pombe Are Highly Variable Genetic Loci

MOLECULAR AND CELLULAR BioLOGy, Aug. 1993, p. 4578-4587 Vol. 13, No. 80270-7306/93/084578-10$02.00/0Copyright © 1993, American Society for Microbiology

Centromeres of the Fission Yeast Schizosaccharomycespombe Are Highly Variable Genetic Loci

NOEMI C. STEINER, KAREN M. HAHNENBERGER,t AND LOUISE CLARKE*Department ofBiological Sciences, University of California, Santa Barbara, California 93106

Received 30 November 1992/Returned for modification 11 February 1993/Accepted 30 April 1993

Gross variations in the structure of the centromere of Schizosaccharomyces pombe chromosome II (cen3)were apparent following characterization of this centromeric DNA in strain Sp223 and comparison of thestructure with that ofcen3 in three other commonly used laboratory strains. Further differences in centromerestructure were revealed when the structure of the centromere ofS. pombe chromosome H (cen2) was comparedamong common laboratory strains and when the structures ofcen2 and cen3 from our laboratory strains werecompared with those reported from other laboratories. Differences observed in cen3 structure includevariations in the arrangement of the centromeric K repeats and an inverted orientation of the conservedcentromeric central core. In addition, we have identified two laboratory strains that contain a minimal cen2repeat structure that lacks the tandem copies of the cen2-specific block of K-L-B-J repeats characteristic ofSp223 cen2. We have also determined that certain centromeric DNA structural motifs are relatively conservedamong the four laboratory strains and eight additional wild-type S. pombe strains isolated from various foodand beverage sources. We conclude that in S. pombe, as in higher eukaryotes, the centromere of a particularchromosome is not a defined genetic locus but can contain significant variability. However, the basic DNAstructural motif of a central core immediately flanked by inverted repeats is a common parameter of the S.pombe centromere.

The centromere (cen) locus is a specialized region ofeukaryotic chromosomal DNA that acts in conjunction withspecific proteins to mediate chromosome segregation duringmitotic and meiotic cell divisions. Centromere functionsinclude maintenance of sister chromatid attachment duringmeiosis I and interaction, via the spindle attachment site orkinetochore, with mitotic and meiotic spindles, therebyensuring proper segregation of chromosomes during celldivisions.Centromere DNAs of the budding yeast Saccharomyces

cerevisiae have been extensively characterized, are onlyabout 125 bp in length, and contain no repeated DNAsequences (5, 8). Although analogous with respect to func-tion, they appear structurally to be quite unlike the cen-tromeres of higher eukaryotes, which in many cases containextensive regions of heterochromatic repeated DNA, whoserole is as yet unclear (2, 25). The genome of the fission yeastSchizosaccharomyces pombe is organized into only threechromosomes and provides an excellent alternate modelsystem for studying the role of the centromere in celldivision. The centromeres of S. pombe and those of highereukaryotes have significant similarities that may be a reflec-tion of their common mode of cell division. S. pombecentromeric regions are large, spanning approximately 40 to100 kb of DNA, and consist in part of repetitive, nontran-scribed DNA sequences that have been shown to play a rolein centromere function (4, 7, 9, 12, 20). In addition, S. pombecentromeres are organized into a large inverted repeat (4, 7,12) which bears structural resemblance to higher eukaryoticcentromeric heterochromatin, based on the observationmade 50 years ago that chromomeres in the pericentricheterochromatin of many plant chromosomes are arranged

* Corresponding author.t Present address: Molecular Devices Corp., Menlo Park, CA

94025.

in a bilaterally symmetrical pattern about a central point(17).The three functional centromeric DNAs (cenl, cen2, and

cen3) from S. pombe Sp223 have been cloned on SalIfragments (65, 100, and 150 kb, respectively) into yeastartificial chromosomes (YACs) (11). The structures of cenl(-40 kb) and cen2 (-80 kb) of strain Sp223 have beenextensively analyzed by restriction mapping, DNA sequenc-ing, and determination of chromatin structure (7, 12, 15, 26).Recently, S. pombe cen3 (-100 kb) from other laboratorystrains has been structurally characterized by a long-rangegenomic mapping strategy in conjunction with cosmid walk-ing (4, 20). That study indicates that cen3 is composed of aninverted repeat structure with centromere-specific repeatsdg and dh (also designated together as repeat K [6]) orga-nized roughly symmetrically and in tandem on each arm ofthe inverted repeat.A large portion of the central core region ofcenl (ccl [Fig.

1]) is homologous by Southern analysis to sequences con-tained on the 150-kb genomic cen3 SalI fragment from strainSp223 (12). This homology was utilized with a plasmidintegration-excision strategy (7) to initiate the structuralcharacterization of cen3 from strain Sp223 described in thisreport. In the course of this analysis, differences among thestructures of cen3 in different laboratory and wild-typestrains of S. pombe were revealed. These differences, as wellas variations in centromere structure in earlier reports fromother laboratories (4, 7, 12), led us to analyze also thestructures of cenl and cen2 among different S. pombestrains. We have found that in S. pombe, as in highereukaryotes, the centromere of a particular chromosome isnot a defined genetic locus but can contain significantvariability. Certain structural elements of the S. pombecentromere are relatively conserved, while other elementsare highly variable. All S. pombe centromeres analyzed arecomposed of a central core region flanked by an adjacent

4578

VARIABILITY OF S. POMBE CENTROMERE STRUCTURE 4579

P.

K' L K B' 1c B' K L K'I ceni

H- 38kb-|-00 - p .-

BJ K L BJ K LB cc2 B L K B

$W3g Icen2

|. 65kb -*|-laZ 49.7 kb 9-

BstEII BstEI

*- *+ Z *444*

M K LI cc3 LI K K K K K K K K K K K M_cI~~ cen3

NcoI 97kb

25kb 80kbSailI NcoI NcoI Sail

near Sal site far Sal site

FIG. 1. Schematic representation of the centromeric DNAs from chromosome I (cenl), II (cen2), and III (cen3) of S. pombe Sp223. Thestructure of cenl from strain Sp223 was published previously (12). Characterization of the structure of cen2 in strain Sp223 is described inResults. The 49.7-kb BstEII fragment that includes a portion of cc2 and the left arm ofcen2 is shown. The structure of cen3 was characterizedby plasmid integration-excision strategies and indirect end-labeling methods with cen3 probes. All three centromeres consist of a chromosomespecific central core flanked by centromere-specific repeated sequences arranged in an inverted fashion. The 6.4-kb K repeats in cen3 aresimilar to the K repeats in cen2 with respect to orientation and structure. The L' repeat in cen3 is in the same orientation as the L repeat incen2, but L' is a truncated (-3-kb) version of L. The M repeats are largely cen3 specific but are thought to contain tRNA genes (seeDiscussion). Thus, they may be somewhat analogous to the B and B" repeats of cen2 and cenl, respectively (15). The 5.2-kb core-associatedrepeats in cen3 (represented by black boxes) that immediately flank the 5.4-kb central core (cc3) are chromosome specific, as are the repeatsdirectly flanking cc2 and ccl (7, 12). cc3 contains approximately 4 kb of homology to ccl (20). Sall and NcoI restriction sites within cen3 areshown, along with the location of an NcoI site immediately adjacent to the proximal SalI site.

inverted repeat. The size of this basic structural unit isgenerally conserved. However, the orientation of the centralcore sequences with respect to flanking markers as well asthe orientation and number of certain classes of centromere-specific repeats are variable.

MATERIALS AND METHODSStrains, transformations, and genetic manipulations. The S.

pombe laboratory strains used in this study are 972 (h-s) and975 (h+"), which are the heterothallic isolates derived fromthe original Osterwalder Swiss S. pombe strain liquefaciensand introduced into the laboratory by Leupold in 1950 (16),and the auxotrophic strains Sp223 (h-s leul-32 ura4-294ade6-216; gift from D. Beach) and SBP120390 (h-s ade6-704ura4-294 leul-32), both of which are derivatives of 972 and975. The S. pombe wild-type strains, isolated from a varietyof locations and referred to here as WT1 to WT8 (gift fromH. Levin), have previously been described in detail byZimmer et al. (32). Briefly, they are as follows: IGC 2769(WT1; Portugal), NCYC 132 (WT2; England), CSIR Y-457(WT13; Swaziland), 0202 (WT4; Germany), 0209 (WT5; Ger-many), 0342 (WT6; Japan), EF1 (W17; Czechoslovakia),and EF4 (WT8; Japan). They were obtained from an assort-ment of foods (cane sugar molasses [WT6 and WT8J and redcurrant jelly [WT5]) and drink (Bantu sorghum beer [WT31).The S. cerevisiae strain used in this study is GTSC14 (a ura3his4 trpl leu2, provided by G. Tschumper and J. Carbon).GTSC141A contains the YAC pSp(cen3)-1OC (11), whichcarries the 150-kb cen3 SalI fragment from S. pombe Sp223.Growth media and conditions for S. pombe (10) and S.cerevisiae (27) were as previously described. DNA transfor-mations were carried out as previously described (13).Plasmids were integrated by site-directed homologous re-

combination (24) into either S. pombe Sp223 or S. cerevisiaeGTSC141A.Enzymes, DNA isolation, plasmid rescues, and FIGE. Re-

striction enzymes, T4 DNA ligase, and calf intestinal alka-line phosphatase were purchased from New England Bio-Labs or Boehringer Mannheim Biochemicals and were usedaccording to the vendors' instructions. Procedures for Esch-erichia coli transformations, cloning techniques, and plas-mid isolations were as described by Maniatis et al. (18). S.pombe DNA was prepared by the method of Beach and Klar(la), using cutoff micropipette tips for all procedures.Spheroplasting of cells was accomplished by using Zymol-yase lOOT purchased from ICN Biomedicals Inc. Plasmidswere excised from genomicDNA prepared from S. pombe orS. cerevisiae integrant strains by the method of Clarke andBaum (7). Excised plasmids were recovered in E. coli DH5a(recAl; Bethesda Research Laboratories). Field inversiongel electrophoresis (FIGE) was carried out as describedpreviously (3) in 1% agarose gels at 180 V for 17 h with alinear ramp that was varied depending on the sizes of thefragments being separated. High-molecular-weight DNAmarkers from Bethesda Research Laboratories were used assize standards.

Partial enzymatic digestions and Southern analyses.Genomic DNAs were restricted for Southern analyses firstby complete digestion with 10 to 50 U of restriction enzymeper jig of DNA for 3 h at 37C and then by partial digestionwith serial dilutions of a second enzyme and incubation for20 min at 37°C (18). Thus, DNA samples which weredigested to completion with one enzyme and partially di-gested to various degrees with a second enzyme weregenerated. The series of restricted DNAs was subjected toFIGE and Southern blotted, and data from the samples

VOL. 13, 1993

4580 STEINER ET AL.

A.

M K L' L' Kcc3

KS KSC K Xb S CCl1 II Il

BgEEBgNBg N E

2A

1-K-opp

1-S

2-S

B C Nc S C B CC S Xb KII I I II )

I I I II 1III kBg Bg E N BgN BgEEBg

2B

1-K

1 kb

B.

K K M

EI

K

Hkb Bt1kb 3B

E

Nc Nc Nc S

3-S

FIG. 2. Restriction map of 49 kb of the cen3 locus of S. pombe Sp223. (A) Four sites of integration (indicated by vertical arrows) were

used in a plasmid integration-excision strategy that led to the characterization of 40.5 kb of cen3 sequences flanking cc3. All integrations(except 3A and 3B) were into an S. cerevisiae YAC containing the 150-kb cen3 SalI fragment (11), and excision plasmids were rescued in E.coli. A 2.3-kb EcoRI-HindIII fragment from ccl (see Fig. 1) was used to target plasmids to integration site 1, leading to the generation of theKpnI excision plasmids 1-K and 1-K-opp and the Sail excision plasmid 1-S. A 2.3-kb NheI fragment from excision plasmid 1-K was used totarget integrations to sites 2A and 2B, and the SphI excision plasmid 2-S was generated from the 2A integrant. A 1.6-kb EcoRI fragment fromexcision plasmid 1-S was used to target integrations to sites 3A and 3B in Sp223 genomic DNA. (B) Approximately 8.5 kb of cen3 sequences(from the region indicated by an open box in Fig. 1) were characterized following integration into site 3B and excision of plasmid 3-S withSphI. Restriction sites: Bg, BglII; C, ClaI; E, EcoRI; K, KpnI; N, NheI; Nc, NcoI; S, SphI; Sal, Sall; Xb, XbaI.

giving the most informative partial digestion patterns arepresented here. FIGE gels were blotted to Zeta-probe GTblotting membranes purchased from Bio-Rad Laboratories,and solution conditions used for blotting were as specified bythe membrane manufacturer. A Posiblot pressure blotterpurchased from Stratagene Cloning Systems was used forDNA transfers. Blotting was for 1 h at 75 mm Hg. Trans-ferred DNA was UV cross-linked to membranes by using a

GS Gene Linker purchased from Bio-Rad Laboratories.32P-labeled probes were prepared from gel-purified frag-ments, isolated with Geneclean purchased from Bio 101, andlabeled by random priming using a Prime-it kit from Strata-gene Cloning Systems.

RESULTS

Use of a plasmid integration-excision strategy to map 49 kbof Sp223 cen3 DNA. The plasmid integration-excision strat-egy (7) involves site-directed integration by homologousrecombination of a pBR322-derived plasmid carrying an S.pombe selectable marker into the host chromosome or anartificial chromosome at a region of interest. Total DNAfrom the integrant strain is then digested with variousrestriction enzymes to excise the plasmid and surroundingsequences, which are subsequently circularized by ligationin vitro, rescued in E. coli, and analyzed. This strategy hasbeen used to determine the structural organization of the S.pombe cenl (12) and cen2 (7) regions. Because cen3 fromstrain Sp223 was known to contain tandem repeats (6), aunique integration-excision scheme was devised in order to

facilitate the subcloning of sequences from this large, par-tially repetitive chromosomal locus.We constructed an integration plasmid, pSp(cenl)HBL,

that contains a 2.3-kb EcoRI-HindIlI fragment from ccl (12)(Fig. 1) as well as unique SalI and KIpnI sites. The 2.3-kbEcoRI-HindIlI fragment had previously been shown bySouthern analysis to share homology with sequences in the150-kb cen3 SalI fragment (12). We postulated that these cclsequences were homologous to sequences in cc3 (Fig. 1).The integration of pSp(cenl)HBL was targeted to cen3sequences specifically (Fig. 2A, integration site 1) by usingthe linearized plasmid to transform S. cerevisiaeGTSC141A, which carries the 150-kb S. pombe cen3 Sallfragment cloned into a YAC vector (11; Materials andMethods). Because the integration plasmid contains a uniqueSall site, the integrity of transformants could be checkedinitially by mapping the newly introduced SalI site in thecloned 150-kb SalI fragment (7). The transformant DNA wasthen used to generate a 20-kb excision plasmid with therestriction enzyme ICpI. This excision plasmid, 1-K (Fig.2A), was rescued in E. coli for subsequent analysis.To walk in the opposite direction along the cloned cen3

DNA, we constructed a derivative of the integration plasmidpSp(cenl)HBL in which the homologous cenl-derived se-

quences used for site-directed recombination into cen3 weresubcloned into the integration plasmid in the opposite orien-tation. This second plasmid, pSp(cenl)HBL-opp, was inte-grated into S. cerevisiae GTSC141A (Fig. 2A, integrationsite 1), and the integrity of the integration event was verifiedby mapping the newly introduced SalI site as described

CCKN ,,,,1

Sal E Bg E E

t3A

MOL. CELL. BIOL.

!

VARIABILITY OF S. POMBE CENTROMERE STRUCTURE 4581

above. Transformant DNAwas digested with Sall orKpnI inorder to generate excision plasmid 1-S (30 kb) or 1-K-opp (20kb), respectively (Fig. 2A), both of which were rescued in E.coli and subsequently mapped. Together, the three excisionplasmids permitted the analysis of 40.5 kb of cen3 DNAstructure. This 40.5-kb region was further characterized bySouthern analysis using probes derived from the region aswell as probes from previously cloned S. pombe centromericDNA repeats (7; data not shown).The Sp223 cen3 region mapped by the plasmid integration-

excision strategy consists of a 5.4-kb central core flanked oneach side by 12.4 kb of inverted repeat, including a cen3-unique core-associated repeat and a K repeat and part of anL repeat that are common to all three S. pombe centromeres(Fig. 2A). The L repeat unit found in cenl and cen2 (Fig. 1)has also been designated repeat dh by Nakaseko et al. (22).In cen3, we have designated this repeat L' because it is atruncated (-3.0 kb) derivative of the 5-kb centromeric Lrepeat. Additionally, the region analyzed includes sequencesdirectly adjacent to the Sall site on excision plasmid 1-S(Fig. 2A; 1.8-kb SalI-KpnI fragment, indicated by an openbox), which are unique and, based on functional studies ofcenl and cen2 (7, 12), are likely to be noncentromeric (datanot shown). Because the centromere is asymmetrically situ-ated on the 150-kb cen3 Sall fragment, with the central core25 kb from one Sall site and approximately 125 kb from theother, we refer to the SalI site that is closer to cc3 as the nearSal site and the site that is farther from cc3 as the far Sal site(Fig. 1).

It was evident from the mapping data that the homologybetween ccl and cen3 lies in the middle of the central core ofcen3. Because the map of cc3 (Fig. 2A) was derived fromtwo separate integration events, each of which generatedexcision plasmids that extended in opposite directions fromthe NcoI site in the middle of cc3 toward the arms of therepeats, it was necessary to confirm that genomic centralcore sequences around the NcoI integration site (Fig. 2A,integration site 1) are represented correctly by these excisionplasmids. A strategy was devised in which integration intoone of the L' repeats of cen3, using a 2.3-kb NheI fragmentfrom the excision plasmid 1-K (Fig. 2A, integration site 2A),was followed by an excision with SphI that released plasmid2-S (Fig. 2A). The integration was carried out in GTSC141A,the S. cerevisiae strain containing the S. pombe cen3 YAC,so that only the L' repeats in cen3 were targeted. Theintegration resulted in only two classes of integrants (Fig.2A, integration sites 2A and 2B), both of which representedintegration into the L' repeats directly flanking cc3, andsuggested that these L' repeats are the only copies in cen3.The excision plasmid 2-S spans the NcoI integration junctionsituated in the middle of cc3, and the restriction map ofplasmid 2-S verifies the maps generated previously for theexcision plasmids 1-K, 1-S, and 1-K-opp.To show that the DNA sequences contained in the cen3

excision plasmids were not rearranged with respect togenomic sequences, we carried out a series of multiplerestriction digests and compared the mobilities of a varietyof restriction fragments by Southern analysis from bothexcision plasmid and Sp223 genomic DNAs (data notshown). cen3 sequences (Fig. 2A, the 1.8-kb NcoI-SphIfragment) and sequences adjacent to the near Sal site (Fig.2A, the 1.8-kb SalI-KpnI fragment) were used as probes. Inthese analyses, no differences could be detected between thearrangement of sequences in the excision plasmids and theSp223 genome. Additionally, digestion of Sp223 genomicDNA with SalI and NcoI followed by Southern analysis

1 2... ..

C)+ cls), N CM O

CL 1o

r- D- r-Z

aCl) 0)31 2 34

-80 kb

.t:4 .ib

u-"Fj-17 kb

-8.6 kb

-6.2 kb13.4 kb- **Ob'

A_ -3.4 kb

A

-19.8 kb

B

FIG. 3. Differences in the organization of cen3 tandem K repeatsamong S. pombe strains. (A) The location of the cen3 tandem Krepeats of strain Sp223 was determined by complete NcoI digestionfollowed by either partial XbaI digestion (lane 1) or partial ClaIdigestion (lane 2) of genomic DNA. Conditions for complete andpartial digestion are described in Materials and Methods. Therestriction enzymes ClaI and XbaI cut only once in the K repeat;thus, partial digestion of tandem K repeats with either of theseenzymes generates a 6.4-kb ladder. DNAs were subjected to FIGEand Southern blotted. The 1.8-kb NcoI-SphI fragment from cc3 wasused as a probe (see Fig. 2A). Lanes 1 and 2 are taken from differentgels, which explains the slight discrepancy between the mobilities ofthe 6.2-kb band in the two lanes. The 6.2-kb band in lanes 1 and 2 isa result of cross-hybridization of this probe to ccl sequences. (B)The location of the tandem K repeats in different commonly usedlaboratory strains was analyzed by complete SalI digestion followedby partial XbaI digestion as described in Materials and Methods.The 1.8-kb SalI-KpnI fragment proximal to the near Sal site (see theopen box in Fig. 2A) was used as probe.

using the 1.8-kb NcoI-SphI cc3 probe or the unique 1.8-kbSall-KpnI end probe revealed the presence of either anapproximately 80-kb hybridizing band or a 25-kb hybridizingband, respectively (data not shown). Additional bands couldnot be detected with any other cen-specific probes used,including repeats J, K, L, B, and M (see below). It was thusconcluded that cen3 contains only two NcoI sites, one in themiddle of the central core and the other approximately 80 kbaway and proximal to the far Sal site (Fig. 1). This conclu-sion is supported by recently published data which appear tobe derived from a composite of S. pombe strains (20).

Restriction mapping reveals the presence of approximately12 tandem K repeats in Sp223 cen3. Because previous studieshad shown that S. pombe cen3 contains at least three tandemK repeats (6), chromosome walking through this region byan integration-excision strategy was predicted to be prob-lematic. Therefore, an indirect end-labeling approach forlong-range mapping of the tandem K repeats in Sp223 cen3was adopted. The two restriction enzymes ClaI andXbaIeach cut only once within the K repeat. We performed twoindirect end-labeling experiments (Fig. 3A) in which Sp223genomic DNA was digested completely with the enzymeNcoI and then partially digested with either ClaI or XbaI.Restricted DNAs were separated by FIGE, Southern blot-ted, and hybridized to a cc3 probe (Fig. 2A, the 1.8-kbNcoI-SphI fragment). When either Clal orXbaI was used forpartial digestion, a 6.4-kb ladder spanning approximately 75

VOL. 13, 1993

4582 STEINER ET AL.

kb of sequences was visualized following autoradiography.This 6.4-kb ladder indicates the presence of approximately12 tandem K repeats on the right arm of the inverted repeatin cen3 (Fig. 1). This asymmetrical arrangement of tandem Krepeats in Sp223, with 1 K repeat on one side of the cc3 and12 tandem K repeats on the other side of cc3, differssignificantly from the structure of S. pombe cen3, derivedwith data from several different strains and published byChikashige et al. (4), which depicts a symmetrical arrange-ment of 6 tandem K repeats on either side of the cc3, or amore recent version from the same laboratory that ascribes4 copies of K to one side of cc3 and 9 copies to the other (20).The M repeat is a new class of centromeric repeat that is

specific to the cen3 region. We attempted to develop a uniqueend probe proximal to the far Sal site in order to characterizefurther the number and organization of K repeats in strainSp223. Of interest were sequences from the S. pombegenomic cosmid library clone cSpK22 (6) that map specifi-cally to cen3 and are situated on the cosmid adjacent toportions of two tandem K repeats. When these cen3-specificsequences were used as probes on Southern-blotted Sp223genomic DNA digested with a combination of Sall and NcoI(data not shown), two hybridizing bands of 25 and 80 kbwere seen. Because this is the result expected from arepetitive cen3-specific probe (Fig. 1), it was postulated thatthese sequences, termed the M repeat, represent a new classof centromeric repeat unique to cen3. While the repetitivenature of these sequences made them of no use for indirectend-labeling experiments, we nonetheless sought to charac-terize them further. By Southern analysis of excision plas-mid 1-S DNA, one copy of the M repeat was localized to aregion proximal to the near Sal site (Fig. 2A). We speculatedthat another copy was located proximal to the far Sal siteand adjacent to the block of tandem K repeats (Fig. 1), asthis is the sequence configuration in cosmid cSpK22 (6). Thelocations of the M repeats were confirmed by targeting anintegration plasmid to the M repeats in Sp223 (Fig. 2,integration sites 3A and 3B), followed by isolation andmapping of excision plasmids (data not shown). Figure 2Bshows a map of one of the excision plasmids (plasmid 3-S)and the M repeat region proximal to the far Sal site.The structure of cen3 in strains Sp223, 972, 975, and

SBP120390 varies with respect to the organization of thetandem K repeats and the orientation of the central coresequences. Because structures of S. pombe centromeres varyin reports from different laboratories, the structure of cen3 instrain Sp223 was compared with the structure of cen3 inseveral other commonly used heterothallic laboratorystrains, including SBP120390h-s ade6-704 ura4-294 leul-32), 972 (h-s), and 975 (h' ). Strains 972 and 975, theisolates derived from the original Osterwalder S. pombestrain liquefaciens, are the standard heterothallic, prototro-phic strains used in most S. pombe research. Many S. pombeauxotrophic strains, including Sp223 and SBP120390, arederivatives of 972 and 975. The location of the tandem Krepeats in each of these different laboratory strains wasanalyzed by complete SalI digestion of genomic DNAsfollowed by partial XbaI digestion (Fig. 3B). The Southern-blotted DNAs were probed with the 1.8-kb SalI-KpnI frag-ment derived from unique DNA sequences proximal to thenear Sal site (open box in Fig. 2A). The displacement of thetandem K ladder in strain SBP120390 (Fig. 3B, lane 4) withrespect to the ladders in the other strains tested (lanes 1 to 3)indicates a different organization of the cen3 tandem Krepeats in strain SBP120390. Extensive mapping of strainSBP120390 DNA was carried out by Southern analyses to

+ Ce,0)I + C90 r)I

Oc CMc) o- o CMlC) 0LO clj qLou C\6u C\rl X- CQ r, F- a- (ucr uz - cn m CD '-

t 2 3 4 5 6 7 812345678:.

80kb-

25kb-

FIG. 4. Evidence that the cc3 regions of some S. pombe strainsare in the opposite orientation with respect to that of Sp223. In lanes1 to 4, NcoI-digested genomic DNAs from different S. pombe strainswere subjected to FIGE, Southern blotted, and probed with the0.8-kb BglII-NcoI fragment located to the left of the NcoI site in cc3(see Fig. 2A). In lanes 5 to 8, the same Southern blot was furtherhybridized to the labeled 1.8-kb NcoI-SphI fragment located to theright of the cc3 NcoI site (see Fig. 2A).

verify the cen3 structure in strain SBP120390 (data notshown). A model for the organization of the cen3 tandem Krepeats in strains Sp223, 975, 972, and SBP120390 is sum-marized in Fig. 9. However, it should be noted that the exactnumber of K repeats that occur in tandem at the cen3 locusin each of these strains has not been precisely established.

Further comparison of the cen3 loci of the four S. pombelaboratory strains was carried out by Southern analysis ofNcoI-restricted DNAs, using the 0.8-kb BglII-NcoI fragmentlocated to the left of the NcoI site in cc3 (Fig. 2A and Fig. 4,lanes 1 to 4) as a probe. Predictions were made concerningthe sizes of the Nco fragments that should hybridize to thisprobe on the basis of the proposed location of the cen3tandem K repeats in the four strains being studied and theknown locations of the NcoI sites in the cen3 regions ofstrains Sp223 and SBP120390 (Fig. 1). Because the exactnumber of K repeats organized in tandem in cen3 is notknown, only approximate sizes of the cen3-derived NcoIfragments containing tandem K repeats are given.Data identical to those shown in Fig. 4 were obtained

when genomic DNAs from the four laboratory strains weredigested with Ncol and SalI in combination (data notshown). Furthermore, when genomic DNA from strainSp223 or SBP120390 was digested with NcoI, Southernblotted, and hybridized with a DNA probe from the left sideof cc3 (Fig. 4, lanes 2 and 7), the result is identical to thatobtained when genomic DNA from these strains was di-gested with NcoI and SalI in combination, Southern blotted,and hybridized to the cen3 unique end probe (Fig. 2A, the1.8-kb SalI-IKpnI fragment) or the probe from the left side ofthe Ncol site in cc3 (data not shown). Thus, we concludedthat in strains SP223 and SBP120390, for which we hadpreviously mapped the NcoI site in cc3, there is an NcoI siteimmediately distal to the near Sal site (Fig. 1). We thenpostulated that there are NcoI sites at similar positions instrains 972 and 975. Therefore, assuming that there is an

MOL. CELL. BIOL.

VARIABILITY OF S. POMBE CENTROMERE STRUCTURE 4583

+ ° CoN

N c NN-N En

1 23 4

150 kb -100 kb -

65 kb -

+Icm CM 0

L_ rC L CM0) ) CO) -

5 6 7 8

-49.7 kb -1k^ -49.7 kb

10.4kb- ¢ ~~~10.4 kb

A BFIG. 5. Differences in the number of K-L-B repeat units present

in cen2 regions of various S. pombe strains. (A) SalI-digestedgenomic DNAs from different S. pombe strains were subjected toFIGE and Southern blotted as described in Materials and Methods.A 32P-labeled 6.4-kb ClaI fragment from the cen3 tandem K repeatregion was used as a probe. (B) Indirect end-labeling experimentswere carried out to determine the nature of the structural differencesin cen2 among different strains. Genomic DNAs were digested tocompletion with BstEII and then partially digested with SphI asdescribed in Materials and Methods. Restricted DNAs were sub-jected to FIGE and Southern blotted. A 2.3-kb SphI-BstEII cc2fragment adjacent and to the left of the cc2 BstEII site was used asa probe (7) (hatched box in Fig. 1). Lanes 5 and 6 are derived fromone gel and lanes 7 and 8 are derived from another, which explainsdiscrepancies in mobilities of the fragments.

NcoI site immediately distal to the near Sal site (data notshown and Fig. 1), the 0.8-kb BglII-NcoI fragment waspredicted to hybridize to a 25-kb NcoI DNA fragmentfrom strains Sp223, 972, and 975. However, because ofthe different arrangement of the tandem K repeats inSBP120390, the 0.8-kb BglII-NcoI probe was predicted tohybridize to an approximately 80-kb fragment from theDNA of this strain. The size of this fragment was veri-fied by hybridization of NcoI-digested, Southern-blottedSBP120390 DNA to a unique end probe (1.8-kb SalI-KpnIfragment; Fig. 2A) that flanks the left arm of the cen3 locus(data not shown). The use of the unique end probe to verifythe size of the 80-kb NcoI fragment provides additional datato support the data shown in Fig. 4, lane 7.

In Fig. 4, Sp223 (lane 2) and 972 (lane 4) DNAs both showthe predicted 25-kb band. In a separate experiment, the cen3unique end probe (1.8-kb SalI-KpnI fragment; Fig. 2A)hybridized to a 25-kb band on a Southern blot of NcoI- andSalI-digested genomic Sp223 DNA (data not shown), whichsupports the data presented for this strain in Fig. 4, lane 2.However, the hybridizing DNA fragments from strains 975(lane 1) and SBP120390 (lane 3) are approximately 80 and 40kb, respectively. These data from strains 975 and SBP120390can be explained if an inverted central core exists in thesetwo strains, relative to the orientation of the central core instrains Sp223 and 972. If an inverted central core is presentin strains 975 and SBP120390, then hybridization of NcoI-restricted DNAs to a probe from the right side of the NcoIsite in cc3 (1.8-kb NcoI-SphI fragment; Fig. 2A) shouldreveal hybridizing bands originally predicted for the probefrom the left side of the NcoI site in cc3 (0.8-kb BglII-NcoIfragment; Fig. 2A). In lanes 5 to 8 of Fig. 4, the Southern blotshown in lanes 1 to 4 was further hybridized to the 1.8-kbNcoI-SphI fragment from the right side of cc3. Sp223 (lane 6)

0) + C'I') +r N r_c N 't

1 23 4 5 6 7 8

150kb- - -

65 kb- - -t

30 kb -

t N-r- a

9 10 11 12

".

.j_'-IWO

FIG. 6. Variability in the sizes of the cenl- and cen3-containingSail fragments in various wild-type S. pombe strains. Strains from avariety of sources (see Materials and Methods) were digested withSal, subjected to FIGE, Southern blotted, and probed with the1.8-kb NcoI-SphI fragment from cc3 (see Fig. 2A). The differencesin band intensities are a result of differences in DNA concentrationsof individual samples.

and 972 (lane 8) DNAs show the approximately 80-kbhybridizing NcoI fragment that is predicted given the orga-nization of the tandem K repeats and orientation of thecentral core in these strains. The appearance of the 25-kbband in lane 5 (975 DNA) and an approximately 80 kb bandin lane 7 (SBP120390 DNA) supports the hypothesis that thecc3 sequences of strains SBP120390 and 975 are inverted inorientation with respect to the cc3 sequence in Sp223 and972. In Fig. 9, we present a model that summarizes theorientation of cc3 in the four laboratory strains studied.

In cen2, differences exist in the number of K-L-B repeatunits present among S. pombe strains. Because differenceswere found among the structures of cen3 in different strains,we proceeded to look for differences in the structures ofcen2in these same strains. In Fig. SA, SalI-digested genomicDNAs from the four S. pombe laboratory strains wereSouthern blotted and hybridized to a probe containing Krepeat sequences. As expected, centromeric repeat K se-quences hybridize to a 100-kb cen2 Sall fragment of DNAfrom strains Sp223 and 972 (lanes 3 and 4, respectively).However, these sequences hybridize to an approximately70-kb cen2 SalI fragment in DNA from strains 975 andSBP120390 (lanes 1 and 2, respectively). The cen2 identity ofthe 70- and 100-kb hybridizing Sail fragments shown in Fig.SA is verified in Fig. 6, lanes 1 to 4, in which a probe thathybridizes to both the cenl and cen3 Sail fragments is used,and the cen2 70- and 100-kb Sall fragments are not evident.Slight variations were noted in the sizes of the 150-kb cen3hybridizing fragment, and no variation in the size of the65-kb cenl hybridizing fragment was seen. We did not lookfurther into structural differences in cenl among the strainsbecause of the conserved size of the cenl-specific Sallfragments and because it appears that cenl has a minimalstructure that probably would not lead to significant struc-tural differences among strains (see Discussion).The nature of the structural differences in cen2 among

VOL. 13, 1993

4584 STEINER ET AL.

different strains is further characterized in Fig. SB. Initialindirect end-labeling experiments (data not shown) revealedthat the structure to the right of cc2 (Fig. 1) is conservedamong different strains. To analyze the structure of cen2 tothe left of cc2, genomic DNAs were digested to completionwith BstEII and then partially digested with SphI. BstEIIcuts once in cc2 and again in the noncentromeric sequencesflanking the block of tandemly repeated K-L-B units to theleft of cc2 (Fig. 1). SphI cuts once in every L and K repeat;therefore, a partial SphI digest would reveal the number of Land K repeats situated to the left of cc2. A 2.3-kb cc2SphI-BstEII fragment that extends to the left of the cc2BstEII site (7) (hatched box Fig. 1) was used as a probe. The2.3-kb cc2 SphI-BstEII fragment was shown to be conservedamong the four laboratory strains, because genomic DNAsfrom these strains that were digested completely with BstEIIand partially with SphI, Southern blotted, and probed withthe 2.3-kb SphI-BstEII fragment revealed a 2.3-kb hybridiz-ing band (data not shown). The pattern of bands seen inDNAs from strains 972 (lane 6) and Sp223 (lane 7) stronglysuggests the presence of two L repeats and two K repeats tothe left of cc2 (4, 7).The initial indirect end-labeling experiments referred to

above (data not shown) also revealed the presence of three Brepeats to the left of cc2 in strain Sp223. In addition, the49.7-kb hybridizing band in Fig. 5B is a BstEII fragment thatis thought to mark the end of the centromeric sequences(Fig. 1). From these data, it was concluded that the structureof cen2 originally reported for the diploid strain SBPD400 (7)(see Fig. 9), in which there are three L and three K repeatsto the left of cc2, differs from the structure of cen2 reportedhere by an additional L and K repeat unit.The structure of cen2 in strains 975 and SBP120390 was

investigated in lanes 5 and 8, respectively, of Fig. SB, byusing the 2.3-kb BstEII-SphI cc2 probe. Only three hybrid-izing fragments are seen in lanes 5 and 8, the smallest two ofwhich strongly suggest the presence of a single L and a singleK repeat on the left arm of the inverted repeat. The top bandis thought to be a BstEII fragment which spans the end of thecentromeric sequences. Thus, in strains 975 and SBP120390,the cen2 region is composed of only one K-L-B unit on eitherside of the central core and resembles in overall form thestructure of cenl. A model summarizing the proposed vari-ations in the cen2 structure of S. pombe laboratory strains isdetailed in Fig. 9.

Several wild-type S. pombe isolates show variability in thesize of the centromere-specific Sail fragments, but the corestructures of the centromere loci, composed of a central coreflanked by inverted repeats, are conserved. Once differencesamong centromere structures of commonly used laboratorystrains were identified, the centromere structures of a num-ber of S. pombe wild-type strains isolated from locationsworldwide were analyzed (32) (Materials and Methods). Toinvestigate initially the gross differences in centromere struc-ture among these strains, genomic DNAs were digested withSall, Southern blotted, and hybridized with the 1.8-kbNcoI-SphI fragment from cc3 (Fig. 2A and 6), which hybrid-izes to both the cen3 and cenl Sall fragments. As shown inFig. 6, the cc3 probe hybridizes to a 65-kb cenl Sallfragment and to an approximately 150-kb cen3 Sall fragmentin the four standard laboratory strains (lanes 1 to 4), aspredicted from data shown in Fig. 3 to 5, but DNA fromwild-type strains (lanes 5 to 12) gave a highly polymorphicpattern of hybridizing bands. The results indicate that thereis a great deal of variability in the sizes of the centromere-specific SalI fragments among recently isolated S. pombe

N (D tt Lo (Co - CO

1 2 3 4 5 6 7

4qA

- 0*

cs+ CY

LF-

1 N

8 9 10 11

Ar4w., - CC3(29.8 kb)

WI(-'ccl(15 kb)

B M * * CC2(33.2 kb)

FIG. 7. The core structure of centromeric DNA, composed of acentral core flanked by inverted repeats, is conserved among S.pombe strains. DNAs from a variety of strains were digested withKpnI, subjected to FIGE, Southern blotted, hybridized to 32p_labeled probe, and autoradiographed as described in Materials andMethods. The only centromeric KpnI sites are in the K repeats;thus, digestion of genomic DNA with Kpnl releases fragmentscontaining the central core and a portion of the flanking invertedrepeats, including part of the K repeat. (A) The Southern blot washybridized with the 1.8-kb NcoI-SphI fragment from cc3 (see Fig.2A). This cc3 fragment hybridizes to KpnI fragments spanning ccland cc3. (B) The same Southern blot was rehybridized with the2.3-kb BstEII-SphI fragment from cc2 (7). The differences in bandintensities are a result of differences in DNA concentrations ofindividual samples.

strains. This variability in SalI fragment sizes could be dueto SalI site polymorphisms or to gross structural differenceswithin the centromeric loci. Because of the extreme levels ofpolymorphisms seen in these wild-type DNAs, we chose todetermine what conserved characteristics, if any, could befound among the centromeres of the strains studied.A common element among all S. pombe centromere

structures characterized to date is a core structure composedof a nonrepetitive central core region directly flanked byinverted repeats. To determine whether this core structure isconserved in the wild-type isolates, DNAs from a variety ofstrains were digested with Kpnl, Southern blotted, andhybridized with central core-specific probes (Fig. 7). Be-cause the only centromeric KpnI sites are in the K repeats,digestion of genomic DNA with KpnI releases fragmentscontaining the central core and flanking inverted sequences,including a portion of the K repeat. In Fig. 7A, the Southernblot of KpnI-digested DNAs was hybridized with the 1.8-kbNcoI-SphI fragment from cc3 (Fig. 2A). This cc3 fragmenthybridizes to KpnI fragments spanning ccl and cc3. In Fig.7B, the same Southern blot was rehybridized with a frag-ment from cc2. It is clear from the data presented in Fig. 7that in the strains studied, the size of the KpnI fragmentdetected by hybridization to central core sequences is con-served. Because this KpnI fragment presumably contains thecentral core with flanking inverted repeats, we concludefrom these data that the centromeric core structure isrelatively conserved among a variety of S. pombe strains.Once it was established that a conserved centromeric core

structural motif occurs in the various strains studied, weexamined whether the size of the central core itself isconserved among strains. DNAs from different strains weredigested with enzymes that excise the central core se-quences on each chromosome in an intact single fragment,and central core probes were used on Southern blots toelucidate the sizes of the fragments produced. In Fig. 8A, aSouthern blot containing DNAs digested with SphI washybridized to the 1.8-kb NcoI-SphI cc3 probe (Fig. 2A) inorder to detect centromeric fragments containing ccl se-

MOL. CELL. BIOL.

VARIABILITY OF S. POMBE CENTROMERE STRUCTURE 4585

0

Clf + (3

F c o N C)

F-

0 1-7 8 9 10 11 12

CC 1(9.6 kb)

--CC2(9.8 kb)

c ____ CC3_I (5.4kb)

FIG. 8. The size of the central core is conserved among variousS. pombe strains. (A) DNAs from various S. pombe strains were

digested with SphI, whose sites flank ccl. The 1.8-kb NcoI-SphIfragment from cc3 (Fig. 2A) was used as a probe to detect cclsequences. (B) DNAs were digested with NcoI, which cuts close tothe borders of cc2, and the 2.3-kb BstEII-SphI fragment from cc2 (7)was used as a probe. (C) DNAs were digested with ClaI, and the1.8-kb NcoI-SphI cc3 fragment was used as a probe. The cc3 ClaIfragment polymorphism seen in lanes 1, 2, and 7 is the result of a

0.5-kb deletion in the cc3 sequences of WT1, WT2, and WT7. Inpanels A and C, the hybridizing bands not indicated by an arrow are

fragments from cc3 and ccl, respectively, which cross-hybridize tothe cc3 probe used. The differences in band intensities are a result ofdifferences in DNA concentrations of individual samples.

quences. The enzyme SphI was used because it cuts at sitesflanking ccl. In Fig. 8B, DNAs were digested with NcoI,which cuts close to the borders of cc2, and cc2 sequences

were used as a probe. Finally, in Fig. 8C, DNAs were

digested with ClaI, and the 1.8-kb NcoI-SphI cc3 fragmentwas used as a probe. The cc3 ClaI fragment polymorphismseen in lanes 1, 2, and 7 is the result of a 0.5-kb deletion inthe cc3 sequences of WT1, WT2, and WT7. This deletionwas localized to the 0.8-kb NcoI-BglII fragment located tothe left of the NcoI site in cc3 (Fig. 2A and data not shown).In Fig. 8A and C, the hybridizing bands not indicated by an

arrow are fragments from cc3 and ccl, respectively, whichcross-hybridize to the cc3 probe used. The data in Fig. 8show that the sizes of the restriction fragments containingccl, cc2, or cc3 sequences and lacking flanking repeats isconserved among most strains, and we thus conclude thatthe sizes of the centromeric central cores, and very likely thenucleotide sequences of these regions, are likewise relativelyconserved among the wild-type strains studied.

DISCUSSION

The fission yeast S. pombe is an attractive model systemfor the study of centromere structure and chromosomesegregation (4, 7, 12, 20). Here we describe plasmid integra-tion-excision strategies in combination with Southern anal-yses that have been used to map 49 kb of the cen3 locus in S.

pombe Sp223. In addition, we have used Southern analysesand indirect end labeling with cen-specific probes to com-

pare the structures of cen3 DNA in four commonly used S.pombe laboratory strains and have found that this locus

differs among strains. Likewise, we have found differencesamong the structures of S. pombe cen2 in different strains.A model for the structures of cen2 and cen3 in strains 972,

975, Sp223, and SBP120390 is shown in Fig. 9. The differentcentromere structures that we have observed include twoorientations of the cc3 sequences with respect to flankingmarkers, asymmetric and symmetric arrangements of thecen3 tandem K repeats with respect to the central core, andvariable numbers of cen2 K-L-B tandem repeats. The vari-ations observed in centromere structure among differentstrains is not surprising, as there is considerable variation inthe organization of centromere-specific repeats among thethree centromere loci in Sp223 alone. For example, centro-meric repeat K, a portion of which is necessary but notsufficient for centromere function (12, 28), occurs twice incenl and approximately 13 times in cen3, and the orienta-tions of the copies of repeat K with respect to the centralcore sequences are different in cenl and cen2.We have also compared the centromere structures in the

four commonly used laboratory strains with the structures ineight wild-type S. pombe strains from sources and locationsaround the world. We have found certain relatively con-served elements in the centromeres of these 12 S. pombestrains, including a core structure composed of a central coreimmediately flanked by inverted repeats that contain aportion of centromeric repeat K and a central core itself,which is of a relatively conserved size. The conservation ofthese regions is expected, as some central core sequencesand at least a portion of centromeric repeat K have beenshown to be necessary for centromere function in S. pombe(7, 12, 28).We suggest that cenl is probably a minimal centromere

structure in S. pombe, because it is composed only of thesymmetrical core structural motif consisting of the centralcore and immediately flanking inverted repeats (4, 12, 28)(Fig. 9), and thus we do not anticipate much variability at thecenl locus in laboratory strains. Furthermore, we haveconcluded that the number, orientation, and location oftandem repeats in the centromere, and the orientation of cc3with respect to flanking markers, are not parameters of thecentromere that are required for function, because theseelements vary among strains. These conclusions agree withfunctional studies, in which it has been shown that centro-mere-specific repeats play a role in maintenance of sisterchromatid attachment in meiosis I (7, 12, 23, 28), butconsiderable function is maintained if the orientation andlocation of the repeats are altered relative to the central core(1) or if blocks of tandem repeats are deleted from cen2 orcen3 (7, 19, 27a).When cen3 M repeat sequences are used as probes, they

hybridize weakly to a number of small restriction fragmentswhich are noncentromeric on the basis of size. This patternof weak hybridization is reminiscent of the pattern producedwhen B repeats from cenl or cen2 are used as probes (15, 28;unpublished observation). It has been shown that the weaklyhomologous nature of the B repeats is due to the presence oftRNA genes within this repeat (15, 29). The M repeats arealso located at the boundaries of cen3, and likewise, tRNAgenes are found at the boundaries of cenl and cen2 (29). Wethus postulate that the M repeats contain tRNA genes. Thefunctional role of tRNA sequences in the centromeres of S.pombe is not known.

All of the differences that occur at the centromere locisummarized in Fig. 9 could arise via simple recombinationalmechanisms, either in mitosis or in meiosis. For example,the loss of a K-L-B unit in cen2 could arise by a recombi-

- N q

1 2 3 4 5 6

A

B mw,, _ Ab

VOL. 13, 1993

,",A&40

.k. da,

4586 STEINER ET AL.

K' L Km B' B' Km L K'

M K L' L' K K K K K K K K K K K MSP223, 972

cc3

cen3 975

cc3

___ SBP120390,*cc3

FIG. 9. Model summarizing the differences in centromere loci of various S. pombe strains. No differences in the structure of cenl indifferent laboratory strains have been detected, based solely on the size of the cenl-containing Sall fragment in each of the strains. Thestructure of cen2 in strain SBPD400 has been previously published (7). The numbers of tandem K repeats shown in the cen3 structures areapproximate. cen3 structures from strains Sp223, SBP120390, 972, and 975 are depicted here such that the cen3 unique end probe (the 1.8-kbSalI-KpnI fragment; Fig. 2A) is adjacent to the left arm of the centromere. Asterisks indicate structures published by Yanagida and coworkers(4, 20).

national looping out of this region during mitotic division,and the two orientations of the central core observed forcen3 may be a result of a simple recombinational flip acrosshomologous regions of the inverted repeat. Alternatively,unequal crossing over during meiosis could account for somealterations, although meiotic recombination frequencies inS. pombe centromere regions are extremely low (4, 6, 21).The means by which these differences were actually gener-ated and the regulatory pathways controlling these structuralchanges remain unknown, however.

Nevertheless, it is clear that S. pombe centromeres arehighly variable loci. This also appears to be the case forcentromere regions in animal cells, where, for example, thesize of the region of centromeric repeats may vary dramat-ically and certain classes of repeated DNAs appear to beorganized in a chromosome-specific manner (14, 30, 31).This variability is only one of the known similarities betweenthe centromeres of S. pombe and the centromeres of highereukaryotes, and such similarities serve to strengthen therelevance of the S. pombe system as a model for the study ofcentromere structure and function.

ACKNOWLEDGMENTS

We thank John Carbon, Mary Baum, Vivian Ngan, and LauraMarschall for helpful discussions and critical reading of the manu-script. We also thank Henry Levin for providing recently isolatedwild-type S. pombe strains.

This research was supported by Public Health Service grantGM-33783 from the National Institutes of Health. N.C.S. wassupported by a National Science Foundation predoctoral fellowship.

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cenl {

cen2 {

K L B B L K B

cc2

SP223, 972, 975,SBP120390, *

SBPD400

SP223, 972, *

975, SBP120390

b k\\'VIcc///

Rm~~~~

K L BJ K L BJ

&,U./l/M F...

MOL. CELL. BIOL.

VARIABILITY OF S. POMBE CENTROMERE STRUCTURE 4587

15. Kuhn, R. M., L. Clarke, and J. Carbon. 1991. Clustered tRNAgenes in Schizosaccharomyces pombe centromeric DNA se-quence repeats. Proc. Natl. Acad. Sci. USA 88:1306-1310.

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26. Polizzi, C., and L. Clarke. 1991. The chromatin structure ofcentromeres from fission yeast: differentiation of the centralcore that correlates with function. J. Cell Biol. 112:191-201.

27. Sherman, F., G. R Fink, and J. B. Hicks. 1986. Laboratorycourse manual for methods in yeast genetics, p. 143-144. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

27a.Steiner, N. C., and L. Clarke. Unpublished data.28. Takahashi, K., S. Murakami, Y. Chikashige, H. Funabiki, 0.

Niwa, and M. Yanagida. 1992. A low-copy-number centralsequence with strict symmetry and unusual chromatin structurein fission yeast centromere. Mol. Biol. Cell 3:819-835.

29. Takahashi, K., S. Murakami, Y. Chikashige, 0. Niwa, and M.Yanagida. 1991. A large number of tRNA genes are symmetri-cally located in fission yeast centromeres. J. Mol. Biol. 218:13-17.

30. Wevnick, R., W. C. Earnshaw, P. N. Howard-Peebles, and H. F.Willard. 1990. Partial deletion of alpha satellite DNA associatedwith reduced amounts of the centromere protein CENP-B in amitotically stable human chromosome rearrangement. Mol. CellBiol. 10:6374-6380.

31. Wevrick, R., and H. F. Willard. 1989. Long range organizationof tandem arrays of a satellite DNA at the centromeres ofhuman chromosomes: high frequency array-length polymor-phism and meiotic stability. Proc. Natl. Acad. of Sci. USA86:9394-9398.

32. Zimmer, M., F. Welser, G. Oraler, and K. Wolf. 1987. Distri-bution of mitochondrial introns in the species Schizosaccharo-myces pombe and the origin of the group II intron in the geneencoding apocytochrome b. Curr. Genet. 12:329-336.

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