Methylation of parental and progeny DNA strands in Physarum polycephalum

Although 5-methylcytosine comprises 4 to 8% of the cytosine residues in the major nuclear DNA of Physarum polycephalum (Evans &; Evans, 1970), only 1 % of the cytosine residues of progeny DNA become methylated during replication. Further methylation occurs during the same and subsequent mitotic cycles, so that 6 to 7 cycles after its synthesis, 5-methylcytosine comprises 5 to 7% of the DNA-cytosine residues of a single generation of DNA. The extent of methylation occurring during the S period has been measured by the determination of the specific activity of the precursor (S-adenosylmethionine) and the product (DNA-5-methylcytosine) and by comparison of the radioactivity in DNA-cytosine and DNA-5-methylcytosine after incorporation of [¹⁴C]deoxycytidine. Continuing methylation of parental DNA has been shown, by density shift experiments and by the conversion of prelabeled DNA-cytosine to DNA-5-methylcytosine. The DNA-5-methylcytosine once formed was found to be stable.     

Periodic organisation of foldback sequences in Physarum polycephalum nuclear DNA.

Nuclear DNA from the slime mould Physarum polycephalum is shown to contain interspersed inverted repeat sequences, such that denatured fragments of DNA containing pairs of these sequences form intra-chain duplexes under appropriate conditions. The organisation and distribution of the nucleotide sequences responsible for the formation of foldback structures in Physarum DNA have been investigated using the electron microscope. The majority of foldback duplexes have sizes ranging up to 800 base pairs, and about 60-80% of DNA molecules 2.2 X 10(4) bases in length contain interspersed foldback elements. The size of individual foldback duplexes, and also the length of the intervening sequences which separate them, are non-random. The results can best be explained by a model in which separate foldback foci in Physarum DNA are spaced periodically at regular intervals. The regions containing foldback foci are thought to contain smaller, tandemly-arranged sequences of discrete sizes, in some cases related to other nucleotide sequences of a similar nature in the same locality in Physarum DNA.     

Distribution of inverted repeat sequences in nuclear DNA from Physarum polycephalum.

Inverted repeat sequences, capable of forming stable intra-chain foldback duplexes, are shown using electron microscopy to be located in over 90% of fragments of nuclear DNA from Physarum polycephalum. A statistical treatment of the data indicates that, on average, foldback sequence foci are spaced every 7,000 nucleotides and that they are distributed uniformly amongst the DNA chains. The majority of inverted repeat sequences give rise to the simple types of foldback structure observed in DNA from other eukaryotic species, but a significant proportion of the DNA fragments also contain novel foldback structures with a more complex appearance, referred to as 'bubbled' hairpins. The latter structures appear to be formed by the annealing of several distinct segments of homologous inverted repeat sequence, each separated by interspersed non-foldback sequences of variable sizes up to 15,000 nucleotides in length. The size, both of the foldback duplexes and of the intervening single-chain segments of DNA, are not random. Instead, they appear to form a regular, arithmetic series of lengths. These observations suggest that the different segments of Physarum DNA from which foldback structures are derived contain nucleotide sequences that share a highly ordered and unform pattern of structural organisation. These regular units of organisation in Physarum DNA in some cases extend over distances up to 50,000 nucleotides in length.     

Characterisation of ribosomal satellite in total nuclear DNA from Physarum polycephalum.

The distinctive properties of satellite DNA molecules containing the genes for ribosomal RNA in Physarum polycephalum permits their identification in total, unfractionated nuclear DNA in the foldback form, after denaturation and fast annealing. Using the electron microscope the location and properties of three characteristic regions containing tandemly-repeated, inverted sequences have been investigated. At least two additional regions, also containing tandem repeats, are shown to be present and located towards each end of the rDNA molecule, at a site adjacent to the segment coding for the 26 S rRNA. All the regions which contain tandem repeats are composed of sequences which, within experimental error, appear to share a common unit repeat length of about 90 nucleotides.     

Sequence organisation in nuclear DNA from Physarum polycephalum. Arrangement of highly-repeated sequences

Recombinant plasmids containing highly repetitive Physarum DNA segments were identified by colony hybridisation using a radioactively-labelled total Physarum DNA probe. A large number of these clones also hybridised to a foldback DNA probe purified from Physarum nuclear DNA. The foldback DNA probe was characterised by reassociation kinetic analysis. About one-half of this component was shown to consist of highly repeated sequences with a kinetic complexity of 1100 bp and an average repetition frequency of 5200. Direct screening of 67 recombinant plasmids for foldback sequences using the electron microscope revealed that about one-half were located in segments of DNA containing highly repetitive sequences; the remainder were present in clones containing low-copy number repeated elements. Analysis of two DNA clones showed that they contained repetitive elements located in over half of all DNA segments containing highly repetitive DNA and that the foci containing these highly repetitive sequences had different sequence arrangements. The results are consistent with the hypothesis that the most highly repeated DNA sequence families in the Physarum genome are few in number and are clustered together in different arrangements in about one-sixth of the genome. Over one-half of the foldback DNA complement in the Physarum genome is derived from these segments of DNA.     

Structural organization of a hypermethylated nuclear DNA component in Physarum polycephalum

Digestion of Physarum polycephalum nuclear DNA using the restriction endonuclease HpaII generates two components, distinguishable on the basis of their molecular size. The high-molecular-weight, HpaII-resistant component, which accounts for 20% of the DNA, contains a fivefold greater concentration of 5-methylcytosine residues than the low-molecular-weight HpaII-digested fraction. Segments of hypermethylated (M+) DNA are largely composed of a single, long, highly repeated sequence, and this major element is sometimes associated with other less highly repetitive sequences in the M+ DNA fraction. Restriction mapping of cloned Physarum M+ DNA segments, and Southern blot analysis of genomic DNA using subcloned segments of M+ DNA as a probe, provide evidence for sequence variation within different copies of the dominant highly repeated element, and possibly the other associated repeats in M+ DNA, and additionally that almost complete tandemly repeated copies of the major repeat are found in some M+ DNA segments.     

Association of newly replicated DNA with the nuclear matrix of Physarum polycephalum.

We have studied the role of the nuclear matrix in DNA replication in a naturally synchronized eucaryote, Physarum polycephalum. When P. polycephalum. When P. polycephalum macroplasmodia were pulse labeled with 3H-thymidine, the DNA remaining tightly associated with the matrix was highly enriched in newly synthesized DNA. This enrichment was found both in nuclei that had just initiated DNA replication as well as in nuclei isolated later during S phase. Pulse chase experiments showed that the association of newly replicated DNA with the matrix is transient, since most of the newly replicated DNA could be chased from the matrix by incubating pulse labeled macroplasmodia in media containing unlabeled thymidine. Studies measuring the size distribution of the matrix DNA supported the hypothesis that replication forks are attached to the nuclear matrix. Reconstitution controls indicated that these results were unlikely to be due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. These results with P. polycephalum in combination with previous studies in non-synchronized rodent cells, suggest that the association of newly replicated DNA with the nuclear matrix may be a general feature of eucaryotic DNA replication.     

Sequence organisation in nuclear DNA from Physarum polycephalum: methylation of repetitive sequences.

Nuclear DNA from the slime mould Physarum polycephalum is digested by the restriction endonuclease HpaII to generate a high molecular weight and a low molecular weight component. These are referred to as the M+ and the M- compartment, respectively. Sequences that are present in the M+ compartment are cleaved by MspI, the restriction enzyme isoschizomer of HpaII, thus showing that the recognition sequences for these enzymes in M+ DNA contain methylated CpG doublets. The distribution of repetitive sequences in the M+ and M- DNA compartments was investigated by comparison of the 'fingerprint' patterns of total Physarum DNA and isolated M+ DNA after digestion using different restriction endonucleases, and by probing for the presence of specific repetitive sequences in Southern blots of M+ and M- DNA by the use of cloned DNA segments. Both types of experiment indicate that many repetitive sequences are shared by both compartments, though some repetitive sequences appear to be considerably enriched, or are present exclusively, either in M+ DNA or in M- DNA.     

DNA polymerase alpha--primase complex of Physarum polycephalum.

DNA polymerase alpha and DNA polymerase alpha--primase complex of Physarum polycephalum were purified by rapid methods, and antibodies were raised against the complex. In crude extracts, immune-reactive polypeptides of 220 kDa, 180 kDa, 150 kDa, 140 kDa, 110 kDa, 86 kDa, 57 kDa and 52 kDa were identified. The structural relationships between the 220 kDa, 110 kDa and 140 kDa (the most abundant form) was investigated by peptide mapping. The 140 kDa form was active DNA polymerase alpha. The 57 kDa and the 52 kDa polypeptides were identified as primase subunits by auto-catalytic labelling. In amoebae, the immune-reactive 140 kDa polypeptide was replaced by a 135 kDa active DNA polymerase alpha.     

ADP-ribosylation of the nuclear matrix in Physarum polycephalum

Less than 10% of the total ADP-ribosylation in isolated nuclei of Physarum polycephalum are bound to the nuclear matrix. In S-phase the matrix-associated ADP-ribosylation is almost twice as high as compared with the G2-period of the cell cycle. Inhibitors of DNA- and RNA-synthesis and the mutagen N-methyl-N′-nitro-N-nitrosoguanidine increase the percentage of matrix-associated ADP-ribosylation.     

Ribosomal DNA in spores of Physarum polycephalum

DNA was isolated from plasmodia, spores and newly hatched amoebae of the slime mould Physarum polycephalum. The DNA preparations were fractionated in CsCl gradients and each fraction hybridised to combined 19 S + 26 S rRNA. In all three DNA preparations hybridisation was found to be limited to satellite DNA (rho = 1.714 g/cm3) and at saturation was found to reach a level of 0.16--0.18 % of total DNA. The main band of nuclear DNA (rho = 1.702 g/cm3) did not hybridise appreciably. Further experiments using analytical CsCl gradients revealed that the ratio of satellite to main band DNA was similar in all three preparations. It is concluded that the genes for ribosomal RNA are equally reiterated in spores, hatching amoebae and in plasmodia. They appear to be similarly organised in all stages of the life cycle examined so far.     

DNA replication in homogenates of Physarum polycephalum

Homogenates of Physarum polycephalum incorporate [³H] dATP into nuclear DNA at an initial rate of approximately 15% of the in vivo rate. To attain this level of synthesis, cultures are homogenized in a medium containing Mg⁺⁺, EGTA, glucose and spermine. Incorporation is strongly stimulated by the addition of ATP and all four deoxyribonucleoside triphosphates to homogenates prior to incubation. Various inorganic cations other than Mg⁺⁺ either do not affect synthesis or are inhibitory. Incorporation is inhibited by a nonionic detergent, Triton X-100. DNA synthesis in this cell-free nuclear system is similar in several respects to that which occurs in vivo: (1) The rate of DNA synthesis in the intact organism at a given time in the mitotic cycle is reflected by the level of synthesis in homogenates prepared from cultures at that time of the cycle; (2) DNA strands labeled in vitro exhibit alkaline sucrose density gradient sedimentation properties similar to those of daughterstrand DNA pulse-labeled in vivo; and (3) Homogenates of cultures which were pre-treated with cycloheximide incorporate [³H]dATP at about 60% of the level observed in homogenates of untreated controls.     

Sequence organisation in nuclear DNA from Physarum polycephalum: Arrangement of highly-repeated sequences

Recombinant plasmids containing highly repetitive Physarum DNA segments were identified by colony hybridisation using a radioactively-labelled total Physarum DNA probe. A large number of these clones also hybridised to a foldback DNA probe purified from Physarum nuclear DNA. The foldback DNA probe was characterised by reassociation kinetic analysis. About one-half of this component was shown to consist of highly repeated sequences with a kinetic complexity of 1100 bp and an average repetition frequency of 5200. Direct screening of 67 recombinant plasmids for foldback sequences using the electron microscope revealed that about one-half were located in segments of DNA containing highly repetitive sequences; the remainder were present in clones containing low-copy number repeated elements. Analysis of two DNA clones showed that they contained repetitive elements located in over half of all DNA segments containing highly repetitive DNA and that the foci containing these highly repetitive sequences had different sequence arrangements. The results are consistent with the hypothesis that the most highly repeated DNA sequence families in the Physarum genome are few in number and are clustered together in different arrangements in about one-sixth of the genome. Over one-half of the foldback DNA complement in the Physarum genome is derived from these segments of DNA.     

Characterization of foldback sequences in Physarum polycephalum nuclear DNA using the electron microscope

An examination of the foldback fraction of nuclear DNA from Physarum polycephalum has been carried out using the electron microscope. Results show that the inverted repeat sequences responsible for the formation of foldback DNA range from 150-3000 bases in length, with a number-average size of 340 bases. About one-half of the inverted sequences form looped structures with loop sizes averaging 1200 bases in length. The distance between adjacent foldback sequences is estimated to be in the range 100-1500 bases.     

DNA synthesis by the isolated nuclear matrix from synchronized plasmodia of Physarum polycephalum

Nuclear matrices were isolated from plasmodia of a true slime mold, Physarum polycephalum, and the DNA synthetic activity in vitro was examined. These matrices isolated in S-phase catalyzed DNA synthesis requiring Mg2+, deoxyribonucleoside 5'-triphosphates and ATP, without exogenous templates. The activity changed during S-phase with the rate of in vivo DNA replication. Product analysis by gel electrophoresis revealed that the matrices produced Okazaki fragments. These results suggest that DNA synthesis partially reflects in vivo DNA replication. DNA synthesis was sensitive to aphidicolin, heparin and N-ethylmaleimide, indicating involvement of the alpha-like DNA polymerase of Physarum. Exogenous addition of activated DNA stimulated DNA synthesis 4-10-fold and suggested that only some of the existing enzymes are involved in endogenous DNA synthesis. Matrices isolated in G2-phase were also associated with a similar DNA synthetic activity, but they did not produce Okazaki fragments in vitro. It is, therefore, concluded that nuclear matrices are associated with alpha-like DNA polymerase throughout the cell cycle, and that some of the enzymes participate in in vivo DNA replication in S-phase; thus, DNA replication is possibly controlled by this process. The relationship between DNA synthetic activities by the isolated nuclei and matrices was also discussed.     

DNA replication-dependent formation of joint DNA molecules in Physarum polycephalum

Two-dimensional neutral/neutral agarose gel electrophoresis is used extensively to localize replication origins. This method resolves DNA structures containing replication forks. It also detects X-shaped recombination intermediates in meiotic cells, in the form of a typical vertical spike. Intriguingly, such a spike of joint DNA molecules is often detectable in replicating DNA from mitotic cells. Here, we used naturally synchronous DNA samples from Physarum polycephalum to demonstrate that postreplicative, DNA replication-dependent X-shaped DNA molecules are formed between sister chromatids. These molecules have physical properties reminiscent of Holliday junctions. Our results demonstrate frequent interactions between sister chromatids during a normal cell cycle and suggest a novel phase during DNA replication consisting of transient, joint DNA molecules formed on newly replicated DNA.