Steen Holmberg

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.

Genetic differences between Saccharomyces carlsbergensis and S. cerevisiae II. Restriction endonuclease analysis of genes in chromosome III

Chromosome III in a haploid Saccharomyces cerevisiae strain has been previously substituted for by its homeologue from S. carlsbergensis. With this chromosome substitution line the two homeologous chromosomes were shown to undergo crossing over only in a limited region, and to differ in nucleotide sequence at theHIS4 locus. In the present study the S. carlsbergensis chromosome III was compared to its S. cerevisiae homeologue at several additional loci.Cloned DNA from the S. cerevisiae lociHML, HIS4, LEU2, MAT andSUP-RL1 was used as hybridization probes in the analysis of nucleotide sequence homology at these loci andHMR. Virtually no differences were detected atSUP-RL1 andHMR, located in the region where the two chromosomes recombine, whereas considerable differences were found in the non-recombining part. The data are consistent with the assumption thatHML, MAT andHMR of the S. carlsbergensis chromosome are organized as in S. cerevisiae Segments X and Z1, which are involved in mating type interconversion, were closely homologous to their S. cerevisiae counterparts, whereas Y α as well as sequences outside theHML andMAT cassettes were substantially different.

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.

A mutant of Saccharomyces cerevisiae temperature sensitive for flocculation. Influence of oxygen and respiratory deficiency on flocculence

A flocculent strain of Saccharomyces cerevisiae, containing the dominant gene for flocculenceFL04, was mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine. Non-flocculent mutants were isolated with a selection procedure based on the slower sedimentation of non-flocculent cells. One closer studied mutant was due to an unlinked suppressor mutation forFL04. This suppressor gene is designatedsufl. The genesufl is neither centromere linked nor linked tohis4 or the mating type locus. The genesufl behaves as a recessive in some diploids and as a dominant in others, illustrating the genetic complexity of the flocculation phenomenon.The mutant was found to be non-flocculent after growth at 30°C whether aerated or not. At 22°C the mutant was flocculent in the absence of aeration during growth, but non-flocculent with aeration or supplementation of the growth medium with ergosterol and unsaturated fatty acids. None of several inhibitors of mitochondrial functions had any effect on the expression of flocculence. A number of petites induced in the mutant strain with ethidium bromide had altered flocculation phenotypes.A method for measuring flocculence using the spectrophotometer is described.

Transfer of chromosome III duringkar mediated cytoduction in yeast

We describe the transfer, at low frequency, of a limited number of nuclear markers duringkarl mediated cytoduction of the RHO+ factor. By selection for a chromosome III marker inKAR1 HIS4[RHO+]×karl his4 [rho−] crosses, strains disomic for chromosome III were isolated. Markers carried on five other chromosomes in theHIS4 donating strain could be shown to be absent from the disomic strains. When these disomic strains were force-mated to haploid tester strains the rate prototrophic products were sporulators with good spore viability. These observations suggest that only one or possibly a few chromosomes were transferred to the recipient strain during cytoduction of the RHO+ factor.

Regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae.

SummaryThe threonine deaminase gene (ILV1) of Saccharomyces cerevisiae has been designated “multifunctional” since Bollon (1974) indicated its involvement both in the catalysis of the first step in isoleucine biosynthesis and in the regulation of the isoleucine-valine pathway. Its role in regulation is characterized by a decrease in the activity of the five isoleucine-valine enzymes when cells are grown in the presence of the three branched-chain amino acids, isoleucine, valine and leucine (multivalent repression). We have demonstrated that the regulation of AHA reductoisomerase (encoded by ILV5) and branched-chain amino acid transaminase is unaffected by the deletion of ILV1, subsequently revealing that the two enzymes can be regulated in the absence of threonine deaminase. Both threonine deaminase activity and ILV1 mRNA levels increase in mutants (gcd2 and gcd3) having constitutively derepressed levels of enzymes under the general control of amino acid biosynthesis, as well as in response to starvation for tryptophan and branched-chain amino acid imbalance. Thus, the ILV1 gene is under general amino acid control, as is the case for both the ILV5 and the transaminase gene. Multivalent repression of reductoisomerase and transaminase can be observed in mutants defective in general control (gcn and gcd), whereas this is not the case for threonine deaminase. Our analysis suggests that repression effected by general control is not complete in minimal medium. Amino acid dependent regulation of threonine deaminase is only through general control, while the branched-chain amino acid repression of AHA reducto isomerase and the transaminase is caused both by general control and an amino acid-specific regulation.

Mutational analysis of isoleucine-valine biosynthesis in Saccharomyces cerevisiae. Mapping ofilv2 andilv5

Fifty-five isoleucine-valine requiring mutants were selected in a haploid strain of Saccharomyces cerevisiae after treatment with ethyl methanesulfonate. All the mutants could be assigned to the known complementation groups,ilv1 (threonine deaminase),ilv2 (acetohydroxyacid synthetase),ilv5 (acetohydroxyacid reductoisomerase) andilv3 (dihydroxyacid dehydrase). Intragenic complementation occurs in bothilv2 andilv5. The levels of α-acetohydroxyacids formed by these four types of mutants were consistent with the previous notion that the mutants define the structural genes coding for these enzymes.Chromosome assignment by the Rec (spo11) mapping method showed thatilv2 was located on chromosome XIII, andilv5 on chromosome XII. The locations were confirmed by mitotic analyses, using eitherrad52 homozygosis or benzimidazol carbamic acid methyl ester. Tetrad analysis placedilv2 on the right arm of chromosome XIII, 36 cM distal tolys7, andilv5 was positioned on the right arm of chromosome XII, distal to the ribosomal RNA gene cluster (rDNA) and proximal toura4.

Transformation of yeast without the use of foreign DNA

Isolated circular molecules of the yeast plasmid 2-micron DNA were converted to linear molecules with restriction endonuclease PstI and ligated with T4 DNA ligase to PstI restriction fragments of total yeast DNA. A haploid strain of Saccharomyces cerevisiae carrying a deletion in thehis4 locus was transformed with the ligated DNA mixture to histidine prototrophy. One unstable histidine prototrophic transformant was obtained. Grown in the absence of histidine, 70–75% of the cells were auxotrophic and this number increased in non-selective medium. The histidine auxotrophic variants carried a deletion in thehis4 locus which had patterns of complementation and UV-induced mitotic recombination identical to the originalhis4 deletion. When the transformant was crossed to ahis4 strain and sporulated, the unstable histidine prototrophy segregated in a non-mendelian way: All five possible segregations from 0∶4 to 4∶0 were observed. When strains carrying the transformed character were crossed to ahis4 karl strain a low frequency of cytoduction of the unstable histidine prototrophy was observed. Nucleic acid from the transformant was able to transform a strain which carried another deletion in thehis4 locus. Treatment of the transformant with ethidium bromide caused an extensive induction of petites without any observable change in the frequency of histidine prototrophic cells.It is concluded that theHIS4 gene function in the transformant is not stably associated with any chromosome. We take the instability as indication that only one or a few copies of the gene conferring the prototrophy are present in the prototrophic cells. The data are consistent with the assumption that the transformant contains a 2-micron DNA in which is inserted a chromosomal DNA region containing theHIS4 gene. A derivative of the transformant with increased stability has been isolated.

Regulated overproduction and secretion of yeast carboxypeptidase Y

SummaryCarboxypeptidase Y (CPY) is a glycosylated yeast vacuolar protease used commercially for synthesis of peptides. To increase the production of CPY in Saccharomyces cerevisiae we have placed its coding region (PRC1) under control of the strongly regulated yeast GAL1 promoter on multicopy plasmids and introduced the constructs into vpl1 mutant strains. Such mutants are known to secrete CPY. High levels of CPY production were obtained by induction of the GAL1 promoter when the cells had left the exponential phase, resulting in a growth-phase-dependent CPY production similar to that cells with PRC1 under the control of its own promoter. Introduction of a high copy number 2μ-URA3-EU2d plasmid with GAL1p-PRC1 fusion in a vpl1 strain resulted in a 200-fold increase of secreted CPY (about 40 mg/l) as compared to a vpl1 mutant carrying a single copy of the wild-type PRC1 gene. The overproduced, secreted CPY was active and had the normal N-terminal sequence. Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed two forms of active CPY, probably due to different levels of glycosylation.

Isolation and characterization of a polypeptide absent from non-flocculent mutants of Saccharomyces cerevisiae

Alkaline cell extracts obtained from whole cells of a flocculent strain of Saccharomyces cerevisiae, containing the dominant gene for flocculenceFL04, and a nonflocculent mutant (FL04, fsul) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mutant lacked a low molecular weight (13,000) polypeptide present in the extract from the parent strain. This polypeptide difference was also observed when five other independently isolated non-flocculent mutants of the parent strain were analyzed. One of these five mutants was characterized genetically and also in this case was non-flocculence shown to be due to an unlinked suppressor mutation ofFL04. This suppressor gene is designatedfsu2. Analysis of petites with different flocculation phenotypes further extended the correlation between non-flocculence and the absence of the polypeptide.The polypeptide was isolated by gel filtration in the presence of sodium dodecyl sulfate followed by ionexchange chromatography on DEAE-cellulose. By electrophoretic analysis the purity of the preparation was estimated to be 95%. From amino acid analysis the polypeptide was calculated to consist of 121 residues with a molecular weight of 12,900 daltons.Differential extraction of proteins iodinated in situ by lactoperoxidase suggested an external location of the polypeptide.