Figure 2

Identification of the transcription start site of Smtnl1 by rapid amplification of cDNA 5'-ends (5'-RACE) and ribonuclease protection assay (RPA). 5'-RACE reactions were performed on cDNA synthesized from smooth (A) or skeletal (B) muscle mRNA with primers specific for Smtnl1. Representative ethidium bromide-stained agarose gels of PCR products are shown. A 5'-RACE CDS primer (Stratagene) and three Smtnl1 gene specific primers (GSPs) were used with expected product sizes: GSP1 (1479 bp), GSP2 (176 bp) and GSP3 (83 bp). The 5'-RACE product from each reaction was used as template in a subsequent nested PCR reaction. The expected sizes of the nested PCR products were: NGSP1 (63 bp) and NGSP2 (823 bp). The arrowhead in (B) indicates the PCR product visible during the primary amplification when skeletal muscle cDNA was used as template for 5'-RACE with GSP1. The DNA ladder markers are indicated on the left. In (C), ribonuclease protection assay (RPA) analysis was performed on 10 μg of total RNA from smooth (SM) and skeletal (SKM) muscle. A biotin-labeled Smtnl1 riboprobe was generated to span -112 bp to the TSS, joined to exon 1 and exon 2 from the Smtnl1 mRNA, up to + 319 bp. Total RNA from S. cerevisiae (10 μg) was used instead of muscle RNA as a negative control. The band appearing with skeletal muscle mRNA is marked by an arrow. The DNA ladder markers are indicated on the left. In (D), the promoter region, eight exons (E1-E8) and 7 intronic DNA sections are shown for the mouse Smtnl1 gene. The positions of the start codon (ATG), transcriptional start site (TSS) defined by 5'-RACE, the TATA box and initiator sequence (Inr) are also indicated. The nucleotides identified by sequencing the cDNA clones (n = 5) obtained from 5'-RACE are underlined.