Advertisement

Current Genetics

, Volume 29, Issue 2, pp 122–129 | Cite as

The cloning and sequencing of thealcB gene, coding for alcohol dehydrogenase II, inAspergillus nidulans

  • Gary D. Hunter
  • I. Gwyn Jones
  • Heather M. Sealy-Lewis
Original Paper

Abstract

Alcohol dehydrogenase II (ADH II, structural genealcB) was purified from a strain H1035,biA1; alcE1; alc500 alcD1, which produces 100-times more ADH II activity than thealcAalcR deletion strain (alc500). Antibodies were raised against this ADH, and were used to screen a cDNA library in γgt11. We have isolated the gene for an ADH which is over-expressed in H1035, and which we believe to be thealcB gene; cDNA and genomic clones were sequenced. The sequence contains three introns and encodes a protein of 367 amino acids. This protein shows a clear level of identity to a range of alcohol dehydrogenases, but is no more closely related to the ADH I and ADH III previously described inA. nidulans than to the ADHs ofS. pombe andS. cerevisiae. The significance of consensus sequences found in the 5′ region of the gene is discussed in relation to the regulation of the gene.

Key words

Alcohol dehydrogenase II Aspergillus nidulans 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Atkinson PN, King JA, Hynes MJ (1985) Identification of theaciA gene controlled by theamdA regulatory gene inAspergillus nidulans. Curr Genet 10:133–138CrossRefPubMedGoogle Scholar
  2. Bailey C, Arst HN Jr (1975) Carbon catabolite repression inAspergillus nidulans. Eur J Biochem 51:573–577CrossRefPubMedGoogle Scholar
  3. Ballance DJ (1986) Sequences important for gene expression in filamentous fungi. Yeast 2:229–236CrossRefPubMedGoogle Scholar
  4. Bennetzen JL, Hall BD (1982) The primary structure of theSaccharomyces cerevisiae gene for alcohol dehydrogenase I. J Biol Chem 257:3026–3025PubMedGoogle Scholar
  5. Branden CI, Eklund H, Cambillau C, Pryor AJ (1984) Correlation of exons with structural domains in alcohol dehydrogenase. EMBO J 3:1307–1310PubMedGoogle Scholar
  6. Bruchez JPJ, Eberle J, Russo VEA (1993) Regulatory sequences in the transcription ofNeurospora crassa genes: CAAT box, TATA box, introns, poly(A) tail-formation sequences. Fungal Genet Newslett 40:89–96Google Scholar
  7. Clutterbuck AJ (1974)Aspergillus nidulans. In: King RC (ed) Handbook of genetics. Plenum Press, New York, pp 447–510Google Scholar
  8. Cove DJ (1966) The induction and repression of nitrate reductase in the fungus,Aspergillus nidulans. Biochem Biophys Acta 113:51–56PubMedGoogle Scholar
  9. Creaser EH, Porter RL, Britt KA, Pateman JA, Doy CH (1984) Purification and preliminary characterisation of alcohol dehydrogenase fromAspergillus nidulans. Biochem J 225:449–454Google Scholar
  10. Cubero B, Scazzocchio C (1994) Two different, adjacent and divergent binding sites are necessary for CREA-mediated carbon catabolite repression in the proline gene cluster ofAspergillus nidulans. EMBO J 13:407–415PubMedGoogle Scholar
  11. Devereux AU, Haeberli J, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395PubMedGoogle Scholar
  12. Duester G, Smith M, Bilanchone V, Hatfield GW (1986) Molecular analysis of the human class-I alcohol dehydrogenase gene family and nucleotide sequence of the gene encoding the b subunit. J Biol Chem 261:2027–2033PubMedGoogle Scholar
  13. Felenbok B, Sealy-Lewis HM (1994) Alcohol metabolism. In: Martinelli SD, Kinghorn JR (eds) Aspergillus: 50 years on. Elsevier, Amsterdam, pp 141–179Google Scholar
  14. Felenbok B, Sequeval D, Mathieu M, Sibley S, Gwynne DI, Davies RW (1988) The ethanol regulon inAspergillus nidulans: characterisation and sequence of the positive regulatory genealcR. Gene 73:385–396CrossRefPubMedGoogle Scholar
  15. Fidel F, Doonan JH, Morris MR (1988)Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes aγ-actin. Gene 70:283–293CrossRefPubMedGoogle Scholar
  16. Goa J (1953) A microbiuret method for protein determination. Determination of total protein in cerebrospinal fluid. Scand J Clin Lab Invest 5:218–222PubMedGoogle Scholar
  17. Gurr SJ, Unkles SE Kinghorn JR (1987) The structure and organisation of nuclear genes of filamentous fungi In: Kinghorn JR (ed) Gene structure in eukaryotic microbes. IRL Press, Oxford, pp 93–139Google Scholar
  18. Gwynne DI, Buxton FP, Sibley S, Davies RW, Lockington RA, Scazzocchio C, Sealy-Lewis HM (1987) Comparison of the cis-acting control regions of two coordinately controlled genes involved in ethanol utilization inAspergillus nidulans. Gene 51:205–216CrossRefPubMedGoogle Scholar
  19. Hynes MJ (1978) Multiple independent control mechanisms affecting the acetamidase ofAspergillus nidulans. Mol Gen Genet 161:59–65CrossRefPubMedGoogle Scholar
  20. Jones IG, Sealy-Lewis HM (1989) Chromosomal mapping and gene disruption of the ADH III gene inAspergillus nidulans. Curr Genet 15:135–142CrossRefPubMedGoogle Scholar
  21. Jornvall H (1970) Horse liver alcohol dehydrogenase. The primary structure of the protein chain of the ethanol-active isozyme. Eur J Biochem 16:25–40CrossRefPubMedGoogle Scholar
  22. Jornvall H (1977) Differences between alcohol dehydrogenases. Structural properties and evolutionary aspects. Eur J Biochem 72:443–452CrossRefPubMedGoogle Scholar
  23. Jornvall H, Eklund H, Branden C (1978) Subunit conformation of yeast alcohol dehydrogenase. J Biol Chem 253:8414–8419PubMedGoogle Scholar
  24. Kaiser R, Fernandez MR, Pares X, Jornvall H (1993) Origin of the human alcohol dehydrogenase system: implications from the structure and properties of the octopus protein. Proc Natl Acad Sci USA 90:11222–11226PubMedGoogle Scholar
  25. Kelly JM, Drysdale MR, Sealy-Lewis HM, Jones IG, Lockington RA (1990) Alcohol dehydrogenase III inAspergillus nidulans is anaerobically induced and post-transcriptionally regulated. Mol Gen Genet 222:323–328CrossRefPubMedGoogle Scholar
  26. Kulmburg P, Sequeval D, Lenouvel F, Mathieu M, Felenbok B (1992a) Identification of the promoter region involved in the autoregulation of the transcriptional activator ALCR inAspergillus nidulans. Mol Cell Biol 12:1932–1939PubMedGoogle Scholar
  27. Kulmburg P, Sequeval D, Lenouvel F, Mathieu M, Felenbok B (1992b) Specific binding sites for the activator protein, ALCR, in thealcA promoter of the ethanol regulon ofAspergillus nidulans. J Biol Chem 27:21146–21153Google Scholar
  28. Kulmburg P, Mathieu M, Dowzer C, Kelly J, Felenbok B (1993) Specific binding sites in thealcR andalcA promoters of the ethanol regulon for the CREA repressor mediating carbon catabolite control inAspergillus nidulans. Mol Microbiol 7:847–857PubMedGoogle Scholar
  29. Lockington RA, Scazzocchio C, Sequeval D, Mathieu M, Felenbok B (1987) Regulation ofalcR, the positively acting regulatory gene of the ethanol-utilization regulon ofAspergillus nidulans. Mol Microbiol 1:275–281PubMedGoogle Scholar
  30. Mathieu M, Felenbok B (1994) TheAspergillus nidulans CREA protein mediates glucose repression of the ethanol regulon at various levels through competition with the ALCR-specific transactivator. EMBO J 13:4022–4027PubMedGoogle Scholar
  31. McKnight GL, Kato H, Upshall, A, Parker MD, Saari G, O'Hara PJ (1985) Identification and molecular characterisation of a third alcohol dehydrogenase inAspergillus nidulans. EMBO J 4:2093–2099PubMedGoogle Scholar
  32. Oakley CE, Weil CF, Kretz PL, Oakley BR (1987) Cloning of theriboB locus ofAspergillus nidulans. Gene 53:293–298CrossRefPubMedGoogle Scholar
  33. Pateman JA, Doy CH, Olsen JE, Norris U, Creaser EH, Hynes MJ (1983) Regulation of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) inAspergillus nidulans. Proc R Soc Lond B 217:243–264PubMedGoogle Scholar
  34. Pickett M, Gwynne DI, Buxton FP, Elliott R, Davies RW, Lockington RA, Scazzocchio C, Sealy-Lewis HM (1987) Cloning and characterisation of thealdA gene ofAspergllus nidulans. Gene 51:217–222CrossRefPubMedGoogle Scholar
  35. Roberts T, Martinelli SD, Scazzocchio C (1979) Allele-specific, gene-unspecific suppressors inAspergillus nidulans. Mol Gen Genet 177:57–64CrossRefPubMedGoogle Scholar
  36. Russell PR, Hall BD (1983) The primary structure of the alcohol dehydrogenase gene from the fission yeastSchizosaccharomyces pombe. J Biol Chem 258:143–149PubMedGoogle Scholar
  37. Saleeba JA, Cobbett CS, Hynes MJ (1992) Characterisation of theamdA-regulatedaciA gene ofAspergillus nidulans. Mol Gen Genet 235:349–358CrossRefPubMedGoogle Scholar
  38. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  39. Samuelson L (1986) A thin-layer technique for improved resolution in two-dimensional immunoelecrophoresis. Int Arch Allergy Appl Immunol 79:140–144PubMedGoogle Scholar
  40. Sanger F, Nicklen S, Coulsen AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedGoogle Scholar
  41. Sealy-Lewis HM (1990) The identification of mutations inAspergillus nidulans that lead to increased levels of ADH II. Curr Genet 18:65–70CrossRefPubMedGoogle Scholar
  42. Sealy-Lewis HM, Fairhurst V (1992) An NADP+-dependent glycerol dehydrogenase inAspergillus nidulans is inducible by D-galacturonate. Curr Genet 22:293–296CrossRefPubMedGoogle Scholar
  43. Sealy-Lewis HM, Lockington RA (1984) Regulation of two alcohol dehydrogenases inAspergillus nidulans. Curr Genet 8:253–259CrossRefGoogle Scholar
  44. Tilburn J, Scazzocchio C, Taylor GG, Zabicky-Zissman JH, Lockington RA, Davies RW (1983) Transformation by integration inAspergillus nidulans. Gene 26:205–221CrossRefPubMedGoogle Scholar
  45. Woloshuk CP, Payne GA (1994) The alcohol dehydrogenase geneadh1 is induced inAspegillus flavus grown on medium conducive to aflatoxin biosynthesis. Appl Envir Microbiol 60:670–676Google Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • Gary D. Hunter
    • 1
  • I. Gwyn Jones
    • 1
  • Heather M. Sealy-Lewis
    • 1
  1. 1.Department of Applied BiologyUniversity of HullHullUK

Personalised recommendations