Gerald H. Rank

Gene transfer in industrial Saccharomyces yeasts

Abstract Industrial strains of Saccharomyces cerevisiae used in the brewing, baking, distilling, wine and sake industries have been uniquely selected within each industry for optimum performance. Nevertheless, the genetic structure of industrial strains is sufficiently closely related to haploid laboratory strains of Saccharomyces cerevisiae to utilize laboratory based vectors for single gene transfer. We describe gene transfer systems based on markers that have the potential for use in industrial strains and indicate when these vectors have been used in industrial yeasts. Alternative gene transfer systems based on selection for carbohydrate utilization and acquisition of killer immunity are discussed. In addition, the limitations of current vector systems are reviewed with a view to improving gene transfer systems that meet the requirements of industry.

Cloning of industrial Saccharomyces 2-μm plasmid variants by in vivo site-specific recombination

Southern analyses defined several industrial Saccharomyces yeast strains with extensive 2-microns DNA polymorphism. Variants included insertions and deletions up to several hundred base pairs. To facilitate the investigation of yeast plasmid evolution we developed a novel method of cloning 2-microns plasmids by taking advantage of 2-microns circle in vivo site-specific recombination and an SMRI gene as a dominant selectable marker. This method can be applied to other organisms for the isolation of plasmid variants and provides a new approach to in vivo plasmid construction.

Evidence forCis- andTrans-acting element coevolution of the 2-μm circle genome inSaccharomyces cerevisiae

SummaryWe compared the DNA sequence of the yeas 2-μm plasmidcis-actingSTB andtrans-actingREP1 partition loci of laboratory haploid and industrial amphiploid strains. Several industrial strains had a uniqueSTB sequence (type 1) sharing only 70% homology with laboratorySTB (type 2). Type 1 plasmids had a REP1 protein with 6–10% amino acid substitutions when compared to REP1 of type 2 plasmids. All 2-μm variants that shared a similarSTB consensus sequence exhibited a high degree ofREP1 nucleotide and amino acid sequence conservation. These observations suggest molecular coevolution oftrans-acting elements with cognate target DNA structure. Based on DNA sequencing and Southern hybridization analyses, we classified 2-μm variants into two main evolutionary lineages that differ atSTB as well asREP1 loci. The role of molecular coevolution in yeast intra- and interspecies plasmid evolution was discussed.

Sequence diversity of yeast 2 μm RAF gene and its co-evolution with STB and REP1

Abstract Despite the extensive study of yeast 2 μm plasmid, the exact function of plasmid-encoded RAF gene is not clear. Variants of 2 μm plasmids from industrial Saccharomyces cerevisiae yeasts were isolated and characterized. Sequencing of RAF alleles revealed about 8 % nucleotide and 10% amino acid diversities between 2 μm variants of closely related strains. RAF sequence variations were correlated with STB-REP1 sequence diversity. We also used restriction fragment length polymorphism linkage to screen a large number of yeast strains from different fermentation industries. The results clearly show a tight linkage of STB-REP1-RAF variations. Thus, our observations suggest that plasmid-borne cis- and tram-acting elements co-evolved to form an optimal molecular parasite and that RAF may play a role in active plasmid partitioning.

Antisense RNA regulation of the ILV2 gene in yeast: a correction

A previous report of the use of antisense RNA to regulate gene expression in yeast is incorrect.

An improved method for yeast 2μm plasmid curing

SMR1-410, a dominant resistance marker, was cloned into the FLP gene of 2 microns DNA to produce the chimeric YEp vector pWX823B. Selection for SMR1-410 at high concentrations of sulfometuron methyl maintained pWX823 at high copy number and resulted in the rapid and efficient loss of native 2 microns DNA. Using this protocol approximately 15% of the cells monitored showed loss of 2 microns DNA. The curing methodology is more efficient and convenient than previous methods and has the added advantage of being applicable to wild-type prototrophic cells.

Transpogenes: the transposition-like integration of short sequence DNA into the yeast 2μ, plasmid creates the STB locus and plasmid-size polymorphism

The type-2 2 mu plasmid of industrial yeast strains exhibits extensive size polymorphism in the STB (plasmid stability) locus and IR (inverted repeat)-right region. Comparative DNA sequence analyses of STB alleles identified a 38-bp sequence flanked by a 25-bp direct repeat as the underlying structural motif. Variable unequal recombination within the direct repeat accounted for the observed polymorphism of STB alleles. IR-right polymorphism was observed to result from tandem duplication of a 22-bp sequence flanked by a 9-bp direct repeat. The flanking direct repeats marked both loci as originating from the transposition-like integration of short DNA fragments. We call these structures transpogenes and note that these are hybrid structures of host and foreign DNA which can evolve into functional loci.