Fig. 3. Detection of the actin gene and its nascent transcripts in situ. (A) Ten different probes, each conjugated to six CY3 fluorochromes, were targeted to intron sequences of the template strand of the beta-actin gene (12). The probes do not detect nascent beta-actin RNA. Most cells in the population show two sites of signal, which represent the two beta-actin alleles. (B) Different unique probes, each labeled with five FITC fluorochromes, were targeted to the 5'-UTR (one probe), splice junctions (five probes), and 3'-UTR (five probes) for beta-actin RNA. Represented here are two sites of intense signal in the nucleus, indicating sites of active transcription in response to serum stimulation. (C) Both signals, detecting the DNA and nascent RNA, respectively, colocalize upon superimposition. (D) A frequency histogram shows an abrupt truncation at the TFI for the 10 fluorescent probes hybridized to DNA, indicating the detection of a single copy of the beta-actin gene. The histogram for nascent RNA shows a broad profile of hybridization, indicating multiple copies at each gene site (truncated at 50). Bar = 1 µm.