No abstract

An improved pulsed-f ield electrophoresis program was developed to study differently sized chromosomes within the genus Saccharomyces. The number of chromosomes in the type strains was shown to be nine in Saccharomyces castellii and Saccharomyces dairenensis, 12 in Saccharomyces servazzii and Saccharomyces unisporus, 16 in Saccharomyces exiguus and seven in Saccharomyces kluyveri. The sizes of individual chromosomes were resolved and the approximate genome sizes were determined by the addition of individual chromosomes of the karyotypes. Apparently, the genome of 5. exiguus, which is the only Saccharomyces sensu lato yeast to contain small chromosomes, is larger than that of Saccharomyces cerevisiae. On the other hand, other species exhibited genome sizes that were 10-25% smaller than that of 5. cerevisiae. Well-defined karyotypes represent the basis for future genome mapping and sequencing projects, as well as studies of the origin of the modern genomes.

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 carrying karl as well as the chromosome V markers canl, ura3, his1, ilvl, and rad3. 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. cadsbergensis 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 region CAN1 -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 the CAN1 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 probes CAN1, URA3, CYC7, and ILV1 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 ~ 0.1 xSSC), hybridization of the probes to DNA from the strain with the homoeologous chromosome was only observed in the case oflLVl, where weak hybridization was found, indicating a considerable difference in nucleotide sequence.To further study the extent ofnucleotide sequence inhomology, the two different ILV1 genes ofS. carlsbergensis were cloned in ~ vectors. Mapping of 16 restriction enzyme sites showed identity between the allele located on the recombining chromosome and the ILV1 gene of S. cerevisiae. The nucleotide sequence of the ILV1 gene of the non-recombining chromosome was by restriction site mapping found to be very different from that of the S. cerevisiae allele.

No abstract

We describe the transfer, at low frequency, of a limited number of nuclear markers during karl mediated cytoduction of the RHO § factor. By selection for a chromosome III marker in KAR 1 HIS4 [RHO + ] • karl his4 [rho-] crosses, strains disomic for chromosome III were isolated. Markers carried on five other chromosomes in the HIS4 donating strain could be shown to be absent from the disomic strains. When these disomic strains were force-mated to haploid tester strains the rare 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.

During alcoholic fermentations, the off-flavour compound diacetyl is formed non-enzymatically from acetolactate leaking out from the cells. Acetolactate is an intermediate in the biosynthesis of valine. In beer fermentation, the amount of diacetyl is reduced to acceptable levels during maturation. A reduction of the time needed for maturation may be achieved by the use of a brewing yeast that produces less diacetyl. Saccharomyces cerevisiae laboratory strains with an inactive ilv2 gene can not form acetolactate, while ilv5 strains, blocked in the subsequent step, leak acetolactate in high amounts. Induction of recessive mutations in production strains of Saccharomyces carlsbergensis has not yet been achieved, as the yeast is polyploid and possibly a hybrid between S. cerevisiae and another Saccharomyces species. Thus, all chromosomes investigated so far are present in at least two genetically different versions. Genetic and molecular analysis has shown that the brewing yeast is structurally heterozygous for ILV2 and ILV5. Genetic modification of brewers' yeast to reduce diacetyl formation is being carried out by mutation of ILV2. Deletion mutations in both ILV2 alleles have been constructed in vitro to be used for gene replacement in the brewing strain. In addition, partial inactivation of the ILV2 function is carried out by selecting spontaneous dominant mutations resistant to the herbicide sulfometuron methyl. Among these mutants some produce only half the amount of diacetyl compared to the parental strain. An alternative way to reduce diacetyl production might be to increase the activity of the ILV5 gene product. Model experiments in S. cerevisiae show that the presence of the ILV5 gene on a 2-micron based multi-copy vector can reduce the diacetyl production by half.

Chromosomal DNA is considered a priori to be a target for the induction of numerical (whole chromosome) aneuploidy in mitotic cells. If true, DNA repair would be expected to contribute to genome stability. One type of repair that appears to play an important role in the response of many organisms to DNA-damaging agents involves recombination. Using the yeast Saccharomyces cerevisiae containing a pair of DNA divergent (homoeologous) chromosomes, we have been able to determine the importance of recombinational repair of DNA damage in the maintenance of chromosome number. Specifically, the induction of aneuploidy by ionizing radiation has been examined in diploids that had one chromosome in replaced by a divergent chromosome from Saccharomyces carlsbergensis. The chromosomes are functionally equivalent but lack precise DNA homology over one-half their length. The absence of homology, and thus the opportunity for recombinational repair (presumably of DNA double-strand breaks) in the divergent chromosomes, results in high levels (5-10%) of aneuploidy for chromosome III at doses of radiation resulting in almost no killing. For homologous chromosomes, the frequency of loss is 20-50 times lower.

In a previous study, genetic transformation in yeast was carried out with a mixture of the yeast plasmid 2-micron DNA and total yeast DNA, which was treated with restriction endonuclease Pstl and DNA ligase. Strains were derived that contain a plasmid which carries the H1S4 gene. In the present study new plasmids have been constructed by combining the HIS4 carrying yeast plasmid, as well as parts of it, with bacterial plasmids. The hybrid plasmids were propagated in bacteria and analysed. From the data obtained the original plasmid was inferred to be a 2-micron DNA circle with a 9.4 kb insertion carrying the HIS4 gene. This structure was confirmed by restriction endonuclease analysis of plasmid DNA from the original transformant, using molecular hybridization to detect fragments which contain sequences of the 9.4 kb insert. The insert, which is bordered by two PstI sites, was mapped with restriction endonucleases EcoRl, Hindlll, and Sail. No BamHl site is present in the insert.Abbreviations: BSA = bovine serum albumin, cc = closed circles, ccDNA = closed circular DNA, kb = kilobases, oc = open circles.