We have cloned previously a 22-kb rat gene which codes for heart 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase from an ATG located in exon 2. To characterize the promoter of the gene, we have now cloned and sequenced 1.9 kb of its 5’ region and show here that it has an unusual structural and functional organization. By SI nuclease mapping and primer extension we found that this region contains the first, noncoding, exon of a mRNA that we call R3. The sequence upstream from this exon behaved as a promoter in transient transfection assays. These assays also suggested that the gene possesses more than one promoter. Indeed, by reverse transcription-polymerase chain reaction techniques we identified three additional mRNAs that differ by their 5’ noncoding exons upstream from the common, coding, exon 2. mRNA R1 contains two 5’ noncoding exons located upstream from the first exon of mRNA R3. mRNA R2 contains one 5’ noncoding exon located upstream from, and partially overlapping with, the first exon of mRNA R3. mRNA R4 contains one 5’ noncoding exon located downstream from the first exon of mRNA R3 but overlapping partially with it. The distribution of these mRNAs in rat tissues was evaluated by reverse transcription-polymerase chain reaction. We conclude that the gene contains four more exons than the 16 previously described and at least three promoters, two of which correspond to exonic sequences. The gene gives rise to at least four mRNAs which are expressed not only in heart but also in most tissues.
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