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1 National Institute for Basic Biology and 2 The Graduate University for Advanced Studies, School of Life Science, 38 Nishigonaka, Myodaijicho, Okazaki, 444-8585 Japan
3 The Graduate University for Advanced Studies, School of Advanced Science, Hayama, 240-0193 Japan
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Abstract |
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). In the rdn strain, HOT1 transcription was increased about 14 times compared to wild-type. Recombination activity stimulated by HOT1 in this strain was also elevated, about 15 times, compared to wild-type. These results indicate that the level of PolI transcription in HOT1 determines efficiency of the recombination. Moreover, Fob1p, which is essential for both the recombination stimulation activity and transcription of HOT1, was dispensable in the rdn strains. This suggests that Fob1p is functioning as a PolI transcriptional activator in the wild-type strain. |
Introduction |
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Originally, HOT1 was isolated as a 4.6 kb BglII restricted fragment including the non-transcribed spacer region (NTS1 and NTS2), the 5S rRNA gene and a part of the 35S rRNA gene (Fig. 1A, lower panel). The fragment stimulates mitotic recombination when inserted at novel locations in the genome. Subsequent research identified two dis-continuous elements that are the essential fragments for HOT1 recombination stimulation (HOT1 activity; Voelkel-Meiman et al. 1987). For example, when HOT1 is integrated in one of the repeated his4 genes (Fig. 5A), this fragment enhances recombination between the repeats 
100 times (Voelkel-Meiman et al. 1987). One of the two HOT1 elements is the I-element which corresponds to the 35S rRNA gene promoter region and is transcribed by RNA polymerase I (PolI); the other is the E-element which overlaps the enhancer for PolI transcription originally identified by Elion & Warner (1984; Fig. 1). Thus, HOT1 activity appears to be causally related to stimulation of transcription by PolI. Indeed, it was shown that in a PolI defective mutant, HOT1 activity was completely abolished (Huang & Keil 1995). However, in the PolI mutant other phenotypes are also observed (e.g. change of the nucleolus shape (Oakes et al. 1998), and contraction (decrease) of the rDNA repeats (Kobayashi et al. 1998)). Therefore it is not clear whether the transcription itself activates the HOT1 recombination, or whether another factor does.
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Fob1p is also required for recombination in the rDNA. The EXP site is thought to be a hotspot for rDNA recombination (Fig. 1A) and was identified as a region required for amplification of the rDNA (Kobayashi et al. 2001). EXP is dependent on Fob1p for this rDNA recombination. Recently, it was shown that this sequence increases the integration frequency of a plasmid to the rDNA (Benguria et al. 2003). Although Fob1p and its binding site, RFB, are required for both HOT1 and the EXP activities (Kobayashi et al. 1998, 2001; Defossez et al. 1999; Merker & Klein 2002; Johzuka & Horiuchi 2002), the role of them seems to be different for these two recombination hotspots. PolI transcription and the enhancer element (EcoRI-HindIII region in the E-element, Fig. 1A) are not required for recombination in the rDNA, although they are required for HOT1 recombination (Kobayashi et al. 1998, 2001; Wai et al. 2001). In addition, Wai et al. (2001) found that FOB1 was required for the PolI transcription of HOT1, although FOB1 does not affect transcription of the 35S rDNA repeats at the original rDNA locus. Therefore, HOT1 recombination seems to be transcription-dependent while the rDNA recombiantion may be replication fork blocking-dependent (Kobayashi et al. 1998).
To investigate the molecular mechanism by which transcription enhances recombination at HOT1, we here use a strain in which the rDNA repeats are deleted. In this strain, HOT1 transcription was highly stimulated (14 times) and the rate of recombination was elevated about 15 times as compared with that of a wild-type strain. Therefore, we conclude that transcription level itself governs HOT1 recombination efficiency.
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Results |
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HOT1 recombination activity is dependent on PolI (Huang & Keil 1995; Wai et al. 2001). Therefore, it may be possible to elevate the recombination activity by enhancing HOT1 transcription. Wai et al. (2001) reported that a strain whose rDNA was deleted showed hyper-transcription (400x) from a plasmid-cloned PolI promoter. This activation was observed when growth is supported by a multicopy helper plasmid (pNOY353) containing the 35S rRNA coding region fused to a strong PolII promoter, the GAL7 promoter (Oakes et al. 1998). However, the activation was not observed when another multicopy helper plasmid containing the 35S rRNA coding region with original PolI promoter was used. Therefore, the authors speculated that in the strain with the rDNA deleted (rdn), excess PolI transcription machineries elevate ectopic PolI transcription. Here we use the rdn strain with pNOY353 to stimulate transcription of HOT1. We crossed a haploid strain (SER001) in which the HOT1 construct is present at the leu2 locus (Fig. 1B) with the rdn strain (SER015). From the resulting diploid strain (SER002, RDN/rdn), a haploid rdn strain with the HOT1 construct was isolated (SER003, for detail see Experimental procedures). The growth of SER003 was poor, although the parent rdn strain (SER015) which does not have the HOT1 construct grew well (Fig. 2A), thereby indicating this phenotype is dependent on the HOT1 construct. Using the strains (SER001 and SER003), we also mated and established diploid strains with the HOT1 construct (RDN/RDN and rdn/rdn; the growth of the rdn/rdn strain is much better than the haploid rdn strain; data not shown, for detail see Discussion).
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haploid strain (SER003), we could not perform this selection because of the poor growth. Therefore, as the HOT1 constructs have been lost in some cells, the transcript level is likely to be underestimated in this strain. In fact, from Southern analysis (Fig. 6, lane 4), the proportion of bands that do not have the HOT1 construct (i.e. all the 4.1 kb band and half of the 15 kb band) is estimated to be about 60% of the total number of leu2 genes in the population. However, as the transcript level is much higher than other strains (as shown below), this effect does not affect the results significantly. RNA isolated from these strains, was used to measure the rate of HOT1 transcription by Northern analysis using a LEU2 probe (P1; Fig. 1B). The results are shown in Fig. 2B. In lane 1 ‘RDN’ without HOT1 construct, only leu2 transcripts were detected around 1.1 kb. In lane 2 ‘RDN’ (SER001), another band around 3 kb appeared and a lower band was enhanced. Transcript level in the rdn strain (SER003) was much greater than that of ‘RDN’ (SER001). The reason for the smear band is that there is no PolI transcription termination sequence in this HOT1 system. Therefore, transcripts of several lengths were produced. In the diploid strains in which one of chromosome III has the HOT1 construct, though the amount of transcripts are somehow less than those of the haploid strains, the similar pattern was observed (lane 4–6). These results indicate that in the rdn strains, transcription of HOT1 was enhanced.
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strains
The FOB1 gene was isolated as an essential gene for HOT1 recombination activity (Kobayashi & Horiuchi 1996). Wai et al. (2001) demonstrated that FOB1 was necessary for HOT1 transcription, although the gene is not required for transcription of the chromosomal rDNA repeats. To test whether this gene plays a role in the hyper-transcription of HOT1 in the rdn, we performed primer extension assays to determine the level of the HOT1 transcripts. We disrupted FOB1 in HOT1 strains used in the Northern analysis and established several fob1 mutant strains, shown in Fig. 3B. Total RNA from the HOT1 strains was isolated and transcribed by reverse-transcriptase with an end-labelled primer (HOTp) to produce complementary DNA, indicated in Fig. 3A. We note that for this assay we used the same HOT1 strains as used in the previous section (Fig. 1B, bottom, except the haploid rdn strains). We can recognize the HOT1 transcripts from the PolI promoter by the length of the transcript (102 bp). The results are shown in Fig. 3B. Lanes 1–4 are sequencing reactions using the same labelled primer to determine the length of the transcripts. In the wild-type strain (lane 5) we could detect HOT1 transcripts which were initiated at the native 35S rRNA start site (the A at position +1 indicated by an arrow). However, transcripts were almost completely lacking in the fob1 mutant (lane 6). A similar FOB1-dependency was observed in the RDN/RDN diploid strain (lanes 9, 10). Therefore, the HOT1 transcription is dependent on FOB1 in the wild-type, as previously described (Wai et al. 2001). In contrast, when FOB1 was disrupted in the rdn haploid strain, the signal intensity of the transcript did not change so much (lanes 7, 8) and when FOB1 was disrupted in rdn/RDN and rdn/rdn diploid strains, the transcript level was reduced in the fob1 mutants (lane 12, 14) but the band was still visible in the mutant. The signal intensities were measured by a phosphorimager and the values were plotted on a graph (Fig. 3C). The amount of transcript in the rdn strain was more than 100–120 times higher than that of the RDN fob1 strain and more than 10–14 times higher than that of the wild-type RDN strain whose rDNA is not deleted (Fig. 3C). The value was not significantly affected by a fob1 mutation in the rdn strain. In the rdn/RDN diploid strain, HOT1 transcripts increased 2.5 times more than that of the wild-type, and with disruption of FOB1 the level was about 45% reduced (lanes 11 and 12 in Fig. 3C). But the level is still higher than that of the RDN/RDN strain (lanes 9 and 12 in Fig. 3C). Similarly, in the rdn/rdn strain a fob1 mutation reduced the transcription activity, but the activity is still high (lanes 13 and 14 in Fig. 3C). This indicates that in the rdn strains FOB1 affects the level of HOT1 transcription but is not essential for HOT1 transcription itself.
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As PolI is essential for HOT1 recombination, the rate of HOT1 transcription is thought to affect the rate of HOT1 recombination (Stewart & Roeder 1989; Huang & Keil 1995). Therefore, the recombination rate is expected to be elevated in the rdn strain. We measured the recombination rate of HOT1 by determining the loss of an ADE5,7 marker which was inserted in the leu2 tandem repeats (Fig. 1B). The results are shown in Fig. 4A. Recombination is not stimulated in a HOT1-less strain, therefore cells form red colonies due to an ade2 background mutation (Fig. 4A,1). When cells lose the ADE5,7 marker by recombination, the cell colour becomes white because the red pigment cannot be produced in an ade2 ade5 genetic background (Lin & Keil 1991). As shown in Fig. 4A,2, cells with HOT1 form red and white sectored colonies because of frequent loss of ADE5,7. The higher the rate of recombination, the larger the portion of the colony that is white. For example, in one of the rdn strains, ‘RDN/rdn’, the colonies are largely comprised of white portions (Fig. 4A,8). The recombination rate was determined quantitatively by a half-sectoring assay that measures recombination only in the first cell division after plating. In a colony with more than a continuous half of the colony being white, recombination has occurred in the first cell division in most cases. Therefore, the recombination rate can be determined by counting the proportion of half sector colonies. The results of this quantitative assay are shown in Fig. 4B.
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strain, most of the colonies stopped growing when the size was so small that sectors were not observed (Figs 4A,4 and 2A). As this growth deficiency is dependent on the HOT1 construct (Fig. 2A, compare SER003 with SER015), hyper-recombination induced by HOT1 is likely to be the cause of the deficiency. However, some of the white colonies grew well. They are thought to have lost the HOT1 construct during a recombination event where the HOT1-less leu2 gene remains. Some dark red colonies with few or no sectors were also observed. Moreover, with disruption of FOB1 in the rdn strain, the growth became somewhat better, but the colonies were still too heterogeneous in site to recognize sectors (Fig. 4A,5). This heterogeneous growth rate made it impossible to measure the recombination rate in the rdn strain.
In the rdn haploid strain, HOT1 recombination is enhanced about 100 times
To determine the rate in the rdn strain, we used a URA3 marker instead of the ADE5,7 marker. As shown in Fig. 5A, the URA3 gene is inserted between two his4 genes in ura3 defective genetic background. After cells were cultured in SG complete medium lacking uracil, the frequency of Ura- recombinants was determined by spotting aliquots of 10-fold serial dilutions of the cultures on SG plates with and without 5-FOA. The results are shown in Fig. 5B, and the recombination rates were calculated (Fig. 5C). In the rdn strain the recombination rate was about 100 times enhanced compared with the wild-type. Interestingly, in the URA3 system, the growth deficiency observed in the rdn strain with the ADE5,7 system was not detected. As the method and locus examined are both different in the ADE5,7 and URA3 systems, it is difficult to compare the recombination rates obtained by the two systems. From previous work, it is known that in the same URA3 system, HOT1 enhances recombination about 100 times compared with the HOT1-less strain (Voelkel-Meiman et al. 1987; Wai et al. 2001). This compares to the ADE5,7 system, where the enhancement was 15 times, as we showed in Fig. 4B (lane 2). Therefore, we speculate that in the rdn strain with the URA3 system the 100-fold enhancement of recombination rate would be equivalent to a 15-fold enhancement of recombination rate in the ADE5,7 HOT1 recombination system. So as the wild-type cells lose the marker at a rate of 2.2 times per 100 cell divisions, we can roughly estimate that the recombination rate in the rdn strain is 33 loss of the marker per 100 cell divisions. In other words, in the rdn strain in the ADE5,7 HOT1 system the cells lose the marker once per 3 cell divisions.
We used the ADE5,7 HOT1 recombination system to test a diploid strain in which one of the two chromosome III has the HOT1 construct. In the RDN/RDN strain the recombination stimulation was dependent on FOB1 as observed in the haploid strain (Fig. 4A,6&7). The rdn/RDN strain showed the highest recombination rate in this assay. The rate of ADE5,7 deletion was 5 per 100 cell divisions. Interestingly, although the recombination rate was reduced when FOB1 is disrupted, the value is still high when compared with that of the RDN/RDN fob1/fob1 strain (compare 9 with 7 in Fig. 4). In the rdn/rdn strain the growth inhibition was not as serious as that in the haploid rdn strain. However, there were still many abnormal heterogeneous colonies. Therefore, although we could do the half-sectoring assay, the recombination rate is possibly under-estimated. In the fob1 mutant (rdn/rdn) the recombination rate was a little reduced (compare lane 10–11 in Fig. 4B), but still high compared with that of the RDN/RDN fob1/fob1 strain (compare lane 11 with 7 in Fig. 4B). Taken together, FOB1 affects the recombination rate but it is not necessary for recombination, just as observed for HOT1 transcription. As shown in the right side of Fig. 4B, recombination stimulation by HOT1 correlates with the level of HOT1 transcription stimulation. Therefore, we conclude that the recombination rate is affected by the level of transcription.
Analysis of HOT1 recombination products
We also analysed the DNA structure of HOT1 recombination products by Southern analysis. DNA was isolated from strains used in the sectoring assay, restricted with SacI and subjected to Southern analysis. The results are shown in Fig. 6. DNA fragments were identified with probe P1 (Fig. 1B). Each strain basically has three discrete bands (Fig. 6, lane 1). Upper bands (15 kb) correspond with pre-recombination constructs which have ADE5,7 in the leu2 repeats, and the lower two bands (4.7 and 4.1 kb) are post-recombination constructs with and without HOT1, respectively (Fig. 1B). In the RDN fob1 (lane 2) strains, lower bands were not detected because of no recombination activation as expected from the sectoring assay. In the rdn strains (lanes 3, 4) the pre-recombination constructs were still visible although the recombination rate is extremely high. We presume that the well-growing dark red colonies, which were observed in Fig. 4A,4&5, represented a minor portion of the DNA isolation culture. In diploid strains, the leu2 gene in another chromosome III are overlapping with the 4.1 kb bands. Therefore, although HOT1 is not active in the RDN/RDN fob1/fob1 strain (lane 6), only the 4.1 kb band is detected. In the rdn/RDN strain, which showed highest recombination rate in diploid strains, pre-recombination constructs were not visible. It should be noted that the assay is not quantitative for recombination efficiency because the ratios of the bands are affected by the starting ratios of pre- and postrecombination constructs. However, in this assay we could confirm that in the rdn strains HOT1 induces recombination in a similar manner to the wild-type strain.
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Discussion |
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HOT1 consists of two elements, I and E (Fig. 1). The role of the I-element for recombination enhancement is shown to be PolI transcription. In up-stream and down-stream of an actively transcribed gene, it is known that negative and positive torsional stresses are accumulated, respectively (Lui & Wang 1987). Such a stress is resolved by activity of topoisomerases. In fact, loss of the topoisomerases’ function confers hyperrecombination in the rDNA (Christman et al. 1988; Wallis et al. 1989). Therefore, in the rdn strain, the torsional stress may be too high to be resolved by the topoisomerases and a similar situation as observed in the topoisomerase defective mutants may be taking place.
The E-element contains the replication fork barrier (RFB) site which inhibits replication fork progress. Recently, we found that Fob1p directly binds to the RFB site (Kobayashi 2003). The protein specifically bound to two separated regions, RFB1 and RFB3, which correspond to cis-essential regions for HOT1 activity identified previously (Stewart & Roeder 1989; Huang & Keil 1995). Therefore, in the wild-type strain the association of Fob1p with the E-element may be necessary for the function of HOT1. As mentioned in Introduction, Ward et al. (2000) demonstrated the RFB activity itself was not related to the recombination stimulation in the wild-type. Moreover, in Fig. 3, a fob1 mutation reduced the HOT1 transcript level to one seventh of the wild-type level (Fig. 3C). Taken together, these result suggest that the Fob1p/E-element complex is required for effective PolI transcription in HOT1, and the transcription is necessary for the recombination stimulation (Wai et al. 2001). In contrast, in the rdn strain the dependency on FOB1 for transcription was much reduced (Fig. 3). In the primer extension assay, the number of HOT1 transcripts are similar in both FOB1 and fob1 strains in the rdn background. Therefore, in the rdn strains the Fob1p/E-element complex is dispensable for PolI transcription in HOT1. One possible reason for the dispensability is that there are excess PolI transcription machineries in nucleus because of reduced rDNA copy number. These excess machineries would then be able to transcribe ectopic PolI promoters, such as HOT1. This in turn suggests that the Fob1p/E-element complex act as an activator of HOT1 transcription by localizing HOT1 to the nucleolus where the PolI transcription machineries are located, as originally proposed by Wai et al. (2001).
In contrast, FOB1 did affect HOT1 transcriptional efficiency in the rdn/rdn diploid strain, in which excess PolI transcription machineries are also expected (Fig. 4). However, in this strain HOT1 transcription is not as enhanced as in the rdn haploid strain. We speculate that one reason for the reduced transcriptional stimulation in the rdn/rdn diploid strain may be related to the cell mass. The diploid strain has around double the mass compared with the haploid rdn strains, thus suggesting that the diploid strain requires more 35S rRNA molecules. The molecules are supplied from a multicopy helper plasmid (pNOY353) containing the 35S rRNA coding region fused to a PolII promoter (GAL7 promoter). In the diploid strain the amount of 35S rRNA from the helper plasmid may not be enough. Therefore, non-genomic rDNA, such as extra-chromosomal rDNA circles (ERCs) which have been popped out from the rDNA repeats before deletion of the rDNA, may help produce the extra rRNA molecules. The copy number of ERC in the rdn/rdn diploid strain is about four copies per cell (our unpublished observation). Although this number is less than expected, it may be possible that such a small number of ERC molecules forms a core that sequesters the excess PolI transcription machineries in the nucleolus. If so, Fob1p would still be working to help localize HOT1 to the nucleolus as in the wild-type strain. Further study is required to resolve this problem.
A similar phenomenon may be occurring in the RDN/rdn diploid strain. In this strain, we expect a similar level of transcriptional enhancement to the rdn haploid strain because it has double the number of transcription machineries and just the haploid level of rDNA copies. However, as shown in Fig. 3C, only a 2.5-fold enhancement of transcription was observed (lanes 9 and 11 in Fig. 3C). We speculate that the transcription level of the rDNA on one of the two chromosome XIIs will about double because of the large cell mass, and therefore many PolI transcription machineries will be required in nucleolus. In this case, we would also not expect such a stimulation of HOT1 transcription as a result of excess transcription machineries in this strain.
Transcription-mediated recombination is known from yeast cells (for review, see Aguilera 2002). However, the molecular mechanisms are still unclear. Recently, we reported that collision between the transcription and replication forks is a cause of recombination (Takeuchi et al. 2003). In a fob1 defective strain whose rDNA copy number is reduced to 20 (about one eighth of the wild-type copy number), inhibition of replication fork progression in the rDNA was observed by two dimensional gel (2D) analysis. In a strain with normal rDNA copy number such inhibition was not observed, therefore increased transcription in the low-copy rDNA strain seemed to stimulate replication inhibition resulting in increased recombination. In this strain, rDNA amplification was often detected, suggesting that this form of recombination could be coupled with replication, and replication fork inhibition by collision is a trigger of amplification, as shown in the RFB-dependent rDNA amplification model (Kobayashi et al. 1998). We actually tried to find amplification of the ADE marker gene in non-sectoring red colonies from the HOT1 recombination assay reported in this study. However, we could not detect such amplification (data not shown). Moreover, we could not detect any replication fork inhibition in the rdn fob1 strain by 2D analysis, although HOT1 is still active (data not shown). Therefore, we believe that HOT1 recombination is not coupled with replication inhibition. This speculation is consistent with the observation that FOB1-dependent replication blocking (RFB) activity in the E-element does not contribute to HOT1 recombination at all. As the RFB activity has a polarity, if the RFB activity is somehow related to HOT1 recombination, direction of the replication fork should affect HOT1 recombination. However, Ward et al. (2000) results and other genetic experiments (Voelkel-Meiman et al. 1987) show that direction of replication and direction of the E-element do not affect HOT1 recombination at all. In addition, it is known that in the rDNA locus the RFB site itself is not enough to induce recombination. For the induction, the flanking sequence that is not involved in the replication fork blocking activity (the right side of HpaI in the EXP, Fig. 1A) is necessary (Kobayashi et al. 2001; Benguria et al. 2003). Therefore, in HOT1, replication and replication inhibition do not seem to be related to the recombination. Instead, transcription seems to be the key factor in HOT1 recombination.
In a haploid rdn strain with the ADE5,7 HOT1 construct, the growth was poor and heterogeneous (Figs 2A and 4). In this strain, observation by microscopy shows that most of the cells seem to arrest at G2/M phase (data not shown). This suggests that continuous stimulation of recombination induces the checkpoint control to stop the cell cycle. Further analysis is required to understand the mechanism by which the checkpoint control is triggered.
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Experimental procedures |
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SD is a synthetic glucose medium (Kaiser et al. 1994). SG is the same as SD, except that 2% glucose is replaced by 2% galactose. Both SD and SG were supplemented appropriately with amino acids and bases to satisfy nutritional requirements and also to retain unstable plasmids (Kaiser et al. 1994), and are called SD complete (SC) and SG complete, respectively.
Yeast strains and plasmids are listed in Table 1. All strains were established from W303. Strains containing the ADE5,7 marker were constructed by crossing, using strains SER001, SER004 and SER015. SER005 was made from SER001 by removing HOT1 by a conventional gene replacement method with the LEU2 gene. Disruption of FOB1 was previously described (Kobayashi & Horiuchi 1996). For diploid strains, both the FOB1 genes were disrupted simultaneously with the LEU2 marker in one step. For a haploid rdn strain with HOT1 construct, we crossed SER001 with SER015 and made SER002. The FOB1 genes in SER002 were disrupted (SER009) to prevent loss of the ADE5,7 marker during sporulation. The diploid strain SER009 (rdn/RDN, fob1/fob1) was sporulated and SER010 (rdn, fob1) was obtained by tetrad analysis. SER003 (rdn) was obtained by transformation of Yep-FOB1 (Kobayashi et al. 1998) into SER010. For the URA3 marker loss assay, the HOT1 construct containing a part of the HIS4 gene and its upstream sequence (Wai et al. 2001) was transformed into NOY408-1b (RDN) and NOY893 (rdn), and SER013 and SER014 were obtained, respectively.
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For analysis by Northern blot hybridization, 10 ng samples of total RNA were separated on 1% agarose gels and hybridized with 32P-labelled DNA probes at 48 °C. Gel electrophoresis, transfer to the membrane and hybridization were carried out using NorthernMax following the manufacturer's instruction (Ambion). For primer extension analysis and sequencing, a 20-base oligonucleotide primer (designated HOTp in Fig. 3A), complementary to chromosomal sequences located 35–54 bases down from the site of HOT1 insertion was used (83–102 bases from the PolI transcription initiation site). The 5' end of the primer was 32P-labelled using T4 polynucleotide kinase (TAKARA, Japan). For the primer extension reaction, 20 µg of total cellular RNA was annealed with 0.2 pmols of end-labelled HOTp primer in a 15 µL reaction containing 40 mM Tris-HCl (pH 7.5), 20 mM MgCl2, and 50 mM NaCl for 90 min at 55 °C. After annealing, 35 µL of reverse transcription solution containing 200 units of reverse transcriptase (ReverTra Ace TOYOBO, Japan) was added. After 60 min at 42 °C, 100 µL of RNase solution (20 µg/ml RNaseA, 100 µg/ml salmon sperm DNA, 100 mM NaCl) was added, and the mixture was incubated 15min at 42 °C, followed by phenol/chloroform extraction. Ethanol-precipitated extension products were suspended in formamide loading buffer (80% formamide, 10 mM EDTA, 1 mg/ml xylene cyanol FF, 1 mg/ml bromophenol blue), heated to 70 °C for 3 min, and fractionated by electrophoresis on 8 M urea-6% acrylamide gels. To size extension products, a sequence ladder was generated by using the HOTp primer on the PCR product of leu2 involving HOT1. Autoradiograms were quantified with a BAStation (FUJI, Japan).
Measurement of recombination frequency
Recombination frequencies between two tandemly repeated leu2 genes were determined as described by Merker & Klein (2002). The loss of the ADE5,7 marker integrated between duplicated leu2s was used to measure recombination. Half-sectored red and white colonies indicate the ADE5,7 marker was lost in the first cell division following plating. Partially white colonies indicate the marker was lost after the first cell division following plating. The recombination rate was determined by considering only the first cell division after plating, and was calculated by dividing the total number of half-sectored colonies by the total number of colonies (half-sectors plus partial sectors).
The HOT1 recombination system using the URA3 marker was assayed as previously described (Wai et al. 2001). Strains SER013 (RDN, HOT1) and SER014 (rdn, HOT1) were grown in SG complete medium lacking uracil overnight. The frequencies of Ura- recombinants were then determined by spotting aliquots of 10-fold serial dilutions of the culture on SG plates with and without 5-fluoroorotic acid (5-FOA).
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Acknowledgements |
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Footnotes |
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*Correspondence: E-mail: koba{at}nibb.ac.jp
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References |
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Received: 8 December 2003
Accepted: 29 January 2004
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) and the origin of replication (ARS; 
) are located in NTS1 and NTS2, respectively, shown in the lower part. The RFB allows progression of the replication fork in the direction of 35S rRNA transcription, but not in the opposite direction. E and I (boxed) are elements of HOT1. EXP is a sequence essential for rDNA amplification (
