Complementation of Saccharomyces cerevisiae acid phosphatase mutation by a genomic sequence from the yeast Yarrowia lipolytica identifies a new phosphatase

Summary A Yarrowia lipolytica gene library was constructed in vector YRp7 and transformed into a Saccharomyces cerevisiae strain lacking both major acid phosphatase activities. A 2.18 kb genomic sequence restoring the ability to hydrolyze -naphthyl phosphate was isolated. Its sequencing revealed an ORF encoding 358 amino acids without significant homology with any known phosphatase. A putative signal peptide and several possible sites for N-glycosylation were identified. Phosphate-regulated expression of the cloned gene was observed in Y. lipolytica. Disruption data favoured the hypothesis that it might encode a minor phosphatase species.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Two genes encode 7SL RNAs in the yeast Yarrowia lipolytica

Summary We have identified an abundant cytoplasmic 7S RNA in crude extracts of the yeast Yarrowia lipolytica. A cDNA probe was prepared from this RNA and used to screen a genomic library. The DNA sequence of a positive clone was determined and the end positions of the 7S RNA gene established by comparison with the sequence of the extremities of 7S RNA. This gene, designated SCR2, encodes a 270-nucleotide RNA that can be folded into a secondary structure similar to that of 7SL RNAs. This RNA is 94.4% homologous to a previously identified 7S RNA from this yeast, but is encoded by a separate gene with highly divergent flanking sequences.     

UV-induced curing of the double-stranded RNA virus of the yeast Yarrowia lipolytica

Summary Curing of the viruslike particles harbored by a strain of the yeast Yarrowia lipolytica was achieved by UV irradiation. The cured strain was found to be able to maintain the viruslike particles after their re-introduction by crossing or by cytoplasmic fusion. The involvement of a UV-induced mutation of a yeast maintenance gene seems therefore unlikely.Abbreviations UV ultraviolet - dsRNA double-stranded RNA - VLPs viruslike particles, A and B alleles of the mating type locus - TCA trichloracetic acid     

Organization and transcription of the mitochondrial ATP synthase genes in the yeast Yarrowia lipolytica

Mitochondrial gene organization was studied in a dimorphic yeast, Yarrowia lipolytica. The gene order in a sequenced 6.6-kilobase region closely resembles that of the human mitochondrial genome in that ATP synthase subunit 8 and 6 genes are followed by genes for cytochrome c oxidase subunit 3 (which contains an intron), NADH-ubiquinone oxidoreductase subunit 4, and ATP synthase subunit 9. This region also contains tRNA genes decoding AUA, UGA, CUN and CCN codons, suggesting a unique mitochondrial translation. All the above genes are transcribed from the same DNA strand into multigenic RNAs, starting from a nonanucleotide sequence, 5′-ATA-TAAATA-3′, similar to other yeast mitochondrial promoters.     

Cloning and characterisation of the ribosomal RNA genes of the dimorphic yeast,Yarrowia lipolytica

Summary The ribosomal RNA genes of Yarrowia lipolytica have been identified, both in restriction digests of total genomic DNA and in a pBR322 gene bank, by hybridisation with cloned Saccharomyces cerevisiae rDNA. The Y. lipolytica rDNA repeat unit is 8.9 kb in size and contains the genes for the 25S and 18S, but not the 5S, rRNA species. The number of copies of these repeat units is approx. 50 per haploid genome. Several clones were found which did not conform to the standard restriction map due to differences outside the coding region. It appears that there is either heterogeneity of the spacer sequence within a strain or that the Y. lipolytica rDNA genes may be present as a number of separate clusters within this yeast's genome.     

Genetic control of Yarrowia lipolytica fatty acid synthetase biosynthesis and function

Yarrowia lipolytica, like other lower fungi, has a fatty acid synthetase complex (FAS) with an alpha 6 beta 6 molecular structure. Both subunits are multifunctional proteins each with a molecular weight of more then 200,000 daltons. A collection of FAS-deficient) Y. lipolytica mutants was isolated and characterized by both genetic complementation and enzyme activity measurements. It was found that the three acyl transferases (acetyl-, malonyl- and palmityl-transacylation) together with the enoyl reductase domain are located on subunit beta and, therefore, are encoded by the gene locus FAS1. beta-Ketoacyl reductase, beta-ketoacyl synthase and acyl carrier protein functions are part of the FAS2-encoded subunit alpha. Thus, the functional organization of FAS1 and FAS2 is identical in both yeasts, Saccharomyces cerevisiae and Yarrowia lipolytica. Nevertheless, the two yeasts differ significantly with respect to the intragenic complementation characteristics of fas1 and fas2 mutants. This finding is discussed in terms of a specific inter- or intramolecular reaction mechanism within the oligomeric FAS complex. The pentafunctional Y. lipolytica FAS1 gene was isolated from a lambda gt11 expression library using polyclonal antisera against the purified FAS complex. At present, sequencing of FAS1, which is more than 5 kilobases long, is almost completed. Available data indicate approx. 60 percent sequence homology together with an identical order of catalytic domains within subunit beta of the two yeasts, Y. lipolytica and S. cerevisiae.     

Natural zeolite clinoptilolite increases the concentrations of sphingoid bases in the yeast Yarrowia lipolytica

In the present paper, we studied the effect of natural zeolite clinoptilolite on sphingolipid metabolism in the yeast Yarrowia lipolytica. We also investigated if zeolite addition had any impact on cell shape and size, as well as on the pH alterations during the culture growth. High performance liquid chromatography analysis of sphingoid bases obtained by acid hydrolysis of complex sphingolipids from Y. lipolytica showed that their concentrations markedly rose upon the zeolite addition. The largest increase among the identified molecular species of sphingoid bases was seen in C18 phytosphingosine, whose levels rose 6.2-fold and 22.3-fold after culturing cells for 24 and 36 hours respectively in the presence of finely ground zeolite. pH measurements of the culture medium showed a similarity between pH profiles of control and zeolite-supplemented cells, suggesting that ion-exchange capacity was not probably responsible for the observed change in sphingolipid metabolism. Scanning electron microscopy revealed that zeolite affected cell size and shape. Y. lipolytica cells grown in the absence of zeolite were oval-shaped with an average cell size of 0.7-2.7 microns, whereas when cultured with zeolite, they were round-shaped and larger, having an average cell size of 1.3-2.9 microns.     

Dependence of the sphingoid bases concentration on growth phase and temperature in the yeast Yarrowia lipolytica

The aim of this research was to investigate the effect of growth phase and temperature on the concentrations of the individual molecular species of sphingoid based obtained by acid hydrolysis of total sphingolipids from the yeast Yarrowia lipolytica. Our results showed C18 phytosphingosine to be the major long-chain base in Y. lipolytica regardless of growth phase or temperature. We also found Y. lipolytica to contain sphingosine, the predominant mammalian long-chain base that is uncommon for yeast sphingolipids. Among the identified long-chain bases, only C18 phytosphingosine appeared to be influenced by culture conditions. Its concentration was largest in the exponential phase and decreased 2.9-fold when cells entered the stationary phase of growth at 28 degrees C. Following a temperature shift from 28 to 39 degrees C, there was a 2.1-fold decrease in the phytosphingosine concentration, but it rose 1.7-fold after the heat-stressed cells had been returned to 28 degrees C and subjected to prolonged growth. These results might point to the possible involvement of phytosphingosine in the cell growth regulation and in the adaptation of Y. lipolytica cells to stressful culture conditions.     

Spontaneous haploidization in early zygote progeny and its use for mapping in the yeast Yarrowia lipolytica

Summary By means of spontaneous haploidization immediately after conjugation, it is possible to map genes in Y. lipolytica. The already known linkages argA — leuA and metA — lysA were confirmed by means of this method. The mating type locus (MAT) is located on the same chromosome as argA — leuA.(Abbreviations: A and B, alleles of the mating type locus; deg^R, recessive resistance to 2-deoxygiucose (DEG) when acetate is given as carbon source.)