Alteration of the levels of the M-type 6-phosphofructo-1-kinase mRNA isoforms during neonatal maturation of heart, brain, and muscle

作者：Mhaskar Y; Armour G; Dunaway G^*

来源：Molecular and Cellular Biochemistry, 2000, 214(1): 81-87.

DOI：10.1023/A:1007195017569

• phosphofructokinase
• development
• transcription
• alternative promoters
• mRNA isoforms
• Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

During muscle, heart, and brain neonatal maturation, the capacity to utilize glucose in energy metabolism is directly related to the extent of accumulation of the 6-phosphofructo-1-kinase (PFK) M-type subunit. Neonatal development of other organs, such as liver and kidney, which are not characterized by large increases in the capacity to use glucose do not exhibit large increases in the M-type subunit protein. The presence of the M-type subunit in a PFK isozyme pool fosters a higher affinity utilization of carbohydrate and increased responsiveness to the levels of regulatory metabolites. To better appreciate this phenomenon, which is vital for normal development, the different isoforms of the M-type subunit mRNA's and alteration of their levels during maturation have been examined. Further, the potential promoter regions, i.e., the regions upstream from the sites of initiation of transcription, which are involved in expression of the different M-type subunit mRNA isoforms have been isolated, sequenced, and examined for possible transcription factor interaction sites. Using cDNA libraries produced from adult rat brain or skeletal muscle RNA, two primary forms of rat M-type subunit cDNA's were detected. Although the translated regions of these mRNA's were essentially identical, the 5'-untranslated region (5'-UTR) exhibited different lengths (90 or 59 bp) and sequences. Each M-type subunit cDNA had 10 common nucleotides immediately upstream from the initiator ATG, and the remaining 5'-UTR's had insignificant identity. A genomic fragment which interacted with probes complimentary to the sequences of the 5'-UTR of each M-type subunit mRNA isoform was isolated and sequenced by primer walking. It was discovered that the 5'-UTR of one of the mRNA's (proximal mRNA) was located immediately upstream from exon I and was apparently transcribed without splicing. Subsequently, the initial bp in the sequence of the other mRNA isoform (distal mRNA) was located 4010 bp upstream from the ATG in exon 1. Employing Reverse Transcription-Polymerase Chain Reaction using total RNA and scanning densitometry, the relative levels of the proximal and distal mRNA's during neonatal maturation of brain, heart, and muscle were measured. In these tissues, both forms of M-type subunit mRNA's were present, and during maturation tissue-specific differences were noted.

• 出版日期2000-11