Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae.

DNA sequences normally flanking the highly expressed yeast 3-phosphoglycerate kinase (PGK) gene have been placed adjacent to heterologous mammalian genes on high copy number plasmid vectors and used for expression experiments in yeast. For many genes thus far expressed with this system, expression has been 15-50 times lower than the expression of the natural homologous PGK gene on the same plasmid. We have extensively investigated this dramatic difference and have found that in most cases it is directly proportional to the steady-state levels of mRNAs. We demonstrate this phenomenon and suggest possible causes for this effect on mRNA levels.     

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.     

Multiple copies of the pyruvate kinase gene affect yeast cell growth

The Saccharomyces cerevisiae pyruvate kinase gene (PYK1) was transformed into yeast using the multicopy vector pJDB207. Growth rates and PYK1 gene expression levels varied considerably amongst the transformants. Yeast transformants expressing the PYK1 gene at high levels formed small colonies compared with those expressing the gene at relatively low levels. Slow-growing transformants 'reverted' at high frequency to more rapid growth, and this correlated with decreases in PYK1 gene copy number and PYK1 mRNA abundance. This apparent selection against PYK1 over-expression was disrupted by the introduction of a stop codon at the 5'-end of the PYK1 coding region, thus confirming that the growth effects were mediated by the PYK1 gene. However, massive overproduction of pyruvate kinase in yeast, using multiple copies of a PGK:PYK gene fusion, had no significant effect upon cell growth. This suggests that the deleterious effect upon the host yeast cell is mediated by abnormally high levels of the wild-type gene or PYK1 mRNA, rather than by increased pyruvate kinase levels.     

Expression in yeast of antisense RNA to ADE1 mRNA

The utility of antisense RNA as a means of regulating gene expression in yeast has been explored by inserting into a high copy number yeast expression vector an ADE1 gene fragment in such an orientation so as to produce antisense RNA in vivo which could hybridize to natural ADE1 mRNA. Northern blotting analysis of total cellular RNA extracted from transformed yeast cells confirmed the presence of high levels of antisense RNA to ADE1 mRNA within cells. However the high level of expression of antisense RNA did not result in production of Ade- cells.     

Expression of a yeast glycolytic gene is subject to dosage limitation

The Saccharomyces cerevisiae pyruvate kinase-encoding gene (PYK1) has been transformed back into yeast using a derivative of the multicopy vector, pJDB207. High levels of PYK1 expression in these transformants are limited by at least two separate mechanisms. Pyruvate kinase assays and polysome analyses demonstrate that the translation of the PYK1 mRNA is inhibited as its abundance increases. The abundance of the PYK1 mRNA per gene copy also decreases as the copy number of the PYK1 gene increases. This is the first report which demonstrates that a eukaryotic glycolytic gene is subject to dosage limitation at the translational level.     

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.