<br/>The aim of this thesis is studying the genetics of <em>Phaffia</em> and to develop a genetic transformation system for this yeast. The genetic properties of <em>Phaffia</em> were studied on the gene and genome level.<p>As a first step the molecular structure of the <em>Phaffia</em> actin gene was analyzed. Actin genes are highly conserved throughout nature, and as such they have been used for the classification of significantly diverging eukaryotic groups, like (in-)vertebrates, plants and fungi.<p>We anticipated that the analysis of the primary structure of <em>Phaffia</em> actin gene and comparison with the actin genes from fungi, including 2 ascomycetous filamentous fungi, 2 basidiomycetous yeasts and 5 ascomycetous yeasts would provide further phylogenetic information on this yeast.<p>It was found that the <em>Phaffia</em> actin gene encoded a protein consisting of 375 amino acids. In addition 4 (non-coding) intervening sequences were present. Comparison of both the coding DNA sequence and its predicted protein product with their fungal counterparts, revealed that least homology was found with the ascomycetous yeasts, like <em>Saccharomyces cerevisiae</em> and <em>Kluyveromyces lactis</em> . It was also shown, that based on these comparisons <em>Phaffia</em> is closer related to the filamentous ascomycetous fungi <em>Thermomyces lanuginosus</em> and <em>Aspergillus nidulans</em> , whereas most homology was found with the basidiomycetous yeast <em>Filobasidiella neoformans</em> (perfect stage of <em>Cryptococcus neoformans</em> ).<p>In addition to the phylogenetic analysis of the actin exons, the architecture of the introns (splice site consensus sequences, size, position in the gene) was compared. it was shown that the <em>Phaffia</em> introns most resembled that of <em>Filobasidiella neoformans</em> where as least resemblance occurred with the ascomycetous yeasts. This result was in agreement with the actin exon homology studies. Furthermore, the presence of multiple introns in the <em>Phaffia</em> actin gene resembled the situation in the actin genes from <em>F. neoformans</em> and the filamentous fungi, whereas the ascomycetous yeasts only carry one intron in their actin genes.<p>Similar results were obtained by (phylo-)genetic analysis of the five introns containing <em>Phaffia</em> glyceraldehyde-3-phosphate dehydrogenase gene.<p>The genomic organization of the multiple rDNA genes in <em>Phaffia</em> was elucidated. It was found that <em>Phaffia</em> carries the rDNA genes in three clusters, of 12, 14 and 35 copies, on three different chromosomes. In the ascomycetous yeasts and fungi the rDNA is mainly present on one chromosome.<p>The significant differences on the gene and genome level with the ascomycetous yeasts affected the strategy for the development of a transformation system for <em>Phaffia</em> . Whereas several marker gene sequences or sequences for plasmid replication and maintenance can be readily interchanged between most ascomycetous species as a result of high homologies, it was shown that this was not the case for <em>Phaffia</em> . Therefore an almost entirely homologous transformation sytem was developed using plasmids carrying the dominant G418 resistance gene (Km <sup>R</SUP>), driven by either the <em>Phaffia</em> actin or the gpd promoter and a <em>Phaffia</em> ribosomal DNA (rDNA) fragment for homologous integration.<p>It was found that the rDNA clusters could serve as a target for high copy number integration. This integrative transformation system was used to determine the ploidy of <em>Phaffia</em> , strain CBS 6938, by monitoiring chromosomal shifts as a result of multiple integrations. It was found that this strain was haploid.<br/>Plasmids carrying the gpd promoter driven Km <sup>R</SUP>gene transformed <em>Phaffia</em> with significant higher efficiencies than constructs with the actin promoter. Furthermore, the plas-mid copy number and transformation efficiencies of the first were found to be influenced by the presence of the gpd terminator downstream the Km <sup>R</SUP>gene.<p>It was shown that plasmid amplification occurred independent from selection pressure to an extend that appeared to be negatively related to the effectiveness of expression of the Km <sup>R</SUP>gene. This observation indicated that the rising metabolic burden, as a consequence of amplification, imposes limits to the number of plasmid copies.<p>The effectiveness, stability, and plasmid amplifying properties of the <em>Phaffia</em> transformation system offer possibilities for the use of recombinant DNA technology in developing industrially attractive <em>Phaffia</em> strains.
|Qualification||Doctor of Philosophy|
|Award date||24 Oct 1997|
|Place of Publication||S.l.|
|Publication status||Published - 1997|