Transient assay, by [3H]guanine incorporation of Escherichia coli xanthine-guanine phosphoribosyl transferase (GPT) in transfected human fibroblasts

Geve, 40 (1985) 93-98

93

Elsevier GENE

1461

Transient assay, by [3H]guanine incorporation of Escheric~ia coli xanthine-guanine transferase (CPT) in transfected human fibroblasts (Recombinant

DNA;

long terminal

repeat)

transient

expression;

promoter;

mycophenolic

acid; SV40 virus;

phosphoribosyl

Rous sarcoma

virus;

J *F . Burke”*, M.H.L. Greenb and J.E. Loweb

(Received

July 24th, 1985)

(Accepted

September

23rd.

1985)

SUMMARY

We have used [ ‘Hlguanine incorporation as a rapid and sensitive assay of x~thine-guanine ph~sphoribosyl transferase (GPT) activity in SV40 transformed human tibroblasts. The SV40 early promoter is more efficient than the Rous sarcoma virus long terminal repeat for transient expression of the gpt gene. The assay works well in a derivative of ATSBIVA which lacks hypoxanthine-guanine phospho~bosyl transferase (hprt _ ) and to work in the hprt + ATSBIVA

we show here how the assay has been adapted

parent. --

INTRODUCTION

The Escherichia coli gene gpt has been used in a number of systems as a basis for the selection of established cell lines which have taken up DNA by transfection (Mulligan and Berg, 1981). It offers

* To whom

correspondence

and

reprint

requests

should

be

addressed. CAT, chloramphenicol G, guanine; guanine

GMP,

acetyltransferase; guanosine

phosphoribosyl

transferase;

hypoxanthine-guanine monophosphate; essential

LTR,

phenyloxazolc;

repeat;

mycophenolic

RSV, Rous sarcoma

sulfate; TG, thioguanine~

transferase;

long terminal

MPA,

GPT, xanthine-

H, hypoxanthine;

phosphoribosyl

medium;

FCS, foetal calf serum;

monophosphate;

HPRT,

IMP, inosine MEM,

acid;

minimal

PPO,

X, xanthine;

XMP, xanthosine

phosphate.

0378-I 119:85:X03.30

0

2.5di-

virus; SDS, sodium dodecyl

1985 Elsevier

Science

Publishers

mono-

advantages over HPRT (Szybalska and Szybalski, 1962), thymidine kinase (Pellicer et al., 1978), and adenine phosphoribosyltransferase (Lowy et al., 1980) in that it is a dominant

marker which confers

a selectable activity lacking in normal mammalian cells. As a consequence it can be used in a wide variety of genetic back~ounds. To date, however, a simple assay for the determination of GPT activity in mammalian cells has not been available. Existing procedures require gel electrophoresis (Mulligan and Berg, 198 1) or thin-layer chromatography (Chu and Berg, 1985). CAT can also be assayed 48 h after transfection by thin-layer chromatography (Gorman et al., 1983), but cannot be used to establish stable transfectants in mammalian cells since no suitable selection system exists. In this communication we describe a simple and sensitive assay for the measurement of the E. co/i GPT activity in a human cell line transiently ex-

pressing the gpt gene. This assay relies on the uptake

containing

of [3H]G,

and its

cytidine

activity

plates

a substrate

incorporation

for the GPT enzyme,

into RNA

and DNA.

can be readily demonstrated by scintillation adapting

counting

the selection

in HPRT-negative

system of Mulligan

hprt+ human

cell line ATSBIVA.

scribed

here

makes

unique

in that

selection

activity

By

The method

de-

can be measured human

colonies. cloning

Single

between clones

100 and

were

picked

1000 Gpt’ using

glass

cylinders.

(c) Transient hrpt- cells

assay for incorporation

of [3H]G in

system

and the system can also be used for the of stable transformed

and 5 pg glycine. 20 days after transfection, contained

and Berg

in a wild-type

the gpt transfection

enzyme

cells

or autoradiography.

(198 l), GPT activity can be measured

transiently

GPT

per ml 25 pg MPA, 10 pg X, 15 c(g deoxy-

cells.

A modification

of the procedure

of Melvin et al.

(1979) was used. Cells were seeded in j-cm containing

a maximum

of four

glass

dishes

coverslips

(9 mm x 35 mm) at a density of 1 x lo5 cells/dish. After 24 h to allow the cells to settle and resume MATERIALS

AND METHODS

(a) Cell lines and plasmids ATSBIVA is an SV40-transformed fibroblast line derived from the primary fibroblast strain ATSBI, which was established from a patient with ataxiatelangiectasia (Taylor et al., 1975). It was the kind gift of Dr. L. Toji. Line 1251 was a spontaneous TG-resistant derivative of ATSBIVA, isolated by S.A. Harcourt in this laboratory. It does not incorporate detectable amounts of exogenous G, X or H and is presumed hprt . SC was a stable gpt + transfectant of 1251 isolated by JEL. 67 is a radioresistant gpt + transfectant of ATSBIVA containing a single copy of the gpt gene. Its isolation is described elsewhere (Green et al., 1985). Plasmids pSV2gpt and pLl0 were generous gifts from Dr. Paul Berg; pRSVgpt was a generous gift from Dr. C. Gorman. All plasmids were prepared using the alkaline/SDS lysis procedure of IshHorowitz and Burke (1981). (b) DNA mediated gene transfer For the selection ofgpt+ cell lines 5 x lo5 ATSBIVA cells were seeded onto 9-cm plates. Two days later DNA transfection was carried out by the calcium phosphate precipitation method (Graham and Van der Eb, 1974) using 20 pg of genomic DNA as carrier and 10 pg of pSV2gpt plasmid DNA. After 16 h the DNA-containing medium was removed, replaced with fresh medium, and 24 h later this medium was in turn replaced with selective medium

growth, 0.5 ml of calcium phosphate precipitate containing 10 pg ofplasmid DNA was added directly to the cells in growth medium. After an overnight exposure to the DNA at 37 ‘C, the medium removed was and replaced with fresh MEM + lo”,, FCS. Coverslips were removed from petri dishes with fine, sterile forceps and 20 111of MEM containing FCS and 1 pCi of [‘H]G (dissolved in HCI and neutralised in NaOH) was spotted onto each coverslip. The coverslips were then incubated overnight at 37 “C in a humidified incubator. After incubation with [ ‘H]G for 16 h, coverslips were rinsed briefly in Dulbecco ‘A’ buffer then in methanol-acetic acid (3 : 1 v/v) and then rinsed twice in ethanol. After air drying, the coverslips were placed in 5 ml of ToluenePPO scintillant and the radioactivity incorporated into the cells was measured by liquid scintillation counting. (d) Transient assay in hprt+ cells Cells were transfected as described above except that 16 h after transfection medium was replaced with MEM plus 100 pg MPA/ml and 15 big H/ml. Cells were maintained in this medium throughout the subsequent time of the experiment.

RESIJLTS

AND DISCUSSION

(a) Incorporation

of purines in human cells

Table I shows incorporation

of ‘3C-labelled

G, X

TABLE

I

Relative uptake of G, X and H by ATSBIVA Uptake

of [‘%Z]G by Four stable

pressed

as femtomol

undetectable

.4T5BIVA,

(0.05 &i/ml,

material removed Cells

of the purine

experiments

(7 for line 67 and

with

at

1PM) with or without unlabelled

approx.

were incubated lysed

immediately,

for 24 h at 37’C.

Medium

with Dulbecco

0.3 ml/plate

of neutral

‘A’ buffer. SDS

buffer

pH 7.4). Cells

were scraped

pipetted

off the plates

j”<) trichloroacetic scintillation activity

and 0.1”ml samples

3MM paper squares. counting.

approx.

International, Cell line

acid

and twice in ethanol

[i4C]G,

[‘“C]X

50 mCi/mmol)

prior

and [“‘C]H

were obtained

on to

3 times with

4

to liquid

(all specific

from Amersham

Buckingshamshire. Genotype

flprl+

67

These were washed

8

was

( 1I“, SDS. 200 mM Tris HCI, 100 PM EDTA, Whatman

-

12

After 24 or 48 h, label was

and the plates were washed

were

for the

in 5 cm dishes

(28 @M). One set of plates was sampled

the remainder

cpm. -2 x10

lines are approximate,

3 for line SC). Cells were plated

10“ cells per dish in 3 ml MEM.

added

amounts

between

since they are based on separate x

in MEM exover 24 h. The

( - ), this is less than 0.1 Y, of the value obtained

cell line 67. The comparisons

3

cell lines grown

of G per cell incorporated

cell line 125 I incorporated indicated

and its derivatives

Uptake

(fm/cell/day)

ABCD 48-72h

G

X

H

zTf)f +

1.6

0.13

0.30

transfected

Fig. 1. I!~corporation control;

SC

hJFr[-

@:Pt+

0.22

0.12

0.02

with pRSVgpt;

AT5BIVA

hprr *

0.30

0.03

irpri -

2.4 -

measured

1251 .-_

z@ -gp1--

-

and H into nucleic acids by ATSBIVA (hpvc’ gpt - ) and its derivatives 1251 (hprt- gpt-), SC (hprt- gpr + ), and 67 (hprt” gpt + ). As expected, the cells which contain the gpt gene incorporate more X than untransfected cells. Indeed this is the basis of the selection protocol of Mulligan and Berg (1981). Cells which can utilize X will grow in a selective medium in which the only source of purine is exogenous X. Untransfected ceils are unable to salvage sufficient purines from X to survive, so that X should be the logical purine with which to assay gpt gene activity. However, as Table I shows G is incorporated more efficiently in gpt+ cells than X. As [ 3H]G is commercially available at high specific activity and is more efficiently incorporated we have used this as a basis for transient expression. (b) Transient assay of GPT in hprtFig 1 shows the inco~oration

fection.

tells

of [ 3H]G into acid

of [3H]G

by 1251 (hprt _ )

(B) transfected

(D) transfected

transiently

with pLl0;

with pSV2mz.

over two different

[“WIG (specific

Amersham

ABCD 72-96h

International,

activity

periods

5 Ci/mmol)

ceils. (A) non(C) transfected

Incorporation

is

following trans-

was obtained

from

Buckinghamshire.

insoluble material following the transfection of various gpt constructs into a TG-resistant derivative of ATSBIVA (1251). Maximal activity is observed with the vector pSV2gpt (Mulligan and Berg, 1981) in which the gpt gene is under control of the SV40 promoter. A construct in which the gpt gene is under control of the RSV LTR (pRSVg~~) (Gorman et al., 1983) gives only half as much activity. Cells transfected with pLl0 which has the gpt gene cloned into pBR322 and no eukaryotic promoter sequence fail to incorporate any [ 3H]G. (c) Transient assay of GPT in hprt+ cells In cells which contain a functional hprt gene G is incorporated in nontransfected cells, because of the ability of the human HPRT to salvage G from the medium (Table I). To block the incorporation of G we have tested various components of the Mulligan and Berg selection system. Fig. 2 shows that H in

.-.:’ :-. . .. (:. ;*.:. ..:.. .‘.

.

*.

. l-l :.

: :

.’ . . . :;:.

:‘:.

:: I. I ,.

;y:;

’

I.

_:. ..., . .._. . __.. . .. ....‘.

8

. .

I *

H

.:

\

to the standard MA.1 l~KIAt.S

. .

I.(. .‘,

f.‘.

:

.‘:

..I

by non-transfected

.

,:

:

H + MPA

. ,a’

~ :.

’ . ..*

of [‘H]G

I

,..*~

Control ATSBIVA

’ ) cells (open bars) or cells 50-68 h post-transfcction

pSV2gpplir(stippled in

: . .

MPA

Fig. 2. Incorporation (heat

:. .

. : n’rcl

5 “’ ,. . .

: f. _: _. f .. .I

*: : . .*

4-

..’ *..

.‘. :

*

. .

‘* . ., .: :.. . . ‘. ’

.:. ..

..

. . . .

. . . ‘

I,

::e.

‘.

:

I.

‘..

‘.,.

with

bars). H and MPA were added or not (control)

MEM at the times and concentrations AND

METIIODS,

SeCtiOn

indicated

C.

the medium reduces the incorporation of [ ‘H]G by a factor of more than 100 in both control and pSV2gpt transfected ATSBIVA cells. However, little preferential incorporation of [3H]G in pSV2gpt transfected cells could be observed. If MPA alone is added to the standard MEM, cells transfected with pSV2gpt show a slightly higher (two-fold) increase in [“H]G incorporation over control cells. By adding both II and MPA, control cells incorporate [“H]G as they would in the presence of H alone whereas inco~oration by pSV2gpt transfected celis is ten-fold greater than controls and can be used as a reliable assay for the gpt activity in the hprt + human cell line ATSBIVA. Similar results have obtained with the SV40 transformed fibroblast line MRCSV I although in this case the control background is higher. The kinetics of expression of the gpt gene by hprt + and hprt -- cells transfected with pSV2gpt is similar, maximal incorporation being observed within 48 h of transfection. It should be noted however that the absolute amount of activity in the two systems is different, maximal activity in hprt cells being 17.5 “/, of that in hprt’ cells after a 16 h labelling period.

Fig. 3. Autoradiography WI 72-96

was for 10 days. shows

of

h post-transfection.

two

(b) part of (a) at higher

cells,

one

(c) ATSBIVA (hprr+). two days. removed

After

Kodak

incorporating

Magnification

liquid

from scintitlant,

for I-10 days

scintillation

magnification.

[‘H]G,

the

counting,

to Kodak

NTB2

(Gurr,

emulsion.

This

other

as for(b). Exposure

not.

was for

coverslips

rinsed briefly in methanol

DlY X-ray developer

and May-Gr~n~a~d

cells expressing transiently (a) Line 1251 (hprt-). Exposure

were

and exposed

After developing

in

the cells were stained with Giemsa Poole. U.K.).

(d) Autoradiograp~y

of transfected ceils

fore impossible The addition

Measurement

of gpt activity

ration is a determination activity in the population

by [3H]G

incorpo-

of the total amount of of cells. To determine

whether the activity measured

was due to a few cells

synthesizing a large amount of enzyme or a larger number of cells with less activity. coverslips with either control cells or cells transfected were taken

and autoradiographed.

show 125 1 (ATSBIVA with pSV2gpt.

From

hprt -gpt-

with pSV2gpt Figs. 3a and 3b

) cells transfected

the autoradiographs

we esti-

mate that approx. 2-5% of the cells are expressing the gpt gene. Autoradiography of ATSBIVA (hprt + ) cells transfected with pSV2gpf again gives a clear distinction between cells expressing or not expressing gpt {Fig. 3~). Incorporation is limited to about 5% of cells. No labelled cells are seen on the control slide. This suggests that H and MPA inhibit incorporation of G via the HPRT pathway very efficiently in all cells in the population and that the activity detected by scintillation counting arises from a minority of cells in the population.

by HPRT

to detect

via gpt.

G incorporation

of H and MPA blocks the uptake of G

and the conversion

of IMP derived from

H to XMP so starving the cell of G and allowing the sensitive ration

of gpt activity

measurement

by incorpo-

of [3H]G.

Using gpt+ derivatives

we have shown that this

system can be used to assay the activity of promoters tr~siently.

The ability to assay a promoter

ly and select cells expressing parison

transient-

the gene allows a com-

to be made of the activity of the same gene

extrachromosomally chromosome.

and when integrated

Potentially

the system

map sites of constitutive gene.

into the

allows one to

expression

for a regulated

ACKNOWLEDGEMENTS

We would like to thank Dr. Alan R. Lehmann Dr. R. Angus Harkness for valuable discussions advice.

and and

(ef Conclusions REFERENCES

We have shown that the dominant selectable gene gpt can be assayed simply and with a high degree of sensitivity, both in stable gpt transfectants and also transiently, using [ 3H]G incorporation. Although G is a substrate for both the HPRT and GPT enzymes, the K,,, for the GPT enzyme is several-fold lower, and uptake by HPRT but not GPT, is competitively inhibited by H. Other advantages of G are the availability of high specific activity 3H-labelled material and the negligible levels of G in FCS. X and H may be present in FCS at levels sufficient to give final concentrations in medium of l-2 pg/ml (Simmonds and Harkness, 1981). A second important point is that with hprt + cells, only those transfected with pSV2gpt will incorporate G in the presence of MPA and H. This is best explained by the fact that H is the preferred substrate for human HPRT (Table I) and if H is present in excess, the incorporation of [ 3H]G is reduced both by competition with H for HPRT and by dilution of the radioactive GMP by unlabelled GMP derived from H via IMP. In the absence of MPA it is there-

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