Cloning and DNA sequencing of cytosolic Cu/Zn superoxide dismutase gene from chinese cabbage

A cDNA clone for the cytosolic Cu/Zn superoxide dismutase (Cu/Zn SOD) from Chinese cabbage (Brassica campestris ssp.pekinensis) was isolated and its DNA sequence was determined. The cDNA clone contains a complete coding sequence which encodes a protein of 152 amino acids and a 3-untranslated region including a poly A signal. The deduced amino acid sequence shows that it is highly homologous to the Cu/Zn SODs from other plants (60–90%). The lack of a putative chloroplast targeting transit peptide indicates that the clone represents a cytosolic form of Cu/Zn SOD. Genomic Southern hybridization suggests that cytosolic Cu/Zn SOD genes are present in 1 or 2 copies per genome.     

Schistosoma mansoni: cloning of antigen gene sequences in Escherichia coli

Fischer rat protective antiserum (F-2x) prepared from Schistosoma mansoni-infected rats was used to screen an adult worm cDNA library constructed in a lambda gt11 bacteriophage expression vector. This led to the isolation of several clones yielding proteins reactive with antibodies in the infection serum. Counter-screening of these clones with Wistar-Furth rat nonprotective antiserum (W-2x) enabled identification of clones either uniquely or preferentially reacting with F-2x, in addition to clones of nearly equal reactivity with both antisera. Six clones were further characterized. Five expressed beta-galactosidase/S. mansoni fusion proteins which migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than beta-galactosidase and all were reactive in a Western immunoblot assay. The cDNA insert sizes in the clones ranged from 150 to 900 base pairs. Rabbit antibodies prepared against fusion proteins from three of the clones recognized biosynthetically radio-labeled 4-week worm proteins of sizes 20, 38, and 70 kDa, respectively. The 20- and 38-kDa proteins were among the protein antigens uniquely recognized by the F-2x protective antiserum. These proteins are therefore candidates for protective vaccine antigens and the recombinant lambda clones are now serving as useful reagents for obtaining the corresponding nucleotide gene sequences.     

Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.     

Crystallization and preliminary X-ray analysis of the monomeric Cu,Zn superoxide dismutase from Escherichia coli.

The Cu,Zn superoxide dismutase (Cu,Zn SOD) originally isolated from the periplasmic space of Escherichia coli has been cloned and overexpressed in the E. coli strain BMH 71/18. The protein has been purified as a single component of 17,000 Da, corresponding to one subunit of the common dimeric eukaryotic Cu,Zn SODs. Large crystals of the purified protein have been grown in the presence of polyethylene glycol 4,000 at pH 8.5; the crystals belong to the monoclinic space group P2(1), with unit cell constants a = 33.1 A, b = 52.6 A, c = 43.3 A, beta = 111.4 degrees. One SOD subunit is contained in the asymmetric unit, yielding a Vm value of 2.1 A3/Da; the crystals diffract X-rays beyond 2.0 A resolution.     

In vivo formation of Cu,Zn superoxide dismutase disulfide bond in Escherichia coli

We have found that the in vivo folding of periplasmic Escherichia coli Cu,Zn superoxide dismutase is assisted by DsbA, which catalyzes the efficient formation of its single disulfide bond, whose integrity is essential to ensure full catalytic activity to the enzyme. In line with these findings, we also report that the production of recombinant Xenopus laevis Cu,Zn superoxide dismutase is enhanced when the enzyme is exported in the periplasmic space or is expressed in thioredoxin reductase mutant strains. Our data show that inefficient disulfide bond oxidation in the bacterial cytoplasm inhibits Cu,Zn superoxide dismutase folding in this cellular compartment.     

High-level expression of complementary DNA encoding rat calmodulin in Escherichia coli

We report the production and characterization of a rat calmodulin made in Escherichia coli. To express the rat calmodulin cDNA in E. coli, we have employed an expression vector containing the E. coli trp promoter and trpA terminator. The cDNA was modified so as to delete the 5' nontranslated sequence and to incorporate a consensus sequence for the E. coli ribosome-binding site. Several codons for the N-terminal amino acids were selected to fit the E. coli consensus nucleotide sequence around the translational initiation codon. After induction of expression in E. coli, rat calmodulin accounted for over 30% of total cellular proteins. About 100 mg of recombinant rat calmodulin, purified to over 90% homogeneity by extraction from bacterial lysate followed by phenyl-Sepharose column chromatography, was obtained from 1 liter of E. coli culture. This recombinant calmodulin activated rat brain cyclic AMP phosphodiesterase to the same extent as the native calmodulin purified from rat brain. These results indicate that the overproduction system of the recombinant calmodulin in E. coli facilitates the study of the structure-function relationship by site-specific mutagenesis.     

The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni. Further characterization and gene expression in Escherichia coli

Due to the lack of de novo purine nucleotide biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential enzyme in the human parasite Schistosoma mansoni for supplying guanine nucleotides and has been proposed as a potential target for antiparasitic chemotherapy. While the enzyme can be purified from adult schistosome worms, yields are too low to allow extensive structural and kinetic studies. We therefore cloned and sequenced the cDNA and gene encoding the schistosomal enzyme but were unable to positively identify the amino-terminal sequence of the enzyme from the DNA sequence. Knowledge of the exact amino terminus was necessary before accurate expression of active enzyme could be attempted. Therefore, we purified the HGPRTase from crude extracts of the adult worms. The purified enzyme has a subunit molecular mass of 26 kDa and an amino-terminal sequence of Met-Ser-Ser-Asn-Met. This sequence matched one of the potential initiation sites predicted from the cDNA and gene sequence. We next expressed the correct size cDNA of the S. mansoni HGPRTase in Escherichia coli using a vector that is regulated by a bacterial alkaline phosphatase promoter and uses an E. coli signal peptide for secretion of expressed product into the periplasmic space. Using this expression system, some of the recombinant enzyme is secreted and found to have a correct amino terminus. That remaining in the cytoplasm has part of the signal peptide attached to the amino terminus. The recombinant schistosomal HGPRTase isolated from the periplasm of the transformed E. coli was purified and found to have kinetic and physical properties identical to those of the native enzyme.     

Heavy metal-mediated activation of the rat Cu/Zn superoxide dismutase gene via a metal-responsive element

The Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced in the course of biological oxidations. When placed under the control of the rat SOD1 gene promoter and transfected into human HepG2 hepatoma cells, the activity of a chloramphenicol acetyltransferase reporter gene was found to increase three- to four-fold in the presence of heavy metals (cadmium, zinc and copper). Functional analysis of mutant derivatives of the SOD1 gene promoter and the use of a heterologous promoter system confirmed that the induction of the SOD1 gene by metal ions requires a metal-responsive element (MRE) located between positions −273 and −267 (GCGCGCA). It was also shown by gel mobility shift assays that an MRE binding protein is induced by the exposure of the human liver cell line HepG2 to heavy metals. These results suggest that the MRE participates in the induction of the SOD1 gene by heavy metals. Received: 5 February 1999 / Accepted: 21 May 1999     

Identification of copper-zinc superoxide dismutase gene from enteroaggregative Escherichia coli.

We describe here the identification of sodC gene from enteroaggregative Escherichia coli (EAggEC). A 294 bp gene-specific fragment was amplified from the organism by DNA as well as RT-PCR using primers from bacterial sodC sequences. The metal co-factor present in the protein was confirmed by running samples in native gels and inhibiting with 2 mM potassium cyanide. However, the nonpathogenic E. coli possesses the gene but does not express it. Thus, the presence of copper-zinc superoxide dismutase encoded by sodC was demonstrated for the first time in EAggEC, which means it could be a novel candidate for a virulence marker.     

Heterologous expression of a novel psychrophilic Cu/Zn superoxide dismutase from Deschampsia antarctica

Superoxide dismutase (SOD) catalyzes the conversion of the superoxide radical (O₂⁻) into oxygen and hydrogen peroxide. Deschampsia antarctica is a plant that grows in Antarctica and survives to extreme low temperature and high UV radiation, thus it is an ideal model to study novel antioxidants. A cDNA Cu/Zn-SOD gene from D. antarctica was cloned into a pET vector and expressed in Escherichia coli BL21-SI. 112 mg/L of recombinant Cu/Zn-SOD was attained in batch cultures in bioreactor. Using Ni-affinity gel chromatography, the recombinant Cu/Zn-SOD was recovered with a purity of 90% and a specific enzyme activity of 749 at 25 °C. However, zymogram test showed that the enzyme has more activity at 4 °C. This D. antarctica SOD could be used to reduce the oxidation of refrigerated and frozen foods.     

Architecture and anatomy of the chromosomal locus in human chromosome 21 encoding the Cu/Zn superoxide dismutase.

The SOD-1 gene on chromosome 21 and approximately 100 kb of chromosomal DNA from the 21q22 region have been isolated and characterized. The gene which is present as a single copy per haploid genome spans 11 kb of chromosomal DNA. Heteroduplex analysis and DNA sequencing reveals five rather small exons and four introns that interrupt the coding region. The donor sequence at the first intron contains an unusual variant dinucleotide 5'-G-C, rather than the highly conserved 5'-GT. The unusual splice junction is functional in vivo since it was detected in both alleles of the SOD-1 gene, which were defined by differences in the length of restriction endonuclease fragments (RFLPs) that hybridize to the cDNA probe. Genomic blots of human DNA isolated from cells trisomic for chromosome 21 (Down's syndrome patients) show the normal pattern of bands. At the 5' end of gene there are the 'TATA' and 'CAT' promoter sequences as well as four copies of the -GGCGGG- hexanucleotide. Two of these -GC- elements are contained within a 13 nucleotide inverted repeat that could form a stem-loop structure with stability of -33 kcal. The 3'-non coding region of the gene contains five short open reading-frames starting with ATG and terminating with stop codons.     

Cloning of a superoxide dismutase gene from Listeria ivanovii by functional complementation in Escherichia coli and characterization of the gene product.

Summary A gene encoding superoxide dismutase (EC 1.15.1.1., SOD) was isolated from a plasmid library of chromosomal DNA from Listeria ivanovii by functional complementation of an SOD-negative Escherichia coli host. The nucleotide sequence of the cloned gene was determined and contained an open reading frame which codes for a protein of 202 amino acid residues (calculated molecular weight 22 755 Da including the amino-terminal methionine residue). Comparison of the deduced amino acid sequence of L. ivanovii SOD with previously reported SOD amino acid sequences revealed considerable homologies with Fe- and Mn-dependent SODs. Enzymatic analyses using cell lysates and the purified recombinant enzyme indicated that this SOD is manganese-dependent. The recombinant SOD accounted for up to 30% of the total soluble protein in recombinant E. coli and protected sodA sodB mutants against the toxic effects of paraquat. Subunits of the recombinant Listeria SOD and of both E. coli SODS formed enzymatically active hybrids in vivo.Some of our preliminary observations have been published as a conference report of SOD V (Jerusalem, 1989) in Free Rad Res Commun (1991) 12–13:371     

Xenobiotic-responsive element for the transcriptional activation of the rat Cu/Zn superoxide dismutase gene

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced from biological oxidation and environmental stresses. A number of xenobiotics are toxic because they generate free radicals, such as superoxide and hydroxyl radicals, through a redox cycle. The xenobiotic responsive element (XRE) was located between the nt -268 and -262 region of the 5'-flanking sequence of the SOD1 gene. Functional analyses of this element by deletion, mutations, and heterologous promoter systems confirmed that the expression of the SOD1 gene was induced by a xenobiotic through the XRE. Gel mobility shift assays showed the xenobiotic inducible binding of the receptor-ligand complex to XRE. The cytoplasmic fraction from nontreated HepG2 cells also contains the factor as a cryptic form and prominently reveals its DNA-binding activity by incubation with betaNF in vitro. These results suggest that the XRE participates in the induction of the rat SOD1 gene by xenobiotics.