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...
