DNA-mediated transformation in the aquatic filamentous fungus Achlya ambisexualis

• Published in: Journal of general microbiology Vol. 134; no. 7; pp. 2019 - 2028
• Main Authors: Manavathu, E.K, Suryanarayana, K, Hasnain, S.E, Leung, W.C
• Format: Journal Article
• Language: English
• Published: England Soc General Microbiol 01.07.1988
• Subjects: Achlya ambisexualis; Chytridiomycota - genetics; Culture Media; DNA; DNA, Fungal - genetics; Drug Resistance, Microbial; Freshwater; fungi; gene expression; Gentamicins - pharmacology; Immunoblotting; Oomycetes - drug effects; Oomycetes - genetics; Plasmids; Protoplasts; secretions; Transformation, Genetic
• ISSN: 0022-1287; 1350-0872; 1465-2080
• DOI: 10.1099/00221287-134-7-2019

1 Departments of Medical Microbiology & Infectious Diseases, Edmonton, Alberta T6G 2H7, Canada 2 Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada ABSTRACT Summary: A DNA-mediated transformation system was developed for the aquatic filamentous fungus Achlya ambisexualis using the chimeric plasmid vector pSV2neo2µm. Hyphal colonies resistant to the neomycin analogue G-418 sulphate were regenerated from transformed protoplasts on soft agar. Southern blot analyses of the transformed-cell DNA produced multiple hybridization bands, suggesting integration of vector DNA into the host genome at multiple sites. Northern blot analyses revealed the presence of three APHII-gene-specific transcripts in the trans-formant, indicating that the G-418-resistant phenotype was due to the expression of the APHII gene. The presence of multiple RNA transcripts of unexpectedly large size suggested that RNA initiation and/or termination is under the control of regulatory element(s) other than the SV40 promoter. Plasmid DNAs recovered by transformation of Escherichia coli cells with total DNA preparations from the fungal transformants showed considerable DNA rearrangements. However, at least a portion of the plasmid DNA recovered from each of the transformants carried a functional APHII gene, suggesting that the episomal vector DNA may have played a role in maintaining the G-418-resistant phenotype.