As part of the Saccharomyces Genome Deletion Project, sets of presumably isogenic haploid and diploid strains that differed only by single gene deletions were constructed. We found that one set of 96 strains (containing deletions of ORFs located between YOR097C and YOR192C) in the collection, which was derived from the haploid BY4741, has an additional mutation in the MSH3 mismatch repair gene

Farber, R. A.

Lehner, K. R.

Petes, T. D.

Stone, M. M.

Carolina Digital Repository

Copyright  2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.079368NoteNinety-Six Haploid Yeast Strains With Individual Disruptions of OpenReading Frames Between YOR097C and YOR192C, Constructed forthe Saccharomyces Genome Deletion Project, Have an AdditionalMutation in the Mismatch Repair Gene MSH3Kevin R. Lehner,*,† Megan M. Stone,† Rosann A. Farber†,‡ and Thomas D. Petes*,1*Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710,†Department of Pathology and Laboratory Medicine and ‡Department of Genetics, University ofNorth Carolina, Chapel Hill, North Carolina 27599-7525Manuscript received July 24, 2007Accepted for publication September 13, 2007ABSTRACTAs part of the Saccharomyces Genome Deletion Project, sets of presumably isogenic haploid and diploidstrains that differed only by single gene deletions were constructed. We found that one set of 96 strains(containing deletions of ORFs located between YOR097C and YOR192C) in the collection, which wasderived from the haploid BY4741, has an additional mutation in the MSH3 mismatch repair gene.GROUPS of researchers involved in the Saccharomy-ces Genome Deletion Project generated sets ofhaploid and diploid strains containing individual dele-tions in nearly all nonessential genes in the yeast ge-nome (Winzeler et al. 1999; Giaever et al. 2002). Inthese strains, each individual open reading frame (ORF)was replaced by the KanMX drug-resistance gene. Thiscollection has been screened for a variety of interestingphenotypes, including drug sensitivities, growth in vari-ous types of media, spontaneous mutation rates, abilityto respond to osmotic stress, and sporulation profi-ciency (Winzeler et al. 1999; Deutschbauer et al. 2002;Giaever et al. 2002; Huang et al. 2003). The assumptionin this type of analysis is that the strains are identicalexcept for the single mutation introduced by transfor-mation. Although, in general, this assumption is likelyto be correct, it has been noted that some mutationsresult in genomic instability that leads to additionalchanges. For example, mec1 haploid strains often be-come disomic for chromosome IV (Gasch et al. 2001).In this report, we describe another, and more subtle,problem: the de novo acquisition of a mutation in one ofthe progenitor strains used to construct a subset of thedeletions.Our discovery of this problem was in conjunction withour analysis of the effect of chromosome context onmicrosatellite instability. In a previous study (Hawk et al.2005), we showed that the stability of a microsatellitevaried by 16-fold, depending on genome location,and that most of this variation reflected context-specificvariation in the rate of DNA mismatch repair (MMR).This study was based on the analysis of 10 genomiclocations. To extend this study, we constructed a plasmid(pKRL1, Figure 1) that contained a galactose-inducibleURA3-GT fusion gene (Wierdl et al. 1996) inserted intothe KanMX-coding region of the pFA6-KanMX4 plasmid(Wach et al. 1994). The URA3-GT fusion gene has an in-frame insertion ½a poly(GT) sequence 33 bp in lengthin a nonessential region of the fusion gene. A NotI frag-ment derived from this plasmid, containing this fusiongene and flanking sequences from the KanMX gene,was transformed into derivatives of the haploid strainBY4741 in which different genes had been deleted usingthe KanMX cassette. Transformants in which the re-porter gene was inserted within the KanMX cassette werephenotypically Ura1 and G418 sensitive (Figure 1). Usingthis method, we could insert the same reporter sequenceinto any ORF of the genome that had been previouslytagged with the KanMX cassette.Strains with the URA3-GT fusion gene are sensitive to5-fluoroorotic acid (5-FOA), a compound that kills strainswith a wild-type URA3 gene (Boeke et al. 1984). Sincealterations in the length of the microsatellite that resultin loss of the normal reading frame (for example, adeletion of 2 bp) cause the cells to be 5-FOAR, the rate ofappearance of 5-FOAR derivatives is a measurement of1Corresponding author: Department of Molecular Genetics and Microbi-ology, Duke University School of Medicine, Durham, NC 27710.E-mail: tom.petes@duke.eduGenetics 177: 1951–1953 (November 2007)the rate of microsatellite instability (Hawk et al. 2005).Rates are determined by measurement of the frequen-cies of 5-FOAR derivatives in multiple independent cul-tures and conversion of these frequencies to rates usingthe method of the median (Lea and Coulson 1949).We inserted the URA3-GT construct into 50 deriva-tives of BY4741, representing 50 random genomic lo-cations. The rates of instability observed in differentgenomic locations varied from 1 3 105 to 5 3 104. Twoof the insertions with the highest rates ½YOR108W (5 3104) and YOR185C (4 3 104) were located close to-gether on chromosome XV. To determine whether therewas a region on chromosome XV that had elevated mi-crosatellite instability, we constructed 18 derivatives ofBY4741 with insertions of the reporter gene within ORFson chromosome XV and measured the rates of insta-bility in these strains. As shown in Figure 2, all 12 in-sertions in the region located between YOR097C andYOR192C showed significantly elevated rates of micro-satellite instability relative to the 6 insertions outside ofthis region. The transition between the rate of high in-stability and low instability was abrupt, occurring be-tween ORFs YOR096W and YOR097C at one junctionand between ORFs YOR192C and YOR193W at theother.The 96 strains with disruptions of the ORFs betweenYOR097C and YOR192C were produced by a single lab aspart of the Saccharomyces Deletion Project (A. Chu, per-sonal communication). This same lab also producedtwo additional 96-strain deletion sets. We measured mu-tation rates of our reporter integrated in strains fromthese other sets and found no significant increase inrates (data not shown). These data suggest that the pro-genitor ‘‘wild-type’’ strain used by this lab to generatethe YOR097C–YOR192C set, but not the additional twosets, had acquired a mutation that resulted in elevatedlevels of microsatellite instability. The gene disrup-tions made for ORFs in the two regions flanking theYOR097C–YOR192C chromosome segment were madeby other labs (A. Chu, personal communication), pre-sumably using a different isolate of BY4741.In yeast, loss of the capacity to repair mismatchesgreatly elevates microsatellite instability (Strand et al.1993). In most eukaryotes, there are two systems thatrecognize mismatches resulting from misincorporationerrors or DNA polymerase slippage events (reviewedby Harfe and Jinks-Robertson 2000). A complex ofMsh2p/Msh6p/Mlh1p/Pms1p recognizes base–base mis-matches and insertions and deletions of one base,whereas a complex of Msh2p/Msh3p/Mlh1p/Pms1precognizes insertions and deletions of 1–14 bases (Harfeand Jinks-Robertson 2000). The stability of dinucleo-tide microsatellites is affected much more strongly by anmsh3 mutation than by an msh6 mutation (Sia et al. 1997).Previously, Huang et al. (2003) screened the BY4741deletion collection for mutants that had elevated rates offorward mutation at the CAN1 locus. These mutators didnot map in the YOR097C–YOR192C region. Since wehave previously shown that msh3 strains have very ele-vated instability for dinucleotide microsatellites, but awild-type rate of mutation at the CAN1 locus (Sia et al.1997), these results suggested that the progenitor strainused to make the deletions might have an msh3 mutation.We sequenced the MSH3 gene in five of the strainscontaining deletions of genes located on the right armFigure 1.—Construction of derivatives of the BY4741 col-lection with insertions of the URA3-GT reporter gene. In a pre-vious study (Wierdl et al. 1996), we constructed a fusion genewith a GAL1,10 promoter regulating transcription of a Ura3fusion protein. Into a region of the gene that did not affectgene function, we inserted an in-frame (33 bp) poly(GT)tract. A plasmid with this gene (pMBW1) was treated withKpnI and XhoI, and the fragment containing the fusion genewas treated with the Klenow fragment to create blunt ends,which were then inserted into the NruI site of pFA6-kanMX4.In the resulting plasmid (pKRL1), the URA3-GT fusion gene isinserted into the middle of the KanMX gene; the fusion geneand KanMX are transcribed in opposite directions. To con-struct strains with the fusion gene inserted in different posi-tions, we treated pKRL1 with NotI and transformed strainswith single KanMX insertions replacing individual ORFs.We selected Ura1 transformants and screened for those trans-formants that become G418 sensitive. Shaded boxes indicateKanMX; solid box, GAL1,10 promoter; crosshatched box,LYS2 sequences; stippled box, HIS4 sequences; and openbox, URA3. The dotted lines indicate the chromosomal se-quences flanking the KanMX gene.Figure 2.—Microsatellite mutation rates in deletion strains.Haploid strains with the URA3-GT reporter at different geno-mic locations were constructed as described above. Mutationrates were calculated by fluctuation analysis as described pre-viously (Hawk et al. 2005). Error bars indicate 95% confi-dence limits.1952 K. R. Lehner et al.of chromosome XV. We found a single base-pair de-letion (loss of an A located at position 1551) in MSH3in the three strains containing deletions of genes be-tween YOR097C and YOR192C (YOR097C, YOR153W,YOR192C), but not in those containing deletions ofgenes outside of this region (YOR096W, YOR193W). Theobserved frameshift mutation results in a truncatedMsh3 protein, composed of 521 instead of 1047 aminoacids. To verify that this msh3 mutation was responsiblefor the elevated microsatellite instability, we inserted thewild-type MSH3 gene into strains that had the URA3-GTreporter at YOR097C and YOR192C. In these derivatives,the rates of microsatellite instability were substantiallyreduced (Figure 3), demonstrating that the msh3 muta-tion is responsible for the elevated rates of microsatelliteinstability.Our results illustrate the difficulties of constructingan isogenic collection of yeast strains, as well as the dif-ficulties of detecting departures from non-isogenicity. Itis likely that, when equivalent isogenic knockout collec-tions are constructed in higher eukaryotes, these col-lections will have similar problems. The appearance of ade novo spontaneous mutation in the progenitor strainsused to construct a collection of deletions may seem im-probable; however, because the strains in the YOR097C–YOR192C region share the same four auxotrophic mu-tations as BY4741, it is unlikely that these strains wereconstructed with a contaminating unrelated yeast strainthat had an msh3 mutation. An alternative possibility isthat some genes, such as MSH3, might be more suscep-tible to mutations than the ‘‘standard’’ genes (CAN1 andURA3) usually used to measure forward mutation rates.It is also possible that this mutation arose in BY4741 cellsthat were kept in a mutagenic environmental condition(high temperature, low temperature, stationary phase,etc.). Finally, we point out that the observations reportedin this article do not influence our earlier conclusionthat the efficiency of MMR varies in different regions ofthe yeast genome (Hawk et al. 2005), since this previousstudy did not utilize the knockout collection.We thank J. Lieb and E. Alani for providing deletion strains andthe MSH3-replacement plasmid, respectively. We thank Sue Jinks-Robertson for suggesting that we investigate the construction of thedeletion collection and A. Chu and G. Volckaert for providing detailsabout the construction of deletions in BY4741. The research wassupported by National Institutes of Health grants GM24110 (T.D.P.)and CA63264 (R.A.F.).LITERATURE CITEDBoeke, J., F. Lacrute and G. R. Fink, 1984 A positive selection formutants lacking orotidine-59-phosphate decarboxylase activity inyeast: 5-fluoroorotic resistance. Mol. Gen. Genet. 197: 345–346.Deutschbauer, A. M., R. Williams, A. M. Chu and R. W. Davis,2002 Parallel phenotypic analysis of sporulation and postgermi-nation growth in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci.USA 99: 15530–15535.Gasch, A. P., M. Huang, S. Metzner, D. Botstein, S. J. Elledgeet al., 2001 Genomic expression responses to DNA-damagingagents and the regulatory role of the yeast ATR homolog Mec1p.Mol. Biol. Cell 12: 2987–3003.Giaever, G., A. M. Chu, L. Ni, C. Connelly, L. Riles et al., 2002 Func-tional profiling of the Saccharomyces cerevisiae genome. Nature 418:387.Harfe, B. D., and S. Jinks-Robertson, 2000 DNA mismatch repairand genetic instability. Annu. Rev. Genet. 34: 359–399.Hawk, J. D., L. Stefanovic, J. C. Boyer, T. D. Petes and R. A. Farber,2005 Variation in efficiency of DNA mismatch repair at differ-ent sites in the yeast genome. Proc. Natl. Acad. Sci. USA 102:8639–8643.Huang, M.-E., A.-G. Rio, A. Nicolas and R. D. Kolodner, 2003 Agenomewide screen in Saccharomyces cerevisiae for genes that sup-press the accumulation of mutations. Proc. Natl. Acad. Sci. USA100: 11529–11534.Lea, D. E., and C. A. Coulson, 1949 The distribution of the numberof mutants in a bacterial population. J. Genet. 49: 264–285.Sia, E. A., R. J. Kokoska, M. Dominska, P. Greenwell and T. D.Petes, 1997 Microsatellite instability in yeast: dependence onrepeat unit size and DNA mismatch repair genes. Mol. Cell. Biol.17: 2851–2858.Strand, M., T. A. Prolla, R. M. Liskay and T. D. Petes, 1993 De-stabilization of tracts of simple repetitive DNA in yeast by muta-tions affecting DNA mismatch repair. Nature 365: 274–276.Wach, A., A. Brachat, R. Pohlmann and P. Philippsen, 1994 Newheterologous modules for classical or PCR-based gene disrup-tions in Saccharomyces cerevisiae. Yeast 10: 1793–1808.Wierdl, M., C. N. Greene, A. Datta, S. Jinks-Robertson and T. D.Petes, 1996 Destabilization of simple repetitive DNA sequencesby transcription in yeast. Genetics 143: 713–721.Winzeler, E. A., D. D. Shoemaker, A. Astromoff, H. Liang, K.Anderson et al., 1999 Functional characterization of the S. cer-evisiae genome by gene deletion and parallel analysis. Science285: 901–906.Communicating editor: B. J. AndrewsFigure 3.—Microsatellite mutation rates in deletion strainswith msh3 replaced with MSH3. Strains with integrated URA3-GT at two different sites (YOR97C and YOR192C) were trans-formed with the plasmid pEAI218 (provided by E. Alani,Cornell University) digested with KpnI and PvuII. The result-ing DNA fragment contains a wild-type MSH3 gene with anadjacent LEU2 gene. Transformants were selected on medialacking leucine. Correct integration at the site of the endog-enous MSH3 gene was confirmed by sequencing. Mutationrates in the original strains with the URA3-GT construct andin the MSH3 derivatives are shown by shaded bars and solidbars, respectively. Error bars indicate 95% confidence limits.Note 1953

Ninety-Six Haploid Yeast Strains With Individual Disruptions of Open Reading Frames Between YOR097C and YOR192C, Constructed for the Saccharomyces Genome Deletion Project, Have an Additional Mutation in the Mismatch Repair Gene MSH3

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Ninety-Six Haploid Yeast Strains With Individual Disruptions of Open Reading Frames Between YOR097C and YOR192C, Constructed for the Saccharomyces Genome Deletion Project, Have an Additional Mutation in the Mismatch Repair Gene MSH3

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