INAPPROPRIATE TRANSCRIPTION FROM THE 5' END OF THE MURINE DIHYDROFOLATE-REDUCTASE GENE MASKS TRANSCRIPTIONAL REGULATION

Using the nuclear run-on assay we found that in proliferating cells th e transcription rate in the 5' end of the murine dihydrofolate reducta se (dhfr) gene was approximately ten-fold higher than in the 3' end of the gene, suggesting transcriptional attenuation within the dhfr gene . However, when the transcription rate was measured by pulse-labeling, the rate was uniform throughout the gene, and the 5' dhfr signal was approximately ten-fold lower relative to a control gene signal than in the run-on assay. Previously, the activity of a dhfr promoter linked to a luciferase reporter gene was shown to increase about ten-fold at the G1/S-phase boundary following stimulation of serum-starved cells. To determine if the run-on procedure would detect growth regulation of the endogeneous dhfr gene, serum-starved and -stimulated NIH 3T3 cell s were analyzed. Using a dhfr 5' end probe no difference in transcript ion rate between these growth states was detected and the dhfr 3' end probe did not detect signal above background. In a cell line that was amplified at the dhfr locus, the transcription rate in the 5' end of t he gene increased less than two-fold in stimulated cells, but the rate in the 3' end of the gene increased five- to seven-fold. Therefore, t he dhfr gene is growth regulated at the level of transcription, but th e nuclear run-on assay was only able to detect a difference in transcr iption rate in the 3' end of the gene in amplified cells. We suggest t hat isolation of nuclei may activate dhfr transcription complexes that normally are activated only at the G1/S-phase boundary.