Analysis of the 5'-flanking regions of adult type .BETA.-globin genes in rats.
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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 β-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:<BR>1. Transcription initiation sites of the rat IIβ-, IIIβ-, and 0 β-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.<BR>2. The nucleotide sequences of 5′-flanking regions of each gene (IIβ: -3819/+51, IIIβ: -716/+51, 0β: -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 IIβ-globin gene.<BR>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 IIβ-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.<BR>4. Dot matrix analysis between the 5′-flanking nucleotide sequence of the rat II/β-globin gene and those of adult type β-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 β-major globin gene, which has been reported to contain a possible repressor element.
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