Efficient Targeted Integration at leul - 32 and ura 4 - 294 in Schizosaccharomyces pombe Jill

Homologous integration into the fission yeast Schizosaccharomyces pombe has not been well characterized. In this study, we have examined integration of plasmids carrying the leul+ and ura4' genes into their chromosomal loci. Genomic DNA blot analysis demonstrated that he majority of transformants have one or more copies of the plasmid vector integrated via homologous recombination with a much smaller fraction of gene conversion to leul+ or ura4'. Non-homologous recombination events were not observed for either gene. We describe the construction of generally useful leul' and ura4' plasmids for targeted integration at the leul-32 and ura4-294 loci of S. pombe. T ARGETED integration by homologous recombination is a standard tool of Saccharomyces cerevisiae yeast genetics and is rapidly becoming a standard procedure in the yeast Schizosaccharomyces pombe as well. In S. pombe, however, integration by homologous recombination has presented problems less familiar to S. cerrmisiae geneticists (RUSSELL and NURSE 1986b; RUSSELL 1989). Many integrating vectors currently available for use in S. pombe are based on complementation of S. pombe mutations with S. cerevisiae genes. In single copy, such complementation may be inefficient, favoring multiple integrations. Many singlecopy integrations into S. pombe utilizing the S. cerevisiae marker gene URA3 fail to complement ura4. (RUSSELL 1989). The S. cerevisiae marker gene LEU2 can complement S. pombe leu1 mutations, but often the majority of transformants are double or triple tandem integrants, which can dramatically affect cellular phenotype (RUSSELL and NURSE 1986a, 1987). Very few studies on integration into S. pombe have been published. GRIMM and KOHLI (1988) analyzed integration at the ura4 locus. They examined six stable Ura' transformants receiving linearized ura4containing plasmid DNA into a ura4-294 strain and found that five of these transformants had undergone gene conversion events. The other transformant contained a wild-type hybridizing fragment, as well as an additional band the size of which was inconsistent with simple homologous integration at ura4. Genetic analysis revealed that the ura+ information was located at the ura4 locus. Thus, as the authors state, it is likely that this transformant also arose via gene conversion, and the additional hybridizing band was due to integration of a truncated fragment. They also examined integration of linearized plasmid into a ura4-D6 strain. This deletion eliminates the restriction site that was used to linearize within the ura4 plasmid sequence, but retains homology Genetics 136: 849-856 (March, 1994) to plasmid sequences at the 5'and 3'-regions of the ura4 gene. Six stable Ura+ transformants were analyzed by genomic blot analysis. Four of the transformants contained the expected single or multiple integrations at the ura4 locus; the other two contained multimers integrated at other locations in the genome. Thus, S. pombe, like S. cerevisiae, tends to favor homologous recombination over non-homologous recombination when homology exists between the chromosome and the transforming DNA. However, in this study, gene conversion appeared to be the predominant event when a ura4-294 containing strain was examined. GRALLERT et al. have published a thorough study of one-step gene disruption at the s u c l locus, using ura4 as the selectable marker. They found that 75-90% of the stable ura+ transformants contained the expected disruption, indicating a high rate of homologous integration. We have undertaken a study involving integration at the leu 1-32 locus and find only homologous integration events with a low rate of gene conversion. The same results were obtained in a study of integration at the ura4-294 locus: we obtain a high frequency of single homologous insertions at the desired loci. The frequency of gene convertants obtained at both loci is similar to that reported for integration in S. cerevisiae. As ura4-294 and Zeul-32 are common auxotrophic markers in s. pombe strains, we have constructed convenient plasmids for targeting integration to these loci. MATERIALS AND METHODS Yeast strains and plasmids: S. pombe strains used for integrative transformation are listed in Table 1. The integrative plasmids are described in Table 2 and are diagrammed in Figure 1. Plasmids were prepared by the boiling mini-prep method (HOLMES and QUICLEY 1981) or by Qlagen columns (Qiagen Inc., Chatsworth California). Plasmid pJK4 was constructed by ligating the 1.8-kb HindIII frqgment of ura4 into the HindIII site ofpBSII(KS+). Plasmid pJKl3 was constructed 850 J. B. Keeney and J. D. Boeke