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Nucleic Acids Research, 2003, Vol. 31, No. 16 4822-4827
© 2003 Oxford University Press

The bacterial transposon Tn7 causes premature polyadenylation of mRNA in eukaryotic organisms: TAGKO mutagenesis in filamentous fungi

Clive Lo*,1,2, Kiichi Adachi1, Jeffrey R. Shuster1, John E. Hamer1 and Lisbeth Hamer1

1 Paradigm Genetics, Inc., 108 Alexander Drive, Research Triangle Park, NC 27709, USA and 2 Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China

*To whom correspondence should be addressed at Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China. Tel: +852 2299 0337; Fax: +852 2858 3477; Email: clivelo{at}hkucc.hku.hk
Present addresses:
Kiichi Adachi, Mitsui Global Strategic Studies Institute, 1-2-1 Ohtemachi Chiyoda-ku, Tokyo, Japan
Lisbeth Hamer, Department of Microbiology, North Carolina State University, Raleigh, NC 27695, USA

TAGKO is a Tn7-based transposition system for genome wide mutagenesis in filamentous fungi. The effects of transposon insertion on the expression of TAGKO alleles were examined in Magnaporthe grisea and Mycosphaerella graminicola. Northern analysis showed that stable, truncated transcripts were expressed in the TAGKO mutants. Mapping of the 3'-ends of TAGKO cDNAs revealed that they all contain Tn7 end sequences, regardless of the transposon orientation. Polyadenylation signals characteristic of eukaryotic genes, preceded by stop codons in all frames, are located in both ends of the bacterial transposon. Thus, TAGKO transcripts are prematurely polyadenylated, and truncated proteins are predicted to be translated in the fungal mutants. Depending on the extent of protein truncation, TAGKO mutations in HPD4 (encoding p-hydroxyphenylpyruvate dioxygenase) resulted in tyrosine sensitivity in the two fungi. Similarly, a particular M.grisea CBS1 (encoding cystathionine ß-synthase) TAGKO cDNA failed to complement cysteine auxotrophy in a yeast CBS mutant. TAGKO, therefore, represents a useful tool for in vivo study of truncated gene products in filamentous fungi.


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