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

Abstract

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.

Transcriptional Down-regulation of Peroxisome Numbers Affects Selective Peroxisome Degradation in Hansenula polymorpha

“…Two Mut Ϫ mutants, designated mpp1-1 and mpp1-2 (previously designated ARJ-59; see Ref. 4), were studied further.…”

“…The genomic regions of pANL7 and pREMI-59 were initially sequenced using vector-based primers (4). Sequence analysis showed that the pREMI-Z vector had integrated in mutants mpp1-1 and mpp1-2 at two different locations in the same gene that was designated MPP1.…”

Routing ofHansenula polymorphaAlcohol Oxidase: An Alternative Peroxisomal Protein-sorting Machinery

“…The H. polymorpha strains used in this study are H. polymorpha NCYC 495 (leu1.1; Gleeson and Sudbery, 1988), pex3::URA3 (leu1.1.pex3; Baerends et al, 1996), pex5::URA3 (leu1.1.pex5; Salomons et al, 2000a), and HF295, a DHAS deletion strain (leu1.1.⌬DHAS; van Dijk R. et al, 2001). Yeast cells were grown at 37°C in selective minimal media containing 0.67% yeast nitrogen base without amino acids supplemented with 0.5% glucose (YND) or 0.5% methanol (YNM) or in mineral media (van Dijken et al, 1976) containing 0.25% ammonium sulfate as nitrogen source supplemented with 0.5% glucose, 0.5% methanol, or 0.5% methanol ϩ 0.1% glycerol as carbon sources.…”

Hansenula polymorphaPex20p is an oligomer that binds the peroxisomal targeting signal 2 (PTS2)

“…The H. polymorpha PEX20 gene was identified by RALF mutagenesis (van Dijk et al, 2001). DNA sequencing was performed at Baseclear (Leiden, The Netherlands) using a Licor automated DNA sequencer and dye primer chemistry (LiCor, Lincoln, NB).…”