Plasmid considerations

A variety of expression vectors are available to express foreign genes in S. cerevisiae. Two major issues require consideration when choosing a yeast vector for expressing Bax or other Bcl-2 family members in these organisms. First, Bax needs to accumulate to high levels to cause cell death, thus a strong promoter should be employed. Secondly, vectors with appropriate selectable markers should be chosen depending on the yeast strain used. Two general types of promoters are used to achieve high level gene expression in S.

cerevisiae: (a) constitutive and (b) conditional promoters. Promoters of the alcohol dehydrogenase (ADH1) and glyceraldehyde-3-phosphate dehydrogenase (GPD or GAP) genes are commonly employed strong constitutive promoters. When expressing the bax gene under the control of either the ADH1 or GPD promoter, the diminished number of transformed colonies can be employed as an indicator of the lethality caused by Bax. A parallel transformation with an 'empty' vector is included as a control. This assay, however, has limitations in interpreting the data, because reductions in transforming activity can be a result of cell cycle arrest, cell death, or even some contaminants in DNA that affect transformation efficiency. For this reason, we prefer a regulatable expression system over a constitutive system. Several conditional promoters have been employed for expressing heterologous genes in yeast; the most commonly used is a hybrid promoter combining the UAS (upstream activating sequence) from the GAL1/GAL10 gene and the basal promoter from the CYC1 gene, these are often referred to as the GAL1 or GAL10 promoters. The GAL promoter is regulated by the availability of carbon sources in the growth medium: transcription is repressed when yeast are grown in glucose-containing medium; derepressed when yeast are grown in raffinose-containing medium; and dramatically induced when yeast are grown in galactose-containing medium. Under optimal conditions, inductions as high as a 1000-fold can be achieved when yeast are grown in medium which contains galactose but lacks glucose. The optimal time for inducing transcription from the GAL promoter is strain dependent and thus should be determined empirically. In general, however, we perform assays at 18-24 h after placing cells in galactose-containing medium. The induction can be monitored by immunoblot analysis using antibodies directed against the heterologous protein of interest.

Basic machinery for transcription and translation is highly conserved from yeast to mammals; therefore, a mammalian cDNA, with minor engineering, often expresses well in yeast. By employing a yeast promoter, transcription initiation of the heterologous gene should proceed efficiently. Most expression vectors also include a yeast transcription terminator, such as the terminator for the CYC1 gene, to ensure production of correctly sized mRNAs and to improve transcription efficiency. Translation initiation in mammalian cells occurs most efficiently within the context of preferred sequences flanking the AUG initiation codon, commonly referred to as a Kozak sequence (66). AUG codons that deviate from the Kozak consensus often translate inefficiently. No well-defined equivalent of the Kozak sequence exists in yeast. To achieve efficient translation initiation, it is required that the initiation codon within heterologous cDNAs represents the first AUG from the 5'-end. In addition, it is preferable to eliminate as much of the 5'-untranslated sequences from the introduced cDNA as possible, since translation can be hindered if the 5'-untranslated region of the mRNA has the potential to form secondary structures or contains a high GC content (67).

For our experiments, we employed the GAL promoter-containing vector YEp51 for expressing Bax in S. cerevisiae. YEp51 is a high copy number yeast plasmid bearing the LEU2 gene as a selectable marker. We cloned bax cDNA into YEp51 using the Sail site in the polylinker (i.e. the restriction site closest to the GAL10 promoter), thus keeping the length of the resulting 5'-untranslated region to a minimum (61).

A variety of yeast expression vectors containing either the GAL regulat-able promoter or the ADH1 and GPD constitutive promoters are available from American Type Culture Collection (ATCC, Manassas, VA) (68,69).

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