Determination of some mitochondrial RNAs concentration in adult rat liver

Vol. 118, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Januory 13, 1984

Pages 284-291

DETERMINATION

OF SOME MITOCHONDRIAL RNAs CONCENTRATION IN ADULT RAT LIVER

P. Cantatore,

M.N.

Institute

Gadaleta,

and C. Saccone

of Biological Chemistry, University of Bari, Bari, Italy

Received November 29, 1983

A method has been developed to accurately measure steady state concentrations of mitochondrial transcripts in adult rat liver. Total mitochondrial RNA has been hybridized with an excess of labelled mitochondrial DNA fragments coding for a single gene or a piece of it. The results obtained show that each mitochondrion contains 36 molecules of 165 rRNA, 81 of 125 rRNA and about 8 molecules of mRNAs coding for identified and unidentified reading frames. Thus, the rRNA/mRNA ratio in rat liver differs from that reported in HeLa cell mitochondria. These results are discussed in the light of mitochondrial DWA transcription regulation.

Very animal

information

mtDNA

HeLa

cell

first re

little

expression.

entirely

L-strand

In

(2).

bably

and

The

involved

two in

ed

that,

in

these unstable.

however,

the

this

may mt

of

rRNAs

gene

one

both

In the

of

The

mRNAs

of

mt

in

ileLa

have

by

cell

pro-

mRNA

and

the

de-

are

(3)

synthe-

have

report-

mRNA species

inhibited

Authors

matuthe

been

H-strand

are

transcript-

mRNA species used

same

to for

Attardi

rRNA

conditions of

the

to

are

leading

polycistronic and

mature

mainly

po-int

on

of

strands

H-strand

the

mechanisms

expression.

and

the

Gelfand

stability of

for in

DhA

processed.. initiation

and

cells,

ion, the

rRNA

regulation refer

both

points

Moreover,

metabolically be

two

the

literature

one

initiation

the

respectively.

the

then

Recently

on

cells

and

(1).

transcription

scribed

in

these

transcribed

products

available

Data

mitochondria.

gene

sis,

is

can

change

cell

to

measured

and

regulate

concentration

mitochondria,

by

kinetic

ex-

periments. To re

regulation

physiological

ations Fion nt:

extend of

of

systems, rat

standard nucleotite(s).

liver

mtDNA

we

mtRNAs

abbreviations:

0006-291X/84 $1.50 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

transcription

decided

at mt:

the

to steady

mitochondrial;

284

studies

determinate state.

to the

This bp:

mo-

concentr-

was basepair(

made

BIOCHEMICAL

Vol. 118, No. 1, 1984 by

hybridizing The

total

results

obtained

mitochondrial rat

mtRNA

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

specific

reveal

mRNA species

labelled

consistent

mtDNA fragments.

differences

concentrations

in

between

t'eLa

celis

and

in

liver.

KCTiRIALS

AND METHODS

Liver mitochondria were isolated from an adult rat (150 g b.w.) through differential centrifugations (4). Care was taken to reduce nuclear contamination by centrifuging the postnuclear supernatant several times at low speed. After lysis in 120 mK NaCl, 10 rnN TRIS-HCl ptl 7.4, 1 mM EDTA, 1.2:: SDS, the nucleic acids were isolated through extraction with phenol-chloroformisoanylic alcohol (50/49/l) and repeated ethanol precipitations. The DNA fragments used as probe in the hybridization experiments were isolated by cutting recombinant plasmids containing Eco RI fragments A, 8, C, D and'E of rat liver mtDNA with the appropriate restriction enzymes. After separation on polyacrylamide gel, the fragments were eluted and ethanol precipitated as previously described (5). Probe labelling was carried out by nick translation (6) for 2 h at 15°C in presence of 75 uCi/mmole of c (S2P)dCTP. At the end of the reaction the labelled CNA was isolated by chromatography on a G-50 Sephadex column eluted with 10 mM TRIS-HCl pH 8.0, 1 di EDTA. The DNA size, determined by urea-polyacrylamide gel electrophoresis, ra ged from 200 to 400 bp and its specific activity was around 1.10 7 Cpmlpg. The soluticn hybridization experiments Kere done in a volg me of 40 ul using the conditions described by Casey and Davidson (7). After incubation (20 h at 48°C) the samples were digested with 100 U/ml cf 51 nuclease (Sigma) for 30 min at 45°C. The amount of hybrid formed c~as determined by t.richloroacetic acid precipitation. ihe DKA and RNA concentration was aetermined by colorimetric methods as diphenglamine and orcinol, or (in case of DNA) using the fluorimetric assay developed by Labarca and Paigen Protein concentration was determined through the Waddel me Kd (9). RESULTS The

strategy

used

concentrations

of

termination

of

zation

of

of

it.

This

mtDNA

is of

amount

for

could

be

since

gene

done, and

cells

containing liver

these

mtDNA

cells, and

plasmids

with

several genes

for

total

of the

were

E.coli

the

two

rRNA

285

165

D and

were

obtained The and

rat

liver

chimeric E fragextrac-

separated

on

by cutting

the

probes 125

a

a piece

DNA was

DNAs

were

of

or the

C,

de -

hybridi-

excess of

Total

enzymes. mt

an gene

B,

on the by

harbouring

plasmid

restriction

with

A,

available. probes

formed

organization RI

state

based

a single

E.coli

and

finally

mtRNA

Eco

steady

was

hybrids

of

gradient the

DNA-RNA coding

a CsCl tained

of

fragment

(10)

the

transcripts

quantity

rat

from

measure

mtDNA

pSF2124,

ments ted

the

known

plasmid

accurately

mitochondrial

a known

radioactive

to

and

useo for

confive

Vol. 118, No. 1, 1984 mRNA

the stion

Co111 , FTFase-6,

(CoI,

sentative

BIOCHEMICAL

pattern

of

As

shown

in

16s

rRNA

was

of

pSF2124

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

URFl

a13

mt

Fia.1,

and

URF4)

so

to

obtain

a repre-

transcripts.

the

fragment

the

1304

bp

(I))

with

HhaI

containing

long

one,

and

the

deriving

RI;

Eco

from

the

12s

gene the rRNA

for digegene

ECoRI A 4 > ECoRI

Hpa ll

c

Hpa ll

.I

Hpa II

+2opo

Lpi

Pro

g’u

,URF

u 1

II 1

I I

wtb

“!?”

ECoRI

+i

6ayi

6 URF 5

1111 \

t thr replication

origin

ECoRIB<

hi/;,

-

6 I

IATPase

‘1 fYS URFAG

t

Co III

ilURF3ll

L

ECoRI C

L H

alaayqy& I I III I 11 I t

-WI

1

origin

ECoRI

4

1

L strand “f

co I

ECoRI D <

L H

Twl

l2opo

replication

URF4

k

III Taq I

lopoC4

>

&I URF4L

f Taq-i Hind

--RF2

ECoRI 4

I

dY’

ECoRI 4

4-

H strand

)

II-II

-URF leu

III

t

-C0II-

> Hha I

ECoRl

+, 1500

5p

166 RNA

J

I IURF i

t

I!”

ECoRI E

>

ECoRI 4 L <

59

URF

1

ECoRl &j

>

Physical and transcription B, C, D and E. Only the A two be prepiration are shown. ments that were labelled by nick Hae III and Hinf I sites.

map of restriction headed arrow translation.

286

rat liver mtDNA fragsites used for proindicates the fragA and H are the

Vol. 118, No. 1, 1984

was

contained

BIOCHEMICAL

the

fragment

of

gestion

of

Eco

A with

HpaII.

The

gestion

of

Eco

RI

tained

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

the

and URF4

have

two,

were

prepared

6am

HI and

then

is

specific

with

of

of

labelled

by

ned

the

DNA coextracted

was

not

eliminated

dard

in

Therefore the

and

with

all

the

tion

are

reported

centration to tion per

amount with

of

used.

the

mitochondrion

that

there

rRNA

per

36

ranging

from

of

by

using

of

DXA

tofrag

of

mtRNA

0

are

URF4

and

in

the of levels

at

satura-

used

for

con-

by relating

them

mitochondrial of

frac-

5 DNA molecules in

81

present

mRNA to

species.

saturation

number

rRNA

It

stan-

added

were the

RNA.

activity

reported

16s

mRKAs f

mtRNA

species,

from

results,

The

of

RNA hybridized

a mean of

internal

specific

quantities

each

The

as

DNA was

the

contai-

the

reached

These

molecules

6 molecules

the

also

used

cold

for

pg

assuming

mitochondrion.

in DNA

maximizing

hybridization was

coextracted

(11).

are

out

concentration

The of

RNA and

grown

hybridized.

mitochondria.with

made

Tab.1.

mtCNA

and

calculate

conditions

Hybridization

determination

the

for

quantity

in

III

plasmid

to

carried

in

the

were

probes

were the

used

other

fragment

concentrations

amount

of

probes.

be

were

the

its

equivalent

radioactive

was

The

determine

and

con-

the

Hind

plasmids

di-

ATPase-6

whereas

To

uCi/ml)

was

B fragment. with

di-

the

CoIII,

for

first

to

used from

corrections

gene

the

(7).

preparation since

URFl

RI

I.

translation,

calculation an

blanks

B,

cpm/ug)

fcrmation

from

sites,

containing (0.5

and increasing

hybrid

the

Hinf

Rl!A

RNA-DNA

mtRNA

and

CNA fragment

the

The

RI

the

nick

Eco III

experiments

mitcchondrial

by

resulted

probes

Eco

1000

hybridization

The

Hind

cells

(about

concentrations

ments

III

Hae

originated the

the

two

3H-thymidine

activity

The

E.

digesting

bp,

whereas

from in

E.coli

presence

RI

prepared

contained

concentrations,

tal

Eco

been

probe

I,

Taq

fragment

URF4

the

C with

934

Co1 gene

Tab.1,

show

molecules at

of

lower

1'LS

levels,

16 molecules

of

CcI

mRNA. This direct same

yield

preparation tion cleic

approach

assuming from is

v;as acids

DNA extracted

to that

calculate

mtRi:fi

the

DNA and

mitochondria

and

of

verified

mitochondrial by

extraction from

that

the

is

coextracted

the only.

The

the

Di:A/RriA

ratio

mitochondria.

mitochondria

was 287

the with

Di:F, present

origin

determining from

the

concentration

RNA are

in

first

that

mtRliA

assump-

during

Furthermore, 1% of

more the

extracted

nuthe

Vol. 118, No. 1, 1984

BIOCHEMICAL

Determination

TABLE I the concentration of several species by hybridization

of

RNA

AND BIOPHYSICAL RESEARCH COMMIJNICATIONS

RNA

DNA hybridized per

RNA

RNA molecules mitochondrion

species

w

Pg

12s

10

102 + 2 225 f 7

81.0

+ 3

13 25

72 + 4 82 + 3 145 + 8

36.0

2 4

URF 1

100

82 + 4

8.5

+ 0.5

co1

100

16.0

+ 2

ATPase-6

100

49 + 7

7.1

+ 1.2

co111

100

38 + 8

7.3

+ 1.6

URF 4

100 150

67 + 2 71 + 2

6.4

+ 0.4

20 165

10

185 ~17

The values reported in the third They are the mean of saturation. far each RNA concentration, run was determined by measuring the cubated without mtRNA. The number chondrion has been calculated as

from

the

rature II,

homogenate (11).

was

mtRKA

absent

in

concentration

total

the the

DNA.

mitochondria the

same

column are those obtained at at least three experiments in triplicate. The background inS1 resistance of samples of RNA molecules per mi Odescribed in the text.

same

preparation

as

nuclear

marker

enzyme,

mitochonarial

RNA either

of of

the

were

cellular

ses

of

Finally,

hybridized

re

nitochondrial

to

also the

by

values

and

RNA occurring magnitude as

order

of

Estimates

referring

mitochondrial

The

the

or

normalized during those

of

pmoles

proteins

obtained,

lite

ymerase

RNA po

preparation. made

in

reported

of

to

for

the

the

the reported

extraction, in Tab.1.

measuring

the

10s we

DISCUSSION In tration chondria RNA is

this of are double

paper the

two

the rRi;As

reported. than

165

results and

obtained of

five

by mRNAs

They

show

that

RNA.

This

difference 288

the

in

rat

liver

mito-

concentration could

concen of

be

due

to

125 the

8lOCHEMlCAL

Vol. 118, No. 1, 1984 gene ly

order

in

adjacent

tration

to of

Co!

is

mRNA and

it

only

by

From

the cells

it in

10

to

100

Coi and their

to

data that

ring

early

which

in

of

the

ven

first

by

the

(19).

synthesis

is

cies

are

trationsare ted

this

mtDNA directly

level

their cannot,

addressed

well

of

rat

with clear liver

liver

the

out

absence In

the

cells

at

10

days

at might

decay also

stability. however,

replication takes

of

at

that

their

level.

steady

The rate,

by as

in

on this rat

mRNA could

A regulation

operating

excluded

measurement

of 289

state

and mtRNA

only stability

post is

that

of

when rnt

gi de-

mtRNA

mRXk

state

data

explained

mitochondrial be

of

of

a kinetic

steady and

stabilities

be

with

that

and

RNA syn

weeks

synthesis

du

mtDNk

k:hereas

indicated

de-

organ

birth, tt;ree

a

place

an

age,

the

the

respiratory of

mtDNA mtRNA

RNA decays

of

have

of

first

have

urchi'n

cerebellum,

the

rate

in

sea

after

that

proceeds

of

completely along

the

in

synthesis

suggesting

to

synthesis

carried

(20,21)

elevated of

mtRNA

Authors

inhibited

concentration

increase

rat

up

between

paper

conditionsof creased

in

concentration

Several

reguieted

in

of

of

assuming

its

increased,

concentration

mENAs

in is

mRNAs

seems

almost

By

ratio

gradation

of

it

the

of

peak

order,

and

(12,i3);

constant

(17,18).

con-

rRNA

correlate

(14-16).

synthesis

life

liver 125

systems

even

develops

remainsquite

natal

sp:te

development

protein

thesis

that

synthesis

rats

rat

aa 3 content

Studies

in

protein

the

iieL.a.

fashion.

monstrated

The

sta

repor-

mitochondria. of

rate

po-

steady been

concentration

respiratory

suggest

constitutive

the

cell in

concentration

higher the

the

the

measured

HeLa

cells.

two

for

have

concentration

HeLa

the

higher

the to

Some

in

in

on

Authors in

whereas

lower

except

mRNA genes.

obtained

the

systems

the

respect

mt

results

of

confirming

literature

mRNAs

that

cytochrome

that

related

and

the

times

respective

with

of

concen-

that

mitochondrial

These

of

out

order

immediatg

The

than

transcripts (3).

fact

point. lower

gene

the

and

mRNAs

therefore

in

in

mRNA measured of

rRNA

two

COIII

all of

Attardi

comes

the

for

mitochondrial

comparison

similar

is

same

existing

125

RNA is

order

pattern

and

of

one

independent

data

Gelfand

HeLa

mt

is

125 initiation

about

the

of

centration

is

is

transcription

The

the

transcription

concentration

ted

mtDNA:

mRNAs about

lycistronic

te

liver

the

the

It

rRNAs.

rat

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

spe-

concen-

nRNA:

repor-

mechanism.

In

liver,

the

depend

on

at

in an

promoter

experiments may

settle

Vol. 118, No. 1, 1984

the

problem.

since

the

This

that

their

lism.

Data

from

sibility.

The

tochondrial

genesis

apply,

liver

and is

used

here

number

of per study

could

during

be

development

mitochondrion. thyroid

cells

12s of

could

useful

in

(22)

is

per

cell

The

same

hormone

due or

metabo-

in

in

to

that

on

speci-

out

if

the

liver

at

to

the

rise

of

RNA con-

considerations

action

ml

different of

rat

either

pos-

studying

finding

observed

sug-

this

in or

rRNAs

level

cell

measurements help

the

confirm

development

Concentration

mitochondria of

HeLa

to

eventually

phosphorilation of

however,

independent

will

transcripts

stages

the

not

systems

expression

oxidative

centration

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

concentration other

mitochondrial

to

rat

conditions.

critical

ply

of

gene

increased the

does

approach

physiclogical

of

model

similarity

gests

fic

BIOCHEMICAL

could

mitochondrial

ail bio

(23).

ACKNOWLEDGEMENTS We thank Z. Flagella and G. Pesole of experiments and F. Fracasso for the stance. This work was partially supported Progetto Finalizzato Ingegneria Genetica le Maiattie Ereditarie, CNR - Italy.

for their help in skilfuil technical by a grant from e Basi Molecolari

some assithe del-

REFERENCES 1)

2) 3) 4) 5) 6) 7)

8)

91 10) llj 12) 13)

Attardi, G., Cantatore, P., Chomyn, A., Crews, S., Gelfand, R Montoja, J. and Ojala, 0. (19823 In “Mitoch~n!~~~~'~e~e;" (Sionimski P P. and Attardi, G. Borst, eds) pp. 51-71. Cold Spring'HarLor, New York. T., Levens, D., Rabinowitz, M., Montoja, J., Christianson, and Attardi, G. (1962) Proc. Natl. Acad. Sci. USA 79, 7195-7199. Gelfand, R. and Attardi, G. (1981) Mol. Ceil Biol. 1, 497-511. Saccone, C., Gadaleta, M.N., and Gallerani, R. (1969) Eur. J. Biochem. 10, 61-65. Saccone, C.,Cantatore, P., Gadaleta, G., G., Gallerani, R., Lanave, C., Pepe, G. and Kroon, A'.M. (1981) Nucl. AC. Res. 9, 4139-4147. :igby; PBYo:., Dieckmann, M., Rhodes, C. and Berg, P. (1977) 113, 237-251. Casey,‘J. and Davidson, N. (1977) Nucl. AC. Res. 4, 1539-1552. Labarca, C. and Paygen, K. (1980) An. Biochem. 102, 344-352. Waddei, W.J. (1956), J. Lab. and Clin. Med. 4B,Tl-314. Pepe, G., Holtrop, M., Gadaleta, G., Kroon, A.M., Cantatore, P Gallerani, .R., De Benedetto, C., Quagliariello, C., &a, E. and Saccone, C. (1983) Biochem. Int. 6, 553-563. Neubert, D., Gregg, C.T., Bass, R. and Marker, lT.3. (1975) In "Biochemistry of animal development" (ed. by Weber R.) Vol. III, Academic Press, New York, 387-464. 175-181. Williams, J.N., Jr (1968) Biochem. Bioohys Acta E, Hare, J., Ching, E. and Attardi, G. (1980) Biochem. 19, 2023-2030.

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18)

19) 20) 21) 22) 23)

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Cantatore, P., Nicotra, A., Loria, P. and Saccone, C. (1972) Cell Differentiation 3, 45-53. Gadaleta, M.N., Nicotra, A., Del Prete, M.G. and Saccone, C. (1977) Cell Differentiation a* 85-94. Devlin, R. (19761, Dev. Biol. 2, 443-456. Gadaleta, M.N., Giuffrida, A.M., Renis, M., Serra, I., Del Prete, G., Geremia, E. and Saccone, C. (1979), Neurochem. Res. 4: 25-35. Giuffrida, A.M., Gadaleta, M.N., Serra, I., Renis, M., Del Prete, G. and Saccone, C. (1979) Neurochem. Geremia, E., ", 37-52. Res. Galau, G.A., Lipson, E.D., Britten, R.J. and Davidson, E.H. (1977), Cell 10, 415-432. England, J., Estantino, P. and Attardi, G. (1978) J. Mol. 119, 445-462. Biol. Gadale= M.N., Di Reda, N., Bove, G. and Saccone, C. (1975) Eur. J. Biochem. 51, 495-501. Pollak, J.K. and 'SiTtton, R. (1980) Trends in Biol. Sci. 1, 23-25. Jakovcic, S., Swift, H.H., Gross, N.J. and Rabinowitz, M., (1978) J. Cell Biol. -77, 887-901.

291