Site-specific genomic (SSG) mutagenesis in the USC2 region of the IME1promoter
SSG mutagenesis involves two sequential transformations (Fig 1). For the first transformation, a PCR fragment containing URA3 is integrated into the yeast genome at a site 20–500 bp from the intended site of mutation; for the second transformation, URA3 is replaced with a PCR fragment containing the mutation near one end of the fragment (see Methods).
As a first demonstration of SSG mutagenesis, we introduced a mutation in the UCS2 region of the IME1 promoter, a region critical for transcriptional control of this gene [18]. For the first transformation, URA3 was inserted 36 bp from the intended site of the mutation. In this trial, >100 Ura+ isolates were identified, of which 26 were analyzed by diagnostic PCR. Of these 26 isolates, seven were integrated at the correct location; the remaining 19 transformants were probably gene conversion events at the ura3-1 locus [19]. For the second transformation, the URA3 strain generated in the first step was retransformed with a 707 bp PCR fragment amplified from wild-type genomic DNA (Fig. 2A). As discussed below, it was necessary to cotransform this fragment with a plasmid containing another marker, in this case a high copy plasmid bearing the LEU2 marker. One end of the PCR fragment corresponded to sequences 46 bp upstream of the URA3 insertion site and contained a single mutation 10 bp from this end. The other end corresponded to sequences 661 bp downstream of the URA3 insertion site. After screening transformants on FOA, six FOAr colonies were identified, of which four had deleted URA3. As revealed by diagnostic PCR and subsequent sequencing of the region, all four of these isolates had incorporated the single base pair mutation. Thus point mutations can be efficiently introduced into the genome by two sequential transformations with PCR fragments.
In the above experiment, a single bp mutation was inserted. In a second trial, we attempted to introduce two mutations within a 3 bp region (Fig 2B). In this trial, the site of URA3 insertion (in the first transformation) was 463 bp from the site of insertion used in the first trial. In the second transformation, mutations were targeted 49 and 51 bp from the URA3 site. For this second trial, four FOAr colonies were recovered, and diagnostic PCR revealed that all of these isolates were deleted for URA3. These PCR also indicated that three of the four FOAr isolates contained both mutations. The fourth isolate contained the wild-type sequence.
Requirement for co-transformation in SSG mutagenesis
For the second transformation described above, the PCR fragment containing the mutation was co-transformed with a plasmid vector containing a selectable marker (in this case LEU2). Transformants were first selected for the presence of the plasmid and then either screened or selected for loss of URA3. To determine whether this cotransformation was necessary, we also plated transformed cells from the first SSG mutagenesis trial directly on FOA medium. After five days of growth, we analyzed FOAR colonies for growth on Ura- medium and for loss of URA3 (by diagnostic PCR). Of 102 colonies growing on FOA medium, all were phenotypically Ura-, but none contained a deletion of URA3. We observed similar results in other trials. Thus, transformants must be allowed to grow before they can be screened or selected on FOA. Interestingly, when the Leu- colonies were replica-plated to FOA medium, only a fraction of cells in any of the colonies were FOAR. Thus generation of FOAr cells may be a relatively late event during the growth of the colony.
Introduction of mutation at sites distant from marker
In the two SSG mutageneses described above, mutations were incorporated 36–51 bp from the URA3 insertion site. We next asked whether mutations could be incorporated at greater distances from the URA3 site. For this purpose, in the second transformation we used PCR fragments in which the end containing the mutation(s) was either 250 bp upstream (Fig. 2C) or 539 bp downstream from the URA3 insertion site (Fig 2D). In both of the experiments, as in Fig 2B, the mutated primer contained two mutations within a 3 bp region. In these experiments, we isolated 30–40 independent ura3Δ isolates from a single transformation (Fig 2, first data column). Of these ura3Δ isolates, a significant fraction were found to incorporate the mutation (Fig. 2, second column). The yield of ura3Δ transformants in the experiments shown in Fig. 2A and 2B was lower than in the experiments shown in Fig. 2C and 2D. This difference may result from the long region of homology on both sides of the insertion site in the latter experiments.
Comparing all four of the SSG mutageneses shown in Fig. 2, the frequency of incorporating mutations among the ura3Δ isolates was highest for the mutation closest to URA3 and lowest for the mutation farthest from this marker (Fig. 2, second column). These results suggest that only a portion of the PCR fragment is incorporated into the chromosome. However, the four experiments shown in Fig. 2 differed in several ways, including the site of mutation and the size of the PCR fragment. For this reason, the relationship between the frequency of incorporating mutations and the distance from URA3 was investigated further, as described in the next section.
Incorporation of a multiply-mutated PCR fragment into the genome
Substrates containing multiple polymorphisms are useful for mapping regions of heteroduplex [20]. To directly test the hypothesis that the frequency of incorporating an SSG mutation depends on the distance of this mutation from URA3, we constructed a plasmid (pS660) carrying a 750 bp region of the IME1 promoter that contained 9 mutations (Fig. 3A, i). This plasmid was used as a template to amplify the multiply-mutated UCS2 region, and the resulting PCR fragment was transformed into a strain containing URA3 inserted in this region (Fig. 3A, ii). The UCS2 region was amplified and sequenced in ten independent ura3Δ transformants. Consistent with the results shown in Fig. 2, the frequency of incorporating a particular mutation decreased dramatically with the distance of the mutation from the site of URA3 integration (Fig. 3B, circles). For example, mutations close to the URA3 site were incorporated at 100% efficiency, whereas mutations >400 bp from this site were incorporated in less than 20% of the transformants. Although only 10 transformants were sequenced, the difference between the frequency of incorporating the mutation nearest to the marker and the frequency of incorporating the mutation farthest from the marker is significant (P < 0.005). To verify that the gradient of incorporation frequency was determined by the URA3 insertion site, we transformed the same multiply-mutated PCR fragment into a strain containing a URA3 insertion at a different position in UCS2 (Fig 3A, iii). As in the previous experiment, mutations near the URA3 site incorporated at 100% efficiency, and the efficiency of incorporation dropped with increasing distance from the site (Fig. 3B, triangles). Examining the sequence of 20 transformants yielded further confirmation that the frequency of incorporating mutations decreased as the distance from the marker increased. Each sequence revealed an uninterrupted tract of mutations, starting at the site nearest the marker and extending in both directions for variable distances from this site.
Effect of genome deletions on the frequency of incorporating mutations
As described above, only a portion of the PCR fragment is incorporated into the target region of the genome. One means to ensure that the majority of a PCR fragment is incorporated into the genome is to limit the homology between the fragment and the chromosome to the ends of the fragment. To limit homology to the fragment ends, we modified the method for creating strains in the first transformation such that the targeted region of the chromosome was deleted at the same time that URA3 is inserted in the genome.
To verify that the entire deleted region would be incorporated when homology was limited to the ends, we generated a strain in which the entire UCS2 sequence was replaced with URA3 (Fig 3A, iv). This strain was transformed with the multiply-mutated UCS2 fragment described above. The fragment contained 200–300 bp of homology on either side of the deletion. Six FOAR colonies were chosen, and the UCS2 region from these isolates were amplified and then sequenced. We found that all transformants had deleted URA3 and incorporated all 9 mutations (Fig. 3B, open circles). Thus deletion of genomic sequence in the first transformation allowed mutations to be introduced efficiently throughout the deleted region.
Random domain-localized (RDL) mutagenesis
Based on the results described in the previous section, we developed a variation of SSG mutagenesis, termed random domain-localized (RDL or "riddle") mutagenesis, to produce random mutations constrained within one domain of a gene at its native locus. In brief, RDL mutagenesis, like SSG mutagenesis involves two sequential transformations (Fig 1A, right side). The first transformation deletes the targeted region and replaces it with URA3. The second transformation replaces URA3 with a randomly-mutagenized copy of the targeted region. These randomly-mutagenized fragments were synthesized under error-prone PCR conditions such that each PCR fragment will contain on average one mutation (see Methods).
As an initial test of RDL mutagenesis, we targeted the 750 bp UCS region of the IME1 promoter for RDL mutagenesis. After the second transformation, we screened 60 FOAR isolates by diagnostic PCR. We found that 37 of these isolates had replaced the URA3 gene with the PCR fragment. The UCS2 region in eight of these isolates was sequenced. We found that all of these isolates contained from 1–4 mutations in the UCS2 region, and each isolate contained different mutations. Thus RDL mutagenesis is an efficient method for targeting mutations at random to a single domain of the yeast genome.