Molecular characterization of an Aspergillus parasiticus dehydrogenase gene, norA, located on the aflatoxin biosynthesis gene cluster.
An Aspergillus parasiticus cDNA library was screened with monoclonal antibody raised against a purified A. parasiticus 43-kDa protein demonstrating norsolorinic acid reductase (NOR) activity. One immunopositive clone contained a cDNA insert of 1,418 bp. DNA sequence analysis of this cDNA identified an open reading frame of 1,167 bp that represented the norA gene. The deduced amino acid sequence of the norA coding region consisted of 388 residues capable of encoding a polypeptide of 43.7 kDa. Southern blot analysis of genomic DNA from A. parasiticus indicated that there may be an additional copy of norA. Western blot (immunoblot) analysis of crude protein extracts of A. parasiticus mycelia demonstrated a band of reactivity at 43 kDa only when the fungus was grown in a medium conducive to aflatoxin biosynthesis. Northern (RNA) blot analysis of total RNA from the fungus demonstrated a band of hybridization at about 1.5 kb. As observed with the fungal NORA protein, the norA transcript was present only when the fungus was grown in medium conducive to aflatoxin biosynthesis. Hybridization of the norA cDNA with cosmid DNAs known to encompass a major portion of the A. parasiticus and Aspergillus flavus aflatoxin biosynthetic pathway gene cluster placed the norA gene coding region just upstream of the ver-1 gene. The deduced amino acid sequence of norA had 49% amino acid identity with that of an aryl-alcohol dehydrogenase (aad) gene from Phanerochaete chrysosporium.
Cary
J W
JW
Southern Regional Research Center, U.S. Department of Agriculture, New Orleans, Louisiana 70179, USA. jcary@nola.srrc.usda.gov
Wright
M
M
Bhatnagar
D
D
Lee
R
R
Chu
F S
FS
D000348:Aflatoxins / Q000096:biosynthesis / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000276:immunology; D000445:Aldehyde Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology / Q000235:genetics; D002874:Chromosome Mapping; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics; D005810:Multigene Family; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; D016366:Open Reading Frames; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000348:Aflatoxins; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D009097:Multienzyme Complexes; C085655:AAD protein, Clostridium acetobutylicum; D000429:Alcohol Oxidoreductases; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; C062609:norsolorinic acid ketoreductase; D000445:Aldehyde Oxidoreductases