Aldolase from Maize Derived from cDNA

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. Anaerobiosis of maize seedlings results in the selective synthesis of cytoplasmic aldolase and at least 19 other proteins, including two alcohol dehydrogenase isozymes, pyruvate decarboxylase, glucose phosphate isomerase and sucrose synthase (C Bennett, personal communication) (9, 12, 13, 15, 23). This selective synthesis of the anaerobic proteins of maize is the result of the selective translation ofmRNA coding for the anaerobic proteins (24) and the accumulation of anaerobic specific mRNA (9). Among a set of cDNA clones shown to be anaerobic specific, a 160 bp3 cDNA, pZMX71 was found to hybrid s'.lect mRNA encoding a protein of approximately 40,000 mol wt that was selectively precipitated by an antiserum specific for maize cytoplasmic aldolase (9). We have synthesized a maize anaerobic cDNA library and purified a set of aldolase specific cDNAs. We have determined the nucleotide sequence of these cDNAs and a derived amino acid sequence corresponding to a protein of mol wt of 38,61 1. We have compared the sequence ofthis maize aldolase to several vertebrate and invertebrate aldolases and found significant overall homology, 55% with rabbit aldolase A. Specific regions ofthe protein, such as the active site, showed much higher homology. MATERIALS AND METHODS Preparation and Characterization of mRNA. Approximately 2000 seeds (Zea mays Berkeley Fast) were soaked 8 h in distilled ' Supported by grant GM-32344 from the National Institutes of Health. 2 Present address: School of Biological Sciences, University of Nebraska-Lincoln, 348 Manter Hall, Lincoln, NE 68588-0118; phone 402472-1683. 'Abbreviations: bp, base pair; Adhi, alcohol dehydrogenase gene 1; b, base; ds, double stranded; ss, single stranded. H20 at 23C, then germinated on moist paper towels at 27C for 4 d. Seedlings were collected and submerged 20 h in 15 L of distilled H20. Primary roots were cut off and frozen in liquid N2. Frozen roots were used immediately or stored at -80°C. mRNA was isolated by the method ofChirgwin et aL (4) with the following modifications. Thirty g of frozen roots (from 2000 seedlings) were pulverized to a fine powder with a mortar and pestle and suspended in 150 ml offreshly prepared: 6 M guanidine thiocyanate, 5 mM sodium citrate (pH 7.0), 0.14 M 2-mercaptoethanol, and 0.5% (w/v) sarcosyl. This solution was mixed 5 min at 50% normal speed in a Waring Blendor. The resulting slurry was filtered through four layers of cheese cloth then centrifuged at 6,000 rpm in a Sorvall SS-34 rotor for 5 min at 4C. One g of CsCl was added for each 2.5 ml of the supematant fraction and layered onto a 10 ml cushion of 5.7 M CsCl, 0.1 M EDTA. RNA was pelleted by centrifugation for 12 h at 25,000 rpm in a Beckman SW 27.1 rotor at 20C. The supernatant fraction was discarded and the RNA pellet was dissolved in 500 Ml distilled H20. The RNA was made 0.25 M with sodium acetate and precipitated at -80C with 2.5 volumes of ethanol. Precipitated RNA was solubilized in distilled H20 and was further purified by chromatography on oligo-dT cellulose, as described in Maniatis et al. (18) with the exception that SDS was not used. Construction of the cDNA Library. All steps involving recombinant DNA were done according to NIH directive. A cDNA library was constructed using methods described by Maniatis et al. (18). The dC-tailed cDNA was fractionated over a Sepharose CL-4B column (0.5 x 7 cm) equilibrated in annealing buffer. Fractions of about 250 Ml were taken and annealed with Pst I restricted and dG-tailed pUC8 (28) in equimolar ratios to a final concentration of 1 ng/Ml. Annealed DNA was stored at -20C. Annealed cDNA (2 Mg) was used to transform 0.3 ml of Escherichia coli strain JM83 or JM103 competent cells and plated on LB plates containing 35 gg/ml isopropyl thiogalactoside and 0.0033% (w/v) 5-bromo-4-chloro-3-indolyl galactoside. Plasmid DNA was purified from eight randomly chosen transformants derived from individual Sepharose fractions. The library derived from the leading fraction with an average insert size of 1100 bp was used to isolate aldolase cDNA clones. Isolation of cDNA Clones. The cDNA library was plated on nitrocellulose filters (Schleicher and Schuell, BA85) at a density of about 1000 per plate. Two copies of the original were made. These two copies were baked, the filters were then soaked in 6xSSC (2OxSSC is 3 M NaCl, 0.3 M sodium citrate adjusted to pH 7.0) for 5 min, then washed 2 h at 42°C in 50 mt Tris-Cl (pH 8.0), 1 M NaCl, and 0.1% (w/v) SDS. The washed filters were prehybridized 12 h at 65°C with 50 mm sodium phosphate buffer, 5x Denhardts, 1 M NaCl, 0.2% (w/v) SDS, 1 mm EDTA, 140 gg/ml salmon sperm DNA, 50 ,ug/ml poly(A), 50 ug/ml poly(C), and 6.25 ,g/ml sonicated pUC8. Gel purified insert DNA was nick translated, heat denatured at 100°C for 10 min, then added to the prehybridization mix and incubated with filters 1076 www.plant.org on January 6, 2016 Published by www.plantphysiol.org Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved. SEQUENCE OF MAIZE ANAEROBIC ALDOLASE DNA for 12 h at 65°C. These filters were then washed for 10 min three times in 2xSSC + 0.1% (w/v) SDS at room temperature and 1 h three times in 2xSSC + 0.1% (w/v) SDS at 65°C. The filters were radioautographed, colonies were located on the original filter and rescreened. Plasmid DNA was isolated as previously described (7). The library was also screened using a radiolabeled deoxyribo-oligonucleotide specific for maize aldolase under the same conditions as above except incubations were at 50°C and washes were at 42°C in 0.2xSSC. Sequence Analysis. Purified insert from cDNA clones was digested with Pst I, Taq I, Alu I, Hae III, Kpn I, or Sst I and subcloned into Ml 3mp8 and/or Ml 3mp9. Alternatively, cDNA clones were digested with restriction endonucleases and individual restriction fragments were gel purified before cloning into M13 vectors. pZM 1085 was sequenced by the method of Sanger et al. (25) by subcloning Taq I, Alu I, and Hae III total digests into M 3mp8. pZM 1 154 was sequenced by subcloning a Kpn I/ Pst I digest into Ml 3mpl 8 or Ml 3mp19 and by internal priming using synthetic deoxynucleotides as primers. The sequence for pZM205 was obtained by subcloning restriction fragments obtained from Sst I and Kpn I/Pst I restriction digestions and by internal priming. The location of the primers and their sequence is shown in Figure 2. Certain compressed regions ofthe sequence were resolved by substitution of 7-deaza-dGTP for the dGTP in the reaction mixtures. This reagent precludes certain intrastrand base pair interaction (26). The cDNA insert from pZMX7 1 was subcloned into Pst I cut pUC8, then cut with EcoR I and Hind III and the fragment containing the cDNA insert was sequenced using the method ofMaxam and Gilbert (20). Computer analysis of DNA and protein sequences was accomplished using the programs of H. Martinez (University of California, San Francisco) with the University of California, Berkeley VAX/UNIX system. Amino Acid Composition. Maize cytoplasmic aldolase was purified from a Black Mexican Sweet suspension cell line as described previously (13). Protein was hydrolyzed in 5 N HCI for 1 h at 150C, then derivatized with 3-phenyl-2-thiohydantoin and separated by HPLC (27). The protein was unreactive during Edman degradation, indicating that the amino terminal residue was blocked. N-Terminus of CNBr Peptides. Maize aldolase was reduced and alkylated with 4-vinylpyridine, the carboxyl groups were amidated with dimethylethylenediamine, and the protein was cleaved with cyanogen bromide. The resulting mixture of peptides was subjected to N-terminal sequence analysis by five cycles of Edman degradation (27).