The bacterial transposon Tn7 causes premature polyadenylation of mRNA in eukaryotic organisms: TAGKO mutagenesis in filamentous fungi.
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 beta-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.
Lo
Clive
C
Paradigm Genetics, Inc., 108 Alexander Drive, Research Triangle Park, NC 27709, USA. clivelo@hkucc.hku.hk
Adachi
Kiichi
K
Shuster
Jeffrey R
JR
Hamer
John E
JE
Hamer
Lisbeth
L
D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000235:genetics; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003541:Cystathionine beta-Synthase / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D020082:Magnaporthe / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D011061:Poly A / Q000235:genetics* / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D012331:RNA, Fungal; D012333:RNA, Messenger; D011061:Poly A; D015111:4-Hydroxyphenylpyruvate Dioxygenase; D003541:Cystathionine beta-Synthase