CEP3 encodes a centromere protein of Saccharomyces cerevisiae.

We have designed a screen to identify mutants specifically affecting kinetochore function in the yeast Saccharomyces cerevisiae. The selection procedure was based on the generation of "synthetic acentric" minichromosomes. "Synthetic acentric" minichromosomes contain a centromere locus, but lack centromere activity due to combination of mutations in centromere DNA and in a chromosomal gene (CEP) encoding a putative centromere protein. Ten conditional lethal cep mutants were isolated, seven were found to be alleles of NDC10 (CEP2) encoding the 110-kD protein of yeast kinetochore. Three mutants defined a novel essential gene CEP3. The CEP3 product (Cep3p) is a 71-kD protein with a potential DNA-binding domain (binuclear Zn-cluster). At nonpermissive temperature the cep3 cells arrest with an undivided nucleus and a short mitotic spindle. At permissive temperature the cep3 cells are unable to support segregation of minichromosomes with mutations in the central part of element III of yeast centromere DNA. These minichromosomes, when isolated from cep3 cultures, fail to bind bovine microtubules in vitro. The sum of genetic, cytological and biochemical data lead us to suggest that the Cep3 protein is a DNA-binding component of yeast centromere. Molecular mass and sequence comparison confirm that Cep3p is the p64 component of centromere DNA binding complex Cbf3 (Lechner, 1994).

dure was based on the generation of "synthetic acentric" minichromosomes. "Synthetic acentric" minichromosomes contain a centromere locus, but lack centromere activity due to combination of mutations in centromere DNA and in a chromosomal gene (CEP) encoding a putative ce__ntromere p_rotein. Ten conditional lethal cep mutants were isolated, seven were found to be alleles of NDCIO (CEP2) encoding the l l0-kD protein of yeast kinetochore. Three mutants defined a novel essential gene CEP3. The CEP3 product (Cep3p) is a 71-kD protein with a potential DNA-binding domain (binuclear Zn-cluster). At nonpermissire temperature the cep3 cells arrest with an undivided nucleus and a short mitotic spindle. At permissive temperature the cep3 cells are unable to support segregation of minichromosomes with mutations in the central part of element III of yeast centromere DNA. These minichromosomes, when isolated from cep3 cultures, fail to bind bovine microtubules in vitro. The sum of genetic, cytological and biochemical data lead us to suggest that the Cep3 protein is a DNA-binding component of yeast centromere. Molecular mass and sequence comparison confirm that Cep3p is the p64 component of centromere DNA binding complex Cbf3 (Lechner, 1994). C HROMOSOME segregation in mitosis requires the presence of a specialized chromosomal structure that facilitates binding of chromosomes to the microtubules (mt) ~ of the mitotic spindle. Functionally, this structure could be defined as a linker between DNA and microtubules, and is known as the centromere, or kinetochore (used interchangeably here). Structural organization of centromeres varies dramatically among evolutionary remote species and can be extremely complex (Brinkley et al., 1992;Grady et al., 1992). This complexity makes the structurefunction analysis of a centromere a very difficult challenge.
Fortunately, the centromere of the budding yeast, Saccharomyces cerevisiae, (Clarke and Carbon, 1980) provides an excellent model for studying centromere organization and activity. The centromere DNA (CenDNA) of S. cerevisiae is less than 150 bp in length (Bloom and Carbon, 1982), drastically smaller than CenDNA of other well studied eukaryotes (Pluta et al., 1990). The CenDNA in yeast thus provides a space for binding of only limited number of protein molecules. Also, only a single microtubule is attached to each kinetochore in the cells of budding yeast (Peterson and known. The genes encoding p58 (CTF13) (Doheny et al., 1993) and pll0 (NDC10/CBF2/CTF14) (Doheny et al., 1993;Goh and Kilmartin, 1993;Jiang et al., 1993a) have recently been characterized. The p58 and pll0 proteins (henceforth, referred to as Ctfl3p and Ndcl0p, respectively) are indispensable for viability of yeast cell. The cells of the temperature-sensitive (ts) ndclO-1 mutant do not exhibit cell cycle arrest at nonpermissive temperature, instead they undergo an abortive mitosis, leaving most of chromosomal DNA unsegregated but associated with an asymmetric spindle (Goh and Kilmartin, 1993). In contrast, the only known ts allele of ctf13 (Doheny et al., 1993) causes cells to arrest at nonpermissive temperature in G2-M with an undivided nucleus and a short mitotic spindle.
Clearly, the identification of additional protein components of yeast kinetochore is necessary to understand its molecular structure and function. We expect such kinetochore proteins to have several characteristic features exemplified by CP1, Ctfl3p and Ndcl0p. First, they should be physically associated either directly or indirectly with CenDNA and/ or the spindle microtubules. Second, cells' deprived of a kinetochore protein should show some cytological defects consistent with the inability to segregate chromosomes properly. Third, the trans-mutations in the genes encoding centromere components should show genetic interaction with cis-CenDNA mutations (cen). These features provide at least three criteria to recognize a particular protein as a kinetochore component. Some other proteins satisfy at least one of these criteria and so, are the potential candidates for kinetochore components: p64 (Lechner and Carbon, 1991), Mckl (Shero and Hieter, 1991), Csel and Cse2 (Xiao et al., 1993), Cbf5 (Jiang et al., 1993b), Top2 (Jiang et al., 1993b), Kar3 (Middleton and Carbon, 1994), and Mif2 (Brown et al., 1993). However, the DNA binding proteins essential for assembly of functional kinetochore remain unidentified. Particularly, proteins which make direct contacts with CDEIII are important, as CDEIII is absolutely required for centromere function. Based on our belief that the yeast centromere is a complex multisubunit structure, with many structural components yet to be found, we have designed a genetic screen aimed directly at identifying proteins involved in the formation of yeast centromeres.

Strains and Genetic Techniques
S. cerevisiae strains ($288C background) are listed in Table I. Strain YPH102 was used for mutagenesis, YPH499 was used for backcrosses of centromere proteins mutants (cep). Strain $42 (Doheny et al., 1993) was used as a tester for ndclO allelism. Media, incubation conditions, and strain manipulations were according to published protocols (Sherman et al., 1986). Escherichia coli strains DH5t~ (BRL), TOP10 (Invitrogen, San Diego, CA) and SCSI10 (Stratagene, La Jolla, CA) were used for plasmid propagation. Standard methods of yeast genetics were done as described (Guthrie and Fink, 1991;Sherman et al., 1986). Mitotic stability of plasmids, defined as the fraction of cells in a culture that retain the plasmid when the cells are grown under selective conditions, was estimated as described previously (Strunnikov et al., 1993). As this parameter correlates well with the efficiency of plasmid transmission per cell division (Hieter et al., 1985a;Koshland et al., 1985), it was used as a routine estimate for the efficiency of minichromosome transmission. The disruption allele (cep3-A/) of cep3 was constructed by transforming the AS260 diploid strain with pAS450 (see below) digested with EagI and Xbal, giving AS270. Disruption of one copy of CEP3 gene with HIS3 marker was confirmed by Southern blot hybridization. After sporulation, segregation of two viable (His3-) to two inviable (His + ) spores was observed in every tetrad. The His3 + spores were recovered only after transformation of AS270 with pAS452 prior to tetrad analysis. Haploid His3 + strains (cep3-Al) always carried the pAS452 (CEP3) plasmid.

Nomenclature
Standard nomenclature (Jones et al., 1992) for yeast genes and proteins was used. As most of yeast centromere factors and corresponding genes are known under redundant names (CP, Cep, Cpf, Cbf, Ctf, Ndc) we chose the existing "CEP" acronym (Baker and Masison, 1990) for the genes isolated in our screen. Centromere mutations where designated using the locus name, CDE number, and particular mutation name, e.g., cen6-(llI)15t. The CDEIII mutations used are shown in Fig. 1 B.

The Screening Procedure for Isolation of cep Mutants
A schematic view of the selection procedure is given in Fig. 1. The first stage of screening included monitoring of plasmids with different reporter CenDNA to identify those suitable for large scale mutagenesis. Strain YPH102 was transformed with corresponding plasmid containing leu2-d gene and a CenDNA of interest. The cultures were mntagenized with EMS (50-90% viability). Cells where plated to form single colonies in the absence of selection for leucine prototrophs. The plates were then replicaprinted onto media lacking leucine and incubated for 48 h. at 23°C. The frequency of Leu + colonies was determined for each reporter CenDNA tested, and the corresponding colonies were picked from master plates. The trans-mntations were distinguished from other mutant classes via retransformation with the same minichromosome. The plasmid pAS97 (see below), which exhibited the highest rate of induced trans-mutations and, at the same time, the lowest rate of spontaneous Leu +, was chosen for largescale mutagenesis. Only mutants reproducibly showing Leo + phenotype and temperature sensitivity where chosen for further analysis. All mutant alleles were back crossed at least twice, to demonstrate that the generated mutations are single mutations of nuclear origin, and that temperature sensitivity cosegregates in meiosis with the accumulation of pAS9Z
Several plasmids, containing the CEP3 gene from the original isolate pAS300, have been constructed, pAS409 (pRS414 backbone) contains the entire genomic insert of pAS300 as 9-kb EagI-BamHI fragment, pAS420 is pRS414 with 3.2-kb BssHII-EcoRI fragment of pAS300. HincII-HincII fragment of pAS420 ( Fig. 2 A) was introduced into pR406, pRS425, and pRS416 cut with Pvull producing pAS451, pAS452 and pAS461, respectively. To construct a deletion of CEP3, two PCR fragments corresponding The Journal of Cell Biology, Volume 128, 1995 To construct the epitope-tagged versions of CEP3 the pAS451 plasmid was cut with either SpeI or HindIII (Fig. 2 A). In-frame insertion of six tandem tag sequences encoding the c-myc epitope (Strunnikov et al., 1993) into SpeI site produced pAS454 (cep3::myc allele). In-flame insertion of a triple tag sequence (contribution of M. Rose, Princeton University, Princeton, NJ; HindIII-ends introduced by PCR) encoding a hemagglutinin epitope (ha) into HindIII site of pAS451 produced pAS464 (CEP3::ha allele). Correct boundaries between CEP3 and the tag-encoding fragments were confirmed by DNA sequencing.

CEP3 Gene
The genomic DNA library used to isolate the CEP3 gene was the LEU2/CEN library (Spencer et al., 1988) in pSB32 vector. Two ts strains, 14-YPH102 (cep3-1), and 10-YPH102 (cep3-3) were transformed with library plasmid DNA. Cells were spread on plates without leucine and incubated at 23°C for 24 h. The plates were then shifted to 37°C, and temperature-resistant colonies were picked. Total DNA was extracted and used to transform E. coli cells. Two plasmids were found to enable 14-YPH102 and 10-YPH102 to grow at 37°C. These plasmids also complemented the ts-lethal phenotype of back-crossed cep3 strains. Restriction mapping showed, that the clones contain largely overlapping inserts of genomic DNA. The plasmid chosen for subcloning was designated pAS300.
Mapping of the cloned fragment to a chromosome was accomplished using the EagI-BamHI fragment of pAS300 as a hybridization probe for filters containing yeast chromosomes separated by PFGE. A single hybridization signal, corresponding to chromosome XIII, was detected. Preliminary sequencing starting from the vector and also from unique internal restriction sites was used for more accurate mapping of the clone. The cloned DNA fragment contained SMP2 (Irie et al., 1993) and MLHI (Prolla et al., 1994), two known genes on chromosome XIII. CEP3 gene is an immediate neighbor of MLH1 and the order of open reading frames on the fragment is as follows: CEP3>,<MLttl ..... SMP2>.
To determine the nucleotide sequence of CEP3, nested deletions of the pAS420 plasmid were constructed (Henikoff, 1984). The resulting plasmids were sequenced using the fluorescent ddNTP PCR sequencing reaction and automated DNA sequencer (model 370A; Applied Biosystems, Foster City, CA), as recommended by the manufacturer. Both strands of CEP3 were sequenced. Conflicting regions were resequenced using the custom synthesized 18-24-met primers.

Antibodies and Cytological Methods
Indirect immunofluorescence was performed as described (Kilmartin and Adams, 1984). Yeast nuclear DNA was stained by DAPI included into mounting media (Mowiol; Cal Biochem, La Jolla, CA). Microtubules were detected with the mouse monoclonal antibody YOLl/34 (1:200) (Kilmartin et al., 1982) and goat anti-mouse antibodies conjugated to rhodamine (Cappel Laboratories, Cochranville, PA). Mouse monoclonal anti-c-myc antibody 9El0 (Evan et al., 1985) and mouse monoclonal anti-hemagglutinin antibody 12CA5 (BAbCO, Richmond CA) were used to monitor tagged Cep3p on Western blots. The pooled monoclonal antibodies against a 90-KD component of the spindle pole body (Rout and Kilmartin, 1990), were used undiluted for indirect immunofluoreseent staining.

Minichromosome-Microtubule Binding Assays
The microtubule-minichromosome binding assays were performed essentially as described (Kingsbury and Koshland, 1991) with minor modifications (Kingsbury and Koshland, 1993). Binding experiments were done for two or four parallel cultures, always including YPHI02/pDK381 strain as a control. For each minichromosome/strain combination ( Fig. 6) the independent binding experiments have been performed at least twice.

Isolation of cep Mutants
The first extrachromosomal replicon identified in yeast was the endogenous 2 #m plasmid (Beggs, 1978). It is a circular multicopy (20-30) double-stranded DNA molecule which uses its own specialized system to partition effectively during mitotic divisions (Broach and Volkert, 1991). In contrast, artificial minichromosomes with a centromere locus, segregate using the mitotic apparatus designed for chromosomes and are maintained as single copy per cell. The screen for genes encoding putative centromere proteins was based on the observation that the presence of both 2#m plasmid replication/segregation locus (2#M-ORI) and a yeast centromere on the same circular plasmid (2#m/CEN plasmid) creates an epistatic relationship between these two loci (Tschumper and Carbon, 1983). Such a plasmid behaves as a singlecopy minichromosome and not as multicopy 2#m plasmid (Tschumper and Carbon, 1983). Several reports have been published however that compromising centromere function in such a minichromosome can convert it into a multicopy equivalent of the 2#m plasmid, at least in some cells in the population (Apostol and Greer, 1988;Chlebowicz-Sledziewska and Sledziewski, 1985;Schulman and Bloom, 1993). These results provided the primary basis for design of our screen ( Fig. 1 A).
The second rationale for the cep screen was the use of a dose-dependent marker to follow changes in the copy number of the 2#m/CEN plasmid. Such markers have been used previously for other applications and genetic screens (Hieter et al., 1985b;Larionov et al., 1989;Runge et al., 1991;Smith et al., 1990;Toh-E, 1981). In our screen for cep mutants we used the leu2-d gene as that marker. The leu2-d  The schematic map of the minichromosome used to select cep mutants is shown with the cen6-(III)15t reporter. The "cep locus" designation stands for the putative target of mutagenesis in yeast chromosome. After the cep mutation is acquired by a cell, the defective product (Cep-) of the mutated gene interferes with already compromised reporter cen6 DNA, allowing accumulation of minichromosomes and the appearance of Leu + cells.
(B) CDEIII mutations used to assess the function of cep genes. Nucleotides are numbered starting from base 1 of CDEIII. The CDEIII palindrome is marked with the halfarrows and its central base is outlined. The mutant centromere DNA fragments were resequenced to confirm their structure.
fragment has a truncation of the LEU2 promoter, making the expression of the single leu2-d gene insufficient to complement the chromosomal leu2 mutation. Approximately 100 copies of the leu2-d gene per Leu 2-cell are required to confer a Leu ÷ phenotype. That copy number can be easily achieved in yeast cells by the amplification of 2#m based leu2-d plasmid, when the cells are selected for Leu + phenotype (Toh-E, 1981). This amplification is possible due to flexibility in 2/zm plasmid copy number control, allowing transition from normal 20-30 copies per cell to 100-150 copies (under leu2-d selection) without any deleterious effect on cell physiology. Due to the epistatic effect mentioned above, the 2#m/CEN/leu2-d minichromosome can not undergo amplification from one copy per cell to 100 copies per cell without abolishing centromere function completely (Schulman and Bloom, 1993). This loss of function of the plasmid-born CEN locus (reporter centromere) as a result of unlinked (chromosomal) mutation in a cep locus ( Fig. 1 A) is the essence of the screen described below. Mutagenesis of YPH102 cells that contained the 2/~m/ CEN minichromosome with a wild-type CenDNA (CEN4 or CEN6) failed to produce trans-mutations that could inactivate the reporter centromere. The most likely explanation for this failure is that these cep mutations not only inactivated the reporter centromere on the plasmid but also inactivated the chromosomal centromeres. Such cep mutants would not be recovered due to loss of viability. To overcome this problem, we decided to compromise the reporter centromere by using the CenDNA mutations that retain only partial centromere activity in vivo. We rationalized that this modification would allow the isolation of putative cep mutants displaying synergistic loss of the reporter centromere activity ("synthetic acentric" phenotype) due to the compound effect of cis and trans mutations. However, these cep mutants would be viable because the chromosomal centromeres with their wild-type CenDNA would retain enough centromere activity to allow proper chromosome segregation.
To identify CenDNA mutations with the appropriate level of partial centromere activity for our screen, we constructed the strains containing the 2#rn/CEN minichromosomes with different CenDNA mutations. We then tested these strains for the generation of Leu + clones before and after mutagenesis. Mutagenesis of strains harboring 2#m/CEN plasmids with cen6-(1)Sc (dysfunctional CDEI) did not give significant numbers of Leu + clones after the mutagenesis. The activity of this reporter centromere was apparently too similar to wild-type and hence, as discussed above, failed to give cep mutants. Strains that had 2/zm/CEN plasmids with GALl:: CEN3 (tanscriptionally inactivated centromere), cen3-(lll)-BCT1, cen3-(lll)BCT2, cen3-(lI)X35, and cen6-(IIl)14a had a very high rate of spontaneous Leu + clones. These reporter centromeres apparently were compromised so much that they already allowed the reporter plasmid to achieve high copy number in the absence of cep mutants. However strains that contained 2#m/CEN plasmids with either cen6-(lll) 15t or cen6-(lll)19t20 had undetectable levels of spontaneous Leu + clones but numerous Leu + clones after the mutagenesis. These induced Leu + clones were good candidates for cep mutants, indicating that either of these CenDNA mutations would work as a reporter for our screen.
The minichromosome pAS97 ( Fig. 1 A), having cen6-(III)15t as a reporter CEN, was primarily used for the screen described in this report (See Materials and Methods). When the YPH102/pAS97 strain was mutagenized, the estimated frequency of induced Leu ÷ clones carrying cep mutations was 9 × 10 -4. The unwanted Leu + clones, including the cis-mutants and clones with rearranged minichromosomes, originated with a similar frequency. More than 30 cep mutants were isolated. 10 of them were conditional lethal mutants recessive for temperature-sensitive growth. These 10   (Kopetzki et al., 1989), Pdr3p (Genbank/ EMBL/DDBJ accession number X79803), Gal4p (Marmorstein et al., 1992), Haplp (Zhang and Guarente, 1994). The identical residues and conserved cysteins are highlighted.
mutations fell into two complementation groups designated cep2 (seven alleles: cep2-1I to cep2-17) and cep3 (three alleles: cep3-1 to cep3-3). All seven cep2 alleles failed to complement ndc10-42 for temperature sensitivity, indicating that cep2 is allelic to the ndclO gene. Assuming, that the NDC10 gene encodes a centromere protein, isolation of multiple ndclO alleles in our cep-screen indicates our approach was selective for centromere-specific genes.

CEP3 Is a Novel Essential Gene
Both CEP2 and CEP3 genes were cloned by complementation of the corresponding ts-mutations. Plasmids carrying inserts with the putative wild type genes conferred temperature resistant growth to the mutant strains. We isolated two independent DNA fragments which complemented the cep3-1 and cep3-3 mutations. Standard genetic procedures were performed to show that these plasmids indeed contained the CEP3 gene. First, we showed that the integration of URA3 marker into the chromosomal locus corresponding to the cloned fragment always segregated away from the cep3 mutation in the meiotic progeny of diploid cells, heterozygous for both insertion and cep3. This result shows that the gene we cloned corresponds to the chromosomal locus of cep3 ts mutation, and is not an ectopic suppressor gene. Second, using a stepwise subcloning procedure, we localized the complementing activity to a single ORF (see below). Minor truncations of the putative CEP30RF from its 5' and 3' ends abolished complementing activity, indicating that we had identified the authentic CEP3 ORE The cloned gene also complemented the "synthetic acentric" phenotype of all three cep3 mutants.
The DNA sequence of 2,074-bp fragment containing the CEP3 gene was determined. The sequence of CEP3 fragment revealed a 608-codon ORF, potentially encoding a novel protein with molecular mass of 71 kD (Fig. 2 A). The amino terminus of predicted polypeptide contained a binuclear zinc-cluster motif Zn(II)2Cys~ (Pan and Coleman, 1990) (Fig. 2 B) common to several DNA-binding proteins (Dhawale and Lane, 1993). The rest of Cep3p did not show any significant similarity to known protein sequences. According to secondary structure prediction (Rost and Sander, 1994) 56% of the polypeptide aminoacid residues are in alpha-helix, and 37 % form loops. Only one short region of the protein was predicted (Lupas et al., 1991) to form coiledcoil structure with probability higher than 0.3 (Fig. 2 A).
The chromosomal copy of the CEP3 gene was disrupted by substituting the CEP30RF with the HIS3 marker. We failed to recover the resulting allele, cep3-Al, as a haploid strain, thus showing that CEP3 is an essential gene. The essential nature of CEP3 and the presence of a DNA-binding motif suggest that Cep3p is a new DNA-binding component of yeast centromere.
To assess the expression level of CEP3, we introduced two alternative in-frame epitope tags (See Materials and Methods) into the CO•H-terminal part of Cep3p (Fig. 2 A). The cep3::myc construct did not complement cep3-1, cep3-2, or cep3-Al defects. This result indicaties that the essential region of Cep3p is not limited to the putative Zn-binding cluster. However the alternative CEP3::ha construct did complement the cep3-1 and cep3-Al mutations. The Cep3p is a rare protein as the Cep3p-ha protein can only be detected on Western blots when concentrated by immunoprecipitation (data not shown).

Cytological Analysis of cep3 Mutants
Detailed cytological analysis of the cep3 mutants was limited to cep3-1 and cep3-2, as cep3-3 cells showed practically identical characteristics in all preliminary tests. When the wild-type and cep3 cells were shifted to 37°C (the nonpermissive temperature for cep3), their viability remained the same for 4 h. However, by 8 h the viability of cep3 cells dropped below 20 %. Time-course observations showed, that after the 6-h shift to nonpermissive temperature the cep3 cells reach full arrest ofceU division (Fig. 3 A). In the course of incubation at 37°C, the late anaphase cells and smallbudded cells, present at 25°C (Fig. 4 A), disappear. The large-budded (dumbbell) cell with an undivided nucleus at the bud neck and a short spindle (Fig. 4 B) become the dominating class in the cep3 population (Fig. 3 A). Most of the cep3 cells arrested at 37°C have diploid DNA content (Fig 3 B). This complex of features is highly suggestive of G2-M phase arrest. The virtual absence of elongated mitotic spindles, was verified by the spindle pole body staining (Fig.   5 C). Therefore the phenotype of cep3 is more similar to ctfl3-30 cells (Doheny et al., 1993) than to ndclO-1 mutant (Goh and Kilmartin, 1993).
The staining of spindle microtubules in the cep3 cells lacking their mitochondrial DNA also revealed that the dominant class of cells in the arrested cep3 population is not homogeneous. We found some indications of the abnormal distribution of nuclear DNA, including the separation of chromosomal DNA from the mitotic spindle, nuclear DNA cut by the cytokinesis, and nuclear DNA unevenly spread between the mother and daughter cell (Fig. 5, A and B). The latter phenotype is probably due to the spindle movements known as the phenomenon of nuclear transits (Palmer et al., 1989), accompanied by the failure of the majority of chromosomes to attach to the mitotic spindle. The presence of cells with their spindles and nuclear DNA positioned in the different cell bodies (Fig. 5, A and B) suggests that in the arrested cep3 ceils chromosomes fail to maintain attachment to the microtubules of mitotic spindle. Interestingly, at the restrictive temperature some of the examined cep2 (ndclO) mutants arrest like the cep3 cells (data not shown). This finding brings up the possibility that the cep3, ctfl3 (Doheny et al., 1993) and ndclO (Goh and Kilmartin, 1993) cells may display the same kind of mitotic arrest as a consequence of centromere inactivation.

Interaction between cep3 Mutations and Centromere Mutations In Vivo
Given the synthetic destabilization of the reporter centro-  somes without 2#m DNA), that differed only in their CenDNA sequences (Table II). We then tested the mitotic stability of these plasmids as a means to assay the function of their centromeres (Table II). It was evident that the cep3 mutations inactivated centromeres in trans when the minichromosome CenDNA contained two CDEIII mutations (cen6-(III)15t and cen3-(llI)BCT1). However, the cep3 mutations did not inactivate significantly centromeres with another CDEIII mutation (cen6-(lll)lgt20), a CDEI mutation (cen3-(1)8c) or a CDEII mutation (cen3-(ll)X78). Therefore, the cep3 mutations appear to interact synergistically only with those centromeres that have two very specific mutations symmetrically flanking the central base in the CDEIII palindrome ( Fig. 1 B). This specific interaction supports the hypothesis that the CEP3 product is a CDEIII binding protein.
As cep2 (ndclO) mutants were identified in the same screen as cep3 mutants, it was of interest to analyze the mitotic stability of the minichromosomes with mutant CenDNA in cep2 mutants as well. Similar inactivation of centromeres with specific CenDNA mutations was observed in cep2 mutants as was observed in cep3 mutants (Table II). Therefore, the Ndcl0p may also directly or indirectly participate in CDEIII binding (see Discussion). On the other hand, the smcl-1 mutation, which also affects minichromosome transmission (Strunnikov et al., 1993), did not exhibit the synthetic acentric phenotype with any of centromeres harboring these CenDNA mutations (data not shown). Therefore, the synthetic acentic phenotype is not a general property of any mutation that affects minichromosome transmission. The similar pattern of interaction with centromere DNA mutations at the permissive temperature displayed by the cep2 and cep3 mutants suggests that the corresponding proteins act in close proximity in vivo.

The cep3 Mutation Interferes with Centroraere Function In Vitro
The kinetochore of S. cerevisiae acts as a tripartite system (CenDNA-kinetochore proteins-microtubules) which acquires motility at the time of mitosis. Based on this concept, three types of biochemical assays have been applied previously to testing the yeast centromere function in vitro: protein-DNA interaction (Doheny et al., 1993;Ng and Carbon, Figure 5. Arrested population of cep3-1 cells lacking mitochondria. (A) The cep3-1 (1cAS281) culture lacking mitochondrial DNA (rho °) after 6 h at 37°C. The cells where stained with DAPI and anti-tubulin antibodies. The cell marked with an arrow has underwent partial separation of chromosomal DNA; the bulk of chromosomal DNA and the spindle are separated from each other and positioned in different cell bodies, the phenotype never observed in wild type ceils. (B) The rho ° cep3-1 cells (lcAS281) representing the abnormal subtypes of the large budded cells with an undivided nucleus. From top to the bottom: separation of chromosomes from the mitotic spindle; cut-like phenotype; formation of a potential aploid cell. Bar, 10 #m. (C) Spindle pole body staining of the cep3-2 cells. 2bAS282 cells were stained with antibodies against the 90-kD SPB component after 6 h. at 37°C. The position of SPBs shows that corresponding mitotic spindles remain bipolar (no spindle collapse) but do not elongate, even in the cells with chromosomal DNA spread out between the mother and daughter cells. Bars, 10 #m. 1987), centromere-microtubule interaction (Kingsbury and Koshland, 1991;Kingsbury and Koshland, 1993), and motility assays (Hyman et al., 1992;Middleton and Carbon, 1994). To reveal the molecular basis of the "synthetic acentric" phenotype, we carried out experiments testing the fidelity of centromere binding to bovine microtubules in a cellfree system. This minichromosome-microtubule binding assay has been described previously Koshland, 1991, 1993). It involves quantitative precipitation of minichromosomes isolated from yeast cells (in the form of chromatin) by their association with taxol-stabilized bovine microtubules. All experimental data presented in this report were obtained for Cep3 ÷ or Cep3-cells arrested with nocodazole prior to the preparation of the extracts contain- ing minichromosomes. Arrest with nocodazole (G2-M transition), as was previously shown (Kingsbury and Koshland, 1991), confers the highest mt-binding potential for wild type cultures. This provides a wide range for the comparison of experimental data obtained for mutant and wild type extracts and CenDNA. The G2-M transition is also the phase when the function of a centromere is actually required for progression of the normal cell division cycle through mitosis, i.e., when the kinetochore is in its most active state. In fact, comparison of mt-binding activity of cis-mutant centromeres from the asynchronous culture (Kingsbury and Koshland, 1991) to activity from nocodazole arrested cells (Fig. 6) suggests that centromeres assembled with some cis-mutant Cen-DNA (e.g., cen6-(IIl)15t) achieve almost full wild-type activity in the cells arrested in G2-M. Four types of cultures (grown at 23°C) were used for microtubule-binding experiments: Cep3 ÷ cells with wild type CEN 6 minichromosome, Cep3 ÷ cells with mutant Cen-DNA (cen6-(lll)15t) minichromosome, Cep3-cells with wild type CEN6 minichromosome and Cep3-cells with mutant CenDNA (cen6-(Ill)15t) minichromosome (Fig. 6).
These combinations are analogous to ones used for assaying minichromosome stability in vivo (Table U). For both cep3/ CEN6 or CEP3/cen6 combinations, only a slight difference in the capacity of minichromosome-microtubule binding was observed at 23°C (Fig. 6). However, combining the cep3 (trans) and cen (cis) mutation in the same cell produced a striking effect: the microtubule-minichromosome binding became indistinguishable from the background level (i.e., of acentric plasmid precipitation; Kingsbury and Koshland, 1991). This result indicates that there is an intimate link between the structure of CDEIII and the Cep3p function. These cell-free system experiments are in agreement with data obtained in vivo for these minichromosomes. Results of these experiments provide a basis for a hypothesis addressing the mechanism of the "synthetic acentric" phenotype: the synthetic acentrics are probably generated in vivo, due to the inability of cen6-(lll)15t minichromosomes to bind yeast microtubules.
The structural properties of Cep3p, as well as the cytological, genetic, and biochemical data obtained for cep3 mutants, provide a basis for assuming that Cep3 protein is the CDEIII-binding subunit of yeast centromere, indispensable for both kinetochore assembly and function. To summarize, several properties of Cep3p suggest that it is a centromere protein: (a) mutant forms of the protein show allele specific interactions with particular CenDNA mutations; (b) minichromosomes isolated from cep3 cells exhibit a defect in the centromere-dependent binding to microtubules in vitro; (c) the Cep3 protein is essential for cell viability, and the cep3 mutants exhibit complex mitotic defects consistent with a failure of centromere function; and (d) the amino acid sequence of the Cep3 protein is identical to the sequence of p64 found in the Cbf3 complex (Lechner, 1994). The fact, that both Cep3p and Cep2p, are the subunits of Cbf3 complex, shows that our screen was very selective for genes encoding the components of yeast centromere.

Developing a Selective Primary Screen for Kinetochore Components
Centromere proteins have been previously identified in cells of vertebrates as centromere (or kinetochore) antigens (Compton et al., 1991;Earnshaw et al., 1987a, b;Yen et al., 1992). The yeast centromere proteins have been identified by their biochemical properties (CenDNA association) (Jiang et al., 1993a;Lechner and Carbon, 1991) or by secondary screening of existing collections of temperature-sensitive mutants (Goh and Kilmartin, 1993) or mutants with frequent chromosome loss (Doheny et al., 1993). However, the identification of additional centromere components by these approaches has proved limited, mainly because they are labor intensive. In this report we described a new approach for identifying centromere proteins by selecting mutants displaying the "synthetic acentric" phenotype.  CEP3/CEN6, strain YPHI02 transformed with pDK381; CEP3/ cen6-(lll)15t, YPH102 with pDK377 minichromosome; cep3-1/ CEN6, ldAS255 with pDK381 minichromosome; cep3-1/cen6-(lll)15t, ldAS255 with pDK377 minichromosome. Binding at mt concentrations, higher than required for saturation of binding in wild type strain (Kingsbury and Koshland, 1991), was done to exclude the possible rescue of binding activity in cep3 mutant extracts by the excess of rot. coding probable centromere components; one of these genes, NDCIO (CEP2), had been identified previously (Goh and Kilmartin, 1993;Jiang et al., 1993a) while the other, CEP3, is a novel gene.
The improved efficiency of our approach relies on three major principles. First, by requiring the reporter minichromosome to achieve a very high copy number, we selected only those mutants that completely inactivate the reporter centromere, thus eliminating the possible background of mutants, able to accumulate only few extra copies of minichromosome (Larionov et al., 1989). Second, we used a reporter centromere that was already partially impaired by a cismutation. In this way mutant centromere proteins, that inactivated the reporter centromere, would still be active on chromosomal centromeres, allowing the mutant cells to survive. Third, since we assumed that many kinetochore proteins would be essential for viability, we imposed the requirement that the cep mutants be conditionally lethal.
The "synthetic acentric" approach has at least two obvious virtues for the future studies on centromere proteins. First, it provides a very rapid and efficient means to isolate mutant alleles of genes encoding the pll0 and p64 components of the kinetochore. These mutations may serve as powerful tools for dissecting the molecular organization and the cellular role of these proteins. For example, some new alleles of ndclO (cep2) isolated by our synthetic acentric approach (see below) are phenotypically distinct from known ndclO alleles.
Second, although this approach may be saturated for the cen6-(lll)15t reporter, as evidenced by the isolation of multiple alleles of ndclO and cep3, other reporter CenDNA sequences with CDEII or CDEIII mutations may be used to identify genes encoding additional centromere proteins. For example, the cen6-(llI)19t20 mutation is not synthetically acentric when combined with the ndclO or cep3 alleles, yet potential cep mutants were obtained with this centromere as a reporter (Strunnikov, A. V., unpublished results).

The CEP3 Gene Encodes a Putative
CenDNA-binding Protein That Is Necessary for Microtubule-binding Activity of the Centroraere DNA sequence analysis revealed that CEP3 encodes a protein with a predicted NH2-terminal DNA-binding domain Zn(II)2Cys6 (Pan and Coleman, 1990). In other fungal proteins this domain mediates DNA binding in a sequencespecific manner and has been found only in transcription factors-like proteins, dispensable under standard laboratory conditions. The most characterized member of this family, Gal4p, binds a palindromic DNA sequence as a dimer (Marmorstein et al., 1992). Interestingly, CenDNA mutations that interact with cep3 alleles lie in symmetrical positions of the CDEHI palindrome, suggesting that Cep3p could also bind DNA as a dimer. Previously, a limited functional symmetry of CDEIII was described (Jehn et al., 1991) based on data obtained for cis mutations.
Of three major proteins in the Cbf3 complex, Cep3p is the only subunit with an evident DNA-binding motif. Therefore, it is reasonable to assume that Cep3p mediates binding of the Cbf3 complex to CDEIII. Surprisingly, all tested cep2 (ndclO) mutations have similar pattern of interaction with CDEHI mutations as do cep3 alleles (this study). One possible explanation for this phenomenon is that Ndcl0p also binds to the same nucleotides of CDEIII as does Cep3p. However, this seems unlikely, as Ndcl0p and Cep3p do not share significant sequence similarity and Ndcl0p lacks any known DNA-binding motif. More likely, Ndcl0p is a cofactor of Cep3p binding to CDEIII. Alternatively, Ndcl0p could be an unconventional DNA-binding protein affecting Cep3p due to the close proximity of Cep3p and Ndcl0p binding sites. These models are yet to be verified by biochemical studies, however, our genetic data provide the first evidence that Cep3p and Ndcl0p physically interact in vivo.
What would be a hypothetical way to construct a functional centromere, taking into account that Cep3p has the only known DNA-binding motif among the Cbf3 components? One possibility is, that Cep3p Zn-cluster is solely responsible for tethering all other centromere components to the CenDNA. This seems unlikely because the binding of kinetochore proteins to CenDNA should have special requirements to withstand the pulling forces that are exerted during mitosis. The interaction between CDEHI and the relatively simple DNA-binding motif of Cep3p, likely, would not be sufficient for this purpose. Also, since CDEII, CDEIII and flanking sequences form a large special chromatin structure (Bloom et al., 1984), it seems likely that other DNAbinding components exist. Based on the fact, that CDEIII and the proteins bound to it are the key elements for the assembly of this entire complex (mutations in CDEUI can both abolish centromere function [Hegemann et al., 1988;McGrew et al., 1986] and prevent formation of centromere chromatin [Saunders et al., 1988]), we can suggest that the DNA-binding motif of Cep3p first recognizes CDEIII during replication of centromere chromatin ("centromere priming" role) and then nucleates assembly of other centromere proteins. These proteins (including Cep3p) could then be locked on the CenDNA using a special mechanism, yet to be identified. For example, the relative richness of Cep3p in cysteine residues (beyond the Zn cluster) provides a possibility for disulfide bonds which could covalently lock Cep3p as well as other subunits wrapped around CenDNA. Partial DNA-wrapping has been documented for the Gal4 protein previously (Marmorstein et al., 1992).
In addition to the putative DNA-binding properties of Cep3p in vivo, we examined the role of Cep3p in organizing microtubule-binding activity of the kinetochore. We showed that the minichromosomes isolated from cep3 cells exhibit a dramatic defect in centromere-dependent binding of minichromosomes to microtubules in vitro. This observation has three important implications. First, it validates the use of isolated minichromosome as the means to identify and characterize structural components of kinetochore, required for microtubule binding. Second, cep3-1 is the first described mutation in a Cbf3 component that disrupts the microtubule binding activity of centromere, corroborating the importance of Cbf3 complex for centromere structure and function (Lechner and Carbon, 1991;Hyman et al., 1992). Third, the microtubule-binding data suggest a molecular model for the "synthetic acentric" phenomenon. While it is theoretically possible that Cep3p directly binds microtubules, it is more likely that the presence of Cep3p is needed to assemble a multi-protein complex which has mt-binding subunits, in ac-cordance with our model published previously (Kingsbury and Koshland, 1993). This hypothetical "centromere priming" role for the Cep3 protein, if verified by additional biochemical experiments, could help to identify other centromere proteins physically associated with Cep3p.

Cellular Response to the Loss of Centromere Function
The cells of multicellular organisms developed a sensitive mechanism which delays anaphase if even one chromosome fails to achieve its metaphase position (Nicklas and Arana, 1992). It has been suggested that this delay reflects a "checkpoint" that monitors chromosome congression and kinetochore-microtubule attachment. As some aspects of cell cycle regulation in S. cerevisiae may be quite different from multicellular organisms (Nasmyth, 1993), it is possible that loss ofkinetochore function would not generate a cell cycle delay.
In fact no delay was observed in ndclO-1 cells, the first mutants demonstrated to be defective in an essential yeast centromere protein (Goh and Kilmartin, 1993). At restrictive temperature these cells undergo asymmetric elongation of the mitotic spindle leaving most of the chromosomes unsegregated. However, experiments with CenDNA mutations (Spencer and Hieter, 1992)  In conclusion, we described an effective and non-laborious screen for cep genes. This screen allowed isolation of the CEP3 gene, a new essential gene encoding a putative centromere component in yeast, as well as new alleles of the CEP2 (NDCIO) gene, encoding a known centromere protein.
These results bring us closer to understanding the centromere structure, however, more proteins need to be identified. Our system based on the generation of synthetic acentric mutants should be very useful in the identification of these unknown centromere proteins.