Download Centromeres: An Integrated Protein/DNA Complex

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Annu. Rev. Cell Biol. 1991.7 ." 311 36
Copyright © 1991 by Annual Reviews Inc. All rights reserved
CENTROMERESAN INTEGRATED PROTEIN/DNA
COMPLEX REQUIRED FOR
CHROMOSOME MOVEMENT
I. Schulmanand K. S. Bloom
Department of Biology, University of North Carolina, Chapel Hill,
North Carolina 27599-3280
KEY WORDS: kinetochore structure, satellite DNA,nucleosomalarrays, mitosis
spindle attachments
CONTENTS
INTRODUCTION
..............................................................................................................
CENT~,OMEmC
ONA
........................................................................................................
Budding
Yeast
..........................................................................................................
Fission
Yeast
............................................................................................................
Mammalian
Centromeres
.........................................................................................
311
312
312
313
314
STRUCTURAL
ORGANIZATION
OFDNA
IN THE
CELL
..........................................................
ChromatinStructure of FungalCentromeres............................................................
Structural Features of Mammalian
Centromeres......................................................
CENTROMERIC
PROTEINS
................................................................................................
Centromere-Associated
Antigens..............................................................................
Histones
...................................................................................................................
Centromere
DNA-Binding
Factors............................................................................
Satellite DNA-Binding
Proteins................................................................................
316
316
317
REGULATION
ANDASSEMBLY
OFCENTROMERIC
COMPONENTS
...........................................
Chromosome
CopyControlMutants........................................................................
Chromosome
Fidelity Mutants..................................................................................
326
327
329
EVOLUTION
OFTHE
KINETOCHORE
..................................................................................
329
318
318
319
320
324
INTRODUCTION
Centromeres of eukaryotic chromosomes are specific regions along the
chromatin fiber that play a fundamental role in chromosome movement
311
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SCHULMAN
& BLOOM
during mitosis and meiosis. The centromere is a distinct DNAdomain
that appears as the primary constriction along metaphasechromosomes.
The centromeremediates sister chromatidinteraction and nucleates the
assemblyof a kinetochore.Thekinetochoreis a region of structural differentiation that is composedof centromeric DNA
and protein components
and that can be visualized as a trilaminar plaqueor disc on the surface of
the chromosome.
Thecentromere,therefore, is responsible for delineating
a chromosomal
domainthat is accessible to the segregation apparatus as
well as providing a scaffold for assemblyof kinetochorecomponents.The
apparatusthat regulates the distribution of chromosomes
to daughtercells
is broughtabout by the assemblyof protein subunits into spindle tubules.
Thefunctionof the kinetochore,at least in part, is to capturethese spindle
microtubulesand generate polewardforces (Mitchison &Kirschner 1985).
Several reports indicate that kinetochoresfacilitate microtubulebinding
while allowing the microtubules to freely exchangetubulin subunits at
their attached ends (Mitchison et al 1986; Salmon1989). Kinetochores
mayalso be the domain where mechanochemicalmotors required for
chromosome
movements
are localized (Rieder & Alexander1990; Nicklas
1989). Recent studies demonstrate that dynein, a force-producing
molecule, is concentrated along the primary constriction of the chromosome
(Pfarr et al 1990; Steuer et al 1990). A completeunderstanding
of the molecularmechanisms
by whichcentromericregions becomeassociated with the microtubules requires a knowledgeof the organization of
centromeric DNAsequences, centromeric proteins, and other chromatin
componentsspecifically associated with the centromere. This review
focuses on the primaryfolding ofcentromericDNA
sequencesinto nucleoprotein complexes.
CENTROMERIC
DNA
Buddin 9 Yeast
Functional centromere DNA
sequences have been isolated from the budding yeasts, Saccharomyces
cerevisiae (Fitzgerald-Hayeset al 1982;Hieter
et al 1985), S. uvarum(Huberman
et al 1986), and K. lactis (Heuset al
1990). TheS. cerevisiae centromerecan be localized to approximately125
base pairs of DNA.The sequence elements required for function (CDE
centromereDNA
element I, 8 bp; II, 78-86 bp of AT-rich DNA;and III,
25 bp) are conservedin their spatial and sequencearrangementin 13 yeast
centromeres sequencedto date. The spacing between CDEI and III is
maintained by CDEII, which is comprised of greater than 90%A+T
nucleotides. Replacementof virtually every nucleotide has confirmedthe
essential role for specific basesin CDEIII and for the general requirement
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STRUCTURE AND FUNCTION OF CENTROMERES
313
of AT-rich sequences in CDEII (reviewed by Gaudet& Fitzgerald-Hayes
1990). Chromosomes
bearing mutations, in CDEI and II are still maintained at frequencies of 99 per 100 to 9999per 10,000 cell divisions.
However,inversion of CDEIII relative to CDEI and II is deleterious to
centromerefunction. Thus the requirements for proper mitotic function
reside in the primarysequenceof CDEIII and in its spatial orientation
relative to the AT-richCDEII. The requirement for ATsequencesjuxtaposedto a putative recognitiondomainis significant whenone considers
that oligo A base pairs deformDNA
with specific spatial parameters. The
migrationof centromereDNA
in polyacrylamidegels is noticeably altered
in comparisonto bulk DNA
sequences. CDEII has been projected in three
dimensions using the wedgeprogramdescribed by Eckdhal & Anderson
(1987).Thepredictedstructure consists of a rigid backbone
that is deflected
backand forth along its helical axis in a zig-zag fashion, quite distinct
from other structural sequences such as bent DNA,or the bent DNA
locus in kinetoplast DNA
and autonomouslyreplicating sequences. This
structural motif is conservedin 12 CDEII regions that exhibit minimal
sequence homology.Whetherthis sequenceis deformedin accord with a
subjacent protein core, or has a role in specifyingthe exquisite sequence
specificity characteristic of CDE
Ill is not known,but suggestsa role for
structural DNA
in this unique protein/DNAcomplex.
CentromereDNAfrom a related species of Saccharomyes,S. uvarum,
can stabilize minichromosomes
in S. cerevisiae, and it contains the conserved sequenceelementsCDEI-III. It is unlikely that centromerefunction extends past the species barrier, however. CentromereDNA
from
anothergenusof Ascomycetes,
K. lactis, does not function in S. cerevisiae
(Heuset al 1990).K. lactis is a buddingyeast used primarilyin industrial
fermentations with a chromosomenumber(6) intermediate between
cerevisiae (16) and S. pombe(3). Five of the six centromereshavebeen
isolated fromK. lactis bytheir ability to confermitoticstability to plasmids
containing an autonomouslyreplicating sequence. Thefunctional domains
have been localized on DNA
fragments ranging from two to five kilobase
pairs in length. Sequenceanalysis of these centromereswill reveal whether
the K. lactis centromereis intermediatein size betweenthe S. cerevisiae
and S. pombecentromeres, or contains the CDEelements characteristic
of S. cerevisiaein a slightly altered sequenceand/orspatial configuration,
Fission Yeast
Thecentromereof the fission yeast Schizosaccharomycespombe
is approximately two to three orders of magnitudelarger than the S. cerevisiae
centromere (50-150 kb vs 150 bp) (Nakasekoet al 1986; Fishel et
1988; Niwaet al 1989; Clarke & Baum1990; Matsumotoet al 1990).
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SCHULMAN & BLOOM
Distinguishing features are blocks of repeated DNAelements that comprise this centromeric domain. A schematic representation of the sequence
organization of the centromere region from S. pombe is illustrated in
Figure 1. At least three middle repetitive elements K (6.4 kb), L (4.5 kb),
and B (1-2 kb) flank a nonconservedsingle copy central core, cc2 (7 kb
chromosome2). Independent investigations have also shown the S. pombe
centromere to be made up of a unique central domain flanked by repeated
elements dg (3.8 kb), dh (4.0 kb), and yn/tm (less than 1 kb) (Nakaseko
al 1986; Niwa et al 1989; Chikashige et al 1989; Matsumotoet al 1990).
The dh region corresponds to a portion of K-L; dg to K, and yn/tm to B,
respectively. Eachof these elements, or portions thereof, occur at all three
S. pombecentromeres. These repeats are in turn tandemly reiterated to
generate an inverted repeating motif that flanks the central core.
The detailed analysis of such large DNAfragments has only recently
been madepossible through the use of yeast artificial chromosomevectors
(YACs, Burke et al 1987). The large repeated sequences present in
pombecentromeres are unstable in E. coli and are lost or rearranged when
maintained in a bacterial host. However, when repeated domains are
introduced into vectors containing S. cerevisiae centromeres and origins
for DNAreplication, these sequences can be subcloned and propagated
in S. cerevisiae or S. pombe without deletion or rearrangement. Upon
introduction into S. pombehost cells, minichromosomescontaining entire
centromere regions are faithfully segregated both during mitosis and meiosis and are stably maintained in the absence of genetic selection (Hahnenberger et al 1989; Clarke & Baum1990). Deletion analysis of functional
minichromosomesindicates that the central core flanked by the short
inverted repeats (B) and at least one copy of the K (dg) repeat are required
for mitotic function (Matsumoto et al 1990; Hahnenberger et al 1991).
Deletions of large regions of the repeats (K-L-B) have a disproportional
effect on meiotic function, as witnessed by a high degree of precocious
sister chromatidseparation in meiosis I. In no instance has the central core
or the tandem array of repeats been shown to function independently in
mitotic or meiotic cell divisions.
Mammalian
Centromeres
A functional analysis of mammaliancentromeres has not progressed to
the extent described for the yeast systems. Our knowledgeis limited to a
molecular description of the abundant sequences located in or near the
constricted region of mitotic chromosomes. Centromeric DNAis predominantly heterochromatic and rich in highly repetitive satellite DNA.
The primary sequence motif in primates is alpha satellite or alphoid DNA
(Arn et al 1989; Willard et al 1989). Alpha satellite DNAis composed
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STRUCTURE AND FUNCTION OF CENTROMERES
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~
100bp
S. cerevlslae
CEN3
2kb
S.
pombe
cen2
~
170bp
’,~
0.68 - 6kb
H. sapiens
alphoid DNA
~
Fi#ure 1 Schematic representation of the centromere regions from S. cerevisiae (top),
pombe(middle), and H. sapiens (bottom). (Top) The elements of centromere DNAhomology
CDEI and III arc indicated. A~-rowscorrespond to nuclease cutting sites delineating the
centromere core. The central oval represents the protected core, flanked by ordered nucleosomal subunits. The minimal fragment with full segregation function is drawn immediately
below. (Middle) The repeated elements B, L, K are shownrelative to the single copy central
core (cc2). The darkened boxes represent 1.5 kb of inverted repeat sequences that occur only
in this region of the genome(adapted from Clarke 1990). The filled circles along the DNA
represent nuclcosomal subunits. Nucleosomesmaybe masked or absent within the cc2. Their
position with respect to the DNAsequence has not been determined. The minimal active
segment is drawn immediately below. (Bottom) The alphoid repeats are arranged in dimers
or pentamers, which in turn are organized into 0.68-6.0 kb sequences. The larger units are
repeated 100-1000 times along the centromere (Arn et al 1989). Nucleosomesoccupy one
major and multiple minor phases with respect to the repeat sequence. These are simply
indicated by ovals. Note the scale differences in the various organisms.
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SCHULMAN
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tandemarrays of 170 bp polymeric units that exhibit 60-95%homology
betweenindividual units. Dimeror pentamericunits of the alphoid DNA,
as shownin Figure l, are in turn organizedinto larger clusters ranging
from 0.68-6.0 kilobase pairs (Willard &Waye1987). Hierarchical arrays
of these clusters comprise up to millions of base pairs of DNA.DNA
sequencingof cloned repeat units, along with conventionalrestriction
mapping, have revealed numerouspolymorphismsamongdifferent chromosomes
as well as within chromosome-specific
subsets. There is a tendency for monomerson one chromosome
to be more conserved than those
fromdifferent chromosomes.
In addition, the organizationof large blocks
of repeats within a chromosome
are specific to that chromosome
(Wervick
& Willard 1989; Grieg et al 1989; Mahtani & Willard 1990). With the
advent of techniques to manipulate large fragments of DNA
and to construct large mammalian
artificial chromosomes,
the identification of mammalian centromeres is soon likely to follow. The paridigms for DNA
manipulationwithin the’ centromereregion of S. pombewill certainly
be invaluable in subsequent functional analysis of their mammalian
counterparts.
STRUCTURAL ORGANIZATION OF DNA IN THE
CELL
ChromatinStructure of Fungal Centromeres
s. CEREVISIAE
Molecularmappingtechniques have been used to visualize
the chromatinstructure of four S. cerevisiae centromeres(CEN3,CEN11,
Bloom& Carbon 1982; CEN4,Bloomet al 1984a; CEN14,Funk et al
1989). Approximately150 to 200 bp of DNA
are folded into a highly
nuclease-resistantcore structure. Thesingle base changesin the central C
nucleotide of CDEIII, whicheradicate segregation function, completely
disrupt the characteristic protected structure. TheCDE
I and II mutations
that retain partial centromerefunction exhibit chromatinconformations
of altered dimension(Saunderset al 1988). Deletions of CDEI decrease
the fidelity of chromosome
transmissionfive- to tenfold, andthe protected
structures of these altered centromeresare shortenedby 10-20 base pairs.
Mutations in which the length between CDEI and CDEIII is increased
or decreasedexhibit lengthenedor shortenedprotected structures, respectively, and have intermediate effects on chromosome
segregation. The
chromatinparticles associated with these defective centromerescan range
from 145 to greater than 300 bp. Interestingly, mutants with less than
145 bp of centromere DNAincorporate flanking DNAinto protected
structures that span 145 bp of chromatinDNA.
Theplasticity in structural
organization indicates that the chromatincore is likely to represent a
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STRUCTURE AND FLrNCTION OF CENTROMERES
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complexinteraction betweencentromere-specific DNA-binding
proteins,
histones, and centromere DNA.
s. POtaBE
The structural organization of the centromererepeats and the
unique central core have recently been determinedby nuclease digestion
studies (Polizzi & Clarke 1991). The large repeated domains,K and
exhibit nucleosomearrays that typify the bulk of the chromatin DNA.
These nucleosomalrepeats are 155 bp, with an average spacer length of
10 base pairs. Theprecise position of nucleasecutting sites with respect to
the DNAsequence has not been determined. The nucleosomalarrays are
not as ordered as in S. cerevisiae, whichindicates the availability of
alternative registers of nucleosomal
arrays in a givencell. Onesuchordered
array of nucleosome
packagingis illustrated in Figure1. Themoststriking
feature of the chromatinstructure is the absenceof ordered nucleosomes
in the central core. It is unlikely that this regionis completelydevoidof
nucleosomesbecause monomersand dimers can be visualized following
micrococcalnuclease digestion. However,the spacing is randomizedor
maskedto an extent that precludes the appearanceofnucleosomeladders.
Thejunction betweenthe central core and the flanking repeats reveals a
third pattern that is intermediatebetweenthe typical nucleosome
ladders
of K and L and the randomizedcentral core. Thenucleosomeswithin the
B repeat and the core-associated repeats (darkenedblocks, Figure 1) are
less distinct andexhibit higher levels of background
hybridization. While
the central domainlacks an organized nucleosomearray, it appears that
in the junction region a moreorganizedstructure begins to formand by
repeat L is arrayedin multipleregisters distally fromthe centromere.The
lack of nucleosomestructure in the central core does not reflect any
inherent nucleotide sequencethat precludes nucleosome
assembly.Polizzi
& Clarke (1991) demonstratedthe assemblyof nucleosomesubunits within
the central core whenthese sequenceswereintroducedinto S. cerevisiae.
Thusthe functional properties of the centromerein fission yeasts are
intertwinedwith this apparentlyatypical subunit structure. It is possible
that histone subunits are predominantly absent, modified, or occupy
limited sites within this region. Alternatively,the bindingof centromerespecific nonhistoneproteins mayprevent exogenousnucleolytic digestion
of the central core. Thechromatinorganization of the centromereregion
in S. pombeis certainly a complexstructure of several functional domains
and mayrepresent a compound
version of that found in S. cerevisiae.
Structural Features of Mammalian Centromeres
Thechromatinstructure of the alphoid satellite has been studied extensively in the African green monkey.AlphoidDNA
is packagedinto highly
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& BLOOM
organized nucleosomearrays (Zhanget al 1983; van Holde1989), which
are positionedin one of eight phases (one major andseven minorphases).
The observation of multiple phases for nucleosomepositioning is also
apparentin the rat satellite I and mousesatellite chromatin.Suchpatterns
of multiplephasingare at least consistent with the data obtainedin the K
and L repeats in the S. pombecentromere. Nucleosomesare present on
the KandL repeats, but mayoccupyalternate positions in different cells.
The binding of nonhistone chromosomalproteins to specific sequences
maybe an importantfactor in the delineation of various phases in individual cells and the subsequentcompactionof heterochromatin. Several
nonhistone proteins, one of whichis a kinetochore component,bind to
specific regions in the alpha satellite sequence.CENP-B
(Masumoto
et al
1989), HMG-I
(Solomonet al 1986; Disney et al 1989), and D1(Levinger
& Varshavsky1982) bind to A-T rich regions of chromosomes,and are
likely candidates in the compactionof centromericheterochromatin(see
below).
CENTROMERIC
PROTEINS
Centromere-Associated
Antigens
The identification of centromeric proteins in mammalian
cells has been
facilitated by the discoveryof kinetochore-specificautoantibodiesin the
serumof patients with the calcinosis/Raynaud’s phenomenon/esophageal
dysmotility/sclerodactyly/telangiectasia variant (CREST)
of scleroderma.
Thedisease is a progressivesystemicsclerosis that can affect manyorgans
includingskin, subcutaneous
tissue, gastrointestinal tract, heart, lung, and
kidney. Moroiet al (1980) demonstratedthat patients with this disorder
contain autoantibodies that react with the kinetochore of chromosomes.
The characterization of CREST
antigens [centromere proteins (CENP)-A, B, C, D, and E], as well as inner centromereproteins (INCENPs)
and
chromatid-linkingproteins (CLiPs), has been instrumental in delineating
distinct domainswithin the primary constriction of metaphasechromosomes
[Brinkley 1990; Cookeet al 1987; Earnshaw& Rattner 1989 (see
Figure1 therein); Rattner et al 1988;Rattner 1991;Yenet al 1991;Willard
1991]. Theseproteins partition into three distinct regions within the primary constriction, or centromere domain. The kinetochore domainis
assembledon the outer surface of the centromereand is comprisedof five
subdomains:a fibrous corona, an outer, middle, and inner plate, and
subjacent chromatinfibrils. The central domainof the centromerelies
belowthe kinetochore plates and is followed by the innermost pairing
domain,whichis responsiblefor tethering sister chromatidstogether prior
to their separation.
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STRUCTURE
AND FUNCTION
OF
CENTROMERES
319
CENPsA and C are located in, but not restricted to, the inner kinetochore plate. CENP-Ais a histone H3 variant and may be involved in
the subunit organization of the centromeric DNA(Palmer et al 1990; see
below). CENP-Cis restricted to active centromeres (Earnshaw et al 1989)
and is equally distributed between different chromosomes. The microtubule-based mechanochemicalmotor protein, dynein, has also been localized to the kinetochore domain(Pfarr et al 1990; Steuer et al 1990). The
ability ofdynein to translocate along a microtubule lattice has considerable
impact with respect to chromosomesegregation mechanisms. The accessibility of dynein to microtubules is most likely maintained in the kinetochore structure, thus implicating dynein as a primary candidate for the
proteinaceous substance that comprises the outermost fibrous corona of
the kinetochore.
CENP-Bis the most extensively characterized CRESTantigen to date
and binds the s-satellite
DNA(see below). It is localized throughout
centromeric heterochromatin in the central domain beneath the kinetochore (Cooke et al 1990). The distribution of CENP-Bvaries between
different chromosomesand correlates well with the amountof s-satellite
DNAper chromosome. CENP-B-~-satellite
DNAcomplexes may comprise the bulk of the central domainand could well provide an internal
frameworkrequisite for the positioning of the kinetochore domain.
INCENPs
and CLiPs are localized to the inner surface of the centromere
and along the arms where contacts between sister chromatids occur.
INCENPs
and CLiPsare likely to represent at least two classes of proteins,
based from their behavior in anaphase. INCENPs
migrate to the equatorial
zone of the spindle and are destined for the midbody, whereas the CLiPs
are no longer detected following chromatid separation.
Additional centromere-associated proteins have recently been identified
by raising antibodies against mitotic chromosomescaffolds. Comptonet
al (1991) generated antibodies to four classes of centromere-associated
proteins distinguished by their staining characteristics throughout the
chromosomecycle. Several of these antigens localize to centrosomes, the
microtubule organizing centers of the cell, as well as metaphase chromosomes. The organization of centromere and kinetochore domains into
visible structural entities will involve diverse classes of proteins that contribute both to the fidelity of kinetochore assembly as well as to active
chromosome segregation.
Histones
S. CEREVISIAE
Histones are the major structural proteins required for
chromosomepackaging into nucleosomes, and their stoichiometric production is a contributing factor to the fidelity of chromosome
segregation
(Meeks-Wagner
& Hartwell 1986). Analysis of specific roles for histone
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SCHULMAN & BLOOM
chromosome
organizationhas beenfacilitated by the introduction of single
copies of histone H2Bor H4, whoseexpression can be modulateddepending on the carbonsource(Hanet al 1987). Deletionof the aminoterminus
of histone H4is dispensablefor cellular growth,but essential for transcriptional repressionof the silent matingtype loci (Kayneet al 1988).Thus
histone-nonhistoneprotein interactions are likely to be as intricate and
essential as histone-DNAinteractions for chromosomestructure and
function.
Repressionof either H2Bor H4histones also leads to the increased
nucleaseaccessibility of the centromere(Saunderset al 1990). Structural
perturbation of the centromere complexfollowing histone repression
reflects either the presenceof histones at the centromere,or a role for
flanking nucleosomal chromatin in centromere assembly and/or maintenance. Strains harboring an aminoterminal deletion of histone H4(428 aa) as the only source of H4are able to grow; however,bulk chromatin
stru6ture is significantly disrupted(Kayneet al 1988). B. Frediani and
Bloom(unpublished results) demonstratedthat centromere structure
these strains is unaltered.Thuscentromerestructure is not simplyreflective
of bulk chromatin perturbation caused by histone deletions. The dissociation pattern of centromereproteins following salt elution of the
chromatincomplexis consistent with a modelin whichhistones are present
at the centromere (Bloom& Carbon1982). At NaCIconcentrations that
dissociate histones (0.75-1.25M), there is a concomitantdisruption of the
centromere core, while at lower salt concentrations, the centromere
remainsunaffected. In addition, the linking numberof centromereplasmidsisolated fromlogarithmically growingcells is indistinguishable from
plasmids of identical size, whichcontain exclusively nucleosomalDNA
(Bloomet al 1984b). Thesedata led us to consider a histone structural
base for the centromereitself.
MAMMALIAN
CENP-A,the centromere-associated antigen localized to the
inner plate of the kinetochore,is tightly associated with mononucleosome
particles following digestion of chromatin with micrococcal nuclease
(Palmeret al 1989). Peptide sequenceanalysis has identified CENP-A
be a histone H3 variant (Palmer et al 1990). Thushistone proteins,
modifiedhistones are also localized within mammalian
kinetochores.
Centromere DNA-Bindin9 Factors
CENTROMERE-BINDING
FACTOR
1 (CBF1) The isolation of a centromere
DNA-bindingprotein that recognizes the centromere DNAelement I
(CDEI) octamerof S. cerevisiae has beenreported by a numberof groups
(CBF1,Cai & Davis 1989, 1990; centromereprotein 1, CP1,Baker et al
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STRUCTURE AND FUNCTION OF CENTROMERES
321
1989; Baker & Masison 1990; centromere and promoter factor 1, CPFI,
Mellor et al 1990). CBF1is an abundant cellular protein (600 copies per
cell, Bakeret al 1989) that is not restricted to the centromericregion (Bram
& Kornberg 1987). CBF1 binds both centromeres and promoters and
exhibits the helix-loop-helix dimerization motif characteristic of other
DNA-binding proteins involved in transcriptional
control. Complete
deletion of the coding information for CBF1 from the yeast genomeresults
in a five- to tenfold increase in chromosome malsegregation, commensurate with the increase in chromosomeloss upon deletion of the
CDEIoctanucleotide. The pleiotropic nature of the binding interactions is
manifested in a genetic sense as well. CBF1deletion strains are methionine
auxotrophs. Thus both genetic and biochemical criteria indicate that this
protein participates in diverse cellular functions. In addition to the helixloop-helix domain, 20%of CBF1 is comprised of acidic residues, aspartate
and glutamate. The charged nature of CBF1exhibits similarity to the
HMGproteins and the highly acidic domains in CENP-B.Domain mapping of CBF1is in progress, and deletion of two clusters of negative
charges (amino acids 80-88 and 184-194) only increases the chromosome
loss rate by a factor of 1.5 comparedto the wild-type protein (Mellor et
al 1990). The proximity of negatively charged domainsto the centromere
mayreflect a role in facilitating the binding of key nonhistoneproteins, or
other componentsthat must access an otherwise highly compacted region.
CENTROMERE-BINDINGFACTOR 3 (CBF3)
The
difficulty
in demonstrating
the binding of a factor to centromere DNAelement CDEIII by similar
methodological approaches may reflect the low copy number of such
molecules, or the lack of faithfully reproducing the in vivo assembly
pathway and/or chromatin requirements.
Lechner & Carbon (1991)
recently succeeded in identifying centromere DNA-bindingfactors that
specifically recognize the CDEIII element. CBF3is a 240-kd phosphoprotein polymeric complex of at least three monomericspecies (110,
64, and 58 kd; CBF3A,B, C, respectively) that are capable of discriminating wild-type CDEIII from an inactivating point mutation in
CDEIII. The proteins are present in vanishingly small amountsin the cell.
Conservative estimates from the purification procedures show that there
are only 20 copies per yeast cell in a logarithmically growingpopulation.
If these proteins are restricted to a particular stage of the cell cycle,
however, they maybe present in higher quantities. In any case, it remains
that the CBF3complex is at least an order of magnitude less abundant
than CBF1.
CBF3protects a 56 bp region from DNAaseI digestion when complexed
to CENDNA.The complex is asymmetrically distributed over CDEIII;
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SCHULMAN & BLOOM
6 base pairs of CDEII to the left, and 24 base pairs of flanking DNAto
the right are protected in the binding reaction. Perhaps the most striking
feature of the reaction is the requirement for assembly factors. Extracts
from either budding yeast, E. coli, or purified casein from bovine milk,
are necessary but not sufficient to support assembly. Ancillary protein
factors required in assembly of macromolecular structures, such as the
centromere DNA-protein complex, may prevent nonproductive proteinprotein interactions, while promoting more favorable interactions. The
role of proteins, such as nucleoplasmin, in facilitating
nucleosome
assembly sets precedence for invoking a similar requirement in centromere
assembly. The ability of the acidic protein casein to substitute for an
endogenousassembly factor(s) suggests a role for acidic domains in promoting centromere DNA-protein assembly. The structure of the centromere DNAmay contribute to the binding specificity as well. Proteins
that stabilize the natural curvature of DNA,such as histones and high
mobility group (HMG)proteins, may play a role in positioning the centromere DNAelements in a conformation that enhances and/or stabilizes
the binding of critical nonhistone proteins. In this regard it is noteworthy
that while only 6 bp of CDEII are complexed in the CBF3footprint, at
least 20-30 bp of flanking A + T DNAon the left are required for in vivo
function. The additional 15-25 runs of A+Tnucleotide may position
CDEIII around the subjacent protein core, but may not be in direct
contact with CBF3itself.
AFFINITY CHROMATOGRAPHY
OF CENTROMERIC
CHROMATIN
The complexity of form and function of yeast and mammaliankinetochores has
directed efforts in our ownlaboratory towards the isolation of an intact
chromatin complex from yeast cells. Restriction enzymelinkers encoding
the BamHIrecognition site have been ligated to a 289 bp CEN3DNA
segment encompassingthe 220-250 bp protected structure. An entire functional segment of centromeric chromatin can be excised from the chromosomeof S. cerevisiae by BamHIdigestion (Kenna et al 1988). The
ability to excise an intact functional core segment, which retains centromeric proteins, offers the opportunity to analyze this autonomous
DNA-protein complex. Release of this complex from any torsional constraints exerted by the flanking chromosomalarms does not perturb protein binding to centromere DNA.Differential sedimentation in linear
glycerol gradients reveals biochemical differences between naked DNA,a
functional centromere, and a nonfunctional altered centromere. However,
the methodis not sufficiently selective to visualize centromere proteins.
An affinity method was developed for protein-DNA complexes that makes
use of the interaction between the lac repressor and its operator (Figure
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STRUCTURE AND FUNCTION OF CENTROMERES
CEN3 289bp
I
EcoRI
I
lacO
CEN3
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I
EcoRI
lacO
I
BamHI
I
I
EcoRI
I
BamHI
BamHI
323
I
II
EcoRI
BamHI
lacl-lacZ
Anti-lacZ Sepharose
Figure 2 Affinity chromatography of centromeric chromatin. A strategy for affinity purification of centromeric chromatin has been devised that exploits the interaction between the
E. coli lac operator (lacO) and its repressor (lacI) (Saunders 1989; Deanet al 1989).
has been cloned adjacent to centromere DNAand introduced into yeast cells. The IacOCENcassette is excised from yeast nuclei by digestion with EcoRI and subsequently incubated
with lac repressor-fl-galactosidase fusion protein (lacIlacZ). The complexis applied to
sepharose column containing immobilized antibodies directed against the fl-galacatoside
portion of the fusion protein. Treatment of the immobilized complex with IPTGresults in
release of centromeric chromatin.
2). A symmetrical lac operator DNAsequence was synthesized and cloned
adjacent to the centromere DNA.This lac operator-CEN cassette was
introduced into yeast by transformation, and centromeres assembled on
this construct in vivo. The intact cassette was excised from the cell nucleus
with restriction
enzymes and incubated with a fl-galactosidase-lac
repressor fusion protein that specifically binds lac operator. The tripartite
complex of centromere proteins, centromere-lac operator DNA,and
fusion protein was purified by passage through anti-fl-galactosidase
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SCHULMAN & BLOOM
sepharose. The complex was specifically eluted with isopropyl-B-D-thiogalactopyranoside (IPTG), a competitive inhibitor of the lac repressor.
Wehave constructed a similar cassette containing a lac operator-mutant
centromere that is completely deficient in its segregation function and
intend to study its intracellular interactions. Comparisonsbetween the
proteins associated with wild-type and mutant centromeres should allow
definitive identification of in vivo centromere DNA-bindingproteins.
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Satellite
DNA-Bindin# Proteins
HIGHMOBILITY
GROUP
PROTEINS
Several proteins have been isolated that
appear to interact specifically with highly repetitive A-I-T-rich satellite
DNA.Two of these proteins, D1 from Drosophila melano#aster and
HMG-Ifrom mammaliancells, were originally characterized as members
of the high mobility group (HMG)family of nonhistone chromosomal
proteins (Alfagemeet al 1980; Lurid et al 1983). HMGs
are characterized
by their solubility in low salt (0.35 MNaC1)and 5%perehloric acid, and
an abundancy of both basic and acidic amino acids. The basic and acidic
amino acids are distributed in a polar fashion with the amino-terminal
region predominantly basic and the carboxy-terminus acidic (see Johns
1982). Immunofluorescent staining of HMG-Iand D 1 reveals their localization to predominantly satellite heterochromatin, including centromeres
(Alfagemeet al 1980; Disney et al 1989). Morerecently another Drosophila
protein, HP-1, was identified; it is localized to centric heterochromatin
and is responsible in part for position-effect variegation. HP-1is a low
molecular weight protein (18,000 K) that exhibits high levels of acidic and
basic residues typical to HMGs
and has one stretch of six glutamic acids
(James &Elgin 1986; James et al 1989).
Both D 1 and HMG-Ihave been shown to interact with A + T-rich DNA
in vitro (Levinger & Varshavsky 1982; Solomon et al 1986; Reeves
Nissen 1990). Analysis of the amino acid sequences of D1 and HMG-I
has identified a potential DNA-bindingmotif repeated ten times in DI
and three times in HMG-I. The motif consists of Gly-Arg-Pro (GRP)
located within a cluster of basic aminoacids (Ashely et al 1989; Reeves
Nissen 1990). A synthetic eleven amino acid peptide of the consensusbinding domain is able to bind the minor groove of A+T-rich DNA
(Reeves & Nissen 1990). The predicted secondary structure of this peptide
is similar to the structure of A + T-rich DNA-bindingdrugs, netropsin and
distamycin, as well as Hoechst 33258. These compoundsdisplace HMGI from naked DNAand disrupt the structure of kinetochores in whole
cells (Lica et al 1986). Structural studies of HMG-nucleosome
complexes
place HMG-likeproteins along the DNAduplex as it enters and exits the
histone octomer of a single nucleosome. Considering that the specificity
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STRUCTURE AND FUNCTION OF CENTROMERES
325
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for nucleosomepositioning can be reconstituted in purified systems with
alphoid DNAand purified histone octamers (Linxweiler & Horz 1985),
the HMG
proteins are likely to confer additional structural information.
Mutations in HP-1 that directly impinge upon heterochromatin physiology indicate that HMG-likeproteins play important roles in higher
levels of heterochromatin condensation and subsequent formation of the
kinetochore.
CENP-BCENP-Bis an acidic protein that binds to alphoid satellite DNA
(Masumotoet al 1989; Sullivan & Glass 1991). The amino acid sequence
of CENP-Breveals the presence of two highly acidic domains located in
the carboxy-terminalhalf of the protein (Earnshawet al 1987). In addition,
CENP-Bcontains a high percentage of basic amino acids (approximately
12%). Almost half of these amino acids are located in the amino-terminal
quarter of the protein. The overall charge of this region is + 17. The last
50 amino acids of the protein are also highly basic (22%, overall charge
+ 19). Thus CENP-Bhas an HMG-like composition. In addition, CENPB contains one copy of the tripeptide Gly-Arg-Pro (GRP); however, unlike
HMG-Iand D1 DNA-bindingdomains this tripeptide is not imbedded in
a cluster of basic aminoacids. The ability to extract CENP-B
from nuclei
with relatively low salt (0.5 MNaCI; Masumotoet al 1989) is also consistent with an HMG-likecharacterization.
It has been reported that
CENP-Bis an abundant component of the insoluble mitotic scaffold
(Earnshawet al 1984). CENP-B
maybe subject to cell-cycle modifications,
and/or its interphase properties may differ from CENP-B
found in mitotic
preparations. Alternatively, differences in extraction protocols may
account for the enhanced dissociation of these antigens from interphase
cells compared to their mitotic counterparts.
CENP-Bis an abundant protein (20,000-50,000 copies per cell) that binds individual chromosomes according to their alphoid DNAcontent (Earnshaw et al
1987). Thus CENP-Bbinds alphoid regions in a stoichiometric fashion
in vivo.
Injection of antibodies against CENP-Band other kinetochore antigens
into living cells directly implicates their functional role in chromosome
movementas well (Simerly et al 1990; Bernat et al 1990; Yen et al 1991).
Interestingly, injection of the antibodies against predominantly CENP-B
only blocks chromosome movements prior to the metaphase/anaphase
transition. Once the cells traverse this point, the kinetochore antigens
cannot be neutralized by the antibodies. As discussed below, these
data indicate at least a two-step process in the assembly of a kinetochore complex: protein DNArecognition and maturation into an active
segregation complex.
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REGULATION
COMPONENTS
& BLOOM
AND ASSEMBLY
OF CENTROMERIC
The dynamicsof centromerereplication, assembly, and maturation have
been studied in greatest detail at the molecularlevel in the yeast, S.
cerevisiae. CentromericDNA
is replicated early in the S-phaseof the cell
cycle (McCarroll & Fangman1988) and is correlated temporally with
replication of the spindle pole bodies. Theuniquechromatinstructure is
present at all stages of the cell cycle (Yeh1985; E. Yeh& K. Bloom,
unpublishedresults). OnlywhenDNA
replication is allowed to proceed
in the absenceof histone synthesis does the centromerebecomeexposed
to nucleolytic digestion (Saunders et al 1990). Thus the assembly
centromeric chromatin is likely to be coordinated with centromere
DNA
replication. Thestructural assemblyof a limited numberof critical
proteins such as CBF3requires their nonrandomassembly. These data
are therefore consistent with the view that centromeric componentsprovide a template for formation of newstructures as the replication fork
proceeds.
Amaturationstep can be defined as the transition from protein binding
to centromereDNA
to a centromerethat actively segregates chromosomes.
Protein bindingitself is not sufficient fromchromosome
segregation. This
is mostevident fromstudies in whichthe protein-bindingcapacity of the
centromereDNA
has been uncoupledfrom its partitioning function. The
centromere can be conditionally inactivated by transcription from an
adjacent promoter (Hill & Bloom1987). The conditionally inactivated
centromereis organizedinto a protected structure characteristic of wildtype centromere.Determination
of the initiation site and length of adjacent
RNAsupontranscriptional activation reveal that the transcripts do not
penetrate the centromere core (Hill 1988; Russo & Sherman1988). The
terminationsite of the transcripts mapsto the boundariesof the protected
core structure and confirms that a portion of the centromeric proteins
remain bound to the DNA
under conditions of centromere inactivation.
In contrast, particular mutations in the centromere DNAthat disrupt
function, disrupt structure as well (Saunderset al 1988). Thusthe conditional centromereand selected point mutations in the centromereDNA
are indistinguishablein their loss of function, but differ in the mechanism
of inactivation. Theseobservationsprovide a framework
for distinguishing
mutations in protein binding at centromere, from mutations in proteinprotein interactions, or post-translational modifications, whichare
required for the maturation of this complexinto an active partitioning
element.
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Chromosome Copy Control
327
Mutants
Competitionfor the binding of a limited pool of centromereproteins or
other spindle components
is manifestedin the severe limitations for extra
centromeresin yeast, first demonstratedby Futcher & Carbon(1986).
Wild-typeyeast cells contain 16 chromosomes,
and therefore 16 centromeres. Thecells can tolerate 1-2 extrachromosomal
centromereswithout
any measurablegrowthor segregation defect. The application of genetic
pressure to maintain an increased numberof centromeres(greater than
five to ten) results in cell death. Theuse of geneticmarkersthat complement
a deficiency in a gene dosage-dependentfashion provides a meansfor
screening heterogeneity in plasmidcopy numberin a population of cells
in the absenceof geneticselection pressures. Resnicket al (1990)used the
CUP1gene, whichprovides resistance to exogenouscopper, to demonstrate limitations in centromere copy numberof approximatelyten per
cell. Thusboth genetic selection for increased copy numberand indirect
assessmentof copy numberby gene dosage reveal limited tolerance for
centromere DNA.
Relaxation of stringent copy control mechanisms
mayreveal mutations
in critical components
that are otherwiselimiting in the cell. Theuse of
gene dosage markers provides a meansfor determining plasmid copy
number, as well as providing selection for mutations in copy number
control. The drug-resistant marker, G418(Tschumper& Carbon 1987),
and particular mutationsin the leu2 geneexemplifysuch dosage-dependent
markers. The leu2-d allele contains a truncated promoterthat is only
partially functional (Runge&Zakian1989), and therefore is required
high copy number(100-200copies per cell) to efficiently complement
leu2
auxotrophs. Fewer than 10 copies of leu2-d results in poor complementation, or a complete failure to grow, dependingon the particular
strain of yeast. Theleu2-d allele has beenclonedinto plasmidsharboring
a variety of wild-type and mutantcentromeres.Results from quantitative
hybridization to yeast colonies containing these plasmidsreveal a highly
elevated plasmidcopynumberwithout the centromere,low levels of wildtype centromere plasmids, and intermediate levels of plasmids with a
mutant centromere(central C residue to CDEIII changedto A) (Figure
3). This eis mutation in centromere DNA
is clearly not subject to the
stringent copycontrol of wild-typecentromeres,but is regulated compared
to vector sequenceswith no centromere.This quantitative assay for plasmid copy numberreveals measurable protein-binding capacity of centromeremutantsthat are otherwisedefective in their segregationcapacity.
Whena conditional centromerewas inactivated by growthon galactose,
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328
SCHULMAN & BLOOM
Figure 3 Quantitative analysis of minichromosome copy number by colony hybridization. S. cereuisiue cells contain minichromosomes carrying the leu2-dallele (see
text) and no centromere (top); mutant centromeres containing A in place of the central C residue of CDE 111 that inactives
segregation function (middle); or wild-type
centromcrc (bottom). Cells were grown
directly onto nitrocellulose filters in the
absence of leucine. The colonies were lysed
on the filters and hybridized to "P-labeled
pBR322 DNA to visualize exclusively plasmid sequences. After hybridization, filters
were washed and exposed to X-ray film.
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STRUCTURE AND FUNCTION OF CENTROMERES
329
only 10-20 copies accumulated per cell (I. Schulman& K. Bloom, unpublished results). These data are consistent with the observation that conditionally inactive centromeres bind limiting componentsin the cell, but
are uncoupled from their segregation function. The ability to dissect the
cell’s tolerance for centromere DNAfrom its segregation function should
allow us to separate mutations in protein binding from the more complex
partitioning functions characteristic of the centromere.
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ChromosomeFidelity
Mutants
A number of genetic screens have been developed to search for genes
whose products are involved in chromosometransmission. These screens
typically exploit a genetic trait that can be monitored in a visual colony
assay. A numberof genes have been identified and will certainly comprise
proteins involved in replication, assembly, and segregation (McGrewet al
1989; Meeks-Wagneret al 1986; Spencer et al 1988, 1990; Gerring et al
1991). Identification of critical componentsin the segregation machinery
per se will require secondary screens to distinguish regulatory and structural components. Characterization of the gene product of one gene
required for the fidelity of chromosome
transmission reveals protein kinase
domains (Shero & Hieter 1991). Considering the role of phosphorylation
in the binding specificity of CBF3and the activation of cell cycle cascades,
the identification and characterization of protein kinases are important
steps towards understanding processes of assembly and maturation of
centromeric components.
EVOLUTION OF THE KINETOCHORE
Centromeres from both S. cerevisiae and S. pombe contain regions of
single-copy DNAsequence that are required for proper centromere function. At this time there is no evidence for the presence of unique sequence
DNAas a major component of human centromeres (Willard et al 1989).
The current observations suggest that the entire region of humancentromeres maybe permissive for kinetochore assembly. The exact placement
of the kinetochore could be randomwithin the larger centromeric domain
(Radic et al 1987) and guided by the initial deposition of critical kinetochore components. The assembly of the segregation machinery is likely
to represent the compilation of repeating structural units. A threshold of
critical mass wouldbe necessary to ensure the capture and stabilization of
kinetochore mictotubules that emanate from the centrosome.
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330
SCHULMAN & BLOOM
Tandemly repeated arrangements of kinetochore units in mammalian
systems are an attractive mechanismto explain an apparent disparity in
centromere DNAorganization throughout phylogeny (see Brinkley et
al 1989). The coordination of multiple centromeres in mitotically stable
arrangements can be addressed in the artificial chromosomesystems. Many
dicentric chromosomes are unstable during mitosis (McClintock 1939;
Mann& Davis 1983; Oertel & Mayer 1984; Vig & Paweletz 1988; Koshland
et al 1987; Hill & Bloom1987, 1989). However, Koshland et al (1987)
demonstratedthat mitotic instability of small circular dicentric molecules
was a function of the distance between the two centromeres. The closer
the two centromeres were in proximity; the more stable the artificial chromosome. E. Amaya& K. Bloom (unpublished results) juxtaposed two
centromere core structures on a chromosomeand found these molecules
to be very stable. The structural organization of adjacent centromeres
revealed two distinct centromeric cores. Thus it is unlikely that one centromere precludes the other from forming. Rather, the two seem to be
coordinately controlled and attached to the same pole, tandemly arranged
in a physically stable structure.
Stable dicentric chromosomesin mammaliancell are typified by one
functional centromere (primary constriction) and one nonfunctional centromere, recognized by C-band staining and, in some cases, by the CREST
antisera (Merry et al 1985; Earnshaw & Migeon 1985; Zinkowski et al
1986; Peretti et al 1986; Cherry & Wang1988; Vig & Paweletz 1988).
Cytological examination of dicentric chromosomesindicates that in most
cases the inactive centromere appears smaller. Examination ofcentromere
antigens by Earnshaw et al (1989) revealed the presence of CENP-B
both functional as well as nonfunctional centromeres, consistent with its
binding to alphoid DNA.However, another centromere antigen, CENPC, seems to be restricted to the active centromere. The inactive centromere
maybe devoid of key binding sites for segregation function, or altered in
such a way that DNApackaging and/or size precludes visualization of
antibody staining. A mechanismfor the loss of centromeric DNAis suggested from studies in S. cerevisiae that indicate monocentricchromosomes
can be derived from dicentrics, by excision of a small region of DNA
containing one of the centromeres (Hill & Bloom 1989; J. Brock & K.
Bloom, unpublished observations). The breakage-fusion-bridge
cycle
described by McClintock(1939) would also result in centromeric deletions
until segregation function ceases. These remnant centromeres maycontain
a sub-threshold level of repeats that are competent to bind a set of
kinetochore proteins, but unable to attain the critical mass necessary for
capturing microtubules.
Microtubule attachments that persist on individual units would support
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STRUCTURE AND FUNCTION OF CENTROMERES
331
the view of a mammalian centromere comprised of tandem arrays of
smaller functional units. A recent report has demonstrated that kinetochores are comprised of such autonomous elements. Brinkley and his
co-workers (Brinkley et al 1988) utilized caffeine to induce cells blocked
with hydroxyurea to segregate their unreplicated chromosomes[mitosis
with unreplicated genomes (MUGs)]. The kinetochores were fragmented
and physically detached from the bulk of the chromosome in MUGed
cells. Kinetochores retained the characteristic trilaminar ultrastructure
and short fragments of centromeric DNA.Surprisingly, the microtubule
attachments were stable, and the detached kinetochores continued their
migration to the spindle pole. These data extend the observations of Kenna
et al (1988) that excision of yeast centromeres does not perturb protein
binding. Experimental detachment of the centromere region from mammalian chromosomesdoes not perturb their functional properties. The
kinetochore region is certainly autonomous from the chromosomal arms
and is likely to be redundant in its composition.
The ability of single centromeric units to function in budding yeasts
may reflect their continual attachment to the spindle apparatus. Systems
in which kinetochores disassemble following mitosis require a mechanism
to establish kinetochore-microtubule connections. The dynamic nature of
the mitotic spindle ensures that an excess of microtubule ends are available
for kinetochore capture. In fact, microtubule ends will be continually
available until such time that every chromosome
is tethered to the spindle
(see Nicklas 1988). An effective kinetochore must be distinguished from
the surrounding chromatin, however, and perhaps a critical size favors
these chance encounters with microtubules. The large repeating motifs in
S. pombe and mammalianspecies represent a region of differentiation
along the chromosomal DNAthat may serve such a purpose. Wesuggest
that the centromere DNA-protein-microtubule connections are fundamentally similar throughout phylogeny, and vast differences in kinetochore size reflect elaborations and variation in modesof microtubule
capture.
ACKNOWLEDGMENTS
Wethank Elaine Yeh and JoAnn Brock for critical review of the manuscript. Workfrom our laboratory was supported by National Institutes of
Health grant RO1GM32238.K.B. was supported in part by a Research
Career Development Award from the National Cancer Institute of the
National Institutes of Health. I.S. was supported by a postdoctoral fellowship from the DamonRunyon-Walter Winchell Cancer Foundation.
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SCHULMAN & BLOOM
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Literature Cited
Alfageme, C. R., Rudkin, G. T., Cohen, L.
H. 1980. Isolation, properties and cellular
distribution
of DI, a chromosomal protein of Drosophila. Chromosoma78:1-31
Am, P. H., Ketabgian, A. A., Smith, S.,
Schwartz, D. C., Jobs, E. W. 1989. The
macromolecular organization of human
centromeric regions. In Mechanisms of
chromosomedistribution and aneuploidy,
ed. M. A. Resnick, I:l. K. Vig, pp. 9-18.
NewYork: Liss. 400 pp.
Ashely, C. T., Pendleton, C. G., Jennings,
W. W., Saxena, A., Glover, C. V. C. 1989.
Isolation and sequencing of cDNAclones
encoding Drosophila chromosomal protein DI. J. Biol. Chem.264: 8394-840l
Baker, R. E., Fitzgerald-Hayes, M., O’Brien,
T. C. 1989. Purification of the yeast centromere-binding
protein CP1 and a
mutational analysis of its binding site. J.
BioL Chem. 264:10843-50
Baker, R. E., Masison, D. C. 1990. Isolation
of the gene encoding the Saccharomyces
cerevisiae centromere-binding protein
CP1. MoLCell Biol. 10:1863-72
Bernat, R. L., Borisy, G. G., Rothfield, N.
F., Earnshaw, W. C. 1990. Injection of
anticentromere antibodies in interphase
disrupts events required for chromosome
movementsat mitosis. J. Cell Biol. 111:
1519-33
Bloom~K. S., Amaya,E., Carbon, J., Clarke,
L., Hill, A., Yeh, E. 1984a. Chromatin
conformation of yeast centromeres. J. Cell
Biol. 99:1559~8
Bloom, K., Amaya, E., Yeh, E. 1984b. Centromeric DNAstructure in yeast chromatin. In Molecular BiolotTy of the Cytoskeleton,
ed. G. G. Borisy, D. W.
Cleveland, D. B. Murphy, pp. 175-84.
NewYork: Cold Spring Harbor. 512 pp.
Bloom, K. S., Carbon, J. 1982. Yeast centromere DNAis in a unique and highly
ordered structure in chromosomes and
small circular minichromosomes.Cell 29:
305-17
Bram,R. J., Kornberg, R. D. 1987. Isolation
of a Saccharomyces cerevisiae centromere
DNA-binding protein, its human homolog, and its possible role as a transcription
factor. MoLCell Biol. 7:403-9
Brinkley, B. R. 1990. Centromeres and kinetochores: integrated domains of eukaryotic chromosomcs.Curr. Op. Cell Biol. 2:
446-52
Brinkley, B. R., Valdiva, M. M., Tousson,
A., Balczon, R. D. 1989. The kinetochore:
structure and molecular organization. In
Mitosis Molecules and Mechanisms, ed. J.
S. Hyams,B. R. Brinkley, pp. 77-118. San
Diego: Academic. 350 pp.
Brinkley, B. R., Zinkowski, W. L., Mollon,
F. M., Davis, F. M., Pisegna, M., et
al. 1988. Movement and segregation of
kinetochores experimentally detached
from mammalian chromosomes. Nature
336:251-54
Burke, D. T., Carle, G. F., Olson, M. V.
1987. Cloning of large segmentsof exogenous DNAinto yeast by means of artificial
chromosomes. Science 236:806-12
Cai, M., Davis, R. W. 1989. Purification of
a yeast centromere-binding protein that is
able to distinguish single-base
pair
mutationsin its recognition site. Mol. Cell.
Biol. 9:2544-50
Cai, M., Davis, R. W. 1990. Yeast centromere binding protein CBF1, of the
helix-loop-helix family, is required for
chromosome stability
and methionine
prototrophy. Cell 61:437-46
Cherry, L. M., Wang, R.-Y. 1988. Kinetochore staining in multicentric chromosomesof murine cancer lines. Cytobios 53:
173-83
Chikashige, Y., Kinoshita, N., Nakaseko,
Y., Matsumoto, T., Murakami, S., et al.
1989. Composite motifs and repeat symmetry in S. pombe centromeres: direct
analysis by integration of Notl restriction
sites. Cell 57: 739-5l
Clarke, L. 1990. Centromeres of budding
and fission yeasts. Trends Genet. 6:150-54
Clarke, L., Baum, M. P. 1990. Functional
analysis ofa centromerefrom fission yeast:
a role for centromere-specific repeated
DNAsequences. Mol. Cell Biol. I0:186372
Cooke, C. A., Heck, M. M. S., Earnshaw,
W. C. 1987. The INCENPantigens: movement from the inner centromere to the
midbodyduring tnitosis. J. Cell Biol. 105:
2053q57
Compton,13. A., Yen, T. J., Cleveland, D.
W. 1991. Identification
of novel centromere/kinetochore associated proteins
using monoclonal antibodies generated
against
human mitotic
chromosome
scaffolds. J. Cell Biol. 122:1083-98
Dean, A., Pederson, D. S., Simpson, R. T.
1989. Isolation of yeast plasmid chromatin. In Methods in Enzymology, ed. P. M.
Wasserman, R. D. Kornberg, pp. 26-41.
" NewYork: Academic. 683 pp.
Disney, J. E., Johnson, K. R., Magnuson,
N. S., Sylvester, S. R., Reeves, R. 1989.
High-mobility
group protein
HMG-I
localizes to G/Q- and C-bands of human
and mouse chromosomes. J. Cell Biol.
109:1975-82
Earnshaw, W. C., Halligan, N., Cooke, C.
A., Rothfield, N. 1984. The kinetochore is
Annual Reviews
www.annualreviews.org/aronline
Annu. Rev. Cell. Biol. 1991.7:311-336. Downloaded from arjournals.annualreviews.org
by University of North Carolina - Chapel Hill on 11/14/07. For personal use only.
STRUCTURE AND FUNCTION OF CENTROMERES
333
part of the metaphase chromosome
Construction of functional artificial
scaffold. J. Cell Biol. 98:352-57
minichromosomes in the fission yeast
Earnshaw, W. C., Migeon, B. R. 1985. Three
Schizosaccharomyces pombe. Proc. Natl.
related centromere proteins are absent
Acad. Sci. USA 86:577-81
Hahnenberger, K. M., Carbon, J., Clarke,
from the inactive centromere of a stable
isodicentric
chromosome. Chromosoma
L. 1991. Identification of DNAregions
92:290-96
required for mitotic and meiotic functions
Earnshaw, W. C., Ratrie, H., Stetten, G.
within the centromere of Schizosac1989. Visualization ofcentromere proteins
charomyces pombe chromosome I. Mol.
CENP-Band CENP-Con a stable dicenCell. Biol. In press
tric chromosomein cytological spreads.
Han, M., Chang, M., Kim, U., Grunstein,
Chromosoma 98:1-12
M. 1987. Histone H2Brepression causes
Earnshaw, W. C., Rattner, J. B. 1989. A map
cell-cycle-specific arrest in yeast: Effects
of the centromere (primary constriction)
on chromosomalsegregation, replication,
in vertebrate chromosomesat metaphase.
and transcription. Cell 48:589
See Arn et al 1989, pp. 33~42
Heus, J. J., Zonneveld, B. J. M., Steensma,
Earnshaw, W. C., Sullivan, K. F., Machlin,
H. Y., Van den Berg, J. A. 1990. Centromeric DNAof Kluyceromyces lactis.
P. S., Cooke,C. A., Kaiser, D. A., et al.
1987. Molecular cloning of cDNAfrom
Curr. Genet. 18:517-22
CENP-B, the major human centromere
Hieter, P., Pridmore, D., Hegemann,J. H.,
autoantigen. J. Cell Biol. 104:817-29
Thomas, M., Davis, R. W., Philippsen, P.
Eckdahl, T. T., Anderson, J. A. 1987. Com1985. Functional selection and analysis of
puter modelling of DNAstructures
yeast centromeric DNA.Cell 42:913-21
involved in chromosome maintenance.
Hill, A. 1988. Genetic manipulation of cenNucleic Acids Res. 15:853145
tromere function in Saccharomycescerevisiae. PhDthesis. Univ. N. Carolina. 133
Fishel, B., Amstutz, H., Baum,M., Carbon,
J., Clarke, L. 1988. Structural organppization and functional analysis of cenHill, A., Bloom, K. 1987. Genetic maniputromeric DNAin the fission yeast Schizolation of centromere function. Mol. Cell.
saccharomycespombe. Mol. Cell. Biol. 8:
Biol. 7:2397-2405
754~63
Hill, A., Bloom,K. S. 1989. The acquisition
Fitzgerald-Hayes, M., Clarke, L., Carbon,
and processing of dicentric chromosomes
J. 1982. Nucleotide sequence comparisons
in yeast. Mol. Cell. Biol. 9:1368-70
and functional analysis of centromeric
Huberman, J. A., Pridmore, R. D., Jager,
DNAof yeast centromere DNAs. Cell 29:
D., Zonneveld, B., Philippsen, P. 1986.
235-44
Centromeric DNAfrom Saccharomyces
Funk, M., Hegemann,J. H., Philippsen, P.
uvarum is functional in Saccharomyces
1989. Chromatindigestion with restriction
cerevisiae. Chromosoma94:162~58
endonucleases reveals 150-160 bp of proJames, T. C., Eissenberg, J. C., Craig, C.,
tected DNAin the centromere region of
Dietrich, V., Hobson,A., Elgin, S. C. R.
chromosome XIV in Saccharomyces cere1989. Distribution patterns of HP1, a
visiae. Monogr.Gen. Genet. 219:153 60
heterochromatin-associated
nonhistone
Futcher, B., Carbon, J. 1986. Toxic effects
chromosomal
of Drosophila. Eur.
J. Cell Biol. protein
50:170-80
of excess cloned centromeres. Mol. Cell.
Biol. 6:2213-22
James, T. C., Elgin, S. C. R. 1986. IdentiGaudet, A. M., Fitzgerald-Hayes, M. 1990.
fication of a nonhistone chromosomal
The function of centromeres in chroprotein associated with heterochromatin
mosomesegregation. In The Eukaryotic
in Drosophila melanogaster and its gene.
Nucleus, ed. P. R. Strauss, S. H. Wilson,
Mol. Cell. Biol. 6:3862-72
2: 845-81. NewJersey: Telford. 881 pp.
Kayne, P. S., Kim, U.-J., Han, M., Mullen,
Gerring, S. L., Spenser, F., Hieter, P. 1991.
J. R., Yoshizaki, F., Grunstein, M. 1988.
The CHL1 (CTF1) gene product of SacExtremely conserved histone H4 terminus
charomyces cerevisiae is important for
is dispensable for growth but essential for
chromosometransmission and normal cell
repressing the silent mating type loci in
cycle progression in G2/M. EMBOJ. 9:
yeast. Cell 55:27-39
4347-58
Johns, E. W. 1982. History, definitions and
Greig, G. M., England, S. B., Bedford, H.
problems.
In The HMGChromosomal
M., Willard, H. F. 1989. ChromosomeProteins, ed. E. W. Johns, pp. 1 8. Lonspecific alpha satellite DNAfrom the cendon: Academic. 251 pp.
tromere of chromosome 16. Am. J. Hum. Kenna, M., Amaya, E., Bloom, K. S. 1988.
Genel. 45:862-72
Selective excision of the centromere
Hahnenberger, K. M., Baum,M. P., Polizzi,
chromatin complex from Saccharomyces
C. M., Carbon, J., Clarke, L. 1989.
cerevisiae. J. Cell Biol. 107:9-15
Annual Reviews
www.annualreviews.org/aronline
Annu. Rev. Cell. Biol. 1991.7:311-336. Downloaded from arjournals.annualreviews.org
by University of North Carolina - Chapel Hill on 11/14/07. For personal use only.
334
SCHULMAN& BLOOM
Koshland,D. L., Rutledge,L., Fitzgeraldgenes that affect mitotic chromosome
Hayes, M., Hartwell, L. 1987. A genetic
transmissionin S. cerevisiae. Cell 44: 53analysis of dicentric minichromosomes
in
63
Saccharomyces
cerevisiae. Cell 48:801-12 Mellor,J., Jiang, W.,Rathjen,J., Barnes,C.
Lechner, J., Carbon, J. 1991. A 240 kD
A., Hinz, T., et al. 1990. CPF1,a yeast
multisubunit protein complex(CBF3)is
protein whichfunctions in centromeres
major componentof the budding yeast
and promoters. EMBO
J. 9:4017-26
centromere.Cell 64:717-25
Merry, D. E., Pathak, S., Hsu, T. C.,
Levinger,L., Varshavsky,A. 1982. Protein
Brinkley, B. R. 1985. Anti-kinetochore
D1 preferentially binds to A+T-rich
antibodies: use as probesfor inactive cenDNA
in vitro and is a componentof Drotromeres. Am. J. Hum.Genet. 37:425-30
sophila melanogaster nucleosomescon- Mitchison,T. J., Evans,L., Schulze,E., Kirtaining A+T-richsatellite DNA.Proc.
schner, M.W.1986. Sites of microtubule
Natl. Acad. Sci. USA79:7152-56
assemblyand disassemblyin the mitotic
Lica,L. M., Narayanswami,S., Hamakalo,
spindle. Cel145:515-21
B. A. 1986. Mousesatellite DNA,cenMitchison, T. J., Kirschner, M. W.1985.
tromere structure, and sister chromatid
Properties of the kinetochorein vitro. I.
pairing. J. Cell BioL103:1145-51
Microtubulenucleation and tubulin bindLinxweiler, W., Horz, W. 1985. Reconing. J. Cell Biol. 101:755-65
stitution experimentsshowthat sequence- Moroi,Y., Peebles,C., Fritzler, M.J., Steispecific histone-DNA
interactions are the
gerwald, J., Tan, E. M.1980. Autoantibody to centromere (kinetochore)
basis for nucleosomephasing on mouse
satellite DNA.Cell 42:281-90
scherodermasera. Proc. Natl. Acad.Sci.
Lund, T., Holtlund, J., Fredrickson, M.,
USA 77:1627-31
Laland, S. G. 1983. On the presence of Nakaseko,Y., Adachi, Y., Funahashi, S.,
two newmobility group-like proteins in
Niwa, O., Yanagida, M. 1986. ChroHeLa$3 cells. FEBSLett. 152:163-67
mosomewalking shows a highly homMahtani, M. M., Willard, H. F. 1990.
ologousrepetitive sequencepresent in all
Pulsed-field gel analysis of or-satellite
the centromereregions of fission yeast.
DNAat the humanX chromosomecentroEMBOJ.5:i011 21
mere: high frequency polymorphismsand Nicklas, R. B. 1988. Chanceencountersand
array size estimates. Genomics7:607-13
precisionin mitosis. J. Cell. Sci. 89: 283Mann,C., Davis, R. W.1983. Instability of
85
dicentric plasmidsin yeast. Proc. Natl. Nicklas, R. B. 1989. Themotorfor poleward
chromosomemovementin anaphase is in
Acad. Sci. USA80:228-32
Masumoto,H., Masukata, H., Muro, Y.,
or near the kinetochore.J. Cell Biol. 109:
Nozaki, N., Okazaki, T. 1989. A human
2245-55
centromere antigen (CENP-B)interacts
Niwa, O., Matsumoto,T., Chikashige, Y.,
with a short specific sequencein alphoid
Yanagida, M. 1989. Characterization of
Schizosaccharomyces pombeminichroDNA,a humancentromeric satellite. J.
Cell Biol. 109:1963-73
mosome
deletion derivatives and a functional allocation of their centromere.
Matsumoto,T., Murakami,S., Niwa, O.,
Yanagida, M. 1990. Construction and
EMBOJ. 8:3045-52
characterization of centric circular and Oertel, W., Mayer,M.1984. Structure and
mitotic stability of minichromosomes
acentric linear chromosomes
in fission
yeast. Curr. Genet.18:323-30
originating in yeast cells transformedwith
McCarroll, R. M., Fangman,W. L. 1988.
tandem dimers of CENll plasmids. Mol.
Timeof replication of yeast centromeres
Gen. Genet. 198:300-7
and telomeres. Cell 54:505-13
Palmer, D. K., O’Day,K., Margolis, R. L.
1989. Biochemicalanalysis of CENP-A,
a
McClintock,B. 1939. The behavior of successive nuclear divisions of a chromosome centromeric protein with histone-like
properties. SeeAmet al 1989,pp. 61-72
brokenat meiosis. Proc.Natl. Acad.Sci.
Palmer, D. K., O’Day,K., Trong, H. L.,
USA 25:405 16
McGrew,
J. T., Xiao, Z., Fitzgerald-Hayes,
Charbonneau,H., Margolis, R. L. 1990.
Partial sequence analysis of the cenM. 1989. Saccharomyces cerevisiae
tromere specific protein CENP-A
reveals
mutants defective in chromosomesegextensive homology
to histone H3. J. Cell
regation. Yeast 5:271-84
Meeks-Wagner,
D., Hartwell, L. H. 1986.
Biol. 111(5):281(Abstr.)
Normalstoichiometry of histone dimer Peretti, D., Maraschio,P., Lambiase,S.,
sets is necessaryfor highfidelity of mitotic
Curto, F. L., Zuffardi, O. 1986.Indirect
immunofluorescence of inactive cenchromosome
transmission. Cell 44:43-52
tromeresas indicator of centromerefuncMccks-Wagner,
D., Wood,J. S., Garvik, B.,
tion. Hum.Genet. 73:12-16
Hartwell, L. H. 1986. Isolation of two
Annual Reviews
www.annualreviews.org/aronline
Annu. Rev. Cell. Biol. 1991.7:311-336. Downloaded from arjournals.annualreviews.org
by University of North Carolina - Chapel Hill on 11/14/07. For personal use only.
STRUCTUREAND FUNCTION OF CENTROMERES
335
putative protein kinase. GenesDev.5: 549Pfarr, C. M., Coue, M., Grissom, P. M.,
Hays,T., Porter, M. E., Mclntosh,J. R.
60
1990. Cytoplasmicdyneinis localized to Simerly, C., Balczon,R., Brinkley, B. R.,
Schatten, G. 1990. Microinjectedkinetokinetochoresduring mitosis. Nature345:
chore antibodies interfere with chro263-65
Polizzi, C., Clarke, L. 1991. Thechromatin
mosome
movementin meiotic and mitotic
structure of centromeres from fission
mouseoocytes. J. Cell Biol. 111: 1491yeast: differentiation of the central core
1504
that correlateswithfunction.J. Cell Biol. Solomon,M.J., Strauss, F., Varshavsky,A.
112:191-201
1986. A mammalianhigh mobility group
protein recognizesanystretch of six A-T
Radic, M. Z., Lundgren,K., Hamakalo,B.
A. 1987. Curvature of mousesatellite
base pairs of duplex DNA.Proc. Natl.
DNAand condensation
of heteroAcad. Sci. USA83:1276-80
chromatin. Cell 50:1101-8
Spencer,F., Connelly,C., Lee, S., Hieter, P.
1988. Isolation and cloning of conRattner, J. B. 1991. Thestructure of the
mammalian
centromere. Bioessays 13:51ditionally lethal chromosometrans56
mission fidelity genes in Saccharomyces
Rattner, J. B., Kingwell,B. G., Fritzler, M.
cerevisiae. In CancerCells Eukaryotic
J. 1988.Detectionof distinct structural
DNAReplication, ed. T. Kelly, B. Stilldomainswithin the primary constriction
man, 6: 441-52. NewYork: Cold Spring
using autoantibodies. Chromosoma
96:
Harbor Lab.
360-67
Spencer, F., Gerring, S. L., Connelly, C,,
Reeves, R., Nissen, M. S. 1990. The A-THieter, P. 1990. Mitotic chromosome
DNA-binding domain of mammalian
transmission fidelity mutants in Sachigh mobility group I chromosomal
procharomyees
cerevisiae. Genetics 124:237
teins. J. Biol. Chem.265:8573-82
49
Rieder, C. L., Alexander,A. P. 1990. Kine- Steuer, E. R., Wordeman,
L., Schroer, T.
tochores are transported polewardalong
A., Sheetz, M. P. 1990. Localization of
a single astral microtubuleduring chrocytoplasmicdyneinto mitotic spindles and
mosome
attachmentto the spindle in newt
kinetochores. Nature345:266-68
lung cells. J. Cell Biol. 110:8145
Tschumper, G., Carbon, J. 1987. SaeResnick, M. A, Westmoreland,J., Bloom,
charomycescerevisiae mutants that tolK. 1990. Heterogeneity and maintenance
erate centromere plasmids at high copy
of centromere plasmid copy number in
number.Proc. Natl. Acad. Sci. USA84:
Saccharomyeescerevisiae. Chromosoma 7203-7
99:281-88
Sullivan, K. F., Glass, C. A. 1991. CENP-B
Runge,K. W., Zakian, V. A. 1989. Introis a highly conserved mammaliancenductionof extra telomericsequencesinto
tromere protein with homologyto the
Saccharomyces
eerevisiae results in telohelix-loop-helixfamilyof proteins. Chromereelongation. Mol.Cell. Biol. 9: 1488mosoma.In press
97
van Holde, K. E. 1989. Chromatin. New
Russo, P., Sherman,F. 1988. Transcription
York:Springer-Verlag.497 pp.
terminates near the poly(A) site in the Vig, B. K., Paweletz,N. 1988. Sequenceof
CYC1gene of the yeast Saecharomyces
chromosomeseparation: generation of
cerevisiae. Proc.Natl. Acad.Sci. USA86:
unstable multicentric chromosomes
in a
8348-52
rat cell line. Chromosoma
96:275-82
Salmon,E. D. 1989. Microtubule dynamics Wevrick, R., Willard, H. F. 1989. Longand chromosomemovement.See Brinkley
rangeorganizationof tandemarrays of ~tet al 1989,pp. 119-82
satellite DNAat the centromeres of
Saunders, M. 1989. The molecular archihuman chromosomes: High-frequency
tecture of yeast centromeres.PhDthesis.
array-length polymorphismand meiotic
Univ. N. Carolina. 144 pp.
stability. Proc. Natl. Acad.Sci. USA86:
Saunders, M. A., Fitzgerald-Hayes, M.,
9394-98
Bloom,K. S. 1988. Chromatinstructure
Willard, H. F. 1990. Centromeres of
of altered yeast centromeres.Proc. Natl.
mammaliancentromeres. Trends Genet.
Acad. Sei. USA85:175-79
6:410-16
Saunders, M. A., Yeh, E., Grunstein, M., Willard,H. F., Waye,J. S. 1987. Hieratchal
Bloom,K. S. 1990. Nucleosome
depletion
order in chromosome-specific human
alters the chromatin structure of Sacalpha satellite DNA.
TrendsGenet.3: 192charomycescerevisiae centromeres.Mol.
98
Cell. Biol. 10:5721-27
Willard, H. F., Wervick,R., Warburton,P.
Shero, J. H., Hieter, P. 1991. A suppressor
E. 1989. Humancentromere structure:
ofa centromere DNAmutation encodes a
organization and potential role of alpha
Annual Reviews
www.annualreviews.org/aronline
336
SCHULMAN & BLOOM
Annu. Rev. Cell. Biol. 1991.7:311-336. Downloaded from arjournals.annualreviews.org
by University of North Carolina - Chapel Hill on 11/14/07. For personal use only.
satellite
DNA.In Mechanisms of Chroreleased from chro~nosomes at the onset
mosomeDistribution and Aneuploidy, ed.
ofanaphase. EMBOJ. 10:1245-54
M. A. Resnick, B. K. Vig, pp. 9-18. New Zhang, X.-Y., Fittler, F., Horz, W. 1983.
York: Liss. 400 pp.
Eight different highly specific nucleosome
Yeh, E. 1985. PhD thesis. Studies of the
phases and e-satellite DNA
in the African
RNAtranscripts
within the centromere
green monkey. Nucleic Acids Res. I1:
region of yeast chromosomeIII and XI.
4287-4306
Univ. Calif., Santa Barbara. 160 pp.
Zinkowski, R. P., Vig, B. K., Broccoli, D.
Yen, T. J., Compton, D. A., Earnshaw, W.
1986. Characterization of kinetochores in
multicentric
chromosomes. Chromosoma
C., Cleveland, D. W. 1991. CENP-E, a
human centromere associated
protein
94:243-48
Annu. Rev. Cell. Biol. 1991.7:311-336. Downloaded from arjournals.annualreviews.org
by University of North Carolina - Chapel Hill on 11/14/07. For personal use only.
Annu. Rev. Cell. Biol. 1991.7:311-336. Downloaded from arjournals.annualreviews.org
by University of North Carolina - Chapel Hill on 11/14/07. For personal use only.