Gene, 106 (1991) 35-42 0

1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

35

037X-l 119/91/$03.50

06052

An androgen-inducible (Steroid

hormone;

promoter,

human

eukaryote;

Ian J. Purvisa,

expression system for Saccharomyces androgen

receptor;

recombinant

DNA;

cerevisi~e

yeast vectors;

heterologous

gene expression;

regulatable

cloning)

Dipti Chotai”,

Colin W. Dykes”,

Dennis B. Lubahn b, Frank S. French b, Elizabeth M. Wilson b and

Adrian N. Hobden” ” Department of Genetics, Glaxo Group Research Ltd. Gree~f~rd, Middlesex. UB6 OHE (U.K.], and ’ Laboratories for ~eprodu&tiveBiology, DepQrt~ent of Pediatrics, ~ioc~e~~str~ and Pathology, ~ni~~ers~t~? ofNorth Carolina, School ofMediciile, Chapel Hill, NC 27599 (7J.S.A .) Tel. (OIOl-919)966-5159 Received by J.K.C. Knowles: 14 November 1990 Revised/Accepted: 12 February 1991128 June 1991 Received at publishers: 18 July 1991

SUMMARY

A novel controllable expression system for Saccharomyces cerevisiae has been developed. Expression of the gene encoding the human androgen receptor, from a strong yeast promoter, results in transactivation of a hybrid promoter carrying androgen-responsive sequences such that a target gene may be expressed in an ~drogen-dependent manner. By selection of an appropriate combination of androgen receptor level, target-gene copy number and concentration of the androgenic ligand, dihydrotestosterone, the expression level can be set within a 1400-fold range with no detectable effect on normal cell growth.

INTRODUCTION

The budding yeast S. cerevisiae is being used increasingly for the study of regulatory processes more commonly associated with mammalian cells. The relatively rapid growth rate, sexual cycle and avaiiability of a range of plasmids and promoters have made S. cerevisiae a powerful tool for

Correspondence to: Dr. Greenford

I.J.

Rd., Greenford,

Tel. (44-81)422-3434; Abbreviations:

aa, amino

androgen

receptor;

mammary

tumour

Glaxo

Group

Research

Ltd.,

UB6 OHE (U.K.)

Fax (4481)864-7118.

BGal, &-gaiactosidase; estrogen-responsive

Purvis,

Middiesex,

acid(s);

ARE, androgen-responsive

element;

ERE,

bp, base pair(s); DHT, dihydrotestosterone; element;

RAR, gene encoding

kb, kilobase

hAR,

or 1000 bp; MMTV-LTR,

virus - long terminal

oligodeoxyribonucleotide;

PBS,

NaC1/3 mM

KH,PO,/8

KCtj1.5 mM

hAR;

repeat; mM

mouse

nt, nucleotide(s);

phosphate-buffered Na,HPO,

saline

human oligo,

(150 mM

pH 7.3);

PGK,

phosphoglycerate kinase; S., S~~~haro~~ces;SDS, sodium dodecyf sulfate; (d)UAS, (deletion of) upstream activation sequence; wt, wild type.

molecular and cell biologists. However, the use of this organism has been limited to some extent by the lack of tightly controllable expression systems. Although a number of inducible promoters are available, e.g., GAL1 ,lO (reviewed in Johnston, 1987), PI!!05 (Meyhack et al., 19X2), CUP1 (Butt et al., 1984), the changes in growth media used to induce transcription can cause profound effects on cellular metabolism. Furthermore, with most of these systems it is difficult to obtain, reproducibly, intermediate levels of expression between the basal and fully induced levels. Recently, Picard et al. (1990) described the use of the rat glucocorticoid receptor in a yeast expression system which could be controlled by the addition of dexamethasone. A number of other soluble nuclear receptors have also been shown to function in yeast (Metzger et al., 1988; Mak et al., 1989; McDonnell et al., 1989). The aim of present study was to develop a controllable expression system based on the use of the human androgen receptor such that by balancing the level of androgen receptor expression, androgen concentration and copy number of

H+S

I

M13mpPGKAUAS

I

P

t

N NhSrn

t

lb71 1

El

O,,

a

HCS

I

N NhSrn

t

‘rxT,,;+s Li

r

lb61

lnrs

Fig. I. Construction

by transferring

and transformations

the BglII-Sal1

boxes denote PGK promoter

the hGH mRNA

plasmid,

(b6) YIp5 (Struhl

enhancer

at the normal

transcriptional

of ARE positioned

in the MMTV-LTR

UAS site by insertion

pATPGKare-lucZ, and Davis,

(B) Construction

(Geitz

of reporter

renders

1988) digested

induction.

region was transferred

of all three

(Geitz and Sugino,

1988) all digested

was inserted,

to produce

carried

of a was in-frame, with the same enzymes.

was placed

into the BamHI

the vector pATPGKareL.

on M13mpPGKare

used were multiples

construct,

A modified PGK promoter,

vector

hAR was created

The Hind111 to Sal1 fragment

pATPGKare (Pharmacia) constructs.

construct from pMC1871 types of S. cerevisiue reporter-gene

fragment

(a5) An with SmaI + SalI. to a yeast 2~ shuttle

sequences PGK promoter

site. The ARE (Ham et al., 1988). (b3) The modified

initiation genes available pPGK-hAR.

attached

(a6) An androgen-inducible

and Sugino,

gene for androgen of an oligo linker at the BamHI

The BamHI

1984) or (b8) YEplac195

was used as the progenitor

both hAR [see part A (as)] and lucZ coding sequences.

et al., 1979) (b7) YCpSO (Johnston

The resultant

site of pATPGKareL.

gene. This promoter

by transformation.

to YIplac204

translation

site in its place, was cloned from pMA766 (Ogden et al., 1986) into M 13mp18 (bl) between the EcoRI and Sal1 sites. (b2) The resultant

as an androgen-dependent of receiving

into plasmids

from pPGK-hAR

from region

et al., 1990). This places the hAR gene under

region altered to provide optimum

(see part B). (a7) The promoter//&R

vector pPGKare-hAR.

pATPGKareL

by that from the GALlJO

hAR coding

fragment

by Bg111 sites provided

the complete

into the unique Bg/II site of pMA91 (Mellor et al., 1983) producing

at the NcoI site followed

fragment

rrpl locus of BJ1991 by linearisation

the EcoRV-Sal1

was placed

contains

EugI-XhoII

both 5’- and

in this bacterium, of regenerating

of the MR gene bounded

pCMVARcom

coding sequence

into pAT153 between its EcoRI and Sal1 sites. (b4) A short oligo linker was placed into the unique BglII site ofresultant

to function

(b5) This plasmid was now capable

then introduced

15-bp region observed

then had a triple arrangement

lacking the normal UAS but with a unique BamHI

M13mpPGKdUAS

into mutant

was made by transferring

1988) using EcoRI (partial digest) and Sal1 to produce

Geitz and Sugino,

(YEplacl81;

was integrated

construct

the complete

in the linker. Plasmid

et al., 1980) and the 5’-untranslated

UAS has been replaced

the Bg111 fragment

where the normal

from PAT-hAR into the unique BglII site of plasmid

pPGK-hAR1

of the BglII fragment

construct,

form of this expression

by insertion

The resultant

plasmid

integrating

sites present

was cloned into the unique BglII site of pKV49 (Cousens

codon bias (Grantham

the hAR coding sequence

(pPGKgal-hAR),

containing

for maximal

(a4) By a similar manipulation,

of a modified PGK promoter

induction

to galactose

and glucose repression.

transcriptional

control

1987). (a3) The Bg/II fragment

(Cigan and Donahue,

13 aa had been restructured

using the EagI and BamHI

pPGK-lacZ Sites suffixed by an

plasmids replication the internal

box) capable

gene (b/u), and the origin of plasmid (pAT153L)

Control

E2, gene coding sequences,

B2, BglII; E, EcoRI;

respectively.

plasmids.

and pKV49,

(al) An oligo linker (hatched

1980). (a2) Into this plasmid

plasmids.

pMA91

various

et al., 1982). B, BarnHI;

sites upon the various

sites in vectors

the E. coli b-lactamase

of hAR expression

(see Maniatis Xm, XmnI. Blank boxes represent

point to restriction

et al., 1988). Plasmid PAT-hAR now contained

was inserted

conditions

into the homologous

Short arrows

sites of pAT153 (Twigg and Sherrat,

poly(A) region. (A) Construction

EdI1 site to the 3’-XmnI site (Lubahn

pCMVARcom,

the N-terminal

from the upstream

plasmid,

from pATPGKare-lacZ

both wt or modified.

using standard

Sp, SphI; Ss, SstI; X,XhoII;

were completed

ApR and ori are used to represent

fragment

sequences,

of the hAR gene was placed between the HindIII-EagI*

hAR expression

sequences

denotes

by the linker; however,

stretching

were constructed

whilst hGHpolyA

the mammalian

3’-coding

ligations

denote the loss of that site upon ligation. The abbreviations

respectively,

asterisk

and pPGKgal-lacZ

All DNA digestions,

N, NotI; NC, NcoI; Nh, NheI; P, PsrI; R5, EcoRV; S, SalI; Sm, SmaI;

of plasmids.

M,MluI;

both wt or modified. The blackened

Eagl; H,HindlII;

38 the cassette carrying the responsive promoter, expression of the target gene could be set at a desired level within a wide range.

RESULTS

kDa

AND DISCUSSION

Expression of human androgen receptor in S. cerevisiae was assessed by Western blot, in vitro band-shift and ability to transactivate a hybrid MMTV androgen response

PGK promoter carrying elements (Table I).

-69

the -46

(a) Western blots A band co~esponding around

to an immunoreactive

95 kDa was visible

in Western

protein

of

blots of cell-free

extracts, from galactose-grown cells, containing the pPGKgal-hAR (Fig. 1A) plasmid (Fig. 2, lane 3). This size was determined by comparison with protein size standards (rainbow markers, Amersham) loaded onto the gel. This band was not visible in glucose-grown cells containing the same plasmid (Fig. 2, lane 2) nor in host cells alone (Fig. 2, lane 4). The 95-kDa protein was also detected in extracts from cells carrying the 2~ pPGK-hAR plasmid (Fig. 2, lane 1). This immunoreactive material was present both in the supernatant and pellet fractions from cell-free extracts (after a 30 min 100000 X g spin) in approximately equal qu~tities, but was not present in galactose-grown cells containing the host vector, pMA91 (data not shown).

-30

-21

-14 Fig. 2. Expression of&R, in S. cerevtiiae as assessed by immunot blotting. Stationary-phase yeast cells were diluted l/t00 either in synthetic selective medium or rich broth,

using either 2”; glucose (YPD) or 2”,, galac-

tose (YP-GAL)

as carbon

source,

were harvested

at A,,,

were washed by resuspension from Oxoid, trifugation,

(b) Band-shift assays Cell-free extracts from yeast containing hAR expression plasmids were assayed for their ability to bind to doublestranded oligo linkers (67 bp) carrying copies of the ARE from the MMTV-LTR. Protein/oligo complexes should have reduced mobility in polyac~lamide gels relative to the free oligos. Using this assay system, no shift in the mobility of the 32P-labelled oligos was detected after incubation with extracts from host cells alone (Fig. 3, lanes 1 and 6) or from glucose-grown cells carrying the pPGKgal-hAR 2~ plasmid (Fig. 3, lanes 7-10) even in the presence of steroid ligand. However, significant retardation of the linker sequences was observed in galactose-grown cells carrying the above plasmid (lanes 2-5). The observed band shift occurred in the absence of the ~drogenic ligand DHT (lane 5) and the addition of DHT to 100 nm had no apparent effect on the binding (lanes 2-5 and 7-10). The band shift was not observed using extracts from galactose-grown host cells alone (i.e., without an /zAR expression plasmid) and could be almost completely abolished by the addition of a ten-fold excess of unlabelled ARE oligos but not by non-specific DNA. Labelled, dimeric ERE oligo, from the gene encodingxenopus laevis vitellogenin, did not show any retardation with respect to any extract even when either DHT or

-0.8

according

and grown

by low-speed

in 0.5 vol. PBS (prepared

to the manufacturer’s

the cell pellet was suspended

assays) or 0.1 M Tris

overnight

HCl (for Western

protein

was loaded

onto

Cells and

After recen-

blots). An equal volume of glass

treatments,

was vortexed

twice

to cause cell breakage

from the lysate by centrifu-

the cell-free extract.

a 0.1%

x g)

using PBS tablets

instructions).

Glass beads and cellular debris were removed gation at 14000 x gtoproduce

(500

in l/20 vol. of PBS (for enzyme

beads (BDH, 0.4 mm) was added and the suspension for 30 s, with cooling on ice between

at 30°C.

centrifugation

SDS-127,

Approximately

polyacrylamide

10 pg gel after

5 min boiling in Laemmli buffer [250 mM TrisiS?; (w.‘v) SDS/lO?c, (w/v) bluei2”; (w/v) ~-mercaptoethanoi].

glycerol/O.01 “/, (w/v) bromophenol Electrophoresis 0.1 “;,/(w/v) librated

was in SDS-glycine

in 25 mM Tris/193

30 min at 4°C. The proteins by electroblotting agent

buffer [SO mM Tris/25

mM glycine

SDS pH 7.51 at 100 V for 30 min. The gel was then equi-

overnight

gelatin/0.2%

mM glycine 20% (v/v) methanol

pH 7.5 for

were transferred

membrane

to Immobilon-P

at 100 V for 1 h. The filter was then soaked in blocking at 37°C

[SS;, (w/v) bovine

(w/v) Na. azide, all dissolved

in 100 ml of PBS/Tween-20

[O.l”;

30 min in a 1 :500-fold dilution

serum

albumin/2”,,

in PBS].

(w/v)] the filter was incubated

of hAR antisera

(w/v)

After three washes

(Lubahn

for

et al., 1988).

This was followed by 3 x 200 ml washes for 20 min each in PBSTween [O. I”, (w/v)] at room temperature. The filter was gently shaken in antirabbit

antiserum

conjugated

to horse radish

peroxidase

for 2 h at room

temperature. Again, 3 x 200 ml washes of PBS/Tween-20 were performed for 20 min per wash and then substrate added

to develop

the blot [I mM MgCIZ/lOflM

ZnCl,iin

(0.19, w/v) solution was 0.1 M Tris

pH 8.6, containing a-naphthol As-Mx phosphate (Sigma N-5000) fast blue (Sigma F-0500) both at 1 mg/ml]. Once colour development complete

the filter was rinsed in water and allowed

to air dry.

and was

39 TABLE

I

Summary

of expression

and reporter

constructs

used in this study”

Parental

Replication

plasmid’

element d

pPGK-hAR

pMA91

2p

Leu

GLUf

pPGKgal-hAR

pKV49

2p

Leu

GALTGLUJ

pPGKare-hAR

YEplac181

DHT?

Ylplac128

2p integrated

Leu

pPGK-hAR1

Trp

GLU t

pPGKare-lurZ

YEplac195

DHTt

YCpSO

2n CEN4

Ura

pPGKare-1urZC

Ura

DHT 7

pPGKare-lucZ1

YIPS

integrated

Ura

DHTt

pPGK-1acZ

pMA9

2P

Leu

GLU t

pPGKgal-lacZ

pKV49

2n

Leu

GAL t GLU 1

Plasmid’

of S. cerevisiae BJ1991

a Transformants supplemented

1

‘SD’ synthetic

Selection’

prbl-1122,

(a pep4-3,

minimal medium (Sherman

Induction’

ura3-52, leu2, frpl, GAL) were grown

in shaken

liquid culture

ofthe GALIJO promoter,

et al., 1981) at 30°C. For induction

either

in YPD or in

cells were pre-grown

in SD medium

to select for plasmid maintenance, then transferred to YP-GAL medium (or YPD, as control), and were further incubated for three to four generations to an A,,,, of around 1.0. E. coli strain TGl [K-12, d(luc-pro), supE, fhi, hsdD5 [F’ truD36, proA+ B’, IucP, lacZdMlS] was used during plasmid constructions. ligations

These cells were grown at 37°C in Luria broth or agar, supplemented of E. coli, were performed

and tranformation

Beggs (1978). h Plasmids were named according

essentially

to the following criteria:

as described

with ampicillin

by Maniatis

first the basic promoter

(50 ng/ml) as required.

DNA manipulations,

et al. (1982). Yeast cells were transformed

is denoted

including

by the method

(PGK) followed by the UAS, if different

of

from the wt (either

gal or are), and then by the transcript (either IucZ or hAR). A further addition (either I or C) indicates the plasmid yeast replication method when a 2~ replicon was not present. ’ Plasmids pMA91 and pKV49 were supplied by S. Kingsman (Cousens et al., 1990; Ogden et al., 1986). YEplacl81, YIplac204 and YEplac195 were gifts from R.D. Geitz (Geitz and Sugino,

1988). YCp50 (Johnston

and Davis,

1984) and YIp5 (Struhl

et al., 1979) are widely available

general

cloning

vectors. ’ Either 2~ multicopy recombinant

’ The recombinants inability r Arrows

replication

constructs

were selected

to grow on synthetic indicate

whether

on media containing

1

2

CEN4 (low copy number)

origins,

or integration

of the plasmid

into the chromosome

were used to maintain

the

in the cell.

(GLU)

4

5

using either the URA3 or LEU2 prototrophic

in the absence

transcription

glucose

3

and maintained

medium

of the respective

from the promoters

or galactose

6

7

(GAL)

8

aa addition,

listed is induced

as sole carbon

9

(upward

source,

marker

gene. Loss of plasmid

would lead to the cell’s

i.e., either uracil or leucine. arrow)

or by growth

or repressed

(downward

on media containing

arrow)

by growth

of host cells

DHT.

P-estradiol was added. Also unlabelled ERE could not compete ARE band-shifting in extracts with hAR synthesis (data not shown). Thus, the band shift assay indicated that functional hAR was present only in cells carrying the expression system, after induction by galactose.

10

-

cell-free extracts

were prepared

MR after growth containing

medium.

double-stranded,

(lanes

The extracts

ARE-containing,

presence

or absence

mixtures

were then analysed

lowed

from BJ1991 cells harbouring

on glucose-

7-10)

were incubated oligo-linker

of the androgenic

by autoradiography.

or galactose-

by polyacrylamide DHT

was included

2-5)

with a 32P-labelled,

(shown

ligand

pPGKgal(lanes

DHT.

in Fig. 1B) in the The incubation

gel electrophoresis in the incubations

folat

100 nM (lanes 2 and 7), 10 nM (lanes 3 and S), 1 nM (lanes 4 and 9). The Fig. 3. Binding

of hAR, synthesised

in S. cerevisiue, to the ARE. Band-

samples

shown

in lanes 5 and 10 received

shown in lanes 1 and 6 were prepared

shift assays were performed essentially as described by Klein-Hitpass et al. (1989) for the estrogen receptor except that, for some experiments,

MR expression

a polyacrylamide

the lanes, is due to a small amount

annealed

gel concentration

MMTV hormone-responsive

for polynucleotide

was 3.5 % used instead

of 4%

The

element was used as the substrate

kinase (BCL) in the presence

of [y-3ZP]ATP.

Soluble

plasmid.

The uppermost

gel. The arrow indicates the position tration of retarded material.

no DHT

and the samples

from cells which did not carry the band, clearly visible in most of

of free oligo which failed to enter the within the gel of the largest concen-

40 (c) Transactivation

of reporter genes

24

,

The PGKare-laczcassette carried on plasmid pPGKarelacZI was integrated into the S. cerevi~j~e genome at the ura3-52 locus. The resulting strain was then transfected with hAR expression plasmids. Cells carrying pPGKgalMR were grown either in glucose or galactose in the presence of different concentrations of the androgeni~ ligand DHT, then assayed for BGal activity. Cells which had been grown on galactose to induce hAR expression, exhibited a marked DHT-dependent increase in activity (Fig. 4A). No such increase was apparent in cells in which expression of the receptor had been repressed by growth on glucosecontaining medium, and in the absence of DHT only very low levels of activity were detected in cells grown on either carbon source. Using this strain, PGal levels could be varied over a 226fold range (Table II). Intermediate levels of activity could be obtained reproducibly by addition of an appropriate concentration of DHT. The kinetics of DHT-mediated induction was studied using a BJ 199 1 strain carrying integrated copies of both the PGKare-facZ and PGK-MR cassettes. The response to a fixed concentration of DHT, over a 7 h period. is shown in Fig. 4B. Enzyme levels began to increase 30 min after exposure to DHT and continued to increase for about 6 h. The increase in enzyme leveis was preceded by a corresponding increase in lacZ mRNA levels (data not shown). The effective range over which /?Gal activity could be varied could be increased further using different combinations of plasmids. The highest levels ofj3Gal activity were obtained from cells which contained the PGKare-lacZ and PGK-hAR expression cassettes carried on compatible 2~ plasmids (Table II, line 9). Sufficient BGal was produced for the protein to be clearly visible when the cell-free extracts were analysed by SDS/polyacrylamide gel electrophoresis using Coomassie-blue staining (data not shown). In this case the background (uninduced) level of activity was much higher than with the integrated PGKare-1acZ construct, and only a 49-fold induction was obtained. Similar results were obtained when the androgen receptor was synthesised from the PGKare promoter carried on a 2~ plasmid (Table II, line 10). There was a clear relationship between background (i.e., no DHT) PGal activity and copy number of the reporter gene (Table II, lines 1-3). When the PGKare-lacZ cassette was carried on the centromeric plasmid pPGKare-bcZC (l-3 copies per cell), the uninduced enzyme levels were about twice as high as those observed with the integrated pPGKare-lacZ1 construct (1 copy per cell), and about 14-fold lower than those obtained using the 2~ pPGKarelacZ plasmid (30-50 copies per cell). The highest fold-induction (607-fold, Table II, line S} was obtained using a strain carrying an integrated copy of the PGKare-lacZ construct (to achieve a low background

(mins) Fig. 4. Androgen response:

expression

gene and the multicopy minimal tionary

medium phase

the integrated

pPGKgal-hAR

supplemented

at 30°C.

the PGKare

from

BJ1991 cells carrying

promoter.

plasmid

were grown

with tryptophan

too-pi

samples

(0)

as the carbon

indicated.

Cultures

were grown

formed on cell-free extracts induction: carrying

source

(at 50 fig/ml)

an integrated

and the constitutive from samples

at the concentrations

and /?Gal assays

copy of the reporter

construct

cassette

culture

of

of BJ1991

(pPGKare-[ucZ

(pPGK-hAR1).

fiGal levels were determined

taken at different

were per-

c. (B) Time course

to an exponential

hAR production

was on YPD with no selection. extracts

overnight

to staculture

either glucose (m) or

plus DHT

see Table II, footnote

10 nM DHT was added

reporter in synthetic

of the stationary-phase

were diluted into 10 ml of fresh medium containing galactose

(A) Dose-

pPGKare-/ncZI

times after ligand

1)

Growth in cell-free

addition.

level) and the 2~ pPGKare-MR plasmid (to obtain high levels of androgen receptor), although in this case the fully induced expression ievel was lower than that obtained using combinations of 2~ vectors. Intermediate levels of induction were obtained using different combinations of constructs for expression of the reporter gene and androgen

41 TABLE

II

Comparison

of BGal levels obtained

Plasmid reporter

using different Receptor construct

and gene”

promoter-lucZ

h

constructs

Uninduced

Max. induced

Fold

level

level

induction’

(units/mg)

(units/mg)d

(-DHT)’ 1

pPGKare-/acZI

-

0.07 * 0.00

2

pPGKare-lucZC

-

0.13 * 0.02

3

pPGKare-IucZ

-

1.76 + 0.28

NA

NA

4

pPGK-1acZ

-

58.52 f 0.31

NA

NA

5

pPGKgal-facZ

-

0.29 + 0.05

65.81 + 0.92

227

6 1

pPGKare-IacZ

pPGK-/&?I

0.06 + 0.01

6.52 + 0.01

109

pPGKare-lacZ1

pPGKgal-hAR

0.08 + 0.02

18.12 f 0.04

226

8 9

pPGKare-lacZ1

pPGKare-hAR

0.06 k 0.01

36.41 f 0.05

607

pPGKare-facZ

pPGK-hAR

1.71 2 0.03

84.21 f 3.62

49

10

pPGKare-lacZ

pPGKare-hAR

1.71 k 0.08

77.84 f 0.01

46

L1Plasmid

names

h Expression

I

are described

c fiGa activity was determined extracts

were measured

extract

was adjusted

KCl/I mM MgSO,/50 incubated Absorbance

in Table I, footnote

of the hAR gene was achieved using a commercially

was measured represent

’ Enzyme

assays

determination

averaged

yellow colour

/lGal activities

for BGal activity by dividing

had developed,

kit (BioRad)

according

in Fig. IA. plasmids.

= growth enzyme

assays

as described

on YPD; induced activity

curve derived

on extracts

in footnote = growth

by the background

was terminated

enzyme

was produced

of 500 ~1 of

PGal enzyme isolates

level of the equivalent

mM

1 M Na,CO,.

(Sigma),

The figures

of each strain.

in YP-GAL reporter

.2H,O/l

Sigma). The tubes were

by cell growh in the presence

and 7 (cells grown

ofcell-free l-25 ~1 of cell

mM NaH,PO,

(N-1227,

by the addition

from at least four independent

on YP-GAL)

instructions.

.7H,0/40

using commercially-available

c. Induction

Protein concentrations

to the manufacturer’s

(60 mM Na,HPO,

Z buffer

at which time the reaction

to a standard

from duplicate

were performed

the induced

as described

ofhAR expression

pH 7.0) and 67 ~1 of 4 mg/ml of o-nitrophenyl-p-D-galactopyranoside

at 420 nm and was compared

in YPD except for lines 5 (uninduced ’ As measured

protein

promoters or presence

to 50 @I with PBS and mixed with 300 ~1 of prewarmed

at 37°C until a distinct

presented

PGK

after growth in the absence

available

mM /%mercaptoethanol

1 NA

b.

using either wt or modified

in cell-free extracts

0.08 f 0.01 NA

of 100 nM DHT

with or without

DHT).

gene.

NA, not attempted

receptor

(Table II, lines 6,7). Thus, by varying DHT concentration and the copy numbers of the androgen receptor and target gene, /3Gal activities could be set reproducibly within a 1400-fold range (0.06 to 84.21 units; Table II, lines 6 and 9), up to levels comparable to those achieved using PGK promoter derivatives to drive expression of the IacZ gene directly (Table II, lines 4 and 5).

galactose induction mechanisms even incorporating the recently-described regl-50 1 mutation (Hovland et al., 1989). There is no requirement for the control of carbon source and the steroid ligand has no observable intrinsic biological effect upon the yeast cell at the concentrations used.

(d) Conclusions (1) Human androgen receptor can be expressed in S. cerevisiae in a form capable of activating transcription from a promoter carrying androgen-response elements, in an androgen-dependent manner. (2) By varying the ligand concentration and copy numbers of the receptor and target genes, the level of expression from the androgen-inducible promoter can be set reproducibly within a 1400-fold range. The maximum expression level is comparable to that obtained using the wild-type PGK promoter. The controllability of this system should be invaluable in studies involving the expression of proteins detrimental to cell growth or where stoichiometric protein interactions are studied in yeast. The system as described has significant advantages over the most commonly used

ACKNOWLEDGEMENTS

We gratefully acknowledge the kind gifts of plasmids YEplac181, YIplac128 and YEPlac195 from Dr. R.D. Geitz, and pMA91, pKV49 and pMA766 from Dr. S. Kingsman. The preparation of pKV49 was performed under the auspices of the LINK Eukaryotic Genetic Engineering Programme at the Oxford Centre for Eukaryotic Genetic Engineering.

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