Identification of transcription start sites of CIRP cDNA using a 5′ RNA ligase-mediated rapid amplification of cDNA ends method (RLM-RACE). Total RNA was isolated from NIH-3T3 cells cold shocked at 32°C for (A) 24 h and (B) 6 h and subjected to RLM-RACE analysis. Control cells were cultured at 37°C for 24 h before RLM-RACE analysis (B). The appropriate primers are detailed in Table 1. (C) The sequence of the resulting transcripts, the transcription start sites (as indicated by arrows), the ATG translation start site (underlined), and two EST sequences in public databases are indicated on the cDNA aligned to the genomic DNA sequence. The location of the 5′-UTR intron is also depicted (1891 bp). For further details see the text.
