Randomly amplified polymorphic DNAs assess recombination following an induced parasexual cycle in Penicillium roqueforti

Random amplified polymorphic DNAs (RAPDs) were used as a genetic marker system to characterize recombinant strains following the parasexual cycle of Penicillium roqueforti. After protoplast fusion and haploidization of diploid hybrids, segregants characterized by a reassortment of the parental genetic markers displayed specific RAPD fingerprints. The appearance or the loss of RAPD fragments demonstrate that these markers provide an efficient method to analyze recombination and to characterize somatic hybrids.     

Formation of intergeneric hybrids of yeast by protoplast fusion of Yarrowia and Kluyveromyces species

Summary Prototrophic hybrids have been obtained by the fusion of auxotrophic haploid strains of the two yeasts Yarrowia (Saccharomycopsis) lipolytica and Kluyveromyces lactis. The hybrid fusants had a colonial morphology intermediate between that of the two parent strains, were uninucleate, and contained an approximately diploid amount of DNA per cell. The growth rates of all the fusants on a minimal glucose medium were slower than those of the two parents. Two of the fusants studied could utilise a novel range of carbon sources.All of these data suggested that the hybrids contained a diploid nucleus formed by the fusion of the two haploid parental nuclei. However, analytical CsCl density gradient centrifugation demonstrated that the nuclear DNA of the fusants was derived almost entirely from the Y. lipolytica parent. Moreover, an examination of the protein constitution of the fusants by two-dimensional gel electrophoresis showed that their protein patterns were indistinguishable from that of Y. lipolytica. Two possible mechanisms for the formation of a diploid nucleus containing DNA derived almost entirely from one of the haploid parents are discussed.     

Auswirkungen von Mutanteninduktion und Protoplastenfusion auf den DNA- und Proteingehalt von Pichia guilliermondii

The DNA content per cell of two tested auxotrophic mutants of Pichia guilliermondii was diminished in comparison to their wild-type strains. After protoplast fusion between these mutants the majority of hybrids did not show the expected amount of DNA. In some hybrids it was only slightly higher than in the wild-type strains, although heterozygosity for markers of three chromosomes could be established. The protein content per cell of most strains was positively correlated with the DNA content, but generally more affected as the DNA content by the induction of mutations and protoplast fusion. The protein/DNA ratio of the two mutants was strongly reduced, while this ratio was somewhat enhanced in one of the hybrids in comparison to the wild-type strains and the majority of hybrids. Apparently, the reduced protein content per cell in the mutants seemed to be partially compensated in the hybrids.     

Auswirkungen von Mutanteninduktion und Protoplastenfusion auf ausgewählte Dehydrogenase-Muster von Pichia guilliermondii

We investigated the enzyme patterns of two wild-type strains, two auxotrophic mutants and some fusion hybrids of Pichia guilliermondii after the electrophoretic separation of their crude cell extracts in dependence on the cultivation on mineral salt medium with different carbon sources. The wild-type strains showed a constitutive synthesis of one NAD-specific and some NADP-specific enzymes and their ability to oxidize primary alcohols with a chain length from C₂ to C_8. After the induction of mutants auxotroph for amino acids, nicotinic acid or adenine we found different enzyme patterns, too. Prototrophic fusion hybrids obtained via protoplast fusion of the two double auxotrophic mutants showed behaviour either characteristic for the wild-type strains or characteristics of their parental strains and in some cases also combination of parental enzyme patterns. The behaviour of the hybrids seems to be dependent on the quantity of DNA transferred into each fusion hybrid from the parental mutant strains. So the method of protoplast fusion allows to get qualitatively different hybrids.     

Hybridization of Saccharomyces cerevisiae with Candida utilis through protoplast fusion

Summary Auxotrophic mutants of Saccharomyces cerevisiae and Candida utilis were hybridized through protoplast fusion. Spontaneous, UV- and FPA-induced mitotic segregation indicated that after cell fusion, exclusion of the S. cerevisiae nucleus or nuclear fusion followed by preferential loss of S. cerevisiae chromosomes can take place. Some of the hybrids were stable. One of them, expressed mating and sporulation functions of the S. cerevisiae parent. Thus, markers from both parents could be recovered as mitotic and meiotic segregants.     

Hybridization of yeasts by protoplast fusion: Early events after fusion in Pichia guilliermondii

The efficiency of protoplast fusions in haploid strains of P. guilliermondii was dependent on the strain used. In the primary fusion colonies, a large proportion of non-dividing cells was sometimes observed. While in two strains stable hybrids occurred immediately after fusion, one strain showed a longer-lasting heterokaryotic state.     

Orthogonal field alternation gelelectrophoresis (OFAGE) as a means for the analysis of somatic hybrids obtained by protoplast fusion of different Saccharomyces strains

Summary PEG-induced fusion of two haploid Saccharomyces strains resulted in three morphologically different types of fusion products. In order to estimate the respective ploidy, one representative of each type — F ₁, F ₂ and F ₃ — was subjected to a measurement of the cellular DNA content and to a meiotic segregation analysis. The data obtained in these analyses suggested the strain F ₁ to be a haploid cybrid resulting from mere plasmogamy, whereas the hybrids F ₂ and F ₃ were likely to be the products of a successful nuclear fusion of two, respectively three, protoplasts of the parental strains. However, the complete genetic composition of the hybrids could only be revealed by OFAGE experiments, as the genetic data solely referred to a few chromosomes with marker genes. All the results of the OFAGE were in full accordance with the assumptions made in the conventional analysis, thus indicating the OFAGE to become a very promising means for the investigation of hybrids inaccessible to genetic analyses.     

Hybridization of yeasts by protoplast fusion: Ploidy level of hybrids resulting from fusions in haploid strains of Pichia guilliermondii

Hybrids produced by protoplast fusion between auxotrophic mutants of the same strain contained the parental markers heterozygously, as indicated by induced mitotic segregation. Even within the same fusion combination the hybrids differed in their mitotic stability and their induced mitotic segregation pattern. The occurrence of aneuploids was proved. The ploidy of the hybrids did not exceed the diploid level in any case.     

Protoplasts fusion hybrids from Candida albicans morphological mutants

Three pairs of strains, derived from Candida albicans 1001 (wild type) and carrying appropriate selection markers, were crossed by means of protoplast fusion. The corresponding fusion products were regenerated in selection medium and hybrids of rapid growth and more defined characteristics were eventually isolated and analyzed for relevant properties. Hybrids from a cross of two auxotrophic strains with no morphological alteration had a normal morphology but gave rise to rough-filamentous segregants of mostly pseudomycelial appearance, probably through DNA content reduction. The observation was consistent with a previous suggestion that C. albicans 1001 carries recessive mutant alleles that can be unmasked. A permanently filamentous strain (Y- phenotype) when crossed with a mycelium deficient (F-) strain gave rise to wild-type hybrids with a normal capacity for mycelium formation. Finally, two Y- strains, with similar phenotypes consisting of rough colony formation and mostly pseudomycelial morphology of cells under all conditions, also complemented since they gave rise to wild-type hybrids. It is concluded that the determinants of the mutant phenotype must be different in these two strains.     

Parasexual hybridization of yeasts by electric field stimulated fusion of protoplasts

Summary Under the action of a high electric field pulse a pronounced stimulation of yeast protoplast fusion initiated by polyethylene glycol and Ca ²⁺ ions was found. The fusion rate was enhanced by a factor of 200 for different genetically marked strains of the yeast Saccharomyces cerevisiae as compared with fusion without electric field treatment. Genetic evidence for fusion has been obtained by isolating parasexually produced hybrids resulting from combination of the genomes of both mutants.Our method allows the production of a large number of parasexual hybrids for different purposes.     

Microbiological implications of electric field effects III. Stimulation of yeast protoplast fusion by electric field pulses

Prototrophic colonies could be selected on minimal medium after mixing of protoplasts from diauxotrophic mutants of the yeasts Saccharomycopsis lipolytica and/or Lodderomyces elongisporus and treatment with polyethylene glycol (PEG) in the presence of calcium chloride. This is the result of protoplast fusion and complementation of auxotrophic deficiencies. Under identical conditions an electric field pulse in the μs-range applied via an electric discharge to the protoplast-PEG mixture resulted in a drastic enhancement of the protoplast fusion rate. The presence of polyethylene glycol was demonstrated to be a prerequisite for fusion in this case, too. The frequency of hybrid formation detected as prototrophic colonies could be increased in the case of intraspecific fusion at initial electric field strengths between 2.5 and 5 kV · cm^?1. The application of an electric field pulse of proper strength and duration to a yeast protoplast suspension turned out to be a more effective tool in production of fusion products than conventional methods. Large numbers of parasexual hybrids for different selection programmes in yeast genetics and for industrial purpose may be delivered by this technique.     

Temperature-dependent internuclear transfer of genetic material in heterokaryons of Candida albicans

Summary Heterokaryons (hets) of Candida albicans are produced by fusing protoplasts of complementing auxotrophic strains and can be propagated continuously on minimal medium despite their tendency to assort nuclei into monokaryotic blastospores. Most mono-karyons have parental-type nuclei, but some are nuclear hybrids with DNA contents between one and two times that of their parental strains. Evidence is presented that hybrids arise by transfer of a portion of the genetic material of one bet nucleus to another, and that the amount of material conveyed during transfer increases with increasing het growth temperatures over the range 25°C to 41°C. This partial hybridization is a general property of hets and is not determined by the wild-type strain backgrounds of their parental components or by the kinds of auxotrophies forcing heterokaryosis. Frequencies of mitotic recombinants induced in partial hybrids by ultraviolet radiation indicate that nuclei of C. albicans are naturally diploid.     

Transfer of mitochondrial function into a cytoplasmic respiratory-deficient mutant of Saccharomyces yeast by electro-fusion

Summary Electric field-induced fusion was induced between Saccharomyces cerevisiae protoplasts from the ^– heterozygous diploid strain 2114 and the respiratory-competent diploid strain 3441, carrying chromosomal markers. Close membrane contact between the cells of the two different strains (ratio 1:1) was achieved by dielectrophoresis in a weak inhomogeneous alternating field (about 1 kV/cm, 2 MHz). Due to dielectrophoresis pearl chains of two or more cells of the two strains are formed between the electrodes. Cell fusion was induced by application of two single square field pulses sufficiently high to induce reversible electrical breakdown in the membrane contact zone between cells within a pearl chain (about 7 to 8 kV/cm field strength and 40 Ms duration). The two subsequent pulses were applied at an interval of about 10 s.Hybrids could be isolated on selection medium in a high yield (compared with conventional fusion techniques). The hybrids were diploid, respiratory-competent and produced prototrophic spores. Thus, the fused hybrids contained only the chromosomal markers of strain 2114 and the cytoplasmic marker for respiratory competence from strain 3441; electro-fusion thus resulted mainly in plasmogamy.     

Genetic transfer mediated by isolated nuclei in Saccharomyces cerevisiae

Summary Viable hybrids of Saccharomyces cerevisiae were obtained by transfer of isolated diploid nuclei into haploid protoplasts using a polyethylene glycol (PEG) fusion procedure.     

The use of mitochondrial mutants in hybridization of industrial yeast strains

Summary The contributions of each of the parental strains to the genomes of the sporulating and non-sporulating hybrids, Saccharomyces diastaticus (S. cerevisiae) × Hansenula capsulata, S. diastaticus × Hansenula wingei, S. diastaticus × Torulopsis glabrata, S. diastaticus × Candida pseudo tropicalis (Kluyveromyces marxianus) S. diastaticus × Saccharomyces rouxii, S. diastaticus × Saccharomyces kluyveri, and Saccharomyces diastaticus × Saccharomyces bayanus, obtained by protoplast fusion, were determined by the methods of whole nuclear DNA-DNA reassociation. Petite mutants of S. diastaticus NCYC625, and respiratory-competent strains of the other species, were used. In all of the hybrids but one, the DNA from the S. diastaticus parent showed 93.3 to 109.3% homology with the DNA from the hybrids, and the other parents, from –7.7% (S. kluyveri) to 20.0% (S. bayanus). Reassociation between the DNA from S. diastaticus and the DNA from the other parental strains ranges from 4.7 to 19.4%. Reassociation between DNAs from S. diastaticus and that of the S. diastaticus × T. glabrata fusion hybrids were 15.2 and 18.9% respectively. Further investigation of this hybrid is desirable. The fusion products were relatively stable as compared to some fusion hybrids selected by use of nuclear markers, and could be maintained on normal media, with little or no selection pressure, but use of an appropriate carbon source. In most of the hybrids, except for S. diastaticus × T. glabrata, the S. diastaticus parent contributed most of the genome, and only a single chromosome, or a fragment of a chromosome, appeared to be transferred to the Saccharomyces nucleus, to form the genome of the fusion product.     

Genetic analysis of intergeneric hybrids obtained by protoplast fusion in yeasts

Summary Prototrophic hybrids have been obtained by the fusion of various auxotrophic haploid strains of Saccharomyces cerepisiae and Schwanniomyces castellii. The fusion hybrids showed starch fermentability which derived from one of the fusion parents, S. castellii. Surprisingly, these fusion hybrids were found to exhibit excellent sporulation and spore germination. The progenies of these fusion hybrids showed a few aberrant segregations, but mostly normal segregation for auxotrophic genetic markers. They also showed many tetrads with an apparently digenic segregation (2:2, 3:1 and 4:0) for starch fermentation. On the other hand, mating types of segregants of the fusion hybrids were determined by the prototrophic recovery method. Consequently, tetrad types for mating type were mostly 2a:1:1 non-mater and several asci showed tetrad types of 2a:2 non-mater and 2a:2. The 60 prototrophic fusion hybrids and its segregants did not secrete -amylase on the starch agar plate. However, all of the data suggested that fusion hybrid could carry two dominant genes (STAB and STAC) to ferment starch, and that the two genes STAB and STA2 may be identical or allelic as may be the genes STAC and STA3.     

Possible chromosomal location for the killer determinant in Torulopsis glabrata

Summary We have used cytoduction and protoplast fusion to characterise the killer trait in Torulopsis glabrata. Killer trait could not be transferred by cytoduction to a non-killer strain of T. glabrata suggesting that the determinant(s) is/are not present in the cytoplasm. Consistent with this interpretation is the observation that all fusion products, where chromosomes of both the parents are represented, are killers. Non-killer segregants could be isolated from fusion products after treatment with benlate suggesting that loss of the trait is associated with non-disjunction and loss of a chromosome.     

Genetic manipulation of non-conventional yeasts by conventional and non-conventional methods

In recent years, yeasts other than those belonging to the species Saccharomyces cerevisiae and Schizosaccharomyces pombe have become increasingly important in industrial processes. Species such as Pichia stipitis, Hansenula polymorpha, Zygosaccharomyces rouxii, Saccharomyces exiguus, Torulaspora delbrueckii, Yarrowia lipolytica and others whose perfect stage is known, can be manipulated genetically by classical methods, but those belonging to the genera Candida (C. utilis, C. tropicalis, C. bombicola, C. zeylanoides, C. boidinii, etc.), Brettanomyces, Cryptococcus, Rhodotorula, and others of the different form genera, cannot be treated in this way. Some, such as Schwanniomyces and Debaryomyces spp., which have a perfect stage, are still difficult to manipulate by conventional means. Genetic manipulation of these yeasts can be approached from two points of view; the first involving improvement of strains by cross-breeding within one species, and the second, the introduction of desirable genes from unrelated species and even from plants or animals. Two techniques are available for construction of industrially-useful strains from these yeasts: protoplast fusion and transformation with chimaeric plasmids containing the gene(s) it is desired to introduce into the recipient strain. The methods for the latter procedure are well known but can be laborious and time-consuming, especially if it is desired to introduce genes from plant or animal sources for production of enzymes, hormones, vaccines and similar products. Protoplast fusion is a simple technique which can be utilized in most laboratories and used for construction of improved yeast strains for brewing, baking, ethanol production and wine-making, either by the fusion of desirable strains of the same species which do not sporulate, or by introduction of genes from non-Saccharomyces species. Methods for fusion of species from different genera and isolation of the desired hybrids have been improved considerably in recent years. We have developed a method for isolation of strains carrying the desired genes by fusing a non-Saccharomyces species with an auxotrophic strain of Saccharomyces cerevisiae and selecting hybrids having the desired characteristics on appropriate media, after which the genes are transferred to the industrial strain by rare-mating, repeated protoplast fusion, or classical mating as required. The advantages and limitations of the method are under investigation.     

Recombinogenic activity of nalidixic acid for artificial hybrids but not for natural strains of Candida albicans: Evidence for the monoploidy of natural strains

Nalidixic acid induces segregation of auxotrophs from prototrophic hybrids of Candida albicans artifically produced by fusing complementing auxotrophic protoplasts. The auxotrophies recovered are limited to those introduced through the fusion process, and patterns of segregations for linked auxotrophic markers demonstrate the segregants are products mitotic cossing-over. Nalidixic acid does not induce auxotrophies of any sort in clinical isolates of C. albicans. These findings are contrary to a current hypothesis that natural strains of C. albicans are diploid and heterozygous for a variety of auxotrophic mutations.     

Ethanol inhibition of Saccharomyces and Candida enzymes

Summary Ethanol inhibition of several hydrolases (sucrase, maltase, trehalase, melezitase and cellobiase) has been measured in both highly ethanol-tolerant Saccharomyces strains (R) and in Candida strains less tolerant to ethanol (S). Cells were either grown in the presence of ethanol and the activities of the enzymes measured without preincubation in this alcohol (in situ inhibition assay), or the culture was grown in the absence of ethanol and the activities of the enzymes were determined after preincubation and in the presence of this compound (in vitro inhibition assay). Ethanol inhibition (Ki values) of sucrase, maltase, trehalase, and melezitase was quite different for these different enzymes in the same strain (R or S), but similar for the same enzyme in different strains (R and S). The Ki values for cellobiase, which is absent from the R strain, were higher when induced than at the basal level and higher in in vitro assays than in in situ assays. This suggests that the inhibition observed in situ is mainly the result of an inhibition of other proteins related to cellobiase (i.e., those involved in its synthesis) but not a direct inactivation of the enzyme by ethanol. Accordingly, when hybrids between Saccharomyces (R) and Candida (S) strains were constructed by protoplast fusion, and cellobiase was measured in the parental Candida strain and some of the hybrids, there was an increase in the Ki values in the in situ assays from 2.25% ethanol in Candida to 5.5% in some of the hybrids.