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1 Laboratory of Genome Dynamics, National Institute for Basic Biology, Okazaki, 444-8585, Japan
2 School of Life Science, Graduate University for Advanced Studies (SOKENDAI), Okazaki, 444-8585, Japan
3 School of Advanced Science, Graduate University for Advanced Studies (SOKENDAI), Hayama, 240-0193, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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In a haploid S. cerevisiae cell, ~200 copies of rDNA on average are tandemly arrayed on the long arm of chromosome XII. A single rDNA unit comprises a 35S rRNA coding region (35S coding region), 5S rRNA coding region that is transcribed by RNA polymerase III and two intergenic spacer sequences (IGS1 and IGS2). The autonomous replication sequence (ARS) and the replication fork barrier (RFB) site that blocks replication fork progression in a direction opposite to the direction of 35S rRNA transcription by RNA pol I are located within IGS2 and IGS1, respectively (Fig. 1A). Fob1p is known as the replication fork blocking protein that binds to the RFB site in a sequence-dependent fashion and is required for the replication fork blocking event (Kobayashi & Horiuchi 1996; Kobayashi 2003). In addition, FOB1 is required for the recombination and expansion/contraction of the rDNA repeat (Kobayashi et al. 1998; Johzuka & Horiuchi 2002). Thus, FOB1 is considered to be required for the maintenance of average copy number by increasing/decreasing copy number through the regulation of recombination between the repeats. In fob1 cells, however, the long rDNA tandem repeat is still maintained stably without any expansion/contraction. Previously, to analyze the mechanism responsible for the stable maintenance of the rDNA tandem repeat under fob1 defective conditions, we screened mutants that were defective in maintaining long repetitive array and isolated six mutants. In all these mutants, new mutation alleles of the condensin genes, SMC2, SMC4 and BRN1, were identified (Johzuka et al. 2006).
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As previously reported, condensin mutants were defective in carrying a long repeat in fob1 background; therefore, the condensin and fob1 double mutants only possessed greatly contracted short repeat (this is referred as supercontraction; 11 copies in the shortest mutant, i.e., smc2–157). In addition, the slow growth phenotype and the accumulation of mitotic cells in which the nuclei were completely separated but the nucleoli were still in the process of separation were observed in the fob1 smc2-157 double mutant. Since the complete deletion of the chromosomal rDNA repeat in the fob1 smc2-157 double mutant restores slow growth and the accumulation of mitotic cells, it was suggested that the fob1 smc2-157 double mutant has some defects with regard to the segregation process of the chromosomal rDNA repeat. Thus, in cooperation with FOB1, condensin plays an important role in the maintenance and segregation of the chromosomal long rDNA repeat.
Interestingly, the introduction of the wild-type FOB1 gene into the fob1 smc2-157 double mutant restored the length of rDNA repeat and the growth rate of the mutant. Thus, FOB1 suppresses the supercontraction of the rDNA repeat and the defect in the segregation of the chromosomal rDNA repeat in the smc2-157 mutant. Analysis of condensin association with rDNA by chromatin immunoprecipitation (ChIP) assay revealed that in the FOB1+ strain, both Smc4p and Brn1p mainly localized within IGS1, with the major peak of association at the RFB site (Johzuka et al. 2006 and Fig. 1B); this binding peak was observed from S-phase until anaphase and disappeared in G1 phase. In contrast, in the fob1 strain, the association peak of both subunits at the RFB site was greatly decreased (Johzuka et al. 2006 and Fig. 1C). These results indicated that the condensin complex localized at the RFB site in a FOB1-dependent fashion and suggested that the accurate localization of condensin at the RFB site was responsible for the FOB1 suppression of the defects in condensin mutants. However, it remains unclear why the accurate localization of condensin at the RFB site is important for the suppression by FOB1.
In fob1 cells, although condensin localization at the RFB site as well as within IGS1 was greatly decreased, the association signals within the IGSs (IGS1 and IGS2) were higher than those within the 35S coding region (Johzuka et al. 2006 and Fig. 1C). This difference in the condensin association level between the IGSs and 35S coding regions observed in fob1 cells raises the possibility that strong transcription by RNA pol I affects condensin localization within the 35S coding region and in turn results in supercontraction of the rDNA repeat in the fob1 smc2-157 double mutant. In this report, we investigate the effect of transcription by RNA pol I on condensin association with the rDNA region in fob1 cells and on the maintenance of the long rDNA repeat in the fob1 smc2-157 double mutant. We found that RNA pol I transcription interferes with condensin association with the 35S coding region in fob1 cells and causes supercontraction of the rDNA repeat in the fob1 smc2-157 double mutant. During the course of experiments, we also found that the stimulation of transcription by RNA pol II from the non-coding RNA promoter located within IGS1 decreased condensin localization near the RFB site.
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Results |
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In order to analyze the effect of transcription by RNA pol I on the difference in the condensin association level between the IGSs and 35S coding regions observed in fob1 cells, condensin association with rDNA was analyzed by a ChIP assay in cells in which transcription by RNA pol I was eliminated. The gene of the second largest subunit (Rpa135p) of RNA pol I was deleted in fob1 cells to create the fob1 rpa135 double mutant (N2KJY254). This strain lacks a functional RNA pol I holoenzyme; therefore, there is no transcription of 35S rRNA by RNA pol I. However, the strain carries a helper plasmid (pNOY102) that can supply 35S rRNA by Gal-driven RNA pol II transcription, whereby it can be grown only in a galactose medium (Nogi et al. 1991b). Thus, the condensin association with rDNA in the rpa135 mutant was analyzed by a ChIP assay using cells grown in galactose medium. Because the condensin association was analyzed in cells grown in glucose medium in our previous report (Johzuka et al. 2006), we first examined whether the similar association profiles were observed in the FOB1+ and fob1– strains grown in galactose medium. Immunoprecipitation (IP) was performed using strains carrying tandem affinity purification (TAP)-tagged (Ghaemmaghami et al. 2003) SMC4, in which the endogenous copy of the gene was modified at its C-terminus by TAP (Smc4p-TAP). Asynchronously growing cells of the wild-type, fob1 and fob1 rpa135 mutants in galactose medium were used for the ChIP assay. To observe a precise profile of condensin association with rDNA, the relative ratio of the association level at each position (1–8 in Fig. 1A) was calculated using an internal control (base fragment) that was located in the 35S coding region (Fig. 1A). It was observed that Smc4p associated with IGS1, with the major peak coinciding with the RFB site (Fig. 1B), as previously reported. In contrast, in fob1 cells, the association peak at the RFB site greatly decreased (Fig. 1C). Thus, the FOB1-dependent Smc4p association peak at the RFB site was similarly observed both in cells grown in galactose medium and in cells grown in glucose medium. In addition, the difference in the Smc4p association level between the IGSs and 35S coding regions in fob1 cells reproduced in galactose medium. However, in the fob1 rpa135 double mutant, Smc4p evenly associated with the 35S coding region as well as with the IGSs, and higher levels of association with the IGSs than with 35S coding regions were not observed (Fig. 1D). These results indicate that the different levels of association of Smc4p with the IGSs and 35S coding regions is due to the effect of functional RNA pol I in fob1 cells. We also analyzed the Smc4p association level by measuring the actual enrichment ratio of signal intensities of IP samples [signal intensity of the fragment in IP sample/signal intensity of the fragment in whole cell extract (WCE) sample] at IGS1 (fragment 3) and 35S coding region (base) in both fob1 and fob1 rpa135– mutants (Fig. 1E). A decrease in the level of association of Smc4p with the 35S coding region was observed in fob1 cells. In fob1 rpa135 cells, however, the level of association with the 35S coding region was almost similar to that of the IGS1. These results suggest that transcription by RNA pol I decreases the level of Smc4p association with the 35S coding region.
If transcription by RNA pol I actually interferes with the association of condensin with the 35S coding region, the difference in the condensin association level between the IGSs and 35S coding regions is expected to be increased in strains in which the rDNA repeats are in a more actively transcribed state. In order to examine this possibility, Brn1p association with the IGS1 and 35S coding regions was analyzed in rDNA that was in the more actively transcribed state. In normal strains carrying 150~200 copies of rDNA, only a fraction (~60%) of the total copy number is in a transcriptionally active state in rapidly growing cells (Warner 1999). It was reported that in cells with a low copy number of rDNA repeats (30~40 copies), all the rDNA genes were in an actively transcribed state, and the density of RNA pol I per rDNA gene was increased by more than twofold (observed as "Christmas tree" by the Miller spreading method) (French et al. 2003). Overall, the rate of 35S rRNA transcription between cells with normal and low copy numbers of rDNA repeats is equivalent. Thus, RNA pol I transcription per rDNA copy in cells carrying a low copy number of rDNA should be in a more active state than that in cells carrying wild-type level of copies (150~200). We analyzed the difference in the association level of condensin with IGS1 and 35S coding regions between strains carrying normal and low copy numbers of rDNA. The N2KJY285 and N2KJY402 strains carry 138 and 30 copies of the chromosomal rDNA repeats, respectively (Fig. 2A,B). The relative ratio of the level of Brn1p association with IGS1 and 35S coding regions was analyzed using an internal control (base described in Fig. 2C) that was located in the 35S coding region. In the N2KJY285 strain, the level of Brn1p association with the IGS1 region was approximately 1.69-fold higher than that with the 35S coding region (Fig. 2D,E). In contrast, in the N2KJY402 strain (low copy number of rDNA), the level of Brn1p association with IGS1 was at least 2.47-fold higher than that with the 35S coding region. Thus, the difference in the levels of association of condensin with the IGSs and 35S coding regions is increased in strains carrying low copy number of rDNA. In addition to the ratio relative to the base fragment, the actual enrichment ratio of signal intensity of IP samples in the 35S coding region (fragment 1 and base) were decreased in the low copy number of rDNA strain when compared with those in the control strain (Fig. 2F). These results indicated that the association of condensin with the 35S coding region was decreased in strains with low copy number of rDNA in the more actively transcribed state, thereby strongly suggesting that transcription by RNA pol I interfered with condensin association with the 35S coding region.
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The above results suggested a possibility that interference of condensin association with the 35S coding region by RNA pol I in fob1 cells was responsible for the supercontraction of the rDNA repeat observed in the fob1 smc2-157 double mutant. In order to examine this possibility, we analyzed the suppression of the supercontraction of the rDNA repeat in the fob1 smc2-157 double mutant by the rpa135 mutation. The size of the chromosomal rDNA repeat was determined by analyzing the length of chromosome XII separated in pulse-field gel electrophoresis (Fig. 3A). Since FOB1 was dispensable for preventing supercontraction of the rDNA repeat in the case of SMC2+ cells, the length of chromosome XII did not change in the fob1 SMC2+ strain (Fig. 3A, compare lanes 1 and 2). In the smc2-157 mutant, however, the length of chromosome XII dramatically decreased to ~1.2 Mb by the elimination of the FOB1 gene (compare lanes 3 and 4). This drastic shortening of chromosome XII is caused by the supercontraction of the rDNA repeat, as previously reported (the fob1 smc2-157 double mutant carries only 11~12 copies; Johzuka et al. 2006). In the case of the rpa135 mutants (lanes 5–8), although the reason is unknown, the copy number of rDNA is gradually decreased in a FOB1-dependent fashion, as previously reported (Kobayashi et al. 1998). The length of chromosome XII in rpa135 mutants in this experiment was approximately 1.8–2.3 Mb in the SMC2+ strain (lane 5) and 1.8 Mb in the smc2-157 mutant (lane 7). The elimination of the FOB1 gene in rpa135 cells produced a discrete 1.8 Mb (~80 copies) signal of chromosome XII (lane 6), because of the freeze of copy number fluctuation in the fob1 strain that was deficient in the FOB1-dependent expansion/contraction (Kobayashi et al. 1998). The elimination of the FOB1 gene in smc2-157 rpa135 cells produced a similar discrete 1.8 Mb (~80 copies) signal of chromosome XII, and the length of the chromosome did not decrease to 1.2 Mb (lane 8). These results indicated that the rpa135 mutation suppressed the supercontraction of the rDNA repeat in the fob1 smc2-157 double mutant. To confirm that the suppression was really due to deletion of the RPA135 gene, the wild-type RPA135 gene was reintroduced into both the fob1 rpa135 double mutant in lane 6 and the fob1 rpa135 smc2-157 triple mutant in lane 8, and the length of chromosome XII was monitored (Fig. 3B). As expected, supercontraction was observed again only in the cells carrying the RPA135 gene in the fob1 rpa135 smc2-157 triple mutant (lane 11) but not in the same cells carrying the vector plasmid (lane 12). These results indicate that the supercontraction of the rDNA repeat observed in the fob1 smc2-157 double mutant is an RPA135-dependent event.
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We also analyzed the FOB1-dependent condensin association peak at the RFB site in the rpa135 mutant. The rpa135 mutation was introduced into the wild-type strain to create the N2KJY213 strain. The profile of Smc4p association with rDNA was analyzed by the ChIP assay and compared with that observed in the RPA135+ strain (N2KJY105). As shown in Fig. 1B, while the RPA135+ strain exhibited a major peak of the Smc4p association at the RFB site, the ridgeline of its association profile extended to the right side of the RFB site. In contrast, in the rpa135 mutant (Fig. 4A), the association of Smc4p with the region to the right of the RFB site was greatly decreased; however, a high level of association with the RFB site was observed. Thus, in the rpa135 mutant, the shape of the association profile of Smc4p changes dramatically to a very sharp peak at the RFB site. The large difference in the Smc4p association profile between the RPA135+ and rpa135- cells indicates that the activity of a functional RNA pol I enzyme is required for the distribution of Smc4p on the right side of the RFB site; however, the binding of Smc4p to the RFB site is independent of this enzyme.
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Why did the rpa135 mutant exhibit a sharp Smc4p localization peak at the RFB site? One clue was obtained from the Smc4p association profile of the fob1 rpa135 double mutant. As shown in Fig. 1D, although Smc4p evenly associates with rDNA, a small reduction in the association of Smc4p with rDNA was observed only at the region to the right of the RFB site (PCR fragments 4 and 5). The bidirectional promoter for RNA pol II transcription for non-coding RNA (E-pro) was reported to be located within this region (Ganley et al. 2005; Kobayashi & Ganley 2005). In addition, it was reported that the disappearance of RNA pol II transcriptional silencing was observed in rDNA copies in which transcription by RNA pol I was abolished (Cioci et al. 2003). Thus, it is possible that the increase in RNA pol II transcription from E-pro decreases the distribution of Smc4p on the right side of the RFB site in the rpa135 mutant. If this is the case, a similar reduction in the distribution of Smc4p on the right side of the RFB site must be observed in the sir2 mutant in which E-pro transcription by RNA pol II was increased (Kobayashi & Ganley 2005). We examined this possibility by analyzing the Smc4p distribution around the RFB site in the sir2 mutant. As shown in Fig. 4B, the distribution of Smc4p on the right side of the RFB site (fragments 4 and 5) was decreased in the sir2 mutant. This result strongly suggests that the stimulation of RNA pol II transcription from E-pro decreases the distribution of Smc4p on the right side of the RFB site.
In order to confirm the above possibility, we created a strain in which the GAL1/10 promoter was substituted for E-pro in all the rDNA copies (N2KJY431). The GAL1/10 promoter for RNA pol II transcription is a bidirectional promoter that can be stimulated in galactose medium but is repressed in glucose medium (Yocum et al. 1984). In order to examine the changes in the distribution of Smc4p on the right side of the RFB site, the ChIP assay was performed using N2KJY431 grown in glucose and galactose media. As shown in Fig. 5, the association of Smc4p with the region to the right of the RFB site (fragments 4 and 5) was reduced in the GAL1/10 strain in the stimulated transcriptional state (galactose) when compared with the cells in the inactive transcriptional state (glucose) (Fig. 5B,C). In a control experiment using the normal strain (N2KJY105), no significant differences in the association profile at the region to the right of the RFB site was observed in the cells grown in galactose and glucose media (data not shown). These results confirm that the stimulation of RNA pol II transcription decreases the association of Smc4p with the region to the right of the RFB site, but it does not decrease the association with the RFB site.
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Discussion |
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The changes in the condensin association profile in the cells with stimulated RNA pol II transcription suggested that the characteristics of condensin association with the RFB site and with the region to the right of the RFB site were distinguishable. Condensin association with the region to the right of the RFB site was hindered by the stimulation of RNA pol II transcription. In contrast, because of the following reason, condensin association with the RFB site was not affected by the stimulation of RNA pol II transcription. It was reported that there was no specific termination site for RNA pol II transcription from E-pro; therefore, the average length of the leftward transcripts was 1.5 kb (Kobayashi & Ganley 2005). Thus, the leftward transcripts could pass through the RFB site (the distance between E-pro and RFB is 0.32 kb). Although transcripts from the E-pro pass through the RFB site, RNA pol II transcription did not interfere with condensin binding at the RFB site as shown in Figs 4B (compare Figs 4B and 1B) and 5C. This is consistent with the results that both the supercontraction of the rDNA repeat and the slow growth phenotype accompanied with an accumulation of mitotic cells that were observed in the fob1 smc2-157 double mutant were not observed in the sir2 smc2-157 double mutant; however, an increased variation in the rDNA repeat length was observed in the sir2 mutant because of the elimination of recombinational repression (data not shown). Based on these results, we deduced that there are two characteristics of condensin association with the chromatin. One is the stable localization of condensin at the RFB site. The other is the flexible localization of condensin near the RFB site. Similar characteristics of flexible association on the DNA strand influenced by RNA pol II transcription are observed in the cohesin complex. On the chromosome arm, the cohesin association sites were centered in the intergenic regions where two oppositely transcribed genes converged (Lengronne et al. 2004). In addition, it was observed that the stimulation of transcription could relocate cohesin. Since cohesin is observed as a ring-shaped structure (Haering et al. 2002; Gruber et al. 2003), there is a possibility that cohesin encircles sister chromatids and slides along the chromatin. The stimulation of transcription from the GAL1/10 promoter decreases the level of condensin association with the region to the right of the RFB site, whereas it increases the level of association with the RFB site (Fig. 5). Thus, the possibility that condensin also forms a movable structure and the stimulation of transcription from E-pro slides the condensin complex to the RFB site or functions to prevent the diffusion from the RFB site offers an attractive explanation for this result. In contrast, the stable association of condensin with the RFB site suggests that an unidentified mechanism is responsible for recruiting and stabilizing the condensin complex to the RFB site. We are currently analyzing two possible mechanisms of condensin association with the RFB site; one depends on a replication fork-blocking event and the other, on protein–protein interactions between Fob1p and the condensin complex.
The third SMC complex, Smc5/6, is also known to be required for the faithful segregation of repetitive chromosomal regions (Torres-Rosell et al. 2005). Although both the Smc5/6 and condensin complexes are enriched at nucleolus and required for segregation of rDNA repeat, the functional interaction between these two complexes is still unclear. Torres-Rosell et al. reported that the smc6-9 smc2-8 double mutant showed increased growth defect and thermosensitivity compared with either of the single mutants (Torres-Rosell et al. 2005). In addition, they observed an additive increase in the mis-segregation of rDNA repeat in the smc6-9 smc2-8 double mutant. Furthermore, condensin (Smc4p) localization to rDNA was not altered in the smc6-9 mutant.
In this study, we observed that condensin, a central player for chromosome condensation and segregation during mitosis, has a characteristic that its association with the chromatin is interfered by transcription. In the case of the rDNA repeat in S. cerevisiae, RNA pol I transcription continues during mitosis (Elliott & McLaughlin 1979). Direct analysis of the in vivo association of Rpa135p with the 35S coding region by a ChIP assay performed using cells arrested in mitosis with the microtubule-depolymerizing reagent nocodazole also confirmed this result (Nomura et al. 2004). It is likely that FOB1-dependent condensin association with the RFB site, outside the active RNA pol I transcription region, contributes to ensuring the faithful segregation of long rDNA repeats by separating condensin association region from active transcription zone. On the other hand, in higher eukaryotes, all nuclear transcription is repressed during the mitotic stage; this is known as mitotic transcriptional repression (Gottesfeld & Forbes 1997). If the abovementioned characteristic of condensin is conserved in higher eukaryotes, mitotic transcriptional repression may contribute to the unobstructed association of condensin with the entire chromosomal regions; this in turn will result in proper segregation of long chromosomes, with the formation of highly condensed chromosome structure.
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Experimental procedures |
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Yeast-extract-peptone (YEP)-glucose (YPD), YEP-galactose and synthetic media were used as described in Sherman (1991). The strains and plasmids used in this study are summarized in Table 1. The N2KJY213 was constructed by the tetrad dissection of diploid cells created by crossing N2KJY105 and NOY408-1a. Similarly, the N2KJY254 was constructed from diploid cells created by crossing N2KJY133 and NOY408-1a. The N2KJY285 strain was created by the selection of Ura– cells from N2KJY131 on a 5-FOA (1 mg/mL) plate. The N2KJY402 strain was created in the following way. N2KJY285 was first transformed with pRDN-hyg1 using the URA3 marker. The transformants were streaked on a YPD-containing hygromycin (300 µg/mL) plate. Several hygromycin-resistant clones were isolated and then streaked on a 5-FOA plate to select the Ura- clones that had lost pRDN-hyg1. The size of chromosome XII in several Ura- clones was analyzed by pulse-field gel electrophoresis. One of them that carried 30 copies of rDNA was used as N2KJY402. The copy number of rDNA in the N2KJY402 strain was stable because of the fob1 strain. N2KJY431 was constructed by the dissection of diploid cells created by crossing N2KJY105 and TAK2004. The N2KJY186 and 177 strains were created as follows. The diploid strain N2KJY073 was first transformed with the rpa135::LEU2 fragment that was amplified by PCR using the genomic DNA of NOY408-1a as the template, and the correct heterozygote of RPA135 / rpa135::LEU2 was selected. The pNOY353 plasmid was then introduced into the Leu+ transformant, and the resultant diploid cell was dissected. The N2KJY186 and 177 strains were selected by testing a genetic marker, as indicated, from haploid progenies. The N2KJY262 and N2KJY257 strains were created by selecting the 5-FOAR colony of the N2KJY186 and 177 strains, respectively, to eliminate the pKJY181 (FOB1, URA3) plasmid.
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Asynchronous cultures grown in YEP-glucose or YEP-galactose medium were used for the ChIP assay. The preparation of whole cell extract (WCE) and immunoprecipitation (IP) were carried out as described in Claypool et al. (2004) with some modifications as follows. The protein concentration of WCE was adjusted to 5 mg/mL. A 500 µL of WCE was mixed with 35 µL (bed volume) of immunoglobulin G agarose beads (Sigma) and incubated for 2 h at 4 °C. IP samples and WCE (10 µL) were finally suspended in 100 µL of Tris–EDTA after reverse cross-linking. A tenfold dilution of WCE samples was carried out before the PCR reaction. A 1 µL of WCE and IP samples was used for the PCR (20 µL total volume) reaction for 17 cycles. In the case of the experiment shown in Fig. 2, 18 cycles of PCR reaction was performed. The PCR products were separated in 2.5% agarose gels (NuSieve GTG agarose; Cambrex) and stained with SYBR green. Signal intensities of the PCR products were measured using a LAS-100 (Fuji) from at least three independent experiments. The primers used are shown in Table 2.
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Chromosomal DNA preparation in gel molds was performed as described in the instruction manual (Clamped homogeneous electric field, CHEF; Bio-Rad). The chromosomes were separated in a 0.8% pulse-field certified agarose by using CHEF for 72 h at 14 °C with 3 V/cm at a linear pulse of 5–15 min.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail:kishori{at}nibb.ac.jp
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References |
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Accepted: 4 March 2007
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1.2 Mb in length is indicated as a black dot in lane 4. The size of the chromosome standard (H. wingei) is indicated to the left. Lane 1, wild-type (UKJY223); lane 2, fob1 (UKJY225); lane 3, fob1 smc2-157-kanMX carrying pKJ181 (N2KJY075); lane 4, fob1 smc2-157-kanMX (N2KJY173); lane 5, fob1 rpa135 carrying pKJ181 (N2KJY186); lane 6, fob1 rpa135 (N2KJY262); lane 7, fob1 rpa135 smc2-157-kanMX carrying pKJ181 (N2KJY177); and lane 8, fob1 rpa135 smc2-157-kanMX (N2KJY257). (B) The RPA135 gene (pKJ255) or vector (YCplac33) was reintroduced into the cells in lanes 6 (N2KJY262) and 8 (N2KJY257), respectively, and the supercontraction of the rDNA tandem repeat was observed. The panel shows the detection of the signal of chromosome XII by Southern hybridization using the SMC4 probe. Lane 9, N2KJY262 carrying pKJ255; lane 10, N2KJY262 carrying YCplac33; lane 11, N2KJY257 carrying pKJ255; and lane 12, N2KJY257 carrying YCplac33.
