Permeabilized Cell System Research Articles

DNA synthesis in vitro in human fibroblast preparations

When confluent cultures of human fibroblasts were ultraviolet irradiated and either permeabilized or lysed, three types of DNA synthesis were subsequently observed during incubation in vitro: (A) a low level of DNA replication, which ceased after 15–30 min incubation at 37°C; (B) radiation-dependent reparative gap-filling, which also ceased after 15 min at 37°C; and (C) radiation-independent DNA synthesis, which was not semiconservative and proceeded at a linear rate for 1 h at 37°C. Normal and xeroderma pigmentosum fibroblasts displayed different rates of radiation-dependent reparative gap-filling after lysis but similar rates of radiation-independent DNA synthesis. The rates of DNA replication and radiation-independent DNA synthesis were less in the permeable cell system than in the lysed cell system, whereas radiation-dependent reparative gap-filling was the same in both. Preparations of permeable and lysed cells activated radiation-dependent reparative gap-filling at about 15% of the rate estimated for intact cells. No radiation-dependent DNA strand breaks, as assayed by alkaline elution, were observed in the lysed cell preparation. Some radiation-dependent breaks were observed in the permeable cell preparation, but radiation-dependent DNA breakage was less than that seen in intact cells. This inability to incise DNA at damaged sites could account for the low rate of activation of reparative gap-filling in vitro. DNA strand breaks were produced in fibroblast preparations nonspecifically during lysis or permeabilization and incubation in vitro, and this breakage of DNA probably was responsible for the radiation-independent DNA synthesis.

Induction of DNA Strand Breaks and Chromosome Abnormalities by an Imide Derivative of 3-Nitro-1 ,B-naphthalic Acid (Mitonafide) in Chinese Hamster Ovary Cells<xref ref-type="fn" rid="FN2">2</xref>

Mitonafide (5-nitro-2-(2-dimethylaminoethyl)-benzo- [de]isoquinoline-1,3-dione hydrochloride), a new candidate as an anticancer or antiviral agent, inhibited the incorporation of DNA precursors into the acid-insoluble fractions of cultured Chinese hamster ovary (CHO) cells and also into CHO cells made permeable (i.e., "permeabilized") that were supplemented with ATP and deoxynucleoside triphosphates. Mitonafide enhanced the degradation of previously incorporated [3H]thymidine and increased the amount of DNA recovered in fractions containing single-stranded DNA after alkaline denaturation and hydroxyapatite chromatography. At concentrations of 0.01 and 1.0 microM, respectively, mitonafide increased the frequency of sister chromatid exchanges and chromosome aberrations in CHO cells. The inhibition of DNA synthesis in a permeabilized cell system suggested that mitonafide acted on the DNA-synthesizing apparatus without requiring metabolic conversion and interfered with DNA synthesis independent of the cellular metabolism of DNA precursors. Mitonafide induced DNA strand breaks and chromosome abnormalities in cultured cells.

Formation and resealing of intercalator-induced DNA strand breaks in permeabilized L1210 cells without the stimulated synthesis of poly(ADP-ribose).

DNA strand breaks produced by damaging agents such as x-ray generally stimulate poly(adenosine diphosphoribose) (ADP-R) synthesis in mammalian cells. DNA intercalating agents induce the formation of strand breaks which are unusual in that they are associated with tightly or covalently bound protein. In order to determine whether the intercalator-induced strand breaks are associated with poly-(ADP-R) synthesis, L1210 cells were treated with the intercalating agent, 4'-(9-acridinylamino)methanesulfon-m-anisidide. Poly(ADP-R) synthesis, measured by [3H]NAD incorporation following cell permeabilization, was enhanced in x-irradiated cells, but not in cells exposed to 4'-(9-acridinylamino)methanesulfon-m-anisidide at doses which produced equivalent strand breaks frequencies. The permeabilized cell system did not support DNA synthesis and x-ray-induced strand breaks did not reseal. The intercalator-induced strand breaks, however, resealed within 10 min. Hence, the strand breaks observed in intercalator-treated cells may not constitute DNA damage in the usual sense. The resealing of intercalator-induced DNA breaks in the absence of DNA or poly(ADP-R) synthesis is unique among chemical or physical agents which produce DNA scissions.

Ultraviolet hypersensitivity of Cockayne's syndrome fibroblasts: Effects of nicotinamide adenine dinucleotide and Poly(ADP-ribose) synthesis

Four Cockayne's syndrome (CS) fibroblast strains with normal repair capacity were highly sensitive to ultraviolet (UV) killing due to the defective recovery of post-UV DNA synthesis. The enzymatic cycling assay for oxidized nicotinamide adenine dinucleotide (NAD +) revealed that CS cells had reduced levels of cellular NAD + pool. Exogenously supplied NAD + raised the cellular level in CS cells, and its concentrations of 0.5–1.0 mM almost normalized such a high UV lethality and defective post-UV DNA synthesis of CS cells. Highly elevated levels of UV-induced sisterchromatid exchanges in CS cells were also completely normalized with 0.1 mM NAD + in culture medium, but not in the xeroderma pigmentosum complementation group-A cells. However, the CS cells exhibited no obvious defect in the maximally deoxyribonuclase I-stimulated, intrinsic Poly(ADP-ribose) polymerase activity and the nearly normal time-course changes in the UV-induced activity of the enzyme, as assayed by the permeabilized cell system. Therefore, our data indicate that CS cells may have a partial deficiency in cellular NAD + level which results in insufficient or inadequate modifications of nuclear proteins and hence the defective recovery of post-UV DNA synthesis, so that exogenously supplied NAD + can rescue the defective phenomena as such in CS cells.

Complete in vitro replication of SV40 DNA and chromatin in saponin-treated permeable cells.

A permeable cell system has been developed by treatment with saponin for studying in vitro replication of DNA and chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in saponin-treated permeable cells was found to be more efficient than that in digitonin-treated permeable cells. Autoradiography of the agarose-gel revealed that [alpha-32P]dCTP was incorporated into SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated complete replication of SV40 DNA and chromatin with a full number of nucleosomes. The saponin-treated permeable cell system will serve as a useful system for studying in vitro replication of DNA and chromatin in eukaryotic cells.

Complete in vitro DNA replication of SV40 chromatin in digitonin-treated permeable cells.

A permeable cell system has been developed by treatment with digitonin for studying in vitro DNA replication of chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in digitonin-treated permeable cells was analyzed by electrophoresis in agarose-gel. Autoradiography of the agarose-gel revealed that [32P]dCTP was incorporated in SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated the complete replication of SV40 DNA and chromatin with a full number of nucleosomes. The digitonin-treated permeable cell system will serve as a useful system for studying in vitro DNA replication of chromatin.

Prophage induction in a permeabilized cell system: induction by deoxyribonucleases and the role of recBC-deoxyribonuclease.

Permeabilized cells able to induce prophage were obtained by plasmolysis and preincubation of the cells in a reaction mixture which allows protein synthesis. These cells became permeable to low-molecular-weight proteins and oligonucleotides. We found that deoxyribonucleases (pancreatic deoxyribonuclease and micrococcal nuclease) triggered prophage (phi 80) induction. This deoxyribonuclease-triggered induction was completely dependent upon the presence of functional recBC genes in the lysogen, regardless of the recombination proficiency determined by recBC and sbcB genes. The possible role of recBC-deoxyribonuclease in prophage induction and recombination is discussed.

Replicative RNA synthesis and nucleocapsid assembly in vesicular stomatitis virus-infected permeable cells.

A permeable-cell system has been developed to study the replication of vesicular stomatitis virus. When vesicular stomatitis virus-infected BHK cells were permeabilized by lysolecithin treatment, they incorporated nucleoside triphosphates into RNA and amino acids into proteins at nearly normal rates. The viral mRNA's synthesized appeared normal in polarity, size distribution, and polyadenylation, and all five viral proteins were synthesized. Replication of the viral genome proceeded, and full-length RNA strands were synthesized in amounts and polarities resembling those found in intact cells. These full-length RNAs associated with viral N proteins to form RNase-resistant nucleocapsids of normal buoyant density. Permeable cells appear to represent ideal hosts for studying vesicular stomatitis virus replication since they closely mimic in vivo conditions while retaining much of the experimental flexibility of current in vitro systems.

Inactivation of phage repressor in a permeable cell system: role of recBC DNase in induction.

UV light causes inactivation of phage (phi80) repressor molecules in a plasmolyzed, permeable cell preparation of Escherichia coli. Induction without UV irradiation occurs when the permeable cells are incubated in the presence of four deoxyribonucleoside triphosphates and ATP. The induction triggered by dNTP's requires a functional recBC gene product and is associated with degradation of the DNA replication fork. The role of recBC DNase in the induction of prophage and SOS functions in general is discussed.

Synthesis and degradation of poly(A) in permeable cells of Escherichia coli

Poly(A) synthesis and degradation have been examined in Escherichia coli cells made permeable to nucleotides by treatment with toluene. Although newly synthesized poly(A) is normally rapidly degraded in this system, extraction of the soluble portion of the cell effectively eliminates this process without affecting poly(A) synthesis. Poly(A) synthesis in this system displays many properties associated with poly(A) synthesis by purified poly(A) polymerase in vitro including a lag in polymerization, stimulation by increased ionic strength, and a low Mg2+ optimum. As with the purified enzyme, this system uses both ADP and ATP as substrates, requires conversion of ATP to ADP, and is strongly inhibited by dADP, orthophosphate, and pyrophosphate. In contrast to the purified poly(A) polymerase, the permeable cell system displays some properties suggestive of in vivo poly(A) metabolism. Thus, the permeable cells require an endogenous RNA primer for activity, the poly(A) product remains with the cells, and the reaction is greatly stimulated by polyamines. This system should prove extremely useful for studies of poly(A) metabolism in E. coli. A surprising feature of these studies was the finding that mutant strains deficient in polynucleotide phosphorylase were unable to synthesize poly(A). The possible roles of polynucleotide phosphorylase and poly(A) in E. coli are discussed.

Chapter 21 Nucleic Acid Synthesis in Permeabilized Eukaryotic Cells

Permeable cell systems are developed in bacteria and disrupted or permeable cell systems are developed in eukaryotes to allow exogenously supplied deoxynucleoside triphosphates and other reaction components to be supplied directly to the replication complex functioning on its intrinsic DNA template. The technique described for rendering eukaryotic cells permeable to nucleotides has several advantages in the study of nucleic acid synthesis under near physiological conditions. The technique itself is simple; the cells remain in a monodisperse suspension and are easy to pipet quantitatively. The cells are freely permeable to phosphorylated compounds which gain rapid access to the nucleus. Nucleotides are incorporated into DNA without intermediate breakdown and rephosphorylation. DNA synthesis in permeable cells is semiconservative, the products are high-molecularweight DNA intermediates, and DNA synthesis occurs as extensions of replication sites that were active in vivo. This technique provides an assay system in which the polymerases function on their endogenous templates. It should be useful in determining whether agents that affect nucleic acid synthesis in intact cells exert their effects by direct action on the polymerase or on the template. The system should also be useful for studies of the intermediates in nucleic acid synthesis, and finally for studies of physiological changes in the activities of polymerases that occur with metabolic manipulation of the cells. As the addition of Triton X-100 renders the cells permeable to exogenous proteins, this technique may also be useful in complementation studies of eukaryotic cells with genetic defects in DNA synthesis.

Description of a permeable eukaryotic cell system to study agents affecting DNA synthesis: demonstration that cytembena is a direct inhibitor of replicative DNA synthesis.

Cells were made permeable to DNTP's by a 15-minute treatment with 0.01 M Tris/HCl (pH 7.8), 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol, and 4 mM MgCl2 at 4 degrees C. These cells used exogenously supplied dNTP's to carry out semiconservative, replicative DNA synthesis. This system could be used to identify inhibitors of DNA synthesis and to determine whether chemotherapeutic agents affect precursor synthesis or whether they have a direct effect on the DNA replication complex. Thus DNA synthesis in the permeable cells was not inhibited by hydroxyurea or by the nucleoside analogue arabinofuranosyl cytosine. In contrast, the active form of the nucleoside analogue, arabinofuranosyl cytosine triphosphate, and daunorubicin promptly inhibited DNA synthesis in the permeable cells. Cytembena, sodium cis-beta-4-methoxybenzoyl-beta-bromoacrylate, also inhibited DNA synthesis in the permeable cells; this demonstrated that this agent functioned as a direct inhibitor of the DNA replication complex.

Replication of Escherichia coli DNA in vitro: inhibition by oxolinic acid.

Oxolinic acid, a quinolone antibacterial agent, inhibits reversibly the ATP-dependent replicative DNA synthesis in permeable cell systems as well as in cellophane disk lysates. It is about 10-fold more active than the structurally related nalidixic acid. Both drugs have no effect on the ATP-independent DNA repair, but interfere to some extent with RNA synthesis in permeable cells. They appear to interact with the same target since spontaneous nalidixic-acid-resistant mutants of nalA phenotype are also resistant to oxolinic acid. Full sensitivity to oxolinic acid can be conferred to lysates from resistant cells by addition of extracts from sensitive cells.

DNA synthesis in permeabilized mouse L cells

Mouse L cells are rendered permeable to nucleoside triphosphates by a cold shock with a near isotonic buffer. These cells retain their morphologic integrity and use exogenously supplied nucleotides and deoxynucleotides to synthesize RNA and DNA. The newly synthesized DNA is nuclear and is the product of semiconservative replication. Incorporation of deoxynucleotides into DNA by thymidine kinase-deficient cells was used to confirm rigorously that the exogenously supplied deoxynucleotides were incorporated into DNA without intermediate processing through nucleosides. DNA synthesis requires the presence of Na +, ATP, all 4 deoxynucleotides, and Mg 2+. The reaction is inhibited by N-ethylmaleimide, p-hydroxymercuribenzoate and actinomycin D. Hydroxyurea and arabinosylcytosine do not inhibit the reaction whereas cytosine arabinoside triphosphate shows competitive inhibition with the deoxynucleotides. These findings indicate that the permeable cell system can be used for in situ evaluations of the replicative DNA polymerase using the endogenous DNA template.

Translational response of an E. coli “permeabilized” cell system (PCS) to hemoglobin messenger RNA

Translational response of an E. coli “permeabilized” cell system (PCS) to hemoglobin messenger RNA

A permeable cell system for studying DNA replication in synchronized HeLa cells

Treatment of HeLa cells with a hypotonic buffer solution makes them permeable to nucleotides. Cells which are in S-phase at the time of treatment continue to synthesize DNA when supplied with the four deoxyriboside triphosphates, ATP, Mg 2+, and the proper ionic environment. DNA replication extends from sites which were active in the cells prior to treatment. The product is confined to the nucleus and is sensitive to deoxyribonuclease. Under optimum conditions, up to 5% of the HeLa genome can be replicated from exogenous nucleotides. In synchronized cultures the level of DNA replicase activity, as measured in permeable cells at different points in the cell cycle, correlates with the rate of [ 14C] thymidine incorporation measured in the living, untreated cells.

Regulation of RNA synthesis in Escherichia coli I. Characterization of cells subjected to simultaneous temperature and osmotic shock

Cells of Escherichia coli can be made permeable to low molecular weight compounds while retaining a sizeable portion of whole cell capacities for overall RNA and protein synthesis when appropriately supplemented. Inducibility and cyclic 3′,5′-adenosine monophosphate dependence of β-galactosidase synthesis in these cells is similar to that shown by whole cells. Therefore these shocked cellular preparations have capacities for coupled RNA and protein synthesis substantially higher than either cell-free extracts or previously described permeable cell systems in which the acidsoluble pools are washed out by the permeabilization procedure. Shocked cell preparations from strains showing stringent control of RNA synthesis synthesize guanosine 5′-diphosphate 2′ or 3′-diphosphate in response to amino acid deprivation. This property together with the high capacity for RNA synthesis allows an investigation of nucleotide participation in the regulation of RNA synthesis in this permeable cell system.

Transcription and translation mechanisms in a «permeabilized E. coli system

Summary An E. coli «permeabilized cell system (PCS) is described, resulting from 2 M-sucrose plasmolysis in the presence of 10−4 M EDTA. This treatment induces some interesting properties in the cells: permeability to various phosphorylated derivatives without osmotical fragility, depletion from endogenous metabolites and lack of growth on rich medium. This system appears well suited for the study of uncoupled macromolecular synthesis: o (1) in the presence of the usual RNA polymerase substrates (ATP, CTP, GTP and UTP), PCS prominently synthesizes messenger RNA of exceptional great stability (half-life greater than 60 minutes). The reasons of this stability will be discussed; (2) the mRNAs thus formed are active in protein synthesis as it can be shown by a stepwise induction experiment: RNA synthesized in the presence of the four ribonucleoside triphosphates plus IPTG can direct the synthesis of β-galactosidase, after PCS is transferred in an amino acid medium plus actinomycin D; (3) when PCS is supplemented with a complete amino mixture in addition to the four ribonucleoside triphosphates, it synthesizes proteins and also accumulates large amounts of ribosomal and transfer RNA's.