|Harris Coward, Pamela|
Submitted to: ASM Conference
Publication Type: Abstract Only
Publication Acceptance Date: 3/1/2005
Publication Date: N/A
Technical Abstract: Aflatoxins (AF) are highly carcinogenic secondary metabolites produced mainly by Aspergillus flavus and A. parasiticus. The main focus of our lab is to understand the genetic factors that control AF production using A. parasiticus as a model system. Many eukaryotes, including Aspergillus spp., modify their DNA by addition of a methyl group to the five position of selected cytosine residues; and this methylation plays a role in controlling gene expression. In this study, we report the isolation and partial characterization of non-aflatoxigenic isolates obtained with the DNA-demethylating agent 5-azacytidine (5-AZA). Spore color and auxotrophic strains producing AF or pathway intermediates (e.g. br nor ade, wh ver lys) were subjected to 0, 0.5, 1.0, and 2.0 mM 5-AZA treatment for 36, 48, and 60 hrs. in minimal medium (A&M). Resulting mycelia were harvested, washed, and placed on sterile filter paper in deep-dish Petri plates containing 10 ml complex medium (YG). After 48 hr. incubation, the spores were harvested and plated on Czapek's potato dextrose agar and PDA media for colony isolation. In general, two phenotypic classes of isolates were obtained 1) non-toxigenic (NT) with normal to altered morphology and 2) toxigenic (T) with altered morphology. No reversion was found in physiological analyses of representative NT isolates using AF-supporting media (PDA or YES), pH ranges 4-7, and temperature ranges 29-37°C for confirming their stability. Preliminary quantitative RT-PCR detected about 50% decrease in steady state levels of aflR mRNA and 90% decrease mRNA of aflJ in the NTs compared to the wild-type controls at 48 and 64 hrs. (aflR and aflJ are positive regulators of AF). These results are similar to those obtained with non-toxigenic sec- variants, a phenotype of fungal development obtained in our lab by mechanical manipulation using repeated mycelial maceration and transfer. These studies suggest that the sec-variant could result from an alteration in DNA methylation patterns in the strains upon repeated mycelial transfer, patterns similar to those obtained with 5-AZA treatment.