ANALYSIS OF MECHANISMS REGULATING EXPRESSION OF THE VER-1 GENE, INVOLVED IN AFLATOXIN BIOSYNTHESIS

Previous studies have shown that ver-1A encodes an enzyme which is dir ectly involved in the conversion of versicolorin A to demethylsterigma tocystin during aflatoxin B-1 (AFB(1)) biosynthesis in the filamentous fungus Aspergillus parasiticus. In this study, two different tools we re utilized to study the regulation of ver-1A expression at the level of transcription and protein accumulation, First, a ver-1A cDNA was ex pressed in Escherichia coli with the vector pMAL-c2. The resulting mal tose-binding protein-Ver-1A fusion protein was purified and used to ge nerate polyclonal antibodies, Western blot analyses showed that these antibodies specifically recognized the Ver-1 protein (similar to 28 kD a) in cell extracts of Aspergillus parasiticus SU1, Second, a GUS (uid A; encodes beta-glucuronidase) reporter system was developed by fusing the ver-1A promoter and transcription terminator to the GUS gene, Rep orter constructs were transformed into A. parasiticus, resulting in a single copy of the ver-1A-GUS reporter integrated adjacent to the wild -type ver-1A gene (3' end) in the chromosome, Western blot analysis, N orthern hybridization analysis, and a GUS activity assay were used to analyze transformants, The timing of appearance and pattern of accumul ation of GUS transcript and GUS protein in transformants were consiste nt with the timing of appearance and pattern of accumulation of ver-1 transcript and Ver-1 protein, These data suggested that the GUS gene w as under the same regulatory control as the wild-type ver-1 gene and c onfirmed that transcriptional regulation plays an important role in ve r-1A expression. Integration of the ver-1A-GUS reporter construct at t he niaD locus resulted in 500-fold-lower GUS activity, but the tempora l pattern of accumulation of GUS activity was dot affected, Therefore, chromosomal location can play a role in determining the level of gene expression in A, parasiticus and should be an important consideration when analyzing promoter function in this organism.