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H W Sauer

Publications and source records attributed to H W Sauer.

At least 19 recordsLinked to original sources
PubMed2000

Calcium and malate are sporulation-promoting factors of Physarum polycephalum.

Fruiting body formation (sporulation) is a distinctive, irreversible differentiation process in the life cycle of the slime mold Physarum polycephalum. The most important requirement for sporulation of Physarum is a period of starvation, and normally sporulation proceeds in the light. It is shown here that by omitting the liquid sporulation medium and elevating the temperature from 21 to 25 degrees C, sporulation can occur routinely in the dark. It is further shown that this autocrine signaling in the dark requires calcium ions and malate. A putative sporulation control factor was detected in conditioned media derived from plasmodia starved in the dark, which was then identified as polymalate. As an additional role for this previously detected polyanion, specific for the plasmodial state of Physarum, it is suggested that the secreted compound serves as a source for both malate and calcium ions and thus promotes sporulation without light signaling.

Animals↗
PubMed1994

A non-cycling mitotic cyclin in the naturally synchronous cell cycle of Physarum polycephalum.

A universal model of the control of the cell cycle in eukaryotic organisms has emerged from the discovery that MPF (maturation or mitosis promoting factor) is a heterodimer consisting of a catalytic subunit (p34cdc2) and a regulatory subunit (mitotic cyclin) encoded by a pair of conserved genes. A prominent feature of the periodic activation of the protein kinase p34cdc2 is the gradual accumulation of cyclin in interphase and its abrupt degradation in mitosis, which is believed to be required for inactivation of MPF and exit from mitosis. Utilizing the precise natural synchrony of mitosis of the plasmodium of the myxomycete Physarum, the high affinity of the p34cdc2/cyclin B complex to p13suc1 Sepharose beads, and immunological reagents including three different anticyclin B antibodies and the anti-PSTAIR antibody, a transient histone H1 kinase activation but not fluctuation in the abundance of cyclin B have been detected during mitosis. It is argued that cyclin degradation may be required for cytokinesis and/or postmitotic controls of cell proliferation in G1 phase and cell-to-cell signaling in development but not for the inactivation of histone H1 kinase in mitosis.

Amino Acid Sequence↗
PubMed1993

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.

Animals↗
PubMed1993

Research note: differential chemotaxis of Physarum polycephalum to Salmonella gallinarum and Salmonella pullorum.

The chemotactic and chemotropic responses of the plasmodial stage of the slime mold Physarum polycephalum were used to distinguish Salmonella gallinarum and Salmonella pullorum from 10 Salmonella serovars that are commonly isolated from domestic poultry. Utilizing an in vitro plasmodium agar plate assay method, P. polycephalum was attracted to S. gallinarum and S. pullorum, but the organism was repelled by Salmonella derby, Salmonella dublin, Salmonella enteritidis, Salmonella heidelberg, Salmonella minnesota, Salmonella montevideo, Salmonella newington, Salmonella newport, Salmonella reading, and Salmonella typhimurium.

Animals↗
PubMed1992

Intrinsic fluctuations of phosphoinositol levels are involved in the progression of the naturally synchronous cell cycle in Physarum polycephalum.

Using [3H]myo-inositol incorporation, changes in phosphoinositol (PI) metabolism at different cell cycle stages in the myxomycete Physarum polycephalum were examined by column chromatography. Two base levels for the inositol trisphosphate (IP3) fraction were determined: a low one for S-phase and a higher one for G2 phase. Two transient increases of IP3 were also observed, one in S-phase, 70 min after mitosis (no G1 phase in the Physarum cell cycle) and another in G2 phase, 90 min before mitosis. It is concluded that the fluctuations in IP3 levels reflect endogenous events in the cell cycle of Physarum, because they occurred in the absence of any exogenous signals. Pulse treatment with Li+ (10 mM) at the points of the cell cycle, characterized by the IP3 transients, had opposite effects: in early S-phase it caused an acceleration while in late G2 phase it caused a prolongation of the cell cycle duration. The pattern of Li(+)-induced changes in PI turnover is also antagonistic: in most cases the IP3 level would decrease, however, Li+ prevents the cell cycle-dependent reduction in IP3 concentration when applied at early S-phase. The possible implications of the autogenous fluctuations in the IP3 fraction on the progression of the cell cycle through several distinct checkpoints are discussed.

Animals↗
PubMed1989

Replication timing of 10 developmentally regulated genes in Physarum polycephalum.

We have tested the hypothesis which stipulates that only early-replicating genes are capable of expression. Within one cell type of Physarum - the plasmodium - we defined the temporal order of replication of 10 genes which were known to be variably expressed in 4 different developmental stages of the Physarum life cycle. Southern analysis of density-labeled, bromodesoxyuridine-substituted DNA reveals that 4 genes presumably inactive within the plasmodium, were not restricted to any temporal compartment of S-phase: 1 is replicated in early S-phase, 2 in mid S-phase and 1 in late S-phase. On the other hand, 4 out of 6 active genes analysed are duplicated early, with the first 30% of the genome. Surprisingly, the two others active genes are replicated late in S-phase. By gene-dosage analysis, based on quantitation of hybridization signals from early and late replicating genes throughout S-phase, we could pinpoint the replication of one of these two genes at a stage where 80-85% of the genome has duplicated. Our results demonstrate that late replication during S-phase does not preclude gene activity.

Blotting, Southern↗
PubMed1989

Evidence for a homolog of the yeast cell cycle regulatory gene product of cdc2+ in Physarum polycephalum.

Evidence for the presence of a Cdc2-like protein in Physarum polycephalum has been obtained using a peptide antibody directed against a highly conserved amino acid sequence near the N-terminal end of Cdc2, Cdc28 and Cdc2HS. The antibody detected a 34 kDa cytoplasmic protein, similar in apparent size to Cdc2 in yeast and Cdc2Hs in HeLa cells. A 60 kDa nuclear band was also detected in Physarum but not in yeast or HeLa. Evidence is presented that this is not related to the 34 kDa protein nor is it found in HeLa nuclei or yeast cells. The Cdc2-like protein level did not fluctuate over more than 10 h of the naturally synchronous cell cycle of Physarum. Several heat-shock experiments using regimens that either: delayed mitosis and S-phase; prevented mitosis or uncoupled S-phase from mitosis were performed. None had any effect on the level of the Cdc2-like protein. The induction of spherulation by starvation was shown to have no effect on the levels of the 34 kDa Cdc2 analog. The invariant level of the 34 kDa protein during the cell cycle and starvation is consistent with previous results obtained with yeast. Three heat-shock regimens which either delay mitosis, eliminate S-phase or uncouple mitosis from S-phase in Physarum also had no effect on the level of the 34 kDa protein. This result emphasizes the stable nature of this protein.

Amino Acid Sequence↗
PubMed1988

Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis.

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.

Animals↗
PubMed1986

Flow cytometry reveals a high degree of genomic size variation and mixoploidy in various strains of the acellular slime mold Physarum polycephalum.

High-resolution flow cytometry, using avian erythrocytes as an internal standard, was employed to study constitutive genome size variation of G2-phase nuclei of Physarum polycephalum strains during the macroplasmodial stage of their life cycle. Our results document a previously unknown extent of genome size variation and mixoploidy in this organism. The unimodal diploid strain Tu 291 displayed the largest genome of the strains tested; in contrast, the Colonia strain displayed only half of the Tu 291 G2-phase fluorescence, confirming its haploid nature. An additional strain, derived from a recent cross between Lu897 and Lu898 amoebae, must have arisen by selfing (propagation of only one of the parental genomes to the macroplasmodial stage), since its nuclei display close to the haploid G2-phase DNA content. The observation of a small fraction of corresponding diploid nuclei within the haploid population of this strain, while maintained as microplasmodia, supports the notion that meiosis in haploid strains may require the presence of diploid nuclei. Two of the descendants of the prototype haploid Colonia strain, which were kept for extended periods of time in submerse culture, proved to be near diploid and mixoploid. Polyploidization and subsequent loss of DNA thus seems to contribute to the extremes of genome size variation in Physarum. In addition to unimodal fluorescence distributions, a number of diploid strains displayed bi- and even trimodal distributions within harvests of a single G2-phase macroplasmodium. Analysis of these mixoploid strains by means of gaussian curve-fitting suggests that the smaller genome size differences in Physarum may arise in step-wise diminution of DNA in approximate units of 3-5% of the original Tu 291 genome.

DNA, Fungal↗
PubMed1986

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.

Actins↗
PubMed1985

Cloning of Physarum actin sequences in an exonuclease-deficient bacterial host.

A genomic library of Physarum was constructed in the replacement vector EMBL3. Efficient propagation of the recombinant phages occurred only on the recBC-sbcB- host Escherichia coli CES200, which is deficient in the exonucleases I and V. Thirteen different recombinants with actin-related sequences were detected and 10 were purified from 90,000 plaques (the equivalent of 6 Physarum genomes) on strain CES200. Comparison of the plating efficiencies of the library and the actin-related isolates suggests that palindromic DNA sequences are responsible for the instability of Physarum DNA in E. coli. In one of these isolates, lambda PpA10, and in a 2.81-kilobase subclone of that isolate in plasmid pBR322, a deletion of 360 base pairs was detected that led to stable propagation of the recombinant DNA molecules in Rec+ E. coli. Electron microscopic analysis of the 2.81-kilobase fragment, after denaturation and self-hybridization, revealed secondary structures consistent with "foldback" structures. Restriction and DNA blot analysis of lambda PpA10 suggest that the unstable DNA segment is in close proximity to, if not part of, the previously defined actin-gene locus ardA.

Actins↗
PubMed1984

Invariant temporal order of replication of the four actin gene loci during the naturally synchronous mitotic cycles of Physarum polycephalum.

The chronological sequence of replication for the four unlinked actin gene loci of Physarum has been established. Southern hybridization analysis of density-labeled, bromodeoxyuridine-substituted DNA isolated from defined periods of S phase demonstrates that three actin loci (ardB, ardC, ardD) are duplicated early, corresponding to the first 10% of the genome. The fourth locus (ardA) replicates later, between 80 and 100 min into S phase and after 75% of DNA synthesis is completed. Gene-dosage determinations, based on the quantitation of hybridization signals from DNAs isolated from various times during S phase, confirm the results obtained with bromodeoxyuridine-substituted DNA and increase the temporal resolution. The chronological order of replication in the macroplasmodium appears constant through two consecutive cell cycles and after prolonged growth in suspension culture. The precise chronology of DNA synthesis at the gene level extends to the coordinate replication of allele pairs.

Actins↗
PubMed1982

Physical relationship between replicons and transcription units in Physarum polycephalum.

Electron microscope spread preparations of nuclear chromatin derived from early S-phase of Physarum reveal 'beads on a string' for nonreplicated and a portion of newly replicated chromatin. Many of the early replicons contain transcription units as visualized by nascent transcripts. They are, in most cases, arranged in continuous length gradients on both newly replicated strands of chromatin, the putative origin of replication being within the transcription unit. Preferential release of DNA as acid precipitable material by DNAse I and of RNA polymerase B (estimated as release of labeled alpha-amanitin bound to isolated nuclei) is observed in early S-phase, but only if DNA synthesis is not inhibited. Also, generation of a small particle (peak A) by staphylococcal nuclease, characteristic of transcriptionally active chromatin, depends on concomitant replication of early replicons. It is concluded that DNA replication is a prerequisite for its transcription by RNA polymerase B. Thus, the sequential replication of the genome of Physarum dictates the order of transcription during S-phase which may in part control the proliferative mitotic cycle of Physarum.

Chromatin↗
PubMed1981

Different chromatin structures in Physarum polycephalum: a special form of transcriptionally active chromatin devoid of nucleosomal particles.

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 micrometer). 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., 1978 a, b).

Chromatin↗
PubMed1980

More evidence for replication-transcription-coupling in Physarum polycephalum.

Endogenous RNA polymerase activity of isolated nuclei from Physarum polycephalum was determined at high (400 mM KCl) and low (5--100 mM KCl) ionic strength. The activity of RNA polymerase B (alpha-amanitin-sensitive UMP incorporation) and of RNA polymerase A (plus C) (alpha-amanitin-resistant UMP incorporation) was compared in accurately sized nuclear samples derived from macroplasmodia at distinct points of the mitotic cycle. Minimum total RNA polymerase activity was detected in metaphase nuclei. A constant level of RNA polymerase B activity was detected at all other stages of the mitotic cycle, if nuclei were assayed at high ionic strength. However, a high level in S-phase, a low level in G2-phase and again a high level in early prophase were measured, if nuclei were assayed at low ionic strength. Inhibition of DNA synthesis by hydroxyurea in vivo had a selective and drastic effect on in vitro RNA polymerase activity of isolated nuclei derived from S-phase plasmodia, yielding up to 100% inhibition in early S-phase.

Cell Cycle↗