Fig. 4. Sequence conservation and heterogeneous transcription initiation of the Brn1 gene. (A) Conservation of the 5' flanking sequences of mammalian and fish Brn1 genes. The PipMaker program (Schwartz et al., 2000) was used to compare the 10 kb upstream regions of the human and mouse Brn1 genes. Sequence block with 50% to 100% identical sequence (y-axis) are indicated together with their position (x-axis) relative to the translation start codon of Brn1. The conserved elements A,B,C,D and P share 93.2% (D) to 96.5% (B) sequence identity in the two mammalian species (Fig. 6D; see Fig. S1 in the supplementary material). Four of these regions (B, C, D and P) are even conserved between mammals and the pufferfish Fugu rubripes (shown in green). CpG islands with an average GC content of 60% are present in the first 3.8 kb upstream of the Brn1 start codon. (B) Mapping of the transcription initiation region. 5'-RACE amplified PCR fragments of 325 bp from E10.5 head RNA with a primer located downstream of element P (–1098/–1069 relative to start codon). The presence or absence of reverse transcriptase (RT) and the PCR cycles are indicated. (C) Identification of transcription start sites. Sequencing of the 5'-RACE products identified heterogeneous transcriptional start sites within a 17 bp sequence of element P. The number of PCR clones with an identical 5' end is indicated together with the nucleotide positions relative to the start codon.