Figure 6

Replacement of the intron V with a single nucleotide. (a) βc-G was constructed by replacing the intron V-derived 79 nucleotides with a single nucleotide G in βc79. The frameshift generating eighteen PTCs were preserved in βc-G. βc258 was a βc with a single PTC at the 259 th codon. Both βc79 and βc-G had 2238 nucleotides downstream of their termination codon, and βc258 had 2183 nucleotides. (b) βc-G and βc258 were cloned into a pIREShyg2 vector and stably expressed in Ba/F3 cells. The cells expressing βc-G, βc258, βc79 (mE) and wild-type βc (5C) were cultured with or without 100 μg/mL puromycin for 4.5 hours. The amounts of βc-specific transcripts relative to GAPDH transcripts in puromycin-treated cells were divided by the amounts in untreated cells, and are presented as-fold increases of βc/GAPDH. Average values and S.E.M. (error bars) were obtained from three independent experiments. * significantly higher than 5C (p < 0.05). (c) EpoR215 was an EpoR with a termination codon (TAG) at the 215 th codon generating 1099 nucleotides downstram of the termination codon. In wild-type EpoR, there were 190 nucleotides downstream of its normal termination codon. (d) Wild-type EpoR and EpoR215 were stably expressed in Ba/F3 cells using a pIREShyg2 vector, and cultured with or without 100 μg/ml puromycin for 4.5 hours. The ratios of EpoR-specific transcripts to GAPDH transcripts in puromycin-treated cells were divided by those in untreated cells. Average values and S. E. M. (error bars) were obtained from three different experiments. * significantly higher than the value in wild-type EpoR (P < 0.05).