Claes Gjermansen

Genetic differences between Saccharomyces carlsbergensis and S. cerevisiae. Analysis of chromosome III by single chromosome transfer

Tetrad analysis of most Saccharomyces strains used in beer production is impossible due to a low yield of viable spores. The present paper describes the use of single chromosome transfer in the genetic analysis of a brewer’s yeast.The technique employs thekar1 mutation, which reduces karyogamy after conjugation. In rare caseskarl×KAR crosses yield progeny resulting from the transfer of one chromosome or a limited number of chromosomes from a nucleus of one parent to one of the other. S. cerevisiae strains with an extra S. carlsbergensis chromosome III have thus been isolated from crosses between spore derived clones of the brewing strain and haploidkar1 S. cerevisiae strains carrying several auxotrophic markers. When the disomics were crossed to other haploid S. cerevisiae strains a normal spore viability was obtained, allowing tetrad analysis.High functional homology was found between the transferred S. carlsbergensis chromosome and chromosome III of S. cerevisiae. All genes essential for viability on the latter are represented on the former as are alsoHIS4, LEU2, MAT andTHR4. Despite the functional homology, the transferred chromosome had a structure that was substantially different from that of standard S. cerevisiae strains. It did not recombine with S. cerevisiae chromosomes III, except in a certain region, where recombination was normal. Furthermore, restriction endonuclease analysis showed that the variant chromosome has a nucleotide sequence in theHIS4 region different from that of S. cerevisiae. The S. carlsbergensis brewing strain is heterozygous for this sequence variation, containing also aHIS4 region with a sequence identical or close to that of S. cerevisiae.

Construction of a hybrid brewing strain of saccharomyces carlsbergensis by mating of meiotic segregants

A brewing strain of Saccharomyces carlsbergensis yielded upon sporulation 1-, 2- and 3-spored asci. The spore viability was low, but a number of clones could be derived from the spores. Among these a- and α-maters were identified revealing that the parental brewing strain is heterozygous for the two mating type alleles. A number of lines with opposite mating type were crossed pairwise. One of the hybrids produced by mating of two presumably haploid or near haploid clones was equal to the parental strain in brewing performance on a pilot scale. The separation of the nuclear genomes of brewer’s yeast into individual cells and their successful mating into hybrid strains opens the possibility to introduce genetic alterations in brewing strains by mutagenesis and genetic transformation of the haploid lines.

Analysis of chromosome V and theILV1 gene from Saccharomyces carlsbergensis

Chromosome V of the Saccharomyces carlsbergensis lager yeast strain 244, a yeast not amenable to tetrad analysis, was analysed genetically in S. cerevisiae genetic standard strains. This was achieved by crossing meiotic progeny of the lager yeast with S. cerevisiae strains carryingkar1 as well as the chromosome V markerscan1, ura3, his1, ilv1, andrad3. From the transitory heterokaryons formed we selected strains retaining the characteristics of the recipient strain but having become prototrophic for uracil, histidine, and isoleucine. The resulting strains were disomic for chromosome V, having acquired a chromosome V from S. carlsbergensis in addition to the normal S. cerevisiae chromosome complement (chromosome addition strains). They were of two classes: In one class the transferred chromosome hardly recombines with the S. cerevisiae chromosome V in the regionCAN1-RAD3, which covers almost the entire known map. In the other class, the transferred chromosome recombined at normal levels. We conclude that S. carlsbergensis harbors two structurally different chromosomes V; one being homologous and one homoeologous to the S. cerevisiae chromosome. By use of theCAN1 locus, strains were selected which by mitotic chromosome loss had their normal chromosome V substituted by either the homologous or the homoeologous S. carlsbergensis chromosome, showing that these chromosomes are fully functional in S. cerevisiae. Tetrad analysis of the chromosome substitution strains confirmed the very different genetic behavior of the two S. carlsbergensis chromosomes V. In spite of the almost complete absence of recombination between the homoeologous chromosome and the S. cerevisiae chromosome, disjunction at meiosis appears normal, as indicated by high spore viability.Genomic Southern hybridizations with the probesCAN1, URA3, CYC7, andILV1 could not detect any nucleotide sequence differences between these loci on the recombining S. carlsbergensis chromosome and the S. cerevisiae alleles. Under standard stringency (68°C, 0.1×SSC), hybridization of the probes to DNA from the strain with the homoeologous chromosome was only observed in the case ofILV1, where weak hybridization was found, indicating a considerable difference in nucleotide sequence.To further study the extent of nucleotide sequence inhomology, the two differentILV1 genes of S. carlsbergensis were cloned in λ vectors. Mapping of 16 restriction enzyme sites showed identity between the allele located on the recombining chromosome and theILV1 gene of S. cerevisiae. The nucleotide sequence of theILV1 gene of the non-recombining chromosome was by restriction site mapping found to be very different from that of the S. cerevisiae allele.

STP1, A GENE INVOLVED IN PRE-TRNA PROCESSING IN YEAST, IS IMPORTANT FOR AMINO-ACID UPTAKE AND TRANSCRIPTION OF THE PERMEASE GENE BAP2

Abstract The bap1 mutant of Saccharomyces cerevisiae was previously isolated by its reduced uptake of branched-chain amino acids. In the present study, the corresponding wild-type gene was cloned and partial sequencing and subsequent genetic analysis revealed identity to STP1, a gene involved in tRNA maturation. The decrease in amino-acid uptake caused by stp1 mutations is independent of GCN4. It was previously found that the BAP2 promoter can be activated by the presence of amino acids, notably leucine, in the medium. We found that this activation depends on STP1. As a simple hypothesis we propose that Stp1p is a transcription factor which activates BAP2, and probably other amino-acid permease genes.

The pYC plasmids, a series of cassette-based yeast plasmid vectors providing means of counter-selection.

A series of 24 general‐purpose yeast plasmid vectors has been constructed. The plasmid series is composed of inter‐replaceable cassettes, allowing for easy interconversion of plasmid types. In addition to the usual replication origins, selectable markers and multiple cloning sites (MCS), cassettes dedicated to counter‐selection have been constructed. A pair of unique 8 bp restriction enzyme recognition sites flank each type of cassette, FseI in the case of yeast replication origins, AscI in the case of selectable markers, PacI in the case of counter‐selectable markers and NotI in the case of the MCS. Thus, any given cassette can be replaced by another cassette of the same type, facilitating interconversion of any given plasmid from one type to another, even after the insertion of DNA into the MCS. Hence, the plasmids have been named pYC for ‘yeast cassettes’. The cassettes consist of either NONE, CEN4/ARS or 2µ as replication origin, either URA3, MET2–CA (Lg–MET2) or the G418 resistance gene (the apt1 gene from bacterial transposon Tn903, encoding aminoglycoside phosphotransferase) as selectable markers, either NONE, PMET25–PKA3 or PCHA1–PKA3 as counter‐selectable marker, and the MCS, containing recognition sites for AflII, AvrII, BspEI, PmeI, SacII, SalI, SunI, BamHI, EcoRI, HindIII, KpnI, MluI, NarI and SacI (of which the seven first are unique in all plasmids). The counter‐selectable markers consist of the PKA3 gene under control of the conditional MET25 or CHA1 promoters. At activating conditions these promoters express the PKA3 gene at toxic levels, facilitating easy selection for loss of plasmid or ‘loop‐out’ of plasmid DNA sequence after genomic integration. Copyright

Cysteine uptake by Saccharomyces cerevisiae is accomplished by multiple permeases.

Abstract Uptake by Saccharomyces cerevisiae of the sulphur-containing amino acid L-cysteine was found to be non-saturable under various conditions, and uptake kinetics suggested the existence of two or more transport systems in addition to the general amino-acid permease, Gap1p. Overexpression studies identified BAP2, BAP3, AGP1 and GNP1 as genes encoding transporters of cysteine. Uptake studies with disruption mutants confirmed this, and identified two additional genes for transporters of cysteine, TAT1 and TAT2, both very homologous to BAP2, BAP3, AGP1 and GNP1. While Gap1p and Agp1p appear to be the main cysteine transporters on the non-repressing nitrogen source proline, Bap2p, Bap3p, Tat1p, Tat2p, Agp1p and Gnp1p are all important for cysteine uptake on ammonium-based medium. Furthermore, whereas Bap2p, Bap3p, Tat1p and Tat2p seem most important under amino acid-rich conditions, Agp1p contributes significantly when only ammonium is present, and Gnp1p only contributes under the latter condition.

Approaches to the Genetic Analysis and Breeding of Brewer's Yeast

Man has subjected many plants to breeding to suit his needs better. Some plant species have been bred for thousands of years. In modern times plant breeding has become increasingly intentional and systematic. Saccharomyces yeasts belong, according to their use, to man’s old, cultivated plants, but with the exception of pure culture selection (Hansen 1888) they have undergone little intentional or systematic breeding.

Mutagenesis and genetic transformation of meiotic segregants of lager yeast

Auxotrophic mutants have been induced by UV-irradiation or EMS treatment in meiotic segregants of the Carlsberg lager production strain. In complementation tests some of the mutations were allelic toadel, ade2, ade6, his1, leu2, lys5 andura2 of S. cerevisiae.Aleu2 mutant was transformed to leucine prototrophy by pYF91.2, a self-replicating plasmid containingLEU2 from S. cerevisiae. The transformants are unstable for the Leu+ phenotype on non-selective medium and DNA hybridization analysis of one of the transformants, using a radioactively labeled probe, showed the presence of plasmid sequences in the transformant.