Article

• The EMBO Journal (1998) 17, 4503 - 4510
• doi:10.1093/emboj/17.15.4503

A site- and strand-specific DNA break confers asymmetric switching potential in fission yeast

Benoit Arcangioli¹

1. Unité des Virus Oncogènes, URA 1644 du CNRS, Departement des Biotechnologies, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France E-mail: barcan@pasteur.fr

Received 12 February 1998; Accepted 8 June 1998; Revised 8 June 1998

• Keywords:

  • imprinting,
  • nick,
  • recombination,
  • replication,
  • Schizosaccharomyces pombe

Introduction

The budding and fission yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, mitotically switch their mating-types, producing a cell population containing an equal proportion of both mating-types (for reviews, see Herskowitz et al., 1992; Klar, 1992). The switching pattern in both yeast is highly regulated and asymmetrically distributed in a cell lineage (Figure 1). Mating-type switching results in the transfer of genetic information from a silent cassette to a transcriptionally active locus. The mechanism of programmed genetic rearrangement is initiated by DNA cleavage of the transcriptionally active locus. The major differences in both yeast lies in the asymmetric distribution of the mechanism controlling DNA cleavage and in the timing of the gene conversion event relative to DNA replication. In S.cerevisiae the endonuclease HO is asymmetrically expressed in only one of the two sister cells (the mother) at the end of the G₁ phase (Nasmyth, 1983), triggering mating-type switching before S phase. Following mitosis, both sister cells inherit a switched chromatid and then a switched phenotype.

Figure 1.

Mating-type switching pattern of fission yeast. Pattern of switching showing the asymmetric cell divisions. P, M are the mating-types and the suffix 'u' and 's' represent 'unswitchable' and 'switchable', respectively. Schizosaccharomyces pombe cells divide by fission generating nearly equal daughter cells, thus the suffix u or s can be attributed to the dividing cell only a posteriori, when the daughter cell has expressed its mating-type.

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By standard Southern blot analysis and by analogy with the S.cerevisiae mating-type switching system, it was proposed that gene conversion at the mat1 locus (Egel and Gutz, 1981; Egel, 1984) of S.pombe is also initiated by a double-strand break (DSB) (Beach, 1983). However, pedigree analysis has shown that two asymmetric divisions (Figure 1) are required to produce one switched cell among four granddaughters (Miyata and Miyata, 1981). Furthermore, the sister of a newly switched cell is competent for switching during its next division (Egel and Eie, 1987; Klar, 1990). Genetic and molecular approaches have shown that the asymmetric cell division in fission yeast does not require differential localization of a diffusible factor, but it is a consequence of a semi-inheritable chromosomal modification (Egel, 1984; Klar, 1987). This chromosomal imprinting event restricts DNA cleavage, at the mat1 locus, to only one of the two sister chromatids, one generation before switching. This triggers mating-type interconversion to only one of the two sister chromatids, at the time of DNA replication or just after (Klar, 1987). Following mitosis, only one of the two sister cells inherits a switched chromatid and consequently a switched phenotype. In summary, in budding yeast the pattern of switching is dictated by the pattern of the endonuclease expression and in fission yeast by the pattern of the endonuclease-target sequence accessibility.

The properties of DNA cleavage observed at the mat1 locus, in fission yeast, is intriguing in several respects: (i) as observed by Southern hybridization, 20% of chromosome II contained a DSB at the mat1 locus, which is found at a constant rate throughout the entire length of the cell cycle (Egel, 1984). (ii) An imprinting event restricts the cutting machinery to only one of the two sister chromatids (Klar, 1987). (iii) The position of the break was mapped, at the nucleotide level, on only one strand. As the ends of the other strand could not be determined it was proposed that they are protected by a covalently-bound protein (Nielsen and Egel, 1989). (iv) The 160 bp distal to mat1 contains two cis-acting elements, SAS1 and SAS2 (switch-activating sequence) which are essential for either imprinting or the catalysis of break formation and consequently for mating-type switching (Egel and Gutz, 1981; Arcangioli and Klar, 1991; Klar et al., 1991; Stykarsdottir et al., 1993). (v) Genetic evidence suggested that the broken chromosome is sealed, replicated and cleaved again (Klar and Miglio, 1986; Arcangioli and Klar, 1991; Klar et al., 1991). This suggestion was recently reinforced by the characterization of the swi7 gene, required for a wild-type steady-state level of the DSB and encoding for the DNA polymerase (Singh and Klar, 1993). Taken together, the properties of the DSB at mat1 are unusual and without precedent.

These intriguing properties have encouraged us to re-examine the DNA break at the mating-type locus. Our work shows that the mating-type locus constitutes an unprecedented fragile chromosomal site, due to a site and strand-specific nick. The nick is found at a constant rate, throughout the length of the cell cycle. These results implicate a single-strand break (SSB) in the initiation of mitotic and meiotic gene conversion. The implications for the asymmetric switching between sister cells and the strand-segregation model are discussed.

The mating-type locus contains a fragile site

To further investigate the DNA break at the mat1 locus, S.pombe genomic DNA was prepared in solid agarose plugs, as for pulse field gel electrophoresis (Schwartz and Cantor, 1984) rather than by the standard established yeast extraction protocol (Moreno et al., 1991). This method protects the chromosomes from mechanical shearing and nuclease degradation during the purification process. Following the genomic DNA preparation, the DNA was digested with HindIII restriction enzyme, separated by agarose gel electrophoresis and analyzed by Southern hybridization using the 10.4 kb mat1-P–HindIII fragment as a probe. The h⁹⁰ wild-type strain yields the typical bands of 10.4 (mat1), 6.3 (mat2-P) and 4.2 kb (mat3-M) plus the 5.4 and 5.0 kb fragments originating from the cleaved mat1 locus (Figure 2). Surprisingly, the 5.4 and the 5.0 kb fragments were barely visible under these conditions (Figure 3A, lane 1). Approximately 1% of the mat1 locus was cleaved, in contrast with the 20% usually observed with the standard yeast genomic DNA preparation (Beach, 1983; Beach and Klar, 1984).

Figure 2.

Schematic representation of the mating-type region on chromosome II. The size of the HindIII fragments containing the mating-type loci observed after standard genomic DNA preparation are indicated. The proportion of broken HindIII–mat1 fragments is shown. The black boxes labelled H1 and H2 are sequences common to each cassette, whereas H3 sequences are common only to the silent loci. An arrow indicates the position of the break site, mapped at the nucleotide level only on the upper strand for mat1P and mat1M.

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Figure 3.

Southern blot analysis of HindIII-digested wild-type DNA prepared in a solid agar plug and probed with mat1P DNA fragment. (A) Mechanical sensitivity. Lane 1: S.pombe genomic DNA maintained in a solid agar plug. Lanes 2–5: after DNA preparation the agar plug was melted, gently pipetted and vortexed for 1, 5 or 25 s, solidified again before HindIII-digestion, electrophoresis and Southern hybridization analysis. (B) Enzymatic sensitivity. Schizosaccharomyces pombe genomic DNA was prepared in solid agar plugs. Lane 1: control HindIII-digested genomic DNA. Lanes 2–8: following HindIII-digestion the plugs were treated with proteinase K (lane 2), pronase (lane 3), V8 protease (lane 4), ribonuclease H (lane 5), ribonuclease A (lane 6), ribonuclease T1 (lane 7) or mung bean nuclease (lane 8), respectively. Names of the mating-type cassettes and their mol. wts are indicated.

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We investigated the nature of the discrepancy between the two genomic DNA preparation methods. Figure 3A (lanes 2–5) shows that mechanical shearing upon vortexing melted agarose plugs containing S.pombe chromosomes DNA prior to HindIII-digestion strongly increased the yield of the 5.4 and 5.0 kb DNA fragments. Little effect was observed when the DNA was first digested by HindIII and then vortexed (data not shown). This experiment showed that the standard genomic DNA preparation introduced shear forces (phenol extraction, DNA precipitation, etc.) that specifically break a fragile chromosomal site at mat1, which can be avoided by handling the chromosomes in solid agarose plugs.

Figure 3B shows the effects of incubation with several proteases and nucleases on the HindIII digestion pattern. A striking effect was observed only with the single-strand specific mung bean nuclease (Figure 3B, lane 8), producing the two mat1 DNA fragments mat1-proximal and mat1-distal of 5.4 and 5 kb, respectively. Quantitation of the labeled DNA fragments indicated that 27% of the 10.4 kbp mat1 fragment had been cleaved generating the mat1-distal and the mat1-proximal signals. The DSB level obtained after mung bean treatment is similar to the level obtained after vortexing and to the level observed using the standard genomic DNA preparation method (Beach, 1983; Beach and Klar, 1984).

We also analyzed the sensitivity of mat1 DNA from the mat1M smt-0 mutant strain (Engelke et al., 1987) containing a deletion of the cis-acting elements SAS1 and SAS2, which abolishes mating-type switching, while preserving the integrity of the cleavage site sequence (Arcangioli and Klar, 1991). In contrast to the wild-type strain, the mat1 mutant DNA is refractory to vortexing and mung bean nuclease treatment (Figure 4), indicating that the cis-acting regulatory elements are required for the mechanism responsible for the mat1 DNA fragility.

Figure 4.

Cis-acting elements are essential for mat1 fragility. Southern transfer of wild-type (lanes 1–3) and mat1M smt0 mutant (lanes 4–6) strain DNA digested with HindIII and probed with mat1P DNA fragment. Lanes 1 and 4, the DNA was maintained in solid agarose. Lanes 2 and 5, the DNA was digested with mung bean nuclease. Lanes 3 and 6, the agarose block containing DNA was melted and vortexed prior to restriction enzyme digestion.

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mat1 contains a single-strand break

The mat1 mung bean nuclease sensitivity was weaker at 37°C (data not shown) and increased with temperature. Previous observations of Nielsen and Egel (1989) mapped the 5' and 3' ends of mat1 upper DNA-strand, with no missing bases. Based on these findings, we favor the hypothesis that the mat1 locus contains a nick (SSB) and not a gap, leaving an intact lower strand. Consequently, we analyzed SspI-digested S.pombe genomic DNA after electrophoresis through polyacrylamide gels under native and/or denaturing conditions (Figure 5). To improve the clarity of the analysis, DNA from the wild-type h⁹⁰ and the mat1M, mat2,3 mutant strains (in which the two donor loci have been removed and contained a stable M allele at mat1) were compared (Figure 5A and B). Under denaturing conditions (Figure 5B) two additional DNA fragments of 199 and 196 bp and one additional DNA fragment of 196 bp (Figure 5B, lanes 7 and 8) hybridized with the lower strand mat1 DNA probe with the h⁹⁰ strain and with the mat1M, mat2/3 strains, respectively. These fragments were not observed with the upper strand mat1 DNA probe (lanes 5 and 6), or under native conditions, irrespective of the sense of the probes used (Figure 5A, lanes 1–4). Interestingly, the mat1M DNA fragment of 440 bp ran as a doublet under native (lanes 1–4), but not under denaturing (lanes 5–8) conditions. For the two consecutive polyacrylamide gels (2D gel) the first migration was performed under native conditions, then the piece of polyacrylamide containing the DNA fragments was cut out and incubated into denaturing buffer at 95°C and separated through the second gel under denaturing conditions (Figure 5C). A sample of SspI-digested DNA was loaded into the first lane as an internal standard (Figure 5C). Following electrophoresis, the gel was electrotransferred and hybridized with a single-strand labeled probe homologous to the upper mat1 strand (Figure 5D). The 2D gel electrophoresis analysis (Figure 5C) shows that the two spots of 199 and 196 bp (mat1M. Pdist), result from the 1450 bp mat1P and the 440 bp mat1M upon SspI-digestion. A detailed analysis of the 2D gel reveals that the mat1M-dist DNA fragment originates from the faster migrating band observed in the native gel shown in Figure 5A (doublet mat1M, see above). It is probable that the nick on the upper mat1-DNA strand perturbs its migrating properties. Also, we cannot exclude the possibility that some DNA degradation occurred during the DNA manipulation steps. Another interpretation relies on a hypothetical DNA modification which has been proposed to be a precondition for mat1 cleavage (Klar, 1987). The mat1M- and mat1P-proximal upper strands are barely visible, probably due to the single-strand probe used.

Figure 5.

Native and/or denaturing gel electrophoresis analysis of the mating-type region. (A) Autoradiogram of SspI-digested DNA from h⁹⁰ and mat1M mat2/3 strains resolved in a native polyacrylamide gel, electrotransferred and probed with either a labeled lower or upper H1 strand from the mat1P or mat1M loci. (B) Same as (A), but the DNA was resolved in a denaturing polyacrylamide gel. (C) Diagram of the migration pattern of the SspI fragment hybridized with a mat1 lower strand probe analyzed by 2D gel electrophoresis, where the first dimension was performed under native conditions and the second dimension under denaturing conditions (upper panel). Autoradiogram of the second gel, under denaturing conditions, after electrophoretic transfer and hybridization with a labeled lower H1 strand from the mat1M locus (lower panel). (D) Schematic representation of the mat1 locus. The polarity of the probes, names of the mating-type cassettes and their mol. wts are indicated.

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The integrity of the lower strand and the nicked upper strand of mat1-DNA fragment was confirmed by PCR-mediated linear amplification using a single labeled oligonucleotide as a primer. Figure 6A shows that the Taq DNA polymerase primed with the S2 oligonucleotide (lanes 5 and 6) terminated at the DNA break present on the upper strand. This resulted in DNA fragments of 158 and 161 nt in length, from mat1M or mat1P DNA templates, respectively (Kelly et al., 1988; Nielsen and Egel, 1989). On the unbroken sister chromatid the PCR products terminated at the NsiI or SspI restriction sites. Conversely, when the Taq DNA polymerase was primed with the labeled M1 or P1 primers, copying the lower strand, no chain termination was observed and the DNA polymerization terminated only at the NsiI or SspI restriction sites, as indicated. The additional bands may originate from low sequence specificity somewhere else in the genome or star activity on non-canonical SspI sequences (AATATa) or less abundant nick sites, especially into the H1 sequence. This result was confirmed by genomic Southern blot analysis at single nucleotide resolution (data not shown) and correlates with position found by Nielsen and Egel (1989). This experiment excludes the possibility that a protein complex, resistant to proteinase K treatment, protects and joins the ends of the two lower strands of DNA. From these data we concluded that the fragility of chromosome II at mat1 is due to a site-specific SSB on the upper strand.

Figure 6.

Primer extension analysis of genomic DNA. (A) Primer extension products from genomic DNA prepared from wild-type strain, digested with NsiI (odd lanes) or SspI (even lanes). Three labeled primers were used; M1 hybridized to the lower strand of mat1M and mat3M; P1 to the lower strand of mat1P and mat2P; and S2 to the upper strand of mat1M and mat1P distal sequences. The arrows indicate the position of the PCR extended products reaching the position of the restriction site or the nick. The precise size of the products obtained from the silent loci are not known, because of lack of sequence information. (B) The DNA fragments and PCR extended products sizes are indicated and schematically represented.

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The nick at mat1 is stable relative to the cell cycle

The asymmetric mating-type switching observed in single cell pedigree analysis (Miyata and Miyata, 1981) implies that the gene conversion replacing the allele at mat1 does not occur at random during the cell cycle, but during or after mat1 DNA replication (Beach, 1983), allowing only one of the two sister chromatids to switch efficiently to the opposite mating-type. Therefore, the nature of mat1 DNA along the cell cycle was investigated. The mutant strain carrying the temperature-sensitive mutation cdc25 [arrested at the G₂/M transition at the restrictive temperature (Russell and Nurse, 1986)] was analyzed. The cdc25 mutant strain was first grown, in minimum medium, at the permissive temperature (24°C), then shifted to the restrictive temperature (36°C) for 4 h and reincubated at 24°C allowing a synchronous entry into the cell cycle. Samples of cell cultures were harvested at intervals, genomic DNA was prepared in agarose plugs and further analyzed (Figure 7). Cell-cycle position and degree of synchrony were monitored by flow cytometry (FACS) analysis and by determining cell number and septation index (calcofluor staining) as a function of time (Figure 7A and B). The PCR-mediated linear amplification, shown in Figure 7C indicates that the nick is present on the upper DNA strand at a constant level and that the lower strand is intact throughout the cell cycle. Figure 7D shows Southern hybridization of HindIII-digested DNA probed with either mat1-proximal (HindIII–NsiI) or -distal (NsiI–HindIII) labeled DNA. These two probes do not overlap with the mat1 locus sequences. Several transient high mol. wt DNA species appeared periodically with both the mat1-proximal and -distal probes, 80 and 280 min after G₂/M block release, concomitant with the beginning of S phase, the peak of septation and before the cell number doubled (Figure 7). The ARS1 and PCNA (Waseem et al., 1992) probes were also used to follow DNA replication timing (Figure 7D). These data indicate that the modifications of mat1-DNA appear with the S phase and may represent replication–recombination intermediates. This issue was not investigated further. Interestingly, the mat1-distal broken DNA fragment (5.0 kb) increased at the beginning of the two S phases (80 and 280 min after the G₂/M block released). However, the proximal-mat1 DNA fragment (5.4 kb) gave a weaker signal for the same periods (Figure 7D and E) and its level increased later, concomitantly with the mat1 10.4 kb DNA fragment upon HindIII-digestion. Quantification of the mat1-distal and mat1-proximal DNA fragments (Figure 7E) confirmed that mat1-distal DNA increased prior to the proximal region. Taken together, these data suggest that the fork of replication initiates from the distal region of mat1, at least for the nicked chromatid. Similar data were observed using synchronized cdc10 mutant strains (arrested at the G₁/S transition; Aves et al., 1985).

Figure 7.

Analysis of the mating-type region throughout the cell cycle. The five panels show the data obtained from the same experiment. (A) Degree of synchrony of the cdc25 strain after shifting cells from restrictive (4 h at 36°C) to permissive (24°C) temperature in minimal medium. Under these conditions, the peak of septation is concomitant with S phase. (B) DNA content of cdc25 synchronized mutant strain measured by flow cytometry. The phases of the cell cycle are indicated, the drifting of 2C peak at 0 and 180 min is the result of the division of the elongated cells. (C) Primer extension products using template DNA from the synchronized cdc25 mutant strain digested with SspI. The name of the primers are indicated and are the same as in Figure 6. (D) Genomic DNA prepared in a solid agar plug from the synchronized cdc25 mutant strain, HindIII-digested, resolved in agarose gel, blotted and hybridized with either the mat1-proximal, -distal, ARS1 or pcn1 probes. Names of the mating-type cassettes and their mol. wts are indicated. (E) Relative intensity of the mat1-distal and mat1-proximal DNA fragments during the cell cycle.

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The implications of these findings have a profound impact on the molecular mechanism responsible for the chromosomal imprinting hypothesized by Klar (1987). The features of the nick described in this work fulfil all the requirements needed for the 'epigenetic' site and strand-specific chromosomal modification at the mat1 locus.

The demonstration of nicked DNA at the mat1 locus provides an alternative explanation for the discrepancy observed between the yeasts S.cerevisiae and S.pombe when the two silent donor loci are removed. Whereas S.cerevisiae cells die, presumably because they fail to repair the DSB generated by HO endonuclease (Klar et al., 1984), S.pombe cells remain viable (Klar and Miglio, 1986). Viability can be explain by a homologous recombination-dependent DNA replication (Skalka, 1974; Zou and Rothstein, 1997). In addition, when such donor-deleted strains were crossed and sporulation induced, they produced at high frequency 3:1 and 1:3 gene conversions of mat1 in meiotic tetrads (Klar and Miglio, 1986). These factors indicate that the recombination-dependent DNA replication model may also use the homologous chromosome as a template for gene conversion.

In wild-type cells it is possible that the nick at mat1 constitutes the initial recombination event allowing unidirectional gene conversion. Since the nick is located 3' of the convertible mat1 DNA, gene conversion cannot be initiated by conventional 3'-end invasion into one of the homologous donors (mat2P or mat3M). A simple hypothesis is that the nick becomes recombigenic by being transiently transformed to a DSB (Strathern et al., 1982; Szostak et al., 1983). However, the data shown in Figure 7C do not support this model and seem more consistent with a switching process related to a replication-stimulated recombination pathway (Figure 8). When the fork of replication, running in early S phase (80 and 280 min), through the distal-part of mat1, encounters the nick, the replication is interrupted. Consequently, a 5.0 kb distal-mat1 DNA without its complementary 5.4 kb DNA fragment is observed upon HindIII-digestion (Figure 8B). Interestingly, this DNA structure is related to a kind of DSB. The stalled polymerase could be rescued by a recombination–replication-coupled machinery channelling the neosynthetized strand into the H1 homologous sequences of the opposite silent mating-type locus (Figure 8C). The donor-choice mechanism promoting intrachromosomal folding of mat2 and mat3 onto mat1 in a cell type-specific fashion (Thon and Klar, 1993) might prevent strand displacement into the H1 sequence of the sister chromatid (see above) by forcing invasion into one of the silent loci of the opposite mating-type. Following branch invasion into the opposite silent cassette, DNA synthesis can take place. Interestingly, a slow migrating species is observed with the mat1-distal and not with the mat1-proximal probes (Figure 7D) and may reveal the transient gene conversion structure represented in Figure 8C. When reaching the H2 homologous box, the replicating strand returned to the mat1 locus, allowing the progression of the replication fork (Figure 8D). The opposite intact strand can be replicated and nicked, producing an unswitched chromatid which is marked for switching at the following S phase, starting the cycle all over again (Figure 8E). The increase in the level of the mat1-distal DNA fragment (5.0 kb) independently of its complementary mat1-proximal DNA fragment (5.4 kb) is more spectacular when a mutant strain carrying a deletion of the mat2P cassette (h^-s; Beach and Klar, 1984) is used, consistent with a fast and efficient strand invasion into the opposite silent locus, allowing mating-type switching (unpublished data).

Figure 8.

Switching–replication coupled model. H1, H2 and H3 homology boxes flank the mating-type cassettes. The arrows indicate the direction of the replication. Thick and thin lines represent newly synthesized DNA and old DNA, respectively. The mat2P or mat3M silent loci are represented by wavy lines and mat1 by dark lines. (A) Before replication a portion of the mat1 locus contains a nicked upper strand. (B) Early in S phase, the fork of replication approaching from the distal region of mat1 is interrupted (80 min after the G₂/M block release). (C) Strand invasion of the opposite mating-type locus allows DNA synthesis of the opposite mating-type. (D) Gene conversion–replication coupled process allows the mat1 replication to proceed. (E) A switched-intact and an unswitched-nicked chromatin are produced upon replication allowing the switching cycle to begin again, next division. The probes used in Figure 7D and the size of the HindIII-digested mat1 DNA fragments are indicated.

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Additional work is needed to provide the molecular details of mating-type gene conversion, implicating the known swi genes. However, it is expected that mutations in the swi1, 3 and 7 genes previously required for the wild-type level of the DSB (Egel et al., 1984) are in fact required for the SSB.

The data presented here do not explain how the newly switched chromatid is initially cleaved, preparing the chromatid for mating-type switching one cell cycle ahead. The strand segregation model (Klar, 1987) proposed that a strand-specific modification acted as a precondition for producing the cleavage at mat1, probably during replication one generation before switching. In this context it is interesting to note the migration properties of the mat1M-DNA fragment (running as a doublet) upon SspI-digestion, under native conditions (Figure 5A). The roles of the cis- and trans-acting elements causing and/or maintaining the nick on the upper strand of mat1 are still not understood. Increasing evidence indicates that DNA replication plays an active role (Singh and Klar, 1993) in the steady-state level of the SSB at mat1, and future work is needed to reproduce specific SSB formation at mat1 in vitro.

The term 'fragility' used in this work is not related to the fragile sites that appear as non-staining gaps in chromosomes occasionally associated with in vivo chromosome breakage and rearrangements (Sutherland and Richards, 1995). However, it is conceivable that the nick at mat1 might be accidentally transformed to a DSB along the progression of the cell cycle, especially around mitosis when compacting or pulling strength are applied to the chromosomes. Recently, the sequence between the silent mat2 and mat3 loci revealed that 4.3 kb share homology with centromeric repeat elements (Baum et al. 1994; Grewal and Klar, 1997). It is possible that these sequences have been selected as a functional accessory centromere, in addition to their proposed role in silencing (Grewal and Klar, 1996; Thon and Friis, 1997), to prevent mitotic loss of the distal part of chromosome II, if the nick at mat1 has been accidentally broken.

Strains and genomic DNA preparation

The S.pombe strains used are: SP62 h⁹⁰leu1-32 ura4-D18; SP714 mat1M mat2,3::LEU2 leu1-32.; SP807 mat1-Msmt0, leu1-32 ade6-210 (Klar and Miglio, 1986; Engelke et al., 1987). Usually, 10⁷S.pombe spheroplasts in 0.1 ml of 1 M sorbitol buffer were mixed with an equal volume of 1% low melting agarose kept in suffusion at 40°C and allowed to harden into sample molds. Solid samples were extensively digested for 48 h in 1 ml of 1% lauroyl sarcosine and 0.5 mg/ml proteinase K at 50°C, changed 4. The plugs were rinsed overnight in a large volume of 50 mM EDTA pH 8, 100 mM NaCl, 1 mM PMSF and 3 30 min in 1 ml of 10 mM Tris pH 8, 0.5 mM EDTA, 100 mM NaCl. For enzymatic digestion, the plug was incubated in 1 ml of enzyme buffer for 30 min and in 100 l of fresh buffer together with the enzyme for 30 min at 4°C, then incubated at 37°C for 3 h. For the mung bean digestion, the optimum condition was obtained with an incubation at 50°C, allowing a better accessibility of the nicked DNA. The reaction was stopped by 20 mM EDTA, and rinsed as above before secondary treatment or loaded into the well of 0.8% agarose gel. After electrophoresis, the DNA was transferred to Hybond-N+ filters (Amersham) and cross-linked with UV Stratalinker (Stratagen). Following DNA–DNA hybridization the membrane was exposed on a phosphor screen and the signal detected by PhosphorImager 445SI (Molecular Dynamics) and quantified using ImagequantNT. When digested DNA was resolved into polyacrylamide gel, the reaction was frozen at -80°C and filtered by centrifugation through 0.22 m cellulose acetate (Costar) and precipitated.

Probes synthesis and PCR extension

The probes were labeled with Random primed DNA Labelling Kit (Boehringer) and the unincorporated nucleotides were removed by gel filtration. The sequence of the oligonucleotide used were for M1: 5'-TAGTTCTAAGCACTGTAATGCCATA-3', for P1: 5'-CCAATTCCTTCTTGTATATGTTATAC-3' and for S2: 5'-CGAAGCAAATATCTCGTTAGAGG-3'. Oligonucleotides were 5'-end labeled with [³²P]ATP and gel purified. Digested genomic DNA were mixed with one oligonucleotide and PCR amplified (Perkin Elmer). The linear amplification steps were, 95°C, 1 min; 50°C, 5 min and 72°C, 2 min for 10 cycles. The reaction was phenol extracted, ethanol precipitated and electrophoresed through a 6% polyacrylamide denaturing gel. The gel was then dried and exposed. Single-strand probes were prepared by PCR linear amplification using mat1M or mat1P DNA as template, one primer (M1, P1 or S2) and two (³²P) labeled and two cold deoxynucleotides using the same steps as above. Then the probes were purified from the template and the unincorporated nucleotides through acrylamide gel and then eluted.

Synchronized culture

cdc25 wild-type for the mating-type locus was constructed, grown at 24°C in minimal medium, switched for 4 h at restrictive temperature (36°C) and incubated at 24°C. Samples were taken every 20 min and monitored for septation index, cell number, DNA content and genomic DNA were prepared for further analysis.

Flow cytometry

Cells (10⁷) were fixed in 3.6% paraformaldehyde for 30 min at room temperature, harvested by centrifugation, washed twice in 50 mM sodium citrate (pH 7.0), incubated for 1 min in the presence of 1% Triton-X at room temperature and washed again twice in 50 mM sodium citrate. The cells were resuspended in 50 mM sodium citrate and 50 g/ml DNase-free RNase A (Boehringer Mannheim) for 1 h at 37°C. Then incubated 15 min with 5 g/ml propidium iodide (Sigma) in the dark. The cell suspension was analyzed with an Epics XL Flow cytometer (Coulter) and the fluorescence of individual cells were plotted against cell numbers using the WinMDI Software (2.6).

I am grateful to the following people for their various contribution to this work: B.Dujon, C.Fairhead, A.Perrin, M.Pontoglio, J.Weitzman and M.Yaniv. This work was supported by the Association pour la Recherche sur le Cancer.

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