Genetic analysis of methylotrophic yeastCandida boidinii PLD1

This paper reports the initial experiments for genetic analysis of the haploid methylotrophic yeastCandida boidinii PLD1. The collection of multiply marked auxotrophic mutants was obtained after treatment with UV-light or X-rays. Protoplasts from several mutants were fused by the PEG-CA²⁺ technique and five prototrophic hybrids were isolated. The genetic structure of the hybrids was studied by means of spontaneous and induced mitotic segregation. Our data suggest that hybrids are diploids, heterozygous by parental auxotrophic markers. We obtained genetic linkage between mutationslys2-8-met-3 from one hand andade-17-arg-24 from the other. The genetic maps constructed showed similar characteristics concerning both the order of the markers and their map distances.     

Isolation and properties of genetically defined strains of the methylotrophic yeast Hansenula polymorpha CBS4732

Genetically defined strains of the yeast Hansenula polymorpha were constructed from a clone of H. polymorpha CBS4732 with very low mating and sporulation abilities. Mating, spore viability, and the percentage of four-spore-containing asci were increased to a level at which tetrad analysis was possible. Auxotrophic mutations in 30 genes were isolated and used to construct strains with multiple markers for mapping studies, transformation with plasmid DNA, and mutant screening. Various other types of mutants were isolated and characterized, among them mutants that displayed an altered morphology, methanol-utilization deficient mutants and strains impaired in the biosynthesis of alcohol oxidase and catalase. Also, the mutability of H. polymorpha CBS4732 vs H. polymorpha NCYC495 was compared. The data revealed clear differences in frequencies of appearance and mutational spectra of some mutants isolated. Many of the mutants isolated had good mating abilities, and diploids resulting from their crossing displayed high sporulation frequencies and high spore viability. Most of the markers used revealed normal Mendelian segregation during meiosis.The frequency of tetratype spore formation was lower than in Saccharomyces cerevisiae suggesting a lower frequency of recombination during the second meiotic division. The properties of genetically defined strains of H. polymorpha CBS4732 as well as their advantages for genetics and molecular studies are discussed.     

Mitotic segregation in hybrid of methylotrophic yeastCandida pelliculosa

Protoplasts from two autoxotrophic mutants of methylotrophic yeastCandida pelliculosa were fused by the PEG technique, and the genetic structure of the hybrid obtained was studied by means of mitotic segregation. Spontaneous mitotic segregation depended on the composition of the medium and the type of carbon source used. Mechanisms by which segregants appeared were studied by means of UV- and benomyl-induced mitotic segregation. Our data suggest that one large percentage of segregants (Leu ^–) occurred as a result of mitotic chromosome loss. The other large portion of segregants (Ade ^– andLys ^–) are a consequence of mitotic recombination.     

Pyruvate carboxylase is an essential protein in the assembly of yeast peroxisomal oligomeric alcohol oxidase

Hansenula polymorpha ass3 mutants are characterized by the accumulation of inactive alcohol oxidase (AO) monomers in the cytosol, whereas other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. These mutants also have a glutamate or aspartate requirement on minimal media. Cloning of the corresponding gene resulted in the isolation of the H. polymorpha PYC gene that encodes pyruvate carboxylase (HpPyc1p). HpPyc1p is a cytosolic, anapleurotic enzyme that replenishes the tricarboxylic acid cycle with oxaloacetate. The absence of this enzyme can be compensated by addition of aspartate or glutamate to the growth media. We show that HpPyc1p protein but not the enzyme activity is essential for import and assembly of AO. Similar results were obtained in the related yeast Pichia pastoris. In vitro studies revealed that HpPyc1p has affinity for FAD and is capable to physically interact with AO protein. These data suggest that in methylotrophic yeast pyruvate carboxylase plays a dual role in that, besides its well-characterized metabolic function as anapleurotic enzyme, the protein fulfils a specific role in the AO sorting and assembly process, possibly by mediating FAD-binding to AO monomers.