~~Several yeast species/isolates belonging to the genus Saccharomyces were examined for the organization of their mtDNAs and ability to generate petite mutants. A general characteristic for all of the mtDNAs tested was that they were very A+T-rich. However, restriction patterns and inducibility of petite mutations revealed a great diversity in the organization and genetic behaviour of mtDNAs. One group of yeasts, Saccharomyces sensu stricto, contains mtDNA ranging in size from 64 to 85 kb. mtDNAs from these yeasts contain a high number of restriction sites that are recognized by the enzymes Haelll and Mspl, which cut specifically in G+C clusters. There are three to nine o r i h p sequences per genome. These yeasts spontaneously generate respiration deficient mutants. Ethidium bromide (Et-Br), a t low concentrations, induces a majority of cells to give rise to petites. A second group of yeasts, Saccharomyces sensu lato, contains smaller mtDNAs, ranging in size from 23 to 48 kb, and probably only a f e w intergenic G+C clusters and no ori/rep sequences. These yeasts also generate petite clones spontaneously, but Et-Br, even when present a t high concentrations, does not substantially increase the frequency of petites. In most petite clones from these yeasts only a small fragment of the wild-type molecule is retained and apparently multiplied. A third group, represented by Saccharomyces kluyveri, does not give rise to petite mutants either spontaneously or after induction.Keywords : yeast, mitochondrial genome, petite mutation, intergenic sequences, taxonomy INTRODUCTIONA remarkable aspect of mitochondrial genomes of all organisms is that they contain a very similar set of genes. On the other hand, mtDNA molecules among diverse species are highly variable in size and organization (5,36). The yeast Saccharomyces cerevisiae has played the central role in studies of mtDNA heredity (for reviews, see 9, 28). This is due mainly to the property that this yeast is a facultative anaerobe; i.e. it can survive without active mitochondria. The average cell of S. cerevisiae contains as many as 50 mtDNA molecules, but the number varies J. Piikur and others almost half of the genome, and they are usually separated from each other by over 200 G + C clusters (38). A large fraction of G + C clusters contains recognition sites for restriction enzymes splitting target sequences which contain only G and C residues, i.e.HaeIII and HpaIIIMspI (38). A special class of G + C clusters are orilrep sequences which are about 300 bp long and are present in eight copies scattered around the genome (4,40). G + C clusters can be grouped into eight families which presumably originated from a proto-G+ C cluster (38). Therefore, mtDNA contains a variety of short duplications which potentially can be involved in intramolecular recombination.S. cerevisiae spontaneously produces mutants, petites, which are deficient in the ability to respire aerobically. The spontaneous frequency is about 1 %, but upon induction with chemical mutagens, e.g. ethidium bro...

30 bottom fermenting strains, 13 hybrid lager strains, 11 top fermenting strains. In addition, 24 other yeast species and strains have been analysed in this study. Four DNA regions have been monitored by restriction endonuclease fragment pattern polymorphisms. The regions analysed are: RDN1 (encoding cytosolic ribosomal RNA molecules) located on chromosome XII in S. cerevisiae, HIS4 (histidine 4) and LEU2 (leucine 2) both located on chromosome III and the Ty elements which are distributed at different positions in the genome ofS. cerevisiae. Seven Ty fragment patterns have been detected in the bottom fermenting strains. Patterns I, II, IIa, lib, and llc are present in lager strains while pattern III and IV are found in S. carlsbergensis bottom fermenting strain No. I and in S. monacensis, respectively. Hybridization of the Tyl p14 probe from S. cerevisiae to OFAGE separated chromosomes from bottom fermenting strains show that at least five Ty elements are present in the genomes of the lager strains, at least five in S. carlsbergensis bottom fermenting strain No. I and at least three in S. monacensis. From the restriction fragment patterns and the karyotypes of the bottom fermenting strains it is suggested that these strains are closely related and different from the S. bayanus, S. pastorianus, and S. uvarum group as well as from the S. cerevisiae strains. In spite of their differences in Ty element patterns the lager strains are so homogenous that most likely they all originate from a single strain which is related to S. carlsbergensis and S. monacensis type strains. In contrast to the bottom fermenting strains there is a large variability among the top fermenting strains, especially with regard to the LEU2 gene and Ty elements. It seems that S. bayanus, S. inusitatus, S. pastorianus, S. validus, and S. uvarum are closely related, and none of the studied strains from these taxons contained DNA which hybridized to the Tyl probe from S. cerevisiae. This is a clear difference from the DNA of bottom fermenting strains which consistently hybridized strongly to the Tyl probe.Abbreviations: kb = kilo base pairs; OFAGE = orthogonal field alternation gel electrophoresis; SSC = 0.15 M-NaCI, 15 mM-Na citrate; TBE = 0.089 M-Tris-borate, 0.089 M-boric acid, 0.002 M-EDTA; Tris = tris-(hydroxymethyl)-amino methane Springer-Verlag

The region of chromosome XII containing the RDNI gene which encodes the cytosolic ribosomal RNA molecules and the region of chromosome Ill containing the H1S4 (histidine 4) gene were analysed in 30 lager yeast strains, 11 ale strains and 20 strains from a number of different species in the genus Saccharomyces.With the aid of restriction endonuclease fragment patterns and cloned probes to the RDN1 gene ofS. cerevisiae three forms of this gene were identified, two of them corresponding to the previously known forms I and II and a third one characterized by an additional HindlIl site located in the 3' spacer region. A more distantly related form of the RDNlgene containing a single HindlIl restriction site was found in Saccharomyces fermentati and one form without any Hindlll site in a wild yeast contaminant.With the help of the restriction endonuclease fragments derived from the HIS4 region seven genotypes can be recognized. They result from various combinations of three restriction endonuclease fragment patterns designated I, II and III, each pattern represents a chromosome.All lager strains are homozygous for form II of the ribosomal RNA gene and heterozygous for patterns I and II of the HIS4 gene. An exception is one German brewing strain which is homozygous for pattern II.With one exception the ale strains were homozygous for form II of the RDN1 gene and for pattern I of the HIS4 gene. One British strain contains form I of the RDN1 gene. Bakers yeast, S. diastaticus and S. italicus are homozygous for form I of the RDN1 gene and for pattern I of the HIS4 gene. In S. bayanus and S. pastorianus homozygosity for form III of the RDN1 gene was combined with heterozygosity for patterns II and III of the HIS4 gene. S. uvarum is homozygous for both form III of the RDN1 gene and pattern III of the HIS4 gene. Form III of the RDN1 gene and patterns I and ti of the HIS4 gene were combined in a Chinese brewing strain and a strain designated as a type strain ofS. carlsbergensis.The nucleotide sequence polymorphisms are useful markers for strain characterization in addition to the generally used fermentation properties.Abbreviations: bp = basepairs; kb = kilobase; SSC = 0.15 M-NaCI, 15 mM-Na citrate; Tris -tris-(hydroxymethyl)-amino methane.

Single chromosome III transfers have been accomplished from the two species S. bayanus and S. uvarum into genetically marked S. cerevisiae strains. Incompabilities between S. bayanus or S. uvarnm, and S. cerevisiae strains may account for the very few chromosome addition lines obtained. The preliminary genetic map for S. bayanus chromosome III is similar to the genetic map for a S. carlsbergensis chromosome lII from Danish lager strain BK2208. Recombination is absent between the transferred chromosomes and the auxotrophically marked chromosome from S. cerevisiae in the interval HIS4 to LEU2 but recombination does occur in the interval LEU2 to THR4. The mapped S. bayanus chromosome III carries HIS4 pattern III while the mapped chromosome III from the lager strain contains HIS4 pattern II. Transfer of a chromosome III from S. uvarum to S. cerevisiae has been recognised by restriction endonuclease fragment patterns, OFAGE chromosome separations and molecular hybridization, but a genetic map could not be constructed for an S. uvarum chromosome due to instability of the chromosome addition lines. The hypothesis is put forward that the S. carlsbergensis type strain and the lager strains are sibling species and species hybrids both produced by hybridization of an S. cerevisiae top fermenting strain and the bottom fermenting strain S. monacensis. The lager strains and the authentic type strain are designated S. carlsbergensis. From the electrophoretic karyotypes of type strains of Saecharomyces and molecular hybridization patterns it is deduced that S. bayanus, S. inusitatus, S. pastorianus and S. uvarum are closely related but distinct from the bottom fermenting strains. I N T R O D U C T I O NBase composition, DNA-RNA homology, DNA-DNA reassociation kinetics and restriction endonuclease fragment length polymorphisms have proven useful in the taxonomic evaluations of genera and species as well as in the diagnosis of closely related yeast strains (3,4,15,21,23,24,25,26,27,28,30,40). For taxonomic as well as breeding purposes the establishment of genetic linkage groups and their assignment to Abbreviations: OFAGE = Orthogonal field alternation gel electrophoresis; TBE = 0.089 M-Tris-borate, 0.089 M-boric acid, 0.002 M-EDTA; SSC = 0.15 M-NaC1, 15 mM-Na citrate.Springer-Verlag

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