the open reading frames from their normal sequence context ...

volume 16 Number 14 1988 Nucle ic Ac ids Research

Reconstruction of the yeast 2 /im 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, 0X1 3QU, UK and 2Departamento deMicrobiologia, Facultad de Farmicia, Universidad Complutense, Ciudad Universitaria, Madrid-3, Spain

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

ABSTRACTThe e f f e c t of the yeast 2um c i r c l e encoded REP1 and REP2 gene products

on plasmid par t i t i on ing and copy number control was analyzed by removingthe open reading frames from their normal sequence context andtranscrlptional control regions and directing their expression usingheterologous promoters in [cir°] host strains. Both the REP1 and REP2 geneproducts are directly required at appropriate l eve l s 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 s ignals can be used to reconstruct e f f i c i ent plasmidpartitioning even in the absence of FLP-mediated plasmid amplification anda functional I) open reading frame.

INTRODUCTION

The yeast plasmid 2um c irc l e i s an autonomously replicating 6318 bp

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

c e l l in most strains of Saccharomyces cerevlslae (1-5). The 2um plasmid

consists of two unique regions separated by two Inverted repeat sequences

(IRS) of 599 bp (5). Within the yeast c e l l , the 2um c i r c l e e x i s t s in

equimolar amounts of two forms which arise by s i t e specific recombination

across the repeat sequences. The orig in of r e p l i c a t i o n of the plasmid

(0RI) has been l o c a l i z e d to a 75 bp region p a r t i a l l y within one of the

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

Under normal conditions, each copy of the 2um c irc l e replicates once per

c e l l cyc le during S-phase (8) under the control of nuclear genes which

regulate chromosomal DNA replication (9, 10). However, the plasmid a l so

encodes i t s own copy number amplification system which allows It to over-

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

distributes the plasmid randomly between mother and daughter c e l l s at c e l l

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

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Nucleic Acids Research

2um c i r c l e at high copy number with a very low rate of generation of

spontaneous 2um - free [cir ] i solates .

DNA sequencing of the 2um c irc le has revealed four major open reading

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

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

for catalyzing the s i t e specific recombination across the repeat sequences

(6) whi le the REP1 and REP2 open reading frames encode trans-act ing gene

products i n v o l v e d in plasmid part i t ioning (7, 12, 13). Insertion or

d e l e t i o n mutants of 2um c i r c l e s with disruptions of REP1 or REP2 when

transformed into a [ c i r ] host s tra in d isp lay high l e v e l s of mitot ic

ins tabi l i ty and a strong maternal bias in transmission, characteristic of

plasmids containing a chromosomally derived or ig in of r e p l i c a t i o n or

autonomous r e p l i c a t i o n sequence (ARS) (7, 12, 37). Another component of

the 2um c i r c l e r e p l i c a t i o n system, REP3 (7, 14) or STB (12), i s a s i t e

required in c is for mitotic s tabi l i ty but i s physically distinct from the

or ig in of r e p l i c a t i o n . REP3 has been proposed as the s i t e at which the

REP1 and REP2 gene products act to ensure e f f i c i e n t part i t ion ing of the

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

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

the addit ion of REP1, REP2 and REP3 to an ARS plasmid resu l ted in an

increase in mitotic s tab i l i t y but with no overal l increase in plasmid copy

number suggesting that the major r o l e of those 2um c i r c l e l o c i lay in

plasmid partitioning rather than in replication.

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

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

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

reading frames of the 2ym 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 I) open

reading frame i s able to re l ieve this repression (19). Expression of the

FLP gene, and hence copy number control, i s a lso mediated at a further

l e v e l since the combination of the REP1 and REP2 gene products i s able to

repress transcription of both the 1) gene (19) and the REP1 gene I t se l f

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

drops in plasmid copy number, signalled by a reduction in the l e v e l 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 REP1 and REP2 gene

products on plasmld partitioning and copy number control by removing the

open reading frames from their normal sequence context and transcrlptlonal

control regions and directing their expression using heterologous promoters

in [cir ] host strains.

MATERIALS AND METHODS

Bacterial and yeast strains

Strains used were E. co l i AKEC28 (C600, thrC, leuB6, thyA, trpC1117,

hsdRy, hsdMy) and S cerevislae MD40/4C (a, ura2, trpl, leu2-3, Ieu2-U2,

hls3-l l , his3-15). An isogenic [clr°] derivative of MD40/4C was created by

curing the strain of a l l 2um plasmid by the method of Dobson et_ a_l (20).

E^ c o l l 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 e_t al^ (23).

ANALYSIS OF YEAST TRANSFORMANTS

Yeast c e l l s were grown for 16 hrs at 30°C with shaking t o a d e n s i t y of

1-5x10° c e l l s / m l In s y n t h e t i c complete medium lacking the appropriate amino

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

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

was determined by washing the c e l l s 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 cel ls (initial %) in the

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

extraction to determine plasmld copy number while plasmid stabi l i ty was

determined by using these same cultures to inoculate 10 ml non-selective

YEPD cultures at 103 cel ls /ml. The YEPD cultures were allowed to grow

with shaking at 30°C to a final density of 2-5xl07 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, unpublished

results). The proportion of plasmid-containing c e l l s in these non-

select ive cultures was determined as before (final %) and the number of

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

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

instabil ity, i, the rate at which [plasmid]+ ce l l s segregate [plasmid]0

cells per generation was calculated according to Murray and Cesareni (25)

by the formula:

i = 1/n ln[initial % / final %]

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

ENZYMES AND PLASMID CONSTRUCTION

Enzymes were purchased from GIBCO-BRL and Northumbria B i o l o g i c a l s Ltd.

and were used according to the s u p p l i e r s i n s t r u c t i o n s . BAL31 exonuclease

d i g e s t i o n s , blunt-end l i g a t i o n s and other DNA manipulations were performed

as described prev ious ly (27). Cons truc t ions i n v o l v i n g BAL31 and l i n k e r

inser t ions were confirmed by dideoxy DNA sequencing (28). The orientat ion

of fragments was confirmed by appropr ia te r e s t r i c t i o n d i g e s t s . BamHl

s y n t h e t i c o l i g o n u c l e o t i d e l i n k e r s (CCGGATCCGG) were purchased from

Col laborat ive Research Ltd.

PLASMIDS AND HYBRIDIZATION PROBES

P a r t i a l r e s t r i c t i o n maps of expression vectors or plasmids used to

der ive vectors for t h i s study are shown in Figure 1A. The fo l lowing

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 TRP1, arsl and CEN3.

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

TRP1 gene replacing the small 750 bp EcoRI fragment encoding the majority

of the LEU2 gene. In pMA132b, the TRP1 gene has been inserted in the

reverse or ienta t ion . DNA fragments were purif ied from agarose g e l s by

running the fragments into wel ls f i l l e d with gel running buffer, followed

by ethanol precipitation. Probes were label led by nick translation (35)

using [a-32P]dCTP (3000 Ci/mmol, Amersham Internat ional ) to a s p e c i f i c

ac t iv i ty of l-2xlO8 cpm/ug.

PLASMID COPY NUMBER DETERMINATION

T o t a l y e a s t DNA p r e p a r a t i o n s were d i g e s t e d w i t h EcoRI, P s t I or

Hindlll, fractionated by agarose gel electrophoresis and the fragments

transferred to n i t r o c e l l u l o s e 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 plasmld specific probe was common to a l l 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 fil ters 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 c i t ra te , 0.1% f i co l l , 0.1% BSA, 0.1% polyvinylpyrrolidone,

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

salmon sperm DNA at 42°C 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 f i l ters 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 act ivi ty between the probes. Hybridization of the 2pm and

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

used to determine the re la t ive proportions of the two plasmids in co-

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

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

c o l i 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 0J_ 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 REP1 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 IB). The

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

BamHI expression sites of the yeast vectors such that their transcription

was 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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OR] I

B 8

211ATG REP1 TAGl 95

1- X.

B

471 ATG

REP2

REP2

1

A |

B

TGAI3

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e f f i c i e n t l y expressed yeast phosphoglycerate kinase (PGK) gene from -1500

to -2 bases upstream of the PGK ATG i n i t i a t i o n codon upstream of the B g l l l

s i t e and the 3' f l a n k i n g reg ion of the PGK gene downstream of the B g l l l

express ion s i t e . pMA278 and pMA247 are promoter d e l e t i o n d e r i v a t i v e s of

pMA91 with reduced promoter e f f i c i e n c i e s r e l a t i v e to the i n t a c t PGK

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

the promoter reg ion being i d e n t i c a l to pMA91. pMA36 c o n t a i n s the 5'

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

gene (TRP1) from -1500 t o -8 bases upstream of the TRP1 i n i t i a t i o n codon

(30). A l l the 2um based expression vectors used in th i s study contain an

Identical 2.45 kb EcoRI fragment derived from pJDB219B which contains the

2um origin of rep l i ca t ion , only a s ing l e copy of the IRS, and REP3. These

v e c t o r s a l l conta in a d i srupted D open reading frame and do not encode

functional FLP a c t i v i t y . The vector pMA136, with an average copy number of

between one and two c o p i e s per c e l l (data not shown) conta ins a 2.45 kb

EcoRI fragment encoding TRP1, ars l and CEN3. The r e l a t i v e e f f i c i e n c i e s of

the expression vectors used in th i s study are indicated in Table 1 based on

their a b i l i t y to direct expression of a human interferon alpha-2 cDNA in a

[cir ] host where a l l the 2um-based vectors ex i s t at about 100 copies per

c e l l (30, 33).

TRANSFORMATION EFFICIENCY OF REP1 AND REP2 EXPRESSION PLASMIDS

The abi l i ty of the various yeast exression vectors and their

derivatives directing the expression of either REP1 or REP2 to transform

lsogenic [cir+] or [cir0] yeast was assessed and the results are shown in

Table 2. The control plasmlds pJDB219A and pJDB219B which contain a l l the

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

able to transform either [c ir 0 ] or [c ir+] host strains to leucine

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

same LEU2 selectable marker, transforms the [cir+] yeast very efficiently

with colonies v i s ib le after 5 days of incubation at 30°C 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 l ines represent E. co l l 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: _ _ ^KS>SI = IRS, UJ1U" REP3. IgflSfl - PGK promoter sequences, IV-V-1- PGK terminatorsequences, S 3 " TRP1 promoter sequences. Restriction site abbreviationsare as follows: B - BamHI, Bg = Bgl l l , E - EcoRI, V - PvuII, X » Xbal.

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days before colonies can be scored for the [c ir° ] host although the f inal

frequency of transformation obtained in both h o s t s i s s i m i l a r . Growth

inhibition of the [ c i r + ] host transformed with pMA91 i s a l so evident but to

a lesser degree than in the [cir ] host and in both cases probably resul t s

from the very e f f i c i ent expression of the extreme carboxy terminus of the

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

inserted at the expression s i te (Dobson unpublished resu l t s ) . pMA132a and

pMA132b are ab le to transform both the [ c i r + ] and the [ c i r ° l yeast to

tryptophan prototrophy with equal e f f i c i e n c y which i s not a l t e r e d when

e i ther of these v e c t o r s i s d i r e c t i n g the express ion of the REP1 gene,

pMA132a-REPl and pMA132b-REPl respect ively . Insertion of the REP1 BamHI

fragment (Figure IB) at the B g l l l express ion s i t e in pMA91 r e s u l t s in a

plasmid, pMA91-REPl which i s able to transform the [ c i r 0 ] host as

e f f i c i e n t l y as the [ c i r + ] hos t . In contrast the REP2 BamHI fragment

inserted in pMA91 r e s u l t s in a plasmid, pMA91-REP2 which even a f t er

prolonged incubatj.on does not y i e l d any LEU transformants in e i ther a

[c ir + ] or a [ c i r 0 ] background. pMA278-REP2 and pMA247-REP2 which should

express the REP2 gene product about 40-fold and 100-fold l e s s e f f i c i e n t l y

than pMA91-REP2 were a l s o unable to transform [c ir 1 yeast although a small

number of transformants were obtained for the [ c i r + ] y e a s t . pMA36-REP2

which should direct REP2 expression about 1000-fold l e s s e f f i c i e n t l y than

pMA91-REP2, was a b l e to transform both the [ c i r + ] and [ c i r 0 ] yeast to

leucine prototrophy with equal efficiency. To assess whether these resul ts

reflected a tox ic i ty of the REP2 gene product when expressed at high l e v e l s

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

VECTOR EXPRESSION ELEMENT0 MOLECULES OFINTERFERON/CELLC

PGK pMA91pMA278PMA247

TRP1 pMA36

-1500 to -2-1500 to -48-1500 to -83-1500 to -8

2x105x10'2x10*2xlO3

4

a. Data in this table are taken from Kingsman and Kings man (33) andDobson et_ a_l. (30). An identical BamHI fragment containing a humaninterferon-alpha-2 cDNA was expressed in each of these vectors.b. The numbers are in nucleotides where -1 represents the first baseupstream from the authentic initiating ATG.c. Yeast protein extracts were assayed on HEp-2 c e l l s and t i tres areadjusted to an interferon-alpha-2 standard. Calculations are made froma specific activity of 2.0xl08 units/mg/3xl016 molecules.

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

specif ical ly affected the abi l i ty of the plasmid to replicate and/or

segregate, the REP2 BamHI fragment was inserted at the Bgll l expression

s i te in pMA136 to give pMA136-REP2. This plasmid was able to transform

both [cir+] and [cir°] yeast with high efficiency which suggests that the

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

per c e l l is not in and of i t s e l f le thal . Therefore, i t appears that the

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

2um-based vectors.

Selected co-transformations were performed to observe the effect on

transformation efficiency of introducing pre-set levels of REP1 and REP2

into the same yeast c e l l 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°] background whereas pMA132a-REPl and pMA36-REP2 or

TABLE 2: TRANSFORMATION EFFICIENCY OF HYBRID PLASMIDSa

TRANSFORMING PLASMID

PJDB219APJDB219BpMA132apMA132bpMA132a-REPlpMA132b-REPlPMA91PMA91-REP1PMA91-REP2PMA278-REP2PMA247-REP2PMA36PMA36-REP2PMA136-REP2

CO-TRANSFORMING PLASMIDSd

TRANSFORMANTS / Ug OF PLASMID

[CIR°] [CIR+]

2.7 x IO4 1.2 x IO4

1.5 x IO4 1.6 x IO4

1.8 x IO4 8.4 x IO3

1.5 x IO4 5.3 x 10J

2.7 x IO4 2.7 x IO4

4.9 x IO3 1.4 x IO4

Ob 8.0 x IO3

1.6 x IO4 8.6 x 10J

0 00 18C

0 , 17C

3.0 x io3 9.o x io;1.2 x IO4 2.8 x IO4

1.0 x IO4 7.0 x 10J

PMA91-REP1 + pMA136-REP2 0 0pMA132a-REPl + pMA36-REP2 2.1 x 10* 2.0 x 107pMA132b-REPl + pMA36-REP2 9.0 x IO3 1.1 x 10

a. Transformation frequencies were scored after p lates had beenincubated for 5 days at 30°C and represent the mean frequency from fourIndependent transformation experiments.b. After 10 days incubat ion at 30°C, 5 x l 0 3 transformants/ug wereobtainedc. Colonies are very growth inhibited.d. Co-transformation frequencies represent simultaneous s e l e c t i o n 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 [c i r + ]

or [ c i r ] y e a s t to l e u c i n e and tryptophan prototrophy with the same high

eff ic iency. I t appears that overexpression of REP2 at the l e v e l directed

by pMA136-REP2 i s s u f f i c i e n t to disrupt e i t h e r r e p l i c a t i o n and/or

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

establ ished even though individual ly , both plasmids w i l l transform either a

[ c i r + ] or a [ c i r 0 ] yeas t with high e f f i c i e n c y . In c o n t r a s t , 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 30°C to form c o l o n i e s on the transformation

p la tes , (data not shown).

PLASMID STABILITY AND COPY NUMBER IN [c lr° ] YEAST TRANSFORMANTS

[c ir ] yeast transformants from TABLE 2 were grown up under s e l e c t i v e

conditions and the copy number and s t a b i l i t y of the transforming plasmids

analyzed as described in the Materials and Methods. The resul t s are shown

in TABLE 3 and FIGURE 2. The c o n t r o l plasmids pJDB219A and pJDB219B are

extremely s t a b l e and exhibi t high copy numbers per c e l l in a [ c i r ]

background as has been previously reported (20, 24). Those plasmids such

as pMA132a and pMA132b, which have the TRP1 s e l e c t a b l e marker, and their

TABLE 3: PLASMID COPY NUMBER AND STABILITY IN [ c i r 0 ] TRANSFORMANTS

TRANSFORMINGPLASMID

PJDB219APJDB219BPMA91-REP1pMA132apMA132bpMA132a-REPlpMA132b-REPlPMA36PMA36-REP2

CO-TRANSFORMINGPLASMIDS b

pMA132a-REPl

PMA36-REP2

pMA132b-REPl

PMA36-REP2

X [PLASMID+

(INITIAL X)AFTERSELECTIVEGROWTH

100.0100.0

27.0(11.6-33.28.6(11.2-71.50.4(30.4-70.

1

2)0)4)

18.2(1.3-60.0)13.1(10.0-15.22.7(19.0-29.31.0(27.5-35.

67.6(46.4-99.

66.8(50.0-75.

6)9 )6)

1)

1)

CELLSa

(FINAL X )

AFTER 1 5GENERATIONSNON-SELECTIVEGROWTH

100.094.1(82.0-100.0)3.8(0.5-8.0)1.4(1.1-1.7)3.6(1.6-5.7)1.7(0.2-6.0)0.5(0.3-0.7)1.0(0.4-1.3)0.8(0.3-1.4)

44.9(11.3-73.7)

40.5(36.5-44.9)

IX a

X LOSSPERGENERATION

0 . 01.1(0.0-3.0)18.5(11.9-22.19.9(13.8-25.19.0(17.6-20.22.6(11.0-41.20.3(16.7-23.21.5(17.7-24.24.9(20.2-33.

2.9(0.0-8.4)

2.8(1.8-3.7)

7)9)4)1)8 )9 )6)

AVERAGE3

COPY

NUMBERPERCELL

423(384-462)221(93-350)37(29-45)5(4-7)20(9-31)7(5-15)19(11-36)115(113-117)414(239-527)

51(7-123)

161(30-246)

82(39-128)

225(72-405)

NUMBER

OFINDEPENDENT

TRANSFORM-ANTS

ANALYSED

24442

12445

9

4

a. See Materials and Methods IX = 1x10*Minimum and maximum values are indicated in brackets after the average value

b. For the co-transformants, [plasmid] + cel l s are those which are LEIT" and TRP

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REP1 expressing derivatives a l l exhibit high l e v e l s of Instabil i ty and low

copy numbers per c e l l 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 c e l l of population and per plasmld-contalning c e l l than

the vectors based on TRP s e l e c t i o n . The data show that 2um REP3/ORI

vectors expressing either the REP1 or the REP2 gene product alone at these

l e v e l s are defective In the 2um plasmid segregation mechanism.

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

REP1 and pMA36-REP2 and those with pMA132b-REPl and pMA36-REP2, shows a

high l eve l of mitotic s tab i l i ty for the co-transformed phenotype with less

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

- -P1

-T

B

FIGURE 2 Autoradiograms of Southern blots of tota l yeast DNA preparationsdigested with EcoRI in A. and B. and Hindl l l in C. and hybridized with a2.45 kb EcoRI plasmid s p e c i f i c fragment plus a 1.8kb EcoRI ribosomal DNAspecif ic fragment in A and C and a 1.45 kb yeast TRPl genomlc DNA fragmentin B. DNA preparations are from MD4O/4C 1. [ c l r T 2. [ c i r ° ] , 3. [ c l r °PJDB219A], 4. j c l r 0 pMA36-REP2]. 5, 6. two i s o l a t e s of [ c i r ° pMA36-REP2,pMA132a-REPl] and 7,8 two i so lates of [cir° pMA132a-REPl]. R = ribosomals p e c i f i c bands, T = 1.45 kb chromosomal TRPl band, PI - pMA132a-REPlspecific bands, P2 = pMA36-REP2 specific bands. The 2.45 kb EcoRI plasmidspecific bands which have hybridized in lanes 4-8, panel A. a l l have 2.45kb of homology with the 2um-speclflc probe, the PI and P2 bands In panel Chave 2.35 kb of homology with th i s probe, whereas in Panel B, PI and P2have respectively 815 bp and 93bp homology with the 1.45 kb TRPl probe.

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than 3% loss of TRP+ LEU* c e l l s per generation of growth In non-selective

medium. Although t h i s two-plasmld system with heterologous promoters

d i rec t ing the expression of the REP1 and REP2 genes i s a very a r t i f i c i a l

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

that the plasmid segregation machinery has been reconstructed. The average

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

comparison to their l e v e l s in the yeast transformed with only one of the

plasmids alone. The average pMA36-REP2 copy number per c e l l of population

and per plasmid containing ce l l i s much lower in the co-transformants than

in the pMA36-REP2 LEU* transf ormants as one might predict s ince in the

l a t t e r case, the strong s e l e c t i o n against c e l l s with low l e v e l s of LEU

plasmid would favour the accumulation of those c e l l s with a higher plasmid

copy number which would be generated due to the absence of e f f i c i e n t

segregat ion. The pMA132a-REPl and pMA132b-REPl average plasmid copy

numbers per c e l l of population are higher in the co-transformants than in

the s ingle transformants but correcting for the proportion of c e l l s which

do not contain plasmid shows that the copy number per plasmid-bearing c e l l

i s s imi lar . This data demonstrates that re- introduct ion of e f f i c i e n t

plasmid segregation in this system does not significantly affect plasmid

copy number.

DISCUSSION

In t h i s paper, we have demonstrated that both the REP1 and REP2 gene

products are required together at a specif ic l e v e l to re-constitute the 2pm

plasmid partitioning mechanism. Cashmore et_ al (13) have previously shown

that the e f f i c i ency of part i t ion ing 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 l eve l s of the two genes

may not n e c e s s a r i l y have been the same as that indicated by the gene

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

express ion of the REP2 gene at the appropriate l e v e l a l l ows e f f i c i e n t

segregation of vectors containing REP3 and ORI but lacking functional FLP

and JD open reading frames. As has been es tabl i shed for the plasmid

amplification system, i t appears that i t i s a complex of the REP1 and REP2

gene products which mediates 2um plasmid segregation.

The results of the transformation experiments in which the REP2 gene

was overexpressed e i t h e r in the presence or absence of REP1 expression

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raise some in teres t ing questions on the ro l e of the REP2 gene product.

Transformants of the [c ir°] host could not be established for 2um REP3/0RI

type vectors expressing any l ev e l of REP2 more ef f ic ient ly than pMA36-REP2.

It i s unlikely that this ref lects a general toxicity to the c e l l of high

l e v e l s of the REP2 prote in, at l e a s t in the case of the pMA247-REP2

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

REP2 expression from the f u l l PGK promoter at one copy per c e l l . It seems

more l ike ly that the overexpression of REP2 affects the segregation of the

2um REP3/0RI based vectors . In th i s context, It i s i n t e r e s t i n g to note

that the vector pMA36 has a s l ight ly higher mitotic s tab i l i ty than i t s REP2

expressing derivative, pMA36-REP2.

Co-transformants could a l s o not be e s tab l i shed for any 2utn-based

REP3/0RI vector directing REP1 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 REP1 gene product i n h i b i t s the segregat ion and/or

replication of the 2um based vectors although the l e v e l of REP2 expressed

would be much l e s s than for pMA91-REP2. A great deal of caution must be

exercised in interpret ing the r e s u l t s from co-transformation studies In

which one i s as sess ing the e f f ec t on r e p l i c a t i o n or segregat ion of a

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

plasmid in the same c e l l . However, the data does suggest that the l e v e l of

the REP2 gene product supplied may affect the functioning of the REP1/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 l e v e l s per

c e l l of the REP2 gene product may be yet another feedback c i r c u i t in the

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

of segregation might provide a mechanism by which c e l l s with reduced

plasmid copy number could be generated from a parental c e l l 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 l e v e l s

of the REP1 and REP2 gene products i s of p r a c t i c a l importance for the

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

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

data presented here shows the f eas ib i l i ty of constructing [cir ] strains in

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which appropriate levels of KEPI 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 wi l l be of interest to determine

whether 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 -operat ive

grant and in Nottingham by MRC grant G8422850CB. M.M was i n rece ip t of an

award from the E.E.C. for t r a i n i n g i n B i o t e c h n o l o g y . We thank S. Redman

and B. Case for s e c r e t a r i a l and photographic a s s i s tance .

* To whom correspondence should be addressed.

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