This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Skory, C D Articles by Linz, J E Search for Related Content PubMed PubMed Citation Articles by Skory, C D Articles by Linz, J E Agricola Articles by Skory, C D Articles by Linz, J E

 Previous Article  |  Next Article 

Appl Environ Microbiol. 1990 November; 56(11): 3315-3320

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

Department of Food Science and Human Nutrition, Michigan State University, East Lansing 48824.

ABSTRACT

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 microgram of DNA was developed for A. parasiticus by using the 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 [14C]OMP to [14C]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 [14C]OMP to [14C]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.

This article has been cited by other articles:

• Hong, S.-Y., Linz, J. E. (2008). Functional Expression and Subcellular Localization of the Aflatoxin Pathway Enzyme Ver-1 Fused to Enhanced Green Fluorescent Protein. Appl. Environ. Microbiol. 74: 6385-6396 [Abstract] [Full Text]  
• Hammond, T. M., Keller, N. P. (2005). RNA Silencing in Aspergillus nidulans Is Independent of RNA-Dependent RNA Polymerases. Genetics 169: 607-617 [Abstract] [Full Text]  
• Lee, L.-W., Chiou, C.-H., Linz, J. E. (2002). Function of Native OmtA In Vivo and Expression and Distribution of This Protein in Colonies of Aspergillus parasiticus. Appl. Environ. Microbiol. 68: 5718-5727 [Abstract] [Full Text]  
• Takahashi, T., Chang, P.-K., Matsushima, K., Yu, J., Abe, K., Bhatnagar, D., Cleveland, T. E., Koyama, Y. (2002). Nonfunctionality of Aspergillus sojae aflR in a Strain of Aspergillus parasiticus with a Disrupted aflR Gene. Appl. Environ. Microbiol. 68: 3737-3743 [Abstract] [Full Text]  
• Chiou, C.-H., Miller, M., Wilson, D. L., Trail, F., Linz, J. E. (2002). Chromosomal Location Plays a Role in Regulation of Aflatoxin Gene Expression in Aspergillus parasiticus. Appl. Environ. Microbiol. 68: 306-315 [Abstract] [Full Text]