The meiotic recombination hot spot created by the single-base...

The meiotic recombination hot spot created by the single-base substitution ade6-M26 results in remodeling of chromatin structure in fission yeast Ken-ichi Mizuno/' ' Yukihiro Emura/'^* Michel Baur,^ Jiirg Kohli,^ Kunihiro Ohta/'^ and Takehiko Shibata^

^Cellular and Molecular Biology Laboratory, The Institute of Physical and Chemical Research, Wako, Saitama 351-01, Japan; ^Applied Biology, Nihon University, Fujisawa, Japan; ^Institute of General Microbiology, University of Bern, Bern, Switzerland

The G ^ T transversion mutation, ade6-M26, creates the heptanucleotide sequence ATGACTG, which lies close to the 5' end of the open reading frame of the ade6 gene in Schizosaccharomyces pombe. The mutation generates a meiosis-specific recombination hot spot and a binding site for the Mtsl/Mts2 protein. We examined the chromatin structure at the ade6 locus in the M26 strain and compared it to that of the wild-type and hot spot-negative control M375. Micrococcal nuclease (MNase) digestion and indirect end-labeling methods were applied. In the M26 strain, we detected a new MNase-hypersensitive site at the position of the M26 mutation and no longer observed the phasing of nucleosomes seen in the wild-type and the M375 strains. Quantitative comparison of MNase sensitivity of the chromatin in premeiotic and meiotic cultures revealed a small meiotic induction of MNase hypersensitivity in the ade6 promoter region of the wild-type and M375 strains. The meiotic induction of MNase hypersensitivity was enhanced significantly in the ade6 promoter region of the M26 strain and also occurred at the M26 mutation site. The formation of the MNase-sensitive region around the heptamer sequence was abolished by the introduction of single-nucleotide substitutions in the heptamer sequence, which also abolish hot spot activity and binding of Mtsl/Mts2. These data suggest that Mtsl/Mts2 binding to the heptamer sequence results in a chromatin structure suitable for the recruitment of a meiosis-specific recombination function or functions.

[Key Words: recombination; meiosis; yeast; hot spot; chromatin] Received December 20, 1996; revised version accepted February 18, 1997.

It has been demonstrated that meiotic recombination occurs preferentially at defined sites called hot spots on meiotic chromosomes in various eukaryotic organisms (Shiroishi et al. 1993; Smith 1994; Lichten and Goldman 1995). In the budding yeast Sacchaiomyces ceievisiae, most of the meiotic recombination is initiated by the formation of meiosis-specific DNA-double strand breaks (DSBs) at hot spots (Nicolas et al. 1989; Sun et al. 1989; Cao et al. 1990; Zenvirth et al. 1992; de Massy and Nicolas 1993; Goyon and Lichten 1993; Nag and Petes 1993; Wu and Lichten 1994). Meiotic DSBs on chromosomes are preferentially introduced at nucleosome-free regions that show hypersensitivity to nucleases (Ohta et al. 1994; Wu and Lichten 1994; Fan and Petes 1996). Therefore, accessibility of DNA is a primary requirement for hot spot activity. In addition, hypersensitivity to micro-

*K. Mizuno and Y. Emura contributed equally to this work. ^Corresponding author. E-MAIL [email protected]; FAX 81-48-462-4671.

coccal nuclease (MNase) becomes more specific and quantitatively more important at hot spots during early meiotic prophase (Ohta et al. 1994). This implies the action of meiosis-specific functions for chromatin changes at hot spots. However, the existence of meiotic DSBs and the role of chromatin structure at hot spots have not been established in organisms other than S. ceievisiae.

In the fission yeast Schizosaccharomyces pombe, the meiotic recombination hot spot ade6-M26 (M26) (Gutz 1971) is created by a G-> T transversion in the open reading frame close to the 5' region of the ade6 gene (Ponticelh et al. 1988; Szankasi et al. 1988). The mutation creates a nonsense codon and causes up to a 15-fold, meiosis-specific elevation of recombination frequency compared to the ade6~M375 mutation (Gutz 1971; Ponticelh et al. 1988; Schuchert and Kohh 1988). The mutation ade6-M375 (M375) is a G ^ T base substitution that creates a nonsense mutation in the codon preceding M26 (Ponticelh et al. 1988; Szankasi et al. 1988), but it does not show hot spot activity (Gutz 1971), thus pro-

876 GENES & DEVELOPMENT 11:876-886 © 1997 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/97 $5.00

Cold Spring Harbor Laboratory Press on October 1, 2020 - Published by genesdev.cshlp.orgDownloaded from

mailto:[email protected]

http://genesdev.cshlp.org/

http://www.cshlpress.com

chromatin remodeling in ade6-M26 hot spot

viding an excellent negative control for M26. M26 has features similar to hot spots in S. cerevisiae. (1) The M26-carrying chromatid is a preferential recipient of genetic information: It is preferentially converted to wild-type in one-factor crosses (Gutz 1971). This is a typical and predicted feature of the DSB repair model (Szostak et al. 1983). (2) The conversion rates of markers upstream of M26 increase steadily as their distance from M26 decreases. This is concluded from the combination of tetrad data (Gutz 1971) with the physical analysis of conversion tracts (Grimm et al. 1994), suggesting the existence of an initiation site in the 5' region of the ade6-M26 gene. (3) The activity of M26 is dependent on the chromosomal context. Transplacement of the whole ade6-M26 gene to other sites in the genome often abolishes the hot spot activity (Ponticelli and Smith 1992; Virgin et al. 1995), whereas the transplacement of the ade6 gene with M26 sometimes leaves the hot spot active. In addition, M26 enhances recombination between plasmid versus chromosome, only when it is located on the chromosome (Ponticelli and Smith 1992; Virgin et al. 1995). These results imply a role for chromatin structure or higher-order chromosome structure in the control of the M26 hotspot.

The M26 mutation generates the heptanucleotide sequence (5'-ATGACGT-3', M26 mutation underlined). All base substitutions at any of the seven positions in the heptamer abolish hot spot activity (Schuchert et al. 1991). The heterodimeric protein Mtsl/Mts2 was identified as a factor binding specifically to DNA carrying the heptamer sequence (Wahls and Smith 1994). This binding was abolished when single-base substitutions were introduced into the heptamer sequences, showing that there is perfect correlation between the sequence requirements of the heptamer sequence for hot spot activity in vivo and Mtsl /Mts2-binding activity (Schuchert et al. 1991). Thus, the Mtsl/Mts2 protein could have a crucial role in the establishment of the M26 hot spot. However, the reason for the meiosis specificity of M26 still remains unexplained, as the activity of the Mtsl/Mts2 protein is present both in mitotic and meiotic cell extracts (Wahls and Smith 1994). One possibility is that meiotic alteration of chromatin structure may contribute to the establishment of the M26 hot spot.

In this study we have examined the chromatin structure at the ade6 gene before and during meiosis of diploid strains homozygous for M26, M375, or wild-type sequences. We found that the ade6 chromatin is remodeled strikingly in M26 diploids but not in wild-type or M375 diploids.

Results

M26-dependent remodeling of chromatin structure

We examined the meiotic chromatin structure at the ade6 region by an indirect end-labeling method. Chromatin was isolated from diploid strains homozygous for either wild-type, M375, or M26 at various time points (t = 0, 2,3, 4, 6 hr) after transfer of cells into a sporulation

medium. The isolated chromatin was digested with various concentrations of MNase. DNA was purified and examined by agarose gel electrophoresis, followed by ethidium bromide staining. A ladder of several broad bands indicated integrity of nucleosomal repeats in the chromatin fraction (data not shown). The MNase-di-gested DNA was then cleaved by appropriate restriction endonucleases, and the DNA fragments were analyzed by Southern hybridization using short probes for sequences adjacent to one of the restriction sites.

Figure 1 illustrates the MNase-sensitive sites in chromatin of the ade6 locus in wild-type, M375, and M26 mutations at t = 3 hr after transfer of the cells into a sporulation medium. The MNase-digestion patterns of wild-type and M375 were similar. We observed two hypersensitive sites (-210 and -80) in a presumed promoter region that lies in the 5' intergenic region of the adeS locus and a ladder of sensitive sites in the open reading frame of adeS. The regular spacing of average 144 bp (seven nucleosomes at positions 90-1100) represents a phasing of nucleosomes (Bernardi et al. 1991) as revealed by comparison with the digestion patterns of naked DNA. In contrast, the chromatin of M26 was significantly different. In addition to the two hypersensitive sites (-210 and -80) in the presumed promoter region, we detected a novel hypersensitive site at the position of the M26 mutation (170) that is covered by a phased nucleo-some in wild-type and M375 mutations. Furthermore, the regular spacing of -150 bp was not observed and some new MNase-sensitive sites appeared at positions corresponding to MNase-sensitive sites in the naked DNA of the coding region (e.g., bands at 340, 480, and 670).

M26-dependent enhancement of meiotic induction of MNase hypersensitivity Next, we compared the chromatin structure at the ade6 locus in premeiotic and meiotic cells (Figs. 2 and 3). To allow comparison, the radioactivity of each band was quantified. The MNase sensitivity at each site was expressed relative to the sum of all band intensities in the lane. For M375, we found that MNase sensitivity at the presumed promoter increased slightly during meiosis (2.4 ± 1.3-fold at site -210, 3.9 ± 2.2-fold at site -80, n = 3; see Table 1). We observed the same features for the wild type (Table 1). On the other hand, the sensitivity at other sites did not change significantly, for example, 1.3 ± 0.4-fold {n = 3) and 1.2 ± 0.1-fold {n = 2) at site -80 in M375 and wild type, respectively. This phenomenon is similar to the observations made at hot spots for meiotic recombination in budding yeast (Ohta et al. 1994).

The meiotic induction of MNase sensitivity was enhanced by up to 9.3 ± 0.6-fold as a result of M26 at site -80 in the presumed promoter region [n = 3, see Fig. 3; Table 1), whereas the sensitivity at site 80 was not enhanced at all (1.0 ± 0.1-fold, n = 3). MNase sensitivity at the M26 mutation site also increased significantly during meiosis (5.4 ± 2.2-fold, n = 3; see Fig. 3 and Table 1). The increase in MNase hypersensitivity was first de-

GENES & DEVELOPMENT 877




Mizuno et al.

ATG

, Xhol 1-485

-»-220^!

Z ^ ■o "

► — - 4 8 5

-^340^ -*-480-^!

- - 6 7 0 ^ -75fr^

26

B

Eco RI 1231-

— 842 ATG

-«1010»-|

-«1100»-|

a 1469

Xhol -785^ Stul

—1231

8 7 0 - ^ 7 5 0 - ^ 5 8 0 - ^ 4 3 0 - ^ 2 5 0 - ^

9 0 - ^

-80-^

-210-*-

-310-*-

-410

Figure 1. Comparison of chromatin structures at the ade6 locus in wild-type, M375, and M26 strains. The yeast strains were harvested at t = 3 hr after transfer of cells into the sporulation medium. Chromatin and naked genomic DNA (from the M26 strain) were isolated, treated with MNase (50 and 32 U/ml , respectively), and analyzed by indirect end-labeling, as described in Materials and Methods. DNA was digested with Xhol [A] and StuI-£coRI (5), and subjected to Southern hybridization with probes for the sequence adjacent to the Xhol site at +1469 bp from the ATG of the adeS coding region (probe B in A), or for the sequence adjacent to the Stul site at -785 bp (probe C in B]. An interpretation of the nucleosome positions is shown. (Shaded shapes) Nucleosomes; (horizontal arrows) MNase-sensitive sites; (open triangles) the M26 mutation site. Vertical open arrows indicate the position of the coding region for the ade6 locus. The numbers beside the horizontal arrows indicate the distance from the A of the ATG at the beginning of the ade6 coding sequence. Sequences recognized by the hybridization probes are indicated by vertical open rectangles. The DNA size markers are Xhol (1953 bp), Xhol-Hindlll (627 bp), and Xhol-BamHl (1443 bp) for A, and Stul-EcoRl (2015 bp), Stul-ffindlll (1626 bp), and Stul-BamHl (810 bp) for B. Note that the M375 and M26 sequences differ only by single-base substitutions from the wild-type adeS sequence.

Figure 2. Quantitative comparison of pre-meiotic and meiotic chromatin structures at the ade6 locus in the M375 strain. Chromatin [(0 hr) premeiotic; (3 hr) meiotic] of the M375 strain was digested with 0, 7, 50, and 225 U /ml of MNase and examined as described in Fig. 1. DNA was digested with Xhol and hybridized with probe B (see Fig. lA). The radioactivity of each band was quantified by a Fuji BAS2000 system and Bio Image analyzer as described in Materials and Methods. Band intensities in lanes containing samples digested with 50 U / m l of MNase were expressed as percentage of the sum of all band intensities. For further explanations, see Fig. 1. Intensity of a faint band corresponding to the band at 0 in Fig. 1 was not quantified in this study, as the intensity was too low for accurate quantification. (Open bars) Data of t = 0 hr,-(hatched bars) data of t = 3 hr. The numbers beside the horizontal arrows indicate the distance from the A of the ATG at the beginning of the ade6 coding sequence.

0 5 10 15 20 25

Band Intensity (% of the total, in SOU lanes)

878 GENES & DEVELOPMENT





r.<> A-i 4' , . V" T." «^ A"̂ ^ '^

■^'' 0^^ <?>^ ^ '?>'' -^ r^ S^^

^ii^ ^

^ i

i ^ i i

< i ^

^ B -V,

□ Ohr eSB 3hr

M26 0 5 10 15 20 Band Intensity (% of the total, in SOU lanes)

Figure 3. Quantitative comparison of pre-meiotic and meiotic chromatin structures at the ade6 locus in the M26 strain. Chromatin isolated from the M26 strain was digested with 7, 50, and 225 U/ml of MNase and examined as described in Figs. 1 and 2. For further explanations see, Fig. 2. Open triangles show the position of the M26 mutation. Bars with horizontal arrows represent the sites of meiotic enhancement of MNase hypersensitivity. In this experiment the band intensity of some faint bands (bands corresponding to those at 450, 590, and 630 in Fig. 1) was not quantified, as the intensity was too low for accurate quantification. The numbers beside the horizontal arrows indicate the distance from the A of the ATG at the beginning of the ade6 coding sequence.

tected at t ~ 2-3 hr, and the sensitivity was still at a high level at £ = 6 hr (data not show^n). Under the same culture conditions, premeiotic DNA synthesis occurred at t ~ 2 -6 hr as revealed by laser scanning cytometry (Fig. 4) and flow cytometry (data not shown). We also verified that the completion of meiosis I occurred around t = 8 hr by staining nuclei with 4',6-diamidino-2-phenylindole (DAPI) (data not shown). These results suggest that the changes of chromatin structure occur at the premeiotic S phase.

Integrity of the heptamei sequence is necessary for M26-dependent chromatin remodehng

The appearance of a nuclease-sensitive region around the M26 mutation site suggests that the binding of M t s l / Mts2 to the heptamer sequence may contribute to the enhanced DNA accessibility around the heptamer. If this is the case, modification of the heptamer sequence AT-

GACGT should affect M26-dependent chromatin remodeling. To test this idea, we analyzed four strains: two hot spot-active strains with a single-base substitution outside the heptanucleotide (7T and 16C; see Fig. 5), and two hot spot-inactive strains with a single-base substitution within the heptanucleotide (13G and 14C; see Fig. 5) (Schuchert et al. 1991). The Mts l /Mts2 protein binds to double-stranded DNA (dsDNA) probes having either of the substitutions 7T or 16C but not to those with either 13G or 14C in a gel mobility retardation assay (Wahls and Smith 1994).

In the hot spot-positive controls 7T and 16C, we detected MNase-hypersensitive sites at the presumed promoter and around the heptamer (Figs. 5 and 6). The enhancement of the meiotic induction of MNase hypersensitivity in the 7T and 16C strains was also detected at site -80, and the sensitivity increased at the heptamer region (Table 1). On the other hand, the chromatin structures of the ade6 region in I3G or 14C DNA were dif-

Table 1. Quantification of MNase hypersensitivity

Strains Site -210 Site Site M26

Wild type M375 M26 7T 13G 14C I6C

2.910.3(2.7,3.1) 2.4+1.3(2.2,3.7, 1.2) 2.6 + 0.7(3.1,3.0, 1.8) 2.6 + 1.3(3.6, 1.7) 1.9 ±0.4 (2.1, 1.6) 1.9 + 0.4(2.2, 1.6) 2.4 ±0.2 (2.5, 2.3)

4.1 ±2.4 (2.4, 5.8) 3.9±2.2(2.1, 6.3, 3.2) 9.3±0.6(8.7, 9.5, 9.8)

10.2 ±2.0 (11.6, 8.8) 4.1 ±2.0 (5.5, 2.7) 3.0 ±1.7 (1.8, 4.2)

11.5 ±1.6 (12.6, 10.4)

5.4 + 2.2(7.9,3.5,4. 4.7 ±0.5 (5.1, 4.4)

3.6 ±0.2 (3.7, 3.4)

The MNase hypersensitivity in each strain was quantified by a Fuji BAS2000 system and a Bio Image analyzer as described in Materials and Methods and Figs. 2 and 3. Meiotic induction of MNase hypersensitivity is expressed as the ratio of the meiotic hypersensitivity to the premeiotic hypersensitivity at each site including standard deviations. Numbers after ± represent standard deviations of the measurement. Numbers in parenthesis show the data for each experiment. The experiments were repeated three times with the M26 and M375 strains and twice with the wild-type, the 7T, 13G, 14C, and 16C strains. The ratio of the MNase hypersensitivity is basically independent from the concentration of MNase up to 50 U/ml.





Mizuno et al.

§ -e^eg o B

2C4C 2C4C 199 159 119

624 2C4C

.? ^ 1 7 IkittpU

50 100 150 PI fluorescence value




Figure 4. Changes in cell DNA content during meiosis. DNA content per cell was monitored during the meiosis of diploids at 0, 2, 4, and 6 hr after transfer of cells into sporulation medium by quantifying fluorescence of nuclei stained with propidium iodide (PI) using an Olympus LSClOl laser scanning cytometer. The vertical axis represents the number of cells; the horizontal axis indicates the PI fluorescence value per cell. The positions for 2C and 4C DNA content are indicated. The majority of fission yeast cells are usually in the G2 phase under vegetative growth conditions (Bahler et al. 1993). Therefore, the DNA content at t = 0 hr was 4C. After the cells are shifted into meiosis, they traverse M phase and enter Gj phase around t = 1-2 hr and then undergo premeiotic DNA synthesis (t = 2-6 hr). FACS analysis gave identical results (data not shown). From the staining nuclei with DAPI, completion of meiosis I was estimated to occur around t = 8 hr, and elongated or horse-tail nuclei were observed at t ~ 2-8hr (data not shown).

ferent from those in 7T or 16C. As expected, they were almost identical with those of the wild type and M375 cells; that is, the MNase-hypersensitive site at the hep-tamer was absent and meiotic induction of MNase hypersensitivity at site -80 was reduced. The difference in the band patterns in these strains is not attributable to the difference in MNase-sensitive sites on naked DNA, as the heptamer mutations did not affect the band patterns on naked DNA (data not shown). Therefore, there is positive correlation between hot spot activity, binding of Mts l /Mts2 to the heptamer, formation of an accessible DNA region around the heptamer, and enhanced meiotic induction of MNase sensitivity. Interestingly, the 16C construct yielded a novel pattern; that is, the three positioned nucleosomes present in wild type and absent in M26 reappeared in 16C but were displaced in the 5' direction. The novel phasing of these three nucleosomes seems to be directed by the MNase-hypersensitive site at M26 and the additional base substitution I6C (Figs. 5 and 6).

Discussion

Chromatin remodeling by the heptamer sequence and the Mtsl/Mts2 protein

The results presented here indicate that the heptamer sequence ATGACGT affects nucleosome positioning, possibly by facilitating the binding of the Mts l /Mts2 heterodimeric protein. In M26 chromatin, the phasing of nucleosomes was affected around the heptamer and downstream in the open reading frame of the ade6 gene. The MNase-sensitive sites characteristic of naked DNA were observed in the chromatin digestion, and a novel MNase-hypersensitive site also appeared at the heptamer. All of these changes in DNA accessibility were the consequence of the single G ^ T base substitution that created M26 from the wild-type sequence. Our re

sults seem to be inconsistent with the data of Bernardi and coworkers (1991), who found the chromatin in M26 haploid vegetative cells to be very similar to that in wild-type cells. We have confirmed their results by showing that the nucleosome positioning in the ade6 region in the haploid M26 strain during vegetative growth was very similar to the patterns seen in diploid wild-type and M375 strains (K. Mizuno, J. Kohli, and K. Ohta, unpubl.). Therefore, the remodeling of chromatin structure found in the mitotic cells (i.e., premeiotic cells at t = 0 in the meiotic time course in this study) seems to be related closely to ploidy or some other factors. We are currently analyzing this phenomenon.

We found that chromatin remodeling by M26 requires the same integrity of the heptamer sequence as that required for the binding of the Mts l /Mts2 protein (Wahls and Smith 1994). Altered chromatin structure as a consequence of sequence-specific DNA-binding proteins has been reported for transcription factors. The a2 repressor in 5. cerevisiae was shown to affect the positioning of nucleosomes at the promoter through binding to its target DNA sequence in vivo (Shimizu et al. 1991). The GAGA factor in Drosophila was shown to be involved in formation of nuclease hypersensitive sites and remodeling of chromatin at the heat shock-responsive element in vitro (Tsukiyama et al. 1994). Probably, the binding of Mts l /Mts2 to the heptamer influences the binding of nucleosomes to this region, thereby altering nucleosome positioning (Fig. 7). In this respect, it would be interesting to analyze chromatin structure in M26 strains disrupted for the genes coding for Mts l and Mts2.

It is intriguing that a pattern of nucleosome phasing downstream of M26 is restored, when the single-base substitution 16C is added to the heptanucleotide. The resulting chromatin structure, however, differs from the wild type in that the nucleosomes are shifted toward the 5' end. We propose that the 16C sequence forms a boundary for nucleosome positioning. Although the mecha-





Chromatin remodeling in ade6-M26 hot spot

A

B

5'- T G G lA T G A " C } G lA T G A C G T I G

T G C C

7T 13G 14C 16C

] G A -3'

■M26

-M375

7 T 1 3 G 1 4 C 1 6 C strains

b r '^ induction

J3 .S -

inincn ( S f ^ S o S ^ o S r ^ o ^ S MNase(Units) o <s

M26sitet>l <|M26site

MNase hypersensitive site around the M26 site

Figure 5. Integrity of the heptamer sequence is required for M26-dependent chromatin remodeling. [A] The DNA sequence around the M26 heptamer (boxed) is shown together with the additional single-base substitutions (arrows) and the mutation designations. [B] The correlation is shown between hot spot activity in vivo (Schuchert et al. 1991) and the DNA-binding activity of Mtsl/Mts2 to each altered sequence in vitro (Wahls and Smith 1994). The values for the in vivo data are normalized by setting the M26 recombination frequency as 100% and the M375 frequency as 0%. (C) The chromatin structures of the heptamer mutants were analyzed by indirect end-labeling as described in Fig. 1. The strain names {top line), the times of cell harvest [middle line, (0 hr) premeiotic; (3 hr) meiotic], and the conditions for MNase digestion {bottom line, U/ml) are indicated. The position of the M26 mutation is marked by open triangles. The presence (+) or absence (-) of the MNase-hyper-sensitive site at the M26 mutation site is shown below the gel. DNA size markers were as in Fig. lA. The difference of the band patterns in these strains is not attributable to the difference in MNase-sensitive sites on naked DNA, as the heptamer mutations did not affect the band patterns on naked DNA (data not shown).

nism underlying this boundary formation is unclear, the 16C in the context of the local DNA sequence may change the intrinsic DNA structure or recruit another DNA-binding protein to form the boundary (Fig. 6). It

should be noted that the 16C mutation also enhances the binding of Mts l /Mts2 (Wahls and Smith 1994) and increases meiotic recombination frequency beyond the M26 level (Schuchert et al. 1991).

Meiotic induction and M26 enhancement of MNase hypersensitivity: imphcation for the meiosis specificity of the M26 hot spot and a general model for the initiation of meiotic recombination at ade6

The mechanism that creates the meiosis specificity of the M26 recombination hot spot (Ponticelli et al. 1988; Schuchert and Kohli 1988) is not yet fully understood. The Mts l /Mts2 protein and its heptamer-binding activity are present in extracts of both mitotic and meiotic cells (Wahls and Smith 1994). In addition, the MNase-sensitive site at M26 is also detected in mitotic and meiotic diploid cells, suggesting the constitutive interaction of Mts l /Mts2 with the heptamer in vivo and the necessity to invoke the existence of additional factors to explain M26 meiosis specificity.

We have demonstrated here that MNase hypersensitivity increases significantly at the presumed promoter and the heptamer sequence during early meiosis (Table I). The increase in MNase sensitivity was observed from two aspects: (1) Meiotic induction of MNase hypersensitivity is observed in all strains (wild type, M375, and M26) at the two sites, -210 and -80, in the presumed promoter; and (2) in M26, meiotic induction of MNase hypersensitivity is significantly stronger at the -80 promoter site and at the M26 site when compared to those in wild-type and M375 strains. These are the first observations suggesting the interaction of meiosis-specific functions with accessible DNA regions during S. pombe meiosis. We put forward three explanations for the meio-sis-specific functions that cause the meiotic increase in the MNase sensitivity of the M26 hot spot.

The first possibility is that enhanced binding of Mts 1 / Mts2 to the heptamer during meiosis increases DNA accessibility at M26. This is supported by the observation that the MNase hypersensitivity at the heptamer increased about fivefold during meiosis. Although this possibility is likely, it should be noted that the MNase-hypersensitive site at M26 is already present in mitotic cells, in which M26 enhanced homologous recombination could not be detected at all (Ponticelli et al. 1988; Schuchert and Kohli 1988) unless a DSB is supplied at the ade6 region by introduction of HO endonuclease (Os-man et al. 1996). This observation implies that meiotic activation of the M26 hot spot might include other steps such as recruitment of a meiosis-specific nuclease or a recombination protein to accessible DNA regions created by Mts l /Mts2 (see below).

The second possibility is that changes in DNA structure occur at the heptamer and in the presumed promoter region during meiosis (e.g., melting of DNA yielding single-stranded regions). However, in the case of budding yeast, we could not detect a significant change in DNA structure during meiosis at the nucleosome-free regions of the ARG4 hot spot by genomic footprinting analysis using single

GENES & DEVELOPMENT 8S1




Mizuno et al.

heptamer

M26 site

Type A) (5'-AGGACGT-3')

nucleosomes

(-210) (-80) (90) (250) (430) (580) (750) (870) (1010) (1100)

Type B) (5'-ATGACGT-3')

(7)Mts2 AAl^

T T W I T I (340) (450)1 (590)1(670)1 | | |

^ ^

(-210) (-80) (80) (170) (250) (480) (630) (750) (870) (1010) (1100)

Type C) (5'-ATGACGTC-3')

(-210) (-80) (80) (170) (350) (520) (670X760) (870) (980X1090)

Figure 6. A schematic diagram showing nucleosome positions in different ade6 strains. The positions of nucleosomes were estimated from the data on MNase-digested chromatin and digests of naked DNA in Fig. 1. The nucleosome patterns were classified into three types: Type A includes wild-type, M375, 13G, and 14C; type B includes M26 and 7T; and type C includes 16C. Vertical arrows indicate the positions of MNase-sensitive sites. Hatched horizontal arrows represent the ade6 coding region. The numbers in parenthesis give the distances from the first A of the initiation codon of the ade.6 coding sequence. The positions of the heptamer sequences are indicated by thin solid boxes; the nucleosomes are indicated by hatched circles; the Mtsl/Mts2 protein is shown as two kinked ovals. A shaded oval with a question mark in type C represents a putative third factor that may be involved in the boundary formation by the 16C sequence.

stranded DNA (ssDNA)-specific probes such as KMn04, mung bean, and PI nucleases (K. Ohta, A. Nicolas, and T. Shibata, unpubl.). Thus, changes in DNA structure might be too subtle to cause the increase in MNase sensitivity. Assuming that the same mechanism for meiotic enhancement of MNase hypersensitivity is operative in both yeasts, changes in DNA structure, at least formation of ssDNA, is unlikely to explain the meiotic increase in MNase sensitivity of the M26 hot spot.

The third explanation is that the meiotic increase in MNase sensitivity at M26 and then hot spot activity is caused by the recruitment of other proteins to the ade6 gene that, unlike Mts l /Mts2 , would be specific for meiotic recombination (Fig. 7). Such recombination proteins w^ould be recruited to the accessible DNA regions formed by the binding of Mts l /Mts2 protein and also probably by transcription factors bound to the promoter. This model is based on our results showing that MNase hypersensitivity at hot spots increases significantly during meiosis in budding yeast (Ohta et al. 1994). In addition, we carried out genomic footprinting analysis of chromatin at the ARG4, CYS3, and yCi?4Swhot spots at the single-nucleotide resolution level (K. Ohta, A. Nicolas, M. Furuse, A. Nabetari, H. Ogawa, and T. Shibata, unpubl.). Meiotic induction of MNase sensitivity often occurs at sites adjacent to (within a few base pairs) re

gions showing repression of MNase sensitivity during meiosis prior to the formation of meiotic dsDNA breaks. The appearance of meiosis-specific footprints was not detected in a subset of meiotic DSB-deficient mutants such as mrell. Therefore, we speculate that the meiotic induction of MNase sensitivity at hot spots in budding yeast reflects an essential reaction caused by the interaction of recombination proteins with DNA prior to the DSB formation. It is also possible that the meiotic enhancement of MNase hypersensitivity at the ade6-M26 locus may reflect the action of meiotic recombination enzymes. If meiotic recombination mechanisms are conserved between the two yeast species, rad32'^, the fission yeast homolog of the MREll gene (Tavassoli et al. 1995), may be involved in the meiotic enhancement of MNase hypersensitivity. The study of the chromatin structure in yeast cells with a disruption of the iad32* or other recombination genes will be a test of this model.

Is the ATGACGT sequence enhancing a preexisting recombination initiation sitel

In budding yeast, hot spots often coincide with accessible promoter regions that show meiotic induction of MNase hypersensitivity (Ohta et al. 1994). The presence of meiotically enhanced chromatin alterations in the






/ ^ transcription factors nucleosome

promoter

ade6 ORF

B Mts1

M26 heptamer

ade6-M26 ORF

Mtsi

meiosis

Increase in DNA accessibility

IMelosls-speclflc Factor?

fo . t DNA break ?

Figure 7. A model explaining the meiosis specificity of the M26 hot spot. (A) Without the heptamer, phasing of nucleo-somes (hatched circles) occurs over the ade6 open reading frame (horizontal arrow). The presumed promoter region (hatched rectangle) is constitutively open and can act as a natural recombination initiation site. The hatched hexagon indicates putative transcription factors bound to the presumed promoter. [B] In the presence of the heptamer, binding of the Mtsl/Mts2 protein to the heptamer alters the local chromatin structure to form an accessible DNA region. (C) The accessible DNA region (especially at the presumed promoter and the heptamer site) becomes a preferential target for meiosis-specific factors required for initiation of recombination. The recruitment of the meiosis-specific factors causes meiotic enhancement in DNA accessibility (MNase hypersensitivity) at the presumed promoter and the heptamer sites. (D) Events for the initiation of meiotic recombination (such as DNA breaks) occur close to the presumed promoter and/or the heptamer sites.

spot activity is abolished when the ade6 presumed promoter region is deleted (Zahn-Zabal et al. 1995). This finding seems to be compatible with the interpretation that M26 enhances an initiation site localized within the presumed promoter region and that no novel initiation site [single-strand break (SSB) or DSB] is created at the heptanucleotide. However, we cannot exclude the possibility that the deletion might change chromatin structure around the M26 mutation sites to inactivate its hot spot activity. It will be interesting to determine whether the MNase-hypersensitive site at M26 is still present in chromatin isolated from an M26 strain carrying a deletion of the presumed promoter region. If the site at M26 is still found in strains deleted for the presumed promoter, then an examination of the meiotic enhancement of MNase sensitivity at the M26 site in these strains should provide an important clue about the role of the M26 mutation in the hot spot activity.

In Figure 7 we propose an alternative idea that both the presumed promoter and the heptamer regions serve as initiation sites for meiotic recombination at the M26 locus. Assuming that meiotic induction of MNase sensitivity is an obligatory signal for initiation, as is the case in S. cerevisiae, the presence of the induction in MNase hypersensitivity after meiotic induction at the presumed promoter sites and the M26 heptamer is consistent with the notion that both sites are initiation (DSB or SSB) sites. According to this model, the M26 heptamer would play the double role of an enhancer of the background initiation site in the presumed promoter and of a novel hot spot of initiation at the heptamer. If the heptanucleotide is contributing to the enhancement of the initiation site in the presumed promoter, then position effects of the heptanucleotide would be expected. This could be examined by varying the position of the heptanucleotide with respect to the presumed promoter and analyzing for any changes in chromatin structure. In relation to this, it is worthwhile noting that the heptamer still functions as a hot spot when it is moved to downstream positions in the ade6 gene (G. Smith, pers. comm.).

ade6 presumed promoter region is consistent with the interpretation that an initiation site for meiotic recombination exists in the presumed promoter region of the wild-type ade6 gene and the M26 mutant. Previously, two possibilities have been advanced to explain the role of the M26 mutation in the hot spot activity. First, the M26 mutation, and the resulting heptanucleotide that serves as a binding site for Mts l /Mts2 , enhances initiation of recombination at a site in the presumed promoter region of ade6, which is also used for the background recombination levels seen in M375. Second, the introduction of the M26 mutation creates a new initiation site for recombination (Grimm et al. 1994). These two possibilities will not be distinguished until the physical nature and the sites of initiation events in M26 and M375 strains are known.

It has been demonstrated recently that the M26 hot

Materials and methods

DNA and strains The plasmid containing the ade6-M26 sequence (pade6-M26) is a derivative of pASl (Szankasi et al. 1988). The plasmid pade6-M26 was digested with Xhol-EcoRl or Xhol-Stul to form 240-and 300-bp fragments. The fragments were subcloned into the Xhol-EcoRl site (pASI-1) or the Xhol-EcoRY site (pASI-2) in pBluescript(KS+), respectively. Probes for indirect end-labeling were prepared from pASI-1 and pASI-2 by digesting the plasmids with Xhol-EcoRl followed by the purification of DNA fragments in agarose gels. All strains are from the collection at the University of Bem (Switzerland) as summarized in Table 2.

Enzymes and chemicals Restriction endonucleases were purchased from New England Biolabs and Takara Shuzo Co. Ltd. MNase and Ficoll 400 were from Pharmacia. Zymolyase lOOT was from Seikagaku Kogyo





Mizuno et al.

Table 2. Genotypes of strains

Diploid

ELD201 (wild-type)

ELD203 (M375)

ELD205 (M26)

ELD207 (7T)

ELD209 (13G)

ELD211 (14C)

ELD213 (16C!

Haploid

ELHIOI ELH102 ELH103 ELH104 ELH105 ELH106 ELH107 ELH108 ELH109 ELHllO E L H l l l ELH112 ELH113 ELH114

mat

h* h-h* h' h^ h' h^ h' h* h' h^ h-h* h'

ade6

+ +

ade6-M375 ade6-M375 ade6-M26 ade6-M26 ade6-M26/7T ade6-M26/7T ade6-M26/13G ade6-M26/13G ade6-M26/14C ade6-M26/14C ade6-M26/16C ade6-M26/16C

ural

ural-61 +

uial-61 +

uial-61 +

ural-61 +

ural-61 +

ural-61 +

ural-61 +

leu3

+ leu3-155

+ leu3-155

+ leu3-155

+ leu3-155

+ leu3-155

+ leu3-155

+ leu3-155

All strains are from the Bern collection. The construction of the heptamer mutants was described by Schuchert et al. (1991).

Co. Ltd. Proteinase K in solution was from Boehringer Mannheim, RNase A was from Sigma.

Cell culture and sporulation

Standard media, presporulation, and sporulation were as described (Bahler et al. 1991, 1993). Briefly, diploid colonies were selected on minimal medium plates containing phloxin B. Colonies showing efficient and synchronous sporulation were further selected. Cells were grown in 1 liter of PM (Watanabe et al. 1988) containing supplements with vigorous aeration at 30°C to a cell density of 0.8 x 10^ to 0.9 x 10^, washed once in PM-N (Watanabe et al. 1988). Half of the cells were precipitated and frozen in liquid nitrogen, and then stored at -85 °C. The resulting half of the cells were incubated with vigorous aeration at 30°C in 0.5 liter of PM-N containing supplements. About 0.6-1.0 gram (wet weight) of cells was recovered in the standard condition. The progression of cells into meiosis was monitored by counting the number of nuclei in cells and determining the percentage of deformed and elongated nuclei (horse-tail nuclei) (Bahler et al. 1993; Chikashige et al. 1994) by DAPI staining and fluorescence microscopy as described (Bahler et al. 1993). From the staining of nuclei with DAPI (data not shown), completion of meiosis I was estimated to occur around t = 8 hr, and elongated or horse-tail nuclei were observed at t ~ 2-8 hr. Laser scanning cytometry was performed on cells stained with propidium iodide by an Olympus LSClOl setup according to the manufacturer's instructions and Sasaki et al. (1996).

Preparation of chromatin and digestion of chromatin DNA by MNase

The preparation of crude chromatin fractions from S. pombe cells was done according to the method of Bemardi et al. (1991), with some modifications. For the quantitative comparison of MNase sensitivity, samples of chromatin were prepared from a fixed amount of cells (0.6-1.0 gram wet weight) in each experiment. Briefly, 1 gram of cells was incubated in 2 ml of preincubation solution (20 mM Tris-HCl at pH 8.0, 0.7 M 2-mercapto-ethanol, 3 mM EDTA) at 30°C for 10 min and washed once in 5 ml of ice-cold 1 M sorbitol. The gram of cells was then precipitated and resuspended in 5 ml of freshly prepared Zymolyase solution (37.5 mM Tris-HCl at pH 7.5, 0.75 M sorbitol, 1.25% glucose, 0.5% (wt/vol) Zymolyase-lOOT, 6.25 mM EDTA). The

cells were incubated for 15 min at 30°C with gentle agitation and resulting spheroplasts were pelleted. All subsequent steps were done at 0-4°C. The spheroplasts were then washed once in 5 ml/gram of ice-cold 1 M Sorbitol and resuspended well by pipetting in 7 ml/gram of freshly prepared lysis buffer [18% Ficoll-400, 10 mM KH2PO4, 10 mM K2HPO4 at pH 6.8, 1 mM MgCli, 0.25 mM EGTA, 0.25 mM EDTA, 1 mM phenylmethyl-sulfonyl fluoride (PMSF)]. After centrifugation at 20,000g for 40 min, the crude nuclear pellet was resuspended well in 8 ml / gram of buffer A (10 mM Tris-HCl at pH 8.0, 150 mM NaCl, 5 mM KCl, 1 mM EDTA, 1 mM PMSF). One-milliliter aliquots of the crude chromatin suspension were digested with different amounts of MNase (0, 7, 10, 20, 30, 50, and 225 U/ml) for 5 min at 37°C in the presence of 5 mM CaClj. The reaction was terminated by adding 20 mM EDTA, 1 % (wt/vol) SDS, and 20 vig of proteinase K and incubated at 50°C for 2 hr. Insoluble material was removed by microcentrifugation at room temperature at 15,000 rpm for 10 min. The supernatants were extracted twice with phenol/chloroform/isoamyl alcohol, digested with RNase A, and then extracted once with phenol/chloroform/isoamyl alcohol. The extracted DNA was precipitated by ethanol, rinsed in 70% ethanol, and resuspended in 100 pi of TE buffer (10 mM Tris-HCl, 1 mM EDTA at pH 8.0). The typical yield of DNA from 1 gram cells was -200 ]ig.

Digestion of naked DNA

Naked DNA samples were prepared as described above except that addition of MNase was omitted. Nine hundred microliters of buffer A was added to 100 pi of naked DNA (20-30 pg). The diluted DNA was incubated with various amounts of MNase (5-60 U/ml) in the presence of 5 mM CaClj at 37°C for 5 min. The reaction was stopped as described above. The mixture was treated with 5 pg of proteinase K at 50°C for 30 min, extracted with phenol/chloroform/isoamyl alcohol, and precipitated by ethanol. The final precipitate was resuspended in 100 pi of TE buffer. Optimal conditions for MNase digestion were determined by gel electrophoresis.

Indirect end-labeling

Indirect end-labeling was performed as described (Bernardi et al. 1991; Ohta et al. 1994). Briefly, 10 pi of DNA (2-3 pg) was digested completely by restriction endonucleases. The digested





Chromatin remodeling in ade6-M26 hot spot

DNA was ethanol-precipitated, separated by electrophoresis on a 1.2% agarose gel (40 cm long) containing 0.5 pg/ml of ethid-ium bromide in TAE buffer (0.04 M Tris/acetate, 1 mM EDTA at pH 8.0) at 80 V for 15-17 hr and alkali-transferred to nylon membranes (Hybond N+, Amersham) under vacuum using a Vacugene (LKB) apparatus. Membranes were prehybridized for 1 hr and hybridized for 24 hr according to Church and Gilbert (1984) with 0.5-1 ng/ml of labeled probes. Probes were labeled by the random priming method according to the manufacturer (Pharmacia) using 3.7 MBq (100 ijCi) of [a-^^P]dCTP (sp. act. 3000 Ci/mmole) for 100 ng of DNA fragments. Radioactive DNA fragments were quantified using the imaging plates for Fuji BAS2000 Image Analyzer combined with the whole-band program of Bio Image (Bio Image Co. Ltd). Band intensity was expressed as a percentage of the total of all the band intensities of a lane including the unbroken parental fragment. For comparison, the same number of bands in lanes for each sample was quantified by the whole band program. The nucleotide positions of the MNase-sensitive sites were measured according to the auto-band detection algorithm of Bio Image. The nucleotide numbers were estimated from the mean values of at least four independent measurements.

Acknowledgments

We are grateful to Ms. Elisabeth Lehman for the construction of the strains. We also thank Dr. Gerald Smith, Dr. Wayne P. Whals, and Dr. Jeff Virgin for sharing data in advance of publication and stimulating discussions. We thank Yoshimasa Kiyo-matsu (Olympus Co. LTD) for the measurement of cell DNA contents by the laser scanning cytometer. This project was supported by a grant from Human Frontier Science Program (RG493/95), a grant for the Biodesign Research Program from RIKEN, grants from the Ministry of Education, Science, &. Culture, Japan, and grants from the Swiss National Science Foundation and of the Sandoz Research Foundation.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

References

Bahler, J., C. Schuchert, C. Grimm, and J. Kohli. 1991. Synchronized meiosis and recombination in fission yeast: Observations with patl-114 diploid cells. Curr. Genet. 19: 445-451.

Bahler, J., T. Wzler, J. Loidl, and J. Kohh. 1993. Unusual nuclear structures in meiotic prophase of fission yeast: A cytological analysis. /. Cell Biol. 121: 241-256.

Bernardi, F., T. Roller, and F. Thoma. 1991. The ade6 gene of the fission yeast Schizosacchaiomyces pombe has the same chromatin structure in the chromosome and in plasmids. Yeast 7: 547-558.

Cao, L., E. Alani, and N. Kleckner. 1990. A pathway for generation and processing of double-strand breaks during meiotic recombination in S. ceievisiae. Cell 61: 1089-1101.

Chikashige, Y., D.Q. Ding, H. Funabiki, T. Haraguchi, S. Mashiko, M. Yanagida, and Y. Hiraoka. 1994. Telomere-led premeiotic chromosome movement in fission yeast. Science 264:270-273.

Church, G.M. and W. Gilbert. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. 81: 1991-1995.

de Massy, B. and A. Nicolas. 1993. The control in cis of the position and the amount of the ARG4 meiotic double-strand break of Saccharomyces ceievisiae. EMBO J. 12: 1459-1466.

Fan, Q.-Q. and T.D. Petes. 1996. Relationship between nucle-ase-hypersensitive sites and meiotic recombination hot spot activity at the HIS4 locus of Saccharomyces ceievisiae. Mol. Cell. Biol. 16: 2037-2043.

Goyon, C. and M. Lichten. 1993. Timing of molecular events in meiosis in Sacchaiomyces ceievisiae. Stable heteroduplex DNA is formed late in meiotic prophase. Mol. Cell. Biol. 13: 373-382.

Grimm, C , J. Bahler, and J. Kohli. 1994. M26 recombinational hotspot and physical conversion tract analysis in the ade6 gene of Schizosacchaiomyces pombe. Genetics 136: 41-51.

Gutz, H. 1971. Site-specific induction of gene conversion in Schizosacchaiomyces pombe. Genetics 69:317-337.

Lichten, M. and A.S.Fi. Goldman. 1995. Meiotic recombination hotspots. Annu. Rev. Genet. 29: 423-444.

Nag, D.K. and T.D. Petes. 1993. Physical detection of heterodu-plexes during meiotic recombination in the yeast Saccharomyces ceievisiae. Mol. Cell. Biol. 13:2324-2331.

Nicolas, A., D. Treco, N.P. Schultes, and J.W. Szostak. 1989. An initiation site for meiotic gene conversion in the yeast Sacchaiomyces ceievisiae. Natuie 338: 35-39.

Ohta, K., T. Shibata, and A. Nicolas. 1994. Changes in chromatin structure at recombination initiation sites during yeast meiosis. EMBO J. 13: 5754-5763.

Osman, F., E.A. Fortunato, and S. Subramani. 1996. Double-strand break-induced mitotic intrachromosomal recombination in the fission yeast Schizosacchaiomyces pombe. Genetics 142: 341-357.

Ponticelli, A.S. and G.R. Smith. 1992. Chromosomal context dependence of a eukaryotic recombinational hot spot. Proc. Natl. Acad. Sci. 89: 227-231.

Ponticelli, A.S., E.P. Sena, and G.R. Smith. 1988. Genetic and physical analysis of the M26 recombination hotspot of Schizosacchaiomyces pombe. Genetics 119:491-497.

Sasaki, K., A. Kurose, Y. Miura, T. Sato, and E. Ikeda. 1996. DNA ploidy analysis by laser scanning cytometry (LSC) in colorectal cancers and comparison with flow cytometry. Cy-tometiy 23: 106-109.

Schuchert, P. and J. Kohh. 1988. The ade6-M26 mutation of Schizosacchaiomyces pombe increases the frequency of crossing over. Genetics 119: 507-515.

Schuchert, P., M. Langsford, E. Kaeslin, and J. Kohli. 1991. A specific DNA sequence is required for high frequency of recombination in the ade6 gene of fission yeast. EMBO J. 10:2157-2163.

Shimizu, M., S.Y. Roth, C. Szent-Gyorgyi, and R.T. Simpson. 1991. Nucleosomes are positioned with base pair precision adjacent to the a2 operator in Sacchaiomyces ceievisiae. EMBO J. 10:3033-3041.

Shiroishi, T., T. Sagai, and K. Moriwaki. 1993. Fiotspots of meiotic recombination in the mouse major histocompatibility complex. Genetica 88: 187-196.

Smith, G.R. 1994. Fiotspots of homologous recombination. Ex-peiientia 50:234-241.

Sun, H., D. Treco, N.P. Schultes, and J.W. Szostak. 1989. Double-strand breaks at an initiation site for meiotic gene conversion. Natuie 338: 87-90.

Szankasi, P., W.D. Heyer, P. Schuchert, and J. Kohh. 1988. DNA sequence analysis of the ade6 gene of Schizosacchaiomyces pombe: Wild-type and mutant alleles including the recombination hot spot allele ade6-M26. J. Mol. Biol. 204:917-925.

Szostak, J.W., T.L. Orr-Weaver, R.J. Rothstein, and F.W. Stahl. 1983. The double-strand-break repair model for recombination. Cell 33: 25-35.

Tavassoli, M., M. Shayeghi, A. Nasim, and F.Z. Watts. 1995.





Mizuno et al.

Cloning and characterisation of the Schizosaccharomyces pombe rad32 gene: A gene required for repair of double strand breaks and recombination. Nucleic Acids Res. 23: 383-388.

Tsukiyama, T., P.B. Becker, and C. Wu. 1994. ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor. Nature 367: 525-532.

Virgin, J.B., J. Metzger, and G.R. Smith. 1995. Active and inactive transplacement of the M26 recombination hotspot in Schizosaccharomyces pombe. Genetics 141: 33-48.

Wahls, W.P. and G.R. Smith. 1994. A heteromeric protein that binds to a meiotic homologous recombination hot spot: Correlation of binding and hot spot activity. Genes & Dev. 8: 1693-1702.

Watanabe, Y., Y. lino, K. Furuhata, C. Shimoda, and M. Yama-moto. 1988. The S. pombe mei2 gene encoding a crucial molecule for commitment to meiosis is under the regulation of cAMP. EMBO J. 7: 761-767.

Wu, T.-C. and M. Lichten. 1994. Meiosis-induced double-strand break sites determined by yeast chromatin structure. Science 263: 515-518.

Zahn-Zabal, M., E. Lehmann, and J. Kohli. 1995. Hot spots of recombination in fission yeast: Inactivation of the M26 hot spot by deletion of the ade6 promoter and the novel hotspot ura4-aim. Genetics 140: 469-478.

Zenvirth, D., T. Arbel, A. Sherman, M. Goldway, S. Klein, and G. Simchen. 1992. Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae. EMBO }. 11: 3441-3447.





10.1101/gad.11.7.876Access the most recent version at doi: 11:1997, Genes Dev.

K Mizuno, Y Emura, M Baur, et al. fission yeast.

insubstitution ade6-M26 results in remodeling of chromatin structure The meiotic recombination hot spot created by the single-base

References

http://genesdev.cshlp.org/content/11/7/876.full.html#ref-list-1

This article cites 35 articles, 16 of which can be accessed free at:

License

ServiceEmail Alerting

click here.right corner of the article or

Receive free email alerts when new articles cite this article - sign up in the box at the top

Copyright © Cold Spring Harbor Laboratory Press


http://genesdev.cshlp.org/lookup/doi/10.1101/gad.11.7.876

http://genesdev.cshlp.org/content/11/7/876.full.html#ref-list-1

http://genesdev.cshlp.org/cgi/alerts/ctalert?alertType=citedby&addAlert=cited_by&saveAlert=no&cited_by_criteria_resid=protocols;10.1101/gad.11.7.876&return_type=article&return_url=http://genesdev.cshlp.org/content/10.1101/gad.11.7.876.full.pdf



The meiotic recombination hot spot created by the single-base...

Documents

Transcript of The meiotic recombination hot spot created by the single-base...