Genomic organization, 5'flanking region and tissue-specific expression of mouse phosphofructokinase C gene

Abstract

Using a combination of mouse bacterial artificial chromosome (BAC) genomic library screening, long-range polymerase chain reaction (PCR) amplification, genomic walking and DNA sequencing, we have characterized the intron/exon boundaries, the sizes of each intron and 5' flanking region of the mouse PFK-C gene. The gene spans approximately 55 kb and comprises 22 exons separated by 21 introns. All intron/exon splice junctions conform to the GT/AG rule. The mouse PFK-C gene organization is similar to that of the human and rabbit PFK-A and human and mouse PFK-B genes. However, PFK-C has much larger intronic sequences throughout the gene. Anchored PCR was performed to amplify about 1.0 kb of genomic DNA upstream of the translational start site. Sequence analysis of the PFK-C 5' flanking region revealed that it is devoid of TATA and CAAT boxes at the usual positions, but it contained several putative binding sites for transcription factors AP1, GATA1, NKX2.5 and STAT. The 5' flanking region was not enriched in GC dinucleotides and lacked CpG islands and putative binding sites for SP1. Four transcription initiation sites have been identified by full-length RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) between -61 and -32 bp from the translation initiation codon. Reverse transcription-PCR analysis revealed that PFK-A, PFK-B and PFK-C genes were expressed, in all mouse tissues tested, at varying levels. PFK-A mRNA was more abundantly expressed in all tissues than were the PFK-B and PFK-C genes. Based on the mouse PFK-C signal normalized to 18S rRNA, the PFK-C mRNA was expressed at the highest levels in the brain, heart, thymus and testicles, whereas low levels were observed in the kidney, liver, muscle, and lung.