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

We have investigated the feasibility of using gene tagging by restriction enzyme-mediated integration (REMI) to isolate mutants in 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...

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Bibliographic Details
Published in:Molecular genetics and genomics : MGG 2001-12, Vol.266 (4), p.646-656
Main Authors: van Dijk, R, Faber, K N, Hammond, A T, Glick, B S, Veenhuis, M, Kiel, J A
Format: Article
Language:English
Subjects:
Amino acids
Base Sequence
Biosynthesis
DNA Restriction Enzymes - metabolism
Enzymes
Genes
Genes, Fungal
Genetic Markers
Genome, Fungal
Genomes
Genomics
Glucose
Molecular Sequence Data
Mutagenesis
Mutagenesis, Insertional - methods
Pichia - genetics
Plasmids
Recombination, Genetic
Transformation, Genetic
Yeast
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Summary:We have investigated the feasibility of using gene tagging by restriction enzyme-mediated integration (REMI) to isolate mutants in 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 (pdd). 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.
ISSN:1617-4615
1617-4623
DOI:10.1007/s004380100584