Activity of the rat liver-specific aldolase B promoter is restrained by HNF3.

Although it contains binding sites for HNF1, NFY and C/EBP/DBP, the proximal promoter of the aldolase B gene is surprisingly weak when tested by transient transfection in differentiated hepatoma cells. This low activity could be due to overlapping between HNF1 and HNF3 binding sites in element PAB, from -127 to -103 bp with respect to the cap site. Replacement of the PAB region by a consensus HNF1 binding site unable to bind HNF3, results in a 30 fold activation of the promoter, in accordance with the hypothesis that activity of the wild-type promoter is normally restrained by HNF3 binding to PAB competitively with HNF1. Consistently, transactivation of the wild-type promoter by excess HNF1 is very high, most likely due to the displacement of HNF3, while the construct with the exclusive HNF1 binding site is weakly transactivated by HNF1. The inhibitory effect of HNF3 on HNF1-dependent transactivation is clearly due to competition between these two factors for binding to mutually exclusive, overlapping sites; indeed, when HNF1 and HNF3 sites are contiguous and not overlapping, the resulting promoter is as active as the one containing an exclusive HNF1 binding site. A construct in which PAB has been replaced by an exclusive HNF3 binding site is weakly expressed and is insensitive to HNF3 hyperexpression. DBP-dependent transactivation, finally, is independent of the nature of the element present in the PAB region.     

Expression of three mRNA species from a single rat aldolase A gene, differing in their 5' non-coding regions

The complete nucleotide sequence of the rat aldolase A isozyme gene, including the 5' and 3' flanking sequences, was determined. The gene comprises ten exons, spans 4827 base-pairs and occurs in a single copy per haploid rat genome. The genomic DNA sequence was compared with those of three species of rat aldolase A mRNA (mRNAs I, II and III) that have been found to differ from each other only in the 5' non-coding region and to be expressed tissue-specifically. It revealed that the first exon (exon M1) encodes the 5' non-coding sequence of mRNA I, while the second exon (exon AH1) encodes those of mRNAs II and III and the following eight exons (exons 2 to 9) are shared commonly by all the mRNA species. These results allowed us to conclude that mRNA I and mRNAs II, III were generated from a single aldolase A gene by alternative usage of exon M1 or exon AH1 in addition to exons 2 to 9. S1 nuclease mapping of the 5' ends of their precursor RNAs suggested that these three mRNA species were transcribed from three different initiation sites on the single gene.     

A comparison of the different DNA binding specificities of the bZip proteins C/EBP and GCN4.

The bZip proteins GCN4 and C/EBP differ in their DNA binding specificities: GCN4 binds well to the pseudopalindromic AP1 site 5'-A4T3G2A1C0T1C2'A3'T4'-3' and to the palindromic ATF/CREB sequence 5'-A4T3G2A1-C0*G0'T1'C2'A3'T4'-3'; C/EBP preferentially recognizes the palindromic sequence 5'-A4T3T2G1C0*G0'C1'A2'-A3'T4'-3'. According to the X-ray structures of GCN4-DNA complexes, five residues of the basic region of GCN4 are involved in specific base contacts: asparagine -18, alanine -15, alanine -14, serine -11 and arginine -10 (numbered relative to the start point of the leucine zipper, which we define as +1). In the basic region of C/EBP position -14 is occupied by valine instead of alanine, the other four residues being identical. Here we analyse the role of valine -14 in C/EBP-DNA complex formation. Starting from a C/EBP-GCN4 chimeric bZip peptide which displays C/EBP specificity, we systematically mutated position -14 of its basic region and characterized the DNA binding specificities of the 20 possible different peptides by gel mobility shift assays with various target sites. We present evidence that valine -14 of C/EBP interacts more strongly with thymine 2 than with cytosine 1' of the C/EBP binding site, unlike the corresponding alanine -14 of GCN4, which exclusively contacts thymine 1' of the GCN4 binding sites.     

Isolation and nucleotide sequence of a full-length cDNA coding for aldolase B from human liver.

Two recombinant clones, pA2 and pA3, containing cDNA sequences for human aldolase B have been isolated from a full length human liver cDNA library. The larger one, pA3, has been subcloned in M13 phage and completely sequenced with the chain terminator method. The sequence covers 1,600 nucleotides including the whole coding region (1,050 nucleotides), 67 nucleotides from the 5' non-coding region and the whole 3' non-coding region, 440 nucleotides long, down to the poly-A tail. Comparison with rabbit aldolase A and with a partial sequence of rat aldolase B, shows a homology of about 76% for aldolase A and of about 94% for aldolase B, which indicates that the sequenced cDNA codes for the liver isoenzyme. This is the first complete sequence reported for human aldolase B. The pA3 clone strongly hybridizes to 18S mRNA from human adult liver as expected from the size of the isolated cDNA.