Helmut W. Sauer

Different chromatin structures in Physarum polycephalum

Nonnucleolar chromatin from interphase nuclei of Physarum polycephalum plasmodia occurs in two different structural configurations as seen in electron microscopic spread preparations. While the majority of the chromatin is devoid of nascent ribonucleoprotein (RNP) fibrils and compacted into nucleosomal particles, a minor proportion (10–20%) is organized differently and reveals a smooth contour. It is this form of smooth chromatin which is rich in transcription units (mean length: 1.36±0.21 μm). Only occasionally are solitary nascent RNP fibrils observed which are associated with beaded strands of chromatin. In transcribed smooth chromatin nucleosomal particles are not only absent from the transcription units but also from their nontranscribed flanking regions, indicating that this special structural aspect is not merely a direct consequence of the transcriptional process. The existence of ca. 10–20% of Physarum chromatin in the smoothly contoured form is discussed in relation to reports of a preferential digestibility of a similar proportion of Physarum chromatin by DNAse I (Jalouzot et al., 1980) and to the altered configuration of “peak A” chromatin subunits after micrococcal nuclease digestion (Johnson et al., 1978a, b).

Structure of Physarum actin gene locus ardA: a nonpalindromic sequence causes inviability of phage lambda and recA-independent deletions

Previously we reported that approx. 80% of the genome from the plasmodial slime mold Physarum polycephalum, including all the actin genes, can be cloned only in recBC- sbcB- Escherichia coli hosts [Nader et al., Proc. Natl. Acad. Sci. USA 82 (1985) 2698-2702]. We have now sequenced the actin gene locus ardA. The nucleotide sequence of its coding region is flanked by the typical putative regulatory sequences for transcription initiation and polyadenylation. The coding region is interrupted by five introns, all located at novel positions with regard to those of previously analysed actin genes. Within the ardA gene we have located a 360-bp fragment which comprises exon V and parts of its flanking introns. This region suppresses plaque formation of recombinant lambda phages and causes recA-independent deletions in phages and plasmids. In contrast to our previous hypothesis, this sequence is not a DNA palindrome, but consists of five (dA) X (dT)- and (dG) X (dC)-homopolymers. Both termination of replication and partial unwinding of duplex DNA under torsional stress were detected within the unstable 360-bp region in vitro.

Evidence For the Presence of A Cdc2‐Like Protein Kinase In the Dinoflagellate Crypthecodinium Cohnii

ABSTRACT. The unusual nature of mitosis and ancestral organization of the dinoflagellate nucleus prompted the question of whether the cdc2‐like histone H1 kinase, a presumed ubiquitous cell cycle regulator in eukaryotes, is present in these primitive organisms. Western blotting of Crypthecodinium cohnii protein extracts using antibody against the Pro‐Ser‐Thr‐Ala‐Ile‐Arg‐Glu (=PSTAIRE) amino acid sequence motif, conserved in all cdc2 homologues known, revealed one prominent band corresponding to a protein with an apparent relative molecular weight ≈ 34,000, identical in mobility to that from HeLa cells and Physarum polycephalum, higher and lower eukaryotic controls, respectively. Incubation of C. cohnii cell lysates with p13suc1‐sepharose beads, which preferentially, though not exclusively, bind p34cdc2, resulted in precipitation of a 34‐kDa protein which was reactive with anti‐PSTAIRE antibody, selectively competed for by the PSTAIRE peptide and able to phosphorylate histone H1 in vitro. We conclude that the dinoflagellate C. cohnii contains a protein very similar to the cdc2 gene product from fission yeast and its homologues in all eukaryotes studied thus far.

High diadenosine tetraphosphate (Ap4A) level at initiation of S phase in the naturally synchronous mitotic cycle of Physarum polycephalum

Levels of the diadenosine tetraphosphate Ap4A are high during exponential growth of Physarum, decrease during encystment (spherulation) and increase again during excystment. Consistently, a rapid 8-30-fold increase in Ap4A level occurs at entry into S phase of the mitotic cycle and is maintained during the first half of genome replication. The elevated Ap4A level depends significantly on ongoing DNA replication and is completely sensitive to the protein synthesis inhibitor cycloheximide administered either before or after initiation of S phase.

Two early replicated, developmentally controlled genes of Physarum display different patterns of DNA replication by two-dimensional agarose gel electrophoresis

The nature of replication origins in eukaryotic chromosomes has been examined in some detail only in yeast, Drosophila, and mammalian cells. We have used highly synchronous cultures of plasmodia of the myxomycete Physarum and two-dimensional agarose gel electrophoresis to examine replication of two developmentally controlled, early replicated genes over time in S-phase. A single, discrete origin of replication was found within 4.8 kb of the LAV1-5 gene, which encodes a homolog of profilin. In contrast, the LAV1-2 gene appears to be surrounded by several origins. Two origins were identified within a 15 kb chromosomal domain and appear to be inefficiently used. Replication forks collide at preferred sites within this domain. These terminating structures are long lived, persisting for at least 2 h of the 3 h S-phase. Analysis of restriction fragment length polymorphisms (RFLPs) within the LAV1-2 domain indicates that replication of alleles on different parental chromosomes is a highly coordinated process. Our studies of the these two early replicated, plasmodium-specific genes indicate that both a fixed, narrow origin region and a broader zone containing two closely spaced origins of DNA replication occur in Physarum.

RNA Polymerase B levels during the cell cycle ofPhysarum polycephalum

SummaryTritiated 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.

Sequence complexity of poly(A) RNA fromPhysarum polycephalum

SummaryPoly(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.

Differential expression of three actin genes in the cell cycle of physarum polycephalum

Abstract Regulation of expression of 3 isocoding genes of the actin gene family of Physarum was analyzed with gene specific probes. At steady state level, ardB and ardC seem to fluctuate coordinately during the cell cycle; ardA transcripts were not detected. Nuclear run-on assays suggested different transcription rates for ardB and ardC. Late G2 phase nuclei revealed hybridization signlas of in vitro synthesized RNA to ardA sequences, indicating transient activation of this late replicating gene.

Introduction to Physarum

In the following, we attempt to demonstrate that Physarum is an adequate model system for studies of the eukaryotic mitotic cycle, cell differentiation, and motility, A schematic presentation of the life cycle of this organism is shown in Fig. 1-1, at the end of this chapter. (See Sauer and Pierron, 1983, for a recent review).