Vol. 91, No. 4, 1979 December

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

28, 1979

Pages 1352-1357

DNA SYNTHESIS IN A SUB-NUCLEAR PREPARATION ISOLATED FROM PHYSARUM POLYCEPRALUM Eugene Division

Received

N. Brewer

and Peggy A. Busacca

of Radiation Biology, Department Case Western Reserve University Cleveland, Ohio, USA 44106

November

of Radiology

5,1979

SUMMARY

A sub-nuclear preparation capable of substantial levels of DNA synthesis has been obtained from isolated S-phase nuclei of.Physarum &--in vitro Nuclei were disrupted by gentle resuspension in a dextran-free cephalum. medium followed by immediate addition of dextran to stabilize the liberated replication complex. Synthesis continues for at least 120 min, and appears to occur by a semi-discontinuous mechanism. Little DNA synthesis occurs in preparations obtained from G2-phase nuclei. INTRODUCTION DNA synthesis tively

similar

disruption

to that

(l-8).

identification

of other

constituents

which

by the are

mammalian gentle retains

of DNA in

means,

isolation,

of a sub-nuclear

physiological

control

to be qualita-

at the

time

to be quite

(9-18).

However,

DNA replication intact

and Fry

communication fraction

we report

of Physarum

of the DNA replication

which

would

be

of sub-nuclear Toward

this

have demonstrated obtained

the

the characterization

--in vitro.

preparations

in

complex

complex

nuclei,

(20)

of cellular

useful

of the DNA replication

in DNA replication

"chromatin"

appears

cell

proven

from

and Kaftory

In the present

cells.

the intact

eukaryotic

active

(19)

in many cases

have

membrane

of the

and Weissbach synthesis

in

constituents

the nuclear

facilitated

preparations

the

occurring

of certain through

greatly

nuclei

Such preparations

can pass

Knopf

in isolated

recently

from nuclei the preparation,

polycephalum process

end,

of by

which

--in vitro.

METHODS AND MATERIALS Macroplasmodia described previously (third synchronous indicated otherwise. experiments.

of Physarum polycephalum, Cultures were (18,21). metaphase following fusion The interdivision time

0006-291X/79/241352-06$01.00/0 Copyright Ail rights

@ 1979 by Academic Press, Inc. of reproduction in anyform reserved.

1352

strain M3C, were prepared as harvested at 60 min after MI11 of microplasmodia) unless was approximately 8.5 h in these

BIOCHEMICAL

Vol. 91, No. 4, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

For preparation of nuclei, individual plasmodia were homogenated gently by hand in 3 ml of a medium containing 0.028 M magnesium acetate, 0.06 M ethyleneglycol-bis-(S-aminoethyl ether)N,N'-tetraacetic acid, pH 7.8, and 12.5% (w/v) dextran. Nuclear pellets, obtained by centrifugation of homogenates at 4300 x g for 10 min, were resuspended in the same volume of homogenizing medium but without dextran, immediately mixed (gently stirring) with solid dextran to a final concentration of 12.5%, and centrifuged again at 4300 x g. The viscous supernate was removed carefully with a Pasteur pipette. Aliquots (0.5 ml) of the supernate were combined with 0.05 ml of a solution containing 0.22 M ATP, 1.7 mM each of dGTP, dCTP, and dTTP, and 0.23 mM dATP (including 8.3 UM r3H]dATP), pH 7.3, and incubated for 2 h at 35Oc. Incorporation of [3H]dATP into DNA was determined as described previously (5). The alkaline sucrose density gradient profile of the DNA synthesized in such sub-nuclear preparations following a 60 min incubation period was determined by addition of 1.5 vols of Mohberg medium (22) followed by centrifugation at 10,000 x g. The pellet was gently resuspended in the same volume of the latter medium, an 0.7 ml aliquot centrifuged at 10,000 x g, and the pellet resuspended in 0.2 ml of 0.15 M NaCli0.015 M sodium citrate containing 0.005 M EDTA and 2% sarkosyl (pH 7.5), and layered over a 5-20% alkaline sucrose density gradient. After centrifugation in the SW 39 rotor for 90 min at 35,000 rpm, ll-dp fractions were collected, and the DNA acid-washed and counted in a liquid scintillation spectrometer (5,23). Dextran (mol wt 70,000,radioimmunoassay grade), 13H]dATP ATP (ultrapure) and unlabeled doxyribonucleoside triphosphates from Schwarz/Mann, Orangeburg, New York. Sarkosyl was a gift Geigy Corp.

(lo C~/IUUIO~), were purchased from the

RESULTS Centrifugation homogenates

at 4300 x g sediments

of Physarum

fraction

exhibits

nuclear

preparations

to allow

their

or its ability

which

constituents

followed

replication

for

containing

prelabeled

in nuclear

preparations present

is

released

in the original

is

of

however,

or leaching

a concomitant

appears of this

decrease

added

back

in to such

at 4300 x g to remove unbroken supernatant (initial

fraction

rate

approx.

120 min at 35°C (Fig.

DNA revealed

in

supernatant

medium

When dextran

the residual

at least

present

Resuspension

complex,

with

DNA (2,5).

of DNA synthesis

(5).

homogenizing

nuclei,

nuclei

The post-nuclear

by re-centrifugation debris,

levels

continues

of the nuclei

from

all

activity

in the dextran-free of the nuclear

and nuclear

substantial

dextran.

DNA-synthesizing

to synthesize

preparations nuclei

little

disruption

complex

containing

virtually

that

1).

nuclei

nuclear

1353

of rate),

of nuclei

40% of the DNA present

by this pellet

capable

3% of --in vivo Disruption

approximately

from

is

are

treatment. found

Few (