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Abstract
We have isolated and characterized a mouse gene encoding liver (B-type) phosphofructokinase, a key regulatory enzyme in glycolysis. The gene spans approximately 21.5 kilobase pairs and consists of 22 exons. Compared with the muscle (A-type) phosphofructokinase gene, the sizes of the introns are different although exon lengths are highly conserved. Two transcription start sites 10 bases apart were determined by primer extension experiments. The immediate 5' sequence does not possess a TATA or CCAAT box but contains multiple GC boxes (positions -10, -43, -50, -62, and +28 in the 5'-untranslated region) which may be Sp1-binding sites. An unusual feature of 200 base stretches of CT repeats is present at position -480 to -693. In addition, direct repeats of CTCGAAGGAG are found at positions -447 and -478. DNase I footprinting showed five regions where liver nuclear proteins may interact. Two proximal 5'-flanking regions spanning -1 to -20 and -30 to -70, which contain GC boxes. Also protected was a region spanning -70 to -90, which contains an AP-1 like sequence (TCAGTCA). The consensus AP-1 sequence, however, did not inhibit footprinting, indicating involvement of a distinct protein. Two distal regions spanning from -450 to -470 and from -500 to -520 were also protected. The former is positioned between the direct repeats and the latter is at the start of the CT repeats. The rate of transcription of the liver phosphofructokinase gene, as measured by run-on assays, increased 5-fold in livers of previously starved mice fed a high carbohydrate diet compared to starved controls. Administration of dibutyryl cAMP blocked the increase in transcription caused by refeeding. Functional analysis of the promoter region of the gene will be necessary to elucidate the mechanisms of transcriptional regulation by fasting/refeeding and by cAMP. These results provide a useful system for the study of regulatory elements in liver phosphofructokinase gene transcription.
Highlights
We have isolated and characterized a mouse gene of the enzyme is a tetramer with a subunit M, of 80,000
An unusual feature of 200 base We have previously isolated cDNA sequences to both liver stretches of CT repeats is present at position -480 to and muscle type phosphofructokinase [10, 11].Using these
Direct repeats ofCTCGAAGGAG cDNA clones as probes in Northern blot analysis, we have a r e found at positions -447 and -478
Summary
The resulting doublelength cDNA sequence or restriction fragments of the genomicor stranded oligonucleotideswere added for the preincubation. CDNA clones labeled by the random primer method [13]. Restriction solution was added followed immediately by the addition of 2 p1 of fragments of phage DNAs of these genomic clones were subcloned 0.025 units of DNase I solution. Nucleotide room temperature followed by addition of 15 pl of 100 mM EDTA, sequence analysis was carried out on alkali-denatured double- pH 8.0. Samples were extracted with phenol/chloroform, ethanolstranded or single-stranded templates by the chaintermination precipitated, resuspended in 90%formamidecontaining xylene cyano method using Sequenase (USB Biochemicals) and universal primers and bromphenol blue, and separated on 8 M urea, 6% polyacrylamide or oligonucleotides synthesized by standard phosphoamidite tech- sequencing gels. Niques on a BioSearch synthesizer according to thepreviously determined nucleotide sequence. For intron/exon boundaries, oligonucleotidescorresponding to exon sequences according to our cDNA sequences and known
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