Transformation of yeast without the use of foreign DNA

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

Section: Strains and Mediamentioning

confidence: 99%

DNA sequence polymorphisms in the genus Saccharomyces IV. Homoeologous chromosomes III of Saccharomyces bayanus, S. carlsbergensis, and S. uvarum

Pedersen

1986

“…SC, YPD and YPG have been described (5). SC was made without serine, aspartic acid and glutamic acid.…”

Section: Mediamentioning

confidence: 99%

Genetic evidence for nuclear location of 2-micron DNA in yeast

Kielland‐Brandt

Wilken

Holmberg

et al. 1980

Self Cite

Progeny from karl• crosses in Saccharomyces cerevisiae were isolated by selection for a mitochondrial marker from each parent. Haploid progeny (heteroplasmons, cytoductants) were obtained at high frequency because of the kar mutation and were identified by replication of progeny colonies to media differentiating on the basis of chromosomal markers. In some of the crosses nuclei of both parental types were found among the progeny, although the KARl nuclei were in majority.In crosses where only one parent contained the plasmid 2-micron DNA, all progeny inheriting the nucleus from that parent also had the plasmid, whereas only about 25-50 % of the progeny with the other nucleus had the plasmid. In crosses where the size of 2-micron DNA of the parents was different, the haploid progeny had a plasmid size identical to that of the parent from which it had received its nucleus.On the basis of these observations we suggest a nuclear location of 2-micron DNA. We propose that one or a few copies of the plasmid are frequently transferred from one nucleus to another in karl mediated transient heterokaryons.Abbreviations: can = canavanine, ery = erythromycin, leu = leucine, oli = oligomycin, SC --synthetic complete dextrose, SCE = sorbitol citrate EDTA, YPD = yeast extract peptone dextrose, YPG = yeast extract peptone glycerol.

“…The media used for growth of yeast have been described (16). E. coli was grown in LB (1% Difco Bacto tryptone, 0.5% Difco Bacto yeast extract, 1% NaCI, 0.1% glucose).…”

Section: Media and Growthmentioning

confidence: 99%

“…The procedure for transformation of yeast has been described (1,16). The amount of DNA and number of protoplasts used are given in the text for each experiment.…”

Section: Transformationmentioning

confidence: 99%

See 1 more Smart Citation

Molecular characterization of a Saccharomyces plasmid containing theHIS4 gene

Holmberg

Petersen

Nilsson-Tillgren

et al. 1979

Self Cite

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.