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Molecular and General Genetics MGG

, Volume 248, Issue 2, pp 142–150 | Cite as

Efficient gene targeting in the filamentous fungusAlternaria alternata

  • Hiroshi Shiotani
  • Takashi Tsuge
Original Paper

Abstract

To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1) transformants accounted for 30 to 80% of hygromycin B-resistant (HyR) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1 transformants accounted for only 5% of HyR transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.

Key words

Alternaria alternata Gene targeting Melanin biosynthesis gene 

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Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Hiroshi Shiotani
    • 1
  • Takashi Tsuge
    • 1
  1. 1.School of Agricultural SciencesNagoya UniversityChikusa, NagoyaJapan

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