The development of microbodies in the yeast Saccharomyces cerevisiae was studied in response to different conditions of growth. Various strains of S. cerevisiae were investigated, using cells from the exponential growth phase on glucose as an inoculum in all transfer experiments. Electron microscopy, including serial sectioning, revealed that these cells generally contained one to four small microbodies which were localized in the vicinity of the cell wall and characterized by the presence of catalase. Transfer of these glucose-grown cells into media supplemented with various compounds known to induce microbody proliferation in other yeasts--i.e. uric acid, alkylated amines, amino acids, C2-compounds such as ethanol or acetate, in the presence or absence of compounds that induce oxygen radical formation--did not result in a significant change in the number of microbody profiles observed. Marked microbody proliferation was, however, observed after a shift of cells into media containing oleic acid and was associated with the induction of activities of beta-oxidation enzymes. In addition, catalase and isocitrate lyase were present in enhanced levels. Kinetic experiments suggested that these microbodies developed from those originally present in the inoculum cells. In thin sections up to 14 microbody profiles were occasionally observed, often present in small clusters. Their ultimate volume fraction amounted to 8-10% of the cytoplasmic volume.

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.

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