|Skory, Christopher - Chris|
Submitted to: Gene
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/20/1996
Publication Date: N/A
Citation: Interpretive Summary: Aflatoxins are highly toxic and carcinogenic compounds produced by several species of Aspergillus fungi. Producers of agricultural commodities such as corn, peanuts, cassava, and cottonseed are concerned about production of aflatoxins in their crop. An understanding of the mechanisms involved in the synthesis of aflatoxins may provide better ways of preventing fungi from producing the toxin and thus improving the safety of many crops. We have developed a method to introduce foreign DNA into an aflatoxin producing strain using a selection system based on a fungicide commonly employed for control of fungal growth on most plants. The fungal strain, which is normally sensitive to the fungicide, was altered through mutation and selection to be resistant to the fungicide. The altered gene conferring this resistance was then isolated and characterized. It is possible to transfer this resistance to a fungicide sensitive strain by reintroducing the altered gene. By using this system, genes involved in the production of aflatoxin can be inserted into fungi so their function can be studied under various conditions.
Technical Abstract: A genomic DNA library prepared from a benomyl resistant strain of Aspergillus parasiticus was screened with a Neurospora crassa beta-tubulin gene probe. A unique A. parasiticus genomic DNA fragment, thought to carry a mutant beta-tubulin gene (ben(r)), was isolated. Two plasmids, pYT1 and pYTPYRG, carrying the putative ben(r) gene or ben(r) plus a second selectable marker (pyrG), respectively, were used to transform a benomyl sensitive strain of A. parasiticus (CS10) to determine if ben(r) conferred benomyl resistance (Ben(R)). Ben(R) colonies were obtained with pYTPYRG, pYT1 or pYT1 cotransformed with pPG3J which carries a functional pyrG gene. No Ben(R) colonies were obtained without added DNA or with pPG3J only (controls). Southern hybridization analysis of Ben(R) and Ben(S) transformants suggested that plasmid integration occurred most frequently at the chromosomal ben(s) locus; however, evidence for gene conversion and heterologous recombination was also observed. The predicted amino acid sequence of ben(r) displayed a high degree of identity (>93%) with other fungal beta-tubulin genes which confer benomyl resistance. Sequence analysis together with the genetic data suggested that ben(r) encodes a functional mutant beta-tubulin.