Position-dependent regulation of the nor-1 promoter in the aflatoxin gene cluster and immuno-localization of proteins involved in aflatoxin biosynthesis

The objectives of my research were to: (1) study the positional-effects on aflatoxin (AF) gene (nor-1) expression in the gene cluster; (2) generate highly-specific antibodies against an AF protein (anti-VBS) for immuno-localization studies; (3) develop methods to localize AF proteins within a fungal colony; and (4) localize proteins involved in AF biosynthesis in a time-course dependent fractionated colony.; To study positional effects on nor-1 promoter function, a plasmid pAPGUSNP containing the nor-1 promoter sequence fused to a reporter gene (uidA encoding β-glucuronidase, GUS) plus a pyrG selectable marker was constructed. Transformation of pAPGUSNP into CS10 (ver-1, wh-1, pyrG) resulted in homologous recombination at 5′ nor-1, 3′ nor-1, or the pyrG locus. The plasmid integration site was screened by a novel rapid DNA extraction/PCR assay and confirmed by Southern hybridization analysis. The nor-1 promoter was positively regulated in the AF gene cluster while outside the cluster (pyrG locus), nor-1 promoter activity decreased dramatically. The data supported the hypothesis that expression of AF genes is cluster position-dependent.; The second hypothesis was tested by studying localization AF proteins utilizing anti-VBS antibodies raised against recombinant Maltose Binding Protein-VBS expressed in Escherichia coli (generated in this study), and other antibodies available in the laboratory (anti-Nor-1, Ver-1, and OmtA). A 72 h-old colony was fractionated based on 24 h intervals to correlate the time of growth with AF protein distribution. Colony fractions were fixed and embedded in paraffin to generate 4–5 μm sections for immuno-fluorescence labeling. Confocal Laser Scanning Microscopy (CLSM) demonstrated that Nor-1, Ver-1, OmtA, and VBS were evenly distributed among different cell types and not concentrated in conidiophores, contrary to the hypothesis. These proteins were not evenly distributed throughout a 72 h-old colony. Among three fractions, colony fraction S2 (24 to 48 h growth) exhibited the highest abundance of Nor-1, Ver-1, OmtA, and VBS (analyzed by CLSM and quantitative fluorescence intensity analysis), the highest levels of AFB₁ (determined by enzyme-linked-immunosorbent-assay), and the highest numbers of newly-developed conidiophores. These data support a spatial and temporal association between AF protein expression/AF production and sporulation at the colony level. (Abstract shortened by UMI.)...