© 2001 Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No. 24 2737–2743

A functional assay for mutations in tumor suppressor genes caused by mismatch repair deficiency

Hanlee P. Ji and Mary-Claire King*

Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA 98195-7720, USA

Received May 25, 2001; Revised and Accepted September 19, 2001

The coding sequences of multiple human tumor MMR-deficient strains of S.cerevisiae has increased our suppressor genes include sequences understanding of the role of MSI in human tumors (5–7). that are prone to mutations. Saccharomyces cerevisiae In cancers from individuals with HNPCC, the elevation in strains deficient in DNA mismatch repair (MMR) can mutation rates causes inactivation of specific tumor suppressor be used to determine de novo mutation rates of these gene, producing a selective growth advantage and thus human tumor suppressor genes as well as any other contributing to stepwise neoplastic progression (8). Multiple genes have been identified with microsatellite mutations gene sequence. in human TGFBR2, including TGFBR2, IGFR2, MSH3, MSH6, PTEN, BAX and PTEN and APC genes were placed in yeast vectors MBD4 (8–15). The majority of these microsatellites consist of and analyzed in isogenic yeast strains that were wild- mononucleotide repeats which accumulate 1 bp deletions or type or deletion mutants for MSH2 or MLH1. In MMR- insertions. deficient strains, the vector containing the (A)10 Observational studies have identified these microsatellite microsatellite sequence of TGFBR2 had a mutation mutation hotspots; candidate genes are sequenced for micro- rate (mutations/cell division) of 1.4 × 10–4, compared satellite mutations in a series of tumors and cell lines exhib- to a mutation rate of 1.7 × 10–6 in the wild-type strain. iting MSI. Recently, criteria have been developed to In MMR-deficient strains, mutation rates in PTEN and standardize diagnostic testing of MSI in primary human APC were also elevated above background levels. tumors, permitting comparison among genes that display high PTEN mutation rates were higher in both MSI (MSI-H) versus those with mutations attributable to back- ground levels (MSS or MSI-L) (16). Whereas these tumor (4.4 × 10–5) and strains (2.3 × 10–5). APC muta- × studies accurately identify mutations and their frequency tion rates in the msh2 strain (2.4 10–6) and the mlh1 among the tumor sample sets, they lack the ability to quantify × strain (1.7 10–6) were also significantly, but less rates of mutation caused by MMR deficiency (17). In addition, dramatically, elevated over background. Mutations there are inherent difficulties in analyzing de novo mutations in selected for in the yeast screen were identical to coding region microsatellites. Fluctuation analysis with those previously observed in human tumor samples mammalian cell lines has been developed for artificial dinucleo- with microsatellite instability (MSI). This functional tide tracts and is technically challenging (17–20). As an added assay has applicability in providing quantitative data complication of tumor studies, comparing the effects of about microsatellite mutation rates caused by MMR individual MMR genes on MSI is difficult because tumor deficiency in any human samples and cell lines are frequently not isogenic and often- times have not been fully characterized for loss of MSH2 or sequence. It can also be applied as a genetic screen MLH1. to identify new genes that are vulnerable to such We have adapted a plasmid frameshift assay to evaluate MSI microsatellite mutations and thus may be involved in within the coding regions of human tumor suppressor genes in the neoplastic development of tumors with MSI. yeast strains lacking either msh2 or mlh1 (5). Relying upon the highly conserved function of MMR genes between humans INTRODUCTION and S.cerevisiae, this genetic screen permits the quantitative analysis and identification of de novo mutations caused by Hereditary non-polyposis (HNPCC) is a MMR deficiency in any human microsatellite sequence. familial cancer syndrome caused by germline mutations in Several plasmids (pHJ series) were constructed in which a DNA mismatch repair (MMR) genes such as hMSH2 and portion of the tumor suppressor cDNA sequence was placed hMLH1 (1). The loss of these MMR genes causes micro- upstream and in-frame of URA3. These vectors were trans- satellite instability (MSI), a phenomenon where mutations formed into three isogenic haploid strains: wild-type, msh2 occur at significantly elevated rates in microsatellite tracts of deletion mutant and mlh1 deletion mutant. De novo mutations genomic DNA (2). The study of MMR genes in Saccharomyces in the tumor suppressor sequence lead to frameshifts that cerevisiae was vital in defining the role of MMR in human prevent the appropriate translation of the URA3 marker. These cancer (3,4). Similarly, the study of microsatellite tracts in events can be seen as growth on 5-fluororotic acid (FOAR) and

*To whom correspondence should be addressed. Tel: +1 206 616 4294; Fax: +1 206 616 4295; Email: [email protected] 2738 Human Molecular Genetics, 2001, Vol. 10, No. 24 subsequently be scored (21). For fluctuation analysis, the Using the method of the median, fluctuation analysis was method of the median was used to determine mutation rates, repeated independently three to four times for each plasmid to the number of FOAR events per cell division (22). determine mutation rates in each strain and the results were For this initial study, we measured the mutation rates of micro- averaged (22). For all tumor suppressor genes, mutation rates satellite sequences in three tumor suppressor genes, TGFBR2, were significantly higher in MMR-deficient strains in compar- PTEN and APC. Transforming growth factor β-receptor II ison with the wild-type strain (Table 1). The plasmid pHJ9, containing the TGFBR2 (A) tract, showed dramatically inhibits cell proliferation and contains an (A)10 tract that is 10 frequently mutated in MSI-positive tumors such as colon elevated mutation rates in comparison with the other vectors. adenocarcinomas (8). The phosphatase tumor suppressor The TGFBR2 plasmid mutation rates were similar between either msh2 or mlh1 backgrounds. Mutation rates in pHJ3, PTEN contains two (A)6 tracts, one in exon 7 and the other in exon 8, the latter being the primary mutation hotspot in containing the PTEN microsatellites, were slightly higher in endometrial carcinomas with MSI (11,13). The adenomatous msh2 strains than mlh1 strains (1.84 times). For pHJ4, polyposis gene APC plays a key role in sporadic and familial containing APC microsatellites, mutation rates were surpris- colon cancers and contains a large number of microsatellites. ingly low with only 11- and 8-fold increases over wild-type mutation rates for msh2 and mlh1, respectively. We chose a region of APC with two (A)5 and one (T)5 micro- satellites previously shown to have mutations in MSI-positive colon carcinomas (9). Analysis of tumor suppressor microsatellite mutations Mutations caused by MMR deficiency were identified in the RESULTS tumor suppressor regions of the vectors (Table 2). Plasmids were recovered from FOAR colonies and the tumor suppressor Mutation rate analysis of positive control and background regions sequenced. Plasmids were isolated from independent levels in MMR-deficient strains experiments, separate samples and represent independent mutations. FOAR mutations were detected more frequently in As a positive control to test mutation rates in the MMR-deficient TGFBR2 than in PTEN and APC, the difference attributable to strains, we designed a centromeric plasmid (pHJ5) with a significantly higher mutation rates of the TGFBR2 region. dinucleotide tract, (GT)16, upstream and in-frame of URA3 Overall, the types of mutations from the msh2 and mlh1 strains (Fig. 1A). This plasmid also contained a LEU2 marker for had a similar distribution to those found in MSI-positive plasmid retention. As has been demonstrated in previous work, tumors, with 90% being 1 bp deletions. this microsatellite tract mutates at significantly elevated rates Plasmids lacking mutations in the TGFBR2, PTEN and APC in MMR-deficient yeast strains (5,6,23). In our hands, tumor suppressor microsatellites had mutations in URA3 as mutation rates (FOAR/cell division) in msh2 were 4.6 × 10–4, a was determined in a subset that was sequenced. URA3 muta- 71.4-fold elevation over wild-type background and in mlh1 tions occurred mostly in PTEN and APC plasmids, given the were 3.4 × 10–4, a 53-fold elevation over wild-type. lower mutation rates of their microsatellites. These mutations As an additional control, vector alone (pCI-HA) was occurred in three microsatellites tracts in URA3: (A)5 codons transformed into all strains to measure background mutation 58–60, (T)5 codons 67–69 and (T)6 codons 85–87. To account rates which can occur in URA3 alone, leading to FOAR. for the difference caused by mutations occurring in the tumor Vector alone had the lowest mutation rates among all the suppressor sequence versus URA3, we calculated an estimated plasmids tested. In wild-type strains, the mutation rate for tumor suppressor mutation rate using the ratio of plasmids with vector alone was 2.0 × 10–7. Mutation rates were 1.0 × 10–6 tumor suppressor mutations over the total number sequenced in msh2, a 5-fold increase over wild-type and were 0.8 × 10–6 in (Fig. 2). Given that no mutations were identified in the PTEN mlh1, a 4-fold increase over wild-type. These mutation rates and APC plasmids rescued from wild-type background, we –8 are a direct reflection of mutations occurring in URA3. used 1.0 × 10 which is an estimate of the general background mutation rate. In concordance with the plasmid mutation rate, Analysis of mutation rates in tumor suppressor the estimated tumor suppressor mutation rates of the TGFBR2 microsatellites in MMR-deficient strains sequence were significantly higher than mutation rates of the PTEN or APC sequences. A series of constructs (pHJ series) were designed using a low- Mutations revealed by the yeast analysis reflected sites copy number centromeric vector which is stably maintained previously identified in MSI-positive human tumors (Table 2). with one to two copies per cell. Use of centromeric vectors The (A)10 microsatellite of TGFBR2 was found to be highly minimizes the possibility that plasmid copy number could prone to mutations. Mutations in the TGFBR2 microsatellite influence mutation rates among the different plasmids. Each consisted of 1 bp deletions and insertions, matching the loca- construct incorporated an ∼400 bp region, containing tumor tion, type and frequency of TGFBR2 mutations found in suppressor coding region microsatellites and in-frame of a human MSI-positive colon adenocarcinomas (8). The muta- URA3 marker (Fig. 1B). These vectors also contain a LEU2 tions in PTEN were distributed over several microsatellites and selectable marker for plasmid retention. The plasmids pHJ3 consisted mostly of 1 bp deletions (Fig. 3). The majority of (PTEN), pHJ4 (APC) and pHJ9 (TGFBR2) were transformed mutations were found in the exon 8 (A)6 tract, located in into isogenic msh2, mlh1 and wild-type strains. All plasmids codons 321–323, the site frequently altered in MSI-H endo- demonstrated a stable Ura+ phenotype, indicating that the metrial carcinomas (11,13). The other PTEN (A)6 tract in encoded Ura3p was not affected by the N-terminal fusion of codons 265–267 had a lower frequency of mutations. In APC, the selected tumor suppressor. the majority of mutations were found in the (A)5 tract located Human Molecular Genetics, 2001, Vol. 10, No. 24 2739

Figure 1. pHJ microsatellite vectors. (A) The positive control vector pHJ9 contains the DNA sequence (GT)16G in-frame of URA3. (B) The coding region micro- satellite vectors contain a portion of cDNA sequence of a tumor suppressor gene upstream and in-frame of URA3. pHJ3 incorporates a region of the PTEN between codons 226 and 360 with four microsatellites. pHJ4 incorporates a region of APC between codons 733 and 867 with three (five repeat unit) microsatellites. pHJ9 incorporates a region of the TGFBR2 between codons 68 and 201 with one microsatellite. Microsatellites with five or greater repeat units are designated as bars within the gene insert. Codons refer to the location within the tumor suppressor gene ORF.

in codons 756–758 where microsatellite mutations have previ- (A)6 or APC (A)5 compared to the (A)10 microsatellite of ously been found in MSI-positive colon adenocarcinomas (9). TGFBR2. The significantly lower mutation rates of the tested Also, a single substitution (G→T) leading to A762S PTEN and APC microsatellites indicate that they have a was also identified in several independent, separate experi- reduced probability of accumulating mutations than the highly ments for determining mutation rate. mutation prone TGFBR2 (A)10 microsatellite. One can hypothesize that mutations in the TGFBR2 microsatellite may be a relatively early event compared to other microsatellites DISCUSSION (i.e. APC) in MMR-deficient cells. A small difference in mutation Taking advantage of the conservation of MMR between yeast rates between msh2 and mlh1 strains for the PTEN plasmid was and humans, we have adapted S.cerevisiae as a model found, but this may not be statistically significant. Plasmid organism system for examining MSI in human tumor copy number was unlikely to have a significant effect on muta- suppressor genes. Whereas MSI has been thoroughly studied in tion rate given our use of centromeric vectors, but it could be a yeast using plasmid frameshift assays, this work is the first to minor factor leading to some of the smaller differences noted demonstrate that human tumor suppressor microsatellites in the PTEN vector. behave in MMR-deficient yeast strains similarly to MSI-positive Sequencing of the de novo tumor suppressor mutations tumors. showed that the spectrum and location of microsatellite muta- A quantitative analysis for individual tumor suppressor tions in MMR-deficient yeast were the same as those found in microsatellites reveals significant differences in MMR-deficient human tumors, namely 1 bp insertions and deletions. The (A)10 strains. The TGFBR2 plasmid had the highest mutation rates microsatellite of the TGFBR2 was found to accumulate 1 bp among the three tumor suppressor sequences tested, correl- deletions and insertions which match the type and frequency of ating with the high frequency of mutations seen in human TGFBR2 mutations found in human MSI-positive colon aden- MSI-positive tumors. The PTEN and APC plasmids also ocarcinomas (8,24). In the first studies, over 90% of the MSI- showed an increase in mutation rates although not as dramatic positive colon carcinoma samples and cell lines were found to as TGFBR2. In general, the mutation rates in different micro- have deletion and insertion mutations in the (A)10 tract in satellite sequences correlate with the lengths of the mono- TGFBR2 and decreased cell surface expression of the receptor. nucleotide tracts as seen in the shorter length tracts in PTEN The MSI-negative tumors showed normal TGFBR2 expression 2740 Human Molecular Genetics, 2001, Vol. 10, No. 24

Table 1. Mutation rates at human tumor suppressor genes in MMR-deficient strains versus wild-type strains

Tumor suppressor gene Strain genotype Plasmid mutation rate Fold induction over Estimated tumor suppressor Fold induction of estimated (plasmid) (FOAR/cell division) wild-type rate mutation rate rate over wild-type rate TGFBR2 (pHJ9) mlh1 1.4 × 10–4 82 1.2 × 10–4 1.5 × 103 TGFBR2 (pHJ9) msh2 1.4 × 10–4 82 1.3 × 10–4 1.6 × 103 TGFBR2 (pHJ9) WT 1.7 × 10–6 –7.9 × 10–8 – PTEN (pHJ3) mlh1 2.3 × 10–5 40 2.9 × 10–6 290 PTEN (pHJ3) msh2 4.4 × 10–5 77 1.3 × 10–5 1.3 × 103 PTEN (pHJ3) WT 5.7 × 10–7 –1.0 × 10–8 – APC (pHJ4) mlh1 1.7 × 10–6 83.1 × 10–7 31 APC (pHJ4) msh2 2.4 × 10–6 11 1.6 × 10–7 16 APC (pHJ4) WT 2.1 × 10–7 –1.0 × 10–8 –

Figure 2. Distribution of mutations in the microsatellites of PTEN. Mutations from both msh2 and mlh1 strains were collated and shown as a distribution against the region of PTEN in pHJ3. The majority of mutations occurred in the exon 8 (A)6 tract which is located in codons 321–323 of PTEN and is known to frequently contain mutations in MSI-positive endometrial carcinomas. Figure 3. Tumor suppressor mutation rates for individual tumor suppressor sequences. Mutation rates represent FOAR/cell division. Tumor suppressor mutation rates were estimated by multiplying the overall plasmid mutation rate against the proportion of vectors containing mutations in tumor suppressor sequence over the total number sequenced. Rates were plotted on a log scale to (8,24). This finding has since been corroborated in multiple facilitate comparison. studies (25). In comparison with TGFBR2’s microsatellite, mutations were found less frequently in the PTEN and APC micro- satellites, a direct reflection of their significantly lower muta- tion rates and in concordance with tumor studies. Among the APC compared with only 20% of the MSI-negative tumors having APC microsatellites mutations. various PTEN microsatellites, the (A)6 tract located in exon 8 R had the highest frequency of mutations and a similar spectrum In FOA colonies where mutations were not found in the tumor suppressor region, mutations occurred in URA3. Based of 1 bp deletions or insertions as seen in MSI-positive endo- upon our determination of mutation rates in the vector alone metrial carcinomas (11,13,26). Kong et al. (13) showed that control, URA3 mutations account for a relatively low fraction 20% of the MSI-positive endometrial tumors had mutations in of mutations and a minimal background mutation rate. We also the exon 8 (A)6 tract, similar to the results reported by Tashiro calculated an estimate of the mutation rate dependent on et al. (11) who found that 39% of MI-positive endometrial can- tumor suppressor sequence alone, independent of de novo cers has mutations in the exon 7 and exon 8 (A)6 tract. In MSI- mutations in the URA3. These estimated tumor suppressor negative tumors, 10% had mutation in the (A)6 microsatellites mutation rates showed the same general trends as the plasmid (13). Likewise, mutations in the APC sequence only occurred mutation rates with the TGFBR2 microsatellite sequence in microsatellites that previously have been shown to have having dramatically higher mutation rates than the PTEN or mutations in MSI-positive tumors (9). Huang et al. (9) showed APC microsatellites. that 56% of the MSI-positive colon tumors had mutations in Sequence context influences MSI as demonstrated by the mononucleotide and dinucleotide microsatellites located in difference in the number of mutations found in the two (A)6 Human Molecular Genetics, 2001, Vol. 10, No. 24 2741

Table 2. Microsatellite mutations in tumor suppressor genes in pHJ3, pHJ4 and pHJ9

Tumor suppressor Strain genotype Total number of FOAR clones Microsatellite Codon location Microsatellite sequence Mutations (number) gene mutations sequenced arranged by codons

APC mlh1 844(A)5 756–758 AAA CAA AAA del A (4)

(A)5 846–848 GAA AAA GAT del A (1)

(GA)4 853–855 GAG AGA GAA ins GA (3)

APC msh2 344(A)5 756–758 AAA CAA AAA del A (2)

(A)5 846–848 TCT GAA AAA del A (1) APC WT 020––– –

PTEN mlh1 647(A)6 265–267 CTA AAA AAG del A (2)

(A)6 321–323 ACA AAA AAT del A (3) del AA (1)

PTEN msh2 16 53 (A)6 265–267 CTA AAA AAG del A (5) ins A (1)

(T)4 272–274 CAC TTT TGG del T (1)

(A)5 288–290 GAA AAA GTA ins A (1)

(A)6 321–323 ACA AAA AAT del A (5) ins A (3) PTEN WT 012––– –

TGFBR2 mlh1 39 44 (A)10 125–128 GAA AAA AAA AAG del A (37) ins A (2)

TGFBR2 msh2 39 42 (A)10 125–128 GAA AAA AAA AAG del A (38) ins A (1)

TGFBR2 WT 122(A)10 125–128 GAA AAA AAA AAG del A (1)

FOAR refers to growth on 5-fluororotic acid.

tracts in PTEN. Mutation frequency was highest in the exon 8 region microsatellite. Mutation rate data will aid in (A)6 tract, as has been seen with MSI-positive endometrial discriminating true MSI-H targets versus events that are the cancers. This suggests some influence of the surrounding result of background levels of mutation and unlikely to be of sequence that contributes to the exon 8 microsatellites vulner- significance to MSI-related tumorogenesis. Mutation rate data ability to MSI. In APC, the (A)5 tract located in codons 756–758 can lead to inferences about mutation events occurring in had the highest frequency of mutations. Compared to the other HNPCC-related neoplastic development. Using isogenic APC microsatellites, the tract in codons 756–758 may have an strains from collections such as those available from the increased susceptibility to DNA mismatches given the pres- Saccharomyces Genome Deletion Project, the individual ence of an (A)3 tract located 1 bp upstream of the (A)5 region. contribution of different MMR genes to MSI can be deter- A substitution mutation (A762S) was also identified in APC mined and compared (27). This system can evaluate the but its functional significance is unknown pending further influence of sequence context on MSI more readily than investigation. One limitation of this assay in terms of sequence models using human tumor cell lines. Finally, this assay can be context is its inability to account for chromosomal context. adapted as a genetic screen to identify new genes containing Application of yeast artificial containing large hypermutable microsatellite targets involved in the pathogenesis regions of sequence as substrates may prove to of MSI-positive tumors. be useful in studying chromosomal context. This genetic assay is limited in its ability to detect mutations that lead to in-frame MATERIALS AND METHODS deletions or insertions. However, in human tumor samples with MSI mutations, in-frame mutations are almost never found with the vast majority being either 1 bp deletions or Ye ast str ains insertions. Saccharomyces cerevisiae haploid strains were obtained from The use of S.cerevisiae in studying the behavior of tumor the Research Genetics and originate from the Saccharomyces suppressor gene MSI has a number of advantages. The applica- Genome Deletion Project (27). The following strains were tion of this frameshift assay complements human tumor studies used in this work: BY4741 (MATa his3∆1 leu2∆0 met15∆0 by providing mutation rates for critical genes with a coding ura3∆0), YD0750 (MATa leu2∆0 met15∆0 ura3∆0 2742 Human Molecular Genetics, 2001, Vol. 10, No. 24

MLH1::KanMX4 his3∆1) and YD6240 (MATa leu2∆0 (YD0750). Leu+, Ura+ colonies were selected initially. Trans- met15∆0 ura3∆0 MSH2::KanMX4 his3∆1). formed yeast strains were subsequently grown on Leu– media for 3 days. Individual colonies were isolated, suspended in Plasmids water and dilutions were plated on Leu–, FOA plates (to measure FOAR) and Leu– plates (to monitor viable cells). For As previously described, the plasmid, pCI-HA, is a low copy each strain, fluctuation analysis was repeated independently number centromeric vector containing two markers (LEU2 and three to four times and mutation rates were determined by URA3) and has a unique BamHI site located upstream and of averaging the results. Mutation rates represent FOAR events/cell URA3 (28). Gap repair was used to create a series of constructs division. Fold induction over wild-type was calculated by with tumor suppressor microsatellites. PCR primers were dividing the MMR-deficient strain rate over the wild-type ∼ designed to incorporate an 400 bp region encompassing the strain rate. Tumor suppressor mutation rates were estimated by selected microsatellite, upstream and in-frame of URA3. multiplying the overall plasmid mutation rate against the Location of the primer is based on the cDNA sequence ORF. proportion of vectors containing mutations in the tumor To mediate the homologous recombination for gap repair, the suppressor sequence over the total number sequenced. 5′ flanks of each primer are derived from sequence encompassing the BamHI site of pCI-HA. For pHJ3 containing the PTEN Plasmid rescue, PCR and DNA sequencing microsatellites, the primers used were 5′-CCCTATGTCC- R CGGACTATGCAGGATCCTCCTCCAATTCAGGACCCA- FOA colonies were patched on Leu–, FOA plates. Plasmids CACGA-3′ and 5′-ATGAGTAGCAGCACGTTCCTTATAT- were recovered using methods described previously (30). GTGGATCCGCTAGCCTCTGGATTTGACG-3′ (nt 678–1080). Primers specific for the pCI-HA and encompassing the tumor ′ APC suppressor insert region were designed: 5 -GTTCCTGAC- For pHJ4 containing the microsatellites, the primers ′ ′ ′ used were 5′-GCCGGCTATCCCTATGACGTCCCGGACTAT- TATGCGGGCTA-3 and 5 -AATGTCTGCCCATTCTG-3 . GCAGGATCCGCGGCCGCTAGGCCTGCGAAGTACAAGG- The tumor suppressor insert was amplified by PCR using the ′ ′ following protocol: 35 cycles of 94°C for 30 s, 55°C for 30 s 3 and 5 -ATGAGTAGCAGCACGTTCCTTATATGTGGATCC- ° GCGGCCGCCTGTTGCTGGATGGTAGTTG-3′ (nt 2196–2601). and 72 C for 30 s. The PCR product was purified. DNA For pHJ9 containing TGFBR2 microsatellite, the primers used sequencing of pHJ3, pHJ4 and pHJ9 PCR products was performed using the first primer listed and Big Dye terminator were 5′-GCGGGCTATCCCTATGACGTCCCGGACTATGCA- sequencing chemistry as per the manufacturer’s protocol GGATCCATGAGCAACTGCAGCATCAC-3′ and 5′-ATGAG- (Perkin-Elmer). Sequencing reactions were precipitated and TAGCAGCTTCCTTATATGTGGATCCCCAGGTTGAAC- run on an Applied Biosystems 377 DNA sequencer. TCAGCTTCTG-3′ (nt 202–603). PCR templates included the vector pSAR-MT containing the APC ORF and pH2-3F containing the TGFBR2 ORF. PTEN was amplified from a ACKNOWLEDGEMENTS cDNA library derived from human lymphocytes (Clontech). We thank I.Chikashi for the pCI-HA plasmid, T.D.Petes for PCR reactions were subjected to 35 cycles of 94°C for 30 s, the pSH44 plasmid, B.Vogelstein for the pSAR plasmid and 50°C for 30 s and 72°C for 30 s. The vector pCI-HA was cut B.Schiemann for the pH2-3F plasmid. We thank I.Ji for her with BamHI and the linearized vector was co-transformed with help in construction of the pHJ5 vector. We thank M.Vidal, the PCR product into BY4741 (wild-type). Plasmids were E.Swisher and J.Boeke for insightful comments about this rescued from yeast strains showing a Leu+, Ura+ phenotype work. 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