The transition between growth and sporulation in Physarum polycephalum CL

The overall aim of this project was to examine the relationship between the cell cycle and sporulation in Physarum polycephalum: in particular, to determine when sporulation specific genes were transcribed. The work fell into three sections. The first made use of the anti-fungal agent nocodazole to determine if a plasmodium competent to sporulate after 72 h starvation was in the G1 or the G2 phase of the cell cycle. Clear evidence was obtained that differentiation was initiated from the G2 phase of the cell cycle. The effect of blocking mitosis with nocodazole on the subsequent DNA replication was investigated in both growing and starving plasmodia. It had previously been reported, and was shown in this work, that P. polycephalum has no G1 phase in the cell cycle during growth. The fact that growing and starving plasmodia responded similarly to nocodazole with regard to the onset of DNA synthesis indicated that there was no prolonged G1 period during the starvation period in P. polycephalum. It is postulated that nocodazole may interfere with a temperature-sensitive pathway that controls both the increase in thymidine kinase activity and metaphase onset. The second part of the investigation was to approach the problem of pinpointing when in the G2 phase of the cell cycle, there was sporulation specific transcription. It was assumed that this question might be answered by differential screening of a genomic library of P. polycephalum, using as probes radiolabelled copy DNA prepared from poly(A)+ RNA from growing and starving plasmodia. The first requirement was a genomic library of P. polycephalum CL DNA. Of the two phage vectors, Charon 4AP and lambda1059 which were compared, the latter proved to be superior as it was shown that a genomic library prepared in Charon 4AP would be diluted by the presence of a considerable number of non-recombinant phage. To generate libraries of P. polycephalum DNA it was necessary to digest it with suitable restriction endonucleases. P. polycephalum DNA was partially digested with either Sau3A or BamHI and the 15-25 kb fragments were isolated by electroelution. These fragments were then used to generate two genomic libraries. In each case only one type of recombinant phage was created which was derived from lambda1059 and contained a fragment of Physarum DNA. The DNA used to prepare these gene banks was found to be contaminated by a second type of DNA. This contaminating DNA was tentatively identified as mitochondrial in origin. This difficulty was eliminated when Physarum DNA was isolated by the method of Hardman & Jack (1978). DNA was partially digested with Sau3A and the 15-25 kb fragments isolated. A genomic library was prepared in lambda1059 and restriction analysis of a random sample of phage showed that all were derived from lambda1059 and all had restriction patterns different from the parental phage. Hybridization of [32P] nick-translated Physarum DNA to filter replicas of phage identified the inserts as Physarum DNA. The third part of the work involved the isolation of RNA from P. polycephalum. A requirement for screening the library was the preparation of undegraded poly(A)+ RNA from which copy DNA probes could be made. Initially an attempt was made to isolate RNA that was being actively translated on polysomes at the time of isolation. However, all attempts to prepare polysomes in sufficient quantity were unsuccessful. Cytoplasmic RNA was isolated from growing plasmodia but was highly contaminated by a polysaccharide material. This contaminant was removed by cetyltrimethylammonium bromide precipitation. Examination of the RNA, after electrophoresis under denaturing conditions showed that the RNA was very susceptible to degradation even when prepared in the presence of two inhibitors of RNase activity, RNasin and vanadyl ribonucleoside complex. Less degraded RNA was isolated in a buffer containing 4M guanidine thiocyanate, an inhibitor of RNase activity. This total RNA preparation was less degraded than the cytoplasmic RNA. When poly(A)+ RNA was isolated by oligo (dT) cellulose chromatography it directed the synthesis of very short copy DNA. The purest and most undegraded RNA was isolated by a modified version of the method described by Cox & Smulian (1983). After the initial isolation procedure the poly(A)+ RNA was further purified by a cetyltrimethylammonium bromide precipitation and a phenol/chloroform extraction. The poly(A)+ RNA was used as a template for the synthesis of cDNA in vitro which was found to be 200-900 nucleotides in length. This cDNA hybridized to filter replicas of recombinant phage. The overall conclusion from this work was that the molecular genetical techniques applied in this study have a good potential for investigating the detailed sequence of events in sporulation of Physarum polycephalum