Sporulation delay by stable cAMP analogues in the slime moldPhysarum polycephalum.

Plasmodial cells of the slime moldPhysarum polycephalum become competent for sporulation following a prolonged period of starvation in darkness. Then sporulation can be induced by illumination. Microinjections of the stable (Sp)- and (Rp)-diastereoisomers of adenosine cyclic 3',5' monothionophosphate before and during a sensitive period from the start of illumination until 5 h after lead to a significant delay in the sporulation process.Both of the diastereoisomers of cyclic AMP prolong the time for sporangia to form in darkness. However, the (Sp)-diastereoisomer is more effective and causes morphological changes in plasmodia.The experimental data suggest that cyclic AMP is decisively involved in light-induced differentiation in the lower eukaryotoPhysarum polycephalum.

RNA transcription of isolated nuclei and chromatin with exogenous RNA polymerases during mitotic cycle and encystment ofPhysarum polycephalum.

Nuclei have been isolated from plasmodia ofPhysarum. Chromatin has been prepared from these nuclei by lysing them gently with lysolecithin. Both nuclei and chromatin contain endogenous activities of RNA polymerases A and C but not of RNA polymerase B under the conditions specifiied. RNA synthesis of chromatin and nuclei can be stimulated by the addition of purified RNA polymerases. RNA polymerase B is significantly more active than A or the bacterial RNA polymerase fromEscherichia coli. Experiments with specific inhibitors indicate that this additional RNA transcription is due to initiation by exogenously supplied RNA polymerase B. Large RNA products (up to 30 S when analysed under denaturating conditions) are transcribed on chromatin.The template activity of nuclei or chromatin, measured using saturating amounts of added RNA polymerase B, correlates with the in vivo RNA synthesis in three well defined situations. It decreases during starvation when the plasmodium prepares for encystment and is very low during mitosis, however, it is very high during the S-phase of the mitotic cycle when the highest activity of poly(A) + RNA synthesis is detected.

Discrimination of potential and actual RNA polymerase B activity in isolated nuclei during differentiation ofPhysarum polycephalum.

Isolated nuclei ofPhysarum contain endogenous RNA polymerase activity. We provide evidence for four different states of RNA polymerase B: 1. free enzyme (85%); 2. weakly bound enzyme (10%) and 3. tightly bound enzyme (0-4%), which can be solubilized from isolated nuclei with 0.5 M and 1.5 M NaCl respectively; 4. "initiated" enzyme. The latter fraction (1-5% of the total RNA polymerase B) is not soluble in salt extractions, does not accept external templates, shows high salt optimum for transcription (0.4 M NaCl) and produces by elongation RNA molecules of mainly 10 S. Treatment of isolated nuclei from differentiating cultures with Triton X-100 increases the proportion of the "initiated" enzyme at the expense of the tightly bound enzyme fraction. This indicates a potential transcription control mechanism which operates at the chromatin level and results in variable proportions of silent and transcribing RNA polymerase B molecules during differentiation of Physarum.

Levels of RNA polymerase activities during growth, encystment and germination ofPhysarum polycephalum.

Two RNA polymerases, enzyme A and enzyme B, were prepared fromPhysarum plasmodia. They are not located in the cytosol. Isolated nuclei, however, contain only a fraction of the total RNA polymerase activity: 1.5-19% depending on the nuclear preparation method.The level of RNA polymerase B shows little variation at developmental stages of growth and encystment, or in cysts and during germination, whereas RNA polymerase A displays a marked transient decrease in activity during the stationary growth phase before encystment.