DNA replication in Physarum polycephalum

Brewer, E.N.

doi:10.1016/0022-2836(72)90094-0

Cited by 34 publications

(10 citation statements)

References 28 publications

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“…The newly replicated DNA strands contain gaps which are repaired later on. Protein synthesis is necessary for DNA replication (BREWER 1972, MULDOON et al 1971, as had been found for other eukaryotes as well (e. g. TAYLOR 1965). In the presence of cycloheximide, the formation of heavy intermediates may continue, but the maturation process as well as the repair of nicks is completely suppressed (BREWER 1972).…”

Section: Enzymesmentioning

confidence: 59%

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Nuclear Proteins in Physarum polycephalum

Jockusch

1973

Berichte der Deutschen Botanischen Gesellschaft

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Section: Enzymesmentioning

confidence: 59%

“…In experiments with pulse labeling of Physarum DNA in situ and analyzing the products in sucrose gradients, it was found that double stranded intermediates are formed which are then converted into molecules of the parental size (BREWER 1972). The newly replicated DNA strands contain gaps which are repaired later on.…”

Section: Enzymesmentioning

confidence: 99%

Nuclear Proteins in Physarum polycephalum

Jockusch

1973

Berichte der Deutschen Botanischen Gesellschaft

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“…Linear sucrose gradients ( 5 -20% sucrose 4.6 ml) were made up in 0.15 M NaCl, 0.015 mM sodium citrate, 0.5 mM EDTA, 0.5 % sodium sarcosylate, pH 7.2 analysed as described by others [25].…”

Section: Grudien T Cen Tr Ifugut Ionmentioning

confidence: 99%

Circular DNA and Rolling Circles in Nucleolar rDNA from Mitotic Nuclei of Physarum polycephalum

Bohnert

Schiller

Böhme

et al. 1975

European Journal of Biochemistry

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1. About 15 of nucleolar DNA (1.712 g/cm3) from Physarumpolycephalum displaying maximum hybridization to ribosomal RNA, is composed of circular DNA of 3.9 0.2 pm 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 Gz 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.The nucleolus of eucaryotic cells contains the genes for ribosomal RNA which are transcribed by RNA polymerase A [l -31. Ribosomal cistrons are clustered in tandem arrangement at certain chromosomal sites (nucleolus organizers) and extend into the nucleolus during interphase [4,5]. These well-understood eucaryotic cistrons [6] have proved to be a useful system to analyse the mechanisms of gene rectification [7], magnification and reduction [8,9], and selective gene amplification in oocytes [lo, 3 11.At the time of amplification of the genes for ribosomal RNA rolling circles have been found in the ribosomal DNA fraction of Xenopus oocytes [12,13]. In analogy to viral systems [14], these structures are interpreted as DNA replication intermediates. The occurrance of ribosomal cistrons in such structures raised the question of how they originate from the chromosome, and/or whether they might persist extrachromosomally as intranuclear episomes [15,16]. If circular ribosomal cistrons are present in mitotically dividing cells one of these possibly alternative mechanisms might be substantiated.In this paper we demonstrate circular DNA molecules of the size of a ribosomal cistron and multiples thereof as well as possibly rolling circles in nucleolar DNA from plasmodia of Physarum polycephalum.

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Section: Preparation Of Dnamentioning

confidence: 99%

“…Main band DNA (1.700 g/ml) was dialysed against 0.1 standard saline citrate and sheared by sonication (Branson sonifier, 3 min at full intensity). Average molecular weight of the fragments was 3.5 x lo5 of single-stranded DNA [8,9], hyperchromicity 38O/,, T, = 86.5 "C. No DNA was released from hydroxyapatite in 0.12 M phosphate buffer at 62 "C.Reassociation was calculated from co t values obtained by two common methods [l]. …”

mentioning

confidence: 99%

Reassociation Kinetics of Nuclear DNA from Physarum polycephalum

1974

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Nuclear DNA of Physarum is made up of 45O/,, repeated (cotxls = 0.07-0.7 mol -1-1 -s) and 550/, non-repeated base sequences (cotlll = 500-1100 mol * 1-1 * s) and has a complexity of 130 times the Escherichia coli genome. DNA replicated early in the S phase of the cell cycle, contains few repeated base sequences (below loo/,).Reassociation kinetics of denatured DNA has been used as a tool to distinguish repeated from nonrepeated base sequences in DNA from eucaryotes [l]. Further analyses of reassociation data allow the characterization of genomes from different organisms in terms of relative amount of repetitive DNA, repetition frequency and complexity [1 -41.I n this paper we describe the genome of Physarum polycephalurn and some experiments which indicate fewer repeated sequences in DNA which is replicated early during a naturally synchronous S phase. MATERIALS AND METHODS CuZtureaPhysarum was grown in a semi-dehed medium as described previously [5]. Microplasmodia were kept as a shaken suspension and transferred serially every 2 -3 days. Microplasmodia from exponential growth phase were harvested by centrifugation (50 x g , 1 min), pipetted onto filters of 8-cm diameter (Schleicher & Schiill no. 576 or millipore), supported by a stainless steel grid and allowed to fuse into macroplasmodia in which nuclei divide synchronously every 8 -10 h at 26 "C. Mitotic stages were accurately determined by phase-contrast microscopy. Mitosis was immediately followed by DNA replication (S phase), which lasted for about 3 h. Preparation of DNANuclei were isolated using published procedures (200 pCi/ml medium, specific activity 17 000 Ci/mmol, Amersham). DNA was isolated after published methods [7] and purified by CsCl gradient centrifugation. Main band DNA (1.700 g/ml) was dialysed against 0.1 standard saline citrate and sheared by sonication (Branson sonifier, 3 min at full intensity). Average molecular weight of the fragments was 3.5 x lo5 of single-stranded DNA [8,9], hyperchromicity 38O/,, T, = 86.5 "C. No DNA was released from hydroxyapatite in 0.12 M phosphate buffer at 62 "C.Reassociation was calculated from co t values obtained by two common methods [l]. Ultraviolet XpectrophotometrySamples of DNA (up to 400pglml in standard saline citrate, 30°/, formamide, 0.1 sodium dodecylsulfate) were heated to 80 "C in a sealed quartz cuvette of 1-mm optical pathlength, quickly cooled to and maintained a t 38 "C. Loss of hyperchromicity was measured a t 260 nm in a Zeiss spectrophotometer. Hydroxyapatite ChromatographySamples of DNA (5 pg in 5-50 p1 standard saline citrate) were sealed in capillary tubes, heated for 10 min at 100 "C and cooled to and maintained at 62 "C (or 38 "C in the presence of 30°/, formamide) for times up t o several weeks. After incubation over 95O/, of the DNA were recovered from hydroxyapatite columns and 98O/, remained trichloroacetic-acidprecipitable. Relative amounts of single or doublestranded DNA (800 -22 000 counts x min-l x pg DNA-l) were computed from fractions eluted at 0.12 M and 0.35 M phospha...

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DNA replication in Physarum polycephalum

Cited by 34 publications

References 28 publications

Nuclear Proteins in Physarum polycephalum

Nuclear Proteins in Physarum polycephalum

Circular DNA and Rolling Circles in Nucleolar rDNA from Mitotic Nuclei of Physarum polycephalum

Reassociation Kinetics of Nuclear DNA from Physarum polycephalum

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