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FIG. 6. Vero cells treated with FITC-labeled oligonucleotides. (A) Combination of a fluorescence picture with a phase-contrast image of the cells (8). (B) CLSM pictures of Vero cells treated with 10 mg/ml FODN bound to 100 mg/ml DEAE-40 PHCA nanoparticles for 4 h. Sixty optical slices of both channels (green fluorescence: FODNs; red fluorescence: cell boundaries stained with TRITC-labeled concanavalin A) are combined to obtain xz sections demonstrating the intracellular uptake of FODNs. (C) FODN control without nanoparticles. No significant FODN uptake occurred. (See article on p. 286 –295).
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FIG. 2. Analysis in vitro replication products. (A) Replication foci are detected in isolated yeast nuclei on incorporation of DIG-dUTP in vitro. G 1-phase wild-type nuclei were incubated in a wt S-phase extract for 30 min in the presence of 20 mm DIG-dUTP. After fixation with paraformaldehyde, genomic DNA is stained with ethidium bromide and DIG-dUTP is detected on slides with an anti-DIG monoclonal antibody and an anti-mouse IgG coupled to DTAF. Shown is a high-resolution image of a single yeast nucleus obtained with a Zeiss LSM 410 confocal microscope with a 633 Plan-Apochromat objective (1.4 oil). Surface topography profiles of the red (DNA) and green (DIG) channels are calculated with the Carl Zeiss LSM 3.95 software. About 15 replication foci appear as individual peaks above the background level of fluorescence (blue). The threshold is determined for the green and red channels in a 50 3 50-pixel area containing no nuclei. Bar 5 200 nm. (B) Density substitution analysis of genomic DNA replicated in vitro. In a standard reaction, corresponding to late G 1 nuclei incubated in an S-phase extract, most of the newly synthesized DNA (measured as cpm of [ 32P]dCMP incorporated) is found at the heavy-light (HL) position of the gradient (E). However, on addition of 0.1% Triton X-100 to the reaction mixture, only light-light (LL) DNA is observed, corresponding to patch repair (F). (C) Two-dimensional gel analysis of plasmid DNA replication in nuclear extracts. A strong bubble arc is detected on the gel before linearization of the plasmid (arrowhead). However, this bubble is almost completely lost after digestion. See Braguglia et al. (13) for the interpretation of replication intermediates. The plasmid pH4ARS was used here as template. It corresponds to the H4 ARS fragment cloned into a pGEM backbone. (D) Density substitution analysis of plasmid DNA replicated in vitro. Incorporation of [ 32P]dCMP into DNA migrating as either HL or LL DNA was monitored by scintillation counting. Use of negatively supercoiled pH4ARS (sc; E) as template results in a strong HL peak, whereas use of mung bean nuclease-nicked pH4ARS (oc, F) results only in repair synthesis. (See article on p. 368 –376).
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FIG. 7. Significant residues on S. pombe PCNA tertiary structure identified by genetic and biochemical approaches. Residues important for various metabolic functions of S. pombe PCNA were mapped onto a two-dimensional monomer structure of S. cerevisiae PCNA. Light purple dots depict residues that are involved in enhancement of DNA polymerase d processivity, red dots depict residues required for response to UV-induced damage, green dots depict residues important for MMS-induced damage, yellow dots depict residues that might be involved in cell cycle progression, and blue dots depict the locations of cold-sensitive alleles of PCNA. (See article on p. 335– 348).
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FIG. 6. Vero cells treated with FITC-labeled oligonucleotides. (A) Combination of a fluorescence picture with a phase-contrast image of the cells (8). (B) CLSM pictures of Vero cells treated with 10 mg/ml FODN bound to 100 mg/ml DEAE-40 PHCA nanoparticles for 4 h. Sixty optical slices of both channels (green fluorescence: FODNs; red fluorescence: cell boundaries stained with TRITC-labeled concanavalin A) are combined to obtain xz sections demonstrating the intracellular uptake of FODNs. (C) FODN control without nanoparticles. No significant FODN uptake occurred. (See article on p. 286 –295).
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FIG. 2. Analysis in vitro replication products. (A) Replication foci are detected in isolated yeast nuclei on incorporation of DIG-dUTP in vitro. G 1-phase wild-type nuclei were incubated in a wt S-phase extract for 30 min in the presence of 20 mm DIG-dUTP. After fixation with paraformaldehyde, genomic DNA is stained with ethidium bromide and DIG-dUTP is detected on slides with an anti-DIG monoclonal antibody and an anti-mouse IgG coupled to DTAF. Shown is a high-resolution image of a single yeast nucleus obtained with a Zeiss LSM 410 confocal microscope with a 633 Plan-Apochromat objective (1.4 oil). Surface topography profiles of the red (DNA) and green (DIG) channels are calculated with the Carl Zeiss LSM 3.95 software. About 15 replication foci appear as individual peaks above the background level of fluorescence (blue). The threshold is determined for the green and red channels in a 50 3 50-pixel area containing no nuclei. Bar 5 200 nm. (B) Density substitution analysis of genomic DNA replicated in vitro. In a standard reaction, corresponding to late G 1 nuclei incubated in an S-phase extract, most of the newly synthesized DNA (measured as cpm of [ 32P]dCMP incorporated) is found at the heavy-light (HL) position of the gradient (E). However, on addition of 0.1% Triton X-100 to the reaction mixture, only light-light (LL) DNA is observed, corresponding to patch repair (F). (C) Two-dimensional gel analysis of plasmid DNA replication in nuclear extracts. A strong bubble arc is detected on the gel before linearization of the plasmid (arrowhead). However, this bubble is almost completely lost after digestion. See Braguglia et al. (13) for the interpretation of replication intermediates. The plasmid pH4ARS was used here as template. It corresponds to the H4 ARS fragment cloned into a pGEM backbone. (D) Density substitution analysis of plasmid DNA replicated in vitro. Incorporation of [ 32P]dCMP into DNA migrating as either HL or LL DNA was monitored by scintillation counting. Use of negatively supercoiled pH4ARS (sc; E) as template results in a strong HL peak, whereas use of mung bean nuclease-nicked pH4ARS (oc, F) results only in repair synthesis. (See article on p. 368 –376).
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FIG. 7. Significant residues on S. pombe PCNA tertiary structure identified by genetic and biochemical approaches. Residues important for various metabolic functions of S. pombe PCNA were mapped onto a two-dimensional monomer structure of S. cerevisiae PCNA. Light purple dots depict residues that are involved in enhancement of DNA polymerase d processivity, red dots depict residues required for response to UV-induced damage, green dots depict residues important for MMS-induced damage, yellow dots depict residues that might be involved in cell cycle progression, and blue dots depict the locations of cold-sensitive alleles of PCNA. (See article on p. 335– 348).