Lo Clive, Adachi Kiichi, Shuster Jeffrey R, Hamer John E, Hamer Lisbeth
Paradigm Genetics, Inc., 108 Alexander Drive, Research Triangle Park, NC 27709, USA.
Nucleic Acids Res. 2003 Aug 15;31(16):4822-7. doi: 10.1093/nar/gkg676.
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.
TAGKO是一种基于Tn7的转座系统，用于丝状真菌的全基因组诱变。在稻瘟病菌和小麦壳针孢中检测了转座子插入对TAGKO等位基因表达的影响。Northern分析表明，TAGKO突变体中表达了稳定的截短转录本。对TAGKO cDNA 3'末端的定位显示，无论转座子方向如何，它们都含有Tn7末端序列。真核基因特征性的聚腺苷酸化信号，在所有阅读框中都有终止密码子，位于细菌转座子的两端。因此，TAGKO转录本过早地进行了聚腺苷酸化，预计在真菌突变体中会翻译出截短的蛋白质。根据蛋白质截短的程度，HPD4（编码对羟基苯丙酮酸双加氧酶）中的TAGKO突变导致两种真菌对酪氨酸敏感。同样，一个特定的稻瘟病菌CBS1（编码胱硫醚β-合酶）TAGKO cDNA未能在酵母CBS突变体中补充半胱氨酸营养缺陷。因此，TAGKO是研究丝状真菌中截短基因产物的体内有用工具。
