Comparative studies on transcription in isolated nuclei. Effect of homologous and of heterologous cytosol

Vol. 74, No. 3, 1977

BIOCHEMICAL

COMPARATIVE

STUDIES

EFFECT

AND BIOPHYSICAL

ON TRANSCRIPTION

OF HOMOLOGOUS

RESEARCH COMMUNICATIONS

IN

ISOLATED

AND OF HETEROLOGOUS

Christine

NUCLEI.

CYTOSOL

Bastian

Abteilung fiir Medizinische Molekularbiologie Klinisch-Theoretischen Zentrum der Medizinischen Hochschule, Ratzeburger Allee 160, D-2400 Liibeck

Received

December

im (G.F.R.)

13.1976 SUMMARY

In-vitro transcription of cell nuclei isolated from normal rat liver and from a rat hepatoma is stimulated when the nuclei are incubated in homologous cytosol. This stimulating effect can be increased in both types of nuclei by using cytosol from regenerating liver. Isolated

nuclei

preserve

synthesizing pends

RNA

on

vely

the

high

obtained sol

by

of

study

in

adding

rate cytoplasm

eucaryotic

an

important

reports

on

from

normal

and

regenerating

ation

rat

hepatoma

nuclei

were

plasm)

derived nuclear

the

incubated.

RNA

synthesis (3).

might

nuclei in

transcription

are

medium

a system

role

By be

and

deA relatican

be

using

cyto-

appropriate

to

cytoplesm

controlling

described

permits

in-vitro

by Academic in any

of reproduction

the

in

same

Press, Inc.

form reserved.

tissue being et

of

with

cell

and

from

9121).

cytosol

then

McNamara

synthesis

liver

hepatoma

synthesis by

done rat

either

from RNA

experiments

(Morris

incubated

system

0 1977

RNA

for

which

gene

expression

nuclei

isolated

cells.

paper

Copyright All rights

to

between

This

the

nuclear

capability

this

nuclei

of

such

their

of

the

interactions play

time

extent

which

origins,

in-vitro

some

The

prolonged

different

apparently of

(1,2).

medium

and

for

al.

pre-mRNA

1109

In (=

or

in

comparative

studies cyto-

heterologous

was as

devi-

particle-free

measured. (4)

a minimum

well

The

cytosol, cell-free

applied as

of

because

it

rRNA.

This

Vol. 74, No. 3, 1977

system

contains

nents who

in use

sulfate

BIOCHEMICAL

no

contrast

(NH4)2S04 to

a medium

with

in

to

Preparation

order

of

cell

conditions relatively

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

or

other

non-physiological

prefered high

by ionic

activate

RNA polymerase

MATERIALS

AND METHODS

other strength

compoauthors and

(5,6) ammonium

II.

nuclei

In principle the method of Pogo et al. (7) was applied. Nuclei were isolated from the liver of female Wistar-rats (weight 140-160 g) and from Morris hepatomas 9121, which had been transplanted to male AC1 rats (inbred animals, weight about 250 g) into both upper thighs, 4-5 weeks after transplantation the tumors had a diameter of about 4 cm. The fresh tissue was homogenized in 0.32 M sucrose, 3 mM MgCl (Potter-Elvehjem); centrifuged for 7 min.at 800 g; the sedimegt was resuspended in 2.4 M sucrose, 1 mM MgC12 and centrifuged for 1 hour at 55,000 g. The sedimented nuclei were washed twice in a solution containing 0.25 M sucrose, 4 mM MgCl and 0.01 M Tris-HCl (pH 8.3) and subsequently kept in this'medium until being used for the incubation experiments. An aliquot of the nuclear suspension was measured in a Coulter Counter for estimating the number of nuclei per ml. Cytoplasm The various tissues (normal and regenerating rat liver, Morris hepatoma 9121) were homogenized (w/v = 1 : 1) in a solution containing 0.2 M Tris-HCl (pH 8.01, 0.05 M MgCl and 0.03 M B-mercapto-ethanol. The homogenate was centrifuied for 15 min. at 12,000 g, the supernatant again centrifuged for 1 hour at 105,000 g. The 105,000 g - supernatant (= cytosol) was dialyzed overnight against distilled water. Protein content was estimated according to (8). In the case of regenerating rat liver the animals were killed 24 hours after partial hepatectomy (9). RNA

synthesis

Nuclei were incubated in a shaking water bath at 30°C. Total volume per tube 0.5 ml, containing 6 - 8 x 107 nuclei; together with 50 mM Tris-HCl (pH 7.51, 2.5 mM MgC12, 2.0 mM dithiothreito1, 0.5 mM CaC12, 0.3 mM MnC12, 5.0 mM NaCl, 2.5 mM Na HP04, 5.0 mM spermidine, 2.5 mM ATP, 2.5 mM phosphoenolpyruva 2 e, 0.5 mg/ml yeast-RNA, 17.5 units pyruvate kinase, dialyzed cytosol (7 mg protein/ml), 1 mM CTP, 1 mM GTP, 0.4 mM UTP, 25 ul [14C]UTP (60 mCi/m mole); essentially as described by (4). At various times of incubation aliquots of 0.05 ml were taken, precipitated with 2 ml of 10 % T t40n Millipore filters and washed (5 x) with 3 ml of 5 % TCA. C -activity of the filters was measured in a "Low-Background GM-Counter". The chemicals used were purchased from E. Merck, Darmstadt; a-amanitin and non-labe ed nucleoside triphosphates from Boehringer, Mannheim, [ “cJ UTP from Amersham-Buchler, Braunschweig.

E 1

1110

BIOCHEMICAL

Vol. 74, No. 3, 1977

I

I

0

10

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

/

30

20

LO

50

6'0

minutes i:g;Oi; 0 rat

Incorporation .kinetics. Rat liver nuclei , A with rat liver cytosol and a -amanltin rat liver cytosol, 0 with cytosol from

with liver.

incubated (1 pg/ml), regenerating

RESULTS The of

described soluble

cytoplasm

nuclei. and In

system

Nuclei from

was on

were

the

leads

to

nuclei.

Replacement

of

albumin

causes

incorporation when no

(~25

nuclei proteins.

merase

block

(at

are addition

of isolated 3OOC.1,

corporation

incubated

involved

RNA

of

liver

while

in

is

already

still in

in

increase cytosol

of

isolated

from

(10);

normal

RNA from

obtained

pg/ml)

Morris

1111

equivalent of

medium

as

well

the

amount

['4C1UTPplace

containing

as RNA polyin-vitro,

causes

a partial 60

continues hepatomas

30 min.

in

takes

about

synthesis

after

an

synthesis

transcription

inhibition

nuclei

by

salt I

nuclear (1

RNA

incorporation

a buffered

the

of

decrease

lower

a -amanitin

nuclei

influence

hepatomas,

RNA polymerase

synthesis rat

an of

A

Apparently

II

because

In

are

Morris

a considerable

%I.

the

liver.

incubated bovine

examining transcription

from

rat

cytosol

for

in-vitro

derived

regenerating

general,

applied

96, Fig. for maximal

The

amount

1. 45 min. inof

Vol. 74, No. 3, 1977

Table

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I

UTP-incorporation Isolated nuclei as described in In brackets the

Cell

into RNA of isolated cell nuclei: were incubated at 30 o C in homologous cytosol Materials and Methods. Values are given h S.E.M. number of experiments.

Nuclei from

Morris

Isolated

Hepatoma

Rat Liver Regenerating

Table

UTP Incorporated IO6 Cell Nuclei/30

9121 Rat

Liver

p moles/ min.

3.39

2 0.22

(17)

4.32

f

(15)

6.01

+- 0.83

0.37

( 4)

II

Effect of cytosol from regenerating rat liver on UTP-incorporation into isolated nuclei in comparison with incorporation in homologous cytosol. Nuclei were incubated as+described Material and Methods at 300C. Values are given - S.E.M.

Cell

Nuclei from

Rat

Hepatoma

P4C1-labeled on

their

UTP

origin

from

Morris

from

regenerating

labeled sol

stimulates in

nuclei

of

cytosol

9121

incorporated

and

on

hepatomas

precursors (Table

96 Stimulation

Isolated

Liver

Morris

rat

into

their

a fairly

liver

have

into

RNA,

Cytosol

RNA

synthesis

in

hepatoma

cells

from

regenerating

RNA of

metabolic

show

I).

from

in

derived

the

1112

(5) (9)

isolated

is

nuclei

incorporation in

liver II).

depends

prepared

whereby

rat nuclei

The

a very

of

homologous

regenerating rat

nuclei

Nuclei

value,

incubated

(Table liver

0.61

highest

from normal

+,

f, 11.42

state. low

when

12.52 33.48

cyto-

liver as

well

stimulating constant

as effect

one

in

BIOCHEMICAL

Vol. 74, No. 3, 1977

Table

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

III

Effect of cytosol from regenerating rat liver on UTP-incorporation into isolated nuclei from Morris hepatoma compared incorporation in homologous cytosol. Same experimental conditions as in Table II.

Hepatoma Nuclei Grouped according to Stimulation

$4 Stimulation

I

3.5

+

2.5

(4)

II

37.9 86.4

+ +

3.6 0.5

(3)

III

transcription other in

of

hand tumor

this

three

spond

with

cond

group

cause as

of

hepatoma

to

Cytosol

One

a third

derived

from

of

can

which

transcription

on

effect

On the

synthesis

those

and

effects the

RNA

nuclei: in

(2)

cells.

preparations.

increase;

measurable

liver

comparing

increase

III).

compared

when

different

a medium

(Table

not

nuclei

from groups

with

non-growing

varies

a considerable

increase

does

from

nuclei

distinguish

no

nuclei effect

cell

to

re-

rate; one

with

normal

almost

rat

UTP-incorporation homologous

a se-

liver in

tumor

cytosol.

DISCUSSION The

presented

results

transcription The

in

stimulating

effects

probably

tures

higher

than

(11).

Stein

Maybe

and the

these

isolated in

-

Hausen

50° (12)

activity factors

the

a protein; 4S"

of are

that

nuclei,

factor

is

increases

indicate

soluble apparently

cytosol it

is

C its

in

1113

a specific for

thermolabile,

at effect

a cytoplasmic

RNA polymerase or

stimulates

responsible

stimulating

describe

similar

cytoplasm

even

II

at

identical.

these temperagets

factor low

way.

ionic The

lost which strength.

effect

Vol. 74, No. 3, 1977

of

cytosol

BIOCHEMICAL

can

albumin.

Therefore

transcription be

An

incorporation

of

already

is

negligible;

the

The

Morris

I).

tumor.

On the

metabolic even

without

In

contrast

to

of

transcription

the

logous

cytosol

tion

is

from

a normal

partial can

hepatoma is are

report

no

be

direct

on the

liver RNA

of

incorporation

DNA

template

less

than

into

RNA.

nuclei

is the

2 % of

somehow

tissue

origin

a very

slowly

growing

has

fulfil

so

to

synthesis

be

synthesis.

has

in

proof

not as

point

tumor is

However,

this

to

most

many take

yet,

it

nuclei.

In

that

the

and

in tumor

question

1114

the

hetero-

cytosol

this

derived liver

after

nuclei,

it

respect

the

Although

various

causal rates

there factors

of

the

age.

whether

cytosol

not

observa-

liver

growth

from does

rat

behaviour.

differences size

of

by

normal

seems

cytosol

interesting

to

a uniform

inhibition

result

(regenerating

limited

show

an to

effect

transcription

hepatoma

especially tumors,

due

tissue not

(141,

nuclei

The of

is

do

liver

al.

a qualitative

stimulation

effective

et

observed. of

RNA

Dvorkin

rat

proliferating

involved,

important

by

normal not

nuclei

dividual An

the

hepatectomy) also

a

of

is

intensive

possibility

that

qlZl

elongation

growth.

could

exclude

cell

seem

at

be

activity

normal

an

in

cells

isolated

hepatoma

that

to

by

precursors

proliferating

hand,

found

labeled in

other

tasks

was

kind

nuclear

nuclei

possible

this

bovine

proteins.

transcription

of

with

isolated

also

as

of

cytoplasmic

is

it

transcription

(Table

of

such

stimulation

using

conditions

incorporation

proteins

real

presence

RNA without

1

by

systems

nucleotides

applied

correlated

tumor

to

of

the

Apparently,

place

addition

the

Under

total

replaced

by

present

r14 C UTP

be

in-vitro

stabilized

(13).

the

in

the

to

of

partially

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from

rat

in-

Vol. 74, No. 3, 1977

BIOCHEMICAL

liver

gene

might

clarify

effect

this

results

in

nuclei

iisolated

scribed of

all

in

expression

extensive

Preliminary

this

kinds

45 S RNA is

in

hybridization indicate by

paper of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

nuclear

is

experiments

that

cytosol

from

apparently RNA;

hepatoma

however

the

stimulation

regenerating due

to

nuclei. have of liver

an

preferentially

To been

begun.

transcription as

increased

desynthesis

ribosomal

involved. ACKNOWLEDGEMENT

I wish to thank Prof. Dr. E. Harbers for helpful discussions and advice throughout these studies. This work was supported by a Deutsche Forschungsgemeinschaft grant to Prof. E. Harbers. The technical assistence of Mrs. I. Fleischhauer and Ms. M. Unruh is gratefully acknowledged.

REFERENCES

;:

Weiss, S.B. (1960) Proc.Natl.Acad.Sci. USA 46, IOZO-lOgO. Widnell, C.C. and Tata, J.R. (1964) Biochem. J. 92, 313-317. and Zubay, G. (1974) Proc.Natl.Acad.Sci.USA 71, wu, G.-J.

4.

McNamara,

1.

1803-1807.

D.J., Racevskis, J., Schumm, D.E. and Webb, Th.E. Biochem. J. 147, 193-197. Marzluff, W.F., Jr., Murphy, E.C., Jr. and Huang R.C. (1973) Biochemistry 12, 3440-3446. Soll, D.R. and Sussmann, M. (1973) Biochim.Biophys.Acta 319, 312-322. Pogo, O., Alfrey, V.G. and Mirsky, A.E. (1966) Proc.Natl. Acad.Sci.USA 56, 550-557. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J.Biol.Chem. 193, 265-275. Higgins, G.M. and Anderson, R.M. (1931), A.M.A.Arch. Pathol. 12, 186-202. Kedinger, C., Gniazdowski, M., Mandel, J.L., Jr., Gissinger, F. and Chambon, P. (1970) Biochem.Biophys.Res.Comm. 38,

(1975)

5. 6. 7.

8. 9. IO.

165-171. 11. 12.

13. 14.

Yannarell, A., Schumm, D.E. and Webb, Th.E. (1976) Bi0chem.J. 154, 379-385. Stein, H. and Hausen, P. (1970) Eur.J.Biochem. 14, Harbers, E. and Heidelberger, C. (1959) Biochim.Biophys. Acta 35, 381-388. Dvorkin, G.A., Efimova, B.F. and Koganiskaya, L.I. Dokl.Akad.Nauk SSSR 210, 461-463.

1115

270-277.

(19731