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

Abstract

Velocity sedimentation in an alkaline sucrose gradient of newly replicated chromosomal DNA revealed the presence of extrachromosomal DNA that was not replicated by differentiating cells in the elongation zone. The extrachromosomal DNA had a number average molecular weight of 12 x 106 to 15 x 106 and a weight average molecular weight of 25 x 106, corresponding to about 26 x 106 and 50 x 106 daltons, respectively, of double-stranded DNA. The molecules were stable, lasting at least 72 h after being formed. Concurrent measurements by velocity sedimentation, autoradiography, and cytophotometry of isolated nuclei indicated that the extrachromosomal molecules were associated with root-tip cells that stopped dividing and differentiated from G2 phase but not with those that stopped dividing and differentiated from G1 phase.Although it is common in plant roots for cells to stop dividing and differentiate from G1 and G2, the mechanisms responsible for the transition are unknown (5-7, 11). Recent work with synchronized cells of pea roots showed that nascent replicons remain unattached until they reach late S and G2 phases (18). At this time, the nascent molecules are joined and form mature, chromosomal-size DNA chains. Other experiments showed that cells arrested in G2 by carbohydrate starvation also have nascent replicons that are unattached and, when given sucrose, many of these replicons are not joined (23). The presence and persistence of smaller molecules that remain unattached and fail to reach chromosomal size in nuclei of the root tip is the topic of this paper. The experiments were designed to determine whether or not the nascent molecules, resolved under alkaline conditions, were products of single-stranded breaks or gaps that nevertheless remained with the chromosomal duplex, or whether they were products of other processes, such as amplification or nucleases indigenous to cells of the root tip. The results indicated that one or the other of the latter processes is involved, since the molecules are free of chromosomal DNA. It was also shown that cells that differentiate from G2 have extrachromosomal DNA that was not detectable in those that stop dividing and differentiate from G1. These results are the first of their kind in plant cell biology. MATERIALS AND METHODSCulture of root tips. Seeds of Pisum sativum (var. Alaska) were surface sterilized with Clorox, washed with distilled water, and aseptically germinated in petri dishes (150 by 20 mm) that contained three layers of Whatman no. 1 filter paper moistened with distilled water. After 4 days, seedlings with primary roots 2.5 to 4 cm long were selected, and the terminal 1 to 1.5 cm was removed, suspended in 50 ml of White medium with 2% sucrose, and cultured at 22 ± 1°C for up to 72 h on a reciprocal shaker at about 1 cycle per min. Seed germination and root culture were carried out in the dark and were only intermittently exposed to light, when isotope was added to the medium.Labeling of DNA. DNA labeling followed different protocols, depending on the experim...

Excision and replication of extrachromosomal DNA of pea (Pisum sativum).

“…Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 x 106 daltons and another of 7 x 106 daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 x 106 and 7 x 106 daltons.Recently, it was discovered that pea roots have double-stranded extrachromosomal DNA (ex-DNA), with a modal size of 25 x 106 daltons, that is present in cells of the meristematic tip and remains associated with them as they differentiate from the G2 phase and occupy the elongation zone (22). Electron microscopic studies (D. Krimer and J.…”

“…(ex-DNA), with a modal size of 25 x 106 daltons, that is present in cells of the meristematic tip and remains associated with them as they differentiate from the G2 phase and occupy the elongation zone (22). Electron microscopic studies (D. Krimer and J.…”

Extrachromosomal DNA of pea ( Pisum sativum ) root-tip cells replicates by strand displacement

“…Alaska) have been described (1). exDNA samples extracted by the procedure of Hirt (2) were prepared from 50 2-mm root tips.…”

“…Sedimentation experiments indicated that exDNA molecules had a modal molecular weight of about 26 X 106 (1) but these analyses were insufficient to give needed details about the structure of the molecules. Consequently, the exDNA was examined by electron microscopy and we present the results of this examination in this paper.…”

Le méristème racinaire, modèle pour l'étude du cycle cellulaire et de ses contrôles

“…La cytophotométrie des noyaux de méristèmes de Pisum marqués après incubations de 24h dans eH) -TdR puis dans C 4 C) -TdR, séparées par une période de 24h sans isotope, a montré que des cellules à stock d'ADN de 4 C sont arrêtées dans le méristème pendant au moins 24h avant de passer dans la zone d'élongation. Ces cellules G 2 sont caractérisées par un ADN extrachromosomial spécifique synthétisé alors qu'elles étaient encore dans le méristème (V an 't Hof et Bjerknes, 1982). Ces cellules sont donc «différentes» des autres et cette différence pourrait être liée à certains contrôles de la prolifération.…”