Analysis of the 5'-flanking regions of adult type beta-globin genes in rats

Abstract

In order to elucidate the molecular mechanism of the tissue- and stage-specific expression of globin genes, the possible regulatory role of the 5'-flanking region of the rat beta-globin genes was investigated by determining the nucleotide sequence and measuring the CAT transfection activity of a series of deletion mutants. Results obtained in the present study are summarized as follows: 1. Transcription initiation sites of the rat II beta-, III beta, and O beta-globin genes were determined to be 52 base pairs (bp) 5'-upstream of the translation initiation codon (ATG), in each gene by primer extension analysis. 2. The nucleotide sequences of 5'-flanking regions of each gene (II beta: -3819/+51, III beta: -716/+51, 0 beta: -727/+51) were determined. Dot matrix search of the individual sequences revealed the occurrence of about 300 bp of alternating purine and pyrimidine repeats at a region extending from the nucleotide positions -757 to -1049 5' to the transcription initiation site of the II beta-globin gene. 3. CAT transfection assay using constructs with deletions in 5'-flanking regions indicated that a negative regulatory effect occurs in the region between -757 and -1049 of the II beta-globin gene, corresponding to that containing the purine/pyrimidine repeats mentioned above. These results strongly suggest that the presently identified purine/pyrimidine stretch may act as a repressor. 4. Dot matrix analysis between the 5'-flanking nucleotide sequence of the rat II beta-globin gene and those of adult type beta-globin genes of other species showed that the repeat sequence existing in the 5'-flanking region of the rat gene is very homologous to that found in a similar region of the mouse beta-major globin gene, which has been reported to contain a possible repressor element.