Figure 7.

Pre-mRNAs expressed from endogenous genes are subject to SOS. (a) Cultured Syrian hamster 165-28 cells were treated with antibiotics as in Figure 6, and RT–PCR analysis of endogenous CAD RNAs was performed using the b/c primer pair. Latent splicing was elicited only by treatment with pactamycin. For comparison we show the expression of latent CAD mRNA in 293T cells transfected with the wild-type CAD1 construct and treated with pactamycin (lane 1). Cloning and sequencing of the PCR products of primer pair b/c revealed that the lower band in the 296 bp region represents the CAD pre-mRNA, while the upper band represents a non-relevant endogenous gene. (b) Human 293T cells were treated with pactamycin and radioactive RT–PCR analysis of the endogenous IDUA RNAs was carried out using IDUA-specific primer pairs (see Primers in Supplementary material) using the 5′ ³²P-lebeled antisense primer. The IDUA PCR products were revealed by electrophoresis in a denaturing gel and autoradiography.