Molecular cloning of verrucosidin-producing Penicillium polonicum genes by differential screening to obtain a DNA probe.
A differential molecular screening procedure was developed to obtain DNA clones enriched for verrucosidin-related genes that could be used as DNA probes to detect verrucosidin-producing Penicillium polonicum. Permissive and nonpermissive conditions for verrucosidin production were selected to obtain differentiated poly (A)+ RNA for the cloning strategy. P. polonicum yielded the highest amount of verrucosidin when cultured in malt extract broth at 25 degrees C without shaking. These conditions were selected as verrucosidin permissive conditions. When shaking was applied to the verrucosidin permissive conditions, verrucosidin was not detected. Approximately 5000 transformants were obtained for the library of DNA fragments from verrucosidin-producing P. polonicum and hybridized with cDNA probes obtained from poly (A)+ RNA of permissive and nonpermissive conditions. A total of 120 clones hybridized only with the permissive cDNA probes. From these, eight representative DNA inserts selected on the basis of size and labelled with fluorescein-dUTP were assayed as DNA probes in the second differential screening by Northern hybridization. Probe SVr1 gave a strong hybridization signal selectively with poly (A)+ RNAs from high verrucosidin production. When this probe was assayed by dot blot hybridization with DNA of different moulds species, hybridization was detected only with DNA from the verrucosidin-producing strain. The strategy used in this work has proved to be useful to detect unknown genes related to mycotoxins. In addition, the DNA probe obtained should be considered for the detection of verrucosidin-producing moulds.
Aranda
E
E
Higiene de los Alimentos, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain.
Rodríguez
M
M
Benito
M J
MJ
Asensio
M A
MA
Córdoba
J J
JJ
D015152:Blotting, Northern; D003001:Cloning, Molecular; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010407:Penicillium / Q000235:genetics* / Q000378:metabolism; D011753:Pyrones / Q000378:metabolism*
D011753:Pyrones; C048224:verrucosidin