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. 1990 Mar;172(3):1539–1546. doi: 10.1128/jb.172.3.1539-1546.1990

Characterization of the Bacillus stearothermophilus manganese superoxide dismutase gene and its ability to complement copper/zinc superoxide dismutase deficiency in Saccharomyces cerevisiae.

C Bowler 1, L Van Kaer 1, W Van Camp 1, M Van Montagu 1, D Inzé 1, P Dhaese 1
PMCID: PMC208630  PMID: 2407726

Abstract

Recombinant clones containing the manganese superoxide dismutase (MnSOD) gene of Bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. Complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of Escherichia coli, allowed us to define the region of DNA which encodes the MnSOD structural gene and to identify a promoter region immediately upstream from the gene. These data were subsequently confirmed by DNA sequencing. Since MnSOD is normally restricted to the mitochondria in eucaryotes, we were interested (i) in determining whether B. stearothermophilus MnSOD could function in eucaryotic cytosol and (ii) in determining whether MnSOD could replace the structurally unrelated copper/zinc superoxide dismutase (Cu/ZnSOD) which is normally found there. To test this, the sequence encoding bacterial MnSOD was cloned into a yeast expression vector and subsequently introduced into a Cu/ZnSOD-deficient mutant of the yeast Saccharomyces cerevisiae. Functional expression of the protein was demonstrated, and complementation tests revealed that the protein was able to provide tolerance at wild-type levels to conditions which are normally restrictive for this mutant. Thus, in spite of the evolutionary unrelatedness of these two enzymes, Cu/ZnSOD can be functionally replaced by MnSOD in yeast cytosol.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Auffret A. D., Blake T. J., Williams D. H. Mass spectrometric sequence studies of a superoxide dismutase from Bacillus stearothermophilus. Eur J Biochem. 1981 Jan;113(2):333–338. doi: 10.1111/j.1432-1033.1981.tb05071.x. [DOI] [PubMed] [Google Scholar]
  2. Bannister J. V., Bannister W. H., Rotilio G. Aspects of the structure, function, and applications of superoxide dismutase. CRC Crit Rev Biochem. 1987;22(2):111–180. doi: 10.3109/10409238709083738. [DOI] [PubMed] [Google Scholar]
  3. Beauchamp C., Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem. 1971 Nov;44(1):276–287. doi: 10.1016/0003-2697(71)90370-8. [DOI] [PubMed] [Google Scholar]
  4. Bermingham-McDonogh O., Gralla E. B., Valentine J. S. The copper, zinc-superoxide dismutase gene of Saccharomyces cerevisiae: cloning, sequencing, and biological activity. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4789–4793. doi: 10.1073/pnas.85.13.4789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Biliński T., Krawiec Z., Liczmański A., Litwińska J. Is hydroxyl radical generated by the Fenton reaction in vivo? Biochem Biophys Res Commun. 1985 Jul 31;130(2):533–539. doi: 10.1016/0006-291x(85)90449-8. [DOI] [PubMed] [Google Scholar]
  6. Bowler C., Alliotte T., De Loose M., Van Montagu M., Inzé D. The induction of manganese superoxide dismutase in response to stress in Nicotiana plumbaginifolia. EMBO J. 1989 Jan;8(1):31–38. doi: 10.1002/j.1460-2075.1989.tb03345.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bowler C., Alliotte T., Van den Bulcke M., Bauw G., Vandekerckhove J., Van Montagu M., Inzé D. A plant manganese superoxide dismutase is efficiently imported and correctly processed by yeast mitochondria. Proc Natl Acad Sci U S A. 1989 May;86(9):3237–3241. doi: 10.1073/pnas.86.9.3237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  9. Bray R. C., Cockle S. A., Fielden E. M., Roberts P. B., Rotilio G., Calabrese L. Reduction and inactivation of superoxide dismutase by hydrogen peroxide. Biochem J. 1974 Apr;139(1):43–48. doi: 10.1042/bj1390043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bridgen J., Harris J. I., Northrop F. Evolutionary relationships in superoxide dismutase. FEBS Lett. 1975 Jan 1;49(3):392–395. doi: 10.1016/0014-5793(75)80793-9. [DOI] [PubMed] [Google Scholar]
  11. Brock C. J., Walker J. E. Superoxide dismutase from Bacillus stearothermophilus. Complete amino acid sequence of a manganese enzyme. Biochemistry. 1980 Jun 24;19(13):2873–2882. doi: 10.1021/bi00554a009. [DOI] [PubMed] [Google Scholar]
  12. Carlioz A., Touati D. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J. 1986 Mar;5(3):623–630. doi: 10.1002/j.1460-2075.1986.tb04256.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  14. Craigen W. J., Caskey C. T. Expression of peptide chain release factor 2 requires high-efficiency frameshift. Nature. 1986 Jul 17;322(6076):273–275. doi: 10.1038/322273a0. [DOI] [PubMed] [Google Scholar]
  15. Craigen W. J., Cook R. G., Tate W. P., Caskey C. T. Bacterial peptide chain release factors: conserved primary structure and possible frameshift regulation of release factor 2. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3616–3620. doi: 10.1073/pnas.82.11.3616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goscin S. A., Fridovich I. The purification and properties of superoxide dismutase from Saccharomyces cerevisiae. Biochim Biophys Acta. 1972 Dec 7;289(2):276–283. doi: 10.1016/0005-2744(72)90078-2. [DOI] [PubMed] [Google Scholar]
  17. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  19. Hjalmarsson K., Marklund S. L., Engström A., Edlund T. Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6340–6344. doi: 10.1073/pnas.84.18.6340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hodgson E. K., Fridovich I. The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: inactivation of the enzyme. Biochemistry. 1975 Dec 2;14(24):5294–5299. doi: 10.1021/bi00695a010. [DOI] [PubMed] [Google Scholar]
  21. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Koshland D., Botstein D. Secretion of beta-lactamase requires the carboxy end of the protein. Cell. 1980 Jul;20(3):749–760. doi: 10.1016/0092-8674(80)90321-9. [DOI] [PubMed] [Google Scholar]
  23. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  24. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  25. Mielenz J. R. Bacillus stearothermophilus contains a plasmid-borne gene for alpha-amylase. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5975–5979. doi: 10.1073/pnas.80.19.5975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Natvig D. O., Imlay K., Touati D., Hallewell R. A. Human copper-zinc superoxide dismutase complements superoxide dismutase-deficient Escherichia coli mutants. J Biol Chem. 1987 Oct 25;262(30):14697–14701. [PubMed] [Google Scholar]
  27. Ohtsuka E., Matsuki S., Ikehara M., Takahashi Y., Matsubara K. An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions. J Biol Chem. 1985 Mar 10;260(5):2605–2608. [PubMed] [Google Scholar]
  28. Oppenheim D. S., Yanofsky C. Translational coupling during expression of the tryptophan operon of Escherichia coli. Genetics. 1980 Aug;95(4):785–795. doi: 10.1093/genetics/95.4.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Parker M. W., Blake C. C. Iron- and manganese-containing superoxide dismutases can be distinguished by analysis of their primary structures. FEBS Lett. 1988 Mar 14;229(2):377–382. doi: 10.1016/0014-5793(88)81160-8. [DOI] [PubMed] [Google Scholar]
  30. Ravindranath S. D., Fridovich I. Isolation and characterization of a manganese-containing superoxide dismutase from yeast. J Biol Chem. 1975 Aug 10;250(15):6107–6112. [PubMed] [Google Scholar]
  31. Riddle D. L., Carbon J. Frameshift suppression: a nucleotide addition in the anticodon of a glycine transfer RNA. Nat New Biol. 1973 Apr 25;242(121):230–234. doi: 10.1038/newbio242230a0. [DOI] [PubMed] [Google Scholar]
  32. Roth J. R. Frameshift mutations. Annu Rev Genet. 1974;8:319–346. doi: 10.1146/annurev.ge.08.120174.001535. [DOI] [PubMed] [Google Scholar]
  33. Scioli J. R., Zilinskas B. A. Cloning and characterization of a cDNA encoding the chloroplastic copper/zinc-superoxide dismutase from pea. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7661–7665. doi: 10.1073/pnas.85.20.7661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shine J., Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. doi: 10.1038/254034a0. [DOI] [PubMed] [Google Scholar]
  35. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  36. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  37. van Loon A. P., Pesold-Hurt B., Schatz G. A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3820–3824. doi: 10.1073/pnas.83.11.3820. [DOI] [PMC free article] [PubMed] [Google Scholar]

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