the ade6 gene of the fission yeast schizosaccharomyces pombe has the same chromatin structure in the...

YEAST VOL. 7: 547-558 (1991)

The ade6 Gene of the Fission Yeast Schizosaccharomyces pombe has the same Chromatin Structure in the Chromosome and in Plasmids FABIO BERNARDI, THE0 KOLLER AND FRITZ THOMA*

Institut f u r Zellbiologie. ETH-Honggerberg, CH-8093 Zurich, Switzerland

Received 18 December 1990; revised 8 February 1991

We have analysed the chromatin structure of the ade6 gene of Schizosaccharomyces pombe and its flanking regions both in the chromosome and in plasmids. The chromatin structure is independent of the chromosomal or extrachromosomal location. The ade6 gene contains eight precisely positioned nucleosomes on the 5’ half, ‘not positioned’ nucleosomes around the 3’ end and a nuclease-sensitive promoter region. Precisely positioned nucleosomes, but no nuclease-sensitive region were also detected on the ura4 gene in the chromosome and on a plasmid. The results show that S. pombe chromosomal and extrachromosomal genes have chromatin structures similar to those of S. cerevisiae and higher eukaryotes.

KEY WORDS - chromatin, yeast, Schizosaccharomyces pombe, ade6 gene

INTRODUCTION Schizosaccharomyces pombe has become a popular organism to study cell biology. It shares the power- ful combination of well-established classical and molecular genetics methods with the budding yeast Saccharomyces cerevisiae, but it is considered to be more closely related to larger eukaryotes than S. cerevisiae, since the chromosomes condense during mitosis and meiosis (Robinow, 1977; Umesono et al., 1983; Erard and Barker, 1985) and since the structures of the centromeres and autonomous rep- licating sequences (putative origins of replication) are more complicated in S. pombe than those of the budding yeast (Fishel et al., 1988; Chikashige et al., 1989; Maundrell et al., 1988). A convenient molecular genetics approach was initiated by establishing a transformation protocol and by constructing DNA sequences which can replicate extrachromosomally and which can be used as vectors (Beach and Nurse, 1981; Losson and Lacroute, 1983). To understand the molecular basis of gene expression, replication, recombination or DNA repair, we need a detailed understanding of the chromatin structures. In contrast to S. cerevisiae, little is known about the structure and function of S. pombe chromatin. The presence of nucleosomes with a repeat length of approximately 160-1 70 bp was reported (Chikashige *Addressee for correspondence.

0749-503X/991/060547-12 $06.00 0 1991 by John Wiley & Sons Ltd

et al., 1989). The core histone proteins have been identified (Whiteside and Plocke, 1988) and their genes have been cloned (Choe et al., 1985; Matsumoto and Yanagida, 1985). However, to our knowledge, no chromatin structures of genes have been reported, nor is it known whether extrachromosomal DNA is organized in chromatin structures. In this paper, we have analysed the chromatin structure of the ade6 gene (Szankasi et al., 1988) and part of the ura4 gene (Bach, 1987; Grimm et al., 1988), both in chromosomes and in plasmids. They contain the typical features of higher eukaryotic chromatin, namely precisely positioned nucleosomes, ‘not positioned’ nucleosomes and nuclease-sensitive regions (NSR; ade6 only). The chromatin structures of the genes are independent of their chromosomal or extrachromosomal location.

MATERIALS AND METHODS

Enzymes and chemicals

Restriction enzymes, micrococcal nuclease (MNase) and Proteinase K were from Boehringer Mannheim. Ficoll 400 and PEG4000 were from Fluka AG and Zymolyase-IOOT from Seikagaku Kogyo Co. Ltd.

F. BERNARDI, T. KOLLER AND F. THOMA

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ADE6 GENE OF THE FISSION YEAST 549

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Figure 2. Chromatin structure of the genomic ade6 gene and its flanking regions. (A) A restriction map (according to Szankasi el u/., 1988) with the relevant restriction sites and hybridization probes (a, b) is shown. The arrow indicates the open reading frame of ude6. (B) Positioned nucleosomes (circles 1-8). not positioned nucleosomes (overlapping circles), the nuclease-sensitive region at the 5’ end (big arrowheads) and the relevant cutting sites for MNase (arrowheads) are indicated. The approximate positionsoftheMNasecuttingsitesareat 22, 176 ,341 ,481 ,W. 778,965, 1113,1300, 1442. 1608, 1743,1895and 2035 bpfrom the Pvull site (average of two independent chromatin and mapping experiments).

Yeast strains and plusmids

The S. pombe strains used were derived from the wild-type strain 972 h- and were a gift from Dr J. Kohli (Bern, Switzerland): ura4-DI8 (Grimm et al.. 1988); leul-32; ade6-M26 (Gutz, 1971); GP352 (ade6-DII leul-32) constructed by Dr F. Ponticelli (Seattle, USA) contains a 3.7 kb deletion of ade6. The strain GP352 was transformed with pASl plasmid-DNA and called YPFB5. The strain ura4- 018 was transformed with the pCGl plasmid-DNA and called YPFR6. The pASl and pCGl plasmids were a gift from Dr J. Kohli. Schizosuccharomyces pomhe were grown according to Gutz et (11. (1974). Transformation and recovery of plasmid-DNA from S. pomhe cells were performed as described by Moreno et ul. ( 1 99 I ) . The S. cerevisiae strain was YS02 (Thoma and Omari, unpublished).

Preparation oj’crude genomic chromatin

A modified procedure according to Almer and Horz (1986) was used. Yeast strains were grown to optical densities between 1 and 1.5 at 600nm. The cells were harvested by centrifugation and washed twice in water. The volume of the pellet was estimated and the cells were resuspended in two volumes of preincubation solution (0.7 M-P- mercaptoethanol, 3 mM-EDTA (pH 8)) by vortex- ing. The suspension was shaken at 180 rpm at 30°C

for 30 min. The cells were pelleted and washed once in 50 ml 1 M-sorbitol. 5 ml/g (wet weight) of 1 M- sorbitol were used to resuspend the pellet. 2mg Zymolyase-I00 T/g cells were added to convert the yeast cells to spheroplasts (1&15 min at 30°C). All subsequent steps were carried out between 0°C and 4°C. The spheroplasts were pelleted, washed once in 50 ml of 1 M-sorbitol and finally resuspended in 7 ml lysis solution/g cells ( 1 8% Ficoll, 20 mM-potassium phosphate, 1 mM-MgCI,, 0.25 mM-EGTA, 0.25 mM- EDTA, adjusted to pH 6.8 with KOH; PMSF was added to 1 mM immediately before use). After centrifugation at 15,000 rpm at 4°C in a SS34 rotor for 30 min, the crude nuclear pellet was resuspended in 8 ml buffer A/g cells (buffer A: 10 mM-Tris-HC1 pH 8, 150 mwNaC1, 5 mM-KCI, 1 mM-EDTA, 1 mM- PMSF). 2 ml of the suspension were digested with MNase in the presence of 5 mM-CaCI, with different amounts of enzyme (normally 0,7,50,225 units/ml) for 5 min at 37°C. The crude nuclear pellet remained partially insoluble during digestion. The reactions were stopped with 20 mM-EDTA, 1 YO (wiv) sodium dodecyl sulphate and incubated with 1 2 3 pg Proteinase K for 2 h at 50°C. Insoluble material was removed by centrifugation (10 min in a table- top centrifuge). DNA was extracted with phenol, phenol/chloroform and chloroform, treated with RNase A, re-extracted, precipitated twice and resuspended in 200 pI TE ( 1 0 mwTris-HCI, 1 mM- EDTA, pH 8).

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Preparation of deproteinized control D N A

2 g of lysed cells (wet weight) were resuspended in 16 ml buffer A and the DNA was extracted as described above. The DNA was resuspended in 1.6 ml TE. 100 pl of DNA were added to 240 pl of buffer A and digested as described above with 3,7, 10, 15,20, 30,60 units of MNase. The optimal conditions were determined by gel electrophoresis.


genomic chromatin of S. pombe was compared side by side with the nucleosomal repeat obtained from S. cerevisiae cells (Figure IA). The repeat length of the two yeast species was similar.

It is obvious from Figure 1A that the dimer, trimer and oligomer bands of the heavily digested chromatin (225, S. pombe; 105, S. cerevisiae) were running slightly faster than the corresponding bands of the weaker digests (50, S. pombe; 52, S. cerevisiae). To quantitate the nucleosomal repeat length, we have included marker DNA in the chromatin lanes of a S. cerevisiae digest (Figure 1 B). The result shows that the molecular weight markers migrate faster above 453 bp (chromatin lanes 7 and 20) and above 298 bp (chromatin lanes 35, 52 and 105). When the nucleosomal bands were measured by the DIGIGEL program of DNASTAR with respect to the included marker DNA, the di-, tri- and tetranucleosomes of the C52 lane were 322,486 and 622 bp long. This is consistent with a repeat length of approximately 160 bp as determined by Lohr et al. (1977). The heavily digested C105 chromatin, however, yielded 301, 450 and 589 bp, respectively, which reflects a smaller repeat of approximately 150bp. Thus, it cannot be excluded that nucleosomes rearrange or slide together under the strongest digestion conditions. The shortening of the repeat length, however, does not affect the positioned nucleosomes in the aded and ura4 regions (see below), since the same positions were mapped at low and high levels of digestions.

Gel electrophoresis and indirect end-labelling

Indirect end-labelling and gel electrophoresis were performed as described by Thoma et al. (1984): 10 p1 of DNA were cut but a restriction nuclease to completion. The DNA was precipitated, dissolved in loading buffer and loaded on a 1% agarose gel (25 cm long). A molecular weight size marker con- sisting of multiples of 256 bp (Thoma et al., 1984) was always included. The gels were run overnight at 856V h in 1 xTBE/EtBr (89mM-Tris-HC1, 89 mM-boric acid, 20 mM-EDTA, 0.5 pg/ml ethidium bromide). The DNA was blotted to nitrocellulose (Schleicher and Schull) and hybridized to radioactively labelled probes (Southern, 1975; Thoma et al., 1984).

Radioactive probes

DNA fragments were labelled by the random multiprime labelling system according to the manufacturer’s conditions (Amersham).

RESULTS

A small size nucleosomal repeat in the$ssion yeast S. pombe

In order to determine the arrangement of nucleosomes in the chromosomes of the fission yeast S. pombe, cells of the strain YPFB6 were grown on selective media to late log-phase, harvested and converted to spheroplasts using Zymolyase. After lysis in Ficoll, a crude nuclear pellet was obtained by centrifugation and immediately digested with variable amounts of MNase. The purified DNA fragments were separated on a 1 YO agarose gel containing ethidium bromide. At levels of digestion corresponding to 50 and 225 units of MNase, a ladder of several broad bands was detected (Figure IA, lanes 50, 225). The bands were approximate multiples of the smallest DNA fragment, consistent with a regular array of tandemly arranged nucleosomes. The nucleosomal repeat obtained from the

The ade6 gene of S. pombe has the same chromatin structure in the chromosome and in a plasmid

In order to address the question whether nucleosomes can be precisely positioned in S. pombe, we have analysed the structure of the aded gene, which is located on chromosome I11 (Kohli et al., 1977; Figure 2A).

Crude genomic chromatin from ura4-DZ8 cells was isolated and digested with MNase. The presence of nucleosomes (nucleosomal repeat) on the ade6 gene was confirmed by hybridizing to a radioactive probe of the gene region (not shown). The positions of nucleosomes with respect to the underlying DNA sequence were determined in Ieul- 32 cells by comparing the cutting sites of MNase on chromatin (C-lanes, Figure 3) and on protein-free DNA (D-lanes, Figure 3) using the indirect end- labelling technique (Wu, 1980; Nedospasov and Georgiev, 1980). Since MNase preferentially cuts in the linker DNA between nucleosomes, protection

ADE6 GENE OF THE FISSION YEAST 551

Figure 3. Mapping of nucleosome positions on the chromosomal ade6 gene. Crude chromatin (C-lanes) and protein-free DNA (D-lanes) from S. pombe strains leul-32 (A) and ade6-M26 (B) were digested with MNase (0-225 units/ml) and the cutting sites were displayed by indirect end-labelling from the XhoI site using probe b. An interpretation of the chromatin structure is shown. Boxes indicate positions of eight nucleosomes. Arrowheads indicate nuclease-sensitive regions. Strain ade6-M26 contains a mutation in the ade6 gene (*), which maps in a nucleosomal region. The open reading frame of ade6 is indicated (long vertical arrow).

of about 140-200 bp of DNA against cutting by MNase operationally defines a positioned nucleosome (Thoma et al., 1984). Mapping from the XhoI

site revealed eight precisely positioned nucleosomes on the 5' half of ade6 (boxes in Figure 3A) flanked by a NSR at the 5' end of the gene. This region is

552 F. BERNARDI, T. KOLLER AND F. THOMA

ADE6 GENE OF THE FISSION YEAST

characterized by two strong cutting sites for MNase (arrowheads, Figure 3A). Both the positioned nucleosomes and the NSR could be detected at low levels and at high levels of digestion, which indicates that nucleosomes were stable and did not rearrange or disintegrate during digestion. The eighth positioned nucleosome could be detected on longer exposures (not shown).

A similar picture of the chromatin structure was obtained using the ade6-M26 mutant strain (Gutz, 1971; Figure 3B), which indicates that the chromatin structure of the ade6 gene is not strain specific. Furthermore, the ade6-M26 mutation, a hot spot of DNA recombination in meiotic cells (Szankasi et al., 1988), appears to map within a nucleosome (star, Figure 3B) and there is no indication of an altered chromatin structure in this region (at least in mitotic cells). Whether a change occurs in meiotic cells remains to be investigated.

Mapping of the nucleosome positions on the ade6 gene from the XbaI site (Figure 4A) confirmed the precisely positioned nucleosomes and the NSR at the 5’ end. Furthermore, it indicates an array of positioned nucleosomes in the flanking region beyond the 5’ end.

In contrast to the 5’ end of the gene, the 3’ end was not organized in precisely positioned nucleosomes (Figure 4B): mapping of the 3‘ end from the Hind111 site indicated no clear protection of MNase sites in DNA. Hybridization of a nucleosomal repeat from ura4-DZ8 cells with a probe from the 3’ region (HindIII-XbaI fragment a, Figure 2A) yielded mono- and multimers of nucleosomal bands, indicating that nucleosomes were present on the HindIII-XbaI region and on the flanking region (Figure 4C). It must be assumed that these nucleosomes occupy different positions in different cells of the population. We prefer to call that situation ‘not positioned’, in contrast to ‘precisely positioned’ when a nucleosome forms at the same place and protects the same sequence in the great majority of the cells of a population. The proposed chromatin structure of the ade6 gene in chromosome I11 of the fission yeast is summarized in Figure 2B.

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To address thequestions ofwhether extrachromosomal DNA is also packaged in nucleosomes and to what extent the chromatin structure of individual genes is maintained when a gene is placed on a plasmid, we have analysed the chromatin structure of the ade6 gene located in plasmid PAS1 (Figure 5A). PAS1 (Szankasi et al., 1988) contains the ade6 gene of S. pombe, the URA3 gene of S. cerevisiae and a putative origin of replication from pFL20 which allows extrachromosomal maintenance in S. pombe (Losson and Lacroute, 1983). Crude genomic chromatin was isolated from YPFB5 (carrying pAS1) and digested with MNase. Mapping of the nucleosome positions from the XhoI site revealed eight precisely positioned nucleosomes on the coding region of the extrachromosomal ade6 gene (boxes in Figure 5B) flanked by a NSR. This pattern in PAS1 was indistinguishable from the pattern obtained from the chromosomal copy of ade6 (Figure 3) .

Figure 5C shows an overview of the extrachromosomal ade6 structures mapped from the flanking vector sequences (EcoRV, Figure 5A). While the NSR and the positioned nucleosomes can easily be identified in the top part of the gel, the 3’ end of the ade6 gene again does not show any indication of precisely positioned nucleosomes. In conclusion, the structure of the ade6 gene is similar in pASl and in chromosome 111. Furthermore, the experiment demonstrates that the extrachromosomal DNA is packaged into nucleosomes in a similar way as the chromosomal DNA.

Although both extrachromosomally and chromo- somally located ade6 genes showed the same clear protection to MNase in strongly digested samples, we noticed additional bands similar to those of deproteinized DNA in weaker digests of the extrachromosomal ade6 (7 units, Figure 5B). We cannot exclude that some of the high copy number plasmids are not regularly packaged into nucleosomes. A proteolytic degradation, however, is unlikely, since no such phenomenon was observed by mapping the chromosomal copy of ade6 in similar chromatin preparations.

Figure 4. Chromatin structure of flanking regions of the chromosomal ade6 (A, B). The MNasecutting sites are displayed by indirect end-labelling from the XbaI site (A) and the Hind11 site (B; using probe a). The positions of nucleosomes (boxes) and nuclease- sensitive regions (arrowheads) are indicated. Chromatin, C-lanes; protein-free DNA, D-lanes. The presence of a nucleosomal repeat around the 3’ end of ade6 is shown (C). Chromatin digested with 225 units/ml of MNase was loaded on a 1 % agarose gel without restriction enzyme treatment, blotted to nitrocellulose and hybridized to probe a (Figure 2A). DNA size markers are included (M). Chromatin preparations were made from strain uru4-DI8 (A, C), S. pombe led-32 (B).

ADE6 GENE OF THE FISSION YEAST

Evidence for the same chromatin structure of the episomal and chromosomal ura4 gene

As a second example, we have analysed the chromatin structure of the ural gene in chromosome I11 ( S . pombe led-32) and on a plasmid pCGl.pCG1 contains the ural gene of S. pombe (Bach, 1987) inserted in pUC8, and it is maintained extrachromosomally in S. pombe (Grimm and Kohli, 1988). S. pombe ura4-DZ8 was transformed with pCGl to give YPFB6.pCGl is present in low copy numbers (not shown). A restriction map of the ural gene is shown in Figure 6C.

Crude genomic chromatin was isolated from S . pombe leul-32 and YPFB6, digested with MNase and the nucleosome positions were mapped from the EcoRV site (Figure 6A). Four positioned nucleosomes were detected in the 5‘ half of the gene (boxes, Figure 6A), both in the chromosomal copy (C-lanes) and on the plasmid pCGl (C*-lane), which indicates that the structure of the ura4 gene is similar in the chromosome and on the plasmids. Surprisingly, no NSR was detected at the 5‘ and 3’ ends of ura4.

Three nucleosome positions were detected flanking the 5‘ end in the chromosome. Further upstream no clear protection pattern was recorded (compare upper part of C-lanes and D-lanes, Figure 6A), indicating multiple or random nucleosome positions. Furthermore, an indication of three NSRs (black points in Figure 6A) upstream of the ura4 gene was identified. The 3’-flanking region of ural in the chromsosome shows an array of positioned nucleosomes and two NSRs (indicated as three arrowheads in Figure 6B), which presumably belong to an unknown flanking gene.

DISCUSSION

We have shown that S. pombe chromatin contains a nucleosomal repeat similar to that of S. cerevisiae. Our measurements of 160 bp obtained by inclusion of marker DNA in the chromatin lane are consistent with the reported repeat lengths of 160 bp for S. cerevisiae (Lohr et al., 1977) and the estimated size for S. pombe (16C170 bp, Chikashige et al., 1989). Moreover, the local repeat length of the eight pos-

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itioned nucleosomes on ade6 calculated from Figure 2B (2035-778]/8 = 157) is also close to 160 bp.

We noticed a shortening of the repeat length during the strongest digestion conditions which yields mono- and small oligonucleosomes. Although we were careful to use ‘non-sliding’ conditions (low temperature and low ionic strength during preparation and short, but constant times during digestion at 37°C; Spadafora et al., 1979), we cannot formally exclude a rearrangement of nucleosomes under those conditions. We have shown, however, that nucleosome positions in the chromosomal and extrachromosomal ade6 and ura4 genes were stable and not affected by the extent of digestion. It was shown previously that mapping of MNase cutting sites is sufficiently sensitive to detect rearrangement or disintegration of unstable nucleosomes (e.g. in the TRPl gene of the TRPlARSl circle of S. cerevisiae; Thoma et al., 1984).

Assuming that a histone H1-containing nucleosome contains at least 168 bp DNA (McGhee and Felsenfeld, 1980), the DNA of an average nucleosome in S. pombe and S. cerevisiae is too short to accommodate a histone H1. In this light, it appears not surprisingly that no histone H1 -like protein was found either in S. pombe or in S. cerevisiae (Matsumoto and Yanagida, 1985; Perez-Ortin et al., 1989). Although chromosome condensation has been an argument for the presence of H 1, there is no direct experimental evidence to support that hypothesis. The major role of H1 is in organizing the 30 nm chromatin fibre (Thoma et al., 1979).

We have demonstrated that nucleosomes can be positioned in S.pombe (e.g. 5’ region of ade6) or they can occupy multiple or random locations (e.g. 3‘ end of ade6). Furthermore, NSRs were found in the ade6-promoter region and outside of ura4. Based on these few examples, S. pombe chromatin has the characteristic features of eukaryotic chromatin.

In S . cerevisiae, the chromatin structures of the SUC2 and LEU2 genes were demonstrated to be independent of the chromosomal or extrachromo- soma1 location (Perez-Ortin et al., 1987; Martinez- Garcia et al., 1989). Extrachromosomal DNA can be maintained in S. pombe, provided that it contains an S. pombe-specific origin of replication (Beach and Nurse, 1981). We demonstrated that the extrachromosomal DNA is packaged in nucleosomes

Figure 5 . Chromatin structure of the nde6 gene located on a plasmid pASl. Chromatin (C-lanes) and protein-free DNA (D-lanes) were prepared from S.pombe strain WFBS, digested with MNase and the cutting sites are displayed by indirect end-labelling from the XhoI site (B) or EcoRV site (C). A map ofpASl is shown (A). Boxes indicate positioned nucleosomes.

556 F. BERNARDI. T. KOLLER AND F. THOMA

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Figure 6 . Chromatin structure ofura l gene located in the chromosome (strain S.pombe leul-32) or on a plasmid (pCG1; strain YPFB6). Chromatin (C-lanes) and protein-free DNA (D-lanes) were digested with MNase and the cutting sites are displayed using indirect end-labelling from EcoRV (A) and StuI (B), respectively. C- and D-lanes represent the chromosomal copy, C* the extrdchromosoma~ copy of ural . A map of the chromosomal ural gene and the relevant restriction sites (according to Grimm and Kohli, 1988) is given in panel C. The arrow indicates the open reading frame of the ural gene. Black points (A) and arrowheads (B) indicate nuclease-sensitive regions upstream and downstream of the genomic ural gene. Boxes reflect positioned nucleosomes.

ArlEn GENE OF THE FISSION YEAST

and that the extrachromosomally located ade6 and ural genes show chromatin structures indistinguishable from the chromosomal copy. It appears that, in both yeasts, the chromatin structure of a homolo- gous gene is largely independent of its immediately flanking structures. Therefore, plasmids can be used to investigate the molecular mechanisms of gene regulation, replication, transcription and DNA- repair in S . pombe.

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ACKNOWLEDGEMENTS

We thank Drs J. Kohli and C. Grimm for generous gifts of strains and plasmids. We thank Drs Furter-Graves, Lucchini, Sogo and Zatchej for comments during the preparation of the manu- script. We are grateful to H. Mayer-Rosa for the artwork. This work was supported by grants to F.T. from the Swiss National Science Foundation, the Sandoz-Stiftung zur Forderung der Medizinisch- Biologischen Wissenschaften and the Prof. Dr Max Clogtta Foundation.

REFERENCES Almcr. A. and Hbrz, W. (1986). Nuclease hypersensitive

regions with adjacent positioned nucleosomes mark the gcnc houndarics of PHOSIPH03 locus in yeast.

Bach, M.-L. (1987). Cloning and expression of the O M P dccarboxylase gene URA4 from Schizosaccharomyces ponibe. Curr. Ganet. 12,527-534.

Reach. D. and Nurse, P. (1981). High-frequency transformation of the fission yeast Schizosaccharomyces pombe. Nature (London) 290, 140-142.

Chikashige, Y., Kinoshita, N.,Nakaseko,Y.,Matsumoto, T.. Murakami, S., Niwa, 0. and Yanagida, M. (1989). Composite motifs and repeat symmetry in S. pombe centromeres: direct analysis by integration of Not1 restriction sites. Cell57, 739-751.

Choc, J. , Schustcr, T. and Grunstein, M. (1985). Organis- ation, primary structure, and evolution of histone H2A and H2B genes of the fission yeast Schizosaccharomyces pomhe. Mol. Cell. B id . 5, 326 lL3269.

Erard. M. and Barker, D. G. (1985). Electron micro- scopic studies of condensed mitotic chromosomes in the fission yeast Schizosaccharomycespomhe. Biol. Cell. 5 5 2 7 - 34.

Fishel. B., Amstutz, H., Baum, M., Carbon, J. and Clarke. L. (1988). Structural organization and func- tional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pomhe. Mol. Cell. B i d . 8, 754 763.

Grimm, C., Kohli, J., Murray, S. and Maundrell, K. ( 1988). Genetic engineering of Schizosaccharomyces pomhe: a system for gene disruption and replacement

EMBO J . 5,2681-2687.

using the ural gene as a selectable marker. Mol. Gen. Genet. 215.81-86.

Grimm, C. & Kohli, J . (1988). Observations on inte- grative transformation in Schizosaccharomyces pombe. Mol. Gen. Genet. 215,87-93.

Gutz, H. (1971). Site specific induction of gene conversion in Schizosaccharomyces pombe. Genetics 69 ,3 17-337.

Gutz, H., Heslot, H., Leupold, U. and Loprieno, N. (1974). Schizosaccharomyces pornbe. In King, R. C. (Ed.), Handbook ofCenetics. Plenum Press, New York, pp. 395446 .

Kohli, J., Hottinger, H., Munz, P., Strauss, A. and Thuriaux, P. (1977). Genetic mapping in Schizosac- charomycespombe by mitotic and meiotic analysis and induced haploidization. Genetics 87,47 1 4 8 9 .

Lohr, D., Kovacic, R. T. and Van Holde, K. E. (1977). Quantitative analysis of digestion of yeast chromatin by staphylococcal nuclease. Biochemisrry 16 ,46347 1.

Losson, R. and Lacroute, F. (1983). Plasmid carrying the yeast OMP decarboxylase structural and regulatory genes: transcription regulation in a foreign environ- ment. Cell 32,371-377.

Martinez-Garcia, J. , Estruch, F. and Perez-Ortin, J. E. (1989). Chromatin structure of the 5’ flanking region of the yeast LEU2 gene. Mol. Gen. Genet. 217,464470.

Matsumoto, S . and Yanagida, M. (1985). Histone gene organisation of fission yeast: a common upstream sequence. EMBO J. 4.3531-3538.

Maundrell, K., Hutchison, A. and Shall, S. (1988). Sequence analysis of ARS elements in fission yeast.

McGhee, J. D. and Felsenfeld, G. (1980). Nucleosome structure. Ann. Rev. Biochem. 49, 1 1 15- 1 156.

Moreno, S., Klar, A. and Nurse, P. (1991). An introduction to molecular analysis of the fission yeast Schizosaccharomyces pombe. Methods in Enzymology

Nedospasov, S. A. and Georgiev, G. P. (1980). Non- random cleavage of SV40 DNA in the compact mini- chromosome and in free solution by micrococcal nuclease. Biochem. Biophys. Res. Comm. 92,532- 539.

Perez-Ortin, J . E., Estruch, F., Matallana, E. and Franco, L. (1987). Fineanalysisofthechromatinstructureofthe yeast SUC2 gene and of its changes upon derepression. Comparison between the chromosomal and plasmid- inserted copies. Nucleic Acids Res. 15,6937-6954.

Perez-Ortin, J. E., Matallana, E. and Franco, L. (1989). Chromatin structure of yeast genes. Yeast 5,219-238.

Robinow, C. F. (1977). The number of chromosomes in Schizosaccharomyces pombe: light microscopy of stained preparations. Genetics 87,491497.

Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98,503-51 7.

Spadafora, C., Oudet, P. and Chambon, P. (1979). Rearrangement of chromatin structure induced by increasing ionic strength and temperature. Eur. J. Biochem. 100,225-235.

EMBO J. 7,2203-2209.

194,793-823.

558

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

Thoma, F., Koller, T. and Klug, A. (1979). Involvement of histone HI in the organization of the nucleosome and of the salt-dependent superstructure of chromatin. J. Cell. Biol. 83,403427.

Thoma, F., Bergman, L. W. and Simpson, R. T. (1984). Nuclease digestion of circular TRpl ARSl chromatin reveals positioned nucleosomes separated by nuclease sensitive regions. J. Mol. Biol. 177,715-733.


Umesono, K., Hiraoka, Y., Toda, T. and Yanagida, M. (1983). Visualization of chromosomes in mitotically arrested cells of the fission yeast Schizosaccharomyces pombe. Curr. Genet. 7, 123-128.

Whiteside, S. G. and Plocke, D. J. (1988). Identification and isolation of core histones from Schizosaccharo- myces pombe. FEBS Letters 266,250-254.

Wu, C . (1980). The 5’ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I. Nature (London) 286,854-860.

the ade6 gene of the fission yeast schizosaccharomyces pombe has the same chromatin structure in the...

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