MEASUREMENT OF GENE-SPECIFIC TRANSCRIPTION BY NUCLEASE PROTECTION OF PULSE-LABELED NUCLEAR-RNA

A gene-specific transcription assay was developed that is based on pul se-labeled incorporation of [H-3]uridine into nuclear RNA. Transcripti on is quantified by scintillation counting of [H-3]uridine incorporate d into nuclear RNA that is protected from S-1 nuclease digestion by hy bridization with cold gene probes. This assay was dependent upon parti al degradation of nuclear RNA and optimization of hybridization and nu clease digestion conditions. To validate this assay, transcription of beta-actin and c-myc genes was measured in two different human cell li nes using the incorporation assay in parallel with the nuclear run-off assay. Transcription kinetics of the beta-actin and c-myc genes in se rum-stimulated fibrosarcoma HT-1080 cells determined by [H-3]uridine i ncorporation were comparable to that determined by the nuclear run-off method. For beta-actin, there was an approximate 2-fold increase in t ranscription rate within two hours of stimulation that declined to bas al levels by 20 h. The c-myc gene response followed a similar kinetics as for the beta-actin gene except that maximal enhancement was greate r at 6-9-fold. The relative transcriptional activities of the beta-act in gene to that of the c-myc gene were virtually identical using the t wo assay methods. Comparable transcription results using both methods were also observed when beta-actin and c-myc gene transcription were m easured in log-phase HL-60 leukemia cells.