Substitute Teacher

Despite the relative ease of genome manipulation in S. cerevisiae, researchers are always looking to learn still more convenient and rapid methods for substituting yeast promoters. Replacing a gene's native promoter with a heterolo-gous promoter of choice allows regulated expression and simplifies the task of discerning functional relevance. Although a host of clever chromosomal insertion strategies have been described over the years, the advent of the S. cerevisiae Genome Deletion Project provides an incredible resource for a further streamlined workflow. The strategy, explained by Liko et al. on p. 728 is appealingly simple. The genome deletion project resulted in a collection of strains in which a single ORF is replaced with a kanamycin resistance module. Although the purpose of the collection is to have a comprehensive resource of essentially all possible knockouts, the authors point out that for almost any given yeast promoter of interest there will be a strain in which the ORF immediately upstream has been replaced by kanamycin. Therefore, by PCR amplifying the kanamycin-promoter segment with amplification primers designed to be homologous to the targeted insertion site, researchers can generate a cassette suitable for promoter swapping. The authors illustrate the feasibility and ease of this approach by a variety of promoter substitutions. Their system should make promoter replacement a simple matter of a single PCR followed by standard yeast transformation.

The pioneering sequencing of the Saccharomyces cerevisiae genome (1) formed the basis for genome-wide, large-scale approaches to studying gene function. One such project was the S. cerevisiae Genome Deletion Project that systematically replaced each yeast open reading frame (ORF) with a kanamycin resistance module (KanMX), yielding precise start-to-stop codon deletions for more than 95% of the genes (2,3). This replacement is widely used for phenotypic analysis of gene function. We have taken advantage of the thousands of KanMX-promoter fusions generated by the yeast deletion project to create insertion modules for replacing a normal promoter with any one of thousands of different possible yeast promoters.

This method takes advantage of the DNA templates produced by the gene knockout project, each possessing a specific ORF that has been replaced by a KanMX module (Figure 1). Since the flanking regions for the deleted ORFs are left intact, what remains produces a set of strains carrying unique DNA templates in which almost any inter-genic region is flanked by a KanMX marker. For almost any yeast promoter, there exists a strain in which a selectable marker has been inserted immediately 5′ of that promoter. This KanMX marker-intergenic region can be PCR-amplified as a cassette, and flanking sequences can be designed to target the product for insertion at the desired site by homologous recombination (4).

Figure 1. Amplification of a desired promoter region for insertion upstream of your favorite gene (YFG). (Click to enlarge)

The ORF immediately 5′ to the promoter of interest is identified so that the knockout strain for which this ORF has been replaced by KanMX can be used as a source of template DNA. To target the PCR product for insertion, the amplification primers are designed with 5′ ends that are homologous to the intended insertion sites (40–60 bp). This can be accomplished with a single PCR step. In the results shown herein, we used genomic DNA as the template, but we have also used the colony PCR method (5), which could potentially allow high-throughput workflows.

Our schematic shows the KanMX-promoter cassette inserted between the normal promoter and the ORF, but the upstream primer can be designed to cause replacement of the normal promoter sequence. We have successfully used both methods. This concept could also be used to design insertion fragments to replace 3′ untranslated sequences with those from another gene, as well as to make in-frame fusions between practically any two coding regions.

PCR fragments containing the KanMX-promoter cassette were amplified using genomic DNA from the chosen deletion strain (Table 1) purchased from Research Genetics (Huntsville, AL, USA).

Table 1. Yeast Strains (Click to enlarge)

For all reactions, primers are shown in Table 2. Bolded and underlined nucleotides indicate the portion that anneals to sequences flanking the KanMx-promoter fragment, while plain text identifies the portion of the primer required for targeting insertion. Our typical PCR conditions used 0.1 µg purified genomic DNA as the template, 2 mM MgCl₂, 1× Taq DNA polymerase buffer, 5 U Taq DNA polymerase (Promega, Madison, WI, USA), 0.2 mM dNTPs, and 1 µM each primer in a total volume of 50 µL, using a GeneAmp® 9700 (Applied Biosystems, Foster City, CA, USA) thermal cycler with start at 94°C, followed by 35 cycles of 94°C for 30 s, 60°C for 45 s, 72°C for 120 s, followed by 72°C for 7 min. PCR products were purified using QIAquick® PCR columns (Qiagen, Valencia, CA, USA), and 0.5 µg amplified DNA was used for each transformation using a Frozen-EZ Yeast Transformation II™ kit (Zymo Research, Orange, CA, USA). Yeast transformants were allowed to express the resistance marker by plating onto nonselective medium and then replica plated to G418 (0.25 mg/mL) for integrant selection. Proper insertion was confirmed via PCR and sequencing. The rate of successful transformation into the appropriate position was comparable to the integration of commonly used plasmid-generated fragments.

Table 2. PCR Primers (Click to enlarge)

As an example of this technique, we used the CCR4 primer set and template DNA from strain 10388 (carrying a deletion in yal020c) to produce a KanMX-CCR4 promoter fragment that was targeted to replace the normal promoter sequence upstream of the CLN3 ORF. We chose the CCR4 promoter as a potential constitutive promoter because CCR4 messenger RNA (mRNA) levels, encoding a general transcriptional regulator (6), were relatively constant in published microarray experiments (7). Replacement of the CLN3 promoter with that from CCR4 reduced CLN3 mRNA levels and blunted CLN3 induction by nutrients (8) (Figure 2A). Thus, this method was effective in manipulating expression of the target gene.

Figure 2. Examples demonstrating altered gene expression after heterologous promoter insertion. (Click to enlarge)

We also used the LSR1 promoter, which normally drives transcription of the U2 small nuclear RNA (snRNA) (9), as a test promoter. We chose U2 RNA as a loading control because it is constant and abundant. The KanMX-LSR1 promoter cassette was amplified with a DNA template from strain 13371 (carrying a deletion in ybr231c) using the LSR1 primers for insertion immediately 5′ of the RPB4 ORF. That produced overexpression of RPB4 mRNA, which encodes a subunit of RNA polymerase II (10) (Figure 2B).

To make a conditional promoter fusion, we used the SMF3 promoter. SMF3 encodes an iron transporter that is induced in low iron environments (11,12). An SMF3 promoter fusion driving CLN3 was constructed with prior amplification of the KanMX-SMF3 promoter fragment using genomic DNA from strain 16433 as a template (strain 16433 carries KanMX inserted in place of ylr035c). CLN3 mRNA levels were determined in mid-log cultures grown in either the absence or presence of an iron chelator, bathophenanthroline-disulfonic acid (BPS). As expected, BPS increased CLN3 mRNA in the altered strain (Figure 2C). The SMF3 promoter is just one example out of the hundreds of regulated promoters that could be selected for use.

Since expression can also be regulated through translational regulatory elements in the mRNA, we moved the 5′ noncoding region of CPA1 to regulate production of CLN3. Translation of Cpa1 is inhibited by arginine through regulation of ribosomal scanning of an upstream ORF (13,14). The CPA1 promoter driving CLN3 fusion was constructed as above, amplifying the KanMX-CPA1 promoter fragment out of strain 11597 (carrying a deletion in yor301w). The addition of arginine (40 mM) to the medium decreased cell size in the transformants, which was the expected phenotype for decreased Cln3 levels (not shown).

It is commonly useful to drive expression of a gene of interest with a heterologous promoter. While vector-based chromosomal insertion tools for yeast, such as the STIK system (15), have been available for many years, our method takes advantage of the previously accomplished generation of thousands of KanMX-promoter fusions, providing ready-made templates for generating a targeted insert with a single PCR. The availability of these templates obviates the need for the intermediate step of creating a recombinant clone carrying the promoter fused to a marker. The collection of S. cerevisiae deletion strains provides a set of readily amplified KanMX markers lying immediately 5′ to intragenic regions that include approximately 95% of the promoters in S. cerevisiae. This provides a useful set of ordered templates that can be used to substitute almost any yeast promoter into a site driving almost any yeast gene. This method involves only a single PCR amplification and a standard yeast transformation. Furthermore, the growing body of microarray data allows predictions to be made regarding the strength of a given promoter under different circumstances, which should enable custom expression patterns to be readily designed.