1 ____________________________________________________________________________

Selfishness in Moderation: EvolutionarySuccess of the Yeast Plasmid

Soundarapandian Velmurugan, Shwetal Mehta, andMakkuni JayaramSection of Molecular Genetics and Microbiology, University of Texas at Austin,

Austin, Texas 78712

I. Introduction

II. The Origins and Evolution of Selfishness

III. Selfish Nucleic Acids in Prokaryotes and Eukaryotes

IV. The Yeast Plasmids

V. Organization and Regulation of the 2-mm Circle Genome

VI. The Plasmid Partitioning and Amplification Systems

VII. Regulation of Plasmid Gene Expression

A. Mechanisms for Plasmid Partitioning

B. Plasmid Organization, Localization, and Dynamics in the Yeast Nucleus

C. Interactions among the Rep Proteins and the STB DNA in

Plasmid Partitioning

D. Host Factors Required for 2-mm Circle Partitioning

E. A Potential Role for the Yeast Cohesin Complex in Plasmid Partitioning

VIII. The Yeast Cohesin Complex Interacts Specifically with the Rep–STB System

IX. Models for Cohesin-Mediated Plasmid Segregation

X. Summary and Perspectives

XI. Open Questions for Future Research

Acknowledgments

References

The yeast plasmid 2-mmcircle is an extrachromosomal selfishDNAelement

whose genetic endowments are devoted to its stable, high copy propagation.

The mean steady state plasmid copy number of approximately 60 per cell

appears to be evolutionarily optimized at its permissible maximum value. A

plasmid-encoded negative regulatory mechanism prevents a rise in copy

number thatmight imperil normal hostmetabolism and thus indirectly reduce

plasmid fitness. The plasmid utilizes the host replicationmachinery for its own

duplication. A plasmid-encoded partitioning system mediates even distribu-

tion of the replicated molecules to daughter cells, apparently by feeding into

the chromosome segregation pathway. The plasmid also harbors

an amplification system as a potential safeguard against a fall in copy

number due to an occasional missegregation event. The 2-mmcircle provides a

model for howmoderation of selfishness can ensure the successful persistence

of an extrachromosomal element without compromising the fitness of its host.

Current Topics in Developmental Biology, Vol. 56

Copyright 2003, Elsevier Inc. All rights reserved.0070-2153/03 $35.00

1

I. Introduction

The interplay of conflict and cooperation, of selfishness and altruism, has a

strong bearing on the inclusive fitness and overall success of social popula-

tions (Dawkins, 1990; Sundstrom and Boomsma, 2001; Thomas, 2000). In-

sofar as a living cell is made up of societies of interacting individual

molecules, the rules of direct fitness gains through selfishness and indirect,

collective fitness gains through altruism are generally applicable at the

molecular level.

In this review, we shall first outline general considerations regarding the

evolution, spread, and establishment of selfish nucleic acid elements in

prokaryotes and eukaryotes. They may exist as autonomously replicating

extrachromosomal entities (plasmids, for example) or as chromosomally in-

tegrated moieties (lysogenic phage, bacterial insertion sequences, and repeat

families in eukaryotes). Several of these elements, say, plasmids or transpo-

sons harboring antibiotic resistance, provide their hosts with some advan-

tage, at least under certain environmental challenges. Yet others rely on

the hosts for their survival, but apparently oVer nothing in return. Such

elements are truly selfish.

The evolutionary success of a selfish genome depends on how well it can

adjust its degree of selfishness to the biochemical resources available to it.

Unfettered avarice could be self-destructive by the rapid depletion of these

resources. The long-term persistence of an element would be better served

by regulating its metabolic needs so as to not jeopardize the well-being of

its host. In this context, we shall focus on the 2-mm plasmid of yeast to

illustrate some of the strategies that shape the character of a model selfish

DNA element.

II. The Origins and Evolution of Selfishness

The primordial RNA world likely provided the breeding ground for

the earliest selfish genetic elements in the form self-replicating oligoribonu-

cleotide entities (Doudna and Cech, 2002; Joyce, 2002). The naturally occur-

ring ribozymes in present day life forms that assist in RNA processing,

facilitate the replication of certain viral genomes, or catalyze peptide bond

formation are perhaps the evolutionary vestiges from this lost world. On

the other hand, in vitro evolved ribozymes that can synthesize nucleotides

and coenzymes or form amide bonds are likely proxies for some of the

missing links in the transition from RNA-based to protein-based bio-

catalysis. Strikingly, in vitro evolution can recapitulate the fortuitous

2 Velmurugan et al.

emergence of selfish RNA molecules called RNA Z (Breaker and Joyce,

1994). By the appropriation of a promoter sequence, purely by accident, they

become preferred substrates for amplification under the selection pressures

imposed by the experiment.

III. Selfish Nucleic Acids in Prokaryotes and Eukaryotes

DNA/RNA elements that fit the description of ‘‘selfish genomes’’ are wide-

spread among prokaryotes and eukaryotes. They may, however, diVer

considerably in the guile and sophistication of their selfishness.

Classically, bacterial plasmids have provided the paradigm for successful

extrachromosomal elements (Thomas, 2000). They utilize a fairly limited set

of biochemical strategies for replication yet incorporate mechanisms for con-

trolling copy number and coordinating replication with cell growth. The

problem of propagating the replicated molecules to daughter cells is dealt

with in one of two ways. Provided the steady state copy number of the plas-

mid is reasonably high (and there are no constraints to free diVusion), the

chances of a plasmid-free cell arising at any given generation would be quite

low. However, as a rule of thumb, a low copy number would be favored by

reducing the metabolic burden on the host. The potential disadvantage of

plasmid loss by missegregation is circumvented by the acquisition of an

active partitioning system. Finally, the horizontal spread and establishment

of plasmids among bacteria may be promoted or accelerated by the

evolution of functions for mobilization and conjugative transfer.

Similar molecular themes or their variations are revealed by eukaryotic

selfish genetic elements as well. The satellite RNA/DNA molecules associ-

ated with adenovirus or the tobacco ringspot virus have little or no coding

capacity but have established themselves stably by virtue of their active rep-

lication potential (Kado, 1998). The large Ti plasmids of Agrobacterium

(that transpose a segment of their DNA to the plant genome) have not only

manipulated the host bacterial cells for their survival but can induce higher

plant cells to produce biochemical environments conducive for their host

and hostile for those of competitors. Several mammalian viruses that exist

primarily as nonintegrated episomes (papilloma and Epstein-Barr, for

example) ensure their stable propagation by attaching themselves to the host

chromosomes using protein tethers (Ilves et al., 1999; Lehman and Botchan,

1998). The ensemble of repeated DNA families found in eukaryotic genomes

represent, in one sense, the limit case of chromosomal attachment, namely,

covalent integration. However, the load of such integrated elements is tightly

controlled, as exemplified by the limits to the integration of multiple forms

of retrotransposons in mammalian cells.

1. Selfishness in Moderation 3

Figure 1 Organization of the 2-mm plasmid and recombination-mediated copy number

control. (a) In the standard dumbbell representation of the double-stranded circular plasmid,

the parallel lines (the handle of the dumbbell) indicate the inverted repeats (IRs) of the plasmid.

The open reading frames are highlighted, with the arrowheads pointing in the direction of their

transcription. The cis-acting DNA elements in the plasmid are the replication origin (ORI ), the

partitioning locus (STB), and the Flp recombination target sites (FRT ). The STB element can

be subdivided into two regions: ‘‘proximal’’ and ‘‘distal’’ with respect to ORI. STB-proximal

contains the tandem array of 5–6 copies of a 62-bp consensus sequence and is central to plasmid

partitioning. STB-distal is important in maintaining the ‘‘active configuration’’ of STB-

proximal, which is subject to context eVects. Forms A and B of the plasmid are generated by

Flp-mediated recombination at the FRT sites. (b) A recombination-mediated amplification

4 Velmurugan et al.

IV. The Yeast Plasmids

Circular, double-stranded DNA plasmids found in Saccharomyces cerevisiae

and other yeasts have been viewed as evolutionarily optimized ‘‘benign’’

parasitic elements (Broach and Volkert 1991). They do not provide any

obvious benefit to their hosts. Yet, they persist in cell populations at rela-

tively high copy numbers and exhibit almost chromosome-like stability.

They exploit the genetic endowments of the host for their replication

and segregation but adjust their degree of selfishness to avoid overtaxing

the host’s metabolic resources. For example, sophisticated regulatory mech-

anisms guard against large upward or downward deviations from the opti-

mized ‘‘high’’ steady state plasmid copy number. The 2-mm circle, the most

well-characterized member of this class of plasmids, is representative of

the genetic organization and functional strategies that underlie the success

of the yeast plasmid family as selfish DNA elements.

V. Organization and Regulation of the 2-mm Circle Genome

The 2-mm circle is almost ubiquitously present in strains of Saccharomyces

yeasts at an average copy number of 40–60 per cell. The high copy propaga-

tion of the plasmid is achieved through an eYcient partitioning system and

a copy number amplification system. The structural and functional design

of the entire plasmid genome appears to be devoted solely to this end.

In the standard dumbbell representation of the plasmid (Fig. 1a), the par-

allel lines represent two copies of a 599-bp sequence in inverted orientation.

The plasmid codes for a site-specific recombinase Flp; two partitioning pro-

teins, (Rep1p and Rep2p) and a presumed positive regulator for Flp expres-

sion (Raf1p) The cis-acting elements important in the physiology of the

plasmid are the replication origin (ORI ), the targets for site-specific recom-

bination (FRT = Flp recombination target) within the inverted repeats, and

mechanism proposed by Futcher (1986). Bidirectional replication starting at the origin in a

plasmid molecule (a) duplicates the proximal FRT site before the distal one (b). An Flp-

mediated inversion (c) results in two replication forks oriented in the same direction (d).

Movement of the two forks around the circular template amplifies copy number (e). A second

recombination event (f ) restores bidirectional fork movement (g). The products of replication

are a template copy (i) and an amplified moiety containing multiple tandem copies of the

plasmid (h). The tandem multimer can be resolved by Flp recombination (or even homologous

recombination) into plasmid monomers ( j, k). The diagram of the Futcher model shown here

follows its representation by Broach and Volkert (1991). (c) Flp-mediated recombination

between the repeats numbered 1 of a replicating 2-mm circle and a nonreplicating one (left)

results in a replicating plasmid dimer (middle). A second intramolecular recombination between

the repeats numbered 2 produces plasmid monomers joined by the replication forks (right).

1. Selfishness in Moderation 5

the stability-conferring partitioning locus (STB). Yeast cells harbor an

equilibrium mixture of the plasmid forms A and B, formed as a result of

Flp–FRT recombination and present in roughly equal abundance. A care-

fully timed site-specific recombination reaction carried out during plasmid

replication is believed to be the critical event in copy number amplification.

VI. The Plasmid Partitioning and Amplification Systems

The natural stability of the 2-mm circle approaches that of the yeast chromo-

somes (a loss rate of approximately 10ÿ5 to 10ÿ4 per generation) and is

mediated by the partitioning system consisting of the two Rep proteins

and the STB locus. Contrary to what their names might suggest, the Rep

proteins have nothing to do with plasmid replication, which is carried out

by the host replication machinery. Each of the 60 or so molecules duplicates

only once during each cell cycle (Zakian et al., 1979), and the Rep–STB

system ensures that the replicated molecules are partitioned nearly

equally to the daughter cells (Velmurugan et al., 2000). The details of the

partitioning mechanism are only beginning to emerge.

The amplification system is brought into action only when there is a drop

in copy number due to an accidental missegregation event. The Futcher

model for amplification (Fig. 1b; Futcher, 1986) is predicated on the asym-

metric location of the replication origin with respect to the FRT sites. This

unequal spacing is retained in all the 2-mm–like plasmids, suggesting the

conservation of a common recombination-based amplification mechanism

during plasmid evolution. Normally, replication of the 2-mm plasmid pro-

ceeds bidirectionally from the origin and terminates at the opposite end by

convergence of the forks. In the amplification mode (induced by low plasmid

levels), a recombination event between a copy of the duplicated ORI-prox-

imal FRT site and the unreplicated distal one causes the inversion of one

of the two forks. They now chase each other on the circular template to spin

out multiple tandem copies of the plasmid. A second recombination event

and restoration of the bidirectional forks may terminate amplification. The

tandem array of 2-mm plasmids in the amplicon can be resolved into unit size

molecules by Flp-mediated resolution or via homologous recombination.

Consistent with the prediction of the Futcher model, plasmid amplifica-

tion cannot occur when Flp recombination is abolished (Reynolds et al.,

1987; Volkert and Broach, 1986; Volkert et al., 1986). However, there is no

direct proof that amplification proceeds through the intermediates dia-

grammed in Fig. 1b. In fact, potential amplifying moieties called pince-nez

(PN) structures that are made up of linked double rolling circles (Fig. 1c)

have been identified by electron microscopy (Petes and Williamson, 1994).

They can be produced by intermolecular recombination between a

6 Velmurugan et al.

replicating plasmid and a nonreplicating one, followed by resolution of the

plasmid into monomers. Each circle of the PN has a constant size corres-

ponding to that of a plasmid monomer (2-mm), whereas the size of the tether

linking them is variable.

VII. Regulation of Plasmid Gene Expression

Early genetic experiments, together with rather sparse biochemical data,

have led to a model in which the Rep1 and Rep2 proteins form a bipartite

regulator that negatively controls the expression of the FLP, REP1, and

RAF1 loci (Fig. 2; Murray et al., 1987; Som et al., 1988). The REP2 gene

is apparently not subject to this negative regulation; therefore the Rep2 pro-

tein is not limiting for the assembly of the Rep1p–Rep2p repressor. At the

steady state copy number, the level of Rep1p is high enough to establish

the critical concentration of the repressor required to turn oV FLP and

RAF1 and thus keep the amplification system under check. A drop in copy

number causes the repressor to fall below threshold, thus turning on FLP

and RAF1. The Raf1p is believed to antagonize the repressor, thereby accel-

erating the amplification response. As copy number builds up, Rep1p and

the repressor levels are boosted, and eventually the steady state is restored.

Overall, the regulatory circuit is designed to rapidly commission and

Figure 2 Positive and negative controls of gene expression in the 2-mm plasmid. The schematic

diagram depicting 2-mm circle gene regulation is adapted from Som et al. (1988). The putative

bipartite regulator Rep1p–Rep2p (R1–R2) negatively controls expression of the FLP (Flp),

RAF1 (D) and REP1 (R1). As a result, the level of the R1–R2 repressor is controlled as a

function of the copy number, and at steady state the amplification system is essentially turned

oV. The product of the RAF1 gene (D) antagonizes R1–R2, permitting rapid triggering of

recombination-mediated amplification when plasmid copy number needs a boost. The REP2

locus appears to be free from repression by R1–R2. Aside from their role in controlling plasmid

gene expression, the Rep1 and Rep2 proteins interact with the STB DNA to bring about equal

segregation of the plasmid molecules at cell division.

1. Selfishness in Moderation 7

decommission the amplification machinery as demanded by the copy

number status. The copy number, in turn, is indirectly read out as a function

of Rep1p or the Rep1p–Rep2p repressor.

The Rep proteins, in concert with the STB locus, are also required for

plasmid partitioning. It is not clear whether the Rep1p–Rep2p repressor

is also the active entity in partitioning, or whether the repressor and

partitioning functions can be uncoupled from each other.

A. Mechanisms for Plasmid Partitioning

Why does a plasmid that has a copy number of 60 utilize a partitioning

system? Why not rely on random segregation, as is the norm with high copy

bacterial plasmids? In principle, the amplification system can readily make

the adjustments to rectify copy number deficits resulting from this segrega-

tion mode. Autonomously replicating yeast plasmids that lack the Rep–

STB system (ARS plasmids) tend to show a finite segregation bias toward

the mother during cell division (Murray and Szostak, 1983). The 2-mm circle

partitioning system might overcome this bias in one of two ways. It could

either (1) promote random segregation by causing the plasmids to be freely

diVusible or (2) mediate active segregation, say, by attaching plasmids to a

nuclear moiety that is equally partitioned between mother and daughter.

One argument against random segregation is the lack of evidence for the

continuous operation of the amplification system during cell growth. In

density shift experiments, during one generation, essentially all of the plas-

mid fraction sediments at the intermediate density, suggesting a single round

of replication for each molecule (Zakian et al., 1979). Low amplification

levels (that would be consistent with the high plasmid copy number) could

have been easily missed in this assay. However, as described in the following

section, more recent evidence favors the active partitioning model.

B. Plasmid Organization, Localization, and Dynamics in theYeast Nucleus

Fluorescence tagging of STB-containing plasmids via GFP–LacI/LacO

interaction in live cells or by immunostaining in fixed cells indicates that they

are localized in the nucleus as a compact cluster in association with the Rep1

and Rep2 proteins (rows 1 and 2 in Fig. 3a; Velmurugan et al., 2000). An

ARS plasmid (lacking STB) is not confined to the Rep protein zone (row 3

of Fig. 3a). The Rep proteins, and therefore the 2-mm plasmid, are almost

always seen at or close to the spindle poles (Fig. 3b). The compactness of

the plasmid cluster, measured as the width of the plasmid residence zone

8 Velmurugan et al.

Figure 3 Organization and localization of the 2-mm plasmid in the yeast nucleus. (a) Plasmids

are visualized by immunostaining using antibodies to the Lac repressor bound at the Lac

operator repeats harbored by them. Rep1 and Rep2 proteins are visualized by using antibodies

to the native proteins. The STB plasmid is contained within the Rep1p/Rep2p staining region

within the larger DAPI staining area (top two rows). The ARS plasmid dots are often seen lying

outside of the Rep1p/Rep2p zone (bottom row). (b) The localization of the Rep1 protein with

respect to the mitotic spindle is shown in yeast cells at diVerent stages of the cell cycle. Nearly all

of the Rep1 protein (with the associated 2-mm plasmid) is concentrated at or close to the spindle

poles. The same pattern is obtained with Rep2 protein as well. The spindle is displayed using

antibodies to tubulin. (c) An STB-containing plasmid appears as a compact cluster when

examined by Z-series sectioning using confocal fluorescence microscopy in a [cirþ] host strain

providing the Rep1 and Rep2 proteins from the native 2-mm plasmid (top row). In a [cir0] strain,

lacking the Rep proteins, the plasmid cluster is less cohesive (middle row). A similar loosening

of cohesion is observed when the cells are treated with the microtubule depolymerizing drug

nocodazole (bottom row). The plasmid residence zone (PRZ) for each cell examined is

expressed as the ratio of the widths of the plasmid and DAPI fluorescence patches. The values

are averaged from 20 cells for each experimental group.

1. Selfishness in Moderation 9

(PRZ in Fig. 3c) in the nucleus, is considerably loosened when the Rep

system is inactive (in a host strain without native 2-mm circles, [cir0], and

therefore lacking the Rep1 and Rep2 proteins; row 2 of Fig. 3c) or when

the spindle is depolymerized with nocodazole (row 3 of Fig. 3c). The values

at the right in Fig. 3c are normalized by dividing the number of sections

containing green fluorescence (size of the plasmid cluster) into the number

of sections containing blue DAPI fluorescence (size of the nucleus). Time-

lapse microscopy of cells released from �-factor–induced G1-arrest reveals

a rough doubling of the plasmid fluorescence (corresponding to plasmid rep-

lication) during bud growth, followed by separation of the cluster into two in

large-budded cells and the rapid migration of each cluster toward opposite

poles (Fig. 4a). These observations suggest that it is a high-order plasmid–

protein complex that is the partitioning entity. As such, the copy number

Figure 4 (a) Segregation kinetics of the 2-mm plasmid in a wild type host; plasmid association

with chromosome spreads. The fluorescence-tagged 2-mm circle reporter plasmid is followed

from the point of bud emergence (time zero) through one full division cycle. The plasmid

fluorescence is doubled in the 6- to 18-minute period (early S phase) and plasmid partitioning

occurs in the 42- to 48-minute interval (G2/M). The observed timing of segregation is quite

similar to that of a fluorescence-tagged chromosome. (b) In yeast chromosome spreads, the

Rep1 protein and the STB containing reporter plasmid (immunostained with antibodies to

bound Lac repressor) are colocalized within the DAPI staining area.

10 Velmurugan et al.

Figure

5Segregationpatternsofthe2-mm

plasm

idsegregationin

mutanthoststhatmissegregate

chromosomes

atthenonpermissivetemperature.In

themutanthosts,

twotypes

ofplasm

idsegregationpatternsare

seen

inrelationto

chromosomemissegregation.In

type‘‘a’’cells,

theplasm

id

missegregatesin

tandem

with

thebulk

ofthechromosomes.In

type‘‘b’’

cells,

theplasm

idsegregation

islargelyindependentofthatofthe

chromosomes.Thepercentageofcellsthatshowthe‘‘a’’orthe‘‘b’’phenotypes

fortw

oreporter

plasm

ids,oneharboringSTBandtheother

lackingit,

islisted

forthesixmutantstrainstested.Allstrainsare

[cirþ]andhence

providetheRep

proteinsin

trans.

Theresultsshownhererepresentthe

segregationprofile

atthenonpermissivetemperature.Atthepermissivetemperature,chromosomesegregationisnorm

al.

1. Selfishness in Moderation 11

of the plasmid is eVectively unity, and the need for an eYcient partitioning

mechanism is justified.

An STB plasmid can be detected in chromosome spreads prepared from

yeast cells, but only if both Rep1 and Rep2 proteins are expressed in them

(Fig. 4b; Mehta et al., 2002). Either Rep1p alone or Rep2p alone is not

retained in the spreads. Together, the two proteins associate with the

spreads even in the absence of an STB plasmid. Thus, it is the Rep proteins,

acting in concert, that are responsible for the specific localization of the

plasmid. Because of limited resolution, the chromosome spread assays

cannot distinguish direct plasmid–chromosome association from independ-

ent localization of the plasmid and certain chromosomal domains common

to nuclear sites. In synchronously dividing yeast cells, there is a striking

similarity in the segregation kinetics of a fluorescence-tagged plasmid with

a similarly tagged chromosome (Velmurugan et al., 2000). Furthermore, in

several mutant yeast strains that missegregate chromosomes at the nonper-

missive temperature, an STB plasmid also missegregates, and most often

does so in tandem with the bulk of the chromosomes. This missegregation

pattern is represented by the type ‘‘a’’ cells in Fig. 5. When STB is removed

from the plasmid or when either Rep protein is absent, the comissegregation

of the plasmid with the chromosomes is no longer observed. This behavior is

denoted by the large increase in the type ‘‘b’’ cells (compare the ARS plasmid

with the STB plasmid in each mutant host strain) in Fig. 5. Taken together,

the data are consistent with the plasmid and chromosome segregation

pathways being interlinked or coordinately regulated. Perhaps the plasmid

is tethered to the chromosomes. Or, it may exploit components of the host

mitotic machinery for its own equal segregation. Or, the partitioning system

may provide a checkpoint to prevent plasmid entry into a cell that lacks a

full chromosome complement.

C. Interactions among the Rep Proteins and the STB DNA inPlasmid Partitioning

The long-held notion that the interactions of the Rep proteins with STB are

important for plasmid stability has received experimental support only re-

cently. The colocalization of the Rep proteins in the yeast nucleus with plas-

mids harboring the STB DNA, as well as their presence in chromosome

spreads in a strictly partner-dependent manner, is consistent with this idea.

By in vivo dihybrid assays and in vitro aYnity trapping assays, self- and

cross-interactions of the Rep proteins have now been demonstrated (Ahn

et al., 1997; Scott-Drew and Murray, 1998; Sengupta et al., 2001; Velmuru-

gan et al., 1998). Evidence for STB binding by the Rep proteins has been

more diYcult to establish. Partially pure Rep proteins fail to associate with

12 Velmurugan et al.

STB when probed by standard gel mobility shift methods (Y. T. Ahn and

M. Jayaram, unpublished data). Hadfield et al. (1995) showed that urea-

solubilized yeast cell extracts containing Rep1p and Rep2p can bind STB,

suggesting the potential involvement of host protein(s) in binding. By using

a southwestern assay, Sengupta et al. (2001) have shown that the carboxyl–

terminal portion of Rep2p has DNA binding activity. A detailed mutational

analysis of the Rep1 protein (X. M. Yang and M. Jayaram, unpublished

data) supports the functional relevance of Rep1p–Rep2p interaction and

Rep1p–STB interaction in plasmid partitioning. Rep1p variants containing

point mutations that disrupt either of the two interactions (or both) are not

able to support normal plasmid maintenance.

D. Host Factors Required for 2-mm Circle Partitioning

The prospect that a simple tripartite system, consisting of two proteins and a

relatively short stretch of DNA, can confer chromosome-like stability on the

2-mm plasmid would seem highly unlikely. A search by yeast dihybrid and

monohybrid assays for host proteins that interact with the Rep1p/Rep2p

or the STB locus has revealed several candidates, among which at least three

are particularly interesting: the products of BRN1, FUN30, and CST6/SHF1

(Velmurugan et al., 1998; X. M. Yang and M. Jayaram, unpublished data).

Whereas BRN1 is an essential gene, both FUN30 and SHF1 are not. The

Brn1 protein is a component of the yeast condensin complex, which plays

a central role in proper chromosome segregation (Lavoie et al., 2000;

Ouspenski et al., 2000). Brn1p appears to interact with Rep1p independent

of Rep2p, and vice versa. Whether these interactions mirror the requirement

of the condensin complex in 2-mm circle partitioning needs to be verified.

Fun30p interacts with Rep1p directly but interacts only indirectly with

Rep2p, presumably through Rep1p. Both Brn1p and Fun30p can associate

with STB in a Rep-protein–dependent manner. The Shf1 protein binds to

STB directly, as suggested by in vivo monohybrid results and by in vitro

mobility retardation assays (Velmurugan et al., 1998). In the Shf1� back-

ground, there is a modest drop in the stability of a 2-mm circle-derived test

plasmid. Interestingly, independent genetic assays have implicated FUN30

as well as SHF1 (CST6) as being important for chromosome partitioning

(Ouspenski et al., 1999).

The Fun30 protein contains peptide motifs characteristic of the SNF2

family of transcriptional regulators with potential chromatin remodeling

activity (SGD database). The Shf1 protein appears to belong to the ATF/

bZIP class of transcription factors and harbors a consensus CREB

motif (Velmurugan et al., 1998). It is likely that chromatin organization at

the STB locus and/or its transcriptional status may aVect its eYciency in

1. Selfishness in Moderation 13

plasmid partitioning. Consistent with this notion, a recent study demon-

strates the requirement of the Rsc2 protein, which forms part of a chromatin

remodeling complex in yeast, for the normal stability of the 2-mm circle

(Wong et al., 2002). The nucleosome pattern at STB is altered, and the

association between Rep1p and STB is aVected by the absence of Rsc2p. It

is noteworthy that the region of STB proximal to the 2-mm circle origin,

consisting of approximately six copies of a 62-bp repeat unit, is kept free

of transcription by a termination signal located in the ‘‘distal’’ STB segment

(Sutton and Broach, 1985). Also, a 24-bp silencer element, capable of

suppressing the activity of a nearby promoter in an orientation-independent

manner, has been identified within the distal STB (Murray and Cesareni,

1986). Rather surprisingly, the 2-mm origin itself has been shown to function

as a silencer whose activity is dependent on the Sir proteins, the origin

recognition complex (ORC), and the Hst3 protein, a Sir2 histone acetylase

homolog (Grunweller and Ehrenhofer-Murray, 2002).

It is known that transcription through eukaryotic replication origins

and centromeres, as well as the partitioning loci of certain bacterial plas-

mids, can adversely aVect their function (Rodionov et al., 1999). It is likely

that the STB locus is also under a similar constraint, and its native location

appears to have been selected to place the repeat units in a transcription-free

zone. From earlier work we know that the stability of yeast plasmids

(lacking the Rep–STB system) can be enhanced by the presence in cis

of yeast telomere–associated sequences or the silencing element E associ-

ated with the unexpressed yeast mating-type locus HMRa (Ansari and

Gartenberg, 1997; Kimmerly and Rine, 1987; Longtine et al., 1992; Longtine

et al., 1993). The partitioning activity of the subtelomeric repeats is

absolutely dependent on the Rap1 protein, whereas that by the E element

is mediated through the Sir1-4 proteins. The underlying common theme in

both types of plasmid stabilization appears to be the organization of a silent

chromatin domain. It has been suggested that the silencing complex anchors

plasmids to a nuclear component that is symmetrically divided between

daughter cells.

The old and new results can be accommodated by a model in which the

plasmid replication is spatially restricted to a nuclear locale that facilitates

the subsequent partitioning event.

E. A Potential Role for the Yeast Cohesin Complex in Plasmid Partitioning

The plausible connection between chromosome segregation and 2-mm circle

segregation is strengthened cumulatively by several pieces of circumstantial

evidence summarized in the earlier sections. They include (1) cell biological

observations of plasmid dynamics during segregation, (2) similar eVects of

14 Velmurugan et al.

host mutations on chromosome and plasmid partitioning, and (3) the inter-

action between host proteins required for chromosome segregation and the

plasmid partitioning system. Because of the interaction between Brn1p and

the Rep proteins, our immediate attention was centered on the yeast conden-

sin complex. We found, in our collection of Rep1p mutants, one that cannot

interact with Brn1p and fails to support plasmid partitioning (X. M. Yang

and M. Jayaram, unpublished data). The same mutant shows normal inter-

action with Rep2p and STB in dihybrid andmonohybrid assays, respectively.

However, the analysis of condensin in plasmid partitioning is impeded by its

nonspecific association with DNA. The yeast cohesin complex then became

the object of our interest because of its relatedness to condensin in a subset

of its subunits and because of the cooperative roles that these complexes play

during the segregation of sister chromatids during mitosis. In addition, the

binding of cohesin to chromosomal locales is highly discriminatory (Laloraya

et al., 2000). As indicated by the results summarized in the following section,

the shift in experimental strategy has paid oV.

VIII. The Yeast Cohesin Complex Interacts Specifically withthe Rep–STB System

As noted earlier, the duplication of the 2-mm plasmid cluster followed by the

segregation of the two clusters into daughter cells is reminiscent of the dupli-

cation and segregation of sister chromatids. Cohesin plays a central role in

chromosome segregation by establishing sister chromatid pairing during

the S phase and maintaining it until chromosomes are ready to be separated

during anaphase (Carson and Christman, 2001; Cohen-Fix, 2001; Nasmyth,

2001; Nasmyth et al., 2000; Skibbens et al., 1999; Toth et al., 1999; Uhlmann

and Nasmyth, 1998; Uhlmann et al., 1999; Uhlmann et al., 2000; Wang et al.,

2000). Cleavage of the integral cohesin component Mcd1p/Scc1p by the

Esp1 protease dissolves the cohesin bridge, and the sisters, bipolarly at-

tached to the spindle, are rapidly pulled apart. A segregation mechanism

based on cohesin-mediated pairing and unpairing of plasmid clusters would

be expected to mimic chromosome segregation in its timing, as has been ob-

served (Velmurugan et al., 2000).

In chromatin immunoprecipitation assays, the Mcd1 protein associates

specifically with the STB DNA in a Rep1p- and Rep2p-dependent manner

(Mehta et al., 2002). Other regions of the plasmid, including the replica-

tion origin, are not occupied by Mcd1p. A similar association of STB is

observed with other cohesin components as well, e.g., Smc1p and Smc3p.

Furthermore, inactivation of Smc1p or Smc3p by Ts mutations disrupts

Mcd1p–STB association at the nonpermissive temperature. These results

are consistent with the preassembled cohesin complex being recruited

1. Selfishness in Moderation 15

Figure 6 Association of the cohesin complex with the 2-mm plasmid assayed by chromatin

immunoprecipitation; noncleavable Mcd1p in cohesin blocks the separation of duplicated

plasmid clusters. (a) Cells arrested in G1 by � factor are released from pheromone arrest at time

zero and followed by chromatin immunoprecipitation (using antibodies to the cohesin

component Mcd1p), light microscopy (DIC), and FACS analysis. During each cell cycle,

association of cohesin with the STB element occurs early in S phase and lasts until late G2/M.

Note the nearly perfect synchrony between the chromosomes (as indicated by the presence of a

cohesin binding site on chromosome V in the immunoprecipitate) and the plasmid in cohesin

association and dissociation. ‘‘WCE’’ refers to whole cell extract. (b) Small budded cells

16 Velmurugan et al.

by the plasmid partitioning system. The timing and periodicity of

cohesin recruitment to the plasmid during the yeast cell cycle match

nearly perfectly those of cohesin recruitment to the chromosome (Fig. 6a).

Moreover, when cohesin disassembly during anaphase is blocked, the

duplicated plasmid clusters mimic sister chromatids in failing to separate

(Fig. 6b).

It should be emphasized that the mechanism of cohesin association with

STB is clearly distinct from that of cohesin binding to chromosomal loci.

There is no apparent sequence similarity between STB and cohesin binding

sites on the chromosomes, and obviously the Rep proteins are not required

for the chromosomal recruitment of cohesin. Yet the timing of cohesin asso-

ciation and dissociation are well synchronized between the chromosomes

and the 2-mm plasmid. Thus, the Rep–STB system appears to be clever mo-

lecular trickery evolved by the plasmid to feed into the temporal program

that its host has established for the cycle of cohesin association–dissociation

on chromosomes.

IX. Models for Cohesin-Mediated Plasmid Segregation

Assuming that the yeast cohesin complex plays fundamentally similar roles

in the partitioning of yeast chromosomes and the 2-mm plasmid, one or more

segregation models can be considered. It is possible that cohesin facilitates

pairing between the two duplicated plasmid clusters that, in turn, are

tethered to a pair of sister chromatids. The coincident dissolution of the co-

hesin bridge between the sister chromatids and the plasmid clusters would

dispatch each cluster in opposite directions in association with the chromo-

somes. The plasmid-chromosome attachment could be mediated by cohesin

itself or through other factors. If cohesin is the tethering agent, there must be

some mechanism to postpone Mcd1p cleavage within this tether until after

segregation has been completed. Another possibility is that the two postre-

plication plasmid clusters are bridged by the cohesin complex but are not

tethered to chromosomes. Upon disassembly of cohesin, each unpaired

plasmid cluster moves to opposite cell poles without assistance from the

harboring a copy of the native MCD1 gene and one of the noncleavable versions under GAL

promoter are transferred from dextrose to galactose at time zero. They are followed for 150

minutes by time lapse fluorescence microscopy to monitor a tagged chromosome (top two

rows), an STB reporter plasmid (central two rows), or an ARS plasmid (bottom two rows). Of

the 10 cells examined in each case (and arrested at the large budded state), the fractions

exhibiting one chromosomal dot vs two dots and one plasmid cluster vs two clusters are

indicated.

1. Selfishness in Moderation 17

chromosomes. This movement may be mediated by spindle attachment

(a spindle-associated motor protein could be involved), by an active trans-

port system unrelated to the spindle, or by association with a subcellular

entity that is evenly partitioned at cell division.

X. Summary and Perspectives

From the various lines of evidence presented here, the 2-mm plasmid emerges

as a minimalist yet carefully optimized structural design for a selfish nucleic

acid. By harboring a replication origin that is functionally equivalent to the

chromosomal origins, the plasmid enjoys duplication by the host replication

machinery. By pilfering host factors using components of its stability

system, the plasmid apparently gains access to a sophisticated partitioning

mechanism, and by preserving a recombination-mediated amplification

system in readiness, the plasmid ensures that its copy number is maintained

at the steady state value. The apparent conservation of the basic 2-mm circle

paradigm by the other yeast plasmids speaks to its eVectiveness and evolu-

tionary durability as a strategy for benign parasitism through moderation

of selfishness.

Why does yeast still maintain a high copy extrachromosomal element that

apparently makes no contribution to its fitness? The built-in sophistication

of the strategies for plasmid maintenance suggests that the plasmid at one

time might have conferred a significant selective advantage on its host, and

paradoxically, this very sophistication may make it diYcult and slow for

yeast to get rid of the plasmid now.

From one evolutionary perspective, the progenitor of the present day

2-mm family of yeast plasmids might have been an infectious agent (similar

to episomal viral genomes) that established itself in an ancestral host by its

ability to attach to the mitotic spindle or to the chromosomes. An early par-

titioning system, from which the Rep–STB system evolved, could have

assisted plasmid propagation during cell division. Maintenance of the elem-

ent would have been determined by the balance between occasional loss and

reinfection. Later acquisition of Flp, perhaps by an integrative transposition

event, accompanied by the loss of infective coding capacity, might have

quarantined the plasmid in the lineage leading to the yeast strains that cur-

rently possess the 2-mm circle and its relatives. A viable alternative view is

that the high copy plasmid segregated by a random mechanism during its

early evolutionary history, relying on the amplification system for copy

number adjustments. The Rep–STB system may have originated more re-

cently in response to a reduction in the eVective copy number as a result of

plasmid clustering.

18 Velmurugan et al.

XI. Open Questions for Future Research

The regulatory scheme outlined in Fig. 2 no doubt neatly fits into the normal

physiology of the 2-mm plasmid, namely, equal segregation at steady state

with provisions for upregulation in copy number, if required. Nevertheless,

the model has its limitations. Although the FLP promoter can be repressed

by overexpression of Rep1p and Rep2p from an inducible promoter, the

magnitude of the eVect under physiological levels of the Rep proteins is

almost imperceptible. The possibility that high Rep protein levels might ti-

trate out host proteins, including transcription factors, to manifest second-

ary eVects on FLP expression cannot be ruled out. Little attention has

been paid thus far to potential contributions by the host to the copy number

regulation of a DNA element that it stably shelters. For example, are there

cell cycle controls on the expression and/or steady state levels of the 2-mm

circle proteins? Are the target DNA sites for these proteins diVerentially oc-

cupied as a function of the cell cycle? Answers to these and related questions

would be central to understanding how the stability and amplification

systems communicate with each other to establish homeostasis in plasmid

partitioning and copy number maintenance.

The conceptual simplicity and mechanistic parsimony of the Futcher

model for plasmid amplification notwithstanding, the proof for its operation

is incomplete. There is no doubt that plasmid amplification cannot occur

when Flp recombination is abolished (Volkert and Broach, 1986). Yet, there

is no direct evidence that amplification proceeds through the intermediates

diagrammed in Fig. 1b. As pointed out earlier, potential amplifying moieties

called pince-nez (PN) structures (Petes and Williamson, 1994; Fig. 1c) raise

the specter of intermolecular amplification that falls outside the purview of

the Futcher model.

Although the need to amplify plasmid in the event of missegregation

has been given considerable thought, the other side of the issue has been

virtually ignored. What happens to a cell that receives more than its fair

share of plasmids? Is plasmid replication in such a cell dampened to readjust

the copy number to the steady state value? We do not know the answer, and

experimental designs to address this issue have been lacking. What we do

know is that very high plasmid copy numbers, imposed by inducing FLP

expression from a strong promoter, are not relished by host cells (Murray

et al., 1987; Reynolds et al., 1987). Such cells divide very slowly or not

at all. In a growing population, cells with high plasmid load (if they do arise)

will be quickly overtaken by those with normal plasmid density. A chromo-

somal mutation, nib1, causes clonal lethality in yeast in a 2-mm-circle–

dependent manner, giving rise to colonies with a nibbled morphology

(Holm, 1982a; Holm, 1982b). It is possible that the cells destined to die are

1. Selfishness in Moderation 19

the ones in which the plasmid load has crossed a critical value either by

amplification or as a result of missegregation. Because of the selective

disadvantage of the nib1 [cirþ] genotype, plasmid-free cells arising in a

mutant population have a strong growth advantage and tend to establish

their lineage rather rapidly.

While cohesin-mediated partitioning is attractive in explaining the

chromosome-like stability of the 2-mm plasmid, evidence in support of it is

almost all circumstantial. Whereas the recruitment of cohesin by the

Rep–STB system has received strong experimental support, the same cannot

be said for the bridging of plasmid clusters via cohesin. The only evidence

that favors such bridging is negative, namely, the failure of plasmid clusters

to separate when the cohesin complex is assembled using the noncleavable

version of Mcd1p. Because cohesin is essential for chromosome segrega-

tion, one major technical stumbling block is to selectively aVect plasmid

segregation without simultaneously aVecting chromosome segregation.

Perhaps mutations or experimental conditions that specifically disrupt either

plasmid–cohesin association or chromosome–cohesin association, but not

both, can help overcome this impediment.

The replication-dependent loading of cohesin is critical for segregating

two sister chromosomes into opposite cell compartments before cytokinesis.

For a diploid organism, the cohesin-mediated pairing avoids the potential

problem of distinguishing homologues from sisters following DNA replica-

tion. By contrast, the plasmid does not face this dilemma. If cohesin is indeed

required for plasmid segregation, it is interesting to ask whether cohesin-

assisted plasmid pairing is mediated only concomitant with replication or

may occur independent of replication.

Assuming that cohesin provides a counting mechanism, are the plasmids

counted approximately by the pairing of two clusters containing roughly

equal numbers, or are they counted exactly by the pairing of each molecule

with its sister in the duplicated cluster? It is now possible to tackle this issue

experimentally. One may place inside the same cell 2-mm plasmids tagged

with two colors, say, yellow and cyan, and follow them through sequential

division cycles. In the precise counting scheme, the ratio of yellow to cyan

will remain constant at each cell division; in the imprecise scheme, this ratio

will change perceptibly over a set of divisions.

Our current thinking on the mechanisms for plasmid stability is based on

the notion that the Rep proteins serve to recruit chromosomally encoded

partitioning factors to the STB locus. Yet, we still know little about how

the Rep proteins themselves associate with STB. Currently available evi-

dence argues against direct association between either of the Rep proteins

and STB and suggests the involvement of one or more host proteins in this

process. If such accessory proteins do exist, their identities need to be

revealed. In addition, how does the partitioning system interact with a

20 Velmurugan et al.

variety of host proteins, whose list seems to grow steadily? One possibility is

that the Rep proteins and their host, encoded partner proteins polymerize

along the STB locus to form a supramolecular partitioning complex not

unlike the kinetochore complex. An alternative, but not mutually exclusive,

possibility is that the Rep proteins may switch their partners as a function of

the cell cycle to best suit their partitioning needs.

As we have pointed out here, the plasmid cluster is normally positioned in

the proximity of the spindle pole, and depolymerization of microtubules by

nocodazole has a measurable adverse eVect on the compactness of the clus-

ter. Recent unpublished results that extend these observations suggest that

the mitotic spindle itself may directly or indirectly contribute to plasmid par-

titioning. A concerted role for both cohesin and the spindle in plasmid seg-

regation, if upheld, would lend further credence to the suspected mechanistic

connection between chromosome and plasmid partitioning.

The yeast plasmid has provided us with the first glimpse of how an appar-

ently rudimentary partitioning system might raise its level of sophistication

by taking advantage of the mitotic segregation pathway of its host. Under-

standing the finer details of this hitherto unsuspected molecular poaching

must await further work.

Acknowledgments

Work in the Jayaram laboratory on the recombination and partitioning systems of the yeast

plasmid has been supported over the years by funds from the National Institutes of Health, the

National Science Foundation, the Robert F. Welch Foundation, the Council for Tobacco

Research, the Texas Higher Education Coordinating Board, and the Human Frontiers in

Science Program.

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