Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae.

The 5' control region of the yeast phosphoglycerate kinase gene (PGK) was fused to the coding sequence of a human interferon-alpha. This PGK-interferon fusion was then introduced into yeast on a high copy number 2mu-based plasmid vector. Strains containing this plasmid produced a PGK-interferon-alpha fusion protein as 1-2% of cell protein and the expression of interferon activity was regulated by the availability of a fermentable carbon source. The system is capable of making as much as 15 mg of human interferon-alpha per litre of batch culture.     

Characterisation of sequences required for RNA initiation from the PGK promoter of Saccharomyces cerevisiae.

In the phosphoglycerate kinase (PGK) gene of yeast, as in other highly expressed yeast genes, the sequences surrounding the site of RNA initiation have a loosely conserved structure of a CT rich stretch followed by the tetranucleotide CAAG. Using internal deletions and insertions we have identified the elements in the PGK promoter which are required for correct RNA initiation at the CAAG sequence at -39. The results indicate that two different components of the PGK promoter contribute to correct RNA initiation, the TATA homologies, located at -152 and -113, and the sequences at the site of initiation. Both TATA elements can function in RNA initiation. Deletion of the upstream TATA element, TATAI, results in slightly heterogeneous RNA initiation, but the majority of the RNA initiates correctly. Deletion of both the PGK TATA elements results in the majority of the RNA initiating at sites downstream from the wild-type I site, within the structural gene between +40 to +80. The CT rich box is not essential for correct mRNA initiation as shown by deletion analysis. The site of RNA initiation in the PGK promoter appears to be determined by sequences located immediately 5' of the CAAG sequence motif. This short sequence, ACAGATC, when located the correct distance from the TATA elements may be sufficient to determine a discrete initiation site.     

Hypervariable noncoding sequences in Saccharomyces cerevisiae

Compared to protein-coding sequences, the evolution of noncoding sequences and the selective constraints placed on these sequences is not well characterized. To compare the evolution of coding and noncoding sequences, we have conducted a survey for DNA polymorphism at five randomly chosen loci among a diverse collection of 81 strains of Saccharomyces cerevisiae. Average rates of both polymorphism and divergence are 40% lower at noncoding sites and 90% lower at nonsynonymous sites in comparison to synonymous sites. Although noncoding and coding sequences show substantial variability in ratios of polymorphism to divergence, two of the loci, MLS1 and PDR10, show a higher rate of polymorphism at noncoding compared to synonymous sites. The high rate of polymorphism is not accompanied by a high rate of divergence and is limited to a few small regions. These hypervariable regions include sites with three segregating bases at a single site and adjacent polymorphic sites. We show that this clustering of polymorphic sites is significantly greater than one would expect on the basis of the spacing between polymorphic fourfold degenerate sites. Although hypervariable noncoding sequences could result from selection on regulatory mutations, they could also result from transient mutational hotspots.     

Deletion of plasmid sequences during Saccharomyces cerevisiae transformation.

Saccharomyces cerevisiae was transformed with DNA by the lithium acetate method. Mutation of nonselected markers on the transforming vector was observed at a frequency several orders of magnitude higher than spontaneous mutation frequencies. These mutations were shown to be deletions. Linearization of the vector before transformation stimulated deletion formation.     

Dynamics of activation of a galactose-inducible promoter in Saccharomyces cerevisiae.

We have investigated the dynamics of accumulation of the Escherichia coli beta-galactosidase (beta-gal) under the control of a promoter containing the galactose-inducible upstream activating sequence (UASG) in single Saccharomyces cerevisiae cells. The accumulation of beta-gal in single cells following the addition of the inducer, galactose, was determined using an in situ combined DNA and immunofluorescent stain in conjunction with flow cytometry. Two strains were studied, D603/2i, which has two copies of the galactose-inducible fusion gene integrated into its genome, and D603/pLGSD5, which carries a 2 microns-based plasmid containing the fusion gene. Flow cytometry results indicate that accumulation of beta-gal within the first three hours following the addition of galactose is dependent on cell cycle position. Two proposed mechanisms explaining this observed behavior are (1) the cell-cycle-dependent synthesis of the fusion protein or (2) the unequal partitioning of the protein at cell division between mother and daughter cells.     

The promoter of the beta-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae

Summary The relationship between the promoter length of the Kluyveromyces fragilis -glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless -galactosidase gene of Escherichia coli. The removal of a region from position-425 to-232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed -glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the -glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.     

Distribution of telomere-associated sequences on natural chromosomes in Saccharomyces cerevisiae.

Pulsed-field gel electrophoresis was used to examine the distribution of telomere-associated sequences on individual chromosomes in four strains of Saccharomyces cerevisiae. The pattern of X and Y' distribution was different for each strain. At least one chromosome in each strain lacked Y', and in some strains, chromosome I, the smallest yeast chromosome, lacked detectable amounts of both X and Y'.     

Chimeric plasmids for cloning of deoxyribonucleic acid sequences in Saccharomyces cerevisiae.

Two sets of plasmids, each carrying a Saccharomyces cerevisiae gene and a portion or all of the yeast 2-micron circle linked to the Escherichia coli plasmid pBR322, have been constructed. One of these sets contains a BamHI fragment of S. cerevisiae deoxyribonucleic acid that includes the yeast his3 gene, whereas the other set contains a BamHI fragment of S. cerevisiae that includes the yeast leu2 gene. All plasmids transform S. cerevisiae and E. coli with a high frequency, possess unique restriction endonuclease sites, and are retrievable from both host organisms. Plasmids carrying the 2.4-megadalton EcoRI fragment of the 2-micron circle transform yeast with 2- to 10-fold greater frequency than those carrying the 1.5-megadalton EcoRI fragment of the 2-micron circle. Restriction endonuclease analysis of plasmics retrieved from S. cerevisiae transformed with plasmics carrying the 2.4-megadalton EcoRI fragment showed that in 13 of 96 cases the original plasmic has acquired an additional copy of the 2-mcron circle. These altered plasmids appear to have arisen by means of an interplasmid recombination event while in S. cerevisiae. A clone bank of S. cerevisiae genes based upon one of these composite plasmids has been constructed. By using this bank and selecting directly in S. cerevisiae, the ura3, tyr1, and met2 genes have been cloned.     

Sequence variation in dispersed repetitive sequences in Saccharomyces cerevisiae

Two new dispersed repetitive DNA sequences related to the transposable element Tyl have been isolated from the genome of Saccharomyces cerevisiae. One sequence, designated Tyl-17, is present at about six copies per haploid genome, and one copy is located approximately 1000 base-pairs from the LEU2 locus on chromosome III. Tyl-17 is about the same size as Tyl (Cameron et al., 1979) and is flanked by δ sequences, but differs from Tyl by the presence of two large substitutions representing about 50% of the sequence. Tyl and Tyl-17 are found in a ‘head-to-head’ array in at least one cloned region of the yeast genome. Another sequence, designated Tyl-161, is situated about 9000 base-pairs from the PGK locus of chromosome III, and is structurally identical to Tyl except for the presence of a 1200 base-pair insertion near one end of the sequence element.     

Modulation of efficiency of translation termination in Saccharomyces cerevisiae

Nonsense suppression is a readthrough of premature termination codons. It typically occurs either due to the recognition of stop codons by tRNAs with mutant anticodons, or due to a decrease in the fidelity of translation termination. In the latter case, suppressors usually promote the readthrough of different types of nonsense codons and are thus called omnipotent nonsense suppressors. Omnipotent nonsense suppressors were identified in yeast Saccharomyces cerevisiae in 1960s, and most of subsequent studies were performed in this model organism. Initially, omnipotent suppressors were localized by genetic analysis to different protein- and RNA-encoding genes, mostly the components of translational machinery. Later, nonsense suppression was found to be caused not only by genomic mutations, but also by epigenetic elements, prions. Prions are self-perpetuating protein conformations usually manifested by infectious protein aggregates. Modulation of translational accuracy by prions reflects changes in the activity of their structural proteins involved in different aspects of protein synthesis. Overall, nonsense suppression can be seen as a “phenotypic mirror” of events affecting the accuracy of the translational machine. However, the range of proteins participating in the modulation of translation termination fidelity is not fully elucidated. Recently, the list has been expanded significantly by findings that revealed a number of weak genetic and epigenetic nonsense suppressors, the effect of which can be detected only in specific genetic backgrounds. This review summarizes the data on the nonsense suppressors decreasing the fidelity of translation termination in S. cerevisiae, and discusses the functional significance of the modulation of translational accuracy.