Gene Encoding Superoxide Dismutase Research Articles

The iron-containing superoxide dismutase-encoding gene from Chlamydomonas reinhardtii obtained by direct and inverse PCR

The complete sequence of the Fe 2+-containing superoxide dismutase (Fe-SOD)-encoding gene of Chlamydomonas reinhardtii was determined from genomic DNA by use of the direct (PCR) and inverse PCR (IPCR). It was confirmed by reverse transcription-PCR. Primers employed in the PCR represented oligodeoxyribonucleotides corresponding to conserved amino acid (aa) residues of Fe-SOD. From the sequence of the direct PCR product, primers were designed for IPCR. All amplified fragments were cloned and sequenced. Analysis of Fe-SOD reveals that the open reading frame consists of 234 aa which show a high degree of homology to other Fe-SOD. The first 33 aa are predominantly either hydrophobic or basic, and may serve as the signal peptide for this chloroplastic protein. Potential transcription start points and eukaryotic control elements, as well as a polyadenylation site, were identified.

Isolation, sequencing and expression of the superoxide dismutase-encoding gene ( sod) of Nocardia asteroides strain GUH-2

Nocardia asteroides (Na) superoxide dismutase (SOD) has been implicated as a virulence factor that allows the organism to survive intracellular killing by phagocytic cells. A full-length Na sod gene from a pathogenic strain of Na (strain GUH-2) was cloned from a recombinant phage library using the Mycobacterium tuberculosis (Mt) sod gene ( Mt sod) as a probe. The promoter region and structural gene (624 bp) of Na sod was sequenced and nucleotide sequence comparisons reveal 77% homology with Mt sod. The Na sod gene also shares considerable sequence homology with sod of other mycobacterial species. In addition, conserved amino acid (aa) sequences important for metal binding indicate that Mn 2+ is the preferred metal ion ligand for Na SOD. An Na sod expression plasmid, pYEX1, under transcriptional control of the Mt hsp70 promoter (pY6013), produced a 25-kDa protein product which showed SOD activity when stained in a native polyacrylamide gel and reacted with rabbit polyclonal antibody specific for Na SOD by Western blot. pYEX1, via transformation, was able to complement an Escherichia coli double sodAB mutant deficient in SOD production in the presence of paraquat (methyl viologen) which stimulates the production of superoxide radicals.

Cloning and characterization of the murine manganous superoxide dismutase-encoding gene

The murine manganous superoxide dismutase-encoding gene (MnSOD) is highly homologous in both sequence and organization to its rat homolog. MnSOD is encoded by five exons spanning approx. 7 kb of DNA. RNA blot analysis indicated multiple RNA species, with the major RNA corresponding to a 960-bp message. This major transcript is highly inducible by tumor necrosis factor (TNF) in murine fibroblasts. Analysis of other murine tissues demonstrated ubiquitous expression. RNase protection and primer extension assays used to map the 5' end of the gene revealed a series of closely spaced multiple transcription start points. Sequence analysis of the 1.7 kb of 5' flanking DNA showed high homology to the 5' proximal 950 bp of the rat homolog. Within this region, multiple potential regulatory elements are present, including several SP1 sites, two NFκB sites and an antioxidant-response element. However, no TATA box was identified, placing MnSOD in the family of inducible genes that lack consensus TATA-box elements and contain G + C-rich promoter regions.

Characterization of a Cu/Zn superoxide dismutase-encoding gene region in Drosophila willistoni.

A Cu/Zn superoxide dismutase-encoding gene (Sod) from Drosophila willistoni was cloned and sequenced. The gene shows a typical structure for a fruit-fly Sod gene, with a coding region of 462 bp in two exons separated by a 417-bp intron. Comparison of the Sod sequences from D. willistoni and D. melanogaster suggests that these species are only remotely related. Downstream from the Sod gene, there is an ORF on the opposite strand that putatively encodes the last exon of an unidentified gene. The polyadenylation signals of the two genes are separated by only 61 bp in D. willistoni, conforming to the common picture of compact dipteran genomes.

Cloning, nucleotide sequence and characterization of a gene encoding superoxide dismutase from Campylobacter jejuni and Campylobacter coli

Genes encoding superoxide dismutase (SOD: EC 1.15.1.1) were cloned from Campylobacter jejuni NCTC 11351 and Campylobacter coli UA585 by heterologous complementation of a SOD-deficient Escherichia coli mutant. Deletion analysis of the cloned C. jejuni DNA assigned the sod gene to a 1.2 kb insert and this contained an open reading frame of 660 bp. The deduced gene product of 220 amino acids was 71% identical to the E. coli iron-containing SOD and 60% identical to the E. coli manganese-containing SOD. The recombinant SOD was expressed at high levels in E. coli and protected a sodA sodB double mutant from the toxic effects of methyl viologen. Nucleotide sequence analysis of the corresponding gene from C. coli showed it to be 92% identical to that from C. jejuni.

Characterization of the gene encoding Superoxide dismutase of Bordetella pertussis and construction of a SOD-deficient mutant

The Bordetella pertussis gene sodB, encoding Superoxide dismutase (SOD), was cloned by complementation of an Escherichia coli sodAsodB double mutant. The nucleotide sequence of sodB predicted a 21-kDa protein with homology to manganese- and iron-containing SODs from other organisms. Examination of SOD activity on gels suggests that B. pertussis extracts have a single SOD containing Fe + as a prosthetic group. A SOD-deβcient mutant was obtained by insertional inactivation of sodB in B. pertussis, confirming that there is only one SOD in this organism.

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

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 22755 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.

Characterization of a superoxide dismutase gene from the archaebacterium Methanobacterium thermoautotrophicum

A gene encoding superoxide dismutase (SOD) was cloned from the archaebacterium Methanobacterium thermoautotrophicum, the first example from an anaerobic bacterium. The deduced amino acid sequence showed overall similarity to sequences of known Mn- and Fe-SODs from aerobic organisms. Judging from a detailed sequence comparison, the cloned SOD gene is classified as Mn-SOD. By comparison of Mn-SOD sequences among various species it was suggested that archaebacterial superoxide dismutase is a direct descendant of a primordial enzyme. Between a putative promoter and the start codon there is an inverted repeat sequence which is also found in the counterpart of Halobacterium halobium.

Evolution and Regulation of the Gene Encoding Superoxide Dismutase from the Archaebacterium Halobacterium cutirubrum

The gene encoding the manganese-containing superoxide dismutase (SOD) of Halobacterium cutirubrum was isolated and characterized. The gene and 5'- and 3'-untranslated regions were located on a genomic DNA fragment of 1127 nucleotides. The deduced amino acid sequence is 200 residues long and has 39-42% identity with manganese-containing SODs of eubacteria and mitochondria. This homology may be due to either lateral transfer of the gene between eubacteria and archaebacteria or to high amino acid sequence conservation in the enzyme during the separate evolution of eubacteria and archaebacteria. Transcription of the gene initiates only about three nucleotides upstream of the translation initiation codon. The 5' end of the transcript does not contain a purine-rich Shine-Dalgarno sequence, and the promoter region does not contain consensus sequences found in other archaebacterial promoters. Termination of transcription occurs at 5 consecutive thymine residues that are preceded by a GC-rich region. The gene is basally expressed in anaerobically grown cells but is also inducible by paraquat, a generator of oxygen radicals. The same transcription initiation site is used in both types of expression, suggesting that one promoter is responsible for both basal and regulated expression. In addition to the single copy of the authentic SOD gene, the genome of H. cutirubrum contains a sequence that is very closely related to but does not code for the previously purified SOD of this organism.