Mechanism of nuclear DNA replication in radicles of germinating cotton

Abstract

The mechanism of nuclear DNA replication in radicles of germinating cotton (Gossypium barbadense) was investigated. Pulse-labeling with [3HIhthymidine indicates that replication intermediates are of smal molecular weight (4-1OS) and behave as if single-stranded. Prolonged labeling indicates that intermediates are of discrete size, suggesting a mechanism of discontinuous replication. Electron microscopy of nuclear DNA demonstrates a complex architecture which may include "eye forms" and "forks" similar to those reported in prokaryotes and animal systems. The possible universality of DNA replication mechanisms is discussed. The mechanism by which nuclear DNA molecules replicate has received considerable attention. Ioannu (1) proposed a model for replication of double-stranded DNA in which initiation of replication is manifested in the sequential appearance of "microbubbles" followed by formation of "macrobubbles." With electron microscopy, purported replication "bubbles" have been visualized in prokaryotic (2) and eukaryotic cells (3-5). Replication "forks," which may be associated with bubble formation and growth, have been noted in eukaryotes (6-8).Biochemical investigations of replication initiation indicate that radioactivity from pulse labeling appears first in smallmolecular-weight fragments (9-12) which are probably single-stranded (13,14). With prolonged labeling in eukaryotic cells, radioactivity also appears in larger size molecules of discrete size, indicating that replication occurs discontinuously (10)(11)(12)15) through defined intermediates. These data suggest that replication in eukaryotes is initiated through multiple replication sites (9,16,17).In a previous publication, we demonstrated that replicating nuclear DNA in germinating cotton is associated with the nuclear membrane (18). In this communication we provide evidence that the nuclear-membrane-associated DNA replicates through short single-strand "Okazaki-type" (19) intermediates of discrete size. Electron microscopy of DNA isolated from radicle tip nuclei during germination, when DNA synthesis is maximum (20), indicates a complex architecture which contains structures that may be similar to the "loops" and "forks" reported in animal DNA. MATERIALS AND METHODSSeeds of Pima cotton (Gossypium barbadense L.) were germinated and handled as previously described (20). Radioactivity was incorporated into nuclear DNA for pulse-labeling while radicle tips were exposed to 10,4Ci of [3H]thymidine (13 Ci/mmol) in 100 ,pl of H20 per radicle as previously outlined (20,21

Differences in timing of replication and maturation of nascent DNA fragments between satellite and main band DNA during the S phase of the first cell cycle in cultured carrot root explants.

“…When embryonic axes, detached from germinating Vicia faba seeds, were pulse-labeled with 3H-thymidine, short and single-stranded DNA molecules of 5-6S and 8-9S (or lOS) appeared (Sakamaki et al, 1975(Sakamaki et al, , 1976Yakura and Tanifuji, 1980). Such short nascent DNA fragments have been observed also in radicles of germinating cotton seeds (Clay et al, 1976), root tips of pea seedlings (Schvartzman et al, 1981), and cultured soybean cells (Cress et al, 1978). In the cases of soybean and Vicia cells, longer molecules of 20-30S and those of approximately 14S, 19S, and 22S were observed after prolonged labeling or chase incubation.…”