Preparation and Characterization of Aflatoxin B1-1-(O-carhoxymethyl) Oxime

182

123

DNA isolated from the wild-type afiatoxin-producing (Afl+) fungus Aspergiius parasiticus NRRL 5862 was used to construct a cosmid genomic DNA library employing the homologous gene (pyrG) encoding orotidine monophosphate decarboxylase for selection of fungal transformants. The cosmid library was transformed into an Aflmutant, A. parasiticus CS1O (ver-1 wh-l pyrG), deficient in the conversion of the aflatoxin biosynthetic intermediate versicolorin A to sterigmatocystin. One pyrG+ Afl+ transformant was identified. DNA fragments from this transformant, recovered by marker rescue, contained part of the cosmid vector including the pyrG gene, the amp' gene, and a piece of the original genomic insert DNA. Transformation of these rescued DNA fragments into A. parasiticus CS10 resulted in production of wild-type levels of afiatoxin and abundant formation of sclerotia. The gene responsible for this complementation (ver-1) was identified by Northern RNA analysis and transformation with subcloned DNA fagments. The approximate locations of transcription initiation and polyadenylation sites of ver-l were determined by an RNase protection assay and cDNA sequence analysis. The predicted amino acid sequence, deduced from the ver-l genomic and cDNA nucleotide sequences, was compared with the EMBL and GenBank data bases. The search revealed striking similarity with Streptomyces ketoreductases involved in polyketide biosynthesis.

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of a gene from Aspergillus parasiticus associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis

Skory

Chang

Cary

et al. 1992

182

123

“…Production of afla B1 antisera. Afla B, was first converted to afla Bi-o-carboxymethyloxime (afla B,oxime) by the method of Chu et al (5). The oxime derivative was then conjugated to BSA in the presence of 1-ethyl-3,3-diethylamino-propyl-carbodiimide (6), yielding a ratio of 5 mol of B, per mol of BSA.…”

Section: Methodsmentioning

confidence: 99%

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay

Pestka¹,

Gaur²,

Chu³

1980

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins Ml and B2a had no effect on the assay.

“…Aflatoxin analysis. Aflatoxin concentrations were determined by an enzymelinked immunoassay with aflatoxin B 1 monoclonal antibodies (8). The antibody and an aflatoxin B 1 horseradish peroxidase conjugate were kindly provided by J. Pestka (Michigan State University).…”

Section: Methodsmentioning

confidence: 99%

A beta-glucuronidase reporter gene construct for monitoring aflatoxin biosynthesis in Aspergillus flavus

Flaherty

Weaver

Payne

et al. 1995

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and A. parasiticus. Current research is directed at the elimination of these compounds in important food sources. The objective of this research was to develop a method to study the induction and regulation of aflatoxin biosynthesis by examining the expression of one aflatoxin pathway gene, ver1. The promoter region of ver1 was fused to the ␤-glucuronidase (GUS) gene (uidA) from Escherichia coli to form the reporter construct, GAP13. A. flavus 656-2 was transformed with this construct. Aflatoxin production, GUS activity, and transcript accumulation were determined in transformants after shifting the cultures from a nonconducive medium to a medium conducive to aflatoxin biosynthesis. Transformants harboring GAP13 displayed GUS expression only when aflatoxin was detected in culture. Further, the transcription of the uidA gene driven by the ver1 promoter followed the same profile as for the ver1 genes. The results show that the GAP13 construct may be useful as a genetic tool to study the induction of aflatoxin in situ and to identify substances that affect the expression of genes involved in aflatoxin biosynthesis. The utility of this construct to detect inducers of aflatoxin biosynthesis in maize kernels was tested in a bioassay. A heat-stable inducer of aflatoxin with a molecular size of less than 10 kDa was detected in extracts from maize kernels colonized by A. flavus.