526 Aid et al. 1999; West et al., 2001). BDNF gene expression is con- trolled by multiple activity-dependent and tissue-speciﬁc promoters. Four BDNF promoters have been previously identiﬁed in rat (Metsis et al., 1993; Timmusk et al., 1993; Timmusk et al., 1995), each driving the transcrip- tion of BDNF mRNAs containing one of the four 50 noncoding exons (I, II, III, or IV) spliced to the common 30 coding exon. Several transcription factors contributing to the regulation of BDNF promoters have been charac- terized. Among these factors are cAMP-responsive ele- ment binding protein (CREB; Shieh et al., 1998; Tao et al., 1998; Tabuchi et al., 2002) and upstream stimula- tory factors 1/2 (USF1/2; Tabuchi et al., 2002; Chen et al., 2003b), which regulate BDNF promoters I and III. In addition, calcium-responsive transcription factor (CaRF) has been found to mediate BDNF transcription through binding to BDNF promoter III upon neuronal activation (Tao et al., 2002). Chromatin remodeling by DNA methyl- ation and histone deacetylation also plays an important role in cell-speciﬁc and activity-dependent regulation of BDNF gene by recruiting global repressors such as REST/NRSF to promoter II (Palm et al., 1998; Timmusk et al., 1999; Zuccato et al., 2003) and MeCP2 to promoter III (Chen et al., 2003a; Martinowich et al., 2003). Unraveling the regulation of BDNF gene expression is important for understanding its contribution to nervous system function and pathology. Provided that BDNF actions are most frequently modeled in rodents, detailed knowledge of the structural organization of rodent BDNF genes would be imperative. We undertook this study to specify the structure and expression of BDNF gene in mouse and rat. We show that rodent BDNF gene structure and expression are more complex than initially characterized (Timmusk et al., 1993) and that novel, as yet unidentiﬁed regulatory sequences may contribute to cell-speciﬁc and ac- tivity-dependent regulation of rodent BDNF expression. MATERIALS AND METHODS DNA and Amino Acid Sequence Analysis Mouse and rat BDNF gene structure in silico analysis was performed using genomic, mRNA and EST databases (http:// www.ncbi.nlm.nih.gov and http://genome. ucsc.edu). Align- ment tools available at http://www.ncbi. nlm.nih.gov as well as software provided by the BIIT group at the University of Tartu, Estonia, were used for homology searches and analysis. AntiHunter software (available at http://bio.ifom-ﬁrc.it/ANTIHUNTER/) was used to search for opposite-strand transcripts in mouse and rat BDNF genomic region. RNA Isolation, cDNA Synthesis, RT-PCR Total RNA from developing and adult mouse and rat total brain and brain regions and nonneural tissues was puriﬁed by RNAwiz (Ambion, Austin, TX) as recommended by the manu- facturer. DNase treatment of total RNA was perfomed by using a Turbo DNA-Free Kit (Ambion) according to the manufacturer’s instructions. Five micrograms of total RNA from different tissues was used for ﬁrst-strand synthesis using oligo(dT) and SuperScript III First-Strand synthesis system (Invitrogen, Carlsbad, CA). To analyze expression of BDNF transcripts, reverse primer speciﬁc for 30 BDNF coding exon and forward primers speciﬁc for 50 noncoding exons were used. To identify homologues of human antisense BDNF exons in mouse and rat, primers were designed corresponding to mouse and rat BDNF genomic regions that showed signiﬁcant homology with human exons. Total RNA was normalized to the expression of ubiquitously expressed HPRT gene. All primers used in the study are listed below, where m designates mouse, r, rat; h, human; for, forward; rev, reverse; and Arabic numbers BDNF exons as follows: mrBDNFI, GTGTGACCTGAGCAGTGGGCAAAGGA; mrBDNFII, GGAAGTGGAAGAAACCGTCTAGAGCA; mBDNFIII, GCTTTCTATCATCCCTCCCCGAGAGT; rBDNFIII, CCTTTCTATTTTCCCTCCCCGAGAGT; mrBDNFIV, CTCTGCCTAGATCAAATGGAGCTTC; mrBDNFV, CTCTGTGTAGTTTCATTGTGTGTTC; mBDNFVI, G C T G G C T G T C G C A C G G T T C C C A T T ; r B D N F V I , GCTGGCTGTCGCACGGTCCCCATT; mrBDNFVII, CCTGAAAGGGTCTGCGGAACTCCA; mrBDNFVIII, GTGTGTGTCTCTGCGCCTCAGTGGA; mBDNFIXA, CCCAAAGCTGCTAAAGCGGGAGGAAG; rBDNFIXA, CCAGAGCTGCTAAAGTGGGAGGAAG; hmrHPRT for, GATGATGAACCAGGTTATGAC; hmrHPRTrev, GTC- CTTTTCACCAGCAAGCTTG; and mrBDNFrev, GAA- GTGTACAAGTCCGCGTCCTTA. To analyze expression of mouse and rat exons I–IV-, exon VI-, and exon IXA-speciﬁc transcripts, cDNA was ampli- ﬁed in a total volume of 25 ml with 35 cycles of PCR using HotFire polymerase system (Solis BioDyne, Estonia). An anneal- ing temperature of 608C was used for all primer combinations. Because of relatively low expression levels of BNDF mRNAs containing exons V, VII, and VIII, a more robust HotStartTaq Master Mix kit (Qiagen, Chatsworth, CA) was used for cDNA ampliﬁcation for 40–45 PCR cycles. All RT-PCR reactions were performed in triplicate. PCR products were resolved in 1.2% agarose gel and visualized by staining with ethidium bro- mide. PCR fragments were subsequently excised from the gel, cloned by using pCRII-TOPO cloning system (Invitrogen), and subjected to sequence analysis. 50 RACE Analyses of Transcription Initiation Sites To determine the transcription start sites of novel BDNF transcripts, 50 rapid ampliﬁcation of cDNA ends (RACE) was per- formed by using the GeneRacer kit (Invitrogen) according to the manufacturer’s instructions. PCR ampliﬁcation was performed with a HotStartTaq Master Mix kit (Qiagen) and GeneRacer 50 for- ward primer and reverse primers speciﬁc for exons III, V, VII, VIII, and IXA. Then, nested PCR was performed to increase the spec- iﬁcity and sensitivity of RACE by using GeneRacer 50 nested primer and nested primers speciﬁc for exons III, V, VII, VIII, and IXA. RACE products were analyzed in a 2% gel and cloned into the pCRII-Topo vector (Invitrogen) for sequence analysis. Primers used for RACE analysis are listed below: rBDNFIIIR- ACE, TCAATGAAGCATCCAGCCCGGCA; rBDNFIIINested, CGGAACTCTCGGGGAGGGAAAATA; rBDNFVRACE, GAACACACAATGAAACTACACAGAG; rBDNFVIIRACE, CTAAAGAGGTGCGCTGGATGGACAGAG; rBDNFVII- Nested, GGACCTGGAGTTCCGCAGACCCTTT; rBDNF- Journal of Neuroscience Research DOI 10.1002/jnr