530 Aid et al. Fig. 3. Identiﬁcation of the transcription start sites for BDNF new exon III, V, VII, VIII, and IXA mRNAs in rat. 50 Rapid ampliﬁcation of cDNA ends (50 RACE) was performed to determine the transcrip- tion start sites for novel BDNF transcripts. Major transcription initiation sites (short arrows) are located at 152 bp and 230 bp for exon III (arrows marked A and B), at 81 bp for exon V and at 277 bp and 286 bp for exon VIII (arrows marked A and B) upstream of the 30 end of the re- spective exon and at 476 bp and 363 bp upstream of the major splice acceptor site of the coding exon (asterisk) for exon IXA. For exon VII, the 50end of the longest EST is shown as a putative transcription initia- tion site, because 50 RACE did not result in any speciﬁc products as a result of very low levels of exon VII transcripts. Exon sequences are in boldface; intron sequences are in lowercase letters. The locations of pri- mers that were used in 50 RACE are indicated with long arrows. (Fig. 2B). In rodent nonneural tissues, relatively high levels of exon IXA transcripts were observed in heart and lung. Identiﬁcation of the Transcription Start Sites for BDNF New Exons III, V, VII, and VIII in Rat The transcription initiation sites for rat BDNF exons I, II, IV, and VI have been determined earlier (Timmusk et al., 1993). To identify the transcription start sites for novel BDNF transcripts, 50 rapid ampliﬁcation of cDNA ends (50 RACE) from rat hippocampal RNA was per- formed by using antisense primers speciﬁc for exons III, V, VII, VIII, and IXA. Sequencing analysis of different RACE clones showed that major transcription initiation sites are located at 152 bp and 230 bp for exon III, at 81 bp for exon V, at 277 bp and 286 bp for exon VIII upstream of the 30 end of the respective exon, and at 476 bp and 363 bp for exon IXA upstream of the major splice site of this exon (Fig. 3). None of the identiﬁed 50 exons con- tains upstream open reading frames, so the usage of these 50UTRs will apparently not affect amino acid composition Journal of Neuroscience Research DOI 10.1002/jnr