpose, and P46 expression was consequently investigated using isolated RNA. Following hybridization experiments to establish an appropriate method for the quantitative analysis of gene expression during development, expression of the P46 gene was investigated by northern analysis of RNA isolated from developing cells. It was possible to conclude from the results that expression of P46 begins in uninucleate cells that have become committed to plasmodial development. The hybridization assay was insufficiently sensitive to establish the onset of transcription more precisely, and the polymerase chain reaction (PCR) was used in subsequent experiments, to amplify P46 transcripts prior to their detection with radioactively-labelled probes. Using the PCR it was possible to conclude that P46 expression begins within a few hours of commitment. Amplification and sequence analysis of genomic DNA revealed an intron, of 182 base pairs in length, within the P46 gene. Amplification of amoebal RNA generated a fragment of a size corresponding to the genomic P46 fragment, suggesting unprocessed P46 transcripts are transcribed in amoebae.