Most methods for constructing aneuploid yeast strains that have gained a specific chromosome rely on spontaneous failures of cell division fidelity. InSaccharomyces cerevisiae, extra chromosomes can be obtained when errors in meiosis or mitosis lead to nondisjunction, or when nuclear breakdown occurs in heterokaryons. We describe a strategy for constructing N+1 disomes that does not require such spontaneous failures. The method combines two well-characterized genetic tools: a conditional centromere that transiently blocks disjunction of one specific chromosome, and a duplication marker assay that identifies disomes among daughter cells. To test the strategy, we targeted chromosomes III, IV, and VI for duplication.
The centromere of each chromosome was replaced by a centromere that can be blocked by growth in galactose, andura3::HIS3, a duplication marker. Transient exposure to galactose induced the appearance of colonies carrying duplicated markers for chromosomes III or IV, but not VI. Microarray-based comparative genomic hybridization (CGH) confirmed that disomic strains carrying extra chromosome III or IV were generated. Chromosome VI contains several genes that are known to be deleterious when overexpressed, including the beta-tubulin geneTUB2. To test whether a tubulin stoichiometry imbalance is necessary for the apparent lethality caused by an extra chromosome VI, we supplied the parent strain with extra copies of the alpha-tubulin geneTUB1, then induced nondisjunction. Galactose-dependent chromosome VI disomes were produced, as revealed by CGH. Some chromosome VI disomes also carried extra, unselected copies of additional chromosomes.
This method causes efficient nondisjunction of a targeted chromosome and allows resulting disomic cells to be identified and maintained. We used the method to test the role of tubulin imbalance in the apparent lethality of disomic chromosome VI. Our results indicate that a tubulin imbalance is necessary for disomic VI lethality, but it may not be the only dosage-dependent effect.
Any change in chromosome number through the gain and/or loss of part of a haploid set of chromosomes is known as aneuploidy. Aneuploidy leads to defects in the growth and development of an organism (reviewed in [1,2]). In cases of chromosome gain, the phenotype of an aneuploid is influenced by the effects of two phenomena: (1) a general, physiological response to excess protein expression, leading to a slowing of cell proliferation , and (2) protein stoichiometry imbalances specific to genes on the extra chromosome [1–3]. A complete understanding of the complex phenotype caused by any specific aneuploid karyotype requires an ability to manipulate chromosome contents and copy number.
In the yeastSaccharomyces cerevisiae, aneuploids have been isolated in a number of ways over the years. Strains with extra chromosomes have arisen spontaneously among lab strains (for examples, see [4,5]), and have been generated through meiosis of triploids . Specific disomes (haploids carrying an extra chromosome, karyotype N+1) have been isolated by differentially marking two homologs in a diploid, then selecting for meiotic segregants that contain both homologs (for example, ). An alternative method to generate disomes makes use of transient heterokaryons that form during mating betweenkar1- andKAR1+ haploids . At a certain frequency, chromosomes are transferred from one nucleus to another before one nucleus is lost. By differentially marking homologs in the parents and selecting for progeny cells that retain both homologs, haploid progeny carrying a disomic chromosome have been isolated [9,10]. This method, termed chromoduction, was used to select for 14 of the possible 16 disomes of yeast in a recent systematic study of aneuploidy . Although the methods described above are clearly effective at isolating disomic strains of yeast, each of them requires a spontaneous failure of chromosome segregation during cell division. The mechanisms that underlie these failures (the breakdown of nuclear integrity in a cell containing multiple nuclei or the bypass of the spindle assembly checkpoint to allow nondisjunction [11,12]) are not well understood, and may lead to additional, unplanned genetic changes.
We have devised a method for generating disomes that does not rely on spontaneous failure in cell division integrity. Instead, the method specifically blocks mitotic segregation of the target chromosome alone. The method comprises a novel combination of two well-characterized genetic tools, a conditional centromere  and a duplication marker . When these are placed at the centromere of a target chromosome, disjunction of the chromosome can be transiently blocked to generate disomic cells, some of which are selectively identified by the duplication marker.
We report the results of a proof-of-concept test with chromosomes III, IV, and VI. The method efficiently generated disomic III and IV strains, but did not produce disomic VI unlessTUB1 copy number was also increased.
Results and discussion
General strategy to induce and select for a duplicated chromosome
The strategy involves modifying the centromeric region of a target chromosome so that (1) the centromere can be inactivated temporarily to cause nondisjunction and (2) daughter cells that obtained two copies of the target chromosome can be selected. The chromosome modification strategy is outlined in Figure1A and1B. The conditional centromere construct PGAL1-CEN3 URA3  is PCR-amplified with primers that provide homology to sequence flanking the target centromere. The PCR fragment is transformed into yeast and integrated into the target site by homologous recombination, replacing the endogenous centromere  (Figure1A). PGAL1-CEN3 functions as an autonomous centromere when placed into plasmids or chromosomes, and its function can be blocked when galactose inducesGAL1 promoter activity [13,16]. In galactose, many kinetochore proteins do not assemble on the centromere, but within 20 minutes of a switch to glucose, kinetochore assembly is observed . Once the conditional centromere is in place, a marker that can detect changes in ploidy is generated atURA3, following a strategy devised by Chan and Botstein . A plasmid containingHIS3 and an internal fragment ofura3 is transformed into yeast, integrating intoURA3 and disrupting its function (Figure1B, forward arrow). Chromosome duplication is detected based on the properties of this integrated plasmid. The integration creates a 390 bp direct repeat of a portion ofura3. Homologous recombination between the repeats causes excision and loss ofHIS3 and regeneration of functionalURA3 (Figure1B, reverse arrow). (For simplicity, we refer to this event as excision ofHIS3 even though it can occur by a variety of homologous recombination events including excision, gene conversion and unequal sister chromatid exchange .) BecauseHIS3 can be lost andURA3 regenerated, the locus exists in one of two mutually exclusive states: either it will contain an intactHIS3 or an intactURA3, but not both. Duplication of this marker locus, followed by excision ofHIS3, should lead to cells that contain bothURA3 andHIS3. Such cells should exhibit Ura+His+ phenotypes.
The strategy for inducing and selecting disomic cells is outlined in Figure1C. A haploid strain with a chromosome containing the conditional centromere and duplication marker is exposed to galactose during one cell division cycle. The conditional centromere is inactivated and the target chromosome fails to disjoin during mitosis. Among the daughter cells that contain two copies of the target chromosome, a fraction will spontaneously excise one of theHIS3 markers to generateURA3. These cells will be recovered as Ura+His+ colonies on selective medium.
Construction of modified chromosomes as targets for duplication
As a proof-of-concept test, we chose three chromosomes (III, IV, and VI) to modify and target for duplication. Chromosomes III and VI are among the smallest yeast chromosomes (317 and 270 kb, respectively), whereas chromosome IV is the one of the largest (1.53 Mb) . We anticipated that chromosomes IV and VI would pose a challenging test of this method because they were among the least frequently isolated disomes using chromoduction , and disomic strains containing a single extra chromosome VI failed to be isolated by Torres et al. . Rather, colonies selected for chromosome VI disomy also contained extra, unselected chromosomes, suggesting that simple chromosome VI disomes may be inviable . We constructed each modified chromosome twice, independently, and tested for concordance of phenotype. The chromosomes were constructed in diploids (or in haploids that were subsequently mated to wild type) to produce cells heterozygous for the conditional centromere and duplication markers. When the heterozygotes were sporulated, the modified chromosomes segregated 2:2 and produced haploid colonies on rich medium that were indistinguishable from wild-type segregants (Additional file1A). We conclude that the centromeric modifications themselves do not lead to growth phenotypes.
To test whether the conditional centromeres in our strains could be inhibited by galactose to cause nondisjunction, we tested for chromosome loss by constructing diploid strains that were heterozygous for a conditional centromere marked withURA3. Growth in galactose, followed by plating to glucose-containing medium, resulted in the appearance of many Ura- colonies. Consistent with Hill and Bloom's observations , galactose exposure for 1–2 generations led to the loss of theURA3 marker in approximately 50% of the cells (Additional file1B). In the case of diploids carrying modified chromosome IV, most of the galactose-induced Ura- colonies exhibited a severe, slow-growth phenotype, suggesting that the Ura- colonies were the result of losing the target chromosome (Additional file1C). We conclude that the conditional centromere allows for galactose-inducible nondisjunction in our strains.
To characterize theura3::HIS3 duplication marker, we measured the frequency ofHIS3 excision and reconstitution ofURA3 at each modified centromere. Each marker excisedHIS3 to produce Ura+ papillae at a frequency near 10-4 (Table1). Galactose did not alter this frequency. When cultures were placed under selection for Ura+His+ papillae, strains with two copies of the marker produced colonies at the frequency ofHIS3 excision, whereas strains with one copy produced colonies at a much lower frequency (Figure2). To produce these rare Ura+His+ papillae, the strains with a single marker had to undergo spontaneous duplication of the chromosome (or the marker itself) in addition toHIS3 excision. Under selection for this kind of duplication marker, Chan and Botstein found that most events (85%) were likely catastrophic increases in ploidy rather than single chromosome gains . We conclude that theura3::HIS3 marker constructed here should identify cells that have gained an extra copy of the modified chromosome.
Frequency of Ura+ cells in haploid strains containingura3::HIS3 integrated next to a centromere
Galactose exposure prior to selection
Frequency of Ura+
3.4 ± 3.2 × 10-4
1.0 ± 0.0 × 10-4
9.7 ± 1.8 × 10-5
1.3 ± 0.6 × 10-4
Haploid cells were grown overnight in YPD rich medium. 106 cells were plated onto selective medium lacking uracil. Colonies were counted after 3 days. The mean frequency and standard deviation obtained from at least 4 independent cultures are shown.
aOnly 2 independent cultures were tested.
bCultures were grown in supplemented minimal medium containing raffinose. Where indicated, galactose was added to 1.5% and culture was grown for 1.3 doublings prior to plating onto selective medium. Diluted cultures were also plated to YPD to score viable cell density. Ura+ frequency was computed per viable cell plated. A paired t-test indicates that the frequencies were not different (2-tailed, P > 0.1).
Targeted chromosomes are duplicated after nondisjunction is induced
If nondisjunction of the conditional centromere causes chromosome gain as well as chromosome loss, induced disomic cells should be detectable by an increase in the frequency of appearance of Ura+His+ colonies. To select directly for Ura+His+ disomes, haploid cells carrying a modified chromosome were grown to log phase in medium containing raffinose, the cultures were split, and galactose was added to one of the resulting cultures. After 1–1.3 culture doublings, cells were spread to selective plates. In the absence of galactose, all strains produced spontaneous Ura+His+ papillae at a frequency of approximately 10-6 (Figure3). For strains containing modified chromosome III or IV, growth in galactose increased the frequency of Ura+His+ papillae formation (Figure3A), suggesting that many of the papillae had developed from disomes that formed by galactose-induced nondisjunction. In contrast, the strains containing modified chromosome VI showed no increase in the appearance of Ura+His+ papillae when grown in galactose. (We consider this lack of chromosome VI duplication below.)
In addition to direct selection for Ura+His+ colonies, we tested whether a "delayed selection" scheme could identify candidate disomes (Figure4A). Cells carrying a modified chromosome IV were grown in galactose for 1.3 culture doublings, diluted, and plated to rich medium. The resulting colonies were replica-plated to selective medium and screened for colonies that produce numerous Ura+His+ papillae (Figure4B). Among colonies from 7 independent galactose-treated cultures, 4.8% behaved as candidate disomes, whereas only 0.37% of the colonies from cultures that were not exposed to galactose looked like candidate disomes (standard deviations were 2.8% and 0.43%, respectively).
To determine whether Ura+His+ isolates were disomic, we examined chromosome copy number by microarray-based comparative genomic hybridization (array CGH). Among galactose-induced cultures, we tested 3 isolates that targeted chromosome III and 6 that targeted IV (1 selected directly and 5 from the delayed selection protocol). All of these isolates contained a single, extra copy of the target chromosome (see Figure5 for examples of each karyotype). In contrast, among spontaneous Ura+His+ isolates, 1/6 that targeted chromosome IV (1/3 direct selection, 0/3 delayed selection) displayed disomy. We conclude that this method, placing a conditional centromere and duplication marker on a target chromosome, allows for efficient isolation of newly formed disomic strains of yeast.
Extra copies ofTUB1 allow the isolation of chromosome VI disomes
Since galactose exposure did not increase the frequency of Ura+His+ papillae in strains carrying a modified chromosome VI (Figure3A), we did not have confidence that the selected colonies were disomic. Indeed, when 5 isolates were examined by array CGH, aneuploidy was not detected (data not shown). This result is consistent with the notion that chromosome VI disomy is lethal, as suggested by previous studies that found VI disomy to be either rare or absent [3,6,10,20]. For example, disomic haploids are frequently found among the rare, viable spores produced by interspecific hybrids ofSaccharomyces cerevisiae andparadoxus . Hunter et al. examined 300 such spores by CHEF gel analysis and observed no occurrences of chromosome VI disomy among the nine chromosomes detectable by this method, consistent with a possible lethality of disomic chromosome VI . Similarly, in a systematic study of chromoduction, Dutcher found that when chromosome VI disomy was selected, the frequency of its appearance was much lower than other chromosomes of its size, perhaps because there is strong selection against chromosome VI disomes . More recently, Torres et al. used chromoduction to construct and study a nearly complete set of N+1 disomic strains . However, when chromosome VI disomy was selected and the resulting cells were examined by array CGH, the unselected chromosomes I and XIII were also present. The absence of single chromosome VI disomes suggests that such a karyotype may be lethal, and that the presence of chromosomes I and XIII suppresses this lethality [1,3].
If chromosome VI disomy is lethal, expression of one or more genes on the extra chromosome VI may cause stoichiometry imbalances severe enough to prevent viability. There are several well-studied genes on chromosome VI that are known to be deleterious upon overexpression, includingCDC14,ACT1 andTUB2 [21–25]. For example,TUB2, which codes for beta-tubulin, has been shown to be exquisitely dosage-sensitive. Overexpression ofTUB2 causes lethality, even when a single, extra copy is integrated into a haploid genome . This lethality can be suppressed by increased expression of alpha-tubulin in the cell, encoded by the chromosome XIII genesTUB1 andTUB3 [26,27]. The observations of Torres et al. support the idea that the extra dose ofTUB2 contributes to the lethality of disomic chromosome VI: in the viable strains that are disomic for I, VI, and XIII, the extra copy of chromosome XIII can supply the cell with additional alpha-tubulin, eliminating the stoichiometry imbalance caused by the extra copy ofTUB2 .
Clearly, a second copy ofTUB2 in a haploid issufficient to cause lethality, as demonstrated by theTUB2 integration experiments of Katz et al. . But when the entire chromosome VI is duplicated, is the duplication ofTUB2necessary for the extra copy of chromosome VI to cause lethality? If it is, then eliminating the tubulin imbalance alone should allow for the viability of chromosome VI disomes. However, if additional chromosome VI genes cause dosage imbalances severe enough to prevent viability, then simply eliminating the tubulin imbalance should not suppress the lethality of disomic chromosome VI.
We used the chromosome duplication method described here to test whether the tubulin imbalance is necessary for the lethality of VI disomy. We suppliedTUB1-containing plasmids to haploid strains carrying modified chromosome VI, then induced nondisjunction and selected for duplication of theura3::HIS3 marker. In contrast to strains without excessTUB1, many Ura+His+ candidate disomes did appear after exposure to galactose when the cells harboured aTUB1 plasmid (Figure3B). The effect occurred whenTUB1 was carried on a low-copyCEN plasmid or on a high-copy 2-micron plasmid.
Most of the Ura+His+ isolates, although viable, produced slow-growing, tiny colonies (Additional file2). We grew 9 isolates in liquid culture, extracted DNA and performed array CGH. Although one culture did not exhibit aneuploidy, the other 8 were disomic for chromosome VI: 3 isolates were simple disomes, 3 isolates also contained an extra, unselected chromosome XII, and 2 contained extra chromosomes II and XII (see Figure5 for examples of each karyotype). Since a number of colonies were isolated that contained the single, extra chromosome VI, and since these were only isolated when extra copies ofTUB1 were present, we conclude that minimizing (or eliminating) the effect ofTUB2 overexpression allows for the viability of chromosome VI disomes.TUB2 overexpression is therefore essential for the inviability of chromosome VI disomy.
Although viable, the chromosome VI disomes exhibited growth defects (Additional file2). We do not know whether these defects are the result of residual tubulin dosage problems, other gene-specific effects, or a combination of the numerous ways that aneuploidy affects phenotype . Further, it is not clear what role, if any, the unselected chromosomes II and XII play. Since the aim of this report is to describe our method for manipulating chromosome copy number, a complete study of the basis for chromosome VI dosage phenotypes will be reported elsewhere.
We have described a new method for inducing and selecting disomic yeast strains which does not rely on spontaneous errors in chromosome segregation. The method allowed for efficient isolation of disomic strains carrying either chromosome III or IV. We used the method to test a specific dosage relationship between chromosome VI and the alpha-tubulin geneTUB1, and found that chromosome VI disomes could be isolated with this method when plasmid-borneTUB1 was present. Our proof-of-concept test was therefore successful for each of the three chromosomes we tested.
In principle, any strain that already contains a conditional centromere could be supplemented with a duplication marker and used to generate disomic aneuploids. Reid et al., for example, have generated conditional centromeres on all 16 chromosomes for inducing loss of heterozygosity [28,29], and these chromosomes could be further modified as described above for the induction and selection of disomes. The method should be useful in studies investigating the genetic basis of aneuploid phenotypes, and any study that wishes to efficiently duplicate a chromosomede novo.
Media and genetic manipulations
Standard methods were used for growth and genetic analysis of yeast , except that YPD medium was supplemented with 50 mg/l adenine sulfate and 20 mg/l uracil. Sporulation was induced as described . Unless otherwise noted, carbon sources were supplemented to 2% (wt/vol) and cells were grown at 30°C in supplemented minimal media to maintain plasmids or unstable integrations. Cell density was determined using a hemacytometer.
Construction of plasmids
Plasmids are listed in Table2[13,26,32,33].Oligonucleotides were designed with Primer3  and are listed in Additional file3.Standard methods of DNA manipulation were used, unless otherwise noted[30,35].To construct the plasmid pKA52, a 390 bp fragment internal toURA3 was amplified from pGALCEN-JC3-13 template DNA with the use of oligonucleotide primers URA3int_F and URA3int_R in a high-fidelityPfu polymerase chain reaction (PCR) (Stratagene).This fragment was digested withBamHI andEcoRI, then ligated into theBamHI/EcoRI sites of pRS303. The plasmid pKA55 was constructed using homologous recombination in yeast  to replace theURA3 marker of pRB326 withLEU2. A 3.7 kbSphI/PvuII fragment containingLEU2 and flanking vector sequence was cut from pRB327, gel-purified with the Qiaquick gel extraction kit (Qiagen), then combined withSmaI-linearized pRB326 DNA to co-transform aleu2- yeast strain.pKA55 was recovered by isolating DNA from Leu+ transformants and transformingE. coli strain JM109.To confirm that pKA55 contained functionalTUB1,it was transformed into atub1Δ strain containing theTUB1-URA3 plasmid pRB326.Cells were grown in medium supplemented with uracil to allow loss of pRB326,plated to YPD,then replica-plated to medium selecting for Leu+.Colonies were identified that were Leu+Ura-,indicating that the sole source ofTUB1 was from pKA55.
Yeast strains used in this study are listed in Table3. All strains were derived from the S288C-related BY4741 and FY strains [37,38]. KAY519, KAY530 and KAY587 were descended from a cross between BY4741 and the FY3-derived DBY10147.
Haploid yeast strains were constructed to contain a modified chromosome (III, IV, or VI) that harbors a conditional centromere and a set of duplication markers (Figures1A, B and Additional file4). To construct KAY418 and KAY419, which carry a modified chromosome III, DNA containing PGAL1-CEN3 URA3 from plasmid pGALCEN-JC3-13 was transformed into strains DBY8923 and DBY8869, respectively, replacing chromosomalCEN3 as described . To generate theura3::HIS3 duplication marker, these strains were transformed with the integrative plasmid pKA52, which had been cut at the uniqueStuI site within itsURA3 fragment. The strains were then backcrossed. Genetic linkage to chromosome III markers and centromere confirmed theCEN3 location of the integrated DNA. To generate KAY541 and KAY542, the strains were crossed to KAY530 to replaceura3-52 withura3Δ0 as described below.
To construct KAY614 and KAY619, which carry a modified chromosome IV, a 2.6 kb fragment containing PGAL1-CEN3 URA3 was amplified with high-fidelityPhusion PCR (Finnzymes) using the primers CEN4_REPL_F and CEN4_REPL_R and pGALCEN-JC3-13 template DNA. Each primer contains at its 5' end a 50 nt sequence identical to that found adjacent toCEN4. The PCR fragment was gel-purified (Qiaquick gel extraction kit, Qiagen) and used to transform the diploid strain DBY8869 × DBY8925. An independently-amplified fragment was used to transform the isogenic diploid DBY8871 × DBY8923. Each resulting strain was transformed withStuI-digested pKA52 to generateura3::HIS3. To confirm that the conditional centromere integrated atCEN4, DNA from Ura-His+ transformants was amplified by PCR using the primers CEN4_F (located outside the integration site nearCEN4) and URA3_int_R (located in the conditional centromere sequence). To generate haploid strains, the heterozygous, transformed diploids were sporulated and theura3::HIS3 marker segregated 2:2. When these strains were used to select for duplication of chromosome IV (see Induction and selection of N+1 disomes, below), some Ura+His+ derivatives were found in which theura3-52 allele on chromosome V (which consists of the full-lengthURA3 gene with a Ty1 insertion ) had recombined withura3::HIS3 on chromosome IV to generateURA3+ without duplicating the intact target chromosome (data not shown). To prevent this unwanted event,ura3-52 was replaced withura3Δ0 by crossing the haploid strains containing PGAL1-CEN3 ura3::HIS3 to KAY530. Spore clones that containedura3Δ0 were identified by the PCR-amplification of a 550 bp fragment from spore clone DNA using the primers URA3_del_F and URA3_del_R, which flank theURA3 coding region.
KAY539 and KAY568, which carry a modified chromosome VI, were constructed with the same methods as were KAY614 and KAY619, except the primers CEN6_REPL_F and CEN6_REPL_R were used to target PGAL1-CEN3 URA3 to replaceCEN6, and the primer CEN6_F was used with URA3_int_R in a PCR to confirm that the conditional centromere had integrated atCEN6. KAY591 and KAY628 were constructed by crossing KAY539 and KAY568, respectively, to KAY519.
Induction and selection of N+1 disomes
Haploid strains carrying PGAL1-CEN3 ura3::HIS3 at the centromere of the target chromosome were grown to saturation (2 days) in supplemented minimal medium that contained raffinose as its nonrepressing carbon source , diluted at least 2000-fold into fresh medium and grown overnight to obtain log-phase cultures. The cultures were split when the density was 0.5–1 × 107 cells/ml. To one half, galactose was added to a final concentration of 1.5%. Cell density was monitored until cultures had grown approximately 1.3 doublings (2.5-fold increase in density). The cells were pelleted, resuspended in water, diluted and plated to YPD and selective media lacking uracil or lacking both histidine and uracil. Ura+ colonies were scored to determineHIS3 excision frequency. Ura+His+ colonies were scored to determine frequency of duplication and excision, and were picked as candidate disomes (direct selection method). Colonies that grew on YPD were scored to determine viable cell density of plated cultures, and replica-plated to selective plates lacking uracil (to monitor excision) and to plates lacking histidine and uracil (to monitor duplication and excision). Replica-plated colonies on which many Ura+His+ papillae grew were considered candidate disomes and Ura+His+ papillae were picked (delayed selection method).
Microarrays were produced by spotting PCR-amplified DNA fragments from approximately 6200 yeast open reading frames (kindly donated by D. Botstein, Princeton University) onto poly-lysine coated glass slides as described . Genomic DNA was isolated by glass bead lysis according to the protocol of Hoffman and Winston . To label each sample, 2 μg DNA was digested withHaeIII, purified, then resuspended in water. The DNA was boiled in the presence of 15 μg random nonamer nucleotide primers, then cooled on ice. The hybridized primers were extended with the use of 20 units ofexo- Klenow polymerase (New England Biolabs) in a 50 μl reaction containing 180 μM each of dATP, dGTP, dCTP, 72 μM dTTP, 108 μM 5-(3-aminoallyl)-dUTP, 50 mM NaCl, 10 mM Tris (pH 7.9), 10 mM MgCl2, and 1 mM dithiothreitol. After 2 hours at 37°C, EDTA (pH 8.0) was added to 45 mM. The primer-extension products were purified through a DNA Clean and Concentrator-5 spin column (Zymo Research) and resuspended in 50 mM sodium bicarbonate (pH 9). Reactive Cy3 (or Cy5) mono NHS ester dyes (GE Healthcare, Cat. No. PN5661) were coupled to the aminoallyl groups in the DNA as directed by the supplier. The labeled DNA was purified through another spin column and resuspended in 20 μl 10 mM Tris (pH 8.5). Cy3- and Cy5-labeled DNAs were combined and hybridized to the microarrays at 65°C for 18 hours in a solution of 3× SSC, 730 μg/ml PolyA RNA, 240 μg/ml tRNA, 24 mM HEPES buffer (pH 7), and 0.24% SDS. Arrays were washed in 0.05× SSC at room temperature and imaged with a GenePix 4000B scanner (Molecular Devices). Array images were analyzed with ScanAlyze . Data was filtered for signal intensity at least 2-fold above background in both channels, ratios were normalized to average 1 across all unaffected chromosomes, and log ratios were visualized with the Karyoscope viewer of Java Treeview .
The raw microarray data, accession number GSE14377, are deposited at GEO .
List of abbreviations
comparative genomic hybridization
polymerase chain reaction
yeast extract, peptone, and dextrose
yeast extract, peptone, and galactose
We thank Kerry Bloom for the conditional centromere plasmid, David Botstein for strains, plasmids, and materials for constructing microarrays, Lisa Shaffer and Bassem Bajjani for the use of their microarray scanner, Maitreya Dunham for the array CGH protocol and critical reading of the manuscript, and Justin Platon for technical assistance. We also thank the Genomics Consortium for Active Teaching (GCAT) for microarrays and the students of the 2008 Genomics course at Gonzaga University for microarray data and analysis. This work was supported by MJ Murdock Charitable Trust grants 2006256 and 2003198, the Robert and Claire McDonald Work Award Program and the Gonzaga Science Research Program.
Biology Department, Gonzaga University
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