The Replication of Ribosomal DNA in Physarum polycephalum

Abstract

The DNA coding for ribosomal RNA in Physarum polycephalum exists as a collection of extrachromosomal molecules of molecular weight 37 x lo6. We have investigated the replication of rDNA, with the following results. (a) Replication of rDNA is unscheduled. This means that molecules that are replicated at any particular time in one mitotic cycle have an equal probability of replicating again in each time interval in the subsequent cycle. Similarly, in a single cycle, some molecules replicate more than once, and some not at all. (b) Replication forks appear to move bidirectionally from points 45 % or 33 from one end of the DNA. Replicating molecules observed by electron microscopy are all linear.In the acellular slime mold Physarumpolycephalum the DNA coding for ribosomal RNA, which comprises 1 -2 % of total DNA, forms a heavy satellite band when sedimented to equilibrium in CsCl gradients. In contrast to the main-band DNA, whose time of replication defines the S period of the mitotic cycle (0-2.5 h after mitosis), ribosomal DNA (rDNA) replication proceeds throughout the G-2 period as well as through the latter half of the S period [1,2]. There is no G-1 period in Physarum plasmodia, DNA synthesis following immediately after the synchronous mitosis of the nuclei. The facts that rDNA can be purified by equilibrium sedimentation and can be labeled specifically with radioactive precursors in the G-2 period have facilitated studies of its structure. We have shown recently [3] that Physarum rDNA exists as a collection of linear DNA molecules of a unit size, 37 x lo6 M,. These molecules have a rotational axis of symmetry in the center, and thus are palindromes. One ribosomal transcription unit is located at each end of the molecule [4], and the large intervening spacer region contains blocks of short inverted repetitive sequences [3].The curious timing of synthesis of rDNA led us to investigate its replication more closely. We show here that: (a) replication is unscheduled, and (b) replication proceeds by means of bidirectional fork displacement, apparently from fixed points on the linear molecules.Abbreviations. rDNA, ribosomal DNA; BrdUrd, bromodeoxyuridine. MATERIALS AND METHODS Growth and Labeling of PlasmodiaMicroplasmodia of Physarum polycephalum, strain MIIIC, were propagated in shaking cultures at 26 "C in semidefined medium [5]. Macroplasmodia were formed on paper filters by fusion of 0.15 ml of packed plasmodia diluted with an equal volume of water. They were grown over wire grids in the same medium. The time of mitosis was determined by microscopic examination under phase contrast. The time of mitosis I1 (MII) was generally 14 h after fusion. For density labeling, bromodeoxyuridine (BrdUrd, 100 pg/ ml), fluorodeoxyuridine (5 pg/ml), and uridine (100 pg/ ml) were added to the growth medium [8]. In all experiments involving further growth after density labeling, thymidine was added to 100 pg/ml after removal of BrdUrd, fluorodeoxyuridine, and uridine. In the presence of the BrdUrd medium, the mitotic cycle appear...

Inheritance of extrachromosomal ribosomal DNA during the asexual life cycle of Dictyostelium discoideum: examination by use of DNA polymorphisms.

“…During vegetative growth the extrachromosomal rDNA copies replicate autonomously. In Physarum polycephalum replication of rDNA palindromes occurs throughout the S and G2 cell cycle stages, and some, but not necessarily all, rDNA palindromes serve as replication templates (33). Similar changes to those observed in this work in the relative proportions of polymorphic rDNA palindromes and effects on rDNA inheritance were observed during the sexual cycle of Physarum polycephalum (6).…”

Cells of pea (Pisum sativum) that differentiate from G2 phase have extrachromosomal DNA.

“…to isolate extrachromosomal doublestranded rDNA molecules of 38 x 106 daltons from chromosomal DNA (3,24 extrachromosomal molecules, and further examination by electron microscopy revealed that 95% or more of the molecules were linear (data not given).…”

“…The cover slip was removed, and the slide was rinsed in running cold water, placed in 7.5% K3Fe(CN)6 for 20 min at 22°C, rinsed several times with water, placed in 12% Na2S203 for 5 Extraction and sizing of extrachromosomal DNA. Nuclei from 12 2-mm root tips were extracted by the method of Hirt (10), which separates extrachromosomal DNA in the supernatant from chromosomal DNA that precipitates as a pellet (3,24 pl (-20 ,ug) of marker DNA, and 1.65 ml of SSC. The mixture was centrifuged for 16 h at 36,000 rpm at 25°C in a Beckman Ti 65 rotor.…”

Method of mapping DNA replication origins.

“…The clearest evidence for specific origins has been obtained in studies of certain extrachromosomal DNAs: the extrachromosomal rDNA in Physarum polycephalum and Tetrahymena thermophilia (6,42,43) and the 2,1 plasmid of yeasts (30). Evidence that chromosomal origins may occur nonrandomly comes from studies with certain repeated DNAs: the rDNAs of several species (2,3,28) and the amplified dihydrofolate reductase genes of a Chinese hamster cell line (18,19); however, the resolution of these studies has not been sufficient to determine whether initiation occurs at a specific nucleotide sequence or anywhere within a relatively long stretch of DNA.…”

“…3 (3,18) or analysis of replicating molecules identifiable by electron microscopy such as plasmids (30), extrachromosomal DNA (6,42,43), and purified ribosomal DNA repeats (2,3,28). If enough replicating DNA can be obtained, the method described in this paper will allow location of origins in stretches of unique chromosomal DNA.…”