Identification of two promoter regions in the rat B-50/GAP-43 gene

To determine cis-acting elements controlling the rat B-50/GAP-43 gene expression, the genomic DNA encoding exon 1 and the 5′ flanking sequence was isolated. Sequence analysis of 1 kb 5′ untranslated region (UTR) revealed the presence of a (GA)-repeat and a (GT)-repeat. The size of the (GA)-repeat varied due to both an instability of phage lambda λ DNA in E. coli and genomic variation between rats. Transcription initiation sites were mapped in 8-day-old rat brain poly(A)+ mRNA. Primer extension indicated multiple transcription start sites at −159 and −339/ −342 nt upstream of the translation start site; reverse transcriptase coupled PCR showed that the most 5′ transcription start site is located between −465 and −440. Northern blotting demonstrated that ∼90% of the B-50 mRNAs initiates at approximately −50. Promoter analysis by transient transfection assays in undifferentiated and retinoic acid-differentiated P19-EC cells revealed that the rat B-50 gene contains two promoters. P1 (located between −750 and −407) contains commonly observed promoter elements such as a TATA box and CCAAT boxes. P2 (located between −233 and −1) neither contains TATA boxes, CCAAT boxes nor consensus sequences of house-keeping gene promoters like GC-boxes. The activity of P1 is inhibited at neuroectodermal differentiation of P19-EC cells whereas the activity of P2 is stimulated. In 8 day old rat brain the majority of the B-50 mRNA transcripts are derived from P2. It is concluded that at this developmental stage P2 is the most important promoter.