Sporulation competence in Physarum polycephalum CL

Chapman, Aileen J (1981) Sporulation competence in Physarum polycephalum CL. PhD thesis, University of Glasgow.

Abstract

The central aim of this work was to explore the possible value of P. polycephalum CL as a model of eukaryotic differentiation. Initially it was hoped to obtain a method of reproducibly obtaining sporulation and later to analyse sporulation both biochemically and genetically. Microplasmodia of the CL strain were found to yield the highest sporulation frequencies when harvested at the end of exponential growth. Sporulation frequencies of microplasmodia harvested at other points in the growth cycle could be improved by allowing overnight growth, as a surface plasmodium, before exposure to starvation medium. The minimum length of starvation in the dark required before one light pulse would induce sporulation of all plasmodia was found to be 72 h. Plasmodia became committed to sporulation about 4 h after illumination. The time of commitment to sporulation was related to the time of illumination, and not the overall length of starvation, since altering the time of illumination also altered the time of commitment. The importance of DNA replication and mitosis during the process of sporulation was assessed by examining the effects of inhibitors of these events on starving plasmodia. Nocodazole, an inhibitor of microtubule assembly, prevented sporulation if added any time up to 48 h during starvation. It was assumed, therefore, that the last mitosis during starvation occurred at about 48 h. However, nocodazole might also have affected some other event, besides mitosis, which involved microtubules. The DNA synthesis inhibitor, hydroxyurea, prevented sporulation if added at any time up to 24 h during starvation. This suggested that the last DNA replication preceded the last event susceptible to inhibition by nocodazole by some 24 h. By pulse labelling with methyl-HJ thymidine during starvation, the periods of DNA synthesis during the 72 h starvation period were defined. Periods of DNA replication began at about 4 h, 15 h and 24 h during starvation, confirming that the last replication occurred at about 24 h and demonstrating that this was the third replication to occur during starvation. The patterns of DNA replication in a sporogenous and an asporogenous culture were compared in an attempt to further clarify the role of DNA replication during sporulation. In the asporogenous derivative, the third period of DNA synthesis, normally detected in the wild type strain, did not occur, yet the previous two rounds of DNA synthesis took place normally. The asporogenous strain was produced by continuous subculture of microplasmodia in broth medium. Before the strain became fully asporogenous, it showed a delay in response to light before it would sporulate. Thus the strain only sporulated after a light pulse at 96 h instead of the normal 72 h. There was, in this strain, a concomitant delay in the escape of the plasmodium from nocodazole inhibition of sporulation. Thus the final replication at about 24 h during starvation and the nocodazole sensitive event which followed some 24 h later were important for determining the condition of the plasmodium for response to light and advance to sporulation. Microplasmodial cultures were able to grow in the presence of both hydroxyurea and the protein synthesis inhibitor, cycloheximide, at concentrations which normally inhibited growth, if incubated in their presence for extended lengths of time. This may have been due to instability of the drugs at the incubation temperature or P. polyc6phalum may have been capable of breaking these drugs down. Similar results were obtained with nocodazole, but in addition micro-plasmodia also developed resistance to the drug. A method of isolating sporulation deficient mutants was developed and several such mutants were obtained. In a preliminary genetic analysis of sporulation a cross between sporogenous (Spo+) amoebae and asporogenous (Spo-) amoebae was made. Although the diploid Spo+/Spo- plasmodium sporulated, none of the progeny able to form plasmodia in clones (matAh) were able to do so. Sporulation capacity of Spo+/Spo- heterokaryons formed by fusion of plasmodia was also investigated, in these asporogeny was dominant. Although no definite results were obtained from the genetic aspect of this work it has provided a base for further genetical studies on the process of sporulation in P. polycephalum CL.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: J G Coote
• Keywords: Microbiology
• Date of Award: 1981
• Depositing User: Enlighten Team
• Unique ID: glathesis:1981-72611
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Jun 2019 11:06
• Last Modified: 11 Jun 2019 11:06
• URI: https://theses.gla.ac.uk/id/eprint/72611