Vol.

89,

No.

August

28,

4, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1979

Pages

CIRCULAR

DICHROISM

OF

Gokul University Biology

C.

of Tennessee-Oak Division, Oak Ridge

Received

July

NATIVE

Dost

WHOLE

and

Salil

SV40

K.

1267-l

272

CHROMATIN*

Niyogi

Ridge Graduate School National Laboratory,

of Biomedical Sciences, Oak Ridge, Tennessee

and 37830

16,1979

Summary - Circular dichroism properties of SV40 virions, isolated minichromosomes from virions, and SV40 Form I (supercoiled) DNA were studied in a buffer of low ionic strength. The isolated minichromosomes are compact as judged by sedimentation and electron microscopy. The molar ellipticity at 284 nm of the virion, which may be regarded as a is about 1500 deg crr?/dmol phosphate; this value is in minichromosome in its native state, the same range as that reported for core particles (1300-2000) isolated from different there is a small increase in the molar ellipticity sources. When the viral capsid is removed, to about 2000. However, both of these values are much lower than that found for SV40 The results strongly suggest that the linker DNA of the supercoiled DNA (about 8200). native wholse chromatin contributes in a similar fashion to the circular dichroic ellipticity as the core DNA.

units,

DNA

in eukaryotic

each

consisting

of DNA years

and

1 and

2).

conformation

of DNA

inside

the

of biophysical

techniques,

established 8280

results

that

(59),

varying of

is controvelrsy chromatin

when

is reduced.

chromatin

containing

see

measurements over preparations.

interact

Measurements

have

length

(1 l),

molar The

with

ellipticity

their

or core value

eukaryotic

t

Postdoctoral Oak Ridge

sponsored by the Office of under contract W-748%eng-26

spectrum

carried

may its

While

reflect size,

ellipticity at 284 phenylmethylsulfonyl

No. 3322 of Tennessee.

a nonexclusive for governmental

from

the

0006-291

1267

in be

SV, simian fluoride; EDTA,

S.

Biology

X/79/1

there

must

nm;

royalty-free purposes,

Copyright All rights

the

variations

and Environmental Research, U. with the Union Carbide Corporation.

Investigator supported by subcontract National Loboratory to the University

meto-

agreement,

large

is

is modified

(11-16).

which

It

4).

mononucleosomes

in reasonable

of

by a

3 and

lo),

pairs few

on eukaryotic

9,

particles

because

past

of DNA

out (6,

of chromatin

molar PMSF,

Refs.

sub-

base

extensively

see

CD

are

in the

studied

multimers

particles

8284,

The U.S. Government's right to retain to the co,pyright covering this paper,

the

core

chromatin,

Health

been

been

higher

200

methods,

reviews,

DNA

nucleosome

Abbreviations: CD, circular dichroism; virus; Tris,. tris(hydroxymethyl)aminomethone; ethylene diiaminetetroacetic acid. *Research of Energy,

(for

generally

nucleosome

to approximately

has

CD

and

of repeating

biochemical

nucleosome

and

on nucleosomes the

Besides

histones

a chain

complexed

particularly

nucleosomes

DNA

into

of histones

Refs.

the

phase

is pocked

of an octamer

(folp reviews,

number well

chromosomes

Department

Division

is

of

license in and acknowledged.

61267-06$01

.00/O

@ I979 by Academic Press, Inc. of reproduction in any form reserved.

Vol.

89,

No.

BIOCHEMICAL

4, 1979

fragmented

before

generally

it can

adopted

for

Nuclease-digested

this

chromatin

since

hand,

“whole”

chromatin

is often

regions

(7) with With

the

SV40

chromatin,

some

biochemical

isolated

perspective, is identical

criteria

(19,

its superstructure

of as a minichromosome proteins at

the

do not positive

of a model

native

The

results

to that SV40

of core

in its

have

any

band

(presented

native

signal

at

CD

the

also

for

for

cellular

solubilized and

the

first

time

chromatin

termed

the

other

mechanical of free

chromatin

preserved

(21,

22).

The

encapsulated the

time

CD

DNA-like

properties

SV40

CD

in this

paper)

virion

can

to be

be thought

Since

of the can

and

so far

proteins.

spectrum

of

structure

reported

by capsid

band,

the

in its subunit

only

first

be On

by

appearance

is the

the

cannot

be attained.

which

positive

shearing.

signal.

here

state,

18),

are

the

virion

especially

be regarded

as that

chromatin. presented

here

particles

chromatin

MATERIALS

and

and

methods

mechanical 17,

cannot

of nuclei

report

to the 20),

(7,

of superstructure

in the we

nuclei

Two

and

“native”

the

lysis

loss

increase

digestion

called

from

by the by the

concomitant

with

often

RESEARCH COMMUNICATIONS

measurements.

nuclease

yield

isolated

which

intact,

though

accompanied

biophyrical

partiol

a complete

above

for

purpose:

chromatin,

whole

shearing

be solubilized

AND BIOPHYSICAL

does AND

isolated not

show

that

from

various

contribute

to the

8284

of

native

sources molar

whole

(1 l-16)

SV40 and

that

chromatin the

is comparable

superstructure

of

ellipticity.

METHODS

SV40 was grown as described previously (22). Isolation of SV40 minichromosome. Before use, the virus preparations were dialyzed extensively against 10 mM Tris-HCI (pH Minichromosomes were isolated from mature virions by our earlier method (22), with 7.4) . In the present studies the final composition of the disruption buffer a minor modification. was: 0. 15 M Tris-HCI (pH 9.3) containing 20 mM dithiothreitol, 10 mM EDTA, 1 mM PMSF, The virions were kept in this medium for 3 hr at 4OC followed by 2 min and 0.10 M NaCI. The crude chromatin thus obtained was purified by sedimentation in IO-30% at 37°C. sucrose gradients containing 10 mM Tris-HCI (pH 7.4), 1 mM EDTA, 0. 1 mM PMSF, and The appropriate 0. 15 M NaCl in a Beckman SW41 rotor for 1 hr at 39,000 rpm and 4°C. fractions were pooled and diluted to 10% sucrose with the same buffer as used in the gradient and concentrated by sedimentation over a 0.5-ml cushion of 60% sucrose solution in 10 mM Tris-HCI, (pH 7.4) in an SW41 rotor for 12 hr at 39,000 rpm and 4°C. The concentrated minichromosomes were dialyzed extensively against 10 mM Tris-HCI (pH 7.4) before CD measurements. CD measurement. Measurements were performed assisted Jasco Circular Dichrograph SS-10, calibrated ( nucleotides) extinction coefficient at 260 nm of DNA of 8284 after correction of the absorbance for scattering. a precision of k.200 deg cm2/dmol phosphate.

at room temperature in a computeras described (10). The molar was taken as 6500 in the calculations 8284 values were measured with

RESULTS

of the

Characterization

of SV4O

minichromosomes.

minichromosomes

isolated

from

virions

Figure in buffer

I268

1 shows

containing

the

sedimentation

0. 15 M NaCI.

profile The

Vol.

89,

No.

BIOCHEMICAL

4, 1979

AND

0

BIOPHYSICAL

10

RESEARCH

20

FRACTION

COMMUNICATIONS

30

NO.

Fig. 1. Sedimentation profile of 3H-labeled SV40 minichromosome gradient containing 0. 15 M NaCI. The markers KRV virion (S = 31.5) are indicated by single (t) and double arrows (f), respectively.

110)

positions

T7 phage

(24)I

of two run

reference

iln parallel

of the

minichromosome

report

(22) .

checked linker

DNA

spectra.

native

state),

10 mM

Tris-HCI

Figure

3.

similar

to that

about isolated about

that

cm2/dmol

minichromosome 2000.

the

in the

qualitative

present

in the

spectra

of both

spectrum

of cellular

lower-wavelength negative

for

region the

virus

same

peak medium

value

native

compared

and (6, with because

7).

shown

of the

of the

1269

a negative

virus,

associated

no

sulfate-gel

in

DNA

in

scale

in

feature

molar

ellipticity

at 274

in 0284

from

of

nm. 1500

The to

is approximately peak

similar

point

for

chrorratin

and

the

signal

capsid

and

on an enlarged

DNA

minichromosomes crossover

When

elsewhere).

a shoulder

increase

earlier

(minichromosome

a maximum

I (supercoiled) that

our

has a characteristic

Form

The that

I (supercoiledj

S

coefficient

dodecyi

Form

a small

isolated

Sodium

SV40 are

31.5

compact

virus

nm with

it appears

very

SV40

shows for

(25-27).

of the

virion

at 284

by others

published

It reaches

7).

features,

chromatin

presumably

(6,

with

to be

spectra

SV40

agreement

(22).

and of the

DNA,

sedimentation

appear

(details

spectra

of the

same

the

other

CD

positive

estimated

is observed

peaks

spectrum

The

obtained

histones

the

S (23);

minichromosomes

five

chromatin

Among

those

minichromosome,

in the

In contrast,

the

Positive

8200.

the

all

SV40

of eukaryotic

deg

than the

2 presents

7.4).

It appears

1500

higher

110

is in reasonable

nucleosomes for

Figure

(pH

which

microscope,

bands

isolated

by arrows.

110 S,

consecutive

shows

Rat Virus,

shown

it is much

electron

between

[Kilham

are

is about

the

electrophoresis

the

gradients

However,

under

CD

markers

in a lo-30% sucrose and T7 DNA (S =

proteins.

around

300

to that

found

is shifted at

222

nm is in to the

nm is more

Vol.

89,

No.

4,

1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

4 2

.._._. ;,r_-. _-_ ,’

0

10 i

-2

a-

-dP

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