Cloning of cDNA for maize superoxide dismutase 2 (SOD2).

Abstract

A cDNA clone encoding maize cytosolic superoxide dismutase 2 (SOD2) was isolated from a cDNA library constructed in pUC9 plasmids from size-selected poly(A)+ RNA. The library was screened with mixed synthetic oligodeoxynucleotide probes. The sequence of the probes was derived from the amino acid sequence from a region of the protein near the NH2 terminus. One positive clone contained an insertion of a 612-base-pair fragment. The amino acid sequence, deduced from the nucleotide sequence of the cDNA, revealed that the clone contained the coding region for all but the first of the 151 amino acids of the SOD2 protein. Additional 5' and 3' flanking sequences, absent on the pUC9 Sod2 clone, were obtained from a Xgtll clone isolated from a maize leaf library probed with the pUC9 Sod2 insert. Hybrid-selection translation assays also demonstrate that the cDNA clone contains Sod2 sequences.The nuclear-encoded metalloprotein superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) of maize consists of four isozymes: two cytosolic isozymes (SOD2, -4), a chloroplast-associated isozyme (SOD1), and a mitochondrial-associated isozyme (SOD3) (1). The genetic analysis, subcellular location, developmental expression, and biochemical properties of these isozymes have been studied in detail (1-4). These SOD enzymes catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide (5). In performing this reaction, these enzymes provide an important defense against oxygen toxicity in the various cellular compartments of maize (6-8).We report in this paper the isolation, DNA sequence, and amino acid sequence deduced from the cytoplasmic Sod2 cDNA clone. MATERIALS AND METHODSNH2-Terminal Sequence Analysis of SOD2 and -4. Maize SOD2 and -4 proteins were purified to homogeneity from 5-day-old scutellar tissue from the inbred maize line W64A as described (9). The sequence of the first 25 amino acids of the NH2 terminus of each protein was kindly provided by M. Hermodson (Purdue University).Preparation of Radioactive Oligonucleotide Probes. Mixed heptadecamers, corresponding to the amino acid sequence of the SOD2 protein, were synthesized by the solid-phase phosphite chemical method developed by Matteucci and Caruthers (10) (see Fig. 1). The chemically synthesized probe was labeled at the 5' end with [_'y32P]ATP (>7000 Ci/mmol; 1 Ci = 37 GBq; ICN) and T4 polynucleotide kinase (P.L.) by the method described (11). This procedure routinely yielded specific activities of =2 x 109 cpm/,g.RNA Isolation and Size Selection. Poly(A)+ RNA was isolated from frozen 5-day-old scutella by the sodium borate method (12). Briefly, the scutella are homogenized in sodium borate buffer, digested with proteinase K, and precipitated in LiCl. Poly(A)+ RNA was obtained from the total RNA by two sequential passages over an oligo(dT)-cellulose column (13). The poly(A)+ RNA was size-fractionated on methylmercury/agarose gels as described (14).Construction of cDNA Clones from Size-Selected mRNA. A cDNA library was ...

Comparison of Maize (Zea mays L.) F1-Hybrid and Parental Inbred Line Primary Root Transcriptomes Suggests Organ-Specific Patterns of Nonadditive Gene Expression and Conserved Expression Trends

“…qRT-PCR analyses of a subset of genes that was nonadditively expressed in at least two hybrids in microarray experiments identified a superoxide dismutase 2 (GenBank AC P11428) that displayed an expression that exceeded the midparent value in all 12 hybrids. Superoxide dismutases are antioxidative enzymes in plants that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide, which are subsequently degraded by the enzyme catalase to oxygen and water and hence protect cells from the damaging effects of reactive oxygen species (Cannon et al 1987;Scandalios 2005). It could be hypothesized that the enhanced defense from reactive oxygen species could be related to the superior performance of hybrids compared to their parental inbred lines.…”

Peroxisomal Copper,Zinc Superoxide Dismutase (Characterization of the Isoenzyme from Watermelon Cotyledons)

“…+ V-4 --1 1 , 19881, pea (Scioli and Zilinskas, 1988), and pine (Karpinski et al, 1992), and cytosolic CuZn-SODs from spinach (Kanematsu and Asada, 1990), cabbage (Steffens et al, 1986), tomato (Perl-Treves et al, 1988), maize (Cannon et al, 1987), and pine (Karpinski et al, 1992 in a concentration-and time-dependent manner (Fig. 5).…”

Cloning and characterization of a cDNA encoding the chloroplastic copper/zinc-superoxide dismutase from pea.

“…Isozymes of Cu/Zn-SOD have been purified from a variety of plant sources. Amino acid sequences of the spinach chloroplastic (8) and cabbage cytosolic (9) isozymes have been published, as well as the cDNA nucleotide and deduced amino acid sequences of the maize cytosolic SOD 2 (10). Pea (Pisum sativum L.) leaves contain two Cu/Zn-SOD isozymes, one chloroplastic and the other cytosolic, which differ in subunit molecular mass, isoelectric point, and amino acid composition (11).…”