Sequence complexity of poly(A) RNA fromPhysarum polycephalum.

Poly(A) RNA from S phase, G2 phase and starved macroplasmodia of Physarum contain mRNA sequences which when translated in vitro, yield similar patterns of polypeptides after fluorography.Reassociation of nick-translated DNA (Cot) allows the isolation of highly labeled single copy DNA which, after saturation hybridization with poly(A) RNA, gives values of 23% for growth and 17% for starvation.Homologous cDNA/poly(A) RNA hybridization reactions (Rot) indicate that 22-28% of the genome is transcribed during growth and 12% during starvation and that about half of the cDNA reacts with 0.1% of the genome and could represent 50-80 RNA species, each present in about 1,000 copies per nucleus. Up to 25,000 different RNA species, 1-5 copies each per nucleus, are estimated to be present during growth, and about 15,000 during starvation. Heterologous cDNA/poly(A) RNA hybridization reactions (Rot) indicate that the RNA sequences in S and G2 phase of the cell cycle are similar, with RNA sequences being more abundant in G2 phase.During starvation about 25% of the sequences present during growth cannot be detected and those sequences present during growth have become diluted during starvation.

RNA Polymerase B levels during the cell cycle ofPhysarum polycephalum.

Tritiated α-amanitin has been used as a specific and sensitive probe to estimate the number of RNA polymerase B molecules in isolated nuclei, chromatin and nucleoids, obtained from macroplasmodia ofPhysarum polycephalum. During mitosis (metaphase±10 min) there is at least 10-fold less RNA polymerase B than at all phases of the cell cycle, even if DNA replication has been blocked in vivo. It is concluded that many of the RNA polymerase B molecules leave the chromatin during decondensation of the chromosomes in telophase of the synchronous nuclear division ofPhysarum.

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.