Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes

Bánfalvi, Gáspár; Sooki-Toth, A.; Sarkar, Nirmal Kumar; Csuzi, S; Antoni, Ferenc András

doi:10.1111/j.1432-1033.1984.tb08041.x

Cited by 50 publications

(35 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This method originally developed for the reversible permeabilization of lymphocytes isolated from the murine thymus (Banfalvi et al, 1984), was (Banfalvi et al, 1984).…”

Section: Regrowth Of Damaged Monolayer In the Presence Of Antibioticsmentioning

confidence: 99%

Antibiotics delay in vitro human stem cell regrowth

Bánfalvi

Peter

Kukoricza

et al. 2015

Toxicology in Vitro

Self Cite

View full text Add to dashboard Cite

“…This method originally developed for the reversible permeabilization of lymphocytes isolated from the murine thymus (Banfalvi et al, 1984), was (Banfalvi et al, 1984).…”

Section: Regrowth Of Damaged Monolayer In the Presence Of Antibioticsmentioning

confidence: 99%

Antibiotics delay in vitro human stem cell regrowth

Bánfalvi

Peter

Kukoricza

et al. 2015

Toxicology in Vitro

Self Cite

View full text Add to dashboard Cite

“…It was originally described in bacteria that DNA synthesis in permeable cells depends on the presence of ATP, characteristic of replicative DNA synthesis [23]. The ATP- [8,21], while dNTP incorporation in the absence of ATP could be prevented by inhibitors of repair synthesis [21]. These experiments served as a basis to follow the two types of DNA synthesis and chromatin condensation in reversibly permeabilized K562 cells.…”

Section: Resultsmentioning

confidence: 98%

“…This method, originally developed for the reversible permeabilization of murine lymphocytes [8] was adapted to human K562 cells. Briefly, 1 ml of hypotonic buffer was added to 10 6 cells in the presence of Dextran T-150 as a molecular coat to prevent cells from disruption.…”

Section: Reversible Permeabilizationmentioning

confidence: 99%

See 1 more Smart Citation

Preapoptotic chromatin changes induced by ultraviolet B irradiation in human erythroleukemia K562 cells

et al. 2007

Self Cite

View full text Add to dashboard Cite

Exponentially growing human erythroleukemia K562 cells were permeabilized and the dose dependent decrease of DNA synthesis rate was measured after ultraviolet (UV B, 290 nm) irradiation. Cells were able to overcome 2 and 5 J/m2 UV doses, partial recovery was observed at 15 J/m2, while at high (25 J/m2) UV dose replicative DNA synthesis remained suppressed. K562 cells were subjected to synchronization prior to and after UV irradiation (24 J/m2) and 18 fractions were collected by centrifugal elutriation. Cell cycle analysis by flow cytometry did not show early apoptotic cells after UV irradiation. The gradual increase in DNA content typical for non-irradiated cells was contrasted by an early S phase block between 2.2 and 2.4 C-values after UV irradiation. Cell cycle dependent chromatin changes after ultraviolet irradiation were seen as a fine fibrillary network covering the mainly fibrous chromatin structures and incompletely folded primitive chromosomes. Based on observations after UV irradiation and on earlier results with cadmium treatment and gamma irradiation, we confirm that typical chromatin changes characteristic to genotoxic agents can be recognized and classified.

show abstract

“…De novo DNA synthesis can be carried out in permeable cells in the presence of Hg-dCTP in permeable prokaryotic [9] and eukaryotic cells [13]. The mercurated nascent DNA can be isolated by affinity chromatography on thiol-substituted agarose [6].…”

Section: Synthesis and Isolation Of Pulse-labeled Mercuri-dnamentioning

confidence: 99%

DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light

et al. 1987

Self Cite

View full text Add to dashboard Cite

DNA synthesis was followed in vivo and in permeable Escherichiu coli after (a) ultraviolet light irradiation, (b) irradiation and incubation in a growth medium containing chloramphenicol and (c) in unirradiated cells. In vitro, replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo. The DNA was pulse-labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates. dCTP was replaced by 5-Hg-dCTP as a substrate for DNA synthesis. Hg-DNA was separated from cellular nucleic acids on thiol-agarose affinity columns. The 5' termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and [Y-~'P]ATP. DNA synthesis in unirradiated permeable E. coli represents a replicative process dependent on ATP and inhibited by novobiocin. About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5'end. In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin. The 5'-RNA content of this cell population was less than 5%.Ultraviolet light irradiation of Escherichia coli strongly inhibits in vivo DNA synthesis. In damaged bacteria a special type of DNA synthesis called 'stable DNA replication' takes place [l]. Alkali-labile sites found during this type of suppressed DNA synthesis [2] may come from nascent DNA pieces initiated by short RNA primers. Alternatively, alkali lability may be due to pseudo-Okazaki fragments of damage origin.In order to study in vitro DNA synthesis a permeable E. coli system has been developed which shows the characteristics of replicative synthesis [3]. Studies in permeable cells showed that the ATP-dependent synthesis represented discontinuous DNA elongation without chromosome initiation [4] that was sensitive to inhibitors of semiconservative DNA replication such as novobiocin 151.Recently we have developed a novel approach to the study of early replicative intermediates involving pulse-labeling with mercurated nucleotides and the 5'-end analysis of nascent mercuri-DNA that was isolated on thiol-agarose affinity matrix [6]. Here we describe the analysis of 5' ends of short Hg-DNA pieces synthesized in permeable E. coli cells prior to and after ultraviolet light irradiation. Our data indicate an RNA-primed DNA replication in unirradiated E. coli and a drastically reduced level of RNA-linked DNA among short fragments from irradiated cells MATERIALS AND METHODS Chemicals

show abstract

Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes

Abstract: Freshly prepared thymocytes continue to synthesize DNA under hypotonic conditions in the presence of 4.5 % dextran T-150, the four deoxyribonucleoside triphosphates and ATP. Permeable cells could seal the membrane in a serum-enriched medium within a few hours.

Cited by 50 publications

References 23 publications

Antibiotics delay in vitro human stem cell regrowth

Antibiotics delay in vitro human stem cell regrowth

Preapoptotic chromatin changes induced by ultraviolet B irradiation in human erythroleukemia K562 cells

DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light

Contact Info

Product

Resources

About