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Tagging Hansenula polymorpha genes by random integration of linear DNA fragments (RALF)

van Dijk, R., Faber, KN., Hammond, AT., Glick, BS., Veenhuis, M. & Kiel, JAKW., Dec-2001, In : MOLECULAR GENETICS AND GENOMICS. 266, 4, p. 646-656 11 p.

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We have investigated the feasibility of using gene tagging by restriction enzyme-mediated integration (REMI) to isolate mutants in 11(Hansenula polymorpha. A plasmid that cannot replicate in H. polymorpha and contains a dominant zeocin resistance cassette. pREMI-Z. was used as the integrative/mutagenic plasmid. We observed that high transformation efficiency was primarily dependent on the use of linearised pREMI-Z, and that the addition of restriction endonuclease to linearised pREMI-Z prior to transformation increased the transformation frequency only slightly. Integration of linearised pREMI-Z occurred at random in the H. polymorpha genome. Therefore, we termed this method Random integration of Linear DNA Fragments (RALF). To explore the potential of RALF in H. polymorpha. we screened a collection of pREMI-Z transformants for mutants affected in peroxisome biogenesis (pex) or selective peroxisome degradation (padd). Many previously described PEX genes were obtained from the mutant collection . as well as a number of new genes, including H. polymorpha PEX12 and genes whose function in peroxisome biogenesis is still unclear. These results demonstrate that RALF is a powerful tool for tagging genes in H. polymorpha that should make it possible to carry Out genome-wide mutagenesis screens.

Original languageEnglish
Pages (from-to)646-656
Number of pages11
JournalMOLECULAR GENETICS AND GENOMICS
Volume266
Issue number4
Publication statusPublished - Dec-2001

    Keywords

  • gene tagging, genomic integration, mutants, peroxisome homeostasis, yeast, ENZYME-MEDIATED INTEGRATION, DEVELOPMENTAL GENES, METHANOL METABOLISM, PLASMID DNA, YEAST, PROTEIN, TRANSFORMATION, IMPORT, DEGRADATION, MUTAGENESIS

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