In vitro transcription of normal, mutant, and truncated mouse alpha-globin genes

We have transcribed various alpha-globin gene-containing templates by using a simplified in vitro system and have shown that the 5' end of each of the RNA polymerase II-specific transcripts corresponds to that of authentic alpha-globin mRNA. Using a truncated alpha-globin gene fragment, we then localized the promotor region of the adult alpha-globin gene to a 138-base-pair fragment of alpha-globin DNA, 141 nucleotides of which precede the initiation site. In contrast, when two naturally occurring alpha-globin gene mutants were tested as templates, no RNA polymerase II-dependent initiation was detected. Comparison of the nucleotide sequences of the normal and mutant alpha-globin genes has allowed us to focus upon nucleotide positions that might be essential for promoter activity.