Rat pyruvate kinase M gene. Its complete structure and characterization of the 5'-flanking region

Abstract

Genomic clones containing the rat pyruvate kinase M gene, which encodes the M1- and M2-type isozymes, were isolated and their exon sequences were determined. This gene contains 12 exons and 11 introns and is 20 kilobases (kb) long. The sequences specific to the M1- and M2-types exist in exons 9 and 10, respectively (Noguchi, T., Inoue, H., and Tanaka, T. (1986) J. Biol. Chem. 261, 13807-13812). The seventh intron begins with the GC dinucleotide instead of the consensus GT dinucleotide, but other exon-intron boundaries are consistent with the "GT-AG" rule. S1 mapping analysis showed that M1- and M2-type mRNAs had multiple, but the same transcription initiation sites. Thus, the M1- and M2-type isozyme mRNAs are concluded to be produced from the same M gene transcript by alternative RNA splicing. RNA blot hybridization analysis indicated that developmental changes of the isozymes in brain and skeletal muscle were regulated at the level of RNA splicing. The 5'-flanking region of the gene has no "TATA box" or "CAAT box," but contains potential Sp1 binding sites. Bacterial chloramphenicol acetyltransferase assay revealed that a fragment of about 0.5 kb of the 5'-flanking region of the gene was sufficient for promotor activity in the rat hepatoma cell line, dRLh-84. This activity was not present in adult rat hepatocytes, indicating that the 0.5-kb fragment has tissue-specific promoter activity. A processed-type pseudogene that resembles the M2-type pyruvate kinase cDNA was also characterized.