Temperature-sensitive mutants of physarum polycephalum: A search for cell cycle mutants.

Physarum polycephalum is a Myxomycete which grows as uninucleate amoebae or multinucleate plasmodia. The aim of this work was to isolate mitotic cycle mutants in an apogamic strain (CLd) of P. polycephalum and to exploit the natural mitotic synchrony of plasmodia for their analysis. The first stage (Chapter 3) involved isolation of temperature-sensitive growth mutants. Such mutants can be isolated by testing amoebal clones or plasmodia derived from amoebal clones. Testing amoebae is much easier, but previous work indicated that most mutations expressed in the plasmodial phase could only be isolated by testing plasmodia. The evidence, however, was equivocal, so both methods were used to isolate mutants, and their merits compared. It was concluded that over 70% of mutants isolated were expressed in both amoebae and plasmodia. Thus, mutations expressed in plasmodia can be isolated by testing amoebae. The second stage (Chapter 4) involved identification of mitotic cycle mutants amongst the temperature-sensitive mutants. Asynchronous cultures of amoebae or plasmodia of the mutants were transferred from permissive to nonpermissive temperature and observed microscopically. Of more than 100 mutants screened, three had altered mitotic indices and one was defective in cell division. The third stage (Chapter 5) involved preliminary characterization of these putative cell cycle mutants. Synthesis of macromolecules in plasmodia, and the DNA contents of plasmodial and amoebal nuclei were measured. These measurements were complemented by time-lapse cinematography of amoebal cultures (Chapter 6). It was concluded that none of the four mutants was completely blocked in cell cycle progression Despite limited success, the results indicated that future attempts to isolate cell cycle mutants of P. polycephalum should be more successful.