High molecular weight DNA-like RNA in rat thyroid

BIOCHEMICAL

Vol. 25, No. 6, 1966

HIGH

MOLECULAR

J. Hare1

(l),

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

WEIGHT B. Nataf

DNA-LIKE L.

(l),,

RNA

Hare1

IN RAT

(2) and J. Imbenotte

(1) Institut Gustave Roussy (2) Centre de Recherches sur la Cellule Canc6reuse - Villejuif - Val-de-Marne

Received

October

RNA

in animal (dRNA)

in organ

AND were

messenger cells.

on which

RNA

from

material

to study

and the biosynthesis here

fetal

Each

a piece lobes

culture

of dacron

(Nataf,

as described

that

very

rat thyroid

before

to 10 dishes,

to one other dish. 131 I measurements 0.14

on an aliquot ted with

M,

the

of specific

“heavy”

DNA-like

glands

maintained

components prepared

Tris

was floated

(Nataf,

Malaise

in 3 experiments

0.05

M.

Total

from

The

rest

the dried

chromatograms.

and counted

in a scintillation

2-4

dishes

experiment

1966)

32P

to the radioautogral%ic counter.

573

incufor

(100 ye/ml) was ’ added

of the homogenate

1963).

109

isolated were

and Tubiana

to separate

and Chaikoff

corresponding

The

in 0.3

was digeslabelled

Radioautograms

bands

ml

measured

the various

Sections

rat

and

NCTC

to support

1311 (50 PC/ml)

Raghupathy

matograms

(Elias

washed and homogenized 131 I incorporation was

and chromatographed,

(Tong,

method

1965).

the end of each

were

21 day CF Wistar

1 ml of medium

and Chaikoff

: Explants

from glass

contained

of the homogenate.

pronase

glands watch

organdy

elsewhere

was

of NaCl

dish

Rivera

24 hours

added

: Thyroid

by a modified

48 hours,

were

an adequate

It is shown

METHODS

cultured

1965).

bated

Normale et - France

culture.

fetuses,

thyroid

provide

can be isolated

MATERIAL

Rivera

glands

between

proteins

(1)

25, 1966

Thyroid rel.atPnship

THYROID

of the chrowere

cut out

Vol. 25, No. 6, 1966

RNA

preparation

2-C

in

2 ml

: Explants

RNA

with

PO4

0.2

M,

0.01 to

as

M

bentonite

(20

of pronase.

48 hours.

RNA

carrier

was

rRNA

in

comment

U.V.

to

freed

MgCl’ Fig.

flow

dients

(Huppert estimated

tated

with

et al’.

described

labelling

24

RNA

et

for

half 8 hours

of the in

non

Nr

Per cent uptake

1311

Per

1311

distribution

origin I MIT DIT T4 T3

on chromatograms

I Front

1 :

al.

131

by

measuring

were

the

medium

a low

plus

I. 33

2.0

2..9

35.0 17.4

43.6 28.0 1.8 0.2 22.9

I:: 47.0

0.6

0.6

(2 d&terminations

574

back gra-

of which precipi-

experiment,

frozen,

as

after

others PO4

pre-

were (up

to

3

0; 57

1.34

0.45

2.5

2.6

2.0

49.0 26.4 1.6

41; 1 15,5 1,2 0,3 38.5 0.8

38.6 23.5 1.4 0.2 33.9 0.4

0.2 19.7 0.6

in

for

in

glycerol

In

2.2

measurements

indicated

S values

a “chase”

M

as

in the

with

with

determined

35.1 17.2 I,6 O’.l 42.2 1.0

carrier

2

o-47

and

0.002

composition

radioactive

refered

dialyzed

in

1960),

were

of PO4

or

aliquots

performed

Spiegelman

explants

M)

or

on

1963).

$% NaCl

be

MgCl’

1964),

counted

base

washed

coprecipitation

fractions,

and

was

phenol

and

estimated

were

extracted

will

0.1

with

was

2 ml

and

M,

by

1

0.‘51

to

were

Labelled

in

at

M

20

which

0.002

(Hare1

et al.

lC

dissolved

either

their

37

1 % SDS

PO4

was

and

at

RNA

DNA

Hall

ethanol,

were

amounts

1966).

(Hare1

hours,

Experiment

Table

RNA

(Nomura,

viously

cent

M

%

(up

Centrifugations

carrier

reincubated

from

Radioactivity counter.

were

against

0.2 3.

absorbancy.

ground

2*C,

80

citrate

Precipitates at

interphase

with

Sodium

0.002

“Phenol-RNA”

2 hours

extracted

and

cells.

&ml)

in

homogenized

MgCl’

initial

with

and

M,

as

The

for

ethanol

mouse

to

overnight

was

with

from

refered

4 mg

- 76-C

0.002

incubated

“Interphase-RNA”

RNA

be

suspended

0.2

at

(P04)

treatments.

and

NaCl

precipitated

the

M,

collected M,

will

phenol

0.002

frozen

buffer

which

4 successive

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were

of phosphate

bentonite. by

BIOCHEMICAL

each

experiment).

0.01

I$.

Vol. 25, No. 6, 1966

RESULTS tic

BIOCHEMICAL

: Table

medium

are

synthezised the

in

results (Nataf

RNA

yields

500

and : In

of

the

for

63 %

for

very

gradient small

quite

synthe-

amounts

low

as

containjng

of

T 4

compared

insulin

to

and,

or

both

thyroid

RNA

phenol-RNA, for

the

specific

estimated

cpm/pg

Phenol-RNA,

total

and

be

400

for

radioactivity

20

for

Interphase-RNA

RNA. of

pg-lo,

Interphase-RNA, cpm/pg

radioactive

extracted

radio-

(a : 60

11 pg-12,000

experiments

% of in

amount

could

10 pg-II,

4 other

- 23

the

The

8 hours

% for

Phenol-RNA

rRNA

and

chase and

Interphase-RNA.

Phenol-RNA by

the

a purely

1965).

free

a decrease

in

However

medium

2 experiments

14 %

I.

RESEARCH COMMUNICATIONS

cultured

were

another

Chaikoff

In

glands 131

conditions

cpm/pg

represented

fetal

metabolize

present

Interphase-RNA).

in

that

with

pg-11,700

resulted

to

carrier

cpm/pg

b : 49

able

obtained

TSH

activity

I shows

AND BIOPHYSKAL

represented

uniformly

centrifugation proportion

(Fig. sedimented

labelled 1) and above

cpmxlOJ

base

composition

Fig. 1 (left) :4Labelled gradient, PO 0.01 of Spinco ultracentrifuge. Fig. 2 (right) rol gradient, dotted lines

M

: Lgbelled PO 0.01 : A 260 of

Phenol-RNA at 39,000

(Table

as

shown 2).

A

60 S.

cpmx103

00 1 o,-1.0

Fractions

sRNA

3.0,

.

number

rpm

centrifuged for 210

in 5-20 % glycerol minutes in the SW 39

Interphase -RNA centrifuged M for 150 minutes. Solid lines carrier RNA, P : cpm in pellets.

575

in

5-30 ‘$ glyce: radioactivity,

Vol. 25, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

v

I! RNA Fraction

Moles C

per

cent

A

G

A+U C+G

U

4 5

25 -30s sd

28.2 0.3

19.0 0.3

34.6 0;4

18.2 0.3

0.59 0.010

ii

15-20s

26.1

22.9.

29.1

21.9

0.81

f PC

sRNA sd sd

28.0 0.4 0.6

19.3 0.3 0.5

31.3 0.25 0.3

21.3 0’; 2 0.2

1 2 3 4

23.6 23.4 22.0 22.3

24.0 24.0 26.1 22.3

24.7 22.6 25.0 25.0

27.8 30.0 26.9 30.5

1.07 1.17 1.13 1.12

I 2 3

23.7 21,2 22.1

24.6 26.0 27.2

24.6 25.3 23.6

27.1 27.5 27.1

1.07 1.15 1.19

“3

22.4 25.0

21.7 24.1

29.7 26.4

26.2 24.1

0.92 0.95

4

25.7 22.5 23.9 23.5

20.3 20.5 22.9 21.1

27.9 29.8 29.1 27.9

26.1 27.2 24.1 27.5

0.87 0.91 0.89 0.95

1 3 4

21.8 25.4 23.5

25.3 22.5 22.8

29.1 28.1 26.8

23.8 24.0 27.0

0.96 0.87 0.99

90s

60-80s

3 d3

45-60s

3 E &

30-45s

1 5

20-30s

Table

2 : Base

riments, (1,2,3,4

sd : standard : experiment

Interphase-RNA (Fig,

composition

deviation) number).

: A major

2, 3) and had a G-C

Heavier

fractions

and were

distinctly

reported

in Fig.

associated

with

760

P Ph~~oPl-RR~p~;;;;;~ and

proportion content

sedimented lower

S, represented

more 3, high DNA

of 32

DNA

like.

S values

or proteins,

1

than that 15-25

From

or to RNA

576

4 expe-

between

20-45

S

of rRNA

(table

2).

% of total

experiments

did not seem

0.68 0.016 0.012

Interphase-RNA such

to correspond aggregates.

as that to RNA

Vol. 25, No. 6, 1966

BIOCHEMICAL

cpmxl 0

cpmxlO! I.5

A I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

Fig. 3 : A Interphase-RNA and pronase (100 pg/ml, fuged as in Fig. 2. Pooled ethanol, dialysed ovenight B : fraction 1, C : fraction 0.01

M

for

DISCUSSION

: From

sists

of

mostly

nuclear

RNA

indicate

to

of

Darnell

1963)

proportion that dRNA. only

al.

It

noteworthy

(30

been

extracted

contribute

S)

Pulse

from

labelled

sheep

a better

12 %

heavy

knowledge

study

(Cartouzou, of RNA of polycistronic

577

synthesis

of

and

I metabolism,

from

Warner

1965).

acids

dRNA.

poly-

isolated

1965.

et al.

Mantk and

of

been

Kawade

131

estimation

presumably

has

of amino

relative

consisted

the

dRNA,

(Scherrer

the

RNA

allow

and The

permit

dRNA and

correspond

dRNA.

of total

nuclear

Base

Latham

fractions

Fukada

thyroid

S may

represent

incorporation

comparative

30-45

which

erythroblasts

stimulated

Mantieva,

(Scherrer,

heavy

(Yoshikawa, duck

1962).

conditions

of thyroxin,

con-

and

mostly

8 -

Phenol-RNA represents

various

and

that

Interphase-RNA

precursors

of the

in

appears

fractions

ones

that

from

to

and

later,

rRNA

RNA

it

Georgiev

the

heavier

cells

which

Further

and

found.

culture

RNA

in

content

amount

and

RNA,

of nuclear

was

X966),

1965).

G-C

%

cistronic,

study

with

that

- 60 is

present

that

and

a small

growing

the

dRNA

and

50

minutes.

agreement

composition a mixture

225

number

treated with DNAse (2 w/ml, 30 minutes) 60 minutes), reextracted with phenol, centrifractions w4ere precipitated with citrate and against PO 0.01 M, EDTA 0.01 M. 2, centrifuged in 5 - 30 $ glycerol,

cytoplasmic

(in

h

t

Fractions

EDTA

cpmd0 C

O3

“in

et

Nuclear vitro”

has

Lissitsky might

Vol. 25, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES Cartouzou,

G.,

Mant6,

S.,

and

Res. Corn. 20, 212, (1965) Elias, J. J. and Rivera, E.M., Georgiev, G. P. and Mantieva,

153,

Lissitsky, Cancer V. L.,

S., Res. Biochim.

Biochem. 19,

Biophys.

505, Biophys.

(1959) Acta

61,

(1962)

Harel,

J.,

J. Mol. Harel, Biochim. Huppert,

Biol. 1, 645, (1963) L., Harel, J., BoBr, A., Biophys. Acta, 87, 212, J., Lacour, F., Harel,

26,

Harel,

L.,

Lacour,

F.,

BoBr, Imbenotte, (1964) J. and

A.

and J.

Imbenotte, and

Harel,

L.,

J.

Carpeni,

N.

Cancer

Res.

156 (1966)

Nataf,

B.M.

and

Chaikoff,

I.L.,

Biochim.

Biophys.

Acta

m,

422

(1965) Nataf, 263D, Nataf,

35,

B. M., Malaise, 63 (1966) Rivera, B.M.,

E., E.

and M.

and

Tubiana,

M.,

Chaikoff,

IL.,

Comptes-Rendus, Endocrinology

76,

(1965)

Nomura,

306,

M.,

Hall,

B.P.

and

Spiegelman,

S.S.,

J.

Mol.

Biol.

2,

(1960)

Scherrer,

K.,

Gros, F., Scherrer, Sci. U.S.

Proc. Coil. Sot. K., Latham, H. 2, 240, (1963) Raghupathy, E.

Tong,

W.,

931,

(1963)

Marcaud,

Warner, J., Soeiro, R., J’, Mol. Biol. In the press. Fukada, Yoshikawa, M., Acta, 103, 383, (1965).

L.,

Zajdela, Chim. Biol., and Darnell, and

Chaikoff,

F.,

Breckenridge, Marseille J.E., Proc. I. L.,

B.

(1965).In Natl.

the Acad.

Endocrinology

Birnboin, C., Girard, M. and Personnal Communication. T. and Kawade, Y., Biochim.

578

and press.

72, Darnell, Biophys.

J.