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. 1996 Jan;142(1):129–140. doi: 10.1093/genetics/142.1.129

Cloning and Analysis of the Alternative Oxidase Gene of Neurospora Crassa

Q Li 1, R G Ritzel 1, LLT McLean 1, L McIntosh 1, T Ko 1, H Bertrand 1, F E Nargang 1
PMCID: PMC1206941  PMID: 8770590

Abstract

Mitochondria of Neurospora crassa contain a cyanide-resistant alternative respiratory pathway in addition to the cytochrome pathway. The alternative oxidase is present only when electron flow through the cytochrome chain is restricted. Both genomic and cDNA copies for the alternative oxidase gene have been isolated and analyzed. The sequence of the predicted protein is homologous to that of other species. The mRNA for the alternative oxidase is scarce in wild-type cultures grown under normal conditions, but it is abundant in cultures grown in the presence of chloramphenicol, an inhibitor of mitochondrial protein synthesis, or in mutants deficient in mitochondrial cytochromes. Thus, induction of alternative oxidase appears to be at the transcriptional level. Restriction fragment length polymorphism mapping of the isolated gene demonstrated that it is located in a position corresponding to the aod-1 locus. Sequence analysis of mutant aod-1 alleles reveals mutations affecting the coding sequence of the alternative oxidase. The level of aod-1 mRNA in an aod-2 mutant strain that had been grown in the presence of chloramphenicol was reduced several fold relative to wild-type, supporting the hypothesis that the product of aod-2 is required for optimal expression of aod-1.

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Selected References

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  1. Akins R. A., Lambowitz A. M. General method for cloning Neurospora crassa nuclear genes by complementation of mutants. Mol Cell Biol. 1985 Sep;5(9):2272–2278. doi: 10.1128/mcb.5.9.2272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allen J. F. Redox control of transcription: sensors, response regulators, activators and repressors. FEBS Lett. 1993 Oct 18;332(3):203–207. doi: 10.1016/0014-5793(93)80631-4. [DOI] [PubMed] [Google Scholar]
  3. Barath Z., Küntzel H. Induction of mitochondrial RNA polymerase in Neurospora crassa. Nat New Biol. 1972 Dec 13;240(102):195–197. doi: 10.1038/newbio240195a0. [DOI] [PubMed] [Google Scholar]
  4. Bertrand H., Collins R. A. A regulatory system controlling the production of cytochrome aa3 in Neurospora crassa. Mol Gen Genet. 1978 Oct 25;166(1):1–13. doi: 10.1007/BF00379723. [DOI] [PubMed] [Google Scholar]
  5. Bertrand H., Nargang F. E., Collins R. A., Zagozeski C. A. Nuclear cytochrome-deficient mutants of Neurospora crassa: isolation, characterization, and genetic mapping. Mol Gen Genet. 1977 Jun 24;153(3):247–257. doi: 10.1007/BF00431590. [DOI] [PubMed] [Google Scholar]
  6. Bertrand H., Pittenger T. H. Isolation and classification of extranuclear mutants of Neurospora crassa. Genetics. 1972 Aug;71(4):521–533. doi: 10.1093/genetics/71.4.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  8. Drygas M. E., Lambowitz A. M., Nargang F. E. Cloning and analysis of the Neurospora crassa gene for cytochrome c heme lyase. J Biol Chem. 1989 Oct 25;264(30):17897–17906. [PubMed] [Google Scholar]
  9. Edwards D. L., Rosenberg E. Regulation of cyanide-insensitive respiration in Neurospora. Eur J Biochem. 1976 Feb 16;62(2):217–221. doi: 10.1111/j.1432-1033.1976.tb10150.x. [DOI] [PubMed] [Google Scholar]
  10. Edwards D. L., Rsenberg E., Maroney P. A. Induction of cyanide-insensitive respiration in Neurospora crassa. J Biol Chem. 1974 Jun 10;249(11):3551–3556. [PubMed] [Google Scholar]
  11. Edwards D. L., Unger B. W. Induction of hydroxamate-sensitive respiration in Neurospora mitochondria. Transcription of nuclear DNA is required. FEBS Lett. 1978 Jan 1;85(1):40–42. doi: 10.1016/0014-5793(78)81243-5. [DOI] [PubMed] [Google Scholar]
  12. Elthon T. E., McIntosh L. Identification of the alternative terminal oxidase of higher plant mitochondria. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8399–8403. doi: 10.1073/pnas.84.23.8399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Elthon T. E., Nickels R. L., McIntosh L. Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. Plant Physiol. 1989 Apr;89(4):1311–1317. doi: 10.1104/pp.89.4.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gavel Y., von Heijne G. Cleavage-site motifs in mitochondrial targeting peptides. Protein Eng. 1990 Oct;4(1):33–37. doi: 10.1093/protein/4.1.33. [DOI] [PubMed] [Google Scholar]
  15. Gessert S. F., Kim J. H., Nargang F. E., Weiss R. L. A polyprotein precursor of two mitochondrial enzymes in Neurospora crassa. Gene structure and precursor processing. J Biol Chem. 1994 Mar 18;269(11):8189–8203. [PubMed] [Google Scholar]
  16. Habener J. F. Cyclic AMP response element binding proteins: a cornucopia of transcription factors. Mol Endocrinol. 1990 Aug;4(8):1087–1094. doi: 10.1210/mend-4-8-1087. [DOI] [PubMed] [Google Scholar]
  17. Hanssens L., D'Hondt E., Verachtert H. Cyanide-insensitive respiration in Moniliella tomentosa and the effect of drugs on respiration and polyol biosynthesis. Arch Microbiol. 1974 Jul 22;98(4):339–349. doi: 10.1007/BF00425294. [DOI] [PubMed] [Google Scholar]
  18. Hartl F. U., Pfanner N., Nicholson D. W., Neupert W. Mitochondrial protein import. Biochim Biophys Acta. 1989 Jan 18;988(1):1–45. doi: 10.1016/0304-4157(89)90002-6. [DOI] [PubMed] [Google Scholar]
  19. Hendrick J. P., Hodges P. E., Rosenberg L. E. Survey of amino-terminal proteolytic cleavage sites in mitochondrial precursor proteins: leader peptides cleaved by two matrix proteases share a three-amino acid motif. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4056–4060. doi: 10.1073/pnas.86.11.4056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Henry M. F., Nyns E. D. Cyanide-insensitive respiration. An alternative mitochondrial pathway. Subcell Biochem. 1975 Mar;4(1):1–65. [PubMed] [Google Scholar]
  21. Kumar A. M., Söll D. Arabidopsis alternative oxidase sustains Escherichia coli respiration. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10842–10846. doi: 10.1073/pnas.89.22.10842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lambowitz A. M., Slayman C. W. Cyanide-resistant respiration in Neurospora crassa. J Bacteriol. 1971 Dec;108(3):1087–1096. doi: 10.1128/jb.108.3.1087-1096.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lambowitz A. M., Smith E. W., Slayman C. W. Electron transport in Neurospora mitochondria. Studies on wild type and poky. J Biol Chem. 1972 Aug 10;247(15):4850–4858. [PubMed] [Google Scholar]
  24. Liao X., Butow R. A. RTG1 and RTG2: two yeast genes required for a novel path of communication from mitochondria to the nucleus. Cell. 1993 Jan 15;72(1):61–71. doi: 10.1016/0092-8674(93)90050-z. [DOI] [PubMed] [Google Scholar]
  25. Loros J. J., Denome S. A., Dunlap J. C. Molecular cloning of genes under control of the circadian clock in Neurospora. Science. 1989 Jan 20;243(4889):385–388. doi: 10.1126/science.2563175. [DOI] [PubMed] [Google Scholar]
  26. Meyer T. E., Habener J. F. Cyclic adenosine 3',5'-monophosphate response element binding protein (CREB) and related transcription-activating deoxyribonucleic acid-binding proteins. Endocr Rev. 1993 Jun;14(3):269–290. doi: 10.1210/edrv-14-3-269. [DOI] [PubMed] [Google Scholar]
  27. Millar A. H., Wiskich J. T., Whelan J., Day D. A. Organic acid activation of the alternative oxidase of plant mitochondria. FEBS Lett. 1993 Aug 30;329(3):259–262. doi: 10.1016/0014-5793(93)80233-k. [DOI] [PubMed] [Google Scholar]
  28. Minagawa N., Koga S., Nakano M., Sakajo S., Yoshimoto A. Possible involvement of superoxide anion in the induction of cyanide-resistant respiration in Hansenula anomala. FEBS Lett. 1992 May 18;302(3):217–219. doi: 10.1016/0014-5793(92)80444-l. [DOI] [PubMed] [Google Scholar]
  29. Moore A. L., Siedow J. N. The regulation and nature of the cyanide-resistant alternative oxidase of plant mitochondria. Biochim Biophys Acta. 1991 Aug 23;1059(2):121–140. doi: 10.1016/s0005-2728(05)80197-5. [DOI] [PubMed] [Google Scholar]
  30. Raskin I., Ehmann A., Melander W. R., Meeuse B. J. Salicylic Acid: a natural inducer of heat production in arum lilies. Science. 1987 Sep 25;237(4822):1601–1602. doi: 10.1126/science.237.4822.1601. [DOI] [PubMed] [Google Scholar]
  31. Roesler W. J., Vandenbark G. R., Hanson R. W. Cyclic AMP and the induction of eukaryotic gene transcription. J Biol Chem. 1988 Jul 5;263(19):9063–9066. [PubMed] [Google Scholar]
  32. Sakajo S., Minagawa N., Yoshimoto A. Characterization of the alternative oxidase protein in the yeast Hansenula anomala. FEBS Lett. 1993 Mar 8;318(3):310–312. doi: 10.1016/0014-5793(93)80535-3. [DOI] [PubMed] [Google Scholar]
  33. Schwab A. J. Mitochondrial protein synthesis and cyanide-resistant respiration in copper-depleted, cytochrome oxidase-deficient Neurospora crassa. FEBS Lett. 1973 Sep 1;35(1):63–66. doi: 10.1016/0014-5793(73)80577-0. [DOI] [PubMed] [Google Scholar]
  34. Schweizer M., Case M. E., Dykstra C. C., Giles N. H., Kushner S. R. Identification and characterization of recombinant plasmids carrying the complete qa gene cluster from Neurospora crassa including the qa-1+ regulatory gene. Proc Natl Acad Sci U S A. 1981 Aug;78(8):5086–5090. doi: 10.1073/pnas.78.8.5086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Surdej P., Riedl A., Jacobs-Lorena M. Regulation of mRNA stability in development. Annu Rev Genet. 1994;28:263–282. doi: 10.1146/annurev.ge.28.120194.001403. [DOI] [PubMed] [Google Scholar]
  36. Umbach A. L., Siedow J. N. Covalent and Noncovalent Dimers of the Cyanide-Resistant Alternative Oxidase Protein in Higher Plant Mitochondria and Their Relationship to Enzyme Activity. Plant Physiol. 1993 Nov;103(3):845–854. doi: 10.1104/pp.103.3.845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Umbach A. L., Wiskich J. T., Siedow J. N. Regulation of alternative oxidase kinetics by pyruvate and intermolecular disulfide bond redox status in soybean seedling mitochondria. FEBS Lett. 1994 Jul 11;348(2):181–184. doi: 10.1016/0014-5793(94)00600-8. [DOI] [PubMed] [Google Scholar]
  38. Vanlerberghe G. C., McIntosh L. Mitochondrial electron transport regulation of nuclear gene expression. Studies with the alternative oxidase gene of tobacco. Plant Physiol. 1994 Jul;105(3):867–874. doi: 10.1104/pp.105.3.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vollmer S. J., Yanofsky C. Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4869–4873. doi: 10.1073/pnas.83.13.4869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wang Z., Deak M., Free S. J. A cis-acting region required for the regulated expression of grg-1, a Neurospora glucose-repressible gene. Two regulatory sites (CRE and NRS) are required to repress grg-1 expression. J Mol Biol. 1994 Mar 18;237(1):65–74. doi: 10.1006/jmbi.1994.1209. [DOI] [PubMed] [Google Scholar]

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