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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.

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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.