Characterization of distinct mRNAs coding for putative isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Three distinct clones encoding full-length 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) were characterized from a rat liver cDNA library. Clone 22c was 1859 bp long and coded for the 470 amino acids of the bifunctional subunit of the liver homodimer. This polypeptide is phosphorylated on serine 32 by cyclic-AMP-dependent protein kinase. Clone 4c (2681 bp) had a coding region identical to that of clone 22c but it included a putative intron of 959 bp. In clone 5c (1750 bp), the sequence upstream from amino acid 33 differed from that in clone 22c and coded for a unique N-terminal portion of 10 amino acids. Poly(A)-rich RNA from rat tissues was hybridized with cDNA probes corresponding to the unique N-terminal portions of clones 22c and 5c. Dot and Northern blots showed signals indicative of three distinct PFK-2/FBPase-2 mRNAs. There were a 6.8-kb mRNA typical of cardiac tissue, a 2.1-kb mRNA typical of liver, corresponding to clone 22c, and a 1.9-kb mRNA typical of skeletal muscle, corresponding to clone 5c. Primer extension analysis showed that clones 22c and 5c were nearly complete since their respective 5'-untranslated sequences were at most 96/97 bp and 44 bp shorter than the corresponding mRNAs. These data provide a molecular basis for the existence of PFK-2/FBPase-2 isozymes.
Crepin
K M
KM
Hormone and Metabolic Research Unit, Louvain University Medical School, Brussels, Belgium.
Darville
M I
MI
Hue
L
L
Rousseau
G G
GG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D005091:Exons; D007438:Introns; D007527:Isoenzymes / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D014018:Tissue Distribution
D015342:DNA Probes; D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases