Nucleic Acids Research

Figure 1. Schematic overview of the method of gap-repair. For our pilot experiment, we PCR-amplified the hus1⁺ gene using pREP42-hus1⁺ (6) as a template and co-transformed this PCR product with a linearized pREP42 vector (7,8) into a strain deleted for the hus1⁺ gene (6). The ends of the PCR product share sequence with the ends of the linearized pREP42 vector. Homologous recombination in vivo results in a repaired circular plasmid that directs expression of the hus1⁺ gene from the nmt1[prime] thiamine-repressible promoter.

PCR reactions can be made mutagenic by several methods (3). Two well-described approaches are (i) altering the ratio of available deoxynucleotides added to the PCR reaction and (ii) the addition of a sub-optimal cation, often Mn²⁺, to the reaction. One advantage of these approaches is that the level of mutagenesis can be controlled, either by how severely the ratio of nucleotides is skewed or by the concentration of Mn²⁺ added. The error rate of Taq polymerase under standard conditions may be sufficient to obtain a complex mixture of mutant PCR products, for example when amplifying very large genes or amplifying for many cycles (4). Which ever method is used, it is wise to create the collection of mutant PCR products by pooling together several independent PCR reactions rather than to perform one large PCR reaction to minimize the possibility of an early mutagenic event `jackpotting' the entire pool.

A very efficient transformation is required to obtain a large library of colonies to screen. For this purpose, we recommend the high-efficiency protocol of Okazaki et al. (5). For the gap repair transformations, we used 100 ng (0.02 pmol) of gapped vector and 500 ng (0.8 pmol) of PCR product. To test the efficiency of the gap repair reaction, we co-transformed fission yeast deleted for the hus1⁺ gene with a linearized REP42 plasmid (cut with NdeI and BamHI) and a DNA fragment containing the hus1⁺ gene flanked by REP42 vector sequence generated by a non-mutagenic PCR reaction (Fig. 1). In this instance, the gap repair recombination allows expression of the hus1⁺ gene from the repaired plasmid and rescues the HU-sensitive phenotype of the hus1[Delta] strain. Therefore, the proportion of total transformants that were HU resistant gives the proportion of colonies resulting from proper gap-repair reactions. Typical results are listed in Table 1.

Table 1. Efficiency of gap repair and mutagenesis

Phenotype	Number of cells	Percentage of total
Non-mutagenic PCR
Total cells counted	554	100
HU sensitive	75	14
HU resistant (express hus1+)	479	86
Mutagenic PCR
Total cells counted	425	100
HU sensitive	194	46
HU resistant	231	54

Using this assay, we found that ~86% of the transformants expressed the hus1⁺ gene. Our gap repair transformations resulted in ~8 × 10⁴ colonies/µg of gapped vector (a control transformation using uncut vector yielded ~1 × 10⁵ colonies/µg), indicating that gap repair occurs efficiently. In these experiments, the vector and PCR fragment overlap by ~600 bp on either side, but we have used overlaps as short as 200 bp without reducing efficiency. Although we have not sequenced the sites of recombination to assess the precision of the recombination events, we have performed gap repair using only the 3[prime] half of a gene, such that recombination would occur within the coding region. We found that ~70% of the resulting colonies expressed functional protein. It is unclear whether the remaining colonies resulted from imprecise recombination or recombination with other homologous sequences in the genome. Transforming yeast with gapped vector alone resulted in a background of colonies, often one third the number obtained using vector plus insert. Because the vector shares extensive sequence with the nmt1⁺ genomic locus, this background may be due to recombination with those sequences.

To obtain a collection of hus1⁺ mutants, we made our PCR step mutagenic by adding 50 µM MnCl₂ to the reactions. We tested the effectiveness of our mutagenesis by repeating the previous gap repair transformation using hus1⁺ fragments generated with our mutagenic PCR conditions. Again, the proportion of HU-resistant colonies was counted (Table 1). By comparing the proportion of HU-resistant colonies resulting from the mutagenic PCR reaction to the efficiency of gap repair found using non-mutagenic PCR, we estimate that our mutagenesis resulted in an ~37% loss of function rate, showing that our PCR reaction was indeed mutagenic.

We have used our gap repair method to obtain dominant alleles of the hus1⁺ gene. To accomplish this, we performed the gap repair transformation into a wild-type fission yeast strain using the mutagenized hus1⁺ PCR products. This transformation resulted in a large number of colonies (~20 000) of wild-type fission yeast, each potentially containing a plasmid expressing a mutant version of the hus1⁺ gene. Our gapped vector (REP42) includes a thiamine repressible promoter (nmt1[prime]) used to control the expression of our hus1⁺ mutants (Fig. 1). To screen for dominant alleles, we replica plated the colonies to media lacking thiamine to induce the promoter and screened for interesting phenotypes. The plasmids from chosen colonies were rescued in E.coli for re-testing and sequencing. By screening only 20 000 colonies, we obtained 23 HU-sensitive colonies and six colonies inviable in the absence of thiamine (the two phenotypes for which we screened). DNA sequencing showed that each of the mutants contains multiple nucleotide changes, resulting in at least one amino acid change per mutant and several amino acid changes in most mutants. We conclude that our level of mutagenesis was excessive, because the existence of multiple amino acid changes in many mutants complicates structure-function analysis. However, the dominant alleles of our gene have proved to be useful for genetic studies, and we are currently analyzing the phenotypes in greater detail.

The success of the hus1⁺ screen illustrates the value of the gap repair method to obtain novel alleles of known genes. Our group has used gap repair to screen for new alleles of several genes, and we believe it is a broadly applicable technique. One limitation we have encountered is that the gap repair transformation does not occur efficiently in mutant strains with abnormal recombination for obvious reasons, but this limitation should not be relevant to most applications of the technique. As a result of the fission yeast genome project, large numbers of genes with unknown function are being identified. Large-scale targeted disruptions of these genes of unknown function could be made, in the hope that the phenotypes of the disruptions will help identify the functions of the genes. However, these disruptions would be complete loss of function alleles of the genes. It may also be useful to be able to obtain other types of alleles of the genes, particularly in the case of essential genes. The technique described here makes these types of screens very feasible.

REFERENCES

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*To whom correspondence should be addressed. Tel: +1 617 432 0288; Fax: +1 617 432 7663; Email: kostrub@rascal.med.harvard.edu

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