The elusive centromere: sequence divergence and functional conservation

The elusive centromere: sequence divergence and functional conservation Claudio E Sunkel and Paula A Coelho Universidade do Porto, Porto, Portugal The centromere is an essential cis-acting structure present in the chromosomes of all eukaryotes, central to the mechanism that ensures proper segregation during meiosis and mitosis. Molecular characterization of centromeres in the budding and fission yeasts has advanced significantly over the last few years due to their relatively small size and the availability of functional assays. However, identification and characterization of centromeric sequences from multicellular organisms has proven to be slow and difficult in the absence of direct functional tests. Molecular data have recently become available on the centromere of Drosophila, making it possible to bridge a long-standing gap in our knowledge on the general structure of centromeres. An evaluation of the available data from yeast to man suggests that centromere sequence and centromere sequence organization have diverged significantly, even amongst different chromosomes of a single organism; however, overall centromere organization and kinetochore components might be significantly more conserved than thought previously. Current Opinion in Genetics & Development 1995, 5:756--767

Introduction The centromere localizes to the primary constriction of higher eukaryotic chromosomes and to a specific region of the chromosomes o f budding and fission yeasts. It is the primary site for the formation o f functional kinetochores, speciahzed multiprotein complexes that interact with spindle microtubules. As chromosomes condense, spindle microtubules that initiate at the nficrotubule-organizing centers make contact with one sister kinetochore giving rise to mono-oriented chromosomes. Congression proceeds and chromosomes become hi-oriented, developing stable connections with both microtubule-organizing centers and aligning along the metaphase plate. Once a stable interaction between microtubules from both poles and the kinetochore is estabhshed, a number of molecular events appear to take place at the kinetochore. It has been proposed [1] that kinetochore components nfight be part o f a signalling mechanism that integrates the different events o f mitosis, allowing the cell to undergo the metaphase--ganaphase transition. The signals emanating from this checkpoint mechanism might be mediated by an anaphase-promoting complex, that induces anaphase through ubiquitin-conjugated proteolysis [2]. During anaphase, the kinetochore is associated with the movement of the chromatid towards the pole along the kinetochore-to-pole microtubules [31.

In this review, we assess recent data on the sequence organization and kinetochore components o f centromeres from the budding yeast to mammals and compare them, with the aim o f pointing out unifying patterns rather than individual sequence diversity. The DNA sequence structures for the budding and fission yeast centromeres (the C E N elements) have been clearly defined after systematic analysis over a number of years [4-6], This task was facilitated to some extent by the fact that yeast chromosomes are relatively small, do not contain large regions of repetitive DNA, and the minimum DNA fragment upor~ which a functional centromere can be assembled is small. In higher eukaryotes, however, the basic sequence organization o f centromeres has not yet been defined [7-10]. The task is enormous, given the fact that the centromere of higher eukaryotes is embedded within highly repetitive DNA. Most importantly, it has not been possible to design functional tests that could determine whether a given DNA fragment can indeed support in vivo the organization o f a functional kinetochore that mediates normal chromosome segregation. In the absence of such tests, it is difficult to ascertain whether any mammalian centromeric DNA has been identified so far. The comparison of DNA sequence motifs, organization and kinetochore components from yeast to man is beginning to indicate that, although centromeres are

Abbreviations CEN--centromere DNA element; YAC--yeast artificial chromosome. 756

© Current Biology Ltd ISSN 0959-437X

The elusive centromere Sunkel highly variable D N A elements, a conserved pattern of sequence organization and function is emerging. Most importantly, shared motifs amongst kinetochore components in yeast and man suggests that mechanisms of segregating sister chromatids may be conserved.

sential yeast artificial chromosomes (YACs) as marker chromosomes. CDEI deletions cause mild meiosis defects whereas single point mutations cause more consistent meiosis I missegregation phenotypes. It is possible that single point mutations alter the binding of centromere factors causing perturbations throughout the kinetochore whereas deletions do not allow binding and therefore have a less profound effect [11°°]. CDEII is essential for proper meiosis; both sequence composition and length might be involved in inducing a bend in the DNA that is required for normal function [4]. A 31 bp CDEII deletion causes a dramatic increase in meiotic chromosome missegregation with an elevated frequency of precocious sister-chromatid segregation. Sears et al. [11 °°] argued that CDEII might be involved in sister chromatid cohesion. Detailed analysis of CDEIII has shown that with the exception o f the C14 nucleotide of CDEIII, point mutations within this element appear to have only slight effects upon meiotic chromosome segregation [11"]. Indeed, there are marked differences in the mitotic and meiotic requirements for different nucleotides within this element, clearly suggesting that although mitosis and meiosis II are thought to be analogous, there are important differences with respect to CDEIII sequence requirements.

Sequence and organization of centromeric DNA in yeasts 5accharomyces cerevisiae The basic structural organization of the ¢entromere in S. cerevisiae has been the subject of intensive research since it was first isolated and shown to faithfully segregate circular plasmids or linear artificial chromosomes [4,6]. The centromere of budding yeast contains an approximately 160-220bp nuclease-resistant core that is flanked by ordered nucleosomes and consists o f three conserved DNA e l e m e n t s - - C D E I , CDEII and CDEIII (Fig. 1). CDEI is an 8bp sequence that fits the general consensus sequence R T C A C R T G (R is A or C). This is followed by the AT-rich CDEII element and then CDEIII, a 26 bp element that exhibits considerable conservation including dyad symmetry around a central C nucleotide. Deletions of CDEI reduce mitotic stability up to 60-fold and deletions of CDEII cause increased rates of chromosomal loss [4]. The sequence requirements for CDEII have not been fully worked out yet, although available data suggest that both sequence composition and length are important for mitosis [4]. In contrast, CDEIII is essential for nfitosis and a single base pair mutation o f the central C at position 14 completely abolishes CEN activity [4]. CDEIII shows an asymmetric response to single base mutations, indicating possible interactions with other centromere DNA-protein complexes or even between the different elements. Indeed, the effects upon chromosome segregation of double mutants of CDEI and CDEIII are not just additive, but synergistic [4]. The meiotic requirements for the different CDE elements have been partially uncovered using non-es-

Schizosaccharomyces pombe S. pombe chromosomes become condensed during mitosis; however, no discrete kinetochore structure has been described [12]. The centromeres of all three chromosomes have been cloned in both circular plasmids and linear YACs and their general structure determined [5]. The three centromeric DNAs span 40-100 kb and contain a number of centromere-specific repetitive elements arranged into inverted repeats around a central core (4-7 kb) that is unique for cen 1 but partly repeated for cen2 and cen3 (Fig. 2). Centromeres in S. pombe are variable genetic elements, varying both between different chromosomes and between the same chromosome amongst different strains. Nevertheless, the

Saccharomycescerevisiae Nucleosomes

I

155-160bp

I

Nucleosomes

CBF3 © 1995 Current Opinkm in Genetics & Development

and Coelho

Fig. 1. Molecular organization of the centromere of 5accharomyces cerevisiae. The top diagram shows the general organization of the centromere with a normal array of nucleosomes at either side of the three conserved centromere elements. Note the position of the C14 nucleotide at the center of CDEIII. The three elements are shown below in an enlarged form to give an idea of their sequence organization and of the proteins that are known to interact with each element. CPfl is thought to bind CDEI as a dimer. However, the detailed composition of the CBF3 complex is not known yet, except that Cbf3A, Cbf3B, Cbf3C and KAR3 are part of it.

757

758

Genomes and evolution basic motif of a central core with inverted repeats is still conserved [5]. Functional analysis of centromeric D N A has been performed in smaller derivatives (7-38 kb) of all centromeres [5]. All stable derivatives were shown to contain the central core and variable amounts of both arms, giving rise to a variable sized inverted array of repeats. Although relatively stable during mitosis, these smaller derivatives tend to segregate prematurely during the first meiotic division. It is possible that the centromeric repeats might play an important role in holding sister chroinatids together during meiosis I [5]. The minimum centromere fragnnent has now been defined by constructing minichromosomes containing specific core and K-type (K, K", dg) repeat sequences [13"]. The central core appears to have a number of redundant sites distributed across its length capable of participating ill centroinere function. Together with part o f the central core, a 2.1 kb KpnI-Kpnl restriction fragment present in all K-type repeats [13"] was found to be necessary and sufficient to obtain substantial centromere activity [5]. Analysis of the chromatin organization of S. pombe centromeres has shown that packaging of the central core sequences and a small part of the flanking repeats is highly atypical and is maintained throughout the cell cycle [5]. Examination of the chromatin structure in centromere-active or centromere-inactive circular minichromosomes has shown the same type of arrangement and functional correlation [14"]. The key to the proper (centromere-active) central core chromatin organization appears to be the 2.1 kb fraginent from the K-type repeats [13"]. This D N A element, that is capable of specifically altering the chromatin organization of the centromeric central core in an orientation-independent manner at a distance, has been named the centromere enhancer. It is now clear that higher-order chromatin organization is essential for centromere function [5,14"].

This model has been used to interpret recent data in which miniInal circular nfinichromosomes containing only the essential 2.1 kb element and the central core sequences were found to switch from an reactive to an active centromere state at relatively high frequency during mitotic cell divisions [151. Finally, recent data has shown that position effect variegation, the unstable expression of a gene when placed next to or within transcriptionally inactive chromatin (telomeres or centromeric heterochromatin) [16], can be detected in the fission yeast I17]. Once the ura4 or the ade6 genes were placed within the central core domain ofce~l 1, ten2 or cell3, their expression fluctuated between expressed and repressed states. Furthermore, like position effect variegation m Drosophila. expression of the genes was temperature-sensitive and the chromatm organization resembled that of the central core rather than the organized nucleosomal arrays. The expression of ura4 + was also ascertained when inserted within the inverted repeat and found to be sigmificantly repressed [ 18].

Protein components of the centromere in yeasts Much is already known about the sequence determinants for CEN D N A in S. cerevisiae and considerable data are now available on proteins that are required to make up the kinetochore (Fig. i; Table 1). A single CDEI-binding factor (Cpfl) has been described [4,6] and shown to be non-essential, although disruption of its gene leads to a 10-fold higher frequency of chromosome loss. Binding o f Cpfl to CDEI appears to induce a bend of this D N A element; thus the correct spatial arrangement of the C D E I - C p f l complex with respect to CDEII and CDEIII may be important for optimal

Schizosaccharomycespombe ,

[::>

<

/--

KI

L

(BJ)

K

l

(M)

K

l'

K I~

B'

c~l

18 kb

I

B

(~2

37 kb

I

( ~~

47 kb

I

] ~9:) ( LJrPt'nl ( )J)I11~(i11 Irl ( ~I'NL'hl • & I )~'\I'[cq)rNI~rll

Fig. 2. Molecular organization of the centromeres of the three S. pombe chromosomes. The diagram shows the central core (cc) and the different types of repeats organized into a large inverted repeat (open arrows). The size in kb indicated below each centromere corresponds to only half their length. Each chromosome has been shown to contain unique and common repeats. Chromosome one (ccl) contains an inverted repeat made up of K', L, K" and B' repeats and the whole centromeric region spans some 38 kb. The inverted repeat ot chromosome two (cc2) contains repeats K, L and B whereas two more repeats (B,J) are present out side on the inverted repeat and the whole centromere spans 75 kb. The third chromosome (cc3) has the largest centromere, spanning 95 kb, and contains the smallest inverted repeat made up of K and L" repeats and one repeat outside (M).

The elusive centromere Sunkel and Coelho

centromere function [4]. Although CDEII is protected from nuclease digestion or chemical modification in ~,iz,o [4,6], no protein has yet been identified that binds to this element. A protein complex named CBF3 that specifically recognizes CDEIII was isolated and shown to contain at least three major components, Cbf3A ( l l 0 k D a ) , Cbf3B (64kDa) and Cbf3C (58kDa) [19]. Cbf3A, the larger subunit of the CBF3 protein complex, was shown to be encoded bv the essential N D C I O gene, with the protein localizing to the spindle poles and along the length of the spindle [6]. Jiang and Carbon [6] suggest that Cbf3A nfight particTipate both in microtubule binding and also in holding sister chromatids together. Cbt3B was first isolated on the basis of partial amino acid sequence [20 bullet] and later identified as the product of the CEP3 gene in a screen designed to generate synthetic acentric marker chromosomes [21°]. Cbf3B is a rare 71 kDa protein that contains an amino-terminal putative Zn(lI)2Cys6 DNA-binding domain and a short internal coiled coil domain. The gene is essential and Cbt3B appears to interact with both sides of the CDEIII palindrome, suggesting that it might bind as a dimer. Temperature-sensitive nmtants appear to arrest during G 2 / M and do not elongate their spindles. Furthermore, some mutant cells have their spindles and D N A in different cell bodies, indicating that chromosomes fail to maintain proper attachment to the microtubules. The authors suggest [21 bullet] that Cbf3B might be the C E N D N A binding component of the CBF3 complex. Cbf3C was isolated using two secondary screens designed to identify alterations in the integrity of the

kinetochore [22]. It is a novel 56 kDa protein encoded by the CTFI3 gene. This protein contains a run of serines similar to that found in mammalian C E N P - B (see below), although no functional homology has been shown. Mutational analysis indicates that the protein is essential, with temperature-sensitive nmtations (e.g. ct[13-10) leading to chromosome missegregation at the permissive temperature and a transient arrest with large buds and a G2 D N A c o n t e n t at t h e non-permissive temperature. The terminal phenotype includes a proportion of cells that appeared to have attempted cytokinesis without segregating their replicated chromosomes. This would be expected if the kinetochore defect in ctfl3-30 cells is recognized by a cell cycle checkpoint [22]. Recently, other putative kinetochore proteins have also been identified. Cse4 is a new chromatin-associated protein that shares significant homology with histone H3 and the mammalian centromere antigen C E N P - A [23°]. In addition, at non-permissive temperatures, the cse4-1 mutant allele causes chromosome missegregation and, like the ctfl3-30 and cep3-1 mutants, cause a mitosis-specific arrest with a G 2 D N A content and short bipolar spindles. Another candidate for a kinetochore protein that binds CDEIII D N A and has minus-end motor activity is the kinesin Kar3 [24°]. This protein was found to be a motor component associated with the CBF3 complex. However, although kar3 null nmtants show severe abnormalities in chromosome segregation, it is not essential for mitosis. The isolation of MIF2 was also reported recently and it was shown to encode a protein o f 549 amino acids that share two blocks of homology with mammalian C E N P - C and appears to interact with Cpfl [25°].

Table 1. Centomere- and kinetochore-associated proteins. Proteins

Genes

Motifs

Phenotypes*t

CP1 Cbf3A

CBF1/CPF1/CP1 NCD10/CBF2/CTF14

Helix-loop-helix Nucleotide-binding

Cbf3B Cbf3C KAR-3 CSE4 MIF2

CBF3B CTF13 KAR3 CSE4 MIF2

DNA-binding Acidic serine-rich Kinesin Histone H3

Met auxotrophy$ G 2 arrest/haploid and polyploid cells G2 arrest G2 arrest Abnormal nuclear fusion-~ G2 arrest Mis-segregation$

Putative homologs

S. cerevisiae

Mammals CENP-A CENP-B CENP-C CENP-D CENP-E MCAK

Histone H3 Acidic serine-rich DNA-binding Highly basic GTP-binding Kinesin Kinesin

CENP-B CENP-A CENP-C

Cse4 Cbf3C Metaphase arrest

MIF2

*Phenotypes of 5. cerevisiae genes were derived from analysis of mutants. ~Phenotypes of mammalian proteins refer to effect of antibody injections. SNot essential.

759

760

Genomesand evolution To date no proteins have yet been identified that bind S. pombe centromeres; however, gel mobility shift assays using specific K" centromere fragments and crude nuclear extracts revealed the formation o f p r o t e i n - D N A complexes with a number of regions of the repeat [14"]. Amongst these fragments, H-I and L are found within the critical 2.1 kb KpnI-KpnI essential centromeric repeat. Furthermore, all fragments that cause retardation contain a 5'-TGGAAA-Y motif. This motif is present within the central core 0 f t h e 17bp C E N P - B box [26], the binding site for the human and mouse centromeric protein CENP-B (see later discussion on mammalian centromeres).

Molecular organization of centromeres in

Drosophila melanogaster A major problem in studying the centromeres of higher eukaryotes, such as Drosophila melanogaster, is that they are embedded within large regions o f constitutive heterochromatin [27]. Inadequate structural and functional analyses have led to the general belief that heterochromatin is inert or 'junk' DNA. However, a number of essential genes have been found within heterochromatin and it is now generally believed

that heterochromatin is required for sister chromatid cohesion of mitotic chromosomes [28-31]. Pericentric heterochromatin and other sites along the chromosome arms also appear to be involved in the segregation of achiasmate chromosomes during meiosis [32,33]. Simple heterochromatic satellites have been cloned and mapped [34-36]. Other more complex satellites that are chromosome-specific include the 359 satellite on the X chromosome [36]; the dodecasatellite on the third chromosome [37]; and on the second chromosome, R.sp [34], Bari [38] and the KH-4 repeat (P Coelho, D Nurminsky, D Hartl C Sunkel, unpublished data) (Fig. 3). More recently, it has become clear that constitutive heterochromatin in the fly also contains a number of different transposable elements in large blocks ([39",40"]; P Coelho, C Sunkel, unpublished data). It was shown recently that closely linked repeats of the P-element transposon containing a white gene at a euchromatic position undergo classical position effect variegation, suggesting that transposons might be involved in heterochromatin formation [41bullet]. It has not yet been determined whether other aspects of heterochromatin behaviour, such as sister chromatid pairing, can be induced at this site. The amount of heterochromatin in a particular chromosome might vary amongst different D. melanogaster wild-type isolates [42]. Molecular analysis shows that

Fig. 3. Fluorescent in situ hybridization of a wild-type second chromosome of Drosophila melanogaster with a probe to a specific repeat only found within the primary constriction of this chromosome. On the left, a chromosome is shown without the centromeric signal and with arrows to show the location of the primary constriction. On the right is the same chromosome with the oveday in situ hybridization signal to show that this repeat occupies more than half of the primary constriction.

The elusive centromere Sunkel and Coelho chromosome 4 varies in size between 4.5 and 5.2 Mb. This variation can only be attributed to changes in the amount o f heterochromatin, as essential genes located in the euchromatin cannot vary significantly. Also, in Drosophila, it has been shown [43] that a significant reduction in the amount of heterochromatin leading to an abnormal chromatin environment, as in the case o f the free duplication Dp(3;f)Th, causes the centromere to variegate and to become highly unstable during mitosis. A systematic dissection of the smallest known stable chromosome derivative in Drosophila, Dp(1;t)1187 (1.3 Mb), has been reported recently (Fig. 4). As the minichromosome is mai:ked with visible markers (rosy + and yellow +) and is dispensable, it was possible to produce a large collection o f internal deletions and thereby provide an entry point to the most centromere-proximal heterochromatin o f the nfinichromosome. In addition, restriction enzyme maps were produced covering the whole of Dp1187 [44°]. The results indicate that Dp1187 contains alternate blocks of complex and simple satellite DNA within the heterochromatin. Three blocks of complex sequences were mapped and named Tahiti, Moorea and Bora-Bora. Cloning and sequencing part of Tahiti revealed the presence of the retrotransposon D O C I and of the X-chromosome 359 satellite [40",44"]. When the smaller Dp1187 derivatives were tested for chromosome transmission, it was concluded that the 220 kb Bora-Bora island is necessary but not sufficient for normal centromere function [45"]. For complete centromere function in mitosis and meiosis, the essential centromere core (the Bora Bora island) must be flanked by >200 kb of the 1.672 (AATAT) satellite DNA on at least one side [45"]. It has not yet been determined, however, whether the 1.672 satellite provides itself an essential function or whether other repeats might also work. It would be most interesting to identify translocated or deleted minichromosomes that have exchanged the flanking satellite for the more distally located 359 satellite.

Proteins associated with the centromere of

Drosophila A few proteins that bind centric heterochromatin of D. melanogaster have been described. The product of the z w l O gene has been shown to localize to regions of the spindle during early stages of mitosis and metaphase. At anaphase, zwl0 protein can be found associated exclusively with the centromere region of all chromosomes [46]. Mutations o f this gene cause severe chromosome missegregation with lagging chromatids and precocious sister chromatid separation suggesting that zwl0 is important for proper anaphase progression. Recently, three other chromosome-associated proteins named the AF1 complex, were isolated from embryo and Kc cell nuclear extracts using human centromeric DNA affinity columns [47"]. The columns were constructed using oligonucleotides corresponding to the human CENP-B box 17bp motif and the three proteins were found to bind as a complex and specifically occupy the C E N P - B box motif. Indirect immunofluorescence shows that the proteins accumulate preferentially in regions o f heterochromatin, although they can also be detected on the chromosome arms. Finally, the kinesin-like putative microtubule-based motor encoded by no distributive disjunction (nod), that is required to maintain the metaphase position for non-recombinant achiasmate chromosomes during female meiosis, has also been localized along the length of prometaphase and metaphase chromosomes in Drosophila [48"] suggesting that chromosome positioning might involve motor proteins localized not only on the kinetochore but also along the chromosome arms [2]. Using the deletion derivatives ofDp1187, it was also shown that pericentric heterochromatin localized towards the short arm of the minichromosome is the most sensitive to nod gene dosage, even though sequences all along the minichromosome appear to interact with the nod protein [49].

Drosophila melanogaster Tahiti

Moorea

Bora Bora

Dpl 187 -...//-..--.~

I

1000 kb

~V_.~Complex DNA ~ 1.688 Satellite(359) 1.672 Satellite Unknown repeat DOCI Q 1qgg Current ( )pinion it] Geneti( s & Devl!lopment

Fig. 4. Molecular organization of the centromeric heterochromatin of the Drosophila minichromosome Dp(1 ;f)1187 showing the localization of the three islands of complex DNA (Tahiti, Moorea and Bora Bora elements) surrounded by different types of highly repeated satellite DNA. The arrows represent different repeats of the 359 satellite. The minimum region required for normal chromosome segregation includes the whole of the Bora Bora (220 kb) plus some 200 kb of satellite DNA located on at least one side.

761

762

Genomesand evolution Proteins of the mammalian kinetochore The protein components of the manmlalian centromere have been studied extensively [7-9]. The first proteins to be identified were a group of three autoantigens recognized by sera from autoimmune patients with scleroderma spectrum disease. The sera stain the centromere region o f chromosomes from several mammalian species--including primates, mouse, rat kangaroo, Indian Muntjac and Chinese hamster--providing the first evidence of conserved kinetochore constituents among niammals. The first three centromeric proteins to be identified were designated CENP-A (17kDa), C E N P - B (80 kDa) and C E N P - C (140kDa) [9]. Human C E N P - A has been shown to be a centromere-specific histone H3 like protein. A comparison of the amino acid sequences o f human CENP-A, part o f bovine CENP-A, and human histone H3 suggests that C E N P - A is organized into two domains: a ca~:box-y-terminal domain sharing more than 60% sequence identity with histone H3 that is essential for centromeric localization; and an amino-terminal divergent domain [50"]. Epitope-tagged versions of human C E N P - A have been expressed and shown to be localized to the centromere o f chromosomes in Indian Muntjac cells, raising the possibility that CENP-A recognises a conserved mammalian centromeric domain. Human C E N P - B was the first centromeric protein to be cloned [9]. Subsequently, homologues have been identified in the African green monkey (K Yoda, H Masumoto, T Okazaki, personal communication) and Mus musculus [9], and a highly homologous protein must exist in Indian Muntjac cells, as it can be detected with an antibody that recognises the human protein (H Masumoto, T Okazaki, personal communication). C E N P - B is an 80 kDa protein that localizes by inmmno-electron microscopy throughout the centromeric heterochromatin beneath the kinetochore [9]. The amino-terminal region directs the protein to the centromere by binding to (z-satellite DNA (see below) [9]. It binds D N A through its amino-terminal 125 amino acid residues that form four (z-helical secondary structures of a helix-loop-helix (HLH) motif. Recent results [51"] have shown that the HLH DNA-binding motif o f C E N P - B is atypical in that it can bind DNA as a monomer whereas the carboxy-terminal 59 amino acids appear to be responsible for dimerization. These results indicate that CENP-B forms a stable complex containing two DNA molecules and two polypeptides [51"]. Furthermore, biochemical analysis of C E N P - B immunoprecipitated from interphase and metaphase cells suggest that C E N P - B is specifically phosphorylated at metaphase [51"]. The general co-localization of functional centromeres and CENP-B have suggested that this protein is required for proper kinetochore function. However, a number of direct functional tests, including injection of antibodies and expression of truncated versions of CENP-B, have failed to establish an essential role (A Pluta, W C Earnshaw, personal communication).

C E N P - C is a highly basic protein located mainly at the inner kinetochore plate [9]. Indirect imnmnofluorescence with anti-CENP-C antibodies showed that this antigen is present in equal amouuts in all human centromeres but is only present in the functional centroxnere of stable dicentric chromosomes [52]. Also, when anti-CENP-C antibodies are injected during interphase, cells become transiently arrested at the following metaphase and electron microscopic examination of their kinetochores shows them to be reduced in size or diameter. In cells that have been arrested for longer periods, the abnormal kinetochores do not bind microtubules but sister chromatids appear to separate. These findings suggest that C E N P - C is required for kinetochore assembly and/or maintenance o f a functional kinetochore that is able to interact with microtubules [53"]. Other centromere-associated proteins have been identified in mammals. CENP-E was identified by a monoclonal antibody (mAb177) raised against enriched centromere/kinetochore components. This protein localizes to the centromere and seems to be required for the metaphase--ganaphase transition, as microinjection of mAb177 into metaphase cells blocks or delays progression into anaphase. The amino-terminal domain of CENP-E shares extensive homology with the motor domain of all known kinesin-like proteins, containing an ATP and microtubule binding-sites. C E N P - E accumulates during early stages o f mitosis and is specifically degraded at late stages of anaphase [54"]. Recent studies using affinity-purified polyclonal antibodies directed against the kinesin-like domain of C E N P - E showed that they are able to reduce or block the depolymerization-dependent chromosome inotion [55]. Finally, the mitotic centromere-associated protein kinesin (MCAK) from Chinese hamster ovary cells was cloned using antibodies against two conserved regions of the kinesin motor domain [56]. MCAK appears to be associated with centromeres throughout mitosis and localizes primarily at the pairing domain and extends towards the outer kinetochore plate. This protein has the motor region located in the center flanked by two coiled coil domains. Western blot analysis using anti-MCAK monoclonal antibodies indicates that this protein might be present in several species from yeast to mammals.

Centromeric sequences of mammals The human genome comprises about 10% repetitive DNA sequences which are organized in tandem arrays of at least five different classes [10]. To date, alphoid (also called (z-satellite) DNA is the only repeat sequence that has been found at the primary constriction of all normal human chromosonms. It is organized in monomers o f - 1 7 0 bp that are tandemly repeated, yet the nucleotide sequence and monomeric organization may diverge from one monomer to another and from one

The elusive centromere Sunkel and Coelho 763 chromosome to another [10]. Long-range physical maps of the centromere of three human chromosomes have been constructed (reviewed in [10]). A partial map o f the centromere o f chromosome 7 revealed long tandem arrays of 0r-satellite DNA. A complete map spanning the centromere o f the Y chromosome revealed a complex set o f new repeats not found in other centromeres, as well as 0t-satellite D N A flanked at either side by satellites found near other centromeres. A long-range map of the centromere of human chromosome 21 was constructed using a m o u s e - h u m a n somatic cell hybrid [57]. Two 0t-satellite arrays were identified; ct21-I, in which the C E N P - B box appears very frequently, extends some 1.3 M b and was mapped by two-color fluorescent in situ hybridization to the primary constriction and co-localizes with centromere antigens recognized by sera from autoimmune patients with scleroderma spectrum disease ( C R E S T antigens). On the other hand, 0t21-II, which contains few C E N P - B boxes, localizes towards the short arm and is flanked by classical satellite III repeats. It is likely that neither array is interrupted by other satellites; however, it is still unclear whether other, as yet uncloned, satellites are located within the primary constriction between the two 0t-satellite families.

With the exception of the Y, most M. musculus chromosomes appear telocentric, with centromeres at the end of the chromosomes and an absence of detectable short arms [58]. The mouse genome is known to contain two satellite D N A families located at the centromeric region. These are organized in two separate domains. The minor satellite is closer to the telomeres, co-localizing with centromere-associated proteins detected by sera from patients with scleroderma spectrum disease. The major satellite is farther away, flanking the minor satellite and adjacent to the euchromatic long arm o f each mouse chromosome. The minor satellite consists o f tandem arrays of a 120 bp m o n o m e r sequence, has an average size o f 300kb and is predominantly uninterrupted by non-satellite sequences [58,59].

The CENP-B box is a highly conserved motif in mammalian centromeric DNA The sequence of centromeric satellites in mannnals has diverged significantly and little conservation was thought to exist. Moreover, attempts to define consensus motifs on the basis o f sequence analysis of the human 0t-satellite did not provide meaningful answers. It was the centromeric localization of an autoantigen that first suggested a pattern o f conservation within human centromeric DNA. C E N P - B was shown to bind 0t-satellite D N A through a short sequence motif of 17bp called the C E N P - B box [26]. The C E N P - B box was found and partially conserved in ct-satellite repeats derived from all human chromosomes, excluding those found in the Y chromosome. In order to determine the consensus C E N P - B box motif, PClkamplified or-satellite nmnomers were incubated with HeLa nuclear extracts and immunoprecipitated with an anti-centromere serum [61]. Analysis of 92 different monomers showed that 9 bp of the C E N P - B box were conserved (PyTTCGttggAaPuCGGGa). This consensus sequence has now been found in both primates and mouse species. M. musculus minor satellite D N A contains the 17bp binding site for the centromere associated protein CENP-B. Also, it was shown that the 79bp centromeric repeat found in M . caroli contains the minimal C E N P - B consensus binding sequences which would explain C E N P - B binding and the maintenance of interspecies hybrids [60"]. Finally, the C E N P - B box minimal consensus sequence is likely to be present in Indian Muntjac cells, as nuclear extracts induce gel retardation of C E N P - B box oligonucleotides and a CENP-B-like protein is present at the centromere in these cells [61].

What is the role of co-satellite DNA in mammalian centromere organization? The Asian mouse Mus caroli separated from M. musculus about 5-7 million years ago, and although C R E S T antigens have been localized to M. caroli kinetochores, no reactivity was detected with rabbit polyclonal sera raised against the recombinant C E N P - B protein [60°]. Also, neither minor satellite D N A nor the 17bp C E N P - B box were found in its genome. Nevertheless, M. musculusxM, caroli interspecies hybrid cell lines segregating chromosomes from both species have been developed, suggesting that at some level the centromeres of these two species must be conserved. This apparent paradox was recently resolved after two satellite DNAs organized as tandem repeat arrays of either 60bp or 79bp were identified and localized to the M. caroli centromeres [60°].

Although 0~-satellite arrays and their mouse counterpart, minor satellite DNA, are thought to be essential for kinetochore assembly, some conflicting data exist that challenge this simplified view. For example, neither human nor M. nlusculus Y chromosonms have C E N P - B box containing satellites, but they segregate normally [10]. It has also been reported [62] that inactive centromeres present in stable multicentric chromosomes of mouse cell lines do show the presence o f minor satellite DNA. In some cases a characteristic primary constriction can also be formed in the absence of minor satellite D N A and functional centromeres of stable dicentric chromosomes can be formed without minor satellite D N A [62]. Furthermore, immunofluorescence

764

Genomes and evolution

analysis using a serum that recognises both C E N P - B and C E N P - C showed that C E N P - C co-localizes with all active centromeres whereas C E N P - B localizes to both active and inactive centromeres [52]. A more direct test for the role of human ix-satellite D N A in the formation of the centromere was performed when cosmids containing ix-satellite D N A fragments were introduced into African Green monkey chromosomes (from COS7 cells) and stable cell lines established [63"]. The sites of integration of the ix-satellite sequences were found to cause bridges between sister chromatids at anaphase and frequent displacement from the metaphase plate. Although it was shown that the sites of integration were stained by C R E S T autoimmune sera, no specific C E N P - B staining was determined. One possible interpretation of these results is that Ix-satellite D N A provides the basis for the D N A structure and the subsequent binding of protein components required for pairing between sister chromatids. In the presence of another functional centromere, however, these sequences are not sufficient to induce the formation o f a fully functional centromere. The original centromere behaves as a dominant element and the chromosome, as a whole, suppresses the formation of a second functional centromere. Other attempts to test whether specific D N A fragments can provide the basis for a functional centromere have not been successful because significant sequence rearrangement has been found at the sites of integration [64].

Conclusions Conservation of sequence and sequence organization in centromeric DNA Overall, centromeric sequences are clearly not conserved amongst different species, but certain motifs do appear in centromeres from yeast to man. It was suggested that part of the CDEIII core element o f S. cerevisiae shared homology with part of the C E N P - B box [65]. Recently, specific p r o t e i n - D N A complexes have been identified using defined fragments of the K-type repeats present in S. pombe centromeres [15]. A c o m m o n motif identified in these elements also shared homology with part o f the C E N P - B box, although it remains to be determined whether this motif is essential for gel retardation. Finally, in both Chinese hamster and Drosophila, C E N P - B box binding activity has been detected even though no C E N P - B boxes have been identified so far. These findings, together with the overall conservation of the C E N P - B box motif amongst mammals, suggest that either the C E N P - B box or similar D N A motifs have special characteristics that have been selected to play an essential role in determining centromere localization and function.

An analysis of the sequence organization amongst different centromeres also suggests functional similarity. The nfinimum centromere in S. pombe and Drosophila

appears to consist o f a repeated satellite array located in cis with a central core. In the case of S. pombe, specific satellite repeats appear to perform a centromere enhancer function, a feature that has not yet been determined for Drosophila. Other components of centromeres also show functional conservation. For example, position effect variegation, a phenomenon observed in Drosophila, has now been detected in both the centromere and the K-type repeats of S. pombe. Furthermore, the K-type repeats appear to be required for full sister chromatid pairing in a manner similar to pericentric heterochromatin for Drosophila and mouse. Recent data on S. cerevisiae also suggest that one of the three conserved elements, CDEII, plays an essential role in sister chromatid cohesion during meiosis. Whether this element corresponds in function to the K-type repeats or higher eukaryotic pericentric heterochromatin is a tempting hypothesis that remains to be tested.

Conservation of kinetochore components Protein components of the kinetochore of a number o f species have been reported. Some of these proteins appeared to be conserved amongst evolutionary very distant species. Putative homologues of the human C E N P - A have been identified in $. cerevisiae (Cse4) and bovine and epitope tagged protein can localize to the centromere of Indian Muntjac cells. Putative homologues o f the human C E N P - B have been either isolated or just detected in African green monkey, M. musculus, Indian Muntjac, Chinese hamster, Drosophila (AF1 complex) and S. cerevisiae (Cbf3C). More recently, a putative homologue o f C E N P - C was also found in S. cerevisiae. These observations clearly suggest that kinetochore components show a pattern of conservation that correlates well with the conservation of sequence motifs and overall sequence organization amongst centromeres from different species.

Higher-order chromatin structure and centromeres One concurrent theme in the molecular analysis o f centromeres o f many species is the atypical chromatin structure found in and around centromeric DNA. In S. cerevisiae, C E N D N A has a central 125bp nuclease-resistant core. Furthermore, both CDEI and CDEII have been found to show D N A bending. In S. pombe, nucleosome positioning in the central core and part of the flanking repeats is also different from the rest o f the chromatin. Indeed, it has been proposed that the switch of centromeres in circular minichromosomes from an active to an inactive state is due to an epigenetic effect that can confer different functional states to a C E N D N A by altering higher order folding o f centromeric components. In mammals, the centromeric localization of C E N P - A suggests that centromeres differ at the most fundamental level from the rest of the chromatin. Studies in mouse have suggested that overall D N A structure, and not a specific sequence,

The elusive centromere Sunkel and Coelho determines the binding o f specific protein components to heterochromatin and thus plays a major role in determining the special physico-chemical properties o f centromeric D N A [66,67]. These notions are generally supported by models that try to integrate CENP-B binding to or-satellite in the context of the solenoid model of chromatin organization. It has been suggested that CENP-B binding to the 0t-satellite arrays would cause a general disruption o f the solenoid structure as it would have to bind to the interior of the fiber [9]. A general consequence o f these models is that overall centromere structure results in a higher-order organization o f chromatin that differs signific~intly from the bulk of the chromatin. This could be the basis for the observation that stable dicentric chromosomes in which both centromeres are fully active are very rare and have only been clearly documented once [68°]. The underlying concept o f this model is that o f centromere dominance, implying that a chromosome behaves has a unit that in general can support the formation of only one fully active centromere and in which any other site would be repressed. The molecular analysis of centromeric sequences, their organization and the identification of kinetochore components has advanced significantly over the last few years. A comparison of the features already known suggests a pattern of functional conservation that is represented at the molecular level and might provide the basis for identifying unifying principles in centromere structure across species.

Acknowledgements We would like to apologize to all those whose work was cited through other reviews, but limitations on space do not allowed us to cite all the original publications. Our thanks to William Earnshaw, Hiroshi Masumoto, Tuneko Okazaki, David Kipling, Gary Karpen and Cayetano Gonzalez for providing results prior to publication. We are specially thankful to William Earnshaw, Gary Grobsky, Hiroshi Masumoto, Cayetano Gonzalez and Daniel Hartl for comments on the manuscript. The work in the laboratory o f CE Sunkel is supported by grants from JN1CT of Portugal and the European Union.

4.

Hegemann JH, Fleig UN: The centromere of budding yeast. Bioessays 1993, t 5:451-460.

5.

Clarke L, Baum M, Marschall LG, Ngan VK, Staeiner NC: The structure and function of Schlzosaccharomycespombe centromeres. Cold Spring Harbor Syrup Quant Biol 1993, 58:687-695.

6.

Jiang W, Carbon J: Molecular analysis of the budding yeast centromere/kinetochore. Cold Spring Harbor Syrup Quant Biol 1993, 58:669-676.

7.

Brinkley BR: Centromeres, Kinetochores: integrated domains on eukaryiotic chromosomes. Curt Opin Cell Bio/ 1990, 2:446-452.

8.

Pluta A, Cooke FCA, Eamshaw WC: Structure of the human centromere at melaphase. Trends Biol Sci 1990, 15:181-185.

9.

Earnshaw WC, Tomkiel JE: Centromere and kinetochore structure. Curt Opin Cell Biol 1992, 4:86-93.

10.

Tyler-Smith C, Willard HF: Mammalian chromosome structure. Curt Opin Genet Dev 1993, 3:390-397.

Sears DD, Hegemann JH, Shero JH, Hieter P: Cis-acting determinants affecting centromere function, sister-chromatid cohesion and reciprocal recombination during meiosis in Saccharomycescerevisiae. Genetics 1995, 139:1159-1173. Describes a clever experimental design using non-essential humanderived yeast artificial chromosomes to test in detail the individual contribution of the different CEN DNA elements required for faithful meiotic segregation. The results point to the role of CDEII for sister-chromatid cohesion during meiosis. 11. ""

12.

B a u m M, Ngan VK, Clarke L: The centromeric K-type repeat and the central core are together sufficient to establish a functional Schizosaccharomycespombe centromere, g4ol Biol Cell 1994, 5:747-761. Demonstration that the minimum DNA fragment required to establish a functional centromere contains the unique central core and the K-type repeat. The results also provide the first evidence that K-type repeat DNA fragments can bind nuclear proteins in gel retardation assays and that these fragments share some homology with the mammalian CENP-B box. 13. •

Marschall L, Clark L: A novel cis-acting centromeric DNA element affects S. pombe centromeric chromatin structure at a distance. I Cell Biol 1995, 128:445-454. Molecular identification of a small DNA fragment from the K-type repeats found adjacent to all centromeres that can modify the chromatin organization of the CEN DNA when in cis, at a distance and regardless of the orientation. This would identify a classical enhancer and give further evidence that centromeres behave as loci. 14. •

15.

Steiner NC, Clarke L: A novel epigenetic effect can alter centromere function in fission yeast. Cell 1994, 79:865-874.

16.

Karpen G: Position-effect variegation and the new biology of heterochromatin. Curr Opin Genet Dev 1994, 4:281-291.

17.

AIIshire RC, Javerzat J, Redhead NJ, Cranston G: Position effect variegation at fission yeast centromeres. Cell 1994, 76:157-169.

18.

AIIshire RC, Nimmo ER, Ekwall K, Javerzat J, Cranston G: Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation. Genes Dev 1995, 9:218-233.

19.

Lechner J, Carbon J: A 240kD multisubunit protein complex (CBF3) is a major component of the budding yeast centromere. Cell 1991, 64:717-725.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest "• of outstanding interest 1.

Gorbsky GJ: Kinetochores, microtubules and the metaphase checkpoint. Trends Cell Biol 1995, 5:143-148.

2.

Yanagida M: Frontier questions about sister chromatid separation in anaphase. Bioessays 1995, 17:519-526.

3.

Rieder CL, Salmon ED: Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle. J Cell Biol 1994, 124:223-233.

Ding R, McDonald KL, Mclntosh JR: Three-dimentional reconstruction and analysis of mitotic spindles from the yeast, Schizosaccharomycespombe. J Cell Biol 1993, 120:141-151.

Lechner J: A zinc finger protein, essential for chromosome segregation, constitues a putative DNA binding subunit of the Saccharomyces cerevisiae kinetochore complex, Cbf3. EMBO J 1994, 13:5203-5211. Describes the cloning of Cbf38 by partial amino acid sequence and identification of a zinc finger DNA-binding motif. Mutational analysis and demonstration of its DNA binding properties. The results also indicate that alterations within the putative zinc finger lead to significant loss of minichromosomes. 20. •

765

766

Genomes and evolution 21. •

Strunnikov AV, Kingsbury J, Koshland D: CEP3 encodes a centromere protein of Saccharomyces cerevisiae. J Cell Biol 1995, 128:749-760. Identification of CEP3, the locus that encodes Cbf3B, using a screen designed to identify mutants specifically affecting kinetochore function. This was done by constructing a chromosome containing an essential gene required at high copy number and both a silent 2~m-ORI and an active CEN DNA. Only cells bearing mutations that cause loss of kinetochore function in trans would be able to induce 21.tm-ORI activity and maintain the essential gene at a high copy number and allow viability. 22.

35.

Bonaccorsi S, Lobe A: Fine mapping of satellite DNA sequences along the Y chromosome of Drosophila melanogaster. relationships between satellite sequences and fertility factors. Genetics 1991, 129:177-189.

36.

Lohe AR, Hilliker AJ, Roberts P: Mapping simple repeated DNA sequence in heterochomatin of Drosophila melanogaster. Genetics 1993, 134:1149-1174.

37.

Abad JP, Carmena M, Baars S, Saunders RDC, Glover DM, Ludena P, Sentis C, Tyler-Smith C, Villasante A: Dodecasatellite: a conserved G+C rich satellite from the centromeric heterochromatin of Drosophila melanogaster. Proc Nail Acad Sci USA 1992, 98:4663-4667.

38.

Caizzi R, Caggese C, Pimpinelli S: Bari-!, a new transposonlike family in Drosophila melanogaster with a unique heterochromatic organization. Genetics 1993, 133:335-345.

Doheny K, Sorger P, Hyman A, Tugendreich S, Spencer F, Hieter P: Identification of essential components of the S. cerevisiae kinetochore. Cell 1993, 73:761-774.

23. •

Stoler S, Keith KC, Curnick KE, Fitzgerald-Hayes M: A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis. Genes Dev 1995, 9:573-586. Identification of the CSE4 locus from a screen based on a visual marker and designed to isolate mutants that cause missegregation of a mutated extra chromosome III. Cse4 shares significant homology with histone H3 and CENP-A and has DNA-binding properties similar to those of H3. 24. •

Middleton K, Carbon J: KAR3 kinesin is a minus-end-directed motor that functions with centromere binding proteins (CBF3) on an in vitro yeast kinetochore. Proc Nail Acad Sci USA 1994, 91:7212-7216. Using an in vitro gliding assay, the authors identify a motor component associated with the CBF3 kinetochore complex. The assay consisted of a glass perfusion chamber coated with CEN3 DNA binding proteins to which polarity-marked microtubules were bound. The motor activity was activated with Mg-ATP and gliding activity recorded visually. 25.

Meluh PB, Koshland D: Evidence that the MIF2 gene of Saccharomycescerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. Mol Biol Cell 1995, 6:793-807. Cloning of MIF2 showing significant homology with the mammalian CENP-C centromere protein. The results also demonstrate clear genetic interaction in trans with CBF1, the CDEI binding protein, as well as with Cbf3A another member of the CBF3 complex.

•

39. •

Pimpinelli S, Berloco M, Fanti L, Dimitri P, Bonaccorsi S, Marchetti E, Caizzi R, Caggese C, Gatti M: Transposable elements are stable structural components of Drosophila melanogaster heterochromatin. Proc Nail Acad Sci USA 1995, 92:3804-3808. Demonstration that transposable elements are present in large number of copies within the pericentromeric and sometimes centromeric heterochromatin of all Drosophila chromosomes. At least seven different transposable elements were found. The results also show some variation in the distribution of transposable elements amongst geographically distant natural populations. 40. •

Carmena M, Gonzalez C: Transposable elements map in a conserved pattern of distribution extending from beta-heterochromatin to centromeres in Drosophila. Chromosoma 1995, 103:676-684. Shows that transposable elements are present within Drosophila heterochromatin and also maps the transposable element DOCt to the smallest known Drosophila chromosome Dp(1 ;f)l 187.

41. •

Dorer DR, Henikoff S: Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila. Cell 1994, 77:993 1002. This paper shows that a transposable element can induce certain aspects of constitutive heterochromatin when present in tandem duplications within the euchromatin. Using a tranpose-mediated amplification of a P-element carrying a marker gene (lacW) within the euchromatin the authors show white variegation as in classical position-effect variegation. The effect was stronger if the transgene was closer to constitutive heterochromatin and correlated well with the number of copies.

26.

Masumoto H, Masukata H, Muro Y, Nozaki N, Okazaki T: A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite. J Cell Biol 1989, 109:1963-1973.

27.

Cook KR, Karpen GH: A rosy future for heterochromatin. Proc Nail Acad Sci USA 1994, 91:5219-5221.

42.

28.

Lica L, Narayanswami S, Hamkalo B: Mouse satellite DNA, centromere structure, and sister chromatid pairing. J Cell Biol 1986, 103:1145-1151.

Locke J, McDermid HE: Analysis of Drosophila chromosome 4 using pulse field gel electrophoresis. Chromosoma 1993, 102:718-723.

43.

Wines DR, Henikoff S: Somatic instability of a chromosome. Genetics 1992, 131:683-691.

Gonzalez C, Casal J, Ripoll P, Sunkel CE: The spindle is required for the process of sister chromatid separation in Drosophila neuroblasts. Exp Cell Res 1991, 192:10-15.

44. •

29.

30.

Miyazaki WY, Orr-Weaver TL: Sister-chromatid cohesion in mitosis and meiosis. Annu Rev Genet 1994, 28:167-187.

31.

Carmena M, dodecasatellite and can form mitosis. J Cell

32.

McKee BD, Karpen GH: Drosophila ribosomal RNA genes function as a X-Y pairing site during male meiosis. Cell 1990, 61:61-72.

33.

Hawley RS, Irick H, Zitron AE, Haddox DA, Lobe A, New C, Whitley MD, Arbel T, Jang J, McKim K, Childs G: There are two mechanisms of achiasmate segregation in Drosophila females, one of which required heterochromatic homolgy. Dev Genet 1993, 13:440-467.

34.

Pimpinelli S, Dimitri P: Cytogenetic analysis of segregation distortion in Drosophila melanogaster, the cytological organization of the Respooder (Rsp) locus. Genetics 1989, 121:765-772.

Abad J, Viilasante A, Gonzalez C: The sequence is closely linked to the centromere connections between sister chromatids during Sci 1993, 105:41-50.

Drosophila

Le M, Duricka D, Karpen GH: Islands of complex DNA are widespread in Drosophila centric heterochromatin. Genetics 1995, 141:283-303. Molecular mapping of centric heterochromatin within the Drosophila minichromosome Dp(1 ;01187 using end-labelled pulse-field restriction mapping showing the presence of islands of complex DNA distributed within highly repetitive satellite DNA. The authors also isolate fragments from one of the islands of complex DNA and show by sequencing the presence of the transposable element DOCI. 45. Murphy TD, Karpen GH: localization of centromere function • in a Drosophila minichromosome. Cell 1995, 82:599 609. Induction of a large number of deletions derivatives of Dp(1;f)l187, mapping by end-labelled pulse-field electrophoresis as in [44 °] but also determine meiotic stability of the derivatives. The results allow the authors to define the minimum fragment of the minichromosome required for normal segregation. They show that the complex island Bora Bora together with satellite DNA at either side is sufficient for chromosome stability. 46.

Williams BC, Karr TL, Montgomery JM, Goldberg ML: The Drosophila I(1)zwl0 gene product, required for accurate mitotic chromosome segregation, is redistributed at anaphase onset. J Cell Biol 1992, 118:759-773.

The elusive centromere Sunkel and C o e l h o

47. •

Avides MC, Sunkel CE: Isolation of chromosomal-associated proteins from Drosophila melanogaster that bind a human centromeric DNA sequence. J Cell Biol 1994, 127:1159-1171. Isolation of CENP-B box binding proteins from Drosophila nuclear extracts. Three proteins were isolated by affinity chromatography that appear to form a complex (AF1) that specifically binds to the CENP-B box. The proteins were localized to condensed chromosomes by injection of fluoroscently labelled native proteins and also using a polyclonal sera. The proteins are distributed along the chromosome arms but appear to accumulate preferentially within heterochromatin. Afshar K, Barton NR, Hawley RS, Goldstein LSB: DNA binding and meiotic chromosomal localization of the Drosophila nod kinesin-like protein. Cell 1995, 81:129-138. Demonstration that the nod gene product, a kinesin-like protein that as been proposed to push chromosomes towards the metaphase plate during female meiosis, localizes along the whole of the chromosomes during prophase. The fact that it is not restricted to the kinetochore supports the hypothesis that motor-based chromosomal movement might not be restricted to the centromere providing a molecular explanation for the proposed polar ejection forces.

56.

Wordeman L, Mitchison TJ: Identification and partial characterization of mitotic centromere-associated kinesin-related protein that associates with centromeres during mitosis. J Cell Biol 1995, 128:95-105.

57.

Ikeno M, Masumoto H, Okazaki T: Distribution of CENP-B boxes reflected in CREST centromere antigenic sites on longrange co-satellite DNA arrays of human chromosome 21. Hum Mol Genet 1994, 3:1245-1257.

58.

Kipling D, Ackford HE, Taylor BA, Cooke HJ: Mouse minor satellite DNA genetically maps to the centromere and is physically linked to the proximal teleomere. Genomics 1991, 11:235-241.

59.

Kipling D, Wilson HE, Mitchell AR, Taylor 8A, Cooke HJ: Mouse centromere mapping using oligonucleotlde probes that detect variants of the minor satellite. Chromosoma 1994, 103:46-55.

48. "•

49.

Murphy TD, Karpen GH: Interaction between the nod* kinesin-like gene and extracentromeric sequences are required for transmission of a Drosophila minichromosome. Cell 1995, 81:139-148.

50. •

Sullivan KF, Hechenberger M, Masri K: Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere. J Cell Biol 1994, 127:581-592. Molecular cloning of the human CENP-A and partial sequence of the bovine homolog. When compared to histone H3, the human CENP-A protein contains an amino-terminal divergent domain and a carboxy-terminal homologous domain. Introduction of epitope-tagged truncated versions of the protein indicate that the carboxy-terminal homologous domain is responsible for centromere localization.

60. •

Kipling D, Mitchell AR, Masumoto H, Wilson HE, Nicol L, Cooke HJ: CENP-B binds a novel centromeric sequence in the Asian mouse mus caroli. /viol Cell Biol 1995, 15:4009-4020. Molecular cloning of two types of satellite repeats (60bp and 79bp) from Mus caroli centric heterochromatin. The 79 bp repeat was found to contain the conserved CENP-B box consensus sequence providing a molecular explanation for the immunolocalization of CENP-B to the centromere. 61.

Masumoto H, Yoda K, Ikeno M, Kitagawa K, Muro Y, Okazaki T: Properties of CENP-8 and its target sequence in a satellite DNA. In Chromosome segregation and aneuploidy. Edited by Vig 8K. NATO ASI Series H 72. Springer Heidelberg; 1993:31-43.

62.

Vig BK, Latour D, Frankovich J: Dissociation of minor satellite from the centromere in mouse. J Cell Sci 1994, 107:3091-3095.

Kitagawa K, Masumoto H, Ikeda M, Okazaki T: Analysis of protein-DNA and protein-protein interactions of centromere protein B (CENP-B) and properties of the DNA-CENP-B complex in the cell cycle. Mo/Ceil Biol 1995, 15:1602-1612. Characterization of the detailed DNA-binding properties of CENP-B through its amino-terminal domain and of its dimerization carboxy-terminal domain. The results show that CENP-B binds DNA as a monomer but can form homodimers with another CENP-B molecule bound to a different CENP-B box. They also show evidence that CENP-B might be specifically phosphorylated during metaphase.

Larin Z, Fricker MD, Tyler-Smith C: De novo formation of severalfeatures of a centromere following introduction of a Y alphoid YAC into mammalian cells. Hum Mol Genet 1994, 3:689-695. Introduction of yeast artificial chromosomes containing alphoid satellite DNA from the Y chromosome into mammalian chromosomes causes the formation of a cytogenetically visible constriction, immunostaining with anti-centromere sera and disruption of anaphase chromosome movement. The smallest amount of Y alphoid satellite associated with these effects was found to be in the order of 200 kb.

52.

64.

Hadlaczy G, Praznovsky T, Cserp~n I, Kereso J, P~terfy M, Kelemen I, Atalay E, Szeles A, Szelei J, Tubak V, Burg K: Centromere formation in mouse cells cotransformed with human DNA and a dominant marker gene. Proc Natl Acad Sci USA 1991, 88:8106-8110.

65.

Grady DL, Ratliff RL, Robinson DL, McCanlies EC, Meyne J, Moyzis RK: Highly conserved repetitive DNA sequences are present at human centromeres. Proc Natl Acad Sci USA 1992, 89:1695-1699.

66.

Hamkalo B, Lundgren K, Radic MZ, Saghbini M: Molecular features of heterochromatin condensation. In Chromosome segregation and aneuploidy. Edited by Vig BK. Springer: Heidelberg; 1993:151-164. [NATO ASI Series H 72.]

67.

Vig BK: Do specific nucleotide bases centromere? Murat Res 1994, 309:1-10.

51. •

Earnshaw, WC, Ratrie H, Stetten G: Visualization of centromere proteins CENP-B and CENP-C on a stable dicentrlc chromosome in cytological spreads. Chromosoma 1989, 98:1-12.

53. •"

Tomkiel J, Cooke CA, Saitoh H, Bernat RL, Earnshaw WC: CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase. ] Cell Biol 1994, 125:531-545. Demonstration that CENP-C is required for maintaining proper kinetochore size and for metaphase-to-anaphase transition. Injection of antibodies against CENP-C into the nucleus during interphase causes a transient arrest at the following metaphase and the kinetochores of these cells show reduced CENP-C staining and size. Kinetochores of cells arrested for a long time no longer bind microtubules, but surprisingly electron microscopy shows that sister chromatids have partially separated. 54. •"

Brown KD, Coulson RMR, Yen TJ, Cleveland DW: Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis. ] Cell Bio/ 1994, 125:1303-1312. Demonstration that the level of CENP-E varies along the cell cycle accumulating during interphase and reaching a peak during metaphase. At anaphase CENP-E appears to be specifically degraded after degradation of cyclin B. 55.

Lombillo VA, Nislow C, Yen TJ, Gelfand Vl, Mclntosh JR: Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerlzation-dependent motion of chromosomes in vivo. J Cell Biol 1995, 128:107-115.

63. •

constitute

the

68. •

Wandall A: A stable dicentric chromosome: both centromeres develop kinetochores and attach to the spindle in monocentric and dicentric configuration. Chromosoma 1994, 103:56-62. Analysis of a stable dicentric human chromosome by electron microscopy shows that either one or both kinetochores can attach to spindle microtubules. When only one kinetochore attaches to the spindle the other participates in sister chromatid adhesion.

CE Sunkel and PA Coelho, Laborat6rio de Gen&ica Molecular, Centro de Citologia Experimental, R, ua do C a m p o Alegre 823, Porto, Portugal.

767