Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis

Journal Article · Thu Nov 01 00:00:00 EST 1990 · Applied and Environmental Microbiology; (USA)

OSTI ID:5707337

Skory, C D; Horng, J S; Pestka, J J; Linz, J E ^[1]

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1. Michigan State Univ., East Lansing (USA)

The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per {mu}g of DNA was developed for A. parasiticus by using homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert ({sup 14}C)OMP to ({sup 14}C)UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert ({sup 14}C)OMP to ({sup 14}C)UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.

• OSTI ID: 5707337
• Journal Information: Applied and Environmental Microbiology; (USA), Vol. 56:11; ISSN 0099-2240
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AFLATOXINS
BIOSYNTHESIS
ASPERGILLUS
MOLECULAR BIOLOGY
PYRIMIDINES
GENES
CARBON 14 COMPOUNDS
DNA-CLONING
TRACER TECHNIQUES
ANTIGENS
AZINES
CLONING
DNA HYBRIDIZATION
EUMYCOTA
FUNGI
HETEROCYCLIC COMPOUNDS
HYBRIDIZATION
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MATERIALS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PLANTS
SYNTHESIS
TOXIC MATERIALS
TOXINS
550401* - Genetics- Tracer Techniques