Reconstruction of the yeast 2 μ m plasmid partitioning mechanism

Volume 16 Number 14 1988 Nucleic Acids Research

Reconstruction of the yeast 2 itm plasmid partitioning mechanism

M.J.Dobson*, F.E.Yull1, M.Molina2, S.M.Kingsman1 and A.J.Kingsman1

Department of Botany, University of Nottingham, University Park, Nottingham, NG7 2RD,'Department of Biochemistry, South Parks Road, Oxford, OXI 3QU, UK and 2Departamento deMicrobiologia, Facultad de Famicia, Universidad Complutense, Ciudad Universitaria, Madrid-3, Spain

Received May 20, 1988; Revised and Accepted June 22, 1988

ABSTRACTThe effect of the yeast 2um circle encoded REP1 and REP2 gene products

on plasmid partitioning and copy number control was analyzed by removingthe open reading frames from their normal sequence context andtranscriptional control regtons and directing their expression usingheterologous promoters in (cir ] host strains. Both the REP1 and REP2 geneproducts are directly required at appropriate levels of expression toreconstitute the 2um circle partitioning system in conjunction with REP3and the origin of replication. The level of expression of REP2 appears tobe critical to re-establishing proper partitioning and may also play a rolein monitoring and thereby regulating the plasmid copy number. Constitutiveexpression of the REP1 and REP2 open reading frames using heterologousexpression signals can be used to reconstruct efficient plasmidpartitioning even in the absence of FLP-mediated plasmid amplification anda functional D open reading frame.

INTRODUCTION

The yeast plasmid 2um circle is an autonomously replicating 6318 bp

double-stranded circular DNA plasmid found in 20-100 copies per haploid

cell in most strains of Saccharomyces cerevisiae (1-5). The 2um plasmid

consists of two unique regions separated by two inverted repeat sequences

(IRS) of 599 bp (5). Within the yeast cell, the 2um circle exists in

equimolar amounts of two forms which arise by site specific recombination

across the repeat sequences. The origin of replication of the plasmid

(ORI) has been localized to a 75 bp region partially within one of the

repeat sequences but extending into the adjacent unique region (6, 7).

Under normal conditions, each copy of the 2um circle replicates once per

cell cycle during S-phase (8) under the control of nuclear genes which

regulate chromosomal DNA replication (9, 10). However, the plasmid also

encodes its own copy number amplification system which allows it to over-

replicate when the copy number is low (11) and a segregation system which

distributes the plasmid randomly between mother and daughter cells at cell

division. Together these two systems ensure the stable maintenance of the

© I RL Press Limited, Oxford, England. 7103

Nucleic Acids Research

2um circle at high copy number with a very low rate of generation of

spontaneous 2um - free [cir J isolates.

DNA sequencing of the 2um circle has revealed four major open reading

frames (5) all of which have been demonstrated to be involved in plasmid

maintenance. The FLP gene encodes a trans-acting gene product responsible

for catalyzing the site specific recombination across the repeat sequences

(6) while the REP1 and REP2 open reading frames encode trans-acting gene

products involved in plasmid partitioning (7, 12, 13). Insertion or

deletion mutants of 2um circles with disruptions of REP1 or REP2 when

transformed into a [cir l host strain display high levels of mitotic

instability and a strong maternal bias in transmission, characteristic of

plasmids containing a chromosomally derived origin of replication or

autonomous replication sequence (ARS) (7, 12, 37). Another component of

the 2um circle replication system, REP3 (7, 14) or STB (12), is a site

required in cis for mitotic stability but is physically distinct from the

origin of replication. REP3 has been proposed as the site at which the

REP1 and REP2 gene products act to ensure efficient partitioning of the

plasmid (12-14) and it has been demonstrated that chromatin organization of

REP3 is disrupted in a repl mutant (15). Kikuchi (12) demonstrated that

the addition of REP1, REP2 and REP3 to an ARS plasmid resulted in an

increase in mitotic stability but with no overall increase in plasmid copy

number suggesting that the major role of those 2um circle loci lay in

plasmid partitioning rather than in replication.

Recent work (16-18) has shown that the amplification of 2am circle at

low copy numbers is mediated by FLP-promoted recombination across the IRS.

It has been demonstrated that the products of the three remaining open

reading frames of the 2um circle are involved in the regulation of FLP

expression. The REP1 and REP2 encoded proteins acting together are able to

repress transcription of the FLP gene while the product of the D open

reading frame is able to relieve this repression (19). Expression of the

FLP gene, and hence copy number control, is also mediated at a further

level since the combination of the REPI and REP2 gene products is able to

repress transcription of both the D gene (19) and the REP1 gene itself

(38). This elaborate series of regulatory circuits means that very small

drops in plasmid copy number, signalled by a reduction in the level of the

plasmid encoded REP1 and/or REP2 gene products would stimulate FLP

expression, inducing plasmid amplification.

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In this paper, we have analyzed the effect of the REPI and REP2 gene

products on plasmid partitioning and copy number control by removing the

open reading frames from their normal sequence context and transcriptional

control regions and directing their expression using heterologous promoters

in [cir l host strains.

MATERIALS AND METHODS

Bacterial and yeast strains

Strains used were E. coli AKEC28 (C600, thrC, leuB6, thyA, trpC1117,

hsdRk, hsdMk) and S. cerevisiae MD40/4C (a, ura2, trpl, leu2-3, leu2-112,

his3-11, his3-15). An isogenic (cir ] derivative of MD40/4C was created bycuring the strain of all 2um plasmid by the method of Dobson et al. (20).

E. coli were grown in Luria broth (21). Yeast media were prepared

according to Hawthorne and Mortimer (22). YEPD was 1% yeast extract, 1%

bacto-peptone and 2% glucose. Synthetic media consisted of 0.67% DIFCO

yeast nitrogen base minus amino acids, 1.0% glucose and appropriate amino

acid supplements.

YEAST TRANSFORMATION

Yeast were transformed as described by Hinnen et al. (23).ANALYSIS OF YEAST TRANSFORMANTS

Yeast cells were grown for 16 hrs at 300C with shaking to a density of1-5x106 cells/ml in synthetic complete medium lacking the appropriate amino

acid(s). Cell densities were determined by haemocytometer and viability

counts. The number of cells containing plasmid at the time of harvesting

was determined by washing the cells in water and then plating onto

duplicate selective and non-selective plates (200-500 colonies per plate).

The ratio of the colonies on the selective versus the non-selective plates

determines the proportion of plasmid containing cells (initial %) in the

harvested culture. The majority of the culture was imediately used for DNA

extraction to determine plasmid copy number while plasmid stability was

determined by using these same cultures to inoculate 10 ml non-selectiveYEPD cultures at 103 cells/ml. The YEPD cultures were allowed to grow

with shaking at 300C to a final density of 2-5x107 cells/ml, taking care

that they did not start into stationary phase which has a dramatic

stabilising effect on 2um and 2um-based plasmids (Dobson, unpublishedresults). The proportion of plasmid-containing cells in these non-

selective cultures was determined as before (final X) and the number of

7105


generations growth in non-selective media (n) calculated from the viable

counts on the non-selective plates and the dilution factors. The

instability, i, the rate at which [plasmidl+ cells segregate [plasmid]0cells per generation was calculated according to Murray and Cesareni (25)

by the formula:

i = 1/n ln(initial X / final %J

Total yeast DNA was isolated according to Cryer et al. (26).

ENZYMES AND PLASMID CONSTRUCTION

Enzymes were purchased from GIBCO-BRL and Northumbria Biologicals Ltd.

and were used according to the suppliers instructions. BAL31 exonuclease

digestions, blunt-end ligations and other DNA manipulations were performed

as described previously (27). Constructions involving BAL31 and linker

insertions were confirmed by dideoxy DNA sequencing (28). The orientation

of fragments was confirmed by appropriate restriction digests. BamHl

synthetic oligonucleotide linkers (CCGGATCCGG) were purchased from

Collaborative Research Ltd.

PLASMIDS AND HYBRIDIZATION PROBES

Partial restriction maps of expression vectors or plasmids used to

derive vectors for this study are shown in Figure 1A. The following

plasmids have been described elsewhere, pMA91 (29) pMA36 (30), pJDB219A,

pJDB219B (31), pYIRG12 (32), pMA247 and pMA278 (33). pMA136 was constructed

by replacing the double EcoRI fragment of pMA91 which contains the 2um and

LEU2 sequences with a 2.45 kb EcoRI fragment encoding TRPI, arsl and CEN3.

pMA132a consists of pMA91 with an 815 bp EcoRI fragment encoding the

TRPl gene replacing the small 750 bp EcoRI fragment encoding the majority

of the LEU2 gene. In pMAl32b, the TRP1 gene has been inserted in the

reverse orientation. DNA fragments were purified from agarose gels by

running the fragments into wells filled with gel running buffer, followed

by ethanol precipitation. Probes were labelled by nick translation (35)using [a-32P]dCTP (3000 Ci/mmol, Amersham International) to a specific

activity of 1-2x108 cpm/ug.PLASMID COPY NUMBER DETERMINATION

Total yeast DNA preparations were digested with EcoRI, PstI or

HindIII, fractionated by agarose gel electrophoresis and the fragments

transferred to nitrocellulose by the procedure of Southern (36).Nitrocellulose blots were hybridized with a plasmid specific probe, a 2.45

kb EcoRI fragment purified from pJDB219B containing the 2um origin of

replication, REP3 and a single copy of the 2um inverted repeat sequence

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(IRS). This plasmid specific probe was common to all the vectors except

pMA136 used in this study. These nitrocellulose blots were simultaneously

hybridised with a 1.8 kb EcoRI fragment purified from pYIRG12 which encodes

the yeast 18S ribosomal RNA gene. Duplicate nitrocellulose filters were

also hybridised with a 1.45 kb EcoRI yeast genomic DNA fragment containing

the TRP1 gene or pBR322. Hybridizations were carried out in 0.15M NaCl,

0.015M sodium citrate, 0.1% ficoll, 0.1% BSA, 0.1% polyvinylpyrrolidone,

0.1% SDS, 50% formamide, 50mM P04 pH 7.5, 250 ug/ml sonicated denatured

salmon sperm DNA at 420C for 16 hrs. The amount of hybridization to

plasmid, chromosomal TRP1 and ribosomal specific bands was determined by

cutting the bands out of the nitrocellulose filters and analysing by liquid

scintillation counting. The plasmid copy numbers were determined relative

to the internal control of the ribosomal repeat copy number (estimated at

100 copies per haploid genome) (32) and the single copy chromosomal TRP1 by

correcting the counts obtained for differences in amount of homology and

specific activity between the probes. Hybridization of the 2um and

ribosomal specific probes to HindIll digests or pBR322 to EcoRI digests was

used to determine the relative proportions of the two plasmids in co-

transformants (Figure 2C). Hybridization of plasmid specific probes to

PstI digests (PstI restricts all vectors used in this study within the E.

coli vector DNA sequences) was used to confirm that none of the

transformants analysed in this study contained integrated copies of the

plasmids (data not shown).

RESULTS

CONSTRUCTION OF REP1 AND REP2 EXPRESSION PLASMIDS

In order to assess the significance of REP1 and REP2 product dosage in

the absence of complicating normal regulation of these genes, we have

analyzed the effect of directing the expression of the REPI and REP2 open

reading frames using heterologous promoters of varying efficiencies. The

plasmid pJDB219B (Figure 1A) was used as the source of the REP1 and REP2

genes. The open reading frames were tailored for insertion into the yeast

expression vectors by BAL31 double stranded exonuclease digestion followed

by closure on synthetic BamHI oligonucleotide linkers (Figure 1B). The

resulting BamHI fragments were then suitable for insertion at the BglII or

BamHI expression sites of the yeast vectors such that their transcriptionwas now directed by the promoter element upstream of the expression site.

pMA91, pMA132a, pMA132b and pMA136 contain the 5' flanking region of the

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A

EARP3

ORI I

TRF

E1<

ORI I

B

ORI l

v )l xIF REPI

B A

E x

I r-£IREP2 1 I

B B17TG REE2 TGI37108

K 3

3

E


efficiently expressed yeast phosphoglycerate kinase (PGK) gene from -1500

to -2 bases upstream of the PGK ATG initiation codon upstream of the BglII

site and the 3' flanking region of the PGK gene downstream of the BglII

expression site. pMA278 and pMA247 are promoter deletion derivatives of

pMA91 with reduced promoter efficiencies relative to the intact PGK

promoter (Table 1) (33) and with the remainder of the plasmid other than

the promoter region being identical to pMA91. pMA36 contains the 5'

flanking region of the yeast N- (5'-phosphoribosyl)-anthranilate isomerase

gene (TRP1) from -1500 to -8 bases upstream of the TRP1 initiation codon

(30). All the 2um based expression vectors used in this study contain an

identical 2.45 kb EcoRI fragment derived from pJDB219B which contains the

2um origin of replication , only a single copy of the IRS, and REP3. These

vectors all contain a disrupted D open reading frame and do not encode

functional FLP activity. The vector pMA136, with an average copy number of

between one and two copies per cell (data not shown) contains a 2.45 kb

EcoRI fragment encoding TRP1, arsl and CEN3. The relative efficiencies of

the expression vectors used in this study are indicated in Table 1 based on

their ability to direct expression of a human interferon alpha-2 cDNA in a

[cir+] host where all the 2um-based vectors exist at about 100 copies per

cell (30, 33).

TRANSFORMATION EFFICIENCY OF REP1 AND REP2 EXPRESSION PLASMIDS

The ability of the various yeast exression vectors and their

derivatives directing the expression of either REP1 or REP2 to transform

isogenic [cir+J or (cir0l yeast was assessed and the results are shown in

Table 2. The control plasmids pJDB219A and pJDB219B which contain all the

2um sequences but have a disrupted FLP gene, and the vector pMA36 are both

able to transform either [cir l or [cir+l host strains to leucine

prototrophy with about the same efficiency. The vector pMA91, with the

same LEU2 selectable marker, transforms the [cir+l yeast very efficientlywith colonies visible after 5 days of incubation at 300C but requires 10

FIGURE 1. Partial restriction maps of A. the expression vectors used inthis study and B. the tailored REP1 and REP2 fragments which were insertedin these vectors. Thick dark lines represent E. coli vector sequenceswhereas thin single lines represent 2um sequences. Open boxes representyeast chromosomal DNA inserts or 2um open reading frames as indicated.Thin arrows indicate the orientation of the genes while the open arrowsindicate the direction and extent of BAL31 deletion. Numbers are innucleotides. Other symbols are as follows:

1- IRS, 11111- REP3, PGK promoter sequences, - PGK terminatorsequences, - TRP1 promoter sequences. Restriction site abbreviationsare as follows: B - BamHI, Bg - Bglll, E - EcoRI, V - PvuII, X - XbaI.

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days before colonies can be scored for the [ciro host although the final

frequency of transformation obtained in both hosts is similar. Growth

inhibition of the (cir+] host transformed with pMA91 is also evident but to

a lesser degree than in the [cir0] host and in both cases probably results

from the very efficient expression of the extreme carboxy terminus of the

PGK protein from this high copy number vector when no open reading frame is

inserted at the expression site (Dobson unpublished results). pMA132a and

pMA132b are able to transform both the [cir+] and the [ciro] yeast to

tryptophan prototrophy with equal efficiency which is not altered when

either of these vectors is directing the expression of the REP1 gene,

pMA132a-REPl and pMA132b-REPl respectively. Insertion of the REPl BaElI

fragment (Figure 1B) at the BglIl expression site in pMA91 results in a

plasmid, pMA91-REPl which is able to transform the (cir ] host as

efficiently as the !cir+1 host. In contrast the REP2 BamHI fragment

inserted in pMA91 results in a plasmid, pMA91-REP2 which even after

prolonged incubation does not yield any LEU+ transformants in either a

(cir+] or a (cir l background. pMA278-REP2 and pMA247-REP2 which should

express the REP2 gene product about 40-fold and 100-fold less efficiently

than pMA91-REP2 were also unable to transform [cir0l yeast although a small

number of transformants were obtained for the [cir+I yeast. pMA36-REP2

which should direct REP2 expression about 1000-fold less efficiently than

pMA91-REP2, was able to transform both the [cir+l and (cirl yeast to

leucine prototrophy with equal efficiency. To assess whether these results

reflected a toxicity of the REP2 gene product when expressed at high levels

TABLE 1: RELATIVE EFFICIENCY OF PROMOTER ELEMENTS DIRECTING THEPRODUCTION OF INTERFERON ALPHA-2 IN YEAST a

VECTOR EXPRESSION ELEMENTb MOLECULES OFINTERFERON/CELLC

PGK pMA9I -1500 to -2 2x1O6pMA278 -1500 to -48 5xlO4pIMA247 -1500 to -83 2x104

TRP1 pMA36 -1500 to -8 2xl3

a. Data in this table are taken from Kingsman and Kingsman (33) andDobson et al. (30). An identical Bamil fragment containing a humaninterferon-alpha-2 cDNA was expressed in each of these vectors.b. The numbers are in nucleotides where -l represents the first baseupstream from the authentic initiating ATG.c. Yeast protein extracts were assayed on HEp-2 cells and titres areadjusted to an interferon-alpka-2 standard. Calculations are made froma specific activity of 2.Ox1O units/mg/3x1016 molecules.

7110


or whether they indicated an action of the REP2 gene product which

specifically affected the ability of the plasmid to replicate and/or

segregate, the REP2 BamRI fragment was inserted at the BglII expression

site in pMA136 to give pMA136-REP2. This plasmid was able to transform

both [cir+l and [cir0l yeast with high efficiency which suggests that the

level of REP2 expression directed by the full PGK promoter at a single copy

per cell is not in and of itself lethal. Therefore, it appears that the

overexpression of REP2 inhibits the replication and/or segregation of the

2iim-based vectors.

Selected co-transformations were performed to observe the effect on

transformation efficiency of introducing pre-set levels of REPI and REP2

into the same yeast cell and the results are shown in Table 2. Co-

transformants of pMA136-REP2 and pMA91-REPl could not be obtained in either

a [cir+] or a [cir l background whereas pMA132a-REP1 and pMA36-REP2 or

TABLE 2: TRANSFORMATION EFFICIENCY OF HYBRID PLASMIDSa

TRANSFORMING PLASMID TRANSFORMANTS / ug OF PLASMID

[ CIRO] [CIR+I

pJDB219A 2.7 x 104 1.2 x 104pJDB219B 1.5 x 104 1.6 x 104pMA132a 1.8 x 104 8.4 x 103pMA132b 1.5 x 104 5.3 x 103pMA132a-REP1 2.7 x 104 2.7 x 104pMA132b-REP1 4.9 x 103 1.4 x 104pMA91 ob 8.0 x 103pMA91-REPl 1.6 x 104 8.6 x 103pMA91-REP2 0 0pMA278-REP2 0 18cpMA247-REP2 O 17c 3pMA36 3.0 x 103 9.0 x 10pMA36-REP2 1.2 x 104 2.8 x 104pMA136-REP2 1.0 x 104 7.0 x 103

CO-TRANSFORMING PLASMIDSd

pMA91-REPI + pMA136-REP2 0 0pMA132a-REPl + pMA36-REP2 2.1 x 104 2.0 x 104pMA132b-REP1 + pMA36-REP2 9.0 x 10 1.1 x 10

a. Transformation frequencies were scored after plates had beenincubated for 5 days at 300C and represent the mean frequency from fourindependent transformation experiments.b. After 10 days incubation at 300C, 5x103 transformants/ug wereobtainedc. Colonies are very growth inhibited.d. Co-transformation frequencies represent simultaneous selection forboth plasmid markers (i.e. colonies that were LEU+ and TRP+).

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pMA132b-REPl and pMA36-REP2 could be used to co-transform either the [cir+lor [cir ] yeast to leucine and tryptophan prototrophy with the same 'high

efficiency. It appears that overexpression of REP2 at the level directed

by pMA136-REP2 is sufficient to disrupt either replication and/or

segregation of the pMA91-REPI plasmid such that co-transformation cannot be

established even though individually, both plasmids will transform either a

[cir+] or a [cir0l yeast with high efficiency. In contrast, LEU+ TRP+

transformants could be obtained with combinations of either pMA136-REP2 and

pMA91 or pMA136-REP2 and pMA36 although the co-transformants were very slow

growing, taking 10 days at 300C to form colonies on the transformation

plates. (data not shown).

PLASMID STABILITY AND COPY NUMBER IN 1cir0j YEAST TRANSFORMANTS

[ciro] yeast transformants from TABLE 2 were grown up under selective

conditions and the copy number and stability of the transforming plasmids

analyzed as described in the Materials and Methods. The results are shown

in TABLE 3 and FIGURE 2. The control plasmids pJDB219A and pJDB219B are

extremely stable and exhibit high copy numbers per cell in a [cirobackground as has been previously reported (20, 24). Those plasmids such

as pMA132a and pMA132b, which have the TRP1 selectable marker, and their

TABLE 3: PLASMID COPY NUMBER AND STABILITY IN [cir°0 TRANSFORMANTS

TRANSFORMING % [PLASMID+1 CELLSa i% a AVERAGEa NUMBERPLASMID COPY OF

(INITIAL %) (FINAL %) x LOSS NUMBER INDEPENDENTAFTER AFTER 15 PER PER TRANSFORM-SELECTIVE GENERATIONS GENERATION CELL ANTSGROWTH NON-SELECTIVE ANALYSED

GROWTH

PJDB219A 100.0 100.0 0.0 423(384-462) 2pJDB219B 100.0 94.1(82.0-100.0) 1.1(0.0-3.0) 221(93-350) 4pMA91-REP1 27.0(11.6-33.2) 3.8(0.5-8.0) 18.5(11.9-22.7) 37(29-45) 4pMA132a 28.6(11.2-71.0) 1.4(1.1-1.7) 19.9(13.8-25.9) 5(4-7) 4pMA132b 50.4(30.4-70.4) 3.6(1.6-5.7) 19.0(17.6-20.4) 20(9-31) 2pMA132a-REPI 18.2(1.3-60.0) 1.7(0.2-6.0) 22.6(11.0-41.1) 7(5-15) 12pMA132b-REP1 13.1(10.0-15.6) 0.5(0.3-0.7) 20.3(16.7-23.8) 19(11-36) 4pMA36 22.7(19.0-29.9) 1.0(0.4-1.3) 21.5(17.7-24.9) 115(113-117) 4pMA36-REP2 31.0(27.5-35.6) 0.8(0.3-1.4) 24.9(20.2-33.6) 414(239-527) 5

CO-TRANSFORMINGPLASMIDS b

pMA132a-REPI 51(7-123)+ 67.6(46.4-99.1) 44.9(11.3-73.7) 2.9(0.0-8.4)

pMA36-REP2 161(30-246) 9

pMA132b-REP1 66.8(50.0-75.1) 40.5(36.5-44.9) 2.8(1.8-3.7) 82(39-128)+ 4

pMA36-REP2 225(72-405)

a. See Materials and Methods iX - ix1O2Minimum and maximum values are indicated in brackets after the average valVe

b. For the co-transformants, [plasmid]+ cells are those which are LEU+ and TRP+

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REP1 expressing derivatives all exhibit high levels of instability and low

copy numbers per cell of population. Similarly, pMA91-REPl and pMA36-REP2

transformants show a high percentage loss of plasmid per generation during

non-selective growth although these LEU2 selectable vectors have a higher

average copy per cell of population and per plasmid-containing cell than

the vectors based on TRP selection. The data show that 2um REP3/ORIvectors expressing either the REP1 or the REP2 gene product alone at these

levels are defective in the 2um plasmid segregation mechanism.

Analysis of the two co-transformed [cir°] yeast, those with pMA132a-

REPI and pMA36-REP2 and those with pMA132b-REPl and pMA36-REP2, shows a

high level of mitotic stability for the co-transformed phenotype with less

1 2 3 4 5 6 7 8 5 6 7 8 2 5 6

EbIi - P2

P2-

R_ *DM0~~ ~ ~ ~ ~ ~ ~ ~ _

IIIIQ ~~-i -P1

EN -P1

A B C

FIGURE 2 Autoradiograms of Southern blots of total yeast DNA preparationsdigested with EcoRI in A. and B. and HindIll in C. and hybridized with a2.45 kb EcoRI plasmid specific fragment plus a 1.8kb EcoRI ribosomal DNAspecific fragment in A and C and a 1.45 kb yeast TRPI genomic pNA fragmenEin B. DNA preparations are from MD40/4C 1. [cir'], 2. [cir ], 3. [cirpJDB219A], 4. [ciro pMA36-REP2]. 5, 6. two isolates of [cir pMA36-REP2,pMA132a-REPl] and 7,8 two isolates of [cir pMA132a-REPl]. R - ribosomalspecific bands, T - 1.45 kb chromosomal TRP1 band, P1 - pMA132a-REPIspecific bands, P2 - pMA36-REP2 specific bands. The 2.45 kb EcoRI plasmidspecific bands which have hybridized in lanes 4-8, panel A. all have 2.45kb of homology with the 2um-specific probe, the P1 and P2 bands in panel Chave 2.35 kb of homology with this probe, whereas in Panel B, P1 and P2have respectively 815 bp and 93bp homology with the 1.45 kb TRPl probe.

71 13


than 3X loss of TRP+ LEU+ cells per generation of growth in non-selective

medium. Although this two-plasmid system with heterologous promoters

directing the expression of the REPI and REP2 genes is a very artificial

system compared to the normal arrangement of the 2um genes, it does appear

that the plasmid segregation machinery has been reconstructed. The average

plasmid copy numbers in the co-transformed [cir°] yeast are also altered in

comparison to their levels in the yeast transformed with only one of the

plasmids alone. The average pMA36-REP2 copy number per cell of population

and per plasmid containing cell is much lower in the co-transformants than

in the pMA36-REP2 LEU+ transformants as one might predict since in the

latter case, the strong selection against cells with low levels of LEU+

plasmid would favour the accumulation of those cells with a higher plasmid

copy number which would be generated due to the absence of efficient

segregation. The pMA132a-REPl and pMA132b-REPl average plasmid copy

numbers per cell of population are higher in the co-transformants than in

the single transformants but correcting for the proportion of cells which

do not contain plasmid shows that the copy number per plasmid-bearing cell

is similar. This data demonstrates that re-introduction of efficient

plasmid segregation in this system does not significantly affect plasmidcopy number.

DISCUSSION

In this paper, we have demonstrated that both the REP1 and REP2 gene

products are required together at a specific level to re-constitute the 2umplasmid partitioning maechanism. Cashmore et al (13) have previously shown

that the efficiency of partitioning was dependent upon the dosage of the

REP1 gene but appeared to be independent of the REP2 gene dosage. However,

in their integration constructions, the REP1 and REP2 were flanked by their

normal 2um sequence environment and the expression levels of the two genes

may not necessarily have been the same as that indicated by the gene

dosage. In our system, where REP1 expression is not limiting, constitutive

expression of the REP2 gene at the appropriate level allows efficient

segregation of vectors containing REP3 and ORI but lacking functional FLP

and D open reading frames. As has been established for the plasmid

amplification system, it appears that it is a complex of the REPM and REP2

gene products which mediates 2um plasmid segregation.

The results of the transformation experiments in which the REP2 gene

was overexpressed either in the presence or absence of REPM expression

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raise some interesting questions on the role of the REP2 gene product.

Transformants of the [cir l host could not be established for 2um REP3/ORI

type vectors expressing any level of REP2 more efficiently than pMA36-REP2.

It is unlikely that this reflects a general toxicity to the cell of high

levels of the REP2 protein, at least in the case of the pMA247-REP2

plasmid, since transformants could be obtained for pMA136-REP2 directing

REP2 expression from the full PGK promoter at one copy per cell. It seems

more likely that the overexpression of REP2 affects the segregation of the

2um REP3/ORI based vectors. In this context, it is interesting to note

that the vector pMA36 has a slightly higher mitotic stability than its REP2

expressing derivative, pMA36-REP2.

Co-transformants could also not be established for any 2um-basedREP3/ORI vector directing REPI expression in combination with pMA136-REP2

although very slow-growing co-transformants could eventually be obtained

for the vectors without the REP1 open reading frame insert in combination

with pMA136-REP2. These results suggest that REP2 overexpression in the

presence of the REPI gene product inhibits the segregation and/orreplication of the 2um based vectors although the level of REP2 expressed

would be much less than for pMA91-REP2. A great deal of caution must be

exercised in interpreting the results from co-transformation studies in

which one is assessing the effect on replication or segregation of a

plasmid of a gene product being expressed from that plasmid or another

plasmid in the same cell. However, the data does suggest that the level of

the REP2 gene product supplied may affect the functioning of the REPl/REP2

gene product complex which mediates both plasmid segregation by an as yet

undetermined mechanism and plasmid amplification through its regulation of

FLP transcription. The inhibitory effect on segregation of high levels per

cell of the REP2 gene product may be yet another feedback circuit in the

2um system which monitors and controls plasmid copy number. The disruption

of segregation might provide a mechanism by which cells with reduced

plasmid copy number could be generated from a parental cell in which the

copy number was too high.The re-construction of an efficient plasmid partitioning system by the

constitutive expression from heterologous promoters of appropriate levels

of the REPI and REP2 gene products is of practical importance for the

development of stable minimal 2pm derived replicons for use as the basis of

vectors to direct the expression of heterologous proteins in yeast. The

data presented here shows the feasibility of constructing (cirl] strains in

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which appropriate levels of REP1 and REP2 gene products are expressed from

single copy expression cassettes integrated into the chromosomal DNA.

Recent work in this laboratory (Dobson and Molina, unpublished results)

indicates that such strains are able to promote stable high copy number

replication of 2um based vectors containing only REP3, a selectable marker

and the 2um origin of replication. It will be of interest to determinewhether FLP expression is also correctly regulated in these strains and

even whether it is necessary for the stable maintenance of these vectors.

ACKNOWLEDGEMENTS

The work was supported in Oxford by an SERC/Celltech Ltd. co-operativegrant and in Nottingham by MRC grant G8422850CB. M.M was in receipt of an

award from the E.E.C. for training in Biotechnology. We thank S. Redman

and B. Case for secretarial and photographic assistance.

* To whom correspondence should be addressed.

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Documents

Reconstruction of the yeast 2 μ m plasmid partitioning mechanism