Takenaka M, Noguchi T, Inoue H, Yamada K, Matsuda T, Tanaka T
Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Japan.
J Biol Chem. 1989 Feb 5;264(4):2363-7.
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.
分离出了包含大鼠丙酮酸激酶M基因的基因组克隆，该基因编码M1型和M2型同工酶，并确定了它们的外显子序列。该基因含有12个外显子和11个内含子，长度为20千碱基（kb）。M1型和M2型特异的序列分别存在于外显子9和10中（野口，T.，井上，H.，和田中，T.（1986年）《生物化学杂志》261，13807 - 13812）。第七个内含子以GC二核苷酸而非一致的GT二核苷酸开始，但其他外显子 - 内含子边界符合“GT - AG”规则。S1图谱分析表明，M1型和M2型mRNA有多个但相同的转录起始位点。因此，得出结论，M1型和M2型同工酶mRNA是通过可变RNA剪接从相同的M基因转录本产生的。RNA印迹杂交分析表明，脑和骨骼肌中同工酶的发育变化在RNA剪接水平受到调控。该基因的5'侧翼区域没有“TATA盒”或“CAAT盒”，但含有潜在的Sp1结合位点。细菌氯霉素乙酰转移酶测定表明，该基因5'侧翼区域约0.5 kb的片段足以在大鼠肝癌细胞系dRLh - 84中发挥启动子活性。这种活性在成年大鼠肝细胞中不存在，表明该0.5 kb片段具有组织特异性启动子活性。还对一个类似于M2型丙酮酸激酶cDNA的加工型假基因进行了表征。
