Correspondence to: Makkuni Jayaram, Section of Molecular Genetics and Microbiology, Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712. Tel:(512) 471-0966 Fax:(512) 471-5546 E-mail:jayaram{at}icmb.utexas.edu.
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Abstract |
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The efficient partitioning of the 2-µm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host encoded factors are likely to contribute to plasmid segregation. Direct observation of a 2-µmderived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes. Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-µm plasmid and the chromosome are quite similar during the yeast cell cycle. Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus. Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle. Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules. In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem. Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates. They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation.
Key Words: ipl1-2 mutation, tubulin, plasmid cohesion, mitotic spindle, BRN1
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Introduction |
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The successful propagation of the 2-µm circle, a relatively small circular plasmid (6,318 bp) present in most common strains of Saccharomyces cerevisiae at a copy number of ~60 per cell, is accomplished via a partitioning system and an amplification system (reviewed in
Although the apparent copy number of the 2-µm plasmid is high, the requirement of a partitioning system for stable plasmid inheritance suggests that the effective copy number within the cell may be considerably lower. Impediments to free movement of plasmid molecules are likely imposed by their attachment to subcellular sites. Yeast plasmids containing chromosomal ARS elements, but lacking the 2-µm circle partitioning system, have a propensity to be retained in the mother cell during division (
The need for plasmid amplification arises only if and when there is a decrease in copy number below the steady state value. Normally, each plasmid molecule is replicated once, and only once, per cell cycle (
Assuming that the functional copy number of the 2-µm plasmid is considerably <60, perhaps as low as unit copy if the individual plasmid copies are coalesced into a single cluster, how does it achieve its remarkably high stability using a seemingly rudimentary partitioning system? The plasmid molecules are resident in the nucleus as minichromosomes with standard nucleosome phasing (
In this paper, we provide visual evidence that the 2-µm plasmid and the Rep proteins are associated with each other in the yeast nucleus. The Rep proteins (and by inference, the associated plasmid molecules) are clustered near the poles of the mitotic spindle apparatus. The apparent linkage between the spindle poles and the plasmid-Rep complex suggests a plausible mechanism for the equal partitioning of plasmid molecules after replication. We propose that the plasmid has evolved a strategy to exploit at least certain components of the chromosome partitioning machinery to ensure its own stable propagation. Such a mechanism is consistent with the observed correlation between the segregation kinetics of a 2-µmbased plasmid and a yeast chromosome (or a centromere containing plasmid) with respect to the cell cycle. Further support for this mechanism is provided by the finding that the yeast chromosomal mutation ipl1-2 (
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Materials and Methods |
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Strains and Plasmids
The strains and plasmids used in the present study are listed in Table 1. The strain AFS479 was kindly given to us by the laboratory of Andrew Murray (University of California, San Francisco, CA). The [ciro] strains were obtained as described previously (
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The plasmid pAFS59 carrying 256 copies of the lac operator and pAFS144 expressing green fluorescent protein (GFP)-lac repressor from the HIS3 promoter were provided by the Murray laboratory (
More detailed information on strains and plasmids are available upon request.
In Vivo Visualization of Plasmids or Chromosome
The yeast strain containing the expression cassette for GFP-lac repressor was transformed with the appropriate plasmid containing the lac operator repeats. The expression of the hybrid repressor was induced by the addition of 10 mM 3-AT (3-aminotriazole) for 30 min. The lac operator DNA bound by GFP-repressor was visualized by fluorescence microscopy following excitation at the appropriate wavelength. To obtain optimal fluorescence, the pH of the media was maintained at 6.5 by the addition of trisodium citrate (6.5 g/liter). Cells were observed under an Olympus BX-60 microscope with recommended filters for GFP excitation and emission. Images were captured using a Spot Digital Camera from Diagnostic Instruments, and were processed using ImagePro Plus software from Media Cybernetics. Confocal images were taken using the Leica confocal system, TCS4D (Core Facility, Institute for Cell and Molecular Biology, University of Texas, Austin, TX).
Synchronization of Cells in G1 Phase by -Factor
Cells were grown overnight in selective medium, washed, and resuspended in YEPD at an OD600 of ~0.1. The culture was incubated at 30°C for 90 min, and -factor was added to a final concentration of 7 µg/ml. Incubation at 30°C was continued for 3 h and the percentage of cells arrested in G1 was monitored by microscopy (
Assay for Plasmid Segregation in Host Strains Harboring the ipl1-2 Mutation
The yeast strains were grown in appropriate selective media at 26°C, and were arrested in G1 by -factor treatment. After washing away the pheromone, the cells were allowed to recover from growth arrest at 26°C for 90 min. They were then shifted to 37°C and allowed to grow for 4 h. 4', 6'-diamidino-2-phenylindole (DAPI) was added to the growth medium (final concentration of 2 µg/ml), and cells were harvested 30 min later. They were washed with sterile water, fixed in 3.5% formaldehyde (04°C), and observed under the microscope. Roughly 7580% of the cells in the population contained large buds. Plasmid and chromosome segregation data shown in Fig 7 pertain only to the large-budded cells.
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Nocodazole Treatment
An exponentially growing yeast culture was treated with either 1% DMSO (control) or 20 µg/ml nocodazole (Sigma Chemical Co.) in 1% DMSO. The cells were incubated in the presence of the drug at 30°C for 2 h. Examination of the cells under the microscope revealed ~85% of them to be arrested with large buds.
Antibodies
Rep1 and Rep2 polyclonal antisera were generated in rabbits. The antisera were affinity-purified and tested for specificity before use. Antitubulin antibodies were obtained from Serotec. Polyclonal antiserum against the lac repressor protein was purchased from Stratagene.
Immunofluorescence Assays
Yeast cells grown to midlog phase (106 cells/ml) were fixed in 5% formaldehyde solution for 60 min at room temperature. The fixed cells were washed once with PBS, once with 1.2 M sorbitol/1mM EDTA, and resuspended in the same medium to a final density of 108 cells/ml. Spheroplasts were obtained by incubating with 1 mg/ml of zymolyase 100T (US Biologicals) in the presence of 10% ß-mercaptoethanol for 60 min at 30°C. The spheroplasts were washed with PBS, transferred to poly-L-lysinecoated slides, and flattened using methanol (5 min) and acetone (30 s). Immunofluorescence staining was done according to
Estimating the Copy Number from GFP-LacItagged Plasmid Fluorescence
GFP fluorescence intensities from individual plasmid clusters were determined using ImagePro Plus or MetaMorph software (supplied by Media Cybernetics and Universal Imaging Corporation, respectively). Similar estimates were made for the tagged chromosome. The intensities of plasmid fluorescence and chromosome fluorescence were averaged for a large number of cells. The ratio of the averaged values was taken as the mean copy number of the plasmid per cell.
Z-series Sectioning of Yeast Nucleus
The compactness (or the residence zone) of the plasmid clusters was determined by z-series sectioning of the yeast nucleus in the confocal microscope. For each sample, 40 sections at 0.25-µm thickness were examined, spanning 5 µm of total thickness. The start point for scanning was set manually ~23 frames beyond the boundary of fluorescence from the GFP-lac repressor-tagged plasmid. Thereafter, the same number of sections (or the same total distance) was scanned for each sample. In every case, the set range completely covered the limits of the plasmid fluorescence zone. An identical procedure was used to obtain the boundary range of the DAPI staining region in each of the cells examined. The ratio of the green fluorescence range to the blue fluorescence range was calculated for each cell. Values from at least 20 individual cells were pooled to express the mean width (± SD) of the plasmid residence zone.
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Results |
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Direct Visualization of a 2-µmderived Plasmid: Comparison to a Yeast Chromosome or a Centromeric Plasmid
To visualize plasmids in live yeast cells, we have used the recognition between multiple copies of the lac operator sequence harbored by the plasmids and a fluorescent version of the lac repressor expressed from an inducible promoter (-factor (see Table 1 for strain and plasmid descriptions), the fluorescently labeled 2-µmderived test plasmid pSV1 was seen most often as a tetrad cluster within the nucleus (>50% of the time). The results shown in Fig 1 were obtained with
-factortreated cells. The superposition of green plasmid fluorescence (from GFP-lac repressor) and blue nuclear fluorescence (from DAPI) revealed that the plasmid molecules reside within the nucleus (data not shown). At the
-factor concentration used in these experiments (7 µg/ml, in SD medium containing required supplements), the cells did not show the typical shmoo phenotype associated with G1 arrest. However, they did not progress through the cell cycle unless they were washed free of
-factor. The same concentration of
factor in rich medium induced shmooing.
Examination of a large sample of cells revealed occasional deviations from the tetrad pattern of plasmid distribution. In ~20% of the plasmid-containing cells, the clusters consisted of triad or diad patterns (Fig 1B and Fig C, respectively), whereas, in ~15% of the cells, single fluorescent dots were observed (Fig 1 D). Occasionally (15% or less), the plasmid foci were constituted by more than four dots (Fig 1 E). In comparison, a marked yeast chromosome appeared as a single fluorescent dot in >99% of the cells examined (Fig 1 F). Similarly, a centromeric plasmid, pSV2, was also detected as a single fluorescent spot in >95% of the cells (Fig 1 G), with an occasional cell revealing two fluorescent spots (presumably representing two plasmid copies). By contrast, a population of cells grown selectively for the plasmid pSV3, containing a chromosomally derived replication origin (ARS) and none of the components of the 2-µm circle stability system, showed an essentially random distribution of cells containing one to four, and occasionally more than four fluorescent dots. The patterns of plasmid distribution and the frequencies of their occurrence in a G1-arrested cell population are summarized at the bottom of Fig 1.
Z-series sectioning by confocal microscopy indicated that the 2-µm circlederived pSV1 plasmid molecules tend to occupy a relatively restricted zone within the yeast nucleus. In a [cir+] host, the mean range of the plasmid residence zone expressed as a ratio of the nuclear diameter (derived from DAPI staining) was ~0.50 (see Materials and Methods and Fig 6). The relative intensities of fluorescence yielded an average copy number of ~1012 molecules per cell for pSV1 (assuming one copy of the chromosome per cell), and ~12 molecules per cell for pSV2 (the centromere containing plasmid). This estimate of pSV1 copy number refers to cells that contain three or four plasmid foci per cell. Such cells constitute a little over one half the cell population grown selectively (see Fig 1, table). Estimates from Southern hybridization of total DNA from a selectively grown cell population yielded a pSV1 copy number of 810 relative to a single copy gene (data not shown). This value for pSV1 is significantly lower than the steady state copy number of 4060 molecules per cell for the native 2-µm circle. However, assuming that the copy number control mechanisms would operate on the plasmid population as a whole, the relevant steady state copy number in a [cir+] strain would be the sum of the copy numbers of pSV1 and the endogenous 2-µm circles.
Association of the 2-µm Plasmid with the Mother or Bud during the Cell Cycle in a Synchronized Yeast Population
The [cir+] host strain containing the 2-µm test plasmid pSV1, the CEN-plasmid pSV2, or the tagged chromosome III was synchronized in G1 phase using -factor. Following release from
-factor arrest, the plasmid or the chromosome was visualized at various times during cell cycle progression. As indicated by the representative samples displayed in Fig 2, there was a strong correlation between pSV1 and chromosome III (Fig 2, compare A to C), or between pSV1 and pSV2 (Fig 2, compare A to B), with respect to the timing of their appearance in the growing bud. In small-budded cells, pSV1 was almost always associated with the mother cell. As the bud enlarged in size, the plasmid cluster migrated to the bud neck, closely following the dynamics of the chromosome. In large-budded cells, plasmid clusters were almost always detectable in both the mother and the would-be daughter.
To quantitate these observations, we have divided the cell population into four classes (IIV; see the schematic representation in Fig 2) with respect to the cell cycle stage as well as the localization of pSV1 within a growing cell (Fig 3). The relative abundance of each cell type over a period of 180 min after the removal of -factor indicates that cell synchrony was maintained fairly tightly through the first two generations, but began to break down thereafter. The plot of the distribution of pSV1 between mother and bud as defined by cell types IIV (Fig 3, solid line) was nearly superimposable with a similar plot for chromosome III (Fig 3, dashed line). The synchrony between pSV1 and chromosome III in the [cir+] strain was associated with pSV1 being partitioned roughly equally between mother and daughter cells (see Table 2). By contrast, in cell types III and IV of the [ciro] strain, there was a substantial increase in the number buds that contained fewer plasmids than the mother or that were plasmid-free (data not shown; see also Table 2).
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The above sets of data, obtained by directly following the cellular location and distribution pattern of a multicopy 2-µm circle derivative, demonstrate that chromosome and plasmid segregation occur as nearly concurrent events during the yeast cell cycle, at least within the limits of the kinetic resolution of our assays. It is possible that the two processes are mechanistically completely distinct, the observed coordination between them being merely coincidental. Alternatively, the shared timing suggests that the plasmid might utilize at least parts of the chromosomal segregation machinery for its own dispersal.
The 2-µm Circle Partitioning System and the MotherDaughter Symmetry in Plasmid Distribution
The 2-µmderived pSV1 plasmid contains only the cis-acting component, the STB locus, of the plasmid partitioning system. It does not encode either of the two Rep proteins. However, the endogenous 2-µm plasmids in a [cir+] strain can supply these proteins in trans to reconstitute the partitioning system. The Flp protein, required for copy number amplification, is also not encoded in pSV1. Because of the absence of a functional pair of recombination target sites (FRT sites) within it, pSV1 cannot undergo amplification (by the standard Futcher model) even when Flp is provided in trans. The integration of pSV1 (which contains one FRT sequence) into a native 2-µm plasmid via Flp recombination could, in principle, make it a target for amplification. However, the in vivo equilibrium of Flp recombination substantially favors plasmid monomers over dimers, trimers, and higher oligomers. Furthermore, the amplification system would be nonoperative in a [cir+] yeast strain containing pSV1 plus 2-µm circles at steady state levels.
To assess qualitative differences in pSV1 segregation in the presence and absence of a functional partitioning system, the synchronization experiment was repeated in a [ciro] host strain. The data were recorded at 75 min after release from -factorinduced cell cycle arrest, and pertain to type IV cells containing four and three fluorescent plasmid foci in the mother cell compartment. They comprised 69 and 40% of the [cir+] and [ciro] populations, respectively. Note that at this time point, 75% of the cells in both the [cir+] and [ciro] classes were in the large-budded state. The partitioning of pSV1 occurred evenly in the vast majority of the [cir+] cells (Table 2, column 1). By contrast, there was a significantly higher asymmetry of pSV1 segregation in the [ciro] cells (Table 2, column 2). For reference, the 4:n and 3:n segregation patterns of the ARS-plasmid pSV3 are displayed in Table 2, columns 3 and 4. Unlike pSV1, the segregation of pSV3 was unaffected by the [ciro] or [cir+] status of the host strain (Table 2, compare column 4 to column 3). Note that the data in Table 2 were derived from cells grown in selective medium. As expected from previous studies (
An interesting observation was the difference between the [ciro] and [cir+] host strains in the relative abundance of cells harboring pSV1 in the high and low copy patterns (four or three fluorescent plasmid dots in the mother cell for the former type; two or one for the latter). The low copy class of cells was nearly doubled for the [ciro] strain (60%) relative to the [cir+] strain (31%; data not shown). We also noted that plasmid fluorescence in the [ciro] background had a tendency to be spread out as individual dots within the nucleus (results not shown; see also Fig 6). These observations are consistent with a potential role for the Rep1 and Rep2 proteins (supplied by the endogenous 2-µm circles) in anchoring the plasmid to a nuclear substructure as an aggregate. The apparent drop in plasmid density in the [ciro] host may be accounted for by missegregation due to lack of a functional partitioning system. Since pSV1 lacks the Flp-mediated amplification system, restoration of copy number would not have been possible. Our failure to observe a significant fraction of cells with greater than their normal share of plasmids (expected to result from missegregation) suggests that there may be some selection against such cells. Overexpression of Flp with consequent artificial amplification of the native 2-µm plasmid is known to be deleterious to the host (
Combining the results from Fig 3 and Table 2, we conclude that the Rep/STB system is indispensable for the chromosome-like segregation of the pSV1 plasmid.
The Rep1 and Rep2 Proteins Associate Preferentially with 2-µm Plasmid DNA in the Yeast Nucleus
The similarity between the timing of pSV1 and chromosomal partitioning during the cell cycle (Fig 3) observed in the present study raises the intriguing possibility that the Rep/STB system might be involved in coupling plasmid and chromosomal segregation machineries. The nuclear localization of the Rep proteins and their in vivo interactions revealed by mono- and dihybrid assays (
To reveal the localization of the 2-µm plasmid relative to the Rep proteins in yeast cells, immunocytology was employed using mildly fixed cells (Fig 4A and Fig B). The Rep proteins were localized by fluorescein-conjugated secondary antibodies and the 2-µm circlederived pSV1 plasmid by Texas red-conjugated secondary antibodies (to lac-repressor antibody). The red and green fluorescence could be overlaid on each other in >85% of the cells, and occupied a sublocale within the DAPI-staining region. This strong tendency for colocalization was absent in the case of the ARS-containing pSV3 plasmid and Rep1p (Fig 4 C) or Rep2p (data not shown). We observed pSV3 dots that were not coincident with the Rep proteins in 50% of the cells. These findings agree with the in vivo and in vitro evidence for Rep-STB interaction (
Potential Link between the Rep Protein Complex and the Mitotic Spindle of the Host Cell
Intrigued by the similarities between chromosome partitioning and the 2-µm plasmid segregation (Fig 2 and Fig 3), we have probed the subnuclear localization of the Rep protein complex with respect to the spindle apparatus during the yeast cell cycle. In these experiments, the chromosomes were visualized by DAPI staining, and the Rep proteins and tubulin were visualized by indirect immunofluorescence using Texas red- and fluorescein-conjugated secondary antibodies, respectively.
The Rep proteins (and therefore the associated 2-µm circle molecules by inference; see Fig 4) tended to accumulate at or near the poles of the mitotic spindle (Fig 5A and Fig B, rows 2 and 3). In the predominant fraction of stage I and stage IV cells (>90%), there was either perfect or near coincidence between a Rep protein and the spindle pole (Fig 5, columns I and IV and rows 13). In some of the stage II cells (8%), the duplication of the spindle pole and the associated bifurcation of the Rep protein stain were readily discernible (Fig 5 A, column II and rows 13). In the majority of cases (92%), however, a bipartite Rep staining profile was not observed (Fig 5 B, column II and rows 13; also data not shown). In some of these cells, the Rep protein overlapped with both spindle poles as a uniformly stained entity with no detectable discontinuity. In others, the Rep protein remained proximal to one of the two poles of a short spindle. In stage III cells, the bulk of the Rep protein was concentrated at or close to each of the poles with a sharp drop in protein levels towards the center of the spindle (>90%). This preferred gradient of Rep localization can be seen distinctly in the confocal images of stage III cells in Fig 5A and Fig B, row 5. The correspondence between Rep protein staining and chromosomal staining at all cell stages, IIV, together with the proximity between the spindle poles and the rep proteins, suggests that plasmid molecules may be directly or indirectly attached to the spindle.
Effect of Microtubule Depolymerization on the Integrity of the 2-µm Plasmid-Rep Fluorescent Foci
To further examine the potential association between 2-µm circle and the mitotic spindle, we have assayed the pattern of Rep protein localization as well as the organization of pSV1 plasmid (2-µm circle based) in nocodazole- treated [cir+] cells. Under our assay conditions, 8085% of the cells were arrested in the G2/M phase as judged by microscopy. Immunofluorescence staining for tubulin in these large-budded cells revealed nearly complete disassembly of the mitotic spindle, although limited residual fluorescence was detectable at some spindle poles (Fig 6B and Fig D, middle). Along with the disassembly of the spindle, the Rep proteins showed a less compact, more disperse, pattern of nuclear localization (Fig 6B and Fig D, right). Nocodazole did not affect the steady state levels of the Rep1p or Rep2p, as assayed by Western blot analysis of total yeast cell extracts (data not shown). The normal tubulin and Rep protein patterns (in untreated cells at the G2/M phase) are shown in Fig 6A and Fig C, for reference.
In addition to the nocodazole-induced scattering of the Rep proteins, the pSV1 plasmid clusters were more loosely organized after disassembly of the spindle (Fig 6E and Fig F). The assay was performed by confocal z-series sectioning of the yeast nucleus in a large number of cells (see Materials and Methods), and estimating the mean range of the plasmid occupancy zone. The values were then normalized to the range of DAPI fluorescence in the same cells, also estimated by z-series sectioning. In the control [cir+] cells, the range of the plasmid zone was 0.5 ± 0.03 (Fig 6 E). By contrast, this range was nearly doubled in nocodazole-treated [cir+] cells (1.1 ± 0.03; Fig 6 F) or in untreated, but [ciro], cells (1.0 ± 0.03; Fig 6 G). Estimates of cell and nuclear sizes (from scanning DIC and DAPI images, respectively) showed that the cell enlargement or nuclear expansion as a result of nocodazole treatment was no more than 20% (data not shown). Thus, the lack of an intact microtubule array or the absence of a functional Rep system (as in the [ciro] host) has the common effect of slackening the cohesive forces between plasmid molecules.
Based on the sum of the results shown in Fig 5 and Fig 6, we argue that the Rep proteins likely act as bifunctional coupling agents: complexing with the plasmids on one hand, and effectively cross-linking them directly or indirectly to some component(s) of the spindle apparatus.
Molecular Connection between 2-µm Circle Partitioning and Genes Required for Chromosome Segregation: Missegregation of the 2-µm Plasmid in the ipl1-2 Yeast Mutant
To further verify the suspected coupling between chromosomal and 2-µm plasmid segregation, we have examined the partitioning of the plasmids pSV1 (containing the 2-µm circle replication origin and STB) and pSV3 (ARS-based and lacking STB) in a host strain harboring the Ts- ipl1-2 mutation. The product of the IPL1 gene is essential for proper chromosome segregation (
In the experiments depicted in Fig 7 A, chromosomes were identified by DAPI staining and the pSV1 (2-µm circlederived) and pSV3 (ARSderived) plasmids by GFP-repressor fluorescence. Unlike the normal segregation observed in the [cir+] wild-type host at 37°C (Fig 7 A, column 1), the bulk of the chromosomes, along with pSV1, was stuck within the mother or daughter compartment in most large-budded cells from the [cir+] ipl1-2 host (Fig 7 A, column 2). It is known that the ipl1-2 mutation does not impart a mother/daughter bias in chromosome missegregation (
In Fig 7 B, the normal chromosome and plasmid segregation represented by cell type a was contrasted by four types of missegregation represented by cell types be. In b and e, the DAPI fluorescence was completely excluded from one of the two cell compartments. In c and d, the fluorescence partitioning was strongly (though not absolutely) biased: ~90 to 10 in C and 80 to 20 in D. Whereas chromosome segregation and plasmid partitioning (indicated by the green fluorescent dots) were tightly coupled in cell types bd, they were strongly uncoupled in e. The correlation between pSV1 and chromosome location during missegregation events was nearly perfect in the ipl1-2 [cir+] host strain (Fig 7 B, only 4% of type e cells in row 2). In sharp contrast, the segregation of the ARS-based pSV3 in the same ipl1-2 host was not coupled to chromosome segregation (Fig 7 A, column 3; Fig 7 B, 55% type e cells in row 4). A similar degree of uncoupling was also observed for pSV1 in an isogenic, but [ciro], ipl1-2 host (Fig 7 A, column 4; Fig 7 B, 61% type e cells in row 4).
In an unrelated experiment, we have identified the product of the BRN1 gene (open reading frame YBL097W on chromosome II in the yeast genome data base) as an interactor with Rep1p as well as Rep2p (data not shown). BRN1 is an essential gene, and a temperature-sensitive mutation in it leads to increased rate of chromosome loss under the restrictive condition (
Identical partitioning defects in the chromosome and a 2-µm circle based plasmid due to a mutation in a well characterized yeast gene (IPL1) required for chromosomal segregation provides strong circumstantial evidence for a molecular link between the two segregation mechanisms. Notably, the linkage between chromosome and plasmid segregation (or rather missegregation) in the ipl1-2 background is strictly dependent on the Rep1p/Rep2p/STB system. This evidence is further strengthened by the interaction between each of the Rep proteins and a second host gene product, Brn1p, involved in stable chromosome inheritance.
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Discussion |
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The dual strategy employed by the 2-µm circle plasmid for its stable propagation as a high copy benign parasite genome in yeast utilizes an efficient plasmid partitioning system and a copy number amplification mechanism. Whereas the Flp recombination system is responsible for plasmid amplification, the Rep1pRep2p/STB system is responsible for faithful plasmid partitioning. The amplification system is not triggered into action unless the copy number drops below steady state levels. The observation that there is little amplification of the plasmid under normal growth conditions (
Functional Similarities between 2-µm Circle and Chromosome Partitioning
The present study has revealed the near equivalence between the chromosome and the 2-µm plasmid in the timing and pattern of their movement across the cell during the yeast cell cycle and the similarity in their segregation into mother and daughter cells. This chromosome-like behavior is determined by the Rep/STB system. In a [ciro] host, the dynamics of the 2-µm circlederived test plasmid pSV1 are significantly altered, resulting in a high frequency of missegregation (see Table 2). The Flp recombination system is unlikely to have any direct effect on this process. Note that the pSV1 plasmid is not a substrate for intramolecular recombination, yet is efficiently partitioned in a [cir+] host.
Our finding that the Rep1 and Rep2 proteins colocalize with the pSV1 plasmid inside the yeast nucleus is significant, particularly so in conjunction with the preferred accumulation of the Rep proteins near the poles of the mitotic spindle. We interpret this observation to be suggestive of a role for the Rep/STB system in coupling the plasmid DNA to the spindle apparatus. This association could be direct or indirect. It might be mediated by binding to the kinetochore complex, to chromosomal DNA sequences, or to chromosome binding proteins. Consistent with the idea of spindle attachment, we also noted that nocodazole treatment results in a more dispersed distribution of the Rep proteins within the yeast nucleus, with a simultaneous decrease in the population of clustered plasmid foci. Based upon the distribution of the Rep1 and Rep2 proteins in cells at different stages of the yeast cell cycle,
The possibility that the partitioning system functions by freeing plasmid molecules from subcellular attachment and facilitating free diffusion is unlikely. Early observations using density shift experiments had suggested that there is little or no plasmid amplification under steady state growth conditions (
Molecular Components of Chromosome Segregation Function in Plasmid Partitioning
Two observations made in this study provide the first strong indication for a molecular link between plasmid and chromosomal partitioning. First, in an ipl1 yeast strain (
The Ipl1 protein is a kinase that appears to act in association with the Sli15 protein after the sister chromatids have separated from each other (
The Brn1 protein is homologous to the Drosophila barren gene product (
Chromosome segregation requires a number of coordinated steps that include chromosome condensation, sister chromatid cohesion, bipolar mitotic spindle assembly and elongation, kinetochore attachment to microtubules, sister chromatid separation and their movement to opposite poles (
Plausible Models for Chromosome/Plasmid Cosegregation
Within the general scheme of coordinated segregation of chromosomes and the 2-µm plasmid using a common machinery, two plausible models can be envisaged. In one, the plasmid adheres to one or more of the chromosomes and hitchhikes with them during segregation; in the other, plasmid attachment to the spindle is mediated independent of the chromosomes. Precedence for plasmid transmission by tethering to chromosomes has been established in the case of bovine papilloma virus (
The 2-µm Plasmid: A Paradigm for Near-Perfect Optimization of a Selfish Genome
The circular geometry, structural compactness, and functional parsimony of the 2-µm plasmid appear to represent an optimized evolutionary solution for the high copy maintenance of an extrachromosomal selfish DNA element in yeast. By virtue of harboring a replication origin that is functionally analogous to chromosomal replication origins, the plasmid molecules are able to utilize the host DNA replication machinery for their own replication during the S phase of the cell cycle. However, the normal control of cellular replication prohibits more than one round of replication per plasmid template. To counter the eventuality of a potential fall in copy number due to unequal segregation, the plasmid has evolved the Flp site-specific recombination system as a means for amplification. The Futcher model (
The present study has provided a direct, semiquantitative measure of the segregation fidelity of the 2-µm plasmid, and suggests a mechanism that accommodates the high functional competence of the Rep/STB system within its organizational simplicity. As diagrammed in the model in Fig 8, the association of the Rep proteins with the plasmid via the STB sequence, and with the spindle apparatus or the chromosomes via one or more host factors can coordinate and synchronize plasmid and chromosome segregation spatially and temporally. In conclusion, the success of the 2-µm plasmid as a stably propagating extrachromosomal element appears to be founded on the plasmid's ability not only to directly utilize its host's replication apparatus, but also to indirectly exploit the chromosomal segregation machine.
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Footnotes |
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1 Abbreviations used in this paper: [ciro], strains devoid of endogenous 2-µm circles; [cir+], strains containing 2-µm circles; DAPI, 4', 6'-diamidino-2-phenylindole; GFP, green fluorescent protein.
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Acknowledgements |
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We thank A. Murray and A. Straight for strains and plasmids. We are grateful to B. Goettgens (Core Facility, Institute of Cell and Molecular Biology) for help with confocal microscopy. We received excellent technical assistance from Xu-Li Wu.
This work was supported by a grant to M. Jayaram from the Council for Tobacco Research, United States of America, National Sciences and Engineering Research Council of Canada grant 155268-97 to M.J. Dobson, and National Institutes of Health grant GM45185 to C.S.-M. Chan.
Submitted: 16 November 1999
Revised: 22 March 2000
Accepted: 22 March 2000
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References |
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