Hamer L, Adachi K, Montenegro-Chamorro M V, Tanzer M M, Mahanty S K, Lo C, Tarpey R W, Skalchunes A R, Heiniger R W, Frank S A, Darveaux B A, Lampe D J, Slater T M, Ramamurthy L, DeZwaan T M, Nelson G H, Shuster J R, Woessner J, Hamer J E
Paradigm Genetics, 108 Alexander Drive, Research Triangle Park, NC 27709, USA.
Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):5110-5. doi: 10.1073/pnas.091094198. Epub 2001 Apr 10.
Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.
丝状真菌是一大类多样且具有重要经济意义的微生物。由于其基因组较大且转化过程中靶向整合（TI）速率较低，在芽殖酵母中开发的大规模基因破坏策略不适用于这些生物体。我们开发了转座子阵列基因敲除（TAGKO）技术来发现基因，并同时创建基因破坏盒用于后续转化和突变分析。携带细菌和真菌抗药标记的转座子用于在体外诱变单个黏粒或整个文库。通过对转座子插入位点进行DNA序列分析来注释黏粒，并释放黏粒插入片段以指导基因组中的插入诱变事件。基于对黏粒插入片段和真菌黏粒文库中插入情况的饱和分析，我们表明TAGKO可用于快速鉴定和突变基因。我们进一步表明插入可导致基因表达改变，并且我们已使用这种方法研究了两种重要的真菌植物病原体中的氨基酸氧化途径。
