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Hr. post-stimulation

Fig. 2. Reporter transfection assay to determine the possible role of the Topo II 3' UTR in mRNA levels during the cell cycle. (A) FACS analysis of cell cycle position in stably transfected 3T3 cells containing the H^galTopo reporter construct. Asynchronously growing monolayer cells were incubated for 30 hr with medium containing 0.2% (v/v) serum and serum stimulated to reenter the cell cycle. Cells were trypsinized at the time of serum stimulation (0 hr) and 6, 20, and 24 hr after stimulation and fixed in ethanol. Ethanol-fixed cells were treated with RNase A, stained with propidium iodide, and analyzed for DNA content by FACS analysis. (B) Representative Northern blot analysis of H/igalTopo and HJ8galSV40 reporter mRNA levels during the cell cycle. Cells in duplicate dishes were harvested at the indicated times for RNA isolation and hybridized with a radiolabeled jfi-galactosidase probe. Blots were rehybridized with a radiolabeled mouse GAPD cDNA probe.

after serum stimulation. These results are comparable to results obtained with un-transfected control cells (data not shown) and demonstrate that transfection of the reporter construct does not perturb cell cycle progression.

When the total cellular RNA from the H/JgalTopo-transfected cell populations is analyzed by Northern blotting, an mRNA band of approximately 4 kb, representing the H/igalTopo reporter construct, is detected with a radiolabeled ¿8-galactosidase probe (Fig. 2B). H^galTopo reporter mRNA levels are low in G¡ phase (0-6 hr) and increase 12- to 16-fold by late S to G2 phases (20-24 hr). Results with the H/JgalSV40 reporter construct (20-24 hr), which contains the Topo II promoter but not the 3' UTR, indicate a small increase in cell cycle-regulated /S-galactosidase reporter mRNA levels. This can be attributed to the 2-fold increase in Topo II transcription that has been reported previously to occur during late S phase by us and other investigators.4'1517 Because the two reporter constructs differ only in the 3' UTR sequence, the above-described results show a regulatory role for the Topo II3' UTR in cell cycle-coupled expression of Topo II. These results demonstrate the feasibility of the reporter assay, which could be used to determine the possible role of 3' UTRs in the regulation of cell cycle-coupled mRNA levels.

In Vitro RNA-Protein Binding Assay

The 177-nucleotide (nt) cDNA sequence representing the putative protein-binding site (nt 4772-4948; Goswami et al.n) in the Topo II 3' UTR is amplified by PCR from plasmid DNA containing the entire Topo II3' UTR (pTopUTR), using primer pairs 5' TAGTGACCATCTATGGG 3' and 5' CTGCTCTAGTTTTAGCT TAGTGG 3'.11 PCR-amplified Topo II3' UTR cDNA (177 nt) is cloned into transcription vector pGEM-T plasmid DNA (Promega, Madison, WI). /J-Galactosidase transcript is used as nonspecific competitor in the RNA-protein binding assays. ¿0-Galactosidase-coding sequence within the Hpal and Clal restriction sites in pCMV plasmid (Clontech) is cloned into pBS II SK(+) and runoff transcript is generated by using T7 RNA polymerase. Riboprobes representing the sense strand of RNA from each plasmid are transcribed in vitro according to the protocol

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