Contingent replication assay (CRA) procedure for rapid isolation of enhancers

29

Gene, 55 (1987) 29-40 Elsevier GEN 02022

Contingent replication assay (CRA) procedure for rapid isolation of enhancers (Recombinant DNA; protoplast fusion; shotgun DNA library; SV40 shuttle vector; monkey cell host; transfection of E. coli; papilloma virus; murine sarcoma virus)

Haren A. Vasavada *, Peter Lengyel b and Sherman M. Weissmann a Department of Human Genetics, and b Department of Molecular Biophysics and Biochemistry. Tel. (203)785-4473 University School of Medicine, New Haven, CT 06511 (U.S.A.) Received Revised

23 October 14 January

Accepted

Yale

1986 1987

1 February

1987

SUMMARY

A rapid procedure for the isolation of functional enhancer sequences consists of (a) the construction of a shotgun DNA library in SV40-based plasmid shuttle vectors which depend on an enhancer for replication, (b) the replication in monkey (CVI) cells of those vectors into which an enhancer sequence was inserted, (c) the selective cleavage of unreplicated vectors by DpnI and (d) the recovery of the replicated vectors by transfection into Escherichiu coli. We describe conditions for the fusion of protoplasts to CVI cells, under which conditions the probability of only one type of plasmid entering a cell is increased and thus complementation and rescue of enhancer-less plasmids are decreased. The effectiveness of the procedure is demonstrated by the recovery of enhancers from bovine papillomavirus and Moloney murine sarcoma virus.

INTRODUCTION

Enhancers may activate transcription when located on the same DNA segment as a promoter. Correspondence Genetics,

ro: Dr. H.A. Vasavada,

Yale University

New Haven,

CT 06511 (U.S.A.)

Abbreviations: papilloma

Ap,

ampicillin;

bp, base

(see INTRODUCTION;

RESULTS,

Fig. 1); DMEM,

Dulbecco LTR,

long terminal

murine

virus;

nt, nucleotide(s);

replication;

PEG,

Sm, streptomycin; cycline;

pairs;

modified

polyethyleneglycol;

BPV, bovine

CRA, contingent

Km, kanamycin; sarcoma

of Human

333 Cedar Street,

Tel. (203)785-4473.

virus; Cm, chloramphenicol;

tion assay

Department

School of Medicine,

Eagle medium; repeat;

replica-

section b, and d, deletion;

MSV,

Moloney

on’, origin

of DNA

R, resistant;

‘, sensitive;

SV, simian virus; Tag, T antigen;

Tc, tetra-

TE, see legend to Fig. 1; u, unit(s).

0378-I 119/87/$03.50 0 1987 Elseviet

Science Publishers

B.V. (Biomedical

They can exert this effect when inserted in the same or in the opposite orientation as the promoter, and over a considerable distance upstream or downstream (for reviews see Khoury and Gruss, 1983; Gruss, 1984; SertIing et al., 1985). Various procedures have been developed for identifying and localizing enhancers within large DNA segments. One procedure consists of the insertion of different DNA segments (one at a time) into recombinant DNA molecules with an enhancerless gene and the screening of the resulting constructs for expression of the gene (Gorman et al., 1983). This is a laborious procedure. More convenient approaches are available for recovering enhancer-containing DNA segments from random collections of DNA segments by biological selection. One of these Division)

30

approaches involves: (a) the transfection of a collection of DNA segments into cells carrying a selectable marker whose expression requires the insertion of an enhancer, and (b) subsequent selection (Ford et al., 1985). This is a slow approach since it requires the growth of colonies starting from single cells. A further approach depends on the introduction of an enhancerless, but enhancer-dependent viral genomic construct into cells together with a collection of DNA segments and recovering infectious viral variants (Weber et al., 1984). It is expected that some of these variants acquire infectivity as a consequence of the incorporation of an enhancer-containing segment from the collection of DNA segments. This procedure is time consuming since it requires pg amounts of viral DNA from the various types of infectious variants generated. Moreover, the selection pressure exerted on the viral DNA by the packaging constraints, together with the occurrence of multiple cycles of infection and amplification, may result in complex rearrangements. We have been developing a plasmid replication system for identifying enhancer sequences from large pieces of cloned DNA. Our approach (see Fig. 1) involves a plasmid shuttle vector containing a viral ori and the viral gene encoding a protein needed for replication. This gene is under the control of an enhancerless but enhancer-dependent promoter. Short, sonicated fragments of the DNA to be examined for enhancer sequences are ligated into the plasmids upstream from the promoter. The resulting plasmid library is propagated in bacteria and subsequently introduced into mammalian cells by protoplast fusion. In the mammalian cells only those plasmids can replicate into which an enhancercontaining DNA segment has been ligated. After recovering the plasmids from the mammalian cells, these are treated with the restriction enzyme DpnI (Lacks and Greenberg, 1977; Peden et al., 1980). The DpnI-treated plasmid preparation is then transfected into E. coli DHl for propagation (see RESULTS, section b, and Fig. 1). We have demonstrated the effectiveness of the approach outlined above by recovering the enhancers from two mammalian viruses, namely BPV and MSV. Since the replication of our plasmid shuttle vector in mammalian cells requires the insertion of an enhancer sequence, we call the procedure outlined a ‘contingent replication rescue assay’.

While this work was nearing completion a conceptually similar approach for isolating enhancers was reported (Tognoni et al., 1985). We compare the effectivity of the two approaches.

MATERIALSANDMETHODS

(a) Enzymes and chemicals

Restriction enzymes and linkers were from New England Biolabs and were used as recommended by the manufacturer. PEG 1000, obtained from the Koch-Light Laboratory, was purified by treatment with the mixed-bed resin AG 501 X-8 (BioRad Laboratories). Radioisotopes were from New England Nuclear. All other chemicals were either from Sigma Chemical Co. or the BioRad Laboratories. (b) Plasmids and bacteria

For the construction of pATSV40RI (see Fig. 2), SV40 DNA was cut out from the pBR322-SV40 recombinant plasmid by EcoRI digestion and inserted into pAT153 at the EcoRI site. pATSV40ASphI was constructed by complete digestion of pATSV40RI with SphI, trimming of the largest fragment with S 1 nuclease, SmaI linker addition and ligation (Fig. 2). ApSTcR pAT153 was generated by cleaving pAT153 with ScaI + SspI, and ligating the large fragment obtained (designated as pAT153 m in Table 1). Plasmids containing BPV and MSV were provided by Drs. D. DiMaio and R. Narayanan, respectively, of Yale University (Reddy et al., 1981; Chen et al., 1982; Sarver et al., 1982; Narayanan et al. 1984). Plasmid pJYM was a gift from Dr. M. Botchan (Lusky et al., 1983) and pSrm36 was a gift from Dr. L. Laimins (Laimins et al., 1984). Competent E. coli DHl cells (transformation frequency 5 x lO’/pg DNA using pATSV40RI) were prepared according to Hanahan (1983). (c) Probes

For detecting the BPV enhancer, a probe was prepared from pBPVXL, in which the &XI site at nt position 3881 had been converted to an XhoI site. As a probe for the enhancer sequences, the

31

XhoI-BamHI fragment, spanning nt 3881 to 4450 on the BPV map (Chen et al., 1982) was used. For the detection of the MSV enhancer, a 434-bp BamHI fragment was cut out from pSrm36 (Laimins et al., 1984). To generate enhancerless BPV (see Table III, column 5) viral DNA was cleaved with BamHI + XhoI, and the 7.3-kb fragment was isolated from 1% agarose gel. LTR deleted derivative of MSV was generated by cutting viral DNA with X/z01 + ClaI (Reddy et al., 1981). This 3.1-kb XhoICluI fragment, which does not contain any LTR sequences, was used as an enhancerless probe (Table III, column 5). All probes were labeled by nick translation to a specific activity of 10’ cpm/pg DNA as described previously (Maniatis et al., 1982). (d) Mammalian

cells and protoplast fusion

African green monkey kidney cells (CVI) were obtained from The American Type Culture Collection. Cos7 cells (Gluzman, 198 1) (kindly provided by Dr. Wilma Summers of Yale University) were grown in DMEM supplemented with 10% fetal calf serum, 10 u penicillin/ml and 10 pg Sm/ml. Protoplast fusion was carried out essentially as described by Sandri-Goldin et al. (1983). Briefly, protoplasts were prepared from E. coli DHl cells by treatment with lysozyme in a hypotonic medium. The cells were either exponentially growing or, if so indicated, treated with Cm for 12 to 18 h to amplify the plasmid content. Protoplast formation was monitored with a phase contrast microscope. Generally, a lo- to 15-min treatment with lysozyme was needed to convert more than 90% of the E. coli DHl cells to protoplasts. The time needed for the treatment was established separately for each batch of lysozyme used. Protoplasts were diluted to a final concentration of 2 x 109/ml in DMEM containing 10% sucrose and 10 mM MgCl,. Five ml of the protoplast suspension was sedimented on top of a monolayer of l-2 x lo6 mammalian cells in a lOO-mm tissue culture dish, by centrifugation in the TH4 rotor of the Beckman TJ-6 centrifuge at 1500 x g for 15 min. The protoplasts were fused to the cells by treatment with 50% PEG for 2 min. The cells were washed twice with Tris-buffered saline (10 mM Tris, pH 7.4, 150 mM NaCl) and DMEM (without serum) and incubated in DMEM containing 10% fetal calf serum and 10 pg Km.

RESULTS

(a) Construction

of a shuttle vector for the con-

tingent replication assay (CRA)

Since some of our experiments required more than one antibiotic marker, the pBR322 derivative, pAT153, was selected for the construction of the parental plasmid pATSV40RI (Fig. 2). pAT153 contains both ApR and TcR genes. Furthermore pAT153 (Twigg and Sheratt, 1980) also has a deletion between the ori and the PvuII site of pBR322 and thus lacks the poison sequences (from nt 1644 to 2349 on the pBR322 map). These poison sequences have been shown to impair replication in CVI cells (Lusky and Botchan, 1981). The plasmid pATSV40RI (ApRTcR) contains the entire SV40 genome inserted into the EcoRI site of pAT153 (Fig. 2). It contains all of the sequences (early region, ori, and enhancer) necessary for the replication of the plasmid in permissive cells. This plasmid also contains the sequences required for replication and maintenance in E. coli (i.e., ColEl ori and the ApR and TcR genes). pATSV40RI was found to replicate in CVI cells as efficiently as pJYM (Lusky and Botchan, 1981), a pMLl-based shuttle vector (not shown). The cleavage of the pATSV40RI (ApRTcR) with QhI (which cleaves twice within the 72-bp repeat of SV40 and once within the TcR gene) followed by ligation of the largest fragment produced the plasmid pATSV40dSphI (ApRTcS) (Fig. 2). This plasmid contains a portion of the SV40 late region, the entire early region, the SV40 ori, three 21-bp repeats and the ori-proximal 23 bp from the SV40 enhancer sequence (Fig. 2). This construct did not replicate in CVI cells to any detectable level and did not express T antigen as monitored by the indirect immunofluorescence assay. However, cos7 cells, which provide SV40 T antigen in truns, supported the replication of pATSV40dSphI (Table I, compare d and e). These results show that all c&acting functions required for SV40 replication in CVI cell are intact in pATSV40dSphI and the lack of all but 23 bp from the 72-bp repeat (SV40 enhancer) has prevented sufficient T antigen expression for the replication of this construct in CVI cells. Similar observations have been made by Lusky et al. (1983) using a PMLlbased shuttle vector.

32

DNA (virus, cosmid or PFGE fragment)

Sonication, size fractionation to about 200 bp long segments. Trimming ends with Sl nuclease and PolIk.

plasmid pATI

ori

Cleavage with Sma I and dephosphorylation. =-->---

I

1

--

Ligation transfection into E coli DHI and plating LB agar containing ampicillin.

Growth to 5 x 10’ cells/ml. Amplification and prepararation of protoplasts.

Fusion of the protoplasts at various concentrations to CVI cells.

Preparation of Hirt lysate for isolating plasmids. Treatment with I& I to cleave unreplicated plasmids.

I

Transfection EDHI

enhancer

sequences

into

Recovery of enhancer containing plasmids. Sequencing, testing. Fig. 1. Outline ofstrategy

for the CRA to rescue functional

(500 pg/ml in TE) was carried sonicated

DNA,

pBRTaqI

DNA markers.

sampies

out using Branson

at various

is described

sonifier (50% duty cycle, output

times aRer sonication,

were electrophoresed

The time point which gave the majority

control

in RESULTS, at microtip

on 1.2% agarose

section b. Sonication limit). To determine

gel alongside

of DNA in the size range of 200-1000

of DNA the size of

with pBRHael1

bp was selected

or

and the DNA

33

SV40 enhancer

+-

SV40 late genes

plasmid pAT153 ori

I

complete digestion with S&t I, to remove DNA segment 128 through 569, trimming with Sl nuclease and insertion of Sma I linkers.

S V40 ori

unique insertion

pATSV40Am

site

I

plasmid pAT153 ori Fig. 2. The rationale METHODS, is indicated

and the details

of the construction

section b and in RESULTS, by wavy line and pBR322

either on the pBR322

map (Maniatis

of pATSV40RI

section a. pATSV40dSph1,

and pATSV40dSphI

an enhancerless

DNA by a solid line. The number

in parentheses

et al., 1982) or on SV40 map (Tooze, I; LB, Luria broth (Maniatis

(b) Experimental strategy Fig. 1 outlines the principles of the CRA for the rescue of enhancer sequences. The recipient plasmid, fragments Various

were fractionated fractions

on Sephadex

from the column

gradient

in MATERIALS

AND

indicates

nucleotide fragment

location

of restriction

sites

of E. co/i DNA polymerase

et al., 1982).

pATSV40ASphI contains both the SV40 ori and the T antigen gene. The T antigen gene is located downstream from an enhancerless but enhancerdependent promoter region that contains a unique

on 1.2% agarose

electrophoresis.

are described

was used as vector in CRA. SV40 DNA

1982). PolIk, Klenow

G-100. The column was pre-equilibrated

were also monitored

plasmid

with TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA).

gel as described

For other designations

in Maniatis

see Fig. 2.

et al. (1982). PFGE,

pulse field

34

TABLE

I

Effect of protoplast

to cell ratio on the recovery

Ratio of protoplasts

of replicated

to CVI cell

plasmid a

Ratio of carrier

%Tag-positive

Number

of ApR bacterial

protoplast

CVI nuclei

colonies

obtained

to cell

transfection DpnI-cut pATSV40Rl

pATSV40dSphI

pAT153m

ApR TcR

ApR Tcs

Aps TcR

(enhancer

+ )

(enhancer

(1)

- )

(enhancer

(2)

(4)

(5)

10

0

0

10

0

1000

50

0

0

50

0

1000

approx.

1

lib

500

0

0

approx.

1

120b

ND approx.

0 1

3b

ND

0

500

0

1000

l-3

250’

5 000

0

0

5

946’

10000

0

0

10-12

1140’

20 000

0

0

15-20

1308’

20 000

0

ND

20 000

0

ND

(columns

1 and 2). Plasmids

the plasmid

DNA was subsequently

in E. coli DHl the observed controls Carrier

otherwise

(100 ~1 of competent numbers

indicated)

in loo-mm

are shown in Fig. 2.48-96

of colonies

digested

cells, transformation

for the efficiency of fusion. There was a 5-10% protoplasts

were prepared

tissue culture

from bacteria

frequency5 mixture variation

containing

: cell ratio indicated

weight DNA (Hirt lysate) was prepared,

volume for 3-4 h. DpnI treated

plasmid

x lO’/pg DNA using pATSV40RI).

(column

5). The undigested

in the number

ApsTcR

12* 1593’

dish were fused at the protoplast

h after the fusion low molecular

with DpnI in 30 p1 reaction

in total transformation

DNA

- )

(3)

a 1-2 x lo6 cells (CVI unless

by

with

pAT153

portions

of colonies in different (column

The results are presented of Hirt extracts

sets of experiments

3). For the detection

of brightly

ND, not determined;

fluorescing

nuclei using antiserum

to T antigen

raised in hamsters

(a gift from R. Carroll,

(not shown).

of T antigen,

48 h after

b Hirt extract

was prepared

from CVI cells after 96 h, and 20% of the extract

was digested

with DpnI.

was prepared

from CVI cells after 48 h, and 10% of the extract

was digested

with DpnI.

from CVI cells after 48 h, and 50% of the extract

was digested

with Dpnl.

e Hirt extract

(Miles for the

New York University).

0, no protoplasts.

c Hirt extract d Hirt extract

as

were used as

protoplast fusion, the cells were trypsinized and reseeded at a density of about 5 x 104-10s cells per tissue culture slide chamber Laboratory) and indirect immunofluorescence was performed (Banerji et al., 1981). On the average, 20000 cells were tested presence

and

is then transfected

was prepared was prepared

from cos7 cells transfected

with pATSV40dSphI

SmaI site. This site is inserted to facilitate blunt-end cloning. Sonicated fragments (of about 200 bp) of the DNA preparation to be screened for enhancers are cloned into this site and a library is prepared by transfection into E. coli DH 1 cells. The transfectants are converted to protoplasts and fused with CVI cells. In CVI cells only those plasmids will replicate which express T antigen (in consequence of the insertion of an enhancer-containing segment upstream from the promoter). After 48-96 h, a Hirt extract is prepared from the CVI cells and the plasmids are digested with DpnI. This only cleaves DNA molecules in which the dA in the recognition site of

after 48 h, and 10% ofthe extract was digested with DpnI.

the enzyme is methylated. Plasmids which replicated in Dam+ bacteria (but not in mammalian cells) retain methyl residues and are cleaved by DpnI. However, those plasmids which have also replicated in mammalian cell, are unmethylated, and are thus resistant to DpnI (Peden et al., 1980). Therefore, the treatment of the plasmids (recovered from CVI cells) with DpnI, results in the selective cleavage of those plasmids which did not replicate in the mammalian cells. After digestion with DpnI the plasmids recovered from CVI cells are transfected into bacteria. Since cleavage with DpnI diminishes the ability of the plasmid DNA to transfect bacteria, most of

35

the colonies obtained will carry plasmids with enhancer sequences. One of the prerequisites for these experiments was to establish the efficacy of DpnI cleavage of the unreplicated plasmids in Hirt extracts. In a control experiment, pATSV4O~SphI which had been replicated in bacteria, was added to a Hirt extract (prepared from CVI cells, into which no plasmids had been introduced) and was digested with f)pnI. Under these conditions more than 96% of the plasmids were eliminated, as measured by the number of colonies formed after transfection into E. coli DHl (not shown). Further, even when pATSV4O~SphI was transfected into CVI cells (in which they cannot replicate) at a high protoplast : cell ratio, the number of Dp&-resistant colonies recovered from the Hirt extract is rather low (Table Id). These results demonstrate that treatment of the Hirt lysate with DpnI effectively reduces the background of unreplicated plasmids. (c) Effect of protoplast concentrations recovery of the replicated plasmid

on

the

The method of protoplast fusion (Sandri-Goldin et al., 1983) for transferring DNA into CVI cells was selected for these experiments for the following reasons: (1) This technique does not require the isolation and purification of cloned DNA sequences prior to the transformation. (2) It may permit the delivery of several copies of a single type of plasmid to a host cell, although the culture as a whole is transfected with many different types of plasmids bearing random inserts. (3) Several reports indicate that in animal cells linear DNA is more recombinogenie than circular DNA (Dorset et al., 1985). Protoplast fusion delivers circular plasmid molecules to the mammahan cells, thus it is likely that the chances of recombination between plasmids and into the chromosomes are reduced. (4) It has been shown that protoplast fusion transfers DNA into CVI cells at a relatively high frequency (Schaffner, 1981; Russoulazdegan et al., 1982; Sandri-Goldin et al., 1983). One of the limitations of this approach is that plasmids which do not contain any enhancer sequence may also replicate in CVI cells due to complementation by T antigen produced from a co-transfected, enhancer-containing plasmid. This

deficiency can be overcome at least partially by establishing conditions for DNA transfer into CVI cells in which the chances of only a single plasmid getting into every cell is increased. To establish such conditions we varied the ratio of protoplasts to CVI cells in the fusion experiments. We obtained between 1%20% of CVI cells positive for T antigen expression at a ratio of 20000 protoplasts per cell. We observed a significant reduction in the percentage of T antigen positive cells with the decrease in the ratio and at a ratio of 10 this percentage decreased to below 1% (Table I). The recovery of replicated plasmids at various protoplasts: celI ratios followed a pattern similar to T antigen expression in the cells. As the ratio of protoplasts : cell decreased, the number of replicated plasmids (as measured by DpnIresistant colonies) also decreased. Also, our results indicate that when the protoplast : cell ratio was less than 500 the presence of carrier protoplasts (containing modified pAT153) was necessary (Table I). Probably, the presence of carrier protoplasts prevents loss of protoplasts which may occur during the fusion process. Sandri-Goldin (1983) and Rassoulzadegan et al. (1982) have reported that when the Cm amplification step is omitted no stable transformants are obtained by the protoplast fusion method. In contrast, Schaffner (1981) using transient expression assay of SV40 T antigen expression, observed only a twofold enhancement in the number of cells expressing SV40 T antigen when the arnpiification step was used. It was suggested that the higher copy number might be required to achieve stable transformation, while a few copies of a plasmid might be sufficient for obtaining transient-expression (Sandri-Goldin et al., 1983). We compared the efficacy of protoplast fusion with protoplasts prepared from unamplified and Cm-treated bacteria containing pATSV40RI. There was no significant difference, either in the number of CVI cells expressing T antigen or in the number of replicated plasmid recovered from the Hirt extract (not shown). Protoplasts prepared from unamplified cultures of bacteria had a tendency to lyse and the recovery of replicated plasmids was not reproducible. Therefore, protoplasts prepared from the amplified culture of E. coli DH 1 were used in all the subsequent experiments, The recovery of replicated plasmids at different times was also monitored. There was an increase in the number of DpnI-

36

resistant colonies obtained from the Hirt extract prepared at 96 h as opposed to 48 h (not shown).

colonies (Table II) indicating the replication of pATSV40dSphI by ‘complementation’. If one assumes that cells which received two copies of pATSV4ORI produce the same number of plasmids as the cells which received one copy each of pATSV40RI and pATSV40dSph1, then simple statistical analysis predicts that at the lower protoplast : cell ratio, a majority of cells had received a single type of protoplast and would predict that more than two thirds of recovered plasmid replicated because of their own enhancer rather than by complementation. The results in Table II indicate that our experimental conditions allow the recovery of replicated plasmids from a large excess of unreplicated plasmids.

(d) Mixed protoplast fusion

After establishing optimal conditions for protoplast fusion and recovery of plasmids replicated in CVI cells we performed reconstruction experiments to examine (i) the feasibility of recovering small amounts of replicated plasmids from a large excess of non-replicated plasmids and (ii) to observe the extent of ‘complementation’ at various ratios of two different protoplasts. For these experiments, a fvted number of protoplasts (10 OOO/cell) harboring pATSV40dSph1, (the plasmid which cannot replicate unless T antigen is provided) were mixed with amounts of protoplasts harboring varying pATSV40RI (which produces T antigen and can replicate on its own). The mixtures of protoplasts were fused to CVI cells, and 48 to 96 h after the fusion a Hirt extract was prepared and the DpnIresistant, i.e., replicated plasmids were scored by plating on Ap plates. The Ap-resistant colonies were individually picked and checked for Tc sensitivity. ApRTcS colonies indicated the presence of ‘enhancerless’ pATSV40dSphI. As the number of protoplasts containing pATSV40RI increased, there was also an increase in the number of ApRTcS TABLE

MSV

After establishing the conditions under which ‘complementation’ was minimized, we tested the proposed protocol by attempting to recover enhancer sequences from BPV and MSV. The BPV enhancer element referred to in this paper is the only autonomous enhancer known in BPV. It is the distal enhancer located between nt 4390 and 4451, about 200 bp downstream from the polyadenylation site of a set of BPV genes expressed in transformed mouse

II

Mixed protoplast Number

(e) Recovery of enhancer sequences from BPV and

fusion”

of protoplasts

Number

per cell

protoplasts

of carrier per cell

%Tag-positive

Number

of ApR bacterial

Number

nuclei

colonies

obtained

DpnIR Tcs

fection with DpnI-cut

Plasmid: pATSV40R

1

ApR TcR (enhancer

by trans-

pATSV40dSphI

pAT153m

ApR Tcs

Apa TcR

(enhancer

+)

(1)

- )

(enhancer

(2)

(3)

DNA

colonies

- ) (6)

(5)

(4)

10

10000

1000

1

15b

50

10000

1000

ND

64s

8

500

10000

1000

ND

180b

43

2

5 000

10000

1000

ND

482”

174

10000

10000

1000

10-12

970”

388

20 000

10000

1000

12-15

1120”

481

a Mixed (column

protoplast

fusion

5) were individually

was carried picked

of

out as described

in Table I, footnote

up to check Tc sensitivity

(column

a, and in RESULTS,

6); columns

section d, DpnIR ApR colonies

1-5 are described

in Table I.

b Hirt extract

was prepared

from CVI cells after 96 h and 20% of the extract

was digested

with DpnI.

c Hirt extract

was prepared

from CVI cells after 48 h and 10% of the extract

was digested

with DpnI.

31

TABLE Rescue

III of BPV and MSV enhancers” Number

Virus

of

enhancer

vector’

protoplasts

per cell

Number

colonies

enhancer-containing

containing

obtained

by transfection

colonies ’

insert other than

100

18’

2

2100d

82

22’

6

3

1839d

54

1386

of this experiment

of the radioactive

In each case 1000 protoplasts

e To detect fragment

per cell containing

from 25 plates was prepared

d Hirt extract

colonies

in RESULTS,

are described

section e, sonicated

in MATERIAL

ApS TcR plasmid

after 96 h and pooled

from five plates was prepared

enhancer-containing

is described

probes

’

1

100

legend of Fig. 1. Details

viral DNA

1232

1000 a The details and rationale

c Hirt extract

enhancers

DNA

1000 MSV

’

of

of colonies

of ApR

bacterial

with DpnI-cut BPV

Number

Number

library was prepared

AND METHODS,

section

as described

in the

C.

were used as carrier.

for DpnI digestion.

after 48 h.

out of DpnIRApR

colonies,

for BPV. XhoI-BarnHI

fragment

and for MSV 434-bp BumHI

was used in colony hybridization.

f To detect the colonies either enhancerless

containing

viral DNA sequences

BPV or MSV. Details

of these probes

other than the enhancer

sequences

are given in MATERIALS

cells (Sarver et al., 1982; Lusky et al., 1983). The MSV enhancer is the 72-bp repeat derived from the LTR of the virus (Reddy et al., 1981; Laimins et al., 1984, Narayanan et al., 1984). The selection of BPV and MSV to test our protocol was based mainly on two observations. First, enhancers from both viruses have been shown to substitute for the SV40 enhancer in a sensitive, indirect assay for large T antigen gene expression in CVI cells (Levinson et al., 1982). Second, although the BPV enhancer activity has been detected in sensitive transient and stable assays, it is weak and undetectable when assayed directly for its ability to enhance synthesis of Cm acetyltransferase from the SV40 early promoter (Lusky et al., 1983). We were interested in testing whether such a weak enhancer can be detected in our ‘contingent replication’ assay, as readily as the strong MSV enhancer. For these experiments, libraries of sonicated DNA fragments of cloned BPV or MSV DNAs were prepared in pATSV40dSphI as described (Fig. 1). Colonies from these libraries (about 4000) were pooled and protoplasts were made from the amplified culture of these bacteria. Fusions of these protoplasts were carried out in the presence of carrier protoplasts, at two different ratios of protoplasts : cell. Between 48 and 96 h after fusion, Hirt

DpnIRApR

AND METHODS,

colonies

were screened

with

section c.

extracts were prepared and DpnI-resistant colonies were obtained. These colonies were then screened for the presence of enhancer sequences using the appropriate hybridization probes (Table III and MATERIALS

AND

METHODS,

SeCtiOrl

C).

The results followed a pattern similar to those obtained from the mixed protoplast fusion experiments (shown in Table II). At a high protoplast to cell ratio there was not only an increase in the number of plasmids containing enhancers but also in the number of enhancerless plasmids (Table III). We attribute these results to complementation between two plasmids. In addition, a number of colonies did not hybridize to the viral probes and therefore might have arisen from recombination with the large excess of carrier plasmid used in these experiments. It is likely that the colonies with plasmids lacking viral sequences arose either from rearranged carrier plasmids, which although unreplicated, were resistant to DpnI, or from DpnI-sensitive carrier plasmids which escaped DpnI digestion. At any rate, colony hybridization to a single filter with radioactive viral DNA eliminated the background derived from carrier plasmids. Restriction analysis of more than 300 miniplasmid preparations (not shown) revealed that about 3% of the plasmids had undergone gross rearrangements or deletions. Rearrangement and

38

mutagenesis of shuttle vector plasmids after passage into mammalian cells has been demonstrated by other investigators also (Calos et al., 1983; Razzaque et al., 1983; Sarkar et al., 1984). Since the probe we used to detect the enhancer containing plasmids also contained 200 bp of flanking DNA, the colonies that hybridized with these probes were retested by protoplast fusion to CVI cells. In the case of BPV, both of the two enhancer containing colonies obtained at the lower protoplast to cell ratio (Table III, column 4) contained plasmids which could replicate in CVI cells, while in the case of MSV three out of six colonies had plasmids which could replicate in CVI cells. Restriction analysis of plasmids from these colonies confirmed that the plasmids which had acquired the ability to replicate in CVI cells had an insertion of a fragment containing the known viral enhancer. In the case of BPV, both the plasmid which had replicated in CVI cells had an extra BamHI site. The generation of a new BamHI site is possible only if the BPV DNA fragment spanning nt 4450 was inserted. Lusky et al. (1983) have shown that the BPV autonomous enhancer activity resides between nt 4391 and 4451 on the BPV map. In the case of MSV, the plasmid which had replicated in CVI, had an extra PvuII band of about 70 bp (not shown). Occurrence of this band confirms the presence of the MSV enhancer in the plasmid as two PvuII sites (70 bp apart) are known to occur in the MSV enhancer. The shuttle vector pATSV40dSphI did not give any band of this size, on digestion with PvuII. The DpnI-resistant colonies were selected from either the BPV or the MSV library which did not contain inserts of viral DNA (see Table III). Plasmids from these colonies were tested for their ability to replicate in CVI cells. None of these plasmid DNA preparations produced DpnI-resistant colonies and the reason for their resistance to the initial DpnI screen is obscure. Thus the contingent replication system described, allowed the identification of the known autonomous enhancer in BPV and MSV. Furthermore the identification of the relatively weak BPV enhancer by this approach indicates that this method is applicable even for screening of weak enhancers.

DISCUSSION

We have described a CRA procedure for the rapid isolation of functional enhancer sequences from large segments of DNA. The major advantages of this assay over conventional enhancer detection assays or the enhancer trap assay (Gorman et al., 1983; Weber et al., 1984; Ford et al., 1985) are the following: the contingent replication system allows the screening of a large number of DNA fragments in a single experiment, it provides rapid selection, it avoids extreme selection pressures that can select for rare rearranged molecules, and it permits the rescue of enhancer sequences in a wide size range of DNA fragments. Since the ends of the inserted fragments are random and their size may be varied, this approach allows the simultaneous identification of enhancer containing regions and the mapping of the borders of enhancers. Using this protocol it is possible to screen 40-kb DNA molecules for enhancers in 7-10 days. The experiments can be carried out with 50 tissue culture dishes, at protoplast : cell ratios that allow the enrichment in enhancer-containing segments. While the present work was in progress, Tognoni et al. (1985) published a protocol for enhancer rescue that employed the same general strategy as described here. A significant difference between our approach and that presented by Tognoni et al. (1985) is that our approach employs protoplast fusion to introduce DNA into the cells. We used conditions under which the majority of the cells receiving plasmids effectively received plasmids from no more than a single bacterium. This diminished the probability of the occurrence of complementation in which T antigen encoded by mRNA produced from one plasmid supports the replication of other SV40 o&containing plasmids in the same cell, even though the other plasmids cannot produce T antigen mRNA. The importance of avoiding extensive complementation was demonstrated in the experiments in which each cell was transfected with a high multiplicity of protoplasts. Under such conditions there was essentially no enrichment in enhancer-containing plasmids. Consistent with this result, enrichment in enhancercontaining nucleotide sequences was also obtained in the ‘expression selection’ system described by Tognoni et al. (1985) in which DEAE-dextran was used to promote the uptake of plasmid DNA to CVI

39

cells. 200-300-bp DNA fragments were generated from a 3.6-kb plasmid containing 0.9-kb fragment of hepatitis virus DNA, and were tested for enhancer activity. Out of 33 clones identified by the selection system two were found to contain enhancers. This corresponds to about 1.5fold enrichment. The enhancer recovery experiments with both MSV and BPV demonstrate the effectiveness of the CBA procedure in detecting and recovering enhancers, even when they have weak activity in the Cm acetyl transferase assay. With this procedure one can isolate enhancer-containing DNA fragments from larger segments of DNA or by two sequential cycles of transfection and enrichment. There are limitations to the approach. If enhancer elements are closely linked to negative regulatory elements only a rare fragment would separate the negative regulatory sequences and unmask the enhancer. Enhancers whose function depends on trans-acting elements missing from CVI cells would not be detected. For example, BPV is known to have a second enhancerlike sequence whose activity is dependent on the expression of a BPV product that can act in trans. Tissue-specific enhancers would generally not be detected unless tested in appropriate cells. A background of ~~~I-res~st~t plasmids that lacked inserts of test DNA was present but could be eliminated by screening the plasmids recovered from the Hirt supernatant fractions with a probe of radiolabeled DNA of the genome or fragment whose enhancers were being studied. The present method can be extended to studies of enhancer function in differentiated cell lines but each cell line may require optimization of the protoplast fusion conditions. In the case of some cell lines, it may not be possible to carry out protoplast fusion at lower protoplast : cell ratios. Some of these limitations can be overcome by making protoplasts from different pools prepared from the sonicated library and then fusing them separately at high protoplast : cell ratio. The approach of preparing a library of diverse plasmids, introducing multiple copies of a single type of plasmid into a cell, and using selective systems for rapid recovery of replicating molecules is valuable for detecting enhancers in cloned genes or viruses and has several additional potential applications. We are currently attempt~g to develop contingent replication systems suitable for detecting silencer

sequences, tissue-specific enhancers and transactive genes which regulate transcription from specific promoters and enhancers.

ACKNOWLEDGEMENTS

We thank Drs. Michael Chomey, Subinay Ganguly and Edward Schuchman for helpful discussions and for critical comments on the manuscript and Ann M. Mulvey and Anne-Marie Fink for preparing this manuscript. This work was supported by a National Cancer Institute grant CA16038-13.

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