Copyright Ó 2006 by the Society of America DOI: 10.1534/genetics.105.048058

Stability of Large Segmental Duplications in the Genome

Romain Koszul,1 Bernard Dujon and Gilles Fischer2 Unite´ de Ge´ne´tique Mole´culaire des Levures (CNRS URA2171, UFR927 Universite´ Pierre et Marie Curie), De´partement de Structure et Dynamique des Ge´nomes, Institut Pasteur, Paris, 75724 Cedex 15, France Manuscript received July 12, 2005 Accepted for publication February 6, 2006

ABSTRACT The high level of gene redundancy that characterizes eukaryotic genomes results in part from seg- mental duplications. Spontaneous duplications of large chromosomal segments have been experimentally demonstrated in yeast. However, the dynamics of inheritance of such structures and their eventual fixation in populations remain largely unsolved. We analyzed the stability of a vast panel of large segmental duplications in (from 41 kb for the smallest to 268 kb for the largest). We monitored the stability of three different types of interchromosomal duplications as well as that of three intrachromosomal direct tandem duplications. In the absence of any selective advantage associated with the presence of the duplication, we show that a duplicated segment internally translocated within a natural chromosome is stably inherited both mitotically and meiotically. By contrast, large duplications carried by a supernumerary chromosome are highly unstable. Duplications translocated into subtelomeric regions are lost at variable rates depending on the location of the insertion sites. Direct tandem duplications are lost by unequal crossing over, both mitotically and meiotically, at a frequency proportional to their sizes. These results show that most of the duplicated structures present an intrinsic level of instability. However, translocation within another chromosome significantly stabilizes a duplicated segment, increasing its chance to get fixed in a population even in the absence of any immediate selective advantage conferred by the duplicated genes.

ECIPROCAL translocations and large duplications Various mechanisms leading to the duplication of R are major driving forces in genome evolution. The whole or large portions of genomes have been described role played by these chromosomal rearrangements in in fungi. Whole-genome duplication, aneuploidization, shaping the genomic architectures of eukaryotic species extrachromosomal amplification, unequal crossing over, has been notably illustrated by comparative analyses cycle of break-fusion-bridge, nonreciprocal transloca- between related species, including (Fischer et al. tion, and retrotransposition events all result in the 2000; Kellis et al. 2003; Dujon et al. 2004), plants duplication of large DNA sequences (Bainbridge and (Schmidt 2002; Yogeeswaranet al. 2005; Yuet al. 2005), Roper 1966; Sexton and Roper 1984; Whittaker et al. animals, and humans (Dutrillaux 1979; Stanyon 1988; Dorsey et al. 1993; Wolfe and Shields 1997; et al. 1999; Bailey et al. 2002, 2004; Stankiewicz and Moore et al. 2000; Dunham et al. 2002; Dietrich et al. Lupski 2002; Jaillon et al. 2004; Bourque et al. 2005). 2004; Kellis et al. 2004; Schacherer et al. 2004). Several experimental studies have shown that the Duplications of large DNA segments are also commonly fixation of translocations and duplications in popula- found in laboratory strains as suppressors of deletion tions of yeast maintained in continuous culture was ob- mutants (Hughes et al. 2000). In a previous work, we tained in response to growth-limited conditions and/or developed a gene dosage selection assay to recover in competition experiments (Hansche et al. 1978; Adams spontaneous duplications from the right arm of chro- et al. 1992; Brown et al. 1998; Dunham et al. 2002; mosome XV in a haploid yeast strain (Koszul et al. Infante et al. 2003; Colson et al. 2004). Adaptive 2004). We showed that a large variety of inter- and evolution of wine yeast strains also results from trans- intrachromosomal segmental duplications appears spon- locations within their genome (Perez-Ortin et al. 2002). taneously, at an estimated frequency of 109/cell/di- vision (revised from our initial estimate of 1010) and possibly through a new mechanism related to the repli- cation process. All the intrachromosomal events corre- 1Present address: Department of Molecular and Cellular Biology, sponded to the direct tandem duplication (DTD) of Harvard University, Cambridge, MA 02138. large DNA segments ranging in size from 41 to 288 kb. 2Corresponding author: Unite´deGe´ne´tique Mole´culaire des Levures, We also found three different types of interchromo- CNRS URA 2171, UFR 927, De´partement de Structure et Dynamique des Ge´nomes, Institut Pasteur, 25 rue du Docteur Roux, Paris, 75015, France. somal duplications where the right arm of chromosome E-mail: fi[email protected] XV was translocated onto other chromosomes, within a

Genetics 172: 2211–2222 (April 2006) 2212 R. Koszul, B. Dujon and G. Fischer (subtelomerically translocated duplica- For meiotic assays, the haploid strains carrying various tion, STD), or in an internal region (internally trans- duplicated blocks (YKF1050, -1038, and -1057 and YKF1246 oszul located duplication, ITD), or onto a supernumerary from K et al. 2004; the backward translocated strain type II* in Figure 2C) and control strains (BY4741 and BY4742) chromosome corresponding to the fusion between the with no duplication were crossed with either Y2159 (MATa, duplicated parts of two different chromosomes, one of ade1-1, trp1-b) or Y2160 (MATa, ade1-1, trp1-b), two deriva- which is chromosome XV (supernumerary chromosome tives of the Y55 strain. The five resulting diploids, heterozygous duplication, SCD). for a large duplicated segment from chromosome XV, were The study of the intrinsic stability of different dupli- named YKF155, -145, -129, -127, and -187; sporulated; and 158, 249, 300, 98, and 149 tetrads were dissected, respectively. One cation structures has been tackled so far in different hundred tetrads were also dissected for the two control strains. ways. Pioneer works on have The presence/absence of the duplicated blocks in the meiotic revealed that tandem arrays of small repetitive units are products was sought by PCR amplification of the breakpoint subject to amplification and deletion through unequal junctions as previously described (Koszul et al. 2004) or by recombination events (Szostak and Wu 1980; Fogel pulsed-field gel electrophoresis (PFGE) karyotyping. elch ackson ink Estimation of the rate of 5-fluoroorotic acid resistance: The and W 1982; J and F 1985). It is also well rate of appearance of 5-fluoroorotic acid (5-FOA)-resistant established that are dynamic chromo- colonies was determined by a fluctuation test analysis. They somal regions that undergo frequent recombinational were calculated from the average of two independent experi- exchanges (Louis and Haber 1990a; Pryde et al. 1997; ments, each corresponding to 4–12 independent cultures. Cells 3 8 Liti et al. 2005). Finally, disomic chromosomes are were diluted from an overnight preculture (1.5–2.5 10 cells/ml) to 25–50 cells/ml and grown in YPD to 2.5–3 3 108 known to be spontaneously lost mitotically at a rather cells/ml. Cells were diluted in sterile water to the appropriate high frequency (Campbell et al. 1975). However, by concentration and plated on 5-FOA (DUCHEFA F0176, 1 mg/ specifically engineering strains carrying large dupli- ml) -containing medium to select for resistant cells (two plates cated segments that can be spontaneously lost without per culture). After 3 days of incubation at 30°, resistant any detrimental effect, our work aims at giving a colonies were counted and the median value for each set of cultures of an experiment was estimated. For each strain comprehensive picture of the relative stability of differ- the spontaneous rate was then calculated using the ent types of duplicated segments. The large panel of median method (Lea and Coulson 1948). duplicated structures recovered from our previous study PFGE and Southern hybridizations: Plugs of intact chro- gives a unique opportunity to precisely measure and mosomal DNA, PFGE, and transfer onto Hybond-N1 mem- compare the mitotic and the meiotic stability of various branes (Amersham Biosciences, Arlington Heights, IL) as well as probe labeling and hybridizations were carried out as large inter- and intrachromosomal duplicated segments described previously (Fischer et al. 2001). in a given genetic background. Our results show that the combination between translocations and large segmen- tal duplications results in stabilizing newly duplicated RESULTS regions within the yeast genome. Construction of the strains used for mitotic stability assays: Seven large segmental duplications from the MATERIALS AND METHODS right arm of chromosome XV (six of which were pre- viously obtained in Koszul et al. 2004 and a newly isolated Yeast strains: All strains used for the mitotic assays are strain) that all encompass the RPL20B (YOR312c) gene haploid derivatives from Saccharomyces cerevisiae BY4743 were studied: an ITD of a 115-kb segment within (MATa/MATa, his3D1/his3D1, leu2D0/leu2D0, ura3D0/ura3D0, LYS2/lys2D0, MET15/met15D0)(Winzeler et al. 1999). The chromosome III (Figures 1A and 2); two STDs of 230 haploid strains carrying large segmental duplications from and 256 kb at the extremities of chromosome I and V, chromosome XV [encompassing the RPL20B (YOR312c) respectively (Figures 1B and 3); a SCD of 268 kb gene] used in this work are derived from the original strains (Figures 1C and 4); and three DTDs internal to chro- YKF1036, -1038, -1057, -1050, and -1246; from YKF1114 de- mosome XV of 41, 115, and 213 kb, respectively (Figures scribed in Koszul et al. (2004); and from a newly isolated strain YKF2048 carrying a 230-kb duplicated segment from 1D and 5). These large segmental duplications resulted chromosome XV (also encompassing RPL20B) at the extrem- from the selection of a duplication of the RPL20B gene ity of chromosome I (YKF210 in Figure 1B). A wild-type copy of in strains that were deleted for RPL20A. The deletion of the originally deleted RPL20A (YMR242c) gene was reintro- RPL20A produced a severe growth defect that was duced in these strains by crossing and tetrad micromanipula- compensated by the duplication of RPL20B (Koszul tion, resulting in 7 different recombinant haploid strains carrying both RPL20A and a duplicated segment encompass- et al. 2004). The RPL20A gene was reintroduced in the ing RPL20B (YKF166, -210, -211, -179, -181, -164, and -161, see genomes of the seven parental strains used in this study. Figure 1 and results). In each of these strains, one of the two The resulting strains carried one copy of RPL20A and copies of RPL20B was replaced by transformation with a URA3 two copies of RPL20B. One of the two copies of RPL20B marker. The position of the URA3 marker was characterized was subsequently replaced by a URA3 cassette (see precisely by karyotype hybridization and by restriction map- materials and methods ping, resulting in 14 different strains, each carrying URA3 ) without detrimental effect located in either of the duplicated blocks (Figure 1, positions on the growth rate. This resulted in the construction of 1–14 indicated in the schematics). 14 strains, each carrying a rpl20bTURA3 marker in Stability of Segmental Duplications in Yeast 2213

Figure 1.—Different types of large segmental duplications. PFGE karyotypes of WT and mu- tant strains carrying segmental duplications. Chromosomes XV and VII comigrate in the WT strain, resulting in a band with a double intensity on the gel. Relevant chromosome numbers are indicated, and solid arrow- heads show the positions of the modified or additional chromo- somes in the karyotypes. For each duplication type a schematic of the chromosomes involved in the duplication events is shown, and the positions where the rpl20BTURA3 marker was in- serted are indicated (1–14). The extent of the duplicated sequen- ces is indicated by black double- headed arrows.

one or the other duplicated block (Figure 1, positions Stability of an ITD: The rpl20BTURA3 marker was 1–14). inserted in the ITD carried either by chromosome The URA3 gene confers prototrophy to uracil but also XVtIII (Figures 1A and 2B, position 1; Table 1, strain sensitivity to 5-FOA. Thus, the loss of the duplicated YKF173) or by chromosome IIItXV (position 2, strain block containing the rpl20BTURA3 marker confers YKF256). The rate of 5-FOAR mutants formation in both resistance to 5-FOA. The mitotic stability of the dupli- strains presents only a threefold increase compared to cated segments was therefore measured from the rate of wild type (WT) (Table 1). Thirty-two and 27 indepen- 5-FOA-resistant colonies formation by a fluctuation test dent 5-FOA-resistant colonies were isolated from the (Table 1). 5-FOA-resistant cells appear in the control progenies of YKF173 and YKF256 and analyzed by PFGE. strain (BY4743) at a frequency of 107/cell/division The corresponding karyotypes showed a complete as a result of inactivation of the URA3 gene probably stability of the ITD, whose loss was never observed through point . In the duplicated strains, among the 59 strains analyzed (no type I event). For the events responsible for 5-FOA resistance were classi- positions 1 and 2, 27 and 23 cells presented an un- fied in three categories: loss of the whole duplicated modified karyotype compared to the parental strain block carrying URA3 (type I), loss of the rpl20bTURA3 YKF166, respectively (Figure 2B and Table 1). Among locus by with the RPL20B gene as those, hybridization with URA3 as a probe showed that donor located on the other duplicated segment (type 20 and 9 strains presented an absence of the rpl20BT II), and inactivation of URA3 through point muta- URA3 marker for positions 1 and 2, respectively. These tions (type III). Among these three categories, only cases correspond to type II events, reflecting a gene con- type I events correspond to the loss of the duplicated version event between the RPL20B and rpl20BTURA3 segment. loci carried by the two duplicated blocks. The remaining 2214 R. Koszul, B. Dujon and G. Fischer

Figure 2.—Mitotic stability of an internally translocated duplication (ITD). (A) PFGE karyotypes of WTand YKF166 strains. The solid arrowheads show the positions of the chromosomes of interest mentioned in the text. A schematic of the duplicative trans- location in YKF166 including positions 1 and 2 where the rpl20BTURA3 marker was inserted is also presented. (B) Karyotypes of representative 5-FOAR mutant strains recovered for each position and hybridization of the corresponding Southern blot with the URA3 gene as probe. Type II corresponds to the loss of the URA3 marker by a gene conversion event between rpl20BTURA3 and RPL20B located in the other duplicated block. Type II* corresponds to the same gene conversion event but associated with a mitotic crossing over. Type III corresponds the inactivation of URA3 by a point mutation. (C) PFGE karyotypes of WT, YKF173, and type II* 5-FOAR mutant strains and hybridizations of the corresponding Southern blot with subtelomeric probes from chromosomes III (YCR106w) and XV (YOR370c). The right side shows schematics of both the original ITD in strain YKF173 (XVtIII and IIItXV) and the backward translocation corresponding to type II*. strains showing unmodified karyotypes (7 strains for These strains, therefore, carry a block from chromo- position 1 and 14 for position 2) still possess a URA3 some XV internally duplicated within chromosome III, gene (type III, Figure 2B and Table 1) probably inac- resulting in a size increase of 115 kb of this chromosome tivated by point mutations, as supported by the sequenc- [consistent with the observed PFGE pattern, chromo- ing of this gene in five independent 5-FOA-resistant some (chr)IX ¼ 440 kb; chrIII 1 115 kb ¼ 430 kb]. clones (three derived from YKF173 and two from YKF256). Chromosome XV is intact in these strains, consistent These five clones presented point mutations resulting in with its comigration with chromosome VII. A crossing an amino acid substitution (A184P); or in premature stop over associated with a gene conversion event between codons at positions 56, 83, and 150; or in a frameshift RPL20B and rpl20BTURA3 is likely to be responsible for (position 247 due to a single-base deletion, DG741). In the formation of these backward translocations. addition, the proportions of these events (7/32 and 14/ The meiotic transmission of this ITD was analyzed by 27, e.g., frequencies of 0.7 3 107 and 1.5 3 107 event/ crossing the corresponding strain with a strain devoid of cell/division for positions 1 and 2, respectively) are sim- both the duplicated region and the translocation. As ilar to the frequency of point mutation in URA3 in the WT expected, the fertility of the diploid was affected strongly (107/cell/division). by the reciprocal translocation, resulting in an overall Five and four 5-FOAR strains, for positions 1 and 2, spore viability of 47%. This is in good agreement with respectively, presented a karyotype different from the the theoretical fertility of 50% expected from a cross karyotypes of both the parental strain YKF166 and the between two strains heterozygous for a reciprocal trans- WT. Hybridization with URA3 as a probe revealed that location. The backward-translocated strains obtained these strains have lost the reporter gene (type II* events, during the mitotic assay (type II*, see schematic in Figure 2B and Table 1). In these strains, chromosome Figure 2C) were also crossed with the strain devoid of XV comigrates with chromosome VII as in the WTstrain, the ITD. In the resulting diploid, the only structural and chromosome III now comigrates with chromosome difference between the two sets of homologous chro- IX. Hybridizations with subtelomeric probes from mosomes consists of a 115-kb segment from chromo- either chromosome III (YCR106w)orXV(YOR370c) some XV duplicated internally to one chromosome III. revealed a backward translocation between the trans- In this case, the backward translocation increases the located arms of chromosomes III and XV that preserved meiotic fertility of the diploid up to 79% (compared to the duplicated block within chromosome III (Figure 2C). 89% for the control strain with no translocation or Stability of Segmental Duplications in Yeast 2215

Figure 3.—Mitotic stability of a subtelomeri- cally translocated duplication (STD). (A) PFGE karyotypes of WT, YKF210 (top), and YKF211 (bottom) strains. The solid arrowheads show the positions of the chromosomes of interest mentioned in the text. The schematics of the STD event in both YKF210 (top) and YKF211 (bottom), including positions 3, 4, 5, and 6 where the URA3 gene was inserted, are presented. (B and C) Karyotypes of representative 5-FOAR mu- tants recovered for each position and hybridiza- tion of the corresponding Southern blot with URA3 as a probe. Type II and type III correspond to inactivation of URA3 by gene conversion with RPL20B and by point mutation, respectively. Type I corresponds to the loss of the URA3-containing duplicated segment. For position 4, faint addi- tional signal onto chromosome V corresponds to hybridization of the probe with the ura3D851 allele, which corresponds only to a partial dele- tion of the URA3 gene. The right side shows a schematic of the chromosomal structure of type I mutants recovered in the progeny of strains carrying URA3 at either position 4 or 6.

duplication). Therefore, backward translocation par- been inserted either onto chromosome XV (strain tially restores the fertility defect conferred by the initial YKF220, position 3 and strain YKF259, position 5, translocation. Moreover, the duplicated block does not respectively) or onto the STD (strain YKF254, position appear to be subject to excision during as all 4 and strain YKF262, position 6, respectively, Table 1 and 20 tetrads tested presented a 2:2 segregation of the Figure 3). duplicated block. Both strains carrying the rpl20BTURA3 marker onto In summary, among 59 5-FOA-resistant strains ana- chromosome XV present a slight increase in the 5-FOAR lyzed, none has lost the ITD (loss frequency is therefore clone formation compared to that in WT (36 and 312 ,108 event/cell/division). The duplicated translocated for strains YKF220 and YKF259, respectively; Table 1). strains carry a duplicated block of 115 kb encompassing The 32 5-FOAR mutants isolated in the progeny of 53 genes that is stably inherited during both mitosis and YKF220 presented karyotypes identical to that of their meiosis despite the absence of any selective advantage progenitor. For strain YKF259, 4 of the 32 mutants had conferred by the presence of the duplicated region (i.e., lost the STD of 256 kb on chromosome V, illustrating the WTand the duplicated strains show identical growth the high intrinsic instability of this structure (see below). rates in rich medium). The majority of mutants (26/32 and 28/32 for strains Mitotic stability of two STDs: In strains YKF210 and YKF220 and YKF259, respectively; Table 1) have lost YKF211, carrying a STD of 230 kb from the right arm of the URA3 marker through type II events (Figure 3B), chromosome XV in the right subtelomere of chromo- probably as a result of a gene conversion event between some I (chromosome ItXV, Figure 1B) and a STD of RPL20B from the STD and rpl20BTURA3 on chromo- 256 kb from the right arm of chromosome XV in a some XV or a break-induced replication (BIR) event subtelomere of chromosome V (chromosome VtXV, between the translocated segment and the right arm of Figure 1B), respectively, the rpl20BTURA3 marker has chromosome XV. The six and four remaining strains 2216 R. Koszul, B. Dujon and G. Fischer

Figure 4.—Mitotic stability of a supernumer- ary chromosome duplication (SCD). (A) PFGE karyotypes of WT and YKF179 strains. The solid arrowheads show the positions of the chromo- somes of interest mentioned in the text. Sche- matic of the structure of the supernumerary chromosome in YKF179, including positions 7 and 8 where the URA3 gene was inserted, is also presented. (B and C) Karyotypes of representa- tive 5-FOAR mutants recovered for each position and hybridization of the corresponding Southern blot with URA3 as a probe. Type I corresponds to the loss of the supernumerary chromosome. Type II and type III correspond to inactivation of rpl20bTURA3 by gene conversion with RPL20B and by point mutation, respectively.

correspond to type III events, as illustrated by the se- family. The loss of this 230-kb STD located on chromo- quence of the ura3 gene characterized in one of the six some I occurs at a frequency of 2.6 3 108 event/cell/ 5-FOAR clones deriving from YKF220 showing a frame- division. shift at position 146 due to a single-base deletion (DG436). Thirty-two independent 5-FOA-resistant colonies were The strains YKF254 (position 4) and YKF262 (position isolated from the progenies of YKF262, the strain car- 6), carrying URA3 onto the STD located on chromosomes rying the rpl20BTURA3 marker on the STD of 256 kb I and V, respectively, present very different 5-FOAR clone on chromosome VtXV, and analyzed through PFGE. formation rates. Whereas the rate of 5-FOAR mutant All karyotypes but one were consistent with the loss of formation in YKF254 presents only a fourfold increase the STD. This suggests that this block was duplicated compared to that in WT, the rate in YKF262 is 2812-fold in a highly unstable subtelomeric region. The loss of higher than that in WT (Table 1). this 256-kb duplication occurs at a frequency of 2.7 3 For YKF254, the karyotypes of 30 resistant mutants 104 event/cell/division. This STD was mapped at the (among 32 analyzed) were found to be similar to the far extremity of one subtelomere of chromosome V parental karyotype of strain YKF210 (Figure 3C and (Koszul et al. 2004). To pinpoint further the site of Table 1). Hybridization with URA3 as a probe revealed translocation, the junction of the rearrangement was that 25 of them correspond to type II events while the 5 PCR amplified using a chromosome XV-specific primer remaining strains still possess a copy of URA3 and (CAACTAATGAACTCTGGATA) and a primer within a correspond to type III events [point mutations leading Y9 element (TTCGAGCAGAGAAGTTGGAG) and sub- to amino acid substitutions (T119K and V233F) were sequently sequenced. The junction occurred precisely characterized in the ura3 sequences from two clones]. between an internal TG1–3 tract distal to the Y9 ele- The karyotypes of the remaining two resistant strains are ment (TGTGTGGGTGTGGTGTGT) and an interstitial consistent with the loss of the STD, with chromosome I -related sequence of 18 bp from chromosome migrating in the pulse-field gel at a position correspond- XV (TGTGTGTGTATGTGTGTG) located in the inter- ing to a size slightly smaller than the WT chromosome I genic region between YOR273c and YOR274w (coordi- (type I, Figure 3C). As expected from this karyotype, the nates 836471–836488). Therefore, the massive instability new chromosome I does not carry the URA3 marker of this STD probably results from frequent ectopic re- anymore. The terminal structure of the new chromo- combination events between Y9 elements possibly as a discussion some I has not been precisely studied; however, the STD result of the fragility of the TG1–3 tract (see ). loss is likely to result from an ectopic recombination Mitotic stability of a SCD: The strain YKF179 carries event between the PAU7 (YAR020c) located centromere a SCD (XfXV) corresponding to the fusion between proximal to the site of insertion of the original STD 594 kb duplicated from chromosome X (containing the and another member of this large subtelomeric gene centromere) and the terminal 268 kb duplicated from Stability of Segmental Duplications in Yeast 2217

position 8, Figure 4). In the latter case, resistance to 5-FOA appears at a rate of 3.4 3 105 mutational event/ cell/division (corresponding to a 359-fold increase com- pared to WT, Table 1). Among the 32 5-FOA-resistant mutants karyotyped, all presented a karyotype similar to the WT (type I, Figure 4B) in which the supernumer- ary chromosome that carried the rpl20BTURA3 marker was lost. Therefore, supernumerary chromosomes are highly unstable during mitosis. Strain YKF190 presents a frequency of resistance to 5-FOA of 1.2 3 106 (312 compared to that in WT). Among 32 mutants analyzed, 29 have conserved the supernumerary chromosome but lost the URA3 gene on chromosome XV (type II, Figure 4C). Two have kept both the supernumerary chromosome and the URA3 locus and have probably acquired resistance by point mutations, in agreement with their frequency of ap- pearance comparable to WT (type III, Figure 4C). The last strain analyzed has lost both the chimeric chromo- some and the URA3 gene (type I, Figure 4C). Type I and type II strains probably result from gene conversion be- tween RPL20B on chromosome XfXV and rpl20BTURA3 on chromosome XV or from BIR events between these two chromosomes, initiated in a region centromere proximal to RPL20B. Stability of intrachromosomal DTDs: The most fre- quent duplication event at the RPL20B locus recovered in our former analysis consists of DTD (Koszul et al. 2004). Twenty-six to 32 independent 5-FOA-resistant colonies were isolated from the progenies of YKF189, YKF169, and YKF170, corresponding to rpl20BTURA3 at positions 9, 11, and 13, respectively (Table 1, Figures 1 and 5A). Two types of 5-FOA-resistant mutants were found. In type I mutants (the majority of cases, Table 1), Figure 5.—Mitotic stability of direct tandem duplication (DTD). (A) Left, PFGE karyotypes of the strains carrying the PFGE karyotype is similar to WT, consistent with direct tandem duplications on chromosome XV. The solid the loss of the duplicated block (Figure 5A, type I). arrowheads show the positions of the comigrating chromo- Hybridization of these karyotypes with URA3 as a probe somes VII and XV in the WT strain as well as the positions confirms the loss of this gene concomitantly with the of the 41-, 115-, or 213-kb duplication-containing chromo- loss of the duplicated segment. Type II resistant mutants somes XV in strains YKF181, YKF164, and YKF161, respec- present an unmodified karyotype compared to that of tively. Middle, schematic of the six positions where URA3 T was inserted (also see Table 1). Right, karyotypes of represen- their parental strains but an absence of rpl20B URA3 tative 5-FOAR mutants recovered in the progeny of YKF169 marker, as revealed by hybridization (Figure 5A, type II). (URA3 at position 11), and hybridization of the correspond- Type I strains result from mitotic unequal crossing over ing Southern blot with the URA3 gene as probe. Type I corre- (UCO) between the tandemly duplicated segments, by sponds to the loss of the tandemly duplicated block, and type II corresponds to the loss of URA3 by gene conversion with either intra- or interchromatid recombination (Figure RPL20B present in the other duplicated segment. (B) Sche- 5B). For each parental strain, we plotted the frequency matic of unequal crossing over leading to the loss of the of type I mutants as a function of the size of the se- URA3-containing block. (C) Frequency of loss of the dupli- quence flanking the URA3 gene and into which the UCO cated block plotted as a function of the size of the sequence events must have occurred to result in a strain that has available for unequal recombination (indicated in Table 1) (R2 ¼ 0.90). For each size two independent experiments were lost the URA3-containing duplicated segment (Table 1 2 performed. and Figure 5C). A linear relationship is observed (R ¼ 0.9). From the regression curve, the mitotic instability (frequency of loss per cell per generation) of tandemly the right arm of chromosome XV (Figure 1C). The duplicated segments in the yeast genome follows the rpl20BTURA3 marker has been inserted either onto relationship I ¼ C 1 (107 3 S), where S is the size of the chromosome XV (strain YKF190, position 7) or onto duplicated sequence (in kilobases) and C is a constant the supernumerary chromosome XfXV (strain YKF185, value of 2 3 106. According to this equation, theoretical 2218 R. Koszul, B. Dujon and G. Fischer

TABLE 1 Rate of appearance of 5-FOA-resistant mutants in cultures

Parental strain Frequency of Size of the Type of mutants Strain and type 5-FOAR Fold loss of the flanking Positiona name of duplication frequency increase I II (II*) III URA3-containing block sequence (kb)c WT BY4741 — 9.6 3 108 31 1 YKF173 YKF166 (ITD) 3.3 3 107 33 0 25 (5) 7 ,108b 2 YKF256 YKF166 (ITD) 2.9 3 107 33 0 13 (4) 14 ,108b 3 YKF220 YKF210 (STD) 6.1 3 107 36 0 26 6 ,108b 4 YKF254 YKF210 (STD) 4.2 3 107 34 2 25 5 2.6 3 108 5 YKF259 YKF211 (STD) 1.2 3 106 312 ,108b 6 YKF262 YKF211 (STD) 2.7 3 104 32812 32 0 0 2.7 3 104 7 YKF190 YKF179 (SCD) 1.2 3 106 313 1 29 2 3.7 3 108 8 YKF185 YKF179 (SCD) 3.5 3 105 3350 32 0 0 3.5 3 105 9 YKF189 YKF181 (DTD) 2.6 3 106 327 31 1 0 2.5 3 106 9 10 YKF183 YKF181 (DTD) 6.8 3 106 371 6.6 3 106 31 11 YKF169 YKF164 (DTD) 6.5 3 106 368 27 5 0 5.5 3 106 45 12 YKF168 YKF164 (DTD) 1.1 3 105 3115 9.3 3 106 69 13 YKF170 YKF161 (DTD) 1.0 3 106 3104 25 1 0 9.6 3 108 77 14 YKF192 YKF161 (DTD) 1.7 3 105 3174 1.6 3 105 135 a Positions refer to the location of the URA3 gene (see Figures 1–5). b Loss of the duplication segment was never observed. c Size of the sequence flanking the URA3 gene and into which the UCO events must have occurred (see Figure 5B). values for the blocks of 41, 115, and 213 kb are 6.1 3 stability of the block during meiosis is higher if one (or 106, 1.3 3 105, and 2.3 3 105/cell/generation, several) of this (these) meiotic hotspot(s) is (are) en- respectively. compassed in the duplicated sequence. Following the To determine the meiotic stability of large direct generation of a meiotic DNA DSB, crossing over occurs tandem duplications, the three strains YKF1050, -1038, preferentially between homologs rather than sisters and -1057 carrying intrachromosomally duplicated (Schwacha and Kleckner 1997). Recombination be- blocks of 41, 115, and 213 kb, respectively, were crossed tween homologs heterozygous for the tandem duplica- with a strain devoid of segmental duplications and tion would end in a 2:2 segregation of the duplicated tetrad analysis was performed on the resulting diploids. block. However, we showed that the proportion of 3:1 As shown in Figure 6A, the global viability of the meiotic tetrads increases when the size of the duplication in- products decreases when the size of the block increases. creases. In the case of structural heterozygosity inter- This is illustrated by a significant increase in the number homologs DNA exchanges can be rejected and the DSB of tetrads with only three viable spores, the number of ends redirected into intersister pathways (Hunter and tetrads with four viable spores decreasing concomi- Kleckner 2001). However, UCO between sister chro- tantly. This result suggests that large tandem duplica- matids must lead to a tandem triplication in one of tions alter meiotic segregation of chromosomes, which the four spores. We sought for the presence of such may lead to abnormal and unviable meiotic products. structures in the meiotic products of the 3:1 tetrads For each diploid strain, tetrads with four viable spores from YKF129 by PFGE karyotyping and subsequent were further analyzed to follow the segregation of the hybridization with RPL20B as a probe. We found only duplicated segment (Table 2). The proportion of 2:2 one triplication of 18 tetrads (not shown). Thus the segregation of the duplicated block decreases as the size higher level of spore lethality in the progeny of the of the block increases. On the contrary, the proportion diploids heterozygous for the large segmental duplica- of 3:1 segregation corresponding to the absence of the tions would not be due to the triplications being duplication in three of the four meiotic products inviable. In addition, predominance of the 3:1 tetrads increases concomitantly with the size of the block (Table with one spore containing a tandem duplication over 2). Therefore, tandemly duplicated segments of this the 3:1 tetrads with a tandem triplication suggests that region are frequently lost during meiosis. The meiotic intrachromatid recombination leading to the loop out DNA double-strand break (DSB) frequencies in the of one copy of the duplication would be more frequent genome of a S. cerevisiae strain related to BY4743 have than sister-chromatid UCO that would lead to the crea- been monitored previously (Gerton et al. 2000). In the tion of a tandem triplication. Our results suggest that, in region covered by the three tandem duplications, four cases of large tandem duplications, DSB ends would be loci presenting high DSB frequencies (meiotic hot- preferentially redirected into intrachromatid rather spots) have been characterized (Figure 6B). The in- than intersister recombination. Stability of Segmental Duplications in Yeast 2219

study is in good agreement with former results obtained on the stability of duplicated DNA sequences structur- ally related to ours. Thus, a SCD, resulting from the fusion between sequences from two different chromo- somes, is lost mitotically at a high frequency (3.4 3 105), similar to the frequency of loss of an aneuploid chromosome [a disomic chromosome III is lost at a fre- quency of 104 during mitosis (Campbell et al. 1975)]. Furthermore, it is known that subtelomeres are highly recombinogenic (Pryde et al. 1997), which could ex- plain the relative instability of the STDs analyzed here. These losses result probably from BIR events involving the repeated sequences interspersed throughout the subtelomeric regions of the chromosomes. However, we found highly variable rates of loss between the two STDs studied. Discrepancies in loss rates might be related to the number and the types of repeated sequences present within subtelomeres and available for recombi- nation. The less unstable STD (loss rate of 2.6 3 108 event/cell/division) presents a copy of the PAU7 gene flanking the site of insertion of the 230 kb from the right arm of chromosome XV in the right subtelomere of chromosome I. This gene belongs to a family of 23 members, with showing 80–85% identity, most Figure 6.—Meiotic instability of DTDs. (A) Influence of of them showing a subtelomeric localization (Pryde the size of the tandemly duplicated block on the viability of et al. 1997). The highly unstable STD of 256 kb from the meiotic products after sporulation of a diploid strain het- the right arm of chromosome XV (loss rate of 2.7 3 erozygous for the duplication. (B) The three duplicated 104 event/cell/division) has inserted in an interstitial blocks tested (41, 115, and 213 kb) and the meiotic hotspot ORFs they encompassed are mapped on chromosome XV. The telomere-related sequence directly flanking a Y9 ele- number in parentheses corresponds to the rank of each hot- ment in the left subtelomere of chromosome V. The Y9 spot in the yeast genome, according to Gerton et al. (2000). repeated sequence family is composed of long and short elements that reside at 17 of the 32 chromosome ends (Louis and Haber 1990b). Despite several insertions/ DISCUSSION deletions between and within Y9-longs and Y9-shorts, these Segmental duplications present highly variable levels elements share more extensive sequence identity than of stability: In this work, we monitored and quantified the members of the PAU family, which could explain in the spontaneous intrinsic instability of different types of part the higher rate of loss of the STD on chromosome V large segmental duplications in the yeast genome. We than on chromosome I. However, the rate of recombi- showed that most of the structures formed by segmental national interactions involving a particular Y9 was pre- duplications present some levels of instability when no viously estimated to 2 3 106 (Louis and Haber 1990a), selective advantage is conferred by the presence of the 100 times lower than the rate of STD loss observed duplication (i.e., the duplication can be lost without in this study. Intrachromosomal telomeric sequences detrimental effect on the growth rate of the strain). Our have been described as fragile sites in higher eukary- otes (Bouffler 1998; Musio and Mariani 1999; Ruiz- Herrera et al. 2005). In addition, replication forks TABLE 2 pause as they pass through internal tracts of TG(1–3) (Ivessa et al. 2002). The particularly high level of in- Meiotic stability of the tandemly duplicated blocks in four viable spored asci stability of the STD on chromosome V could result from an intrinsic fragility of the TG(1–3) telomere-related se- Segregation of the duplicated blocka quence flanking the Y9 element at the rearrangement breakpoint: the resulting double-strand breaks would Strain Block size (kb) Totalb 21:2 11:3 4 be repaired using an ectopic Y9 and subsequently lead YKF155 41 23 22 1 0 to loss of the STD. YKF145 115 24 19 5 0 We also showed that large DTDs, from 41 to 213 kb in YKF129 213 35 15 16 4 size, are lost during mitosis at frequencies proportional a 1, block present; , block absent. to their size. Unequal crossovers between sister chro- b Total number of tetrads analyzed. matids are likely to be the cause of these losses, as 2220 R. Koszul, B. Dujon and G. Fischer

advantage conferred by the duplicated block exceeds this fitness reduction, the duplication will tend to be lost also during meiosis (Table 2). It was previously shown that loss of a tandemly duplicated block encompassing the yeast HIS4 hotspot occurs frequently during meiosis (Jackson and Fink 1985). Here, tandem block excision frequencies seem also to depend on the positions of recombination hotspots in the genome (Figure 6B). Translocation of the duplicated segment internally onto another chromosome suppresses its intrinsic in- stability (Figure 7, iii and iv), allowing its fixation in the population even in the absence of any associated selective advantage. A twofold reduction in meiotic fertility is expected for such a strain carrying a reciprocal translocation between two chromosomes. But a mitotic backward translocation both preserving the segmental Figure 7.—Dynamics of segmental duplications in the duplication and restoring collinearity between the yeast genome. Schematic of two arbitrary chromosomes chromosomes of the two parental strains has been (open and shaded areas) is shown. The orientated black independently isolated several times in the course of boxes represent the chromosomal segments subject to dupli- this study (Figure 7, v). This backward translocation can cation and loss. (i) Duplication of a chromosomal segment partially restore the fertility defect of the diploid that generates an unstable structure (STD, SCD, and DTD). (ii) Loss of the duplications through UCO or BIR events. heterozygous for the duplicated region (79% of spore (iii and iv) Duplication associated with a reciprocal transloca- viability vs. 89% for the control strain without any tion between nonhomologous chromosomes (ITD). (v) Back- duplication). Thus, the chance of fixation of a segmen- ward translocation restoring the original collinearity between tal duplication both in mating and in asexual popula- chromosomes (see text). tions is favored by reciprocal translocation events. Conciliation of our model with preexisting obser- previously described for much smaller tandem repeats vations and models from higher : Translo- (Szostak and Wu 1980; Fogel and Welch 1982; Welch cation events often occur by ectopic recombination et al. 1990; Paquin et al. 1992; Lambert et al. 1999). between the LTR sequences in laboratory and industrial Moreover, we demonstrated that a duplication inter- strains (Rachidi et al. 1999; Dunham et al. 2002) as well nally translocated onto another chromosome (ITD) is per- as in wild isolates (Fischer et al. 2000). LTRs are also manently stabilized both in asexual and in meiotic cells. commonly found at the breakpoints of direct tandem The intrinsic instability of tandem and subtelomeric duplications (Koszul et al. 2004). Other sequences such duplications favors a translocation-based model to as microhomologous sequences may also be used as reach fixation: Previous results on the spontaneous recombination breakpoints leading to both segmental formation of segmental duplications suggest that, at duplications and translocations (Perez-Ortin et al. each generation, a large chromosomal segment can be 2002; Koszul et al. 2004). Studies have demonstrated spontaneously duplicated (Koszul et al. 2004). This the association of segmental duplications with event is rare but its frequency is such that numerous breakpoints between the human and mouse genomes duplications can occur in a population. The sole (Armengol et al. 2003; Bailey et al. 2004) and between RPL20B gene is duplicated at a frequency of 109 the human and Gorilla gorilla genomes (Stankiewicz event/cell/division in a DNA segment whose median et al. 2001). Our study provides experimental evidence size is 115 kb (Koszul et al. 2004). In most cases, the that segmental duplications can be associated directly duplicated block lies in direct tandem next to the with translocations in a eukaryotic genome, to concom- original block but interchromosomal duplications have itantly duplicate and stabilize dozens of genes. The also been characterized (Figure 7, i). Despite the intrin- breakpoint junctions of the segmental duplications we sic instability of most of these structures, if the dupli- analyzed mapped within low-complexity DNA sequen- cation confers a selective advantage, the duplication ces, replication fork convergence regions, and LTR may become fixed in the population. For instance, sequences associated with tRNA genes (Koszul et al. a DTD of 11 kb has been found in the genome of 2004). These particular regions are scattered through- Kluyveromyces lactis (Dujon et al. 2004). If no selective out the genome and provide numerous possibilities for advantage is associated with the duplication, the present genomic rearrangements. Our study fits with a model of analysis reveals that such duplicated blocks tend to genome evolution intermediate to the random break- be mitotically lost (Figure 7, ii). The meiotic viability age model of Nadeau and Taylor (1984) and to the is slightly decreased if one parent carries a tandem ‘‘fragile site’’ model of Pevzner and Tesler (2003): duplicated segment (Figure 6A). 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