Chromosomes undergo dramatic programmed changes in higher-order structure as a prerequisite to their proper segregation in mitosis. Replicated sister chromatids must remain attached to one another until anaphase, undergo significant condensation, and become disentangled from one another before complete segregation to opposite poles. Although many activities are required to ensure that these processes occur correctly, the characterization of key players remains at an early stage. A number of specialized components have been identified, either biochemically or genetically, and subsequently shown to be conserved among the different experimental systems. One of the more interesting but perplexing is the Barren protein.

Barren was originally discovered as a Drosophila mutation that caused defects in the nuclear division of neuronal precursor cells (Bhat et al., 1996 ). Further examination of the cellular phenotype of barren mutants revealed a morphology similar to that exhibited by DNA topoisomerase II mutants of Saccharomyces cerevisiae (Holm et al., 1985 ). Dividing cells frequently contain chromosomes that appear unable to separate from one another during anaphase; instead, the anaphase chromosomes are frequently stretched across the two daughter cells, and cytokinesis initiates with the plasma membrane pinching in toward the chromatin bridge. Because this stretched DNA phenotype did not appear to result from global defects in chromosome condensation, a role for Barren in topoisomerase II function was inferred from the observation that Barren and topoisomerase II coimmunoprecipitate and interact in a two-hybrid assay. Indeed, purified Barren influences the activity of purified topoisomerase II in vitro, suggesting that Barren may regulate topoisomerase II activity in vivo (Bhat et al., 1996 ). Although this is an exciting possibility, the hypothesis has yet to be tested directly in vivo.

A different model for Barren function was proposed from work in the Xenopus system based on the copurification of Barren with 13S condensin. This five-component complex, in which all of the proteins are conserved from Xenopus to yeast, was initially isolated from mitotic extracts of Xenopus eggs and shown to promote the condensation of chromatin in vitro (Hirano and Mitchison, 1994 ; Hirano et al., 1997 ). Two of the condensin subunits, XCAP-C and XCAP-E, are members of the SMC (structural maintenance of chromosomes) family of proteins (for recent reviews, see Jessberger et al., 1998 ; Strunnikov, 1998 ; Yanagida, 1998 ; Hirano, 1999 ). In vivo, inactivation of the yeast SMC homologues of XCAP-C, Smc2p and cut3, leads to defects in mitotic chromosome condensation (Saka et al., 1994 ; Strunnikov et al., 1995a ). Because Barren is a condensin subunit in the Xenopus system, it has been postulated to be required for chromosome condensation.

Assigning a function for Barren based on the Drosophila or Xenopus studies is complicated by several factors. Because it has not been possible in the Xenopus system to inactivate individual subunits, their specific contribution to this in vitro condensation activity has yet to be established. Although a role for the SMC subunits in this activity is inferred from the condensation defect of yeast smc mutants (Saka et al., 1994 ; Strunnikov et al., 1995a ), the in vivo role of individual non-SMC subunits in chromosome condensation has not been addressed. Thus, it remains an open question whether the non-SMC subunits mediate condensin activity or are involved in auxiliary functions, such as linking condensation with DNA topoisomerase II activity. At first, this latter model might seem particularly appealing given both the in vitro interaction between Drosophila Barren and DNA topoisomerase II and the failure to observe a condensation defect in the Drosophila barren mutants. However, studies in other systems have not revealed interactions between the condensin components and DNA topoisomerase II (Hirano and Mitchison, 1994 ; Hirano et al., 1997 ; Strunnikov, personal communication). Given the apparent discrepancy between the Drosophila and Xenopus observations, it is critical to test the in vivo role of Barren in cell cycle–dependent chromosome metabolism, particularly in chromosome topology and condensation.

With the development of cytological tools in budding yeast and its extremely powerful genetics, Saccharomyces cerevisiae has become an excellent model system for studying the in vivo function of proteins, such as Barren, involved in cell cycle–dependent chromosome dynamics. The coupling of these methods with biochemical analysis of DNA metabolism and topology allows a uniquely broad spectrum of questions in mitosis to be addressed. Here we exploit these approaches to analyze the Barren homologue in budding yeast, addressing its in vivo function in eukaryotic chromosome metabolism.

Sherman et al., 1986

Ausubel et al., 1999

Table 1 S. cerevesiae strains used in this study

Strains lacking mitochondrial DNA ([rho⁰]) were used for flow cytometry and were created by growing [rho⁺] strains in 25 μg/ml ethidium bromide in YEPD to stationary phase. These cells were plated for single colonies on YEPD and tested for their ability to grow on YEP plates containing 2% glycerol (a nonfermentable carbon source). In addition, fluorescence microscopy of DAPI-stained cells confirmed the absence of mitochondrial DNA in the [rho⁰] strains.

Sikorski and Hieter, 1989

Boeke et al., 1984

Muhlrad et al., 1992

Robzyk and Kassir, 1992

To replace the normal chromosomal BRN1 allele with mutant brn1 alleles, the TRP1 gene was inserted just downstream of each plasmid-borne brn1 mutation, the brn1 genes plus flanking DNA were cut out of each plasmid with the use of BamHI and SacII, and the linear DNA was used to transfer the BRN1 gene into strain CH1580 (trp1 BRN1). After selecting the transformants for TRP1, integration was confirmed by PCR. In each case, the strain carrying the transferred brn1 gene was heat sensitive, as expected. Strains bearing integrated brn1 alleles were used for all physiological experiments.

Weinert and Hartwell, 1993

Kilmartin et al., 1982

Koshland et al., 1985

Spell and Holm, 1994

Spell and Holm, 1994

Hutter and Eipel, 1979

Figure 3 Mutant brn1 strains produce cells containing >2C DNA content after two generation times at the restrictive temperature. (A) Production of >2C cells in asynchronously dividing cultures. Strains CH2534 (BRN1), CH2587 (top2-4), CH2535 (brn1-9 (more ...)

Strunnikov et al. [1995a]

Guacci et al. (1994)

Figure 4 Chromosome condensation and chromosome cohesion in brn1 mutants at the restrictive temperature. (A) FISH analysis of rDNA morphology in brn1 mutants. Exponentially growing strains CH2523 (BRN1) and CH2524 (brn1-9) were synchronized in G1, shifted to the (more ...)

For the rDNA condensation maintenance experiment, cells were arrested in G1 with the use of α-factor (as described above) and then released into medium containing nocodazole for 2 h at the permissive temperature (23°C). An aliquot was taken to assess the initial state of rDNA condensation. The remainder of the culture was then shifted to 37°C for 1 h in the continued presence of nocodazole, after which the cells were harvested. Condensation was assessed with the use of DIG-labeled rDNA, as described above.

Boeke et al., 1984

brn1 mutants also exhibit a loss of viability when incubated at the restrictive temperature. When exponentially growing cultures are shifted to the restrictive temperature, ~75% of the brn1 cells become inviable after 3 h (1.5 generation times) and ~95% become inviable after 6 h (3 generation times) (Table 2). This result is quantitatively similar to results observed with smc1 and smc2 mutants, which maintain moderate viability after 1 generation time at the restrictive temperature (Strunnikov et al., 1993 , 1995a ). However, the result is noticeably different from what is observed with top2 mutants, which lose viability much more rapidly at the restrictive temperature (Holm et al., 1985 ).

Table 2 Viability at the restrictive temperature of dividing cells and cells arrested in the cell cycle

Loss of viability at the restrictive temperature could be caused by the requirement for Brn1p at a specific time during the cell cycle, or it could be caused by a more general requirement for Brn1p in cell metabolism. If the former is true, then brn1 mutants should remain viable at the restrictive temperature when they are prevented from traversing the cell cycle; both smc2 and top2 mutants exhibit this phenotype. Exponentially growing brn1, smc1, smc2, and top2 cultures were arrested in G1 phase of the cell cycle with α-factor or in S phase with hydroxyurea. The cultures were divided into two aliquots, and the cells were incubated at either the restrictive or the permissive temperature for an additional 3 h. All cultures maintained good viability (at least 84%) when cell cycle arrest was maintained (Table 2). This result demonstrates that brn1 mutants must pass through the cell cycle to incur irreversible damage. It also demonstrates that arrested G1- and S-phase cells do not require ongoing Brn1p activity.

Our results suggest that Barren is required between the beginning of S phase and the beginning of mitosis. When a synchronously dividing brn1 strain is prevented from entering S phase with hydroxyurea, the viability of the culture remains high, even though it is incubated at the restrictive temperature for 3 h (Table 3). If the strain is instead allowed to pass though S and G2 phases, the viability of the culture decreases to the same extent whether it is treated with nocodazole or not. (The efficacy of the nocodazole treatment was confirmed by observations of cell morphology [our unpublished results].) This result is in sharp contrast with the result observed with a top2 strain, which maintains good viability as long as it is prevented from passing through mitosis by treatment with either hydroxyurea or nocodazole. These results suggest that the activity or assembly of Brn1p is required between early S phase and early M phase.

Table 3 Viability at the restrictive temperature of cells traversing specific landmarks in the cell cycle

Figure 1 Phenotypic characteristics of brn1 mutants at the restrictive temperature. (A) Distribution of cell types. Asynchronous cultures of strains CH2523 (BRN1) and CH2524 (brn1-9) were shifted to 37°C for 3 h, fixed, and stained with DAPI to visualize (more ...)

To characterize the morphology of the large-budded cells more thoroughly, cells from similarly grown cultures were fixed, prepared for indirect immunofluorescence of tubulin, and treated with DAPI to stain DNA. More than half of the large-budded BRN1 cells contained two well-separated nuclei (anaphase and telophase cells) with nuclear microtubules extending along the entire DNA mass (Figure 1, B and C). In contrast, roughly half of the large-budded brn1 cells possessed a dumbbell-shaped DNA mass that extended through the bud neck and was frequently distributed asymmetrically. The nuclear spindles in these cells were intensely stained, only partially extended (4–5 μm), and predominantly localized at or near the bud neck, although typically failing to traverse it. That the cells with stretched nuclei initiate a true anaphase was confirmed by a separate experiment in which hemagglutinin-tagged Pds1p, an inhibitor of the metaphase-to-anaphase transition, was observed to be depleted (our unpublished results). Surprisingly, 62% of brn1 cells with bilobed nuclei displayed aberrant spindle localization, with tubulin staining localized to only one of the two DNA masses. Together, these data suggest that brn1 cells undergo an aberrant anaphase.

Because DNA topoisomerase II is the only cellular enzyme capable of disentangling catenated circles of double-stranded DNA, circular plasmids remain catenated after replication when top2 strains are incubated at the restrictive temperature (Koshland et al., 1985 ). If Barren is required for DNA topoisomerase II activity, as has been suggested in Drosophila, then brn1 mutants might exhibit a similar accumulation of catenated plasmids when they are incubated at the restrictive temperature. Wild-type and mutant strains containing a 14-kb circular minichromosome were synchronized in G1 in medium containing α-factor, then released into nocodazole-containing medium at the nonpermissive temperature. The topological state of purified minichromosomes was then analyzed by agarose gel electrophoresis (Figure 2A). As expected, higher-molecular-weight catenated circles accumulate in nocodazole-treated top2 cells. In contrast, the brn1 mutants display no change in plasmid topology at the restrictive temperature, indicating that brn1 mutants effectively resolve the catenanes produced during replication, even at the restrictive temperature.

Figure 2 Neither catenated plasmids nor broken chromosomes are apparent in brn1 strains at the restrictive temperature. (A) Plasmid catenation in top2 and brn1 mutants. Strains CH2523 (BRN1), CH2524 (brn1-9), CH2525 (brn1-16), CH335 (TOP2), and CH325 (top2-4), (more ...)

When full-length chromosomes are segregated in the absence of DNA topoisomerase II activity, approximately one-third of chromosomes larger than 300 kb suffer a distinctive pattern of breakage (Spell and Holm, 1994 ). If Barren is required for DNA topoisomerase II activity, then brn1 mutants might be expected to exhibit the same kind of chromosome breakage. To test this hypothesis, BRN1, brn1-9, brn1-16, and top2-4 strains were grown exponentially at 25°C, and the cultures were divided and incubated for an additional 5 h at either 25 or 37°C. Although a clear smear of broken chromosomes was visible in the lane with DNA from the top2 culture grown at 37°C, no such smear was visible in the lanes containing DNA from brn1 strains (Figure 2B). Thus, it appears unlikely that Barren is required to activate DNA topoisomerase II in vivo.

Howell et al., 1994

Amin and Holm, 1996

Holm et al., 1989

To examine the timing of this phenomenon, we arrested exponentially growing cells in G1 with α-factor, released them to the restrictive temperature, and then examined them by FACS at various times. For the brn1-16 strain, DNA replication was apparent 1 h after release from α-factor, which is similar to what is observed with BRN1 cells (Figure 3B). The initial appearance of >2C cells occurred 2 h after release, and the population of >2C cells was much more apparent 3 h after release from α-factor (Figure 3B). This cell type appears to be produced during the second cell cycle after release from α-factor, because its production was entirely prevented by treating the culture with either α-factor or hydroxyurea 1.5 h after release from the initial α-factor treatment (Figure 3C; our unpublished results). A similar phenomenon was seen with smc2 mutants, except that progress through the cell cycle was somewhat slower with this strain. Thus, it appears that brn1 and smc2 cells begin to exhibit this unusual staining in the second cell cycle after a shift to the restrictive temperature. One possibility is that this staining pattern reflects aberrant excessive DNA replication (see DISCUSSION).

Hirano et al., 1997

Guacci et al., 1994

To determine whether Barren function is limited to an early stage in chromosome condensation (i.e., as for an assembly factor) or whether ongoing Barren activity is required to maintain mitotic chromosome structure, we inactivated Barren after chromosome condensation had occurred. BRN1 and brn1 cultures were arrested in early mitosis with the use of nocodazole at the permissive temperature to allow rDNA condensation. The cultures were then shifted to the restrictive temperature to inactivate Barren specifically in mitosis, and the state of the rDNA was monitored by FISH with the use of an rDNA probe. As expected, both BRN1 and brn1 chromosomes were condensed at the permissive temperature. In contrast, although the BRN1 chromosomes remained condensed at 37°C, rDNA condensation was disrupted in the brn1-9 strain (Figure 4C). These data indicate that ongoing Barren activity is required for the maintenance of chromosome condensation during mitosis.

To address whether the condensation defects in brn1 mutants are limited to repetitive DNA, we examined chromosome condensation in unique sequences on chromosome XVI. Exponentially growing brn1 and BRN1 cultures were arrested with α-factor, shifted to the restrictive temperature, and then released into nocodazole to block the cells in mitosis. Chromosome condensation was examined by using FISH and measuring the distance between two sequences that map 255 kb apart on chromosome XVI (Figure 4D). In BRN1 nuclei, the average distance between CEN16 proximal and distal probes was 0.75 ± 0.08 μm (experiment 1; Figure 4D) and 0.77 ± 0.09 μm (experiment 2; our unpublished results), which are similar to values reported previously for condensed DNA (Strunnikov et al., 1995a ). In contrast, in brn1-9 nuclei, the average distance between probes was 1.07 ± 0.11 μm (experiment 1) and 0.97 ± 0.11 μm (experiment 2); these values are very similar to what is seen in uncondensed chromosomes in G1 cells (1.0 ± 0.01 to 1.1 ± 0.1 μm; Strunnikov et al., 1995a ). Thus, little or no chromosome condensation occurs on unique DNA sequences in the absence of functional Barren protein.

Because a number of mutations that affect chromosome cohesion also cause alterations in chromosome condensation (Guacci et al., 1997 ; Strunnikov, personal communication), we tested brn1 mutants arrested in mitosis for precocious separation of sister chromatids in the absence of microtubules. Although chromosome condensation was impaired under these conditions, sister chromatid cohesion appeared normal when analyzed with a CEN16 probe (Figure 4E) and a CEN1 probe (our unpublished results). Thus, an underlying defect in cohesion is unlikely to be the source of the observed condensation defects. Instead, brn1 cells are specifically impaired in their ability to establish and/or maintain chromosome condensation at the restrictive temperature. Together, these results strongly suggest that Barren plays an essential role in chromosome condensation in yeast.

To begin to unravel the biological function of Barren in eukaryotic cells, we used genetic, cytological, and molecular biological methods to initiate studies of the Barren homologue in budding yeast. The Barren product in budding yeast is essential for viability and cell division. The analysis of heat-sensitive brn1 mutants revealed that cells compromised in Barren function exhibit partially elongated chromosome masses similar to the anaphase defect observed for barren mutants in Drosophila, which suggests a conserved function for Barren in chromosome transmission between yeast and flies. This chromosome phenotype is also very similar to the phenotype of smc2 mutants of budding yeast, cut3 and cut14 mutants of Schizosaccharomyces pombe, and mix1 mutants of Caenorhabditis elegans (Saka et al., 1994 ; Strunnikov et al., 1995a ; Lieb et al., 1998 ). All of these genes encode homologues of components of Xenopus condensin, a complex essential for chromosome condensation in vitro. Furthermore, we directly show that brn1 mutants are defective in chromosome condensation in vivo, as has been demonstrated for smc2, cut3, and cut14. Consistent with this observation, Barren performs an essential function between early S phase and early M phase, the period of the cell cycle during which condensation is established and maintained. Together, our results reveal that the Barren product of budding yeast is critical for mitotic chromosome segregation and condensation.

The demonstration that yeast Barren functions in vivo in chromosome condensation both corroborates and extends the previous analysis of the role of Barren. In Xenopus, Barren copurifies with the five-subunit condensin complex, and as such it was inferred to play an important role in the condensation process (Hirano et al., 1997 ). Because the contribution of individual condensin subunits to the in vitro condensation reaction was not addressed, however, the physiological significance of Barren's association with condensin was unclear. That yeast Barren is essential for viability and is required for chromosome condensation in vivo suggests that it is an integral component of condensin function. Although we do not know the molecular function of Barren in the condensin complex, our genetic results provide two important constraints. Because loss of Barren function yields uncondensed chromosomes, Barren is likely to be critical to the core function of condensin rather than to serve as a minor auxiliary factor. Second, the absence of chromosome condensation when Barren activity is compromised implies that the loss of Barren function inactivates the cell's condensation machinery, i.e., Barren is not a negative regulatory subunit of condensin. Recent work with the Xenopus condensin complex has shown that, in vitro, the condensin complex wraps DNA in a right-handed manner (Kimura and Hirano, 1997 ; Kimura et al., 1999 ). That this reaction requires phosphorylated Barren and/or XCAP-D2 and/or XCAP-G suggests that some or all of these proteins play a role in the activation of condensin (Kimura et al., 1998 ). Consistent with this suggestion, anti-Eg7 (XCAP-D2) antibodies were used to demonstrate a role for this subunit in the in vitro condensation assay (Cubizolles et al., 1998 ). Additional experiments will be needed to assess whether Barren functions as a regulatory switch or as a direct mediator of altering chromosome topology.

In vivo, ongoing Barren activity is required to maintain mitotic chromosome structure. The most simple interpretation of this result is that Barren plays a structural role in higher-order chromosome organization. Alternatively, the maintenance of chromosome condensation could be a highly dynamic process involving a constant reestablishment of the condensed state. Regardless of the mechanism, it is clear that inactivation of Brn1p in early M causes an untimely loss of chromosome condensation, probably through inactivation of the cellular condensation machinery. Because chromosome condensation is a reversible process that is initiated in early M and reversed in late M/early G1, the regulation of condensin activity through Brn1p could provide an effective mechanism for both condensing and decondensing DNA.

An intriguing question is whether both repetitive and unique DNA condense through similar mechanisms. Although previous work with smc2 mutant cells failed to show a defect in rDNA condensation (Strunnikov et al., 1995a ), this was most likely due to a less stringent inactivation regimen (see Figure 4B). We now show that both Smc2p and Brn1p are required for the condensation of repetitive as well as unique DNA sequences. These data are consistent with a model whereby chromosome compaction of both repetitive and unique DNA sequences occurs through a common molecular machinery and thus through a conserved mechanism. Although defects in rDNA condensation are more dramatic by FISH than those seen with unique DNA probes, this difference may reflect the reiterative nature of the rDNA array and/or a change in the relative distribution of condensin proteins. Indeed, indirect immunofluorescence of Smc4p and Ycd2p, yeast homologues of two additional condensin subunits, suggests that they are enriched on the rDNA (Strunnikov, personal communication; Zheng and Koshland, unpublished results).

It is noteworthy that condensation of both unique and repetitive DNA is perturbed in the first mitosis after Brn1p inactivation. At this time, topological links between sisters are resolved, as judged by the absence of catenated circular minichromosomes and chromosome V breaks. Thus, condensation does not appear to be obligatory in driving out DNA tangles, as has been proposed previously (Wood and Earnshaw, 1990 ). Rather, anaphase itself may be sufficient to promote DNA untangling by topoisomerase II (Holm et al., 1985 ; Holm, 1994 ). However, a modest delay in anaphase is detectable in brn1 mutants at the restrictive temperature (our unpublished results), suggesting that the kinetics of chromosome untangling may be facilitated by condensation, and this effect may be particularly important for the larger chromosomes of higher eukaryotes.

Although a condensation defect is the earliest observable phenotype of brn1 mutants, there is a later appearance of additional phenotypes, including an anaphase failure that is cytologically similar to DNA topoisomerase II mutants. Although earlier in vitro analysis of Drosophila Barren suggested that it might serve as an activator of topoisomerase II function, our results suggest that yeast Barren is probably not an essential activator of topoisomerase II in vivo. Because topoisomerase II is the only enzyme known to disentangle catenated circular plasmids, a failure by the mutant Barren protein to activate topoisomerase II should have led to the accumulation of catenanes. In brn1 mutants at the restrictive temperature, however, catenanes are efficiently resolved and chromosomes are segregated without breakage. Nonetheless, it remains possible that Barren could serve to regulate topoisomerase II activity in a more subtle manner. Barren may serve as an inhibitor of topoisomerase II activity in vivo, as was observed in vitro when Barren was present at high concentrations. Cytological evidence suggests that Barren and topoisomerase II reside in close proximity in condensed and condensing chromosomes (Bhat et al., 1996 ). Perhaps when associated with chromosomes they are members of a large multiprotein complex and perform their functions simultaneously but independently.

An unexpected phenotype of cells defective for Barren function is an accumulation of cells containing >2C DNA content. This phenotype may be general for chromosome condensation mutants, because smc2 mutants also produce >2C cells. When Barren or Smc2p is inactivated at the beginning of the cell cycle, chromosome condensation is perturbed at the time of the first mitosis (Figure 4; our unpublished results). In contrast, the appearance of cells with >2C DNA content occurs much later (Figure 3). In fact, the appearance of >2C cells can be entirely abolished if the brn1 cells are prevented from proceeding through a second cell cycle with either α-factor or hydroxyurea (Figure 3C; our unpublished results). It is unlikely that this phenotype is caused by some aberrant binding of propidium iodide to the uncondensed DNA, because a sharp 2C peak is observable in both mutants in the first M phase. Instead, the >2C FACS profiles are reminiscent of FACS profiles produced by a cdc6 mutant that displays promiscuous initiation of DNA replication (Liang and Stillman, 1997 ). This observation brings up the interesting possibility that improper condensation of the chromosomes in the first cell cycle subsequently leads to inappropriate initiation of DNA replication. Because the prereplication complex is formed at the M-to-G1 transition (Diffley et al., 1994 ), one possibility is that its normal assembly is affected by the condensation state of the chromosomes.

A second unexpected phenotype of brn1 mutants is the absence of spindle and chromosome colocalization. One possible explanation is that decondensation results in intertwining that causes detachment of chromosomes from the spindle when the cell enters anaphase. This possibility seems unlikely, however, because topological links and chromosome breaks do not accumulate in brn1 mutants. Furthermore, top2 mutants complete anaphase by breaking chromosomes, suggesting that kinetochore-spindle attachments resist a challenge from topologically linked DNA (Holm et al., 1989 ). A more attractive model is that Barren (or the chromosome condensation it maintains) plays a novel role in centromere structure and/or function. In support of this idea, cytological observations of mitotic chromosomes suggest an unusual condensed state of centromeric regions (Rattner and Lin, 1985 ). In addition, mutations in Cep3p, an established kinetochore component, result in a similar displacement of the chromosomes from the spindle, presumably resulting from a failure of the chromosomes to maintain a stable attachment to the dynamic spindle (Strunnikov et al., 1995b ). Furthermore, preliminary evidence suggests that Brn1p genetically interacts with a subset of kinetochore proteins (Lavoie and Koshland, unpublished observation). It will be interesting to determine whether this putative role for Barren at the centromere reflects a general role for condensin proteins. However, neither smc2 mutations nor mutations affecting the homologue of XCAP-G cause an accumulation of mid anaphase nuclei (Lavoie and Koshland, unpublished observation). Thus, it is tempting to speculate that specific cellular processes may be differentially sensitive to defects in the functions of condensin proteins. Additional study will be necessary to determine the precise functional roles of these proteins, which play a pivotal role in the condensation of chromosomes.

Note added in proof. In the accompanying paper by Ouspenski et al., brn1 mutants do not exhibit excess DNA replication during the time course of the FACS experiment. This disparity probably reflects the roughly twofold difference in doubling times of the strains used by the two laboratories.

We are indebted to A. Strunnikov for providing the 1aAS330 (smc2-8) strain and for communication of unpublished results. We thank A. Philp and members of our laboratories for critical reading of the manuscript. B.D.L. and K.M.T. contributed equally to this work. This work was supported by grants GM36510 (to C.H.) and GM41718 (to D.K.) from the National Institutes of Health. B.D.L. is the recipient of a Centennial Fellowship from the Medical Research Council of Canada.