Inheritance of extrachromosomal rDNA in Physarum polycephalum.

In the acellular slime mold Physarum polycephalum, the several hundred genes coding for rRNA are located on linear extrachromosomal DNA molecules of a discrete size, 60 kilobases. Each molecule contains two genes that are arranged in a palindromic fashion and separated by a central spacer region. We investigated how rDNA is inherited after meiosis. Two Physarum amoebal strains, each with an rDNA recognizable by its restriction endonuclease cleavage pattern, were mated, the resulting diploid plasmodium was induced to sporulate, and haploid progeny clones were isolated from the germinated spores. The type of rDNA in each was analyzed by blotting hybridization, with cloned rDNA sequences used as probes. This analysis showed that rDNA was inherited in an all-or-nothing fashion; that is, progeny clones contained one or the other parental rDNA type, but not both. However, the rDNA did not segregate in a simple Mendelian way; one rDNA type was inherited more frequently than the other. The same rDNA type was also in excess in the diploid plasmodium before meiosis, and the relative proportions of the two rDNAs changed after continued plasmodial growth. The proportion of the two rDNA types in the population of progeny clones reflected the proportion in the parent plasmodium before meoisis. The rDNAs in many of the progeny clones contained specific deletions of some of the inverted repeat sequences at the central palindromic symmetry axis. To explain the pattern of inheritance of Physarum rDNA, we postulate that a single copy of rDNA is inserted into each spore or is selectively replicated after meiosis.     

Structure of the central spacer region of extrachromosomal ribosomal DNA in Physarum polycephalum

We have analyzed the sequence organization of the central spacer region of the extrachromosomal ribosomal DNA from two strains of the acellular slime mold Physarum polycephalum. It had been inferred previously from electron microscopy that this region, which comprises about one third of the 60 kb³ palindromic rDNA, contains a complex series of inverted repetitious sequences. By partial digestion of end-labeled fragments isolated from purified rDNA and from rDNA fragments cloned in Escherichia coli, we have constructed a detailed restriction map of this region. The 11 kb of spacer DNA of each half molecule of rDNA contains the following elements: (a) two separate regions, one of 1.1 kb and one of 2.1 kb, composed of many direct repeats of the same 30 base-pair unit; (b) a region of 4.4 kb composed of a complex series of inverted repeats of a 310 base-pair unit; (c) another region of 1.6 kb composed of inverted repeats of the same 310 base-pair unit located directly adjacent to the center of the rDNA; (d) two copies of a unique sequence of 0.85 kb, which probably contains a replication origin. Some of the CpG sequences in the spacer resist cleavage by certain restriction endonucleases and thus appear to be methylated. The lack of perfect symmetry about the central axis and the arrangement of inverted repeated sequences explain the complex pattern of branches and forks of the fold-back molecules previously observed by electron microscopy. Comparison of the rDNA restriction maps from the two strains of Physarum suggests that the repeat units in the spacer are undergoing concerted evolution. We propose a model to explain the evolutionary origin of the several palindromic axes in the Physarum rDNA spacer.     

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.     

The blue-light reaction in plasmodia of Physarum polycephalum is coupled to respiration

The influence of inhibitors of energy metabolism (2-deoxy-D-glucose, monoiodoacetate, KCN) as well as various substrates for respiration (sodium acetate, glycine, glutamine, α-ketoglutarate, pyruvate) were investigated with respect to the effect of blue light (450 nm) on contractile behaviour of plasmodial strands of Physarum polycephalum. When the energy metabolism is not experimentally modified, blue light induces a prolongation of the period of the contraction-relaxation cycle. This effect appears within 2–3 min and seems to represent the primary reaction of this organism to blue light. Inhibition of respiration by KCN completely abolished this response to blue-light irradiation. In contrast, an impediment of glycolysis enhanced the effect. This indicates that the reaction to blue light is related to respiration, i.e., to the function of mitochondria. Among different substrates for respiration only α-ketoglutarate combined with pyruvate and applied in the presence of inhibitors of glycolysis showed an enhancement of the photoresponse, i.e., a prolongation of the period and an increase of the amplitude of the force oscillations. This indicates that the pyruvate and α-ketoglutarate-dehydrogenase complexes functioning in mitochondrial respiration are involved in the primary blue-light reaction of plasmodia of Physarum polycephalum.     

DNA replication in Physarum polycephalum: characterization of replication products in vivo.

Synchronous plasmodia of Physarum polycephalum in DNA synthesis were pulse-labelled with [oH]- thymidine for time periods of 15 seconds up to 9 minutes, or given a 30 seconds pulse followed by chase periods of 9 minutes up to 6 hours. Sedimentation analysis in alkaline sucrose gradients revealed at least five species of single stranded DNA14 molecules in the pulse experiments. Co-sedimentation of [14C]-labelled phage-DNA gave relative S-values of 5-7, 13-15, 23-25, 30 and 33-35 for these DNA molecules, all of which can be chased into DNA of higher molecular weight.     

DNA replication in Physarum polycephalum: UV photolysis of maturing 5-bromo-deoxyuridine substituted DNA.

Combinations of 5-bromodeoxyuridine (BrdUrd) and 3H-deoxyadenosine (3H-DAdo) short pulses were given in the synchronous DNA-replication period of Physarum polycephalum. After a chase period, UV-photolysis products were analyzed on alkaline sucrose gradients. This strategy has allowed the following conclusions. a) at the time of master-initiation of DNA replication, points separated by 1.1-2.2x10(7) daltons of single strand DNA may initiate DNA synthesis. b) among these, only selected groups of replicons actually proceed in DNA replication at this time, while others appear to hold (later temporal sets of replicons). The origins of the ones that proceed in replication are separated from each other by a distance corresponding to 1.1-2.x10(7) daltons. c) regions in actual replication are separated from each other by increasing distances (up to 1.5x10(8) daltons single strand DNA) at later times in S.     

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.     

Circular DNA and rolling circles in nucleolar rDNA from mitotic nuclei of Physarum polycephalum.

1. About 15% of nucleolar DNA (1.712 g/cm3) from Physarum polycephalum displaying maximum hybridization to ribosomal RNA, is composed of circular DNA of 3.9 +/- 0.2 mum contour length or multiples thereof. 2. A portion of these circular molecules (25%) contained linear DNA pieces longer than circumference length. In a small fraction of circular DNA linear pieces, shorter than the unit length, were observed. 3. Most nucleolar DNA, [3H]thymidine-labeled or hybridizable to ribosomal RNA was separable from chromosomal DNA during G2 phase, mitosis and S phase of the cell cycle. 4. Ribosomal DNA content was not amplified during the cell cycle, was unchanged during exponential or stationary growth phase and amounted to about 0.11 -- 0.21% of nuclear DNA in diploid and hexaploid strains of Physarum or 100--200 ribosomal genes per diploid genome.     

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.     

Expression of ras-family genes in the cell cycle and during differentiation of the lower eukaryote Physarum polycephalum.

Messenger RNA levels of three ras-family genes (Ppras1, Ppras2, and Pprap1) were measured in different life forms and throughout the cell cycle of the slime mold Physarum polycephalum. All three genes are expressed at constant rates in the uninucleate amoebae and flagellates, regardless of the culture conditions (solid or liquid medium, particulate or dissolved nutrients). In the multinucleate stages (micro- and macroplasmodia) Ppras1 and Pprap1 mRNAs are somewhat less abundant, while Ppras2 is not expressed at all. The early stages of the amoeba-plasmodium transition proceed without any drop in Ppras2 expression. During the synchronous cell cycle in macroplasmodia Ppras1 and Pprap1 are expressed at a constant level.     

DNA replication by a possible continuous-discontinuous mechanism in homogenates of Physarum polycephalum containing dextran.

Nuclear DNA synthesis in homogenates of Physarum is greatly stimulated by the presence of dextran in the homogenizing medium. In this cell-free system, the DNA precursor is incorporated approximately equally into two classes of DNA intermediates. On of these is similar in size to that observed previously in the intact organism, i.e. its sedimentation rate in alkaline sucrose density gradients increases, presumably by chain elongation, as the organism progresses through the S phase. The other class (approx. 10 S) is similar to 'Okazaki' fragments. Thus, nuclear DNA synthesis in homogenates of Physarum may occur by a continuous-discontinuous mechanism. Substantial DNA-synthetic activity is obtained by the addition of dextran to dextran-free homogenates. Maximal activity in this system requires the presence of both the nuclear and post-nuclear supernatant fractions. It is possible that a partial separation and recombination of a DNA polymerase and the endogenous template is effected by this procedure.