Effect of specific inhibitors on mitochondrial DNA replication in HeLa cells

Vol. 60, No. 3,1974

BIOCHEMICAL

L. Laboratory

of

Received

Cell

July

Pica

Mattoccia

Biology,

22,

AND BIOPHYSICAL

C. N.

and

R.,

Via

S.

RESEARCH COMMUNICATIONS

Roberti

Rornagnosi

18

A,

Rome,

synthesis

on

the

bromide

densit)-

Ital>-

1974 SUMMARY

The

effect

of

tochondrial

different

DNA,

sedimentation xyadenosine

velocity, excess,

of mitochondrial difference clear DNA

of 22%) synthesis,

analogy

what

as

to

inhibitors

isolated

by

of

has been hydroxyurea

studied in HeLa and fluorodeoxyuridine

DNA less than while cytosine has a very

happens

expected,only

in

DNA

CsCl-ethidium

cells.

labeling

Thymidine inhibit

that

of the n.uclear DNA (with arabinoside, which strongly limited action on mitochondrial

the

bacterial

mitochondrial

system.

DNA

of

gradient

Ethidium

miand

and deolabeling

the

a maximum inhibits DNA, bromide

nuin

inhibits:

replication.

INTRODUCTICN

study-

The drial

of

replicative

and

cytoplasmic

and

alcohol

xic

The

(m.

Borst

All rights

only

replication

Irorri

lack

of thymiciine

in

to

HeLa

drugs

of

understand

of

cells,

the

the

in

to

isolated

nuclear

extent

into

acid

to

a limited

that

thymidine been

and of

the

bromide;

HU Thy

=.

@ 1974 by Academic Press, Inc. of reproduction in any form resetvd

by

mirochonacton

“1:

of

by

by

An UN-4

= hydroxyurea; thymidine;

882

DNA

not

Pica

block Mattoccia

synthesis

alternative synthesis CA FIJdR

not

after

by of

also

= cy-tosine :? fl-orodeox)-crilir.e

nalidi-

mitochondrial and the

hypothesis but

that

inhibited

concentrations

does

suggested

circular

found

is

high

of

hydroxyurea;

mitochondria,

excess

rn.

labeling

material

extent

block.

indeedblock

the

inhibition

insoluble

of m.

thymidine

that

chick-liver

synchronization

might

found

resistant

has

a double

Eth. br = ethidium d. Ade = deoxjradenosine:

Copyright

dATF

possibility

the

cells

(2))

and

DNA)

interpret

excess

help

relatively

of

phenethyl acid.

(1)

is

incorporation

HeLa

can

Penman DNA

Tershegget

DNA

to different

biogenesis.

Vesco

the

sensiti-Jity

systems

rritochondrial

to

the

Attardi release

was cause arabinoside;

(3) of

that anarrest

the of

BIOCHEMICAL

Vol. 60, No. 3, 1974

AND BIOPHYSICAL

mitochondria

development

at different

stages

cpon

of the

thymidine,

mitochondria

removal

development

and reach

In the

present

of HeLa sine,

excess

cells

the

paper

grown

stage

we investigated

in the presence

fluorodeoxyuridine,

bromide.

That

of DNA

compared

HeLa

(S,

clonal

(Joklik,

strain)

Grand

Island)

ranging

b) Drug

and labeling

treatment

if Samples

C/ml; B one hour

as DNA xyadenosine

ii)

at the

and

ethidium

labeling.

incubated

thymidine

for

(0, 1 PC/ml; and mixed

and

5. 1 OS/ml.

were

two

serum

at 37°C

in the

The

(Merck),

resus-

[methyl-

3H.]

H-G deoxyadenoP I. of the specific inhibitor

presence

isotope.

and

to 4. lO’im1.

with with

following

drugs

hydroxyurea

fluorodeoxyuridine,

were

(Sigma),

cytosine

used deo-

arabinoside

HCI,

HeLa

cells

the

recovery

Sigma) ethidium bromide (Sigma). 7 (1 or 2.10 cells in 50 ml), to be used as of m.DNA,

50, 5 mC/mM,New with

modified

centrifuged

hours

Amersham)or

thymidine

arabino-furanosyl

control

5% calf

of 5. 105,/ml

for

radioactive

inhibitors:

Drug-untreated

in Eagle’s

with

cultures

8 C/mM,

the

in suspension

concentration

(Schwar’zfMann),

an internal

the tritium

were

England

labeled

labeled

with

Nuclear

samples

Corp.)

at the

[2-l*C] for

end of the

about incuba-

period. iii)

and

before

1-p-D

24 hours

DNA

deoxyadeno-

conditions.

7, 5 C/mM,Amersham)

synthesis

(cytosine

of m. DNA

METHODS

4. 1 04/ml

growing

10 pC/ml;

(10

added

tion

medium

(5 or

grown

between

of 10 to 25 ml were

thymidine sine

AND

their

excess

arabinoside

on total

supplemented

of exponentially

in fresh

Aliquots

of labeling

effect

were

at a concentration

pended

resume

the.inhibition thymidine,

so that

cultures.

cells

medium

cycle,

wouid

cytosine

to the

life

asinchronously.

of excess

MATERIALS a) Cells

of their

replication

hydroxyurea,

was

RESEARCH COMMUNICATIONS

The

[2-‘“Cl

radioactivity trichloacetic (matman

incorporation thymidine

into

in samples acid GF/C).

3

of [methyltotal

of the

H] thymidine

cellular total

(TCA).in

the

cold

Samples

labeled

DNA

was

cell

homogenate

and

collected

with 883

tritiated

or

PH-GI

analyzed

deox)-adeno

sine

by- measuring

the

precipitated on glass

fiber

deoxyadenosine

with

5%

filters were

treated

Vol. 60, No. 3,1974

with

NaCH

lize

the

BlOCHEMlCAL

0. 5 N for

15 min

Cytosine-arabinoside

measured

in cell

[5 -3S] c)*osine m. DNA

was

m. DNA

samples

then

first

fraction

was

bromide

gradient

method,

was

The

m. DNA

two

hours

I and RNase

centrifuged

, to ‘a:.-dro-

was

then

tions.

mitochondrial

The

sulfate

(SDS),

digested

for

pellet

30 min

hours

samples

were

(Merck)

chilled

for

recentrifuged

under

the

added

in order

alcohol

were

hours

at -20°C

the

at 14. 000 rev/min

for

in 1 ml

0. 01 M Tris

a two-step

CsCI-ethidium in

bromide)

0. 01

M in

same

was

for

the

buffer Spinco

pH

The

SW

50.

and

gradient

CsCl,

1 rotor

(1 ml

884

M at

buffer

(pH condi-

dodee)-!

and pronase

EGTA. 33,

000

(self-

of 75 rg/ml. concentration

of

at 12.000

rev/min

fraction of cold

acids.

acids

0. 01 M EDTA

0. 01

10 min,

was

93’

After

were

sedimrnted

rev/min

rotor

resuspended throl?gh

g=l.70, with

eth?-1 at least

in the SS 34 Sorvall nucleic

for

for

same

supernatant

the nucleic

centrifuged

7. 2,

7.2)

to a final

and the pelleted

bromide

the

of 1% sodium (pH

digested

fraction

at 4°C

and two volumes

to precipitate

pH 7. 2,

and

0. 01 M Tris

and centrifuged

15 min.

were

cells)

to a concentration added

conditions

samples

buffer

added

0. 05 Xl

rcitochondrial

under

buffer

2,

respectively-)

SS 34 rotor

in 3 ml

30 min

at 4°C

20 min

Tris

lysed

was

CsCl

SS 34 rotor

the

M sucrose,

0. 01 M Tris

in the Sorvall

packed 100 rg/ml

and

as in the first

M MgCl

and recentrifuged was

at 37°C)

at 37”C,

and

of the Storrie

obtained

buffer/ml

in the Sorvall

cells)

mitochondrial

in a CsCl-etbidium

0.0015

treatment

in 0.25

packed

of the

is a modification

100 pg/ml

enzimatic

lysate

fraction,

(4 ml

7.2)

rev/mm

methods.

below,

(pH

0. 01 M EDTA, two

with

(3).

mitochondrial

resuspended

buffer/ml

-Gas

Amersham):

to equilibrium

M sucrose,

the

at 9600

for

14 C/mM,

sulfate

centrifuged

A (10 or

After

labeled

dodecyl

in 0.25

buffer

7. 2) (4 ml

dium

and

nuclear

different

described

(4).

at 37°C.

@ = 1.40

TCA

below.

described

method,

resuspended

DNase

1 M,the

a sodium

as previously

0. 005 M Tris

the pellet

two

into

5 rC/ml,

to two

and then

procedure

After

with

DNA.

method

second

or

as described

according

prepared

The

Attardi’s

was

precipitation

1.1 06/m1)

(2.5

isolated

isolated

i) In the

15 min

(70 ml,

of mitochondrial was

NaCl,

before

incorporation

#j-D-arabinoside

c) Isolation

with

at 37°C

RESEARCH COMMUNICATIONS

RNA.

iiii)

ii)

AND BIOPHYSICAL

3ml

200

CsCl,

yg/ml for

ethi5 hours

Vol. 60, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table 1 Effect of llffmmt DNA synth,,ls Inblbitors on the lrbella6 tf total cdl DNA an1 n.DIA lrolrtrd by CsCI-stbldlum bromide density pradisnt ,

Expt

Ethidlun btoaida

IPlhl

0

: INHIBITIOI m. ONA 100

2 3 4

Hydroxyurer * .

lwSP . 5*10-‘M

95 94 70

02 59 53

5 6

:ytarlne rrbiaosld~ Ho-’ M * *

96 07

0 6

7 0 9

Oetxyrdtntsinc . .

2WM t *

96 67 76

04 71 79

IO I1 12

lkymidine * .

2*lbJP .

65 69 77

67 63 63

I3 I4 I5

Fluorodatxyuridinr * *

Ir#/ml . *

47 77 75

74 54 70

INHIBITOR

no r

l

Table 1 Effect of different DNA synthesis inhi.bitors on the labeling =ell DNA and m. DNA isolated by CsCl-ethidium bromide equilibrium gradient.

of total

density

m. DNA was isolated from HeLa cells labeled with tritiated thymidine (expts. l-9) or deoxyadenosine (expts. 10-15) in th-e presence of specific inhibitors as explained in Materials and Methods section ,b fi) and in the legend of Fig. 1. The labeling of total cell DNA was ca?culateci as 3H ctslmin present in the samples of the cell homogenate precipitated with TCA3(Materials and Methods associated b_ (iii)) while the labeling of m. DNA was calculated as H cts/min with the heavier band of the gradient (pertaining to closed circular DS,Q, The percentages of inhibition of radioactivity incorporation in total cell DNA and m. DNA were calculated relatively to the incorporation in controls.

at 20°C. bottom

After of the

and counted

the tube,

before

were TCA

fractions

precipitated

in a liquid

deoxyadenosine lized

run,

with

scintillation treated

of about

with

120 ~1 were

TCA.

collected

counter. NaOH

precipitation.

885

0.5

Samples N (15 min

collected

from

the

on glass

fiber

filters

labeled

with

[‘H-G]

at 37°C)

and,neutra-

Vol. 60, No. 3,1974

BIOCHEMICAL

RESULTS Effect

of different

a) IX. DNA Table

1 shows

to the

method

cedure

only

seen

that

inhibitors reported the

(2. 1 Oe3M)

of inhibitors, 20%

arabinoside, is very

I of m. DNA

it doesn’t

affect

5. 10W4M),

m. DNA

finally, poorly

equilibrium

of these

inhibitors

corresponds

the

density

circular

or open

circular

strong

labeling.

in Fig.

that

of the 1.

m. DNA DNA

Ml,

For

labeling

of total

cell

by

m. DNA

labeled

pattern

molecules molecules

total

with

analysis

the

and the (mostly

DNA.

thymidine

among

on total

each

inhibits cell

as expected,

effect

The

It can be

of total -3

Hydroxycrea,

verus

pro-

completel>-

(2.10

on m. DNA

expected

B>- this

labeling

inhibit,

labeling.

gradients

is shown

the

expo-

according

can be quantitated.

at all

active

were

isolated

sectionS(

deoxyadenosine

of this

on m. DNA

to closed to linear

inhibition shows

active

less

cells

of 1 pg/ml,

(1 pg/mI)

extent

in the

bromide

pertains

and Methods,

at a concentration

is consistently

difference

m. DNA

labeling.

gradient.

was

labeled

and fluorodeoxyuridine

and to a lesser

densit)-

and the

form

(1.1 Oq3M,

on m-DNA

bromide

in Materials

while

synthesis

in which

bromide

labeling,

Hydroxyurea

of DNA

of a set of experiments

supercoiled

ethidium

m-DNA

DNA

results

RESEARCH COMMUNICATIONS

DISCUSSION

by CsCl-ethidium

the

sed to different

AND

inhibitors

isolated

AND BIOPHYSICAL

cell

this

group

an average

DNA. cell

Cytosine DNA

but it

CsCl-ethidium in the heavier major

presence band

lighter

nuclear

band

contami-

nant s) .

Fig. 1 Analysis m. DNA labeled

b>- CsCl-ethidium in the presence.

bromide

of different

equilibrium inhibitors.

density

gradient

of

HeLa cells were labeled with tritiated thymidine (a- cl) or deoxyadenosine (e, f) as explained in Materials and Methods section b(i). DNA extraction from the mitochondrial fraction and banding in CsCl-ethidium bromide density gradients were carried out as previously described (3). Gradients were centrifuged for 48 hours at 35. K rpm in the SW41 Spinco rotor at 2O”C, fractions of about 70 ~1 were collected from the bottom of the tube and aliquots of 20 VI with TCA, collected on glass (a), 30 pl (b, c ) or 50 ~1 (d, e,f) were precipited fibre filters (Whatman GF/C) and counted in a scintillation counter. Aliquots from gradients e and f were treated with NaOH (0.5 N, 15 min at 37’C) and neutralized before being TCA precipitated., -3 arabinoside a: ethidium bromide 1 pi/ml; b: hydroxyurea 1. 10 M; c;3cytosine 1. 10W5M; d: deoxyadenosine 2. 10W3M; e: thymidine 2.10 M; f: fluorodeoxyuridine 1 pi/ml. . ---. ---. control; . -. inhibitor.

886

BIOCHEMICAL

Vol. 60, No. 3,1974

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

v (bl

HU

:i =F ,

(I)

7 :

Ethbr.

dAda

Fraction

b)

m.

de

two-step

It

DNA

cannot

isolated

by

sedimentation

velocity

no

through

a CsCl-ethidi?lm

m.

determinations

bromi-

gradient. be

significantly small

Thv. (eJ ,

ignored

that

the

precision

by

the

isolation

procedure

(relative

to

affected proportion

of

m.

DNA

of

the

total

887

DNA used,

DNA)

in

in

view

the

cell,

be

ma)-

not but

only also

of of

the its

(d)

Vol. 60, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL

Table Effect

of different

DNA

RESEARCH COMMUNICATIONS

2

synthesis

inhibitors

on the

Iabelinl

of total

cell DNA and m.011 isolated by sedimentation throughaCsCI-ethidium bromide two step-gradient.

cBIc

Expt. lWHlBlTOR no 1 Et hidh brtrtde

*

PERCENTAGE OF INHIBITION TOTAL DNA m. DNA 0 100

2 3

Hydroxyurca

82

65

98

5

96

89 75

83

47

96 95

34 24

iytotine

arabinosik

7 II 9

94

28

10 11

96 97

20 15

12 13

97 96

35 37

14

36

ta

90

60

15

Oeoxyrderosine

16

89

75

11

91

82

70

57

Thymidine

I8 Table

2

Effect

of

and m. two-step

different

the

DNA

DNA isolated gradient.

Cells were labeled (expt.18’) as explained isolated as specified The labeling of total while

100

4

6

cell DNA bromide

20

synthesis

by

inhibitors

sedimentation

on

velocit)-

the

labeling

through

of

total

a CsCl-ethidium

with

tr itiated thymidine (expt s. 1 - 1’7) or deoxyadeno sane s section _b (i) and rr,.DZaq was Materials and Method in section s(ii). cell DNA was calculated as described in legend Table 1 3 H cts/min incorporated into m. DNA was caiculated as in

radioactivity

associated with m. DNA form I (a and b) and form II as separa.ted b>- sedimentation velocity (Fig. 2-4). Normalization for differences in recovery of 14C cts/min present in the same bands. m. DNA was done on the basis of the The percentages of inhibition of incorporation in total cell DNA and m. DSA were calculated relatively to control samples. Each value of inhibition of m. DNA labeling DNA form I and

represents II.

presence

in

accurate

assessment

which

appeared

multiple

the

forms of

to

vary

average

with the over

of

the

different

values

physico-chemical

differential a broad

separate

effect range

888

of of

the

activities,

found

ior

m.

properties. inhibitors

used required

An here, then

to

be

Vol.

60,

No.

BIOCHEMICAL

3,1974

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1500.

100O-

5010. 2 c

v) ',

d

I"

150nl-

100IO

50IO

8

Fraction

Fig. 2 gradient

no

Fraction

Analysis by sedimentation velocity of m. DNA labeled in the presence

HeLa cells (5 pi/ml, 8 1 pi/ml (b). standard for of m. DNA, are described to in the text. .-.-.

no

in CsCl-ethidium bromide of ethidium bromide.

two-step

(8. lo7 cells in 20 ml) were labeled with [methyl-3Hj thymidine C! mM) in the absence (a) or in the presence of etfidium bromide Long-term labeled cells were used as in internal k-l4 C 1 thymidine the recovery of m. DNA (Materials and Methods g (ii)).Isolation conditions of the run and collection of fraction from the gradient in Materials and Methods c(ii). The numbered bands are referred 3H

cts/min;

. -----.

Fig. 3 Analysis by sedimentation gradient of m. DNA labeled in the

14

-----. velocity presence

C cts/min in CsCl-ethidium bromide of different inhibitors.

two,-step

HeLa cells (2: 4. 10’ cells in 20 ml)were labeled with’[methyl-3H] thymidine in the absence (a) and in the presence of: hydroxyurea (5 pi/ml, 8 C/mM) 1. 10-3M (b), cytosine arabinoside 1.1 OW5M (cl, deoxyadenosine 2.1 Oe3M (d) See also legend Fig. 2. 14 .-.3H cts/min . -----. -----. C cts/min

889

Vol. 60, No. 3,1974

verified

BIOCHEMICAL

by another

than

the

method,

classical

Materials

employed

and Methods contaminant

riments

we also

obtained

by mixing

-term

[2-14C]

E (ii), band

introduced the

should

which

the

analytical

obtained

the

bromide

gradient,

with

a band

a faster

merit

forms

lar

m. DNA.

The

From

comparison

effects de,

the

on nuclear

In the

The

effect

possibility

still

that

the labeling

results

of Table

1 and

values

of rr. DNA

form

rence.

In addition, analytical

found

labeling

appeared

derably

smaller

that

artefacts

extraction,

since

that

could

procedure

used,

but not

presence this

source

into

of error,

with

account,

of an internal

was

of m. DNA

form

could

since

widely

(although

such perhaps the

where

second,

b) variable standard

the

c) contamination

with

resolved for

the

in the first difference

of the

any

of the

inhibition

significant

carried

diffe-

OUT b>- the

remaining

in all

consi-

variations

results

cases

could

of closed

not be easily

circles

obtainedwith

nicked

a different bands

during the

molecules

of materials,

of m. DNA

890

of experiments.

on m. DNA

recovery

labeled

type

dif-

arabinosidc

as: a) nicking alter

reproduced.

of cytosine

These

DNA).

bromi-

a considerable

first

show

circu-

differential

well

used,

of experiments

2) the effect

open

to ethidium

calculation

didn’t

m. DNA

DNA.

the

fairly

II (not

of

of oligo-

nuclear

in the

the

bottom

circular

to the

be responsible

a series

on total

to obvious

are

two-step

consists

that

1 (except

illustrate

the

closed

by exposure

than

discarded

to fluctuate than

caused

smaller

(Table

attributed

and taken

but

within

procedure

from

it appears

I and II individually

even

starting

presumably

arabinoside

of m. DNA) 2 was

througha

and thymidine

cytosine

was

of separation

ted

labeling

of such

2-4

m. DNA

to degraded

of long-

in Table

(II) corresponding

tables

of m. DNA

results

Figures

the

,of expe-

amount

reported ).

see,

corresponds

of the two

where

method

same

band

deoxyadenosine

experiments

ferential

band

and m. DNA

hydroxyurea,

data

(lb) which

a second

siowes:

to the

II from

recovery

the

to monomeric

shoulder

of m.. DNA;

2 shows

by sedimenting

corresponding

sedimenting

Table

can

the

in

series

a constant

re-examined

One

I and form

for

samples

sensitive

described

In this

standard

was not

gradient. (Ia)

form

velocity.

cells.

more

procedure,

m. DNA

be compared

fluorodeoxyuridine

RESEARCH COhW&INlCATlONS

and actually

This

experimental

for

CsCl-ethidium

separates

labeled

patterns

above.

and internal

with

which

from,

by sedimentation

thymidine

experiments

independent

method

nuclear

AND BIOPHYSICAL

are

first separa-

because

isotope by nuclear

the

eliminates DSX:

be-

Vol. 60, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL

Fraction

RESEARCH COMMUNICATIONS

II’

Fig. 4 Analysis by sedimentation velocity gradient of m. DNA labeled in the presence

in CsCl-ethidium of thymidine.

bromide

HeLa celis (2, 2. lo7 cells in 15 ml) were labeled withfH-G] in the absence (a) and in the presence (10 @/ml, 7, 5 C/mM) 2. 10m3M. See also legend Fig. 2 14 3H cts/min, . -----. -----. C cts/min . -.-.

cause (one

all

the

experiments

of which

is illustrated

-resistant

radioactivity

a ten-fold

increase

did

not

might drug

whese

cells,

results.

the

inhibition

used

at very

cells

were

incubated

r(iiii)), effect

DNA

high

of cytosine

arabinoside

of ethidium-

employed,

then

that

depends

on mitochondrial

derivative

(ara-CTP),

replication

is also

found

into

on m. DNA

891

tha:

mitochondria these

variations of the

of m. DN.‘A s:.-ntheinabilit>in analog)not

(6). Indeed,

was

found

of action

insensitivity

cytosine

also

to purify

relative

radioactive

no incorporation

absence

characteristics

concentrations

with

bromide

It was

We believe

on the

that

(5) where

bands.

deoxyadenosine (b) of thymidine

of ethidium

a complete

of DNase

dependent

unless

the

2)show

it to its triphosphate

in bacteria

and Methods

the

likely

in the presence

mitochondrial

amount

arabinoside

phosphorylate happens

in part

It is quite

sis to cytosine

bitor,

in the change

be at least

out

in Fig. in the

significantly

itself.

carried

two-step

to what

affected

b:- the

in two

ewgeriments

arabinoside m. DNA does

to

not

inhi-

(Xaterials (Fig, seem

5). in HeLa to depend

on

Vol. 60, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Fig. 5 Analysis by sedimentation velocity in CsCl -ethidium bromide two- step gradient of m. DNA isolated from HeLa cells (7. 1 07ceils in 70 ml) labeled for two ho,urs with [5-31-1] cytosine /j-D-arabinosi.de (2, 5 rC/ml, 14 C/m&:) See also legend Fig. 2.

the

concentration

occur,

(Table

we believe

uncontrolled caused

that

factors

by variable

meabilized

by toluene

concept

cells

is supported

mouse ted

Pica

nuclear

of two

only

into

Our

results,

since

play

a role

cultures):

into

the mitochondria

system.

It has

distinct

can

systems

by the results Kinase

cell

the

effect

shown

that

suppress

in E. coli

by Robberson

s where

radioactive

and

be phs s -

cells,

synthesis

phosphorylation

on

then

arabinoside

DNA

obtained

does

in turn would

of cytosine

of nucleoside

inhibition

(depending

inhibitory

been

in fact

a variable

per-

(7 1. in animal

Cla>-ton

th:-midine

(8) in

is incorpora-

m. DNA.

Mattocia

synthesis

hand,

might

, ara-CTP

L Thymidine

other

dell

leaking

by the

on the

permeability

in the

phorylated

The

i!),

finally

are

and Attardi’to

after

release

in favour interpret

of HeLa

cells

of the

second

the lack from

hypothesis

made

of synchronization

double

tly;-midine

b: of

m. DNA

block.

ACKNOWLEDGEMENTS We are manuscript

grateful to Dr. C. Vesco for advise in the preparation and Mrs. F. Sarracino for her excellent assistance.

of the

REFERENCES 1. 2.

Vesco, C. and Penman, S. Terschegget, J. and Borst,.

Biochem. Biophys. Res. Comn., 2, 249 (1969) P. Biochem. Biophys.Acta, 246, 239 (1971)

892

Vol. 60, No. 3,1974

3. 4.

Pica Storrie,

5. 6.

KarlstrlJm, Lark,

C.

7.

Staub, corr.nl. Robbersor

M., Warner, , 46, 1824 D. L.

8.

3810

Mattoccia, B.

BIOCHEMICAL

and

and

L. and Attardi,

H.O., Lark,

Attardi, G.,

Eur. J. K.G.,

J.

AND BIOPHYSICAL

G., Mol.

Biochem., J. Mol.

H. R. and (1972) and Clayton,

J. Mol. Biol. 17, Bier,

Reichard, D.A.

(1972)

893

RESEARCH COAWJNtCATfONS

Biol., ?I, 177 68(1970) 10, 120 F.,

Biocherr,.

Proc.Nat.Acad.Sci.

64, 465 (1972)

(1972)

(1964) Biophys. U.S.A.,

Res. B