Polyriboadenylate synthesizing activity in chromatin of wheat seedlings

BIOCHEMICAL

Vol. 52, No. 4, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

POLYRIBOADXNYLATE

SYNTHESIZING

ACTIVITY

IN

CHRCMATIN CF '&HEAT SEEDLINGS

Kimiko

Sasaki

Department

of Botany,

Hokkaido Received

April

25,

and Tadashi

Tazawa

Faculty

University,

of

Science

Sapporo,

Japan

1973 SUMMARY

The occurrence

of

poly(A)

polymerase

was demonstrated

in

chromatin of wheat seedlings. The optimum pH was about 8.0. The activi:y was depressed by the treatment of chromatin with pronase or deoxyribonuclease, and it was sensitive to ribonuclease. AiW. ADP. inorganic pyrophosphate, CTP, GTP, UTP, dAl'P, dC1P. dGTP, dTTP, and lysine inhibited the activity, while aspartate stimulated it. Rifampicin significantly, and actinomycin D insignificantly inhibited the activity. Difference of the susceptibilities cates that poly(A) polymerase

There those

is

evidence

required

for

Large

mRNA. ribosomal

binding

liver

have at

(6j,

(3)

nuclear

been shown to the

significance

tory

site

it in

may play translation

of

of

a role

this

present

in

as specific

site

in

in mRDiA

has been demonstrated

(4.5).

reticulo-

(9).

cells

sequence

as a binding

reaction.

than

each cistron

of binding

189 ascite

indi-

other

RNA and mRNA of HeLa cells

sent

but

type

are

serve

and mitochondria

mouse sarcoma

known,

beginning

Poly(A)

and in mRn'A of the

sequences

of amino acids

and other

cytoplasm

tryptophane

RDlA polymerase.

polynucleotide

in HeLa cells.

polysome

time,

is unlike

coding

sites

(1.2).

has been found

cyte

the

segments

bacteriophage

in rat

that

to dTTP and to

The pre-

in mRNA is

site

Recently,

(7,8),

un-

or a regulathe occurrence

BIOCHEMICAL

Vol. 52, No. 4, 1973

of

ribosomal(l0)

Escherichia nent

of

Until

and that

Di'.A-dependent

in wheat

this

poly(A)

paper,

poly(A)

in

chromatin

were carried

M!uka(winter with

wheat).

8% bleachin

in water,

a layer the

and soaked

(about

sterile

has not

been

for

of

of

seedlings.

Triticum

water

for

Soaked

in

L.

rinsed

several

several

hours

var.

experiments,

at

Petri-dishes

or

5,

hours

seeds were placed

sterilized

3,

sterilization

vulgare

seeds were surface-sterilized

30 minutes,

for

occurrence

6 days

at

and

24'C

in

root-eliminated

with

penicillin

the

procedures

on

the

seedlings

and washed with

water.

Isolation between

of 0'

mortar

After

chromatin:

The seedlings

4 volumes

(pH 8.0), filtration

of Millacloth, minutes.

a mixture

1 mM M&12,

the

fluid

The pellet

the

by centrifugation

Further

purification 2 hours

were ground of 0.5

double

in

out

a cold

M sucrose,

layers

was centrifuged

was washed three

at

were carried

50 mM

and 6 mM /3-mercaptoethanol.

50 mM Tri s-HCl

ethanol

x g for

of

through

M sucrose,

30.000

All

and 4'C.

p:ith

Trie-HCl

0.25

use,

paper

following

were used after

(12.13).

wheat

with

2O'C).

seeds were incubated In the

were demonstrated

AND METHODS

in strile

of wet filter

dark.

a compo-

evidence

of

out

Before

g powder

room temperature

is

polymerase

the

MATERIALS Experiments

in

plants.

we describe

polymerase

polymerase

polymerase

RNA polymerase of

RESEARCH COMMUNICATIONS

poly(A)

the poly(A)

the occurrence

demonstrated In

or non-ribosomal(l1)

u,

now,

AND BIOPHYSICAL

of gauze at

times

(pH 8.0).

4,000 with

x g for

20

a mixture

of

and 6 mM p-mercapto-

4.,000 x g for

20 minutes.

was made by ultracentrifugation in a mixture

1441

and a layer

of 1.7

M sucrose,

at 10 mM

BIOCHEMICAL

Vol. 52, No. 4, 1973

Table

1.

AND BIOPHYSICAL

Polynucleotide

Nucleotide

synthesis

added

3H-labeled

RESEARCH COMMUNICATIONS

by chromatin Activity (cpm)

Unlabeled

1.805

ATP CTP GTP UTP ATP CTP GTP UTP

CTP. ATP, ATP, ATP 9

GTP. GTP, CTP, CTP t

147 115 41 914 166

UTP UTP UTP GTP

73 130

The assay mixture (0.5 ml) contained 20 pmoles Tris-HCl (pH 8.0), 2 Imoles I&$12, 0.5 Imole MnC12, 6 pmoles P-mercapto0.2 @mole unlabeled nucleotide. 0.41 nmole 3H-labeled ethanol, nucleotide (10 FCi), and chromatin equivalent to 10 ~g DNA. The chromatin was obtained from 6 day old seedlings. The reaction was carried out at 37OC for 10 min. After addition of 300 pg bovine serum albumin, it was terminated by addition of 2 ml of 10% TCA containing 10 mM sodium pyrophosphate. Acidinsoluble material was collected on Whatman GF/C filter (24 mm), washed with 5% TCA containing 10 mM sodium pyrophosphate, and dried. The radioactivity in the filter was determined by counting in 5 ml of a toluene based scintillation solution. These values were corrected by subtraction of each corresponding initial value.

Tris-EC1

(pH 8.0).

was washed buffer,

with

10 mIkl Tris-HCl

dialyzed

and used

at -2OOC

10 mM Tris-HCl Determination 5% TCA at

against

as "purified

was stored

method

and 6 mM P-mercaptoethanol

the

for

(pH 8.0)

of Giles

for

in

the

same buffer

overnight

at

2-4'C,

In

one case,

30 days until

in

50% glycerine

the

same

chromatin

containing

use.

DNA was extracted

10 minutes,

and Myers

The pellet

suspended

chromatin".

of DNA: 90°C

(pH 8.0).

(14).

then

(15).

1442

it

from

chromatin

was determined

with by the

Vol. 52, No. ,4, 1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0.0 I 7.5

8.0

8.5

PH

Fig.

1.

Optimum pH for of chromatin.

Chemicals:

poly(A) synthesizing Conditions as in

AYIP, ADP, ATP,

and dTTP

were

and RNase pronase

purchased

were

from

purchased

was obtained D were

Rifampicin

was given

Tritium

labeled

ATP,

Schwarz

Bioresearch,

from

by the

Daiichi

GTP,

poly(A)

poly (G ). vity mixture

of

and poly(U)

synthesis

of

GTP,

and RNA from

Sigma Pure

and

Actinomycin

Chemical

I

C and

Co.

Chemicals

Co.,

Ltd. from

AND DISCUSSION

to the %I -ATP

Chromatin extent,

to a lesser

poly(A)

may be due

DNase

Chemicals, Co.

dGTP,

and LJTP were purchased

activity:

to a large

CTP,

dCTP.

Inc.

synthesizing

merize

dATP,

Mannheim.

Kagaku

purchased

CTP,

UTP,

Worthington

Kaken

RESULTS Poly(A)

GTP,

Boehringer

from

from

actinomycin

CTP,

activity Table 3.

competition in

and also extent

was depressed

and UTP

the

was found

(Tables

presence

1443

polymerize

(Table

1).

by the

addition

1 & 3).

between

the of

CTP,

to

poly-

poly(C), The actiof

a

The depression

syntheses

of

poly(A)

GTP,

and UTP.

BIOCHEMICAL

Vol. 52, No. 4, 1973

Table

2.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Effects of various reagents synthesizing activities

Reagent

Concentration

and RNA

Synthesizing

(pmoles/0.5 None 5'-AMP 5'-ADP ATP Phosphate Pyrophosphate

on poly(A)

ml)

activity

($)

Poly(A)

RNA

100 10

100 29 29

8

30

0.1 0.1 0.1

13

107

0.25 0.25

92

50

70

The assay mixture for poly(A) synthesis contained 0.41 nmole 3 H-ATP, unlabeled and that for RNA synthesis contained CTP, GTP, and UTP. The reactions were carried out with or without the reagent at 37'C for 10 min. Other experimental conditions as in Table 1. 3H-ATP,

Such depression

did

not

or poly(U)

poly(G), about

8.0

l

(Fig.1).

shown as 20 to 25'C, 2 to

10°C

Effects

of

phosphate (Table tion

during

Optimum

pH for

dATP,

and the

activity

inhibitors

nucleoside dTTP

poly(C),

synthesis

was

the

synthesis

was considerably

and stimulators: inhibited

The result

poly(A)

(Tables

conformational

for

of

was

high

at

(Fig.2).

which

triphosphate

poly(A) to

are

poly(A) Such

of poly(A) above to poly(A)

the

product

GTP,

inhibi-

from UTP,

synthesizing

ATP dCTP,

dGTP,

activity

of

inhibition

may be due to the

polymerase

by the

listed. synthesis,

1444

pyro-

and RNA syntheses

produced

ADP, CTP,

the

2 & 3).

was specific

the

synthesis.

change

AMP and inorganic

may be related

and dTTP inhibited

chromatin

synthesis

poly(A)

temperature

by AMP and pyrophosphate the

the

Optimum

significantly 2).

during

of

occur

binding

The inhibitory it

did

not

with effect

inhibik

Vol. 52, No. 4, 1973

Table

3.

BIOCHEMICAL

Effects poly(A)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of various nucleoside and RNA synthesizing

Additi.on

triphosphates activities

Concentration (~~‘0.25

None ATP CTP GTP UTP CTP, GTP, UTP dATP dCTP dGTP dTTP dTTP dTTP dTTP dATP, dCTP, dGTP,

on

Synthesizing ml)

activity

Po~Y(A)(~

m)RNA 262

656 258 301 56 203 96 130

144 148 447 149

246

81 10 dTTP

30

Assay mixture (0.25 ml) for poly(A) synthesis contained 10 umoles Tris-HCl (pH 8.0). 2 ymoles MgC12, 0.25 pmoles MnC12, 3 pmoles R-mercaptoethanol, 0.25 nmole 3H-ATP, and chromatin equivalent to 2.5 I.rg DNA. Assay mixture for RNA synthesis contained 0.2 pmole CTP, GTP, and UTP, in addition to the components listed for poly(A) synthesis. The chromatin was prepared from 3 day old seedlings. The reactions were carried out at 24'C for 30 min. Other experimental conditions as in Table 1.

the

RNA synthesis.

regulator cells. of

poly(A)

This

to repress

the

The inhibitory synthesis

finding predominant effect

might

unlabeled

ATP to labeled

inhibited

by the

treatment

suggested

of unlabeled

be related ATP (Tables of

synthesis

chromatin

1445

that

dTTP of

poly(A)

ATP on the

to a dilution 2 & 3). with

is

in activity

effect

The activity pronase

a

of was

or DNase

Vol. 52, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

: 0 x

2.5

-

E 0”

2.0-

e t Q

1.5-

x 8 y

l.O-

o.o-

’ 0

I

I

I

5

IO

I5

I

I

20

30

TEMPERATURE

Fig.

2.

(Table

Optimum activity

This

4).

enzymatic both

temperature for of chromatin.

result

indicated

and DNA-dependent

the

activities

of

to RNase

(Table

4) indicated

degraded

by RNase.

actinomycin

C and D did

the

antibiotics

increased

for

5 to

and actinomycin thesis

more

lysine

inhibited

ses,

while

inhibited

than

that the

aspartate only

ceptibilities RNA polymerases

the

not

(Table

after the

of

treatment,

inhibited

the

activities

of

stimulated

them

RNA synthesis to dTTP and to that

tryptophane poly(A)

polymerase.

1446

were

synthesis,

at

rifampicin

(Table

that

sensitive

chromatin

to 37’c

strongly,

of poly(A)

poly(A)

acids

used,

Tryptophane

Differences

polymerase

syn-

and RNA synthe-

5).

between

an

sensitivity

Of amino

5).

is

polymers

activity

both

(Table

were

The

treatment

3.

hand,

poly(A)

4).

of RNA synthesis.

suggested

other

synthesized the

Table

synthesis

On the

inhibited

After

D weakly

poly(A)

and RNA syntheses

Rifampicin

10 minutes.

poly(A) synthesizing Conditions as in

that

that

but

c

40

(‘Cl

reaction.

poly(A)

I

35

I

25

of

poly(A) is

unlike

susand RNA

Vol. 52, No. 4, 1973

Table

4.

Exp.

BIOCHEMICAL

Effects of synthesizing

AND BIOPHYSICAL

various inhibitors activities

Inhibitor

Cone.

None Actinomycin Actinomycin Actinomycin Actinomycin Rifampicin Rifampicin DNase I RlYase

1

C C D D

and RNA

activity

(5)

RNA

Poly(A)

100 102 102 101

2.5 25.0

100

48

84

49 70 48

25.0

57 37 62

74 64 40

1.0

56

69

2.5 25.0 2.5 25.0

None DNase I Pronase

2

on poly(A)

Synthesizing

(w/o.25ml)

NO.

RESEARCH COMMUNICATIONS

100 2.5 2.5

54 37

The chromatin was prepared from 5 day old seedlings, and the amounts equivalent to 2.8 pg DNA were used. Effects of DNase and pronase were assayed with the chromatin preincubated with DNase or pronase at 37’C for 10 min in Tris-HCl (10 mM, pH 8.0). The reactions were carried out at 20°C for 30 min with or without inhi-bitor.

The finding er than seedlings biological younger Further

the

that

suggests role

that other

polymerizing activity

poly(A) than

in

polymerase

as a component

activity chromatin may play of

was highof

younger

a certain

RNA polymerase

in

plants. experiments polymerase

merase,

and to

living

poly(A)

RNA polymerizing

poly(A)

in

the

must of

evaluate

wheat

be required seedlings

an actual

cells.

1447

is function

to ascertain

whether

a subunit

of RNA poly-

of

poly(A)

polymerase

BIOCHEMICAL

Vol. 52, No. 4, 1973

Table

5.

Exp. No.

Amino

1

2

Effects of synthesizing acid

None Tryptophane Tryptophane Tryptophane Tryptophane None Alanine Arginine Lysine Aspartate Glutamate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

various amino activities

Cont. (do.25

acids

on poly(A)

Synthesizing ml)

and RNA

activity RNA

Poly(A)

(cpm)

(cpm)

5 10 50 100

1518 1'748 2001 1941 1479

1008 927 582 546 411

10 10 10 10 10

4927 5230 4431 2930 5276 4897

262 220 190 185 402 326

The reaction mixture in Exp. 1 contained 0.2 nmole 3-l -ATP and 40 nmoles unlabeled ATP, and the reaction mixture in Exp.2 contained 0.2 nmole 43 -ATP. The reactions were carried out at 20°C for 20 min with or without amino acid. Other experimental conditions as in Table 3.

vice

The authors thank and interesting

to Professor through this

S.Usami study.

for

his

valuable

ad-

REFERENCES 1. A.Steitz, Nature, 224, 957 (1971). 2. K.Shishido, and Y.Ikeda, Biochem.Biophys.Res.Commun., 44, 1420 (1971). 3. A.Hadjivassilon, and G.Brawerman, J.Mol.Efiol., 20, 1 (1966). 4. L.Lim, and E.S.Canellakis, Nature, 227, 710 (1970). 5. R.S.T.Jacob, and D.G.Shindler, Biochem.Biophys.Res.Commun., 48, 126 (1972). 6. J.E.Darnell, R.Wall, and R.I.Tushinski, Proc.Netl.Acad.Sci., U.S.A., 68, 1321 (1971). 1448

Vol. 52, No. 4, 1973

BIOCHEMICAL

7. J.E.Darnell,

L.Phillipson,

Acad.Sci., 8. M.Edmonds. Sci.,

J.Mendecki,

U.S.A., J.Smith,

68,

1331

1336

R.Wall, and M.Adesnik, 507 (1971). and H.Nakazato,

Proc.Natl.

Proc.Natl.Acad.

(2.571)

and G.Brawerman,

Proc.Natl.Acad.Sci.,

(1971).

and H.Alberty,

J.Biol.Chem.,

241,

3525 (1968).

Biochem.Biophys.Res.Commun.,

-46

(1972).

12. 13.

S.Ohasa, S.Ohasa,

14.

(1972). R.C.Huang,

960

74,

and J.T.Augus,

11.. K.J.Gross,

581

68,

U.S.A.,

9. Y,.Lee, 10.

U.S.A.,

M.H.Vaughm,Jr.,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and A.Tsugita, Nature A.Tsugita, and S.Mii, and J.Bonner,

New Biology, 240, Nature New Biology,

Proc.Natl.Acad.Sci.,

(1965).

15. K.W.G.iles. 16. K.Sasaki,

and A.Myers, in preparation.

Nature,

1449

206,

93 (1965).

35 (1972).

U.S.A.,

240, 54,

39