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Kinetics of HCMV immediate early mRNA expression in stably transfected fibroblasts

Department of Molecular Cell Biology, Laboratory for Cytochemistry and Cytometry, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands

View larger version (76K): [in a new window]   Fig. 1. Inhibition of RNA polymerase II activity by DRB or AMD treatment. (A) Integrated fluorescent intensity of HCMV IE transcripts accumulated in the nuclear foci relative to the integration site. Addition of either RNA polymerase II inhibitor resulted in a rapid decrease (half time 2 minutes) in the amount of HCMV IE transcripts accumulated in nuclear foci. (B) Integrated fluorescent intensity of HCMV IE transcripts dispersed through the nucleoplasm relative to negative nuclei. The amount of HCMV IE mRNAs in the remainder of the nucleoplasm decreased at a much slower rate (half time 10 minutes). (C) CSLM images of cells at different times after RNA polymerase II inactivation. After induction of the HCMV IE gene expression by cycloheximide, transcripts were present in nuclear foci and also dispersed throughout the nucleoplasm. These images demonstrate the decrease in the amount of transcripts in the nuclear foci and in the nucleoplasm at different intervals after addition of a RNA polymerase II inhibitor. At all times, HCMV IE transcripts were observed in the cytoplasm.

View larger version (78K): [in a new window]   Fig. 2. Activation of RNA polymerase II by removal of DRB. (A) Integrated fluorescent intensity of HCMV IE transcripts accumulated in the nuclear foci relative to the integration site. Removal of DRB resulted in a rapid accumulation of HCMV IE mRNAs at nuclear foci. (B) Integrated fluorescent intensity of HCMV IE transcripts dispersed through the nucleoplasm relative to negative nuclei. The amount of HCMV IE mRNAs in the remainder of the nucleoplasm increased at a slower rate after DRB removal. Transcripts radiating from the nuclear foci were observed from approximately 5 minutes onwards. (C) CSLM images of cells at different times after RNA polymerase II inactivation. After induction of the HCMV IE gene expression by cycloheximide, transcripts were present in nuclear foci and also dispersed throughout the nucleoplasm. Two hours of DRB inhibition removed virtually all transcripts from the nucleus. Subsequent removal of DRB resulted in a fast increase of fluorescent intensity at nuclear foci (3 minutes and later), followed by the nucleoplasm. Untreated cells were not subjected to cycloheximide or DRB treatment; therefore these cells did not express the HCMV IE genes.

View larger version (74K): [in a new window]   Fig. 3. Effect of incubation temperature on the amount of HCMV IE transcripts after RNA polymerase II inhibition by DRB. (A) Integrated fluorescent intensity of HCMV IE transcripts accumulated in the nuclear foci relative to the integration site. The mRNA amount in the nuclear foci remained relatively constant during incubation of cells with DRB at 4°C. Shifting cells back to 37°C restored the previously observed decrease in integrated fluorescence intensity (half time 2 minutes). (B) Integrated fluorescent intensity of HCMV IE transcripts dispersed through the nucleoplasm relative to negative nuclei. The mRNA amount in the remainder of the nucleoplasm remained relatively constant during incubation of cells with DRB at 4°C. Shifting cells back to 37°C restored the previously observed decrease in integrated fluorescence intensity (half time 10 minutes). (C) CSLM images of cells at different incubation temperatures during intervals of DRB treatment. After induction of the HCMV IE gene expression by cycloheximide at 37°C, transcripts were observed in nuclear foci and in the remainder of the nucleoplasm. These transcripts remained present when incubation with DRB was carried out at 4°C. Subsequent incubation at 37°C resulted in a decrease in integrated fluorescence intensities with similar kinetics to cells treated with DRB at 37°C only.