Abstract.A highly-efficient method for transformation of the methylotrophic yeast Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7 x 106/gg plasmid DNA were obtained by applying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformation was dependent on: (1) pretreatment of the cells with the reducing agent dithiotreito1, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to the mid-logarithmic phase. Important parameters for optimizing the transformation frequencies were field strength, pulse duration, and cell concentration during the electric pulse. In contrast to electrotransformation protocols described for Saccharomyces cerevisiae and Candida maltosa, transformation frequencies (transformants per gg DNA) for H. poIymorpha remained high when large amounts (up to 10 gg) of plasmid DNA were added. This feature renders this procedure pre-eminently advantageous for gene cloning experiments when high numbers of transformants are needed.

Abstract. We describe the cloning of the Hansenula polymorpha PERI gene and the characterization of the gene and its product, PERlp. The gene was cloned by functional complementation of a per1 mutant of H. polymorpha, which was impaired in the import of peroxisomal matrix proteins (Pim-phenotype). The DNA sequence of PER1 predicts that PERlp is a polypeptide of 650 amino acids with no significant sequence similarity to other known proteins. PER1 expression was low but significant in wild-type H. polymorpha growing on glucose and increased during growth on any one of a number of substrates which induce peroxisome proliferation. PERlp contains both a carboxy-(PTS1) and an amino-terminal (PTS2) peroxisomal targeting signal which both were demonstrated to be capable of directing bacterial B-lactamase to the organelle. In wild-type H. polymorpha PERlp is a protein of low abundance which was demonstrated to be localized in the peroxisomal matrix. Our results suggest that the import of PERlp into peroxisomes is a prerequisite for the import of additional matrix proteins and we suggest a regulatory function of PERlp on peroxisomal protein import. UKARYOTIC cells are characterized by the compartmentalization of various metabolic functions into separate subeellular organelles. Each organelle contains a characteristic set of proteins to accomplish specific metabolic functions essential for the cell. Microbodies (peroxisomes, glyoxysomes) represent the most recently discovered class of organelles, which are ubiquitous in higher and lower eukaryotic organisms (Borst, 1989;van den Bosch et al., 1992). They are involved in a variety of metabolic functions (Lazarow and Kindl, 1982;Veenhuis and Harder, 1991;van den Bosch et al., 1992) and in many cases their presence appears to be essential for the cell's viability. Consequently, the organelles have been intensively studied and in recent years the knowledge on the molecular mechanisms of microbody biogenesis and function is rapidly expanding.It is now generally accepted that upon their induction microbodies develop by multiplication of preexisting orAddress all correspondence to M.

Using the bifunctional cloning vehicle pHP13, which carries the replication functions of the cryptic Bacillus subtilis plasmid pTA1060, the effects of BsuM restriction on the efficiency of shotgun cloning of heterologous Escherichia coli DNA were studied. In a restriction-deficient but modification-proficient mutant of B. subtilis, clones were obtained at a high frequency, comparable to frequencies normally obtained in E. coli (10(4) clones per microgram target DNA). Large inserts were relatively abundant (26% of the clones contained inserts in the range of 6 to 15 kb), which resulted in a high average insert length (3.6 kb). In the restriction-proficient B. subtilis strain, the class of large inserts was underrepresented. Transformation of B. subtilis with E. coli-derived individual recombinant plasmids was affected by BsuM restriction in two ways. First, the transforming activities of recombinant plasmids carrying inserts larger than 4 kb, were, in comparison with the vector pHP13, reduced to varying degrees in the restricting host. The levels of the reduction increased with insert length, resulting in a 7800-fold reduction for the largest plasmid used (pC23; insert length 16 kb). Second, more than 80% of the pC23 transformants in the restricting strain contained a deleted plasmid. In the non-restricting strain, the transforming activities of the plasmids were fairly constant as a function of insert length (in the range of 0-16 kb), and no structural instability was observed. It is concluded that for shotgun cloning in B. subtilis, the use of restriction-deficient strains is highly preferable. Evidence is presented that in addition to XhoI other sequences are involved in BsuM restriction. It is postulated that AsuII sites are additional target sites for BsuM restriction.

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