Tobacco lines with high copy number of replicating recombinant geminivirus vectors after biolistic DNA delivery

The feasibility of obtaining clonal lines with replicating, multicopy geminivirus vectors by direct DNA trans-formation of cultured tobacco cells was studied. The replicating vectors pTGA32 and pST31 are based on the tomato golden mosaic virus (TGMV) A genome and encode the neomycin phosphotransferase type II (NPT-II) enzyme that confers kanamycin resistance to plant cells. Following introduction into plant cells, unit-length viral genomes were released from the tandem repeats and replicated. In protoplasts, replication of unit-length pTGA32 and pST31 was about as efficient as replication of unit-length DNA A from plasmid pTGA26, which contains 1.5 copies of wild-type DNA A. Tobacco suspension culture cells were bombarded with the recombinant DNA A constructs and selected for kanamycin resistance. The number of kanamycin-resistant clones per bombardment was about the same when the TGMV DNA A vectors or a non-replicating plasmid (pLC14) which also encodes NPT-II was used. Replicating, unit-length DNA A in up to approximately 1000 copies per cell was found in about 10% of the kanamycin-resistant clones selected following bombardment of cells with TGMV vectors. The results suggest that geminiviruses may serve as useful multicopy vectors in cultured cells.     

Extra perspectives of 5-ethynyl-2′-deoxyuridine click reaction with fluorochrome azides to study cell cycle and deoxyribonucleoside metabolism

Beginning with the pioneering work of Salic and Mitchison (2008), the application of thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) for the detection of cells replicating DNA is actively expanding. Being incorporated into DNA, this nucleoside after click reaction of azide-alkyne cycloaddition with azides of fluorochromes can be easily detected by fluorescence. Recently, protocols of EdU application in combination with click reaction adapted for plant cells appeared, and they are help for a monitoring S-period of the cell cycle in the root meristems and in vitro cultured cells with the help of a microscope and flow cytometer. In this work, we focused some details of developed methods and their modifications and also recommended new protocols. In particular, we suggested combining EdU incorporation into the cells replicating DNA with subsequent isolation of protoplasts from them and their preparation for the microscopic analysis and flow cytometry. In addition, the method of determination of EdU phosphorylation dynamics in the cells in vivo is suggested.     

Replication of a geminivirus derived shuttle vector in maize endosperm cells.

A maize (Zea mays L.) endosperm cell culture has been shown to efficiently replicate DNA sequences derived from wheat dwarf virus (WDV), a monopartite monocot geminivirus. To analyze sequences necessary for viral replication and to verify their application for a plant gene expression vector, we have developed a 3.7 kilobase pairs Escherichia coli--plant cell shuttle vector, pWI-11. The p15A origin of replication, functional in E. coli, was introduced into the viral sequences. We have replaced the coding region of the coat protein gene by that of bacterial neomycin phosphotransferase II (NPT II) gene. The resulting NPT II gene fusion can serve as a selectable marker in both plant and E. coli systems. Into a unique cloning site in this pWI-11 vector, we introduced a gene fusion carrying the bacterial beta-glucuronidase (GUS) coding region under control of the cauliflower mosaic virus 35S (CaMV35S) gene promoter and terminator. By transferring these viral sequences into protoplasts derived from maize endosperm cell cultures, we have demonstrated that the plasmid pWI-11 can replicate in maize endosperm cells, that the GUS reporter gene introduced into pWI-11 can be expressed at high level in the transformed cells, and that the replicating viral DNA can be rescued from endosperm cells by transforming E. coli in the presence of kanamycin. The level of GUS gene expression increased progressively in transformed endosperm cells during a prolonged culture period, coinciding with replication of the viral sequences in these cells.     

Role of RNA synthesis in DNA replication of synchronized populations of cultured mammalian cells

Using the harvesting method of synchronizing L cells, the relationship of RNA synthesis of DNA replication was studied by the use of selective inhibitors of RNA synthesis such as actinomycin D and chromomycin succinate. The synthesis of the early replicating DNA fraction is a process sensitive to the inhibition of RNA synthesis during the G1 period. The synthesis of early replicating DNA was inhibited by chromomycin succinate without affecting the initation of DNA synthesis. However, actinomycin D inhibited the synthesis of early replicating DNA and prevented the initiation of DNA synthesis in 50% of the synchronized cells. However, it was found that the continued synthesis of RNA during the S period is not essential for the synthesis of late replicating DNA. In addition to this specific response of DNA synthesis to the inhibitors of RNA synthesis, another function of early and late replicating DNA was determined relative to the cell viability. Cells synthesizing early replicating DNA were killed more efficiently by chromomycin than at other stages of the cell cycle. This indicates that the early replicating DNA unit plays a more important role in cell reproduction than the late replicating DNA unit.     

Agrobacterium tumefaciens Ribonucleic Acid Synthesis in Tomato Cells and Crown Gall Induction

Purified Agrobacterium tumefaciens deoxyribonucleic acid (DNA) does not produce crown gall tumors in growing plants, conditioned by wounding, as the living bacteria do. Purified bacterial DNA migrates in the plant and replicates, but it is not transcribed in our experimental conditions. On the contrary, when DNA is released naturally from bacteria into plant cells, a bacterial ribonucleic acid (RNA) can be found in these cells. There seems to be a direct relation between the appearance of A. tumefaciens RNA in the plant cells and the induction of the tumor.     

Effect of damage to early, middle, and late-replicating DNA on progress through the S period in Chinese hamster ovary cells

In order to explain sequential replication of DNA in eukaryotic cells, the duplication of a given bank of replicons is proposed to be initiated by specific events coupled to synthesis of the preceding replicons in the sequence. This model predicts that DNA synthesis in mid or late S should depend upon the synthesis and/or integrity of previously replicating DNA, but should not depend upon the integrity of DNA replicating later in the sequence. By incorporating BUdR into DNA during a given short interval of one S period in synchronous Chinese hamster ovary cells, we are able to selectively damage this DNA by irradiation at selected times before or during the next S period. Utilizing this technique, we find that damage to early replicating DNA before entry into the second S phase markedly suppresses DNA synthesis in the entire S period. Damage to mid or late replicating DNA prior to entry into the second S period has no effect on early S, but markedly reduces DNA synthesis commencing in mid or late S, respectively. Furthermore, if early replicating DNA is damaged with light in mid-S, no effect on subsequent DNA synthesis is observed. These results can be fitted to a model in which sequential triggering of replicon synthesis promotes orderly progression through S.     

Topoisomerase II activity in the replicating DNA of irradiated hamster cells.

The activity of DNA topoisomerase II in the replicating DNA of irradiated Chinese hamster ovary cells was estimated by determining protein-linked DNA double-strand breaks generated in the presence of the DNA intercalative drug 4'-(9-acridinylamino) methanesulfon-m-anisidide. In the presence of this drug, DNA double-strand breaks were produced at the same rate, and with the same overall frequency, in both the bulk and the newly synthesized DNA of control cells and cells irradiated with 10 Gy. The results indicate that DNA topoisomerase II is fully active in the replicating DNA of irradiated cells and is distributed at a frequency similar to that in parental DNA.     

Establishment of infection by spleen necrosis virus: inhibition in stationary cells and the role of secondary infection

The relationship of two early events in the establishment of infection by avian retroviruses, the inhibition of viral DNA synthesis in stationary avian cells and the secondary infection which occurs after infection of replicating cells, was investigated. When neutralizing antibody to spleen necrosis virus was used to prevent secondary infection, the amount of unintegrated linear spleen necrosis virus DNA detected was much lower in infected stationary cells than in infected replicating cells. The amount of unintegrated linear spleen necrosis virus DNA in stationary cells was less than one copy per cell even at high multiplicities of infection. Viral DNA synthesis resumed after stimulation of the cells to replicate. The time of this viral DNA synthesis was closely correlated with renewed cellular DNA synthesis. In addition, blocking secondary infection of replicating cells prevented the rate of virus production from reaching the high levels usually associated with a normal productive infection by SNV. Virus production increased if secondary infection was allowed. However, this rise in virus production was not proportional to the amounts of viral DNA integrated after secondary infection.     

The replication behavior of Saccharomyces cerevisiae DNA in human cells

We studied the replication of random genomic DNA fragments from Saccharomyces cerevisiae in a long-term assay in human cells. Plasmids carrying large yeast DNA fragments were able to replicate autonomously in human cells. Efficiency of replication of yeast DNA fragments was comparable to that of similarly sized human DNA fragments and better than that of bacterial DNA. This result suggests that yeast genomic DNA contains sequence information needed for replication in human cells. To examine whether DNA replication in human cells would initiate specifically at a yeast origin of replication, we monitored initiation on a plasmid containing the yeast 2-micron autonomously replicating sequence (ARS) in yeast and human cells. We found that while replication initiates at the 2-micron ARS in yeast, it does not preferentially initiate at the ARS in human cells. This result suggests that the sequences that direct site specific replication initiation in yeast do not function in the same way in human cells, which initiate replication at a broader range of sequences.by J.A. Huberman     

Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus

Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.     

Strand switching during rolling circle replication of plasmid-like DNA circles in the mitochondria of the higher plant Chenopodium album (L.)

The structure of sigma-like mitochondrial DNA molecules prepared from suspension cultured cells of Chenopodium album (L.) was studied by electron microscopy. These molecules were highly variable in size, ranging from about 1 to 104 kb, and had single- and double-stranded regions typical for rolling circle replicating intermediates. Partial denaturation studies confirmed that these structures constitute rolling circles. Close inspection of the circle-tail junctions of the replication fork at high magnification suggests that in circles with a double-stranded tail, both strands of the tail seem to be covalently attached to the circle in about 27% of the molecules. This observation can be explained by a phenomenon called strand switching or strand splippage during rolling circle replication, similar to a mechanism proposed for bacterial replicons or in vitro replicating constructs harboring bacteriophage T4 replication origins.     

Running into problems: how cells cope with replicating damaged DNA

The ability of cells to fully and faithfully replicate DNA is essential for preventing genomic instability and cancer. DNA is susceptible to damage both in resting and in actively replicating cells. Thus, genome duplication necessarily involves replication of damaged DNA. The many mechanism cells use to avoid or overcome the problems of replicating an imperfect DNA template are discussed.     

Transfer of T-DNA from agrobacteria into plant cells through cell walls and membranes

Discusses probable routes of agrobacterial penetration through the plant integumental tissues, cell wall, and plant cell plasmodesma. Analyzes the contribution of extracellular structures of agrobacteria in penetration through barriers of a plant cell, primary contact (adhesion), and during DNA transfer from bacterial (E. coli, A. tumefaciens) to recipient (bacterial or plant) cells. Discusses the relationship between donor cell adhesion to recipient cell surface and the infectious and conjugation processes. Considers the probable role of piles in conjugative transfer of agrobacterial DNA through membranes of donor and recipient (bacterial and plant) cells. Analyzes the contribution of the plant cell cytoskeleton to T-DNA transfer. Suggests a model of transport of T-DNA-VirD2 complex and VirE2 proteins through independent channels consisting of vir-coded proteins.     

Identification of late DNA-replicating regions in Chinese hamster chromosomes using a BrdU-giemsa technique

Asynchronous Chinese hamster cells were labelled with BrdU for 3 h prior to harvesting the metaphase cells. The late DNA replicating sites became unifilarly BrdU-substituted as compared to the earlier replicating sites having a normal DNA constitution. Those late replicating sites were identified by pale coloration or dot formation after treatment with 1.0 M Na-phosphate solution (adjusted to pH 9.0 with supersaturated amount of NaHCO₃ and at a temperature of 69–75° C) and staining with Giemsa dye. Using this technique, nuclei with incorporated BrdU could be distinguished from nuclei that had not incorporated BrdU. — One of the advantages of using this technique for identification of late DNA replicating sites is that cells are treated continuously with BrdU for a short period of time before harvesting and only one sampling, rather than a series of samplings, is required to achieve a clear-cut result.     

Filamentous Bacterial Viruses IV. Fate of the Infecting Parental Single-Stranded Deoxyribonucleic Acid

Some of the infecting fd single-stranded viral deoxyribonucleic acid (DNA) molecules were found to be degraded after having initiated infection. The degradation products were reused for synthesis of DNA, primarily bacterial. Degradation was most extensive when viral DNA was replicating.     

Sizes and rates of elongation of replicons in the frog embryo

Summary DNA fiber autoradiography has shown an increase in size of replicons during early development of the frog embryo. Replicons of endoderm cells were considerably larger than those of dorsal ectoderm and mesoderm cells in tailbud embryos. Late replicating DNA in partially synchronized tailbuds has a more rapid rate of replicon elongation than does early replicating DNA.     

Preferential modification of replicating DNA by benz(a)pyrene)

The nuclei of cells from regenerating rat liver were incubated with benzo(a)pyrene and the concentrations of the metabolites that covalently bound to DNA of different nuclear fractions were compared. It appeared that DNA associated with nuclear matrix (containing replicating DNA) is modified most intensively. The synchronized mouse embryo cells were incubated with benzo(a)pyrene during S phase and the levels of modifications in short and long single-stranded DNA fragments were compared. It has been observed that replicating DNA is represented in short fragments. These short DNA fragments were found to be modified by benzo(a)pyrene 4-9 times more intensively than total DNA. The possible mechanisms of both the increase in the number of DNA modifications in proliferating cells and the reason for the enhancement of carcinogenic effect on dividing cells are being discussed.     

Mechanisms and age-specific characteristics of nuclear DNA replication

Replication mechanisms of nuclear DNA in eucaryotic cells and their changes with the organism aging are discussed. Tge polyrepliconic nature of replicating DNA, discontinuous synthesis of its newly formed chains, enzymic apparatus of replication and regulation of this process are described. Special attention is paid to destabilization of the secondary structure of the replicating molecule. In aging, changes in the DNA replication were found at each level of its regulation, i.e. at synthesis precursors, enzymic replication complex and replicating DNA structure. This may be an indication of the altered regulation of the DNA replication with aging.     

DNA synthesis in polyoma virus infection. III. Mechanism of inhibition of viral DNA replication by cycloheximide.

The formation of viral DNA was inhibited in polyoma virus-infected cells in which protein synthesis had been blocked by cycloheximide. The present studies show the following. (i) The pool of replicating viral DNA molecules was reduced in cycloheximide-treated cells by an amount consistent with inhibition of [3-H]thymidine incorporation into viral DNA, whereas the rate of turnover of the replicating population was not affected. (ii) The rate of conversion of replicating molecules into closed-circular DNA was not affected by cycloheximide. (iii) The rate of elongation of nascent viral DNA fragments into strands of unit genome length was unaffected by cycloheximide. It is concluded that viral DNA synthesis is inhibited in the absence of protein synthesis exclusively at the level of initiation of new rounds of genome replication. Replicating molecules already initiated at the time of addition of cycloheximide matured into progeny closed-circular DNA at a normal rate.