Incorporation of leucine-H3 into subnuclear components of isolated pea nuclei

Vol. 6, No. 3, 1961

BIOCHEMICAL

INCORPORATION

OF

LEUCINE-H3

COMPONENTS Max

L.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF

Birnstiel,

Division

ISOLATED

Margaret

of

Biology,

INTO PEA

Chipchase

California

October

We

have

of nuclei that

pea

nuclei

(Birnstiel

et -2)

(1959),

we

We

now

the

to

the

We of

Methods: axes

the

of

(3.57

“C/FM) an

legend

ATP

nuclear

then

differential Birnstiel

the

in

by

with

50 or

presence

PC/ml

a mixture

of

under

the

incubated

nuclei

were

sucrose, as

1961b).

In

this

in

have

the

stems

--et

within

from

embryonic of a roller-

1961).

The

acids,

described

by the

5-H3

19 amino

further

legend each

in

purified

grinding,

and

of figure fraction

the

components. or

al.,

other

next

been

between

conditions

procedure 161

methods

of D, L-leucine-4,

disrupted

described

appear-

composition.

subnuclear

(Rho

system,

concentrated

’

of

100

by

centrifugation

earlier

al.

sub-

incorporation

pea

--et

chromatin,

differentiating

differential

reported

vitro

distinct

above

remaining

shown

Johnston

chemical

acid

4-day-old

seedlings,

The

-*al

from

of

and

in

of the

--in

various

the

interested

have

characterized

range which

condition

and

methods

each

of amino

as

protein

nucleoli,

of the

that

pure

into

namely:

site

relative

into

in

obtained

centrifugation -et

nuclei

of a study

and

regenerating 2.

Technology

1961)

the

supernatant,

particularly

incubated in

of

sedimentation

pea

of figure

centrifugation

also

1961b),

homogenate

were

by

al.,

were

36-hour-old

nuclei

and

--et

nucleolus

mill-produced

Bonner

-- et al.

acids

elaboration fractionate

in

(Rho

amino

to

findings

are

Nuclei

of

isolation

seedlings

able

investigation

nucleus.

behavior

By

and

the

the

labeled

microscope,

report

applied the

been

fraction

under

and

1961a).

(Birnstiel

ribosomal

James

Calif.

for

incorporate

have

components

methods embryos

al

and

1961

developed

from

such

ance

12,

NUCLEI.

Institute

Pasadena,

Received

SUBNUCLEAR

the by fractionated

1 (see was

examined

,

Vol.6,No.3,1961 under

BIOCHEMICAL

the microscope

abundance

to determine

of nuclei

analytically

for

containing

content

as intermediate

nuclear

components. individual method

corporated successively, cold

NaOH

determined

with

Instrument

Co.

oxazole Results:

this

an automatic

data

leucine

short

an ethanol-toluene

incubation

90 sec.

period,

activity,

is by the nucleoli,

fractions

are

is a mixture decreases

with

minutes

case

from

additional to

Thus

of Packard 2,5-diphenyl-

previously

in contrast

activity 1).

chromatin

a pulse

experiment

and specific These

two 3 which

by other

and with

labeled

during

to fraction

incorporation

but little

incubated

incorporation

total

of nucleoli

label

fractions

increasing

content

and the protein-rich

leucine

into

protein

in which,

after

during

period.

data of figure

2 concern in labeled

leucine

was

tips

of young

added

leucine,

It is clear

a large

to the nuclear

pea stems,

7, 17, or 40 minutes

fractionation.

nuclei

principle

the DNA-rich

incorporate

of incubation

unlabeled

abundance

was

of incorporated

4 and 5 of figure

The

with

(POPOP).

to both

nucleoli,

and nucleoli.

chromatin.

incubation The

respect

(Fractions

decreasing

supernatant

a brief

with

The

in-

leucine,

314-DC

the distribution

at 37°C.

that

washed

containing

of pea embryo

of intact

of nuclei

of DNA-rich nuclear

composed

were

and

Radioactivity

medium

1 concern

than

of unlabeled

No.

obtained of the sub-

other

(5-phenyloxazolyl)-benzene

components for

Model

fractions

by an appropriate

and alcohol.

TRICARB

of figure

the subnuclear

in tritiated

hot TCA,

examined

defined

activity

excess

was

mixtures

the pellets

of a large

and 1,4-bis-Z-

The

all tritium

as the

reproducibly

washed

procedure,

(1 M at 37°C))

and with

(PPO)

among

In this

were

physical

exhaustively

and in presence

TCA,

were

as well

fraction

Well

etc.

which

to remove

protein.

each

and protein.

chromatin,

were

in order

into

RNA

fractions

pellets

or absence

In addition,

of DNA, nucleoli,

as well

rigorous

the presence

and of nucleoli.

principaliy

The

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from

preparation,

The nuclei

in unlabeled the data 162

(1000

were

leucine of figure

fold)

excess

obtained incubated before

being

2 that during

5 of

in this for

an subjected the first

Vol. 6, No. 3‘1961

BlOCHEMICAl SHORT

z e

TERM INCORPORATION OF DL-LEUCINE-H3 INTO NUCLEAR SUBFRACTIONS

IOOO-

k-?

F 2n 0

.A

t

Fig.

AND BIOPHYSICAL RESEARCH COMMUNlCATtONS

SPECIFIC ACTIVITY

‘2C@

AY

I

l50-

.2.3.4.5’6=7.!

9

1

Incubation for 90 set at 37°C with 100 &/ml of DL-leucine-4, 5-H3 under optimal conditions as specified in Fig. 2. The nuclei were purified in a sucrose gradient and the density of the recovered nuclear suspension The nuclei were grouy in the presence of 0.5 mM adjusted to 1. 310 gr/cm3. sodium citrate (pH 7. 2) in a Omnimix (40 V for 2- min) and the suspension (1.260 gr/cm3) was centrifuged differentially (Spike rotor 25) for 20 min at 1,935 g, 4,340 g, 14,500 g, 30,900 g, 48,200 g and for 2 hrs at 72,000 g (Fraction l-6). After 5 fold dilution with a solution of 0. 01 M KGl, 0. 0005 M CaCl and 0.002 M tris (pH 7. 2) the centrifugation was continued (Spinco rotor230) for 25 min at 34,850 g and 6 hrs at 78,410 g (Fraction 7 and 8), yielding a supernatant fraction 9.

7 minutes the that

specific of the

after activity chromatin

addition

of the of

the fraction

unlabeled

protein

leucine

of the remains

nucleoli high.

163

the

Over

rate

of

increase

diminishes,

whereas

the

periods,

longer

of

17

Vol. 6, No. 3, 1961

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

INCORPORATION INTO NUCLEAR FRACTIONS ( 3 TO 5 OF FIG. I) AFTER SHORT DL-LEUCINE -H3 PULSE I

I

I

I

/NUCLEOLI

‘EXTRA NUCLEOLAR NUCLEOPLASM

1

EXCESS OF UNLABELED LEUCINE ADDED I I I IO 20 40 MINUTES ABELED LEUCINE ADDED

30

pr,

0.003 under optimal condition: 5O+/ml of DL-leucine-4, 5-H3; 0.2 M sucrose; 0.005 tris; 0.0001 ATP; 0.02 M phosphocreatine, salt; 50 y/ml creatine kinase; a complete mixture of 19 L-amino 4Oy/ml of each except leucine; 50 y/ml streptomycin; final pH 7.0. aerobically at 37 “C. Fractionation as described in Fig. 1.

Incubation CaC12; sodium acids, Incubation

and

40 minutes,

case

the

specific

microscopically

identified)

accompanied

by

an

nucleoplasm;

an

increase

Discussion:

The

counting and

feel,

ment

for

the

these

experiments

on

increase

-et this

presence

of the

specific

found

principally

basis of represents

* 1961a) as all

well amino

acids,

incorporation

164

the

used

destroys on

the

all the

in

into

preparation

of

each

however

chromatin. of

sRNA-amino

the

is

(in

extranucleolar

DNA-rich

basis that

nucleoli

This

of the in

here

as

of

markedly. activity

procedure

--al

proteins

decreases in

washing

(Birnstiel

we

activity

M

the

acid

require-

incorporated or

into

for

complexes

observed

leucine protein

samples

other

in

Vol. 6, No. 3, 1961

peptide

linkage.

that

of figure

that

the

the

nucleus

to

The

the

amount

gained

by

due

in

sRNA-

several

part

accounted

amino

Although

acid

the

of nuclear

to RNA

nucleolus

as

a significant

an

integral

Peveling

(1961)

of young

results

autographic which

here

techniques

the

support

nucleolus

is

the

site

exact

balance and

the

present

nucleolar

ex-

fraction leucine.

a considerable

to

be present

the

This pool

in

as

pea

of

these

nuclei.

a principal

site

possibility

nucleus -2et

is

in

of

a slower

studies

in

isolation biochemical

that

those

recently

by

the

work

the

nuclei

synthesis

of many Sirlin

protein

subnuclear nature

of protein

addition

apparently

with

with

of nuclear of

196lb),

’

showed

agreement

a site

al

in

of

material.

summarized as

contains

agreement

chromatin

and

of a more principal

the

This

are

and

the

of

(Birnstiel

embedded

of fractionation

quantitative

from

DNA,

nucleolus

nucleolus

itself. it

presented

in

the

exclude

from

fractions

nucleolus

microscope

reviewed

suggest

the

structure.

contain

studies, also

of

electron

cells

know

do not

prepare

its

whose

plant

The

of

we

An

of unlabeled

presence

chromatin

amount

part

the

they

the we

addition

within

product

found

as

2 indicate

incorporation

nucleolar not

such

of figure

chromatin.

into

establish

synthesis, by

the is

which

results

of protein

The

by

complexes,

protein

synthesis

after

for

present

from

incorporation

minutes

that

acid

to

fraction

that

duration

of labeled

principally lost

short as

of amino

movement

chromatin fact

of such

site

activity

the

to the

for

be

principal

nucleoplasm, of

incubations

experiments

suggest

amount

periments

may

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from

pulse

further

remaining

between

from is

and

continues

results

1 and

nucleolus

the

as

BIOCHEMICAL

radio-

(1960),

synthesis.

Our

components,provide for in

the the

view

that

nucleus

the of young

cells.

Ack,nowledgements We been No.

are

supported RG-5143)

grateful

to

in

part

and

the

Robert by

grants

National

J.

Hayes from

for the

Science

his National

Foundation

165

assistance.

This

Institutes (Grant

of No.

work Health

G-7129).

has (Grant

Vol. 6, No. 3, 1961

BIOCHEMICAI.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References Birnstiel, M. L., Chipchase, M. 1. H., and Hayes, R. J. (1961a): Submitted to Biochem. Biophys. Acta. Birnstiel, M. L., Rho, J. H., and Chipchase, M. I. H. (1961b): Submitted to Biochem. Biophys. Acta. Johnston, F. B..,. Setterfield, G., and Stern, H. (1959): J. Biophys. Biochem. Cytol., h, 53. Peveling,“%. (1961): Planta, 56, 530. Rho, J., Birnstiel, M. L. , Chipchase, M. I. H. , and Bonner, J. (1961): Federation Proc., 2, 147f. Sirlin, J. L. (1960): The Cell Nucleus, Academic Press Inc., N. Y.

166