Analysis of genetic markers in new breeding stocks of the yeast Saccharomycopsis lipolytica

For development of breeding stocks of S. lipolytica for genetic investigations we isolated and identified several auxotrophic mutants. After selection of strains with normal meiotic segregation pattern for the markers metA, lysA, argA, leuA, uraA, hisA it was shown that the loci metA and lysA and the loci argA and leuA are linked, crossing over frequencies are 25 ± 0.9% and 30 ± 1.2%, respectively. We identified several complementation groups. The marker uraA is allel to ura3 from strains of the Donner Laboratory (USA). These results are the basis for selection of special mutants for forthcoming investigations. The existence of linkage fragments makes it possible to use these strains as tester strains for genetic analysis.     

Genetic studies on the yeast Saccharomycopsis lipolytica Inactivation and mutagenesis

Spontaneous mutants of Saccharomycopsis lipolytica were selected and partially characterized. Several antibiotics and antimetabolites were used for selection of spontaneous resistant mutants from Saccharomycopsis lipolytica. The frequencies of such mutants were mainly arranged between 1 × 10^?7 and 5 × 10^?6 mutants per cell. But one class of glucosamine resistant mutants (GAM^RA) occurred more frequently. Among the resistant mutants different types of dominant and recessive resistant mutants could be observed. UV light was used for inactivation of cells and induction of mutants from S. lipolytica. Comparing four haploid strains only small differences were detected in sensitivity to UV light. UV light at a dosage of 135 J/m² was applied to increase the mutant frequencies in three haploid strains. Besides auxotrophic, temperature sensitive and colony morphology mutants, some new mutant types like small colony forming mutants, red-brown coloured mutants, allylalcohol, glucosamine, 2-deoxyglucose or nystatin resistant mutants, hitherto not described for S. lipolytica, were isolated and partially characterized.     

Isolation and characterization of mitochondrial DNA from the alkane yeast Saccharomycopsis lipolytica

Summary Mitochondrial (mt) DNA of the alkane yeast, Saccharomycopsis lipolytica, was isolated. Its buoyant density in CsCl was found to be of 1.687 g/cm³, indicating a GC content of 27.5% and its melting point T_m = 79.5 °C, indicating a GC content of 24.9%. The corresponding values for nuclear (n) DNA, are 1.709 g/cm³ (GC: 49.5%) and T_m = 90.5 (GC: 51.7%) respectively. Electron microscopy revealed that mtDNA has a circular structure with a contour length of about 14.5 µm corresponding to 45.5 kb per molecule. The size estimated from restriction analyses performed with 7 endonucleases was 48.35 kb/molecule. A restriction map was constructed, using the cleavage data of 4 endonucleases.     

Genetic analysis of sexual hybrids and protoplast fusion products of the yeast Saccharomycopsis lipolytica

Protoplast fusion of genetically marked Saccharomycopsis lipolytica strains was carried out succesfully. Mitotic segregation was induced to prove the presence of parental markers in fusion products. Methylbenzimidazol-2-yl-carbamate has been shown to induce efficiently mitotic segregation in this yeast. The segregation pattern of genetic markers in a sexual hybrid was compared to that of a parasexual hybrid.     

Substructural studies on sporulation of Saccharomycopsis lipolytica

During sporulation of diploids from crosses between different strains of the yeast Saccharomycopsis (Candida) lipolytica irregular numbers of ascospores per ascus have been observed. Using the serial section method it could be shown now by means of electron microscopy that in one-, two-, and three-spored asci unenclosed “naked” nuclei occur additionally to nuclei incorporated in mature spores. It was demonstrated that the production of less than four spores per ascus in this yeast is not the result of a lack of meiotic products but of the nonutilization of nuclei could be demonstrated which indicate a normal meiotic division. All ascospores derived from asci with different spore numbers are uninuclear. It is assumed that a defect in spore formation caused by structural changes of chromosomes or aneuploidy should give rise to the occurrence of non incorporated nuclei and spore irregularity. It was concluded that meiosis and spore formation in Saccharomycopsis lipolytica seem to represent parallel and coordinated processes which generally resemble those recorded for Saccharomyces cerevisiae and Hansenula species.     

Genetic and biochemical studies of N-alkane non-ultilzing mutants of Saccharomycopsis lipolytica

Summary Alkane non-utilizing mutants of the yeast Saccharomycopsis lipolytica were induced by ultraviolet light. Thirtyfour of the mutants were found to be alkaline-negative and fatty acid-positive (Phenotypes A and C) indicating a defect in n-alkane uptake or in hydroxylase complex activity. The hydroxylase complex is a microsomal aggregate composed of the first three enzymes of n-alkane catabolism. Leaky and non-mating mutants were eliminated leaving 21 mutants which were analyzed genetically. All 21 of the mutations showed a 1:1 pattern of segregation indicating they are chromosomal and all but one were recessive. Analyses of inter-mutant complementation and recombination showed that the 21 mutations represent 18 genes.Several of the mutants had pleiotropic phenotypes in addition to alkane non-utilization. These phenotypes included a loss of mating function, an inability to sporulate, a changed colony and cellular morphology, osmotic sensitivity and a lack of extracellular protease.The hydroxylase complex activities of mutants and wild type were assayed in cell-free extracts prepared by protoplast lysis. A small amount of detergent was necessary for the extraction of hydroxylase complex activity. The hydroxylase complex was inducible by n-decane and incubation was complete by 6 h. Hydroxylase complex activities in the mutants varied from 2.8% to 46.5% of wild type. The hydroxylase complex activities of two temperature sensitive mutants were as stable as wild type at the non-permissive temperature. These mutants showed temperature sensitive induction suggesting that the uptake of n-alkanes is temperature dependent in these strains.     

Cryptic DNA plasmids of the heterothallic yeast Saccharomycopsis crataegensis

Summary Three DNA plasmids, designated pScrl-1, pScrl-2, and pScrl-3 have been found in a strain of the heterothallic yeast Saccharomycopsis crataegensis (NRRL Y-5902). pScrl-l, -2 and -3 are, respectively, 15, 7, and 5 kilobase pairs (kbp) in size. Based on the results of exonuclease digestions, all three plasmids appear to be linear molecules with blocked 5 ends. All three plasmids also have a lower buoyant density than does nuclear DNA of S. crataegensis. The two lower molecular weight plasmids hybridize strongly with one another, but only weakly to the higher molecular weight plasmid. Two of four related S. crataegensis strains surveyed were found to contain two plasmids that are of the same size as the two larger plasmids of Y-5902. Evidence is presented indicating that the plasmids in strain Y-5902 reside in the cytosol since they were found not to be located within the major organelles (mitochondria and nuclei).     

Integrative transformation of the yeast Yarrowia lipolytica

Summary An EcoR1 shotgun of Yarrowia lipolytica DNA was inserted into the plasmid YIp333 which carries the LYS2 gene of S. cerevisiae. The resulting plasmid pool was transformed in both S. cerevisiae and Y. lipolytica. Whereas numerous replicating plasmids could be isolated from the S. cerevisiae Lys⁺ transformants, all transformants of Y. lipolytica so far analyzed were found to result from integrative transformation. This occurred at a frequency of 1 to 10 transformants per g of input DNA. Co-transformation occurred at high frequency and resulted in tandem integration of 2 to 10 copies of the incoming DNA. Structural and segregational stability of the transforming DNA were both high.     

Gene organization of the mitochondrial DNA of yeasts: Kluyveromyces lactis and Saccharomycopsis lipolytica

Summary Restriction fragment maps have been constructed for the mitochondrial DNA from two petitenegative yeasts, Kluyveromyces lactis and Saccharomycopsis lipolytica (Candida lipolytica). On these circular genomes, we localized the sequences homologous to the S. cerevisiae mtDNA fragments carrying known genes. The arrangement of genes for ATPase subunit proteins, ribosomal RNA and 4S RNA shows a common feature with respect to S. cerevisiae mitochondrial genome.Abbreviations bp base pairs - mtDNA mitochondrial DNA - tRNA transfer RNA - rRNA ribosomal RNA     

Multiple-copy integration in the yeast Yarrowia lipolytica

Using an EcoRI-BglII fragment of the G unit of the rDNA of Y. lipolytica and a set of 11 deletions in the URA3 promoter, we have constructed several plasmids to test gene amplification in the rDNA. These plasmids contain the rDNA fragment for integration, defective versions of the URA3 gene, the XPR2 gene encoding alkaline extracellular protease (AEP) as a reporter gene, and part of the pBR322 plasmid for selection and replication in E. coli. Among these plasmids, one corresponds to a deletion which allows multiple integration into the rDNA (plasmid pINA773). Two other plasmids (pINA767 and pINA772) give multiple integration only with a mutated URA3 gene. Transformants carrying these three plasmids were tested for copy number, stability, chromosomal localization and AEP secretion. Transformants containing plasmids pINA767, 772 and 773 displayed an average copy number of 5, 12 and 25–60 copies respectively of the plasmid, as estimated by PCR and DNA hybridization. Integrations occurred in only one chromosome except for transformants containing 60 copies where copies were observed at least in two different chromosomes. Multiple integrations were found both as tandem repeats and as dispersed copies. Plasmid copy number was stable in both minimum and rich media, for strains containing less than ten copies per cells. However, for higher copy number, multiple integrations were stable only when AEP synthesis was not induced, while in inducing medium stability of the multiple integrations was dramatically affected.     

Virus-like particles from the yeast Yarrowia lipolytica

Summary Four out of the 24 strains of the yeast Yarrowia lipolytica we have checked for the presence of virus-like particles (VLPs) proved to contain encapsidated double-stranded RNA (dsRNA) molecules, 4.9 kb long. A major VLP polypeptide of MW 80,000 was observed in all 4 cases, and a second one of MW 77,000 in three cases. dsRNA from the VLPs harboring only the larger polypeptide showed little homology with the 3 others. We have found no homology between VLP dsRNAs and host DNA or dsRNAs from Saccharomyces cerevisiae, and no relationship between the presence of VLPs and a possible killer phenomenon in Y. lipolytica.     

Mating in the alkane-utilizing yeast Yarrowia lipolytica

Mating in Yarrowia lipolytica can be induced by mixing cells of both complementary mating types (A and B) in yeast extract-malt-medium. By means of an inbreeding programme, strains with significantly improved mating frequencies were selected. The main events of the mating process, that have been detected so far, by means of cytological methods, are: formation of mating tubes, cell contact, cell fusion and karyogamy. During the process of conjugation, mating tubes of different lengths as and shapes are formed between haploid parental cells. Under special conditions the preformation of mating tubes was observed. Mating in the dimorphic yeast Y. lipolytica has been shown to be a distant mating and to occur between cells of a different shape and size. Cells involved in conjugation are no changed morphologically, except for the formation of special copulation structures. Several events of mating and the morphology of zygotes are different than those in the case of baker's yeasts. It is supposed that there is in this yeast also an action of diffusible factors that trigger mating.     

Parasexual process in the yeast Yarrowia lipolytica

Genetic studies of several events of the life cycle of Y. lipolytica demonstrated that diploid strains were unstable and produced mitotic segregants by haploidization. A screening system was developed which enabled us to show that parasexual processes can take place in addition to the sexual life cycle. This haploidization occurred through aneuploid intermediates as was proven statistically by the deviations from the segregation pattern as well as by the segregation data of the clones. The direction of the cross, was--with respect to the resistance to 2-deoxyglucose of A- or B-strain--not important for selection of mitotic segregants.     

Characterization of isocitrate lyase from the yeast Yarrowia lipolytica

The active form of isocitrate lyase (ICL) from the yeast Yarrowia lipolytica was eluted as single peak from ion exchange on DEAE-cellulose. The enzyme had a specific activity of 7.4 U/mg. Its molecular mass was estimated to be approximately 200 to 210 kDa by gel filtration chomatography on Sephadex G 200. In SDS-polyacrylamide gel electrophoresis the enzyme was characterized by an unique protein band of about 50 kDa, thus indicating that ICL is a tetramer of identical subunits. The optimum pH was 6.0 in 50 mm phosphate buffer at 30°C. The K_m value of the 35-fold purified ICL for threo-d_s-isocitrate in 50 mm phosphate buffer at pH 6.0 was 0.3 mm. In regulation studies ICL was found to be noncompetitive inhibited by succinate and oxaloacetate. Antibodies against ICL were raised in rabbits. The specificity of the anti-ICL-antibodies was estimated by Ouchterlony tests and by a competitive ELISA on microtiter plates.     

Identification of mutations preventing n-hexadecane uptake among 26 n-alkane non-utilizing mutants of Yarrowia (Saccharomycopsis) lipolytica

Summary Genetic analyses of n-alkane non-utilizing mutants of the yeast Yarrowia (Saccharomycopsis) lipolytica were continued. By analyses of inter-mutant complementation and recombination a total of 26 genetic loci have been identified. Mutations representing these loci have phenotypes characteristic of defects in substrate uptake or in one or more of the enzymatic activities making up the hydroxylase complex. Tests of ¹⁴C n-hexadecane uptake by a set of alkane-negative mutants representing the 26 loci show that 16 of the mutations cause a significant reduction in n-alkane uptake. N-alkane uptake by Y. lipolytica is shown to be inducible and to be inhibited by the metabolic poisons 2–4 dinitrophenol and KCN. The latter observation indicates that n-alkane uptake of Y. lipolytica is due to active transport.     

Gene-protein assignments within the yeast Yarrowia lipolytica dsRNA viral genome

Summary Some strains of the yeast Yarrowia lipolytica possess virus-like particles (VLPs) which encapsidate a double-stranded RNA (dsRNA) genome designated L_y. We report here that these VLPs have two associated polypeptides of molecular weights 83 kd (VL_y-P1) and 77 kd (VL_y-P2). Denatured Ly-dsRNA was used to program a cell-free rabbit reticulocyte translation system, resulting in the appearance of four major products, viz. Ly-P1 (83 kd); L_y-P2 (77 kd); L_y-P3 (74 kd) and L_y-P4 (68 kd). The in vivo viral-associated protein VL_y-P1 co-migrated on SDS-polyacrylamide gels with the in vitro product L_y-P1 and, similarly, VL_y-P2 co-migrated with L_y-P2. Peptide mapping data confirm the identity of the in vivo products (VL_y-P1 and VL_y-P2) and their in vitro counterparts. The conclusion made is that VL_y-P1 and VL_yP2 are almost identical primary translation products of the L_y genome, derived from a single or multiple species of L_y-dsRNA. RNA blot hybridizations using L_1A M₁ and separately, L_2A M₂ probes prepared from appropriate K1 and K2 Saccharomyces cerevisiae killer strains, failed to show any detectable homology to L_y-dsRNA, substantiating the uniqueness of the Ly genome with respect to the K1 and K2 S. cerevisiae dsRNA killer systems.     

Isolation and characterization of cerebrosides of the hydrocarbon-assimilating yeast Yarrowia lipolytica

Yarrowia lipolytica yeast was grown batchwise on n-hexadecane as the carbon and energy source. Two cerebroside species were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative high-performance thin-layer chromatography. The cerebroside content accounted for 1.3% of the total cell lipids. Glucose was detected as the sole sugar constituent in cerebrosides. The fatty acid composition of cerebrosides was characterized by a high proportion of hydroxylated long-chain saturated fatty acids. The major fatty acids were h16:0 and 16:0. The long-chain bases composition shows a preponderance of trihydroxy bases and a small amount of dihydroxy bases. The striking finding was a high proportion of 19-phytosphingosine.     

Temporal relationship of diploidization and haploidization in the yeast Yarrowia lipolytica

By means of special selective conditions we analyzed the temporal relationship of diploidization and haploidization in the yeast Yarrowia lipolytica. These processes could be divided into several steps. Twelve hours after mixing parental strains dissociation products of unstable heterokaryons are selectable. Four hours later karyogamy takes place as shown by isolation of mitotic recombinants. At this time spontaneous haploidization starts which leads to an enrichment of haploid and aneuploid segregants in the cell population. A high portion of chimeric clones which represents the dynamics of segregation were selected at time intervals 16, 23 and 29 hours. We concluded that an unstable heterokaryon should be a connecting link between sexual and parasexual processes in Y. lipolytica.