Rat aldolase A messenger RNA: the nucleotide sequence and multiple mRNA species with different 5'-terminal regions

The nucleotide sequence of aldolase A mRNA in rat skeletal muscle was determined using recombinant cDNA clones and a cDNA synthesized by primer extension. The sequence is composed of 1343 nucleotides (nt) except for the poly(A) tail. Based on the sequence analysis we have deduced an open reading frame with 363 amino acids (aa) (Mr 39134). The sequence suggests several nt polymorphisms in the mRNA population, one of which causes an aa change. The determined sequence of rat aldolase A mRNA was compared with the published ones of rabbit aldolase A or rat aldolase B mRNAs. The homology between rat and rabbit aldolase A mRNA sequences is greater than that between rat aldolase A and B mRNA sequences. Multiple aldolase A mRNAs having different Mrs were detected in the various tissues, and appeared to be expressed in a tissue-specific manner. Further analysis suggests that differences in mRNA length are due to differences in the 5'-noncoding terminal region.     

Rat aldolase isozyme gene

Rat aldolase B mRNA was partially purified from liver polysomes by an immunochemical technique followed by oligo(dT)-cellulose column chromatography. Double-stranded cDNA, synthesized from this mRNA, was inserted into the PstI site of plasmid pBR322 employing the oligo(dC)-oligo(dG) tailing method. Clones containing aldolase B cDNA inserts were selected by colony hybridization using 32P-labeled purified mRNA as a specific probe. Several recombinant plasmids containing 600 to 1000 base pair inserts were isolated. Hybrid selection-translation experiments showed that they hybridize specifically with aldolase B mRNA. By overlapping restriction maps of several individual cDNA inserts, it was found that they spanned 1200 base pairs, which represented about 70% of the aldolase B mRNA sequence. The nucleotide sequence of the cDNA was then determined and the sequence of 180 amino acids from the COOH terminus and the entire 3' untranslatable nucleotide sequence were clarified. Although the complete amino acid sequence of rat aldolase B has not yet been reported, it was found that several amino acids neighboring the COOH-terminal tyrosine obtained by carboxypeptidase digestion completely coincided with those determined from the cDNA sequence; i.e. -Ser-Leu-Phe-Thr-Ala-Ser-Tyr-Thr-Tyr. Furthermore, a putative active site peptide appeared and is extensively homologous to those of rabbit aldolases A and B.     

Tissue-specific expression of rat aldolase A mRNAs. Three molecular species differing only in the 5'-terminal sequences

Three species of aldolase A mRNA (mRNAs I, II, and III) only differing in the structure of the 5'-terminal noncoding region were detected in rat tissues. The cDNA clones for mRNAs II and III were prepared from ascites hepatoma AH60C and sequenced. The mRNA II is 1393 nucleotides long excluding poly(A) tail, while the mRNA III is 1440 nucleotides long, some 50 nucleotides longer than the mRNA II. The mRNAs II and III differ in the sequence between -25 and the 5' termini from the previously reported skeletal muscle aldolase A mRNA (mRNA I, 1343 nucleotides long). By contrast, the residual 5' noncoding sequence (-24 to -1) and the coding and 3' noncoding sequences are common to all the mRNAs. By dot spot hybridization and S1 mapping the distribution of these mRNAs in the various tissues was determined. The mRNA I appears exclusively in a skeletal muscle and some in heart and hepatoma AH60C, whereas the mRNAs II and III appear more or less in all the tissues examined, implying that their appearances are under tissue-specific control. Furthermore, partial nucleotide sequence analysis of the fetal liver aldolase A mRNA supports that aldolase A mRNA that reappeared in hepatoma is really a resurgence of the gene product expressed in the fetus.     

Nucleotide sequence of a cDNA clone for human aldolase: a messenger RNA in the liver

Nearly complete cDNA clones for human aldolase A mRNA were isolated from human liver cDNA library and the nucleotide sequence determined. Using the cDNA clone as a probe the length of human aldolase A mRNAs, isolated from the skeletal muscle, liver and placenta tissues, was measured by RNA blotting and estimated to be 1,600 nucleotides for skeletal muscle mRNA and 1,700 nucleotides for both the liver and placenta mRNAs, indicating that different species of mRNA coding for human aldolase A were expressed in the different tissues.     

The complete amino Acid sequence for the anaerobically induced aldolase from maize derived from cDNA clones

A cDNA library was synthesized from maize anaerobic root mRNA and screened with cDNA specific to the anaerobically induced Zea mays cytoplasmic aldolase. At least 1% of the cDNA of the library corresponded to maize cytoplasmic aldolase. The sequence of four overlapping cDNA clones encoded a protein of molecular weight 38,611 homologous to aldolase. These cDNAs were polymorphic at three bases and one of these cDNAs had a different, shorter 3′-untranslated region. No known eukaryotic poly(A) addition site was detected. The derived amino acid sequences of maize was compared to the sequence of aldolase of trypanosome, Drosophila, and two mammalian isozymes, A and B. Of these, maize cytoplasmic aldolase was found to have the highest homology (55%) with rabbit aldolase A.     

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.     

Structure and regulated expression of genes encoding fructose biphosphate aldolase in Trypanosoma brucei.

Low stringency hybridisation with a rabbit aldolase cDNA was used to select cDNA clones encoding fructose biphosphate aldolase in Trypanosoma brucei. A clone which is almost full length encodes a protein of 41 027 daltons which has 50% identity with rabbit aldolase A and slightly lower homology with B-type aldolases. The homologous mRNA is at least 6-fold more abundant in bloodstream trypomastigotes than in procyclic forms, as expected from measurements of enzyme activity. Genomic mapping results indicate that trypanosomes have four copies of the aldolase gene arranged as two copies of a tandem repeat. The protein has a short N-terminal extension (relative to other known aldolases) which could be involved in the glycosomal localisation of the enzyme.     

Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications

Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle. Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture. The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively. Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence. Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases. Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases. Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme. Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology. The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed.     

Localization of the active gene of aldolase on chromosome 16, and two aldolase A pseudogenes on chromosomes 3 and 10

Summary Southern blot analysis of human genomic DNA hybridized with a coding region aldolase A cDNA probe (600 bases) revealed four restriction fragments with EcoRI restriction enzyme: 7.8 kb, 13 kb, 17 kb and >30 kb. By human-hamster hybrid analysis (Southern technique) the principal fragments, 7.8 kb, 13 kb, >30 kb, were localized to chromosomes 10, 16 and 3 respectively. The 17-kb fragment was very weak in intensity; it co-segregated with the >30-kb fragment and is probably localized on chromosome 3 with the >30-kb fragment. Analysis of a second aldolase A labelled probe protected against S1 nuclease digestion by RNAs from different hybrid cells, indicated the presence of aldolase A mRNAs in hybrid cells containing only chromosome 16. Under the stringency conditions used, the EcoRI sequences detected by the coding region aldolase A cDNA probe did not correspond to aldolase B or C. The 7.8-kb and >30-kb EcoRI sequences, localized respectively on chromosomes 10 and 3, correspond to aldolase A pseudogenes, the 13-kb EcoRI sequence localized on chromosome 16 corresponds to the aldolase active gene. The fact that the aldolase A gene and pseudogenes are located on three different chromosomes supports the hypothesis that the pseudogenes originated from aldolase A mRNAs, copied into DNA and integrated in unrelated chromosomal loci.     

Initiation of translation at a UAG stop codon in the aldolase gene of Plasmodium falciparum.

The gene coding for the key glycolytic enzyme fructose-1,6-diphosphate aldolase of the human malaria parasite Plasmodium falciparum lacks a functional AUG initiation codon for translation. Protein sequences of natural or in vitro translated aldolase include the candidate start methionine residue at internal positions. No additional AUG start codon is found in genomic DNA, cDNA or mRNA sequences. Instead, a UAG chain termination codon is recognized as the start signal of protein synthesis in vivo and in vitro.     

Expression of aldolase A messenger RNAs in human adult and foetal tissues and in hepatoma

3 specific cDNA clones for human aldolase A were isolated from a human muscle library. One of them was subcloned in M 13 phage, then used as a probe to investigate the patterns and the levels of aldolase A mRNA in various human tissues. Two mRNA species differing in length were observed. The lighter one -1550 bases- was found specific to skeletal muscle; its amount increased during muscle development. The heavier aldolase A mRNA -1650 bases- accounted for foetal and ubiquitous presence of aldolase A isozyme. The resurgence of aldolase A in hepatomas occurred through this latter mRNA species.     

A new human species of aldolase A mRNA from fibroblasts

A full-length cDNA aldolase A clone was isolated from a human fibroblast cDNA library and completely sequenced. Excluding the poly(A) tail, the clone covers 1095 base pairs (bp) of the coding region, plus 199 bp downstream for the termination codon and 146 bp upstream for the initiation codon, within a total of 1440 bp. Primer extension experiments performed with human cultured fibroblast mRNA indicate an elongated product of a further 40 bp. These results evaluated together with those obtained in a concurrent study concerning aldolase A mRNA isolated from human liver are direct evidence of aldolase A mRNA multiplicity in man. The data also suggest the existence in mammals of three different classes of aldolase A mRNA, which would account for tissue specificity and resurgence of foetal expression in tumors.     

Human aldolase isozyme gene: the structure of multispecies aldolase B mRNAs.

A complete nucleotide sequence of human aldolase B mRNA was determined with a recombinant cDNA (pHABL120-3). The cDNA insert was composed of 1,652 bases excluding poly(A) tail and the sequence was consistent with the previous results reported by others. However, S1 nuclease mapping and subsequent genomic analysis allowed us to know that the clone possesses two more sites corresponding to 5'-termini in the 5'-noncoding region and another site of polyadenylation in the 3'-noncoding region. In fact, the major aldolase B mRNA species occupying 90% of the total mRNAs initiated at the predominant position corresponding to the position around -82 of the 5'-noncoding sequence in pHABL120-3 and terminated at the distal polyadenylation site. Second species accounting for 9% of the mRNAs initiated at the same site and terminated at the proximal polyadenylation site. The remainings have a longer 5'-noncoding sequence which starts from further upstream region of the major one and pHABL120-3 corresponds to one of these largest clones.     

Fructose 1,6-bisphosphate aldolase of Drosophila melanogaster: comparative sequence analyses around the substrate-binding lysyl residue

The amino acid sequence of a 103 residue segment encompassing the substrate-binding active site lysyl residue of fructose 1,6-bisphosphate aldolase from Drosophila melanogaster is determined. The sequence is identical to more than 70% with the structure of rabbit muscle aldolase and with the known partial sequences of the sturgeon muscle, trout muscle, and ox liver enzymes. The homology of the insect enzyme with the vertebrate aldolases strongly implies a similar tertiary structure folding.