No description available.

No description available.

No description available.

No description available.

The genome of the eukaryotic slime mould <i>Physarum polycephalum</i> contains highly methylated (M+) regions, 20-50kb long, which are resistant to cleavage by methylation sensitive restriction endonucleases. The major sequence comprising the M+ component is an abundant family of repetitive DNA sequences: Tpl elements. Members of the Tpl family are around 8.6Kb in length and have a number of structural characteristics in common with eukaryotic retrotransposons. The direct long terminal repeat (LTR) sequences defining the ends of Tpl elements are a typical feature of well-characterised eukaryotic transposons. The similarity extends to the ends of the LTRs, which are terminated by short inverted repeats. The present study describes some of the properties of the transposon-like Tpl family. Sequence analysis of cloned segments of copies of Tpl has allowed partial characterisation of the element at the nucleotide sequence level. In addition, novel sequence arrangements are described which may represent variant forms of Tpl or could indicate the presence of other, previously unidentified, repetitive sequence families in the <i>Physarum</i> genome. Tpl elements are arranged in scrambled clusters which are believed to have arisen by transpositional insertion of the element into copies of its own sequence. Several putative target sites for transposition have been identified and found to be clustered in specific regions of the Tpl sequence. These sites bear some resemblence to the Chi sequences found in bacteriophage lambda, thus suggesting an alternative mechanism for the rearrangement of Tpl sequences, i.e. homologous recombination. A study of the replication and transcription of Tpl elements was also initiated. The timing of replication during the cell cycle was determined using synchronous plasmodial cultures. Tpl elements were found to replicate at between 60 and 90 min into S-phase. Thus, they are contained within the relatively late-replicating fraction of the genome. Tpl elements were found to be transcribed in amoebae and plasmodia and could be the first examples of late-replicating <i>Physarum</i> sequences which are transcriptionally active. Some evidence of developmental regulation of expression of Tpl elements was also found. The similarity with retrotransposons suggested that an RNA intermediate may be involved in transposition. However, no full-length transcript was detected in this study.

Amoebae of strain CL of Physarum polycephalum undergo apogamic development to form multinucleate plasmodia. During the amoebal-plasmodial transition, large uninucleate cells become irreversibly committed to plasmodium development. The transformation of an amoeba to a plasmodium involves a change in the tubulin isotypes expressed and a radical restructuring of cellular microtubules. During the transition the amoebal cytoplasmic microtubules, centrioles and cytoplasmic MTOC must disappear and the plasmodial-specific tubulin isotypes and intranuclear microtubule organising centre (MTOC) must be acquired. In developing cultures, amoebae lose the ability to flagellate before they become committed. Enriched suspensions of committed cells can be obtained by inducing asynchronous differentiating cultures to flagellate and passing the cells through a glass bead column. The resulting committed cells can be cultured, with some synchrony, to form plasmodia on bacterial lawns or in axenic liquid medium but cannot be cultured on axenic agar medium. During mating, cells lose the ability to flagellate early in plasmodium development. Committed cells from mating mixtures can be enriched in a similar way to committed cells of CL and have similar growth characteristics. Uninucleate committed cells of CL have the same DNA content as amoebae and plasmodia but have 6-10 times the amount of RNA. Apogamic committed cells express tubulin isotypes characteristic of amoebae, but after culture in axenic liquid medium, the cells express plasmodial-specific tubulin isotypes. Results suggest that plasmodial-specific tubulin isotypes are switched on in quadrinucleate cells. The amoebal cytoskeleton persists in binucleate and quadrinucleate cells but has disappeared in larger multinucleate cells. Mitosis in uninucleate committed cells is intranuclear (plasmodial-type). The amoebal MTOCs are eliminated during the first few mitotic cycles after commitment and do not become the plasmodial intranuclear MTOCs. Centriole loss apparently occurs before MTOC loss.

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No description available.

No description available.

A study has been made of the effects of various treatments on the phagocytosis of 14C-labelled E.coli by amoebae of Dictyostelium discoideum. An assay was also developed for the adhesion of amoebae to glass and the effects of a number of inhibitors on this process have been investigated. The phagocytosis of bacteria was inhibited by chelating agents at millimolar concentrations. The effect of chelators was not apparent in the presence of added divalent cations. However, only a small reduction in cell-glass adhesion was seen with EDTA concentrations that caused large reductions in phagocytosis. The calcium ionophore, A23187 abolished phagocytosis at 40 ug/ml. Pretreatment of amoebae with lanthanum ions completely inhibited both phagocytosis and cell-glass adhesion at low concentrations. Both phagocytosis and adhesion to glass are also strongly inhibited by calmodulin antagonists. Neither cytochalasin B or colchicine affected phagocytosis. Concanavalin A strongly inhibited phagocytosis presumably due to a direct interaction with cell surface glycoproteins, since the effect did not occur in the presence of alpha-methyl mannoside. Both phagocytosis and adhesion to glass were greatly reduced on treatment of the amoebae with tunicamycin, again suggesting glycoprotein involvement. Pretreatment of amoebae for 30 min with 1 mg/ml trypsin or pronase had no effect on phagocytosis, although pretreatment with papain at the same concentration caused some reduction. However, phagocytosis became pronase sensitive on exposure to tunicamycin. Beta-glucosidase also caused a small but consistent reduction in phagocytosis and cell-glass adhesion. Phagosomes were isolated from amoebae by two procedures. In the first, cells were allowed to phagocytose 1 um diameter polystyrene beads. The endocytosed beads were then isolated by flotation on a discontinuous sucrose density gradient. In a second procedure, devised during the course of this work, an attempt was made to isolate phagosomes from ingested glutaraldehyde-fixed E.coli. Analysis of these preparations by SDS-polyacrylamide gel electrophoresis showed a number of differences between them. A comparison of these preparations with &quot;bulk&quot; plasma membrane revealed a considerable similarity of the polypeptide profile with that isolated using fixed E. coli.