Molecular and functional characterization of a second copy of the aflatoxin regulatory gene, aflR-2, from Aspergillus parasiticus.
The genes required for the synthesis of aflatoxin (AF) in Aspergillus flavus and Aspergillus parasiticus have been shown to be clustered on a chromosome in these fungi. Transcription of most of these genes is dependent upon the activity of the aflR gene, also present on the gene cluster, which encodes a zinc binuclear cluster DNA-binding protein. While many strains of A. parasiticus have only one copy of aflR (aflR-1), many others contain a second copy of this gene (aflR-2) which resides on a duplicated region of the aflatoxin gene cluster. Targeted disruption of aflR-1 generated a number of non-aflatoxin producing transformants of A. parasiticus SU-1 which still harbored a wild-type aflR-2 gene. Southern and Northern hybridization analyses and ELISA assays demonstrated that aflR-1 had been successfully inactivated in strain AFS10. DNA sequence analysis showed that aflR-2 was capable of encoding a deduced 47 kDa protein. Northern and RT-PCR analysis of RNA from a toxin producing strain indicated that aflR-2 was transcribed at extremely low levels compared to aflR-1. RT-PCR analysis of RNA from AFS10 demonstrated that mRNAs of aflatoxin pathway genes were not processed to their mature forms. Functional analysis of aflr-2 protein in a yeast system showed that it was not activating transcription.
Cary
Jeffrey W
JW
Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Robert E. Lee Blvd., New Orleans, LA 70124, USA. jcary@nola.srrc.usda.gov
Dyer
John M
JM
Ehrlich
Kenneth C
KC
Wright
Maureen S
MS
Liang
Shun-Hsin
SH
Linz
John E
JE
D000348:Aflatoxins / Q000235:genetics*; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics*; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors
C113837:AFLR protein, Aspergillus; C464521:AFLR-2 protein, Aspergillus parasiticus; D000348:Aflatoxins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D014157:Transcription Factors