Origin of DNA and protein in λ DNA infected disrupted cell preparations

Vol.

42,

No.

BIOCHEMICAL

1, 1971

ORIGIN

OF

DNA

AND

AND

PROTEIN

DISRUPTED Roy P. Mackal,

CELL

Betty

Received

November

cells

which

was

DNA

were 15

labels synthesis

described

that

DNA

simply NH4CI

E. A.

Evans,

Illinois

15

was

not

and

D20

DNA

Jr.

68637

DNA

being

protein

.

the

However, Consequently

but

synthesized

bacteria in the

to prepare bacteria and DNA and protein in frozen-

from

synthesis.

synthesized,

to prepare

and D20 of viral

preparations l-3

X DNA.

in newly

and

NH4Cl synthesis

frozen-thawed

net

encapsulated

of viral

and

Chicago,

by isolated

to demonstrate

be excluded

INFECTED

23, 1970

be infected

insufficient

X DNA

COMMUNICATIONS

of Biochemistry

of Chicago,

previously

can

RESEARCH

PREPARATIONS

SUMMARY: By employing the density labels D20 to prepare X bacteriophage, the --de novo thawed cell preparations has been demonstrated. We have

IN

Werninghaus,

Department University

BIOPHYSICAL

and

D20

amount

the

rather virus

virus-resistant

that

of virus

possibility

portions

of the

By employing

to prepare

X bacteriophage,

bacterial

production

could

protein.

frozen-thawed

--E. col i

not

infecting density --de novo

preparations

has been

demonstrated. MATERIALS

AND

METHODS

Preparation X temperate

of density

and

X virulent

labeled

phage

were

prepared

by

substituting

and

bacterial

and

cells-Unlabeled

purified

stocks

as previously

of phage

described.4

Density *

labeled 1900

phage

was

ml batches

of -E. --coli ation

of medium

W-1485

at 370

was

first

and

excessive

prepared

gown

to give

passed

through

a minimum

of 3 x lOlo

*

same the

15

large

CsCl

NH4CI amounts

way

until

filter

plaque

as unlabeled gradients.

lysis

flasks The

for were

2 liter

H20

and

a trap

X virulent

cultures

was

farming lysates,

The

4 except

that

of light

crude

only

unlabeled

medium for labeling compounds.

89

the

The

labeled

phage

air

ml cultures with

used

aer-

for aeration contamination

of 2) were

added

crude

phage appearing

purposes

100

to prevent

lysates

heavy

medium.

incubated dry

D 0 2

(4 to 6 hrs).

units/ml.

Traces

cannot be used with Fraser of other nitrogen-containing

complete

were

(multiplicity

Fraser

with

The

containing

phages

in the

inoculated

of 1 x 1 O9 cells/ml.

4 x 1012

continued

density

at 37O.

a bacterial

(99.86%)

Erlenmeyer

concentration

evaporation. was

fram

wernight

a final

incubation

in the

in 4 liter

D20

due

lysates

contained

were band

purified was

abwe

to the

and

removed the

presence

of

Vol.

42,

major

No.

1, 1971

phage

band

fraction

BIOCHEMICAL

were

occasionally

of unadsorbed Frozen-thawed

fication

of the

method

Feiss,’

prepared

with

and

Nuclear

overnight

with

fuged

and

cells

were

identically

10 min

in a Sorvall

added

ice.

and

and

fuged

5.3

passed

g CsCl

comparison

containing

Tris

position

the

1.4,

1.5

tube

buffer

phage

and

was 5.5

1.6

fractions

The

of either

plating

Gierer

taining

and

fractionator

suitable

-40’

a

frozen

with

were

non-isotopic

prepared

components. preparations

NaCl

and

0.025%

ml.

The

tubes

The

contents

180 and

density,

aliquots

mg CsCl/ml

and

the

density included

then

centrifuged

of the

centrifuged

Each

were

MgS04 were

at

centri-

of each

the

tube

absarbancy

added

were

profile

on the

to centrifuge densities

beads.

Beckman-Spinco

in each

tube.

for tubes

fraction

were

depending

were

were

of infectious

and 2 x 1013

preparation

centrifugation layer

to 680

2 or 5 drops.

was

was placed

DNA-Infectious

Schramm

method.6

0.5

plaque

forming/ml

were

of water-saturated The

and

contents

rpm.

with The

cells

was

recorded. densities,

samples

below

centri-

solution

to -35O.

pg) of X DNA

of 5.5

were

applied

Unlabeled

0.5%

volume

gradient

of phage 620

M Tris,

incubated

supernatant

was then

from

were

then

The

final

to beads,

volume rpm

fractionated

assayed

required density

20 hrs at 39,000

were

tubes

in each

in the

Spinco

by collecting

for

phage

by standard

agar

techniques. Preparation

the

All

at -80’.

Chemical

cultures

at a temperature

24 hrs at 44,000

density

cell

modi-

and

International

of -40’

(15

a final

for

paste

Fangrnan

medium

The

small

by a slight

by

15N,

The

cell

of the

prepared

of this

was prepared

(0.067

to give

nm was

nominal

kept

stored

medium

concentrations

ml.

SW 50 L rotor.

tube

an ISCO of 254

were

buffer

SW 50 L rotor

through

at a temperature and

were

centrifuge.

comparison-Aliquots

Tris

per

flasks.

kept

mortar

growth

Fernbach

The

cells

density

containing

at a wavelength For

the

batches

angle

reisolation

99%

1 liter

COMMUNICATIONS

described

D and

inversion.

blocks

ground

that

in a Spinco

then

steel

The

liter

by

in a small

phage

to tubes

pH7)

drained

ground

except DNA

Inoculated

x 6 for

to stainless

dry

used.

1,7

medium

(99.86%

in 2.5

tube

The

15NH4CI

of the

3350/X,

described.’

and

RESEARCH

as a result

of --E. coli

at 37O

then

powdered

cells

was

each

smal I spatula

bacterial

shaking

at 6,000

discarded

inoculum.

previously

Corp.),

BIOPHYSICAL

observed

phage

D20

AND

phenol,

pH

was then

gently

of the removed in dialysis

mixture

by means bags.

7.

at

All

ml of stock added

were

for

12,000

x g for mouth

preparations

5 min

was

buffer

were

dialyzed

and

(mouth four

5.0

times

con-

ml procedure.

After

centrifuge, orifice

of

buffer

the

movement.

in a Sorvall pipet

in Tris

at 4O throughout

by a rolling

Pasteur

by a modification

of phage

ml of Tris kept

TO min

90

prepared

solutions

to 2.5

solutions

agitated

of a wide The

DNA

the

DNA

2 mm in diameter) in 1 liter

volumes

Vol.

42,

No.

of Tris

1, 1971

buffer

BIOCHEMICAL

at 4’.

Absorbancy

quantity

of DNA

in each

indicator

bacteria

prior

that

Infection added

cell a 370

water

amino

45 set

at 20’

Model

K 500-4). for

10 min

were

As controls, intact

In every experiment training

case

absence

The

by extraneous

for

indicating

that AND

As can incubation

gradients

absence

materials

phage

used

per

The

completely

DNA,

frozen-thawed

cell these

The

pastes, conditions

and were

approconducted

of non-isotopic were

of plaque

to the

mixed

formation

virus.

In each a tube

con-

operations

subsequent

again

components.

cells

experimental

with

subjected

supernatant

by subjecting

the

and

x g for was

experiments

established

to all

Inc.,

techniques

resistant

was

Industries,

removal,

absence

in for

medium

frozen-thawed

plate).

shaking

at 6,000

consisted

of resistant

contamination

under

When

containing

was shaken

labeled

plating

of appro-

gentle

tube

50 rotor.

by standard

were

100 mg of

(vial

(Scientific

after

No.

as required.

were

each

centrifuged

solution,

units

without

and

acids

pg DNA)

with

70 min

speed

in a Spinco

all

cells

but

particles

plating

indicated

that

employed on indicator

contamination

was absent. of phage

3 hrs were

also

made.

density

added Such

was

not

1 A,

when

to the phage

increased

density was

labeled

incubation

indistinguishable

by exchange

from

of D20

medium unlabeled

and phage

or 15NH4Cl.

DISCUSSION be seen

medium

in

are

Fig. density

the

incubation

labeled

(Fig.

1 B).

As shown

80%

or more

that

the

of plaques

initially

indicates

DNA,

of viral

measurements

incubated

RESULTS

to CsCl

infected

virus

Density

rpm

(25

for

was

supernatant

forming

cells,

of DNA

bacteria.

the

ml

to 39 ml of density

material

plaque

that

frozen-thawed

in the

and

infectious

indicated

the

incubated

.05

was added

added

indicated

containing

control

at maximum

aliquots of all preparations 12 (10 plaque forming units

virus case

mixer

on

case

casamino

appropriate

incubated

buffer

was then

The

for

labeled

tube

at 39,000

applied

density

of Tris

mixture

centrifuge

assayed

quantities

with

were

of centrifugation

solutions priate

no DNA)

the

preparations.

plus the

to estimate

directly

in every

5 cm height,

as required,

used

plated

mg of Difco

2 cm diameter,

but

3 hrs at 37O.

in an angle

to 30 min

with

Each

Fifteen

including

were

DNA

COMMUNICATIONS

nm was

formation

in the

samples,

test

preparations

present

The

a Vortex

at 260

of plaque

pastes-

vials,

A 1 ml aliquot

with

DNA

or unlabeled

acids

bath.

wa:

RESEARCH

pg of DNA

absence

cell

labeled

preparations. and

incubated

phage

BIOPHYSICAL

per All

The

bottomed

cells,

DNA paste

viable

flat

frozen

priate

preparation.

of frozen-thawed

to round,

ground

of 0.02

to use.

no contaminating

AND

medium

X temperate

labeled,

the

only

is labeled,

in Table of the

label

I, the

DNA

newly

formed the

81%).

91

phage

synthesized

relative

is derived

infected

density from

the

frozen-thawed is also

heavily

phage

is still

difference incubation

in these medium

cells labeled.

and If

heavily two cases .0188 (mx 100 =

Vol.

42,

No.

BIOCHEMICAL

1, 1971

AND

BIOPHYSICAL

TABLE Density

Difference

Labeled plus

COMMUNICATIONS

I

of X Phage

Unlabeled

RESEARCH

Light

Marker

frozen-thawed bacterial cells labeled incubation medium

Compared

to

Phage

Unlabeled cells plus

frozen-thawed labeled incubation

bacterial medium

X Temperate

Experiment

number

g/cc3

AP

I

Experiment

number

AP

g/cc3

.018

V

.018

II

.026

VI

.025

III

.023

VII

.016

.026

VIII

.016

IV

Average

.0232

+ ,003

Average

.0188

-+ 404

X Virulent

Experiment

number

Experiment

when

g/cc3

.017

XIV

.014

X

.027

xv

.027

.022

Differences in buoyant bead positions versus

A similar contains

AP

IX

Average

density

number

result

no isotope, mixed

with

is found a relatively

light

marker

-+ .005

densities position

in the light phage

Average

.0205

were estimated by plotting density of maximum phage concentrations.

as determined

case

incubation

of X virulent

phage (Fig.

progeny 1C).

92

(Table results

I).

If the

as indicated

by

the

+- .007

by

medium single

peak

,

BIOCHEMICAL

Vol. 42, No. 1, 1971

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

r

+Ol X SllNll

ONlWMOj

3nOV-ld *Ol X SllNtl

-/

, ?

/ 2 .01 X SllNfl

, s SNIWMOJ

, 2

1%

3nOt’ld

93

BNlWkOj

3nOVld

Vol.

42,

Fig.

1

No.

1, 1971

Relative

Phage produced by infection of labeled frozen-thawed cells and unlabeled incubation medium with unlabeled DNA. Light marker phage in approximately equal amount was also added; 5 drop fractions collected.

D.

Comparison lected; ----,

E.

Phage produced by infection of unlabeled frozen-thawed cells and unlabeled incubation medium with labeled DNA; 2 drop fractions collected. Arrow No. 1 indicates the approximate position where phage synthesized exclusively from density labeled infectious DNA would appear. Arrow No. 2 indicates the approximate position where phage containing 50% density labeled infectious DNA would appear.

F.

Phage produced by infection of unlabeled frozen-thawed cells and unlabeled incubation medium with labeled DNA. Light marker phage in approximately equal amount was also added; 2 drop fractions collected.

results

.0135

to

indicate

.015

a large since

that

fraction the

heavy

are

confirmed marker

in medium

containing virus

fractions

for

detecting

all

of its

phage DNA

approximately DNA

from at the

would

have

phages)

were

heavy

point placed

be newly

synthesized.

difference

between

by arrow progeny

original

No. phage

94

that

and

ranging to phage

This

comparison

cells

and

resulting 1 in Fig.

approximately

labeled

under

can

incubation

marker

phage

phage 1E.

(isolated cells

the

phage

the

density (Fig.

1 D).

of the method

progeny peak

had

would

Conservation at the

and

conditions

and

sensitivity

If the

DNA

these

phage

light

experiments,

the

( p

frozen-thawed

heavy

heavy

was high.

DNA,

density

in addition

density

unlabeled

1E shows

in these

be synthe-

density. in which

the

medium that

col-

must to the

frozen-thawed

to infect

than

infecting

the

therefore

Fig.

densities

marked

conclude

less dense

collected

components

in D20

labeled

fractions

is comparable

increased

used

ccl Is and collected.

2 drop

virus

by growth

experiments

was

of intermediate the

of the

labeled

no isotope.

to the

of 2 drops

total

by the

is much

is comparable

We can

to the

-,

experiments

virus DNA must 15 N label in the

slightly

D-labeled

synthesized

difference

of the

proportion

is prepared

1 D).

frozen-thawed 5 drop fractions

marker phage. collected.

in these

X virulent

additional

results

a large

(Fig.

only

and light fractions

increase

g/cc3)

contributes

incubated

the

of heavy 5 drop

marker

These

Since

gradients.

C.

heavy

newly

COMMUNICATIONS

Phage produced by infection of unlabeled incubation medium with unlabeled DNA;

when

medium

in CsCl

RESEARCH

8.

density

be made

of phage

BIOPHYSICAL

Phage produced by infection of labeled frozen-thawed cells and labeled incubation medium with unlabeled DNA; 5 drop fractions collected.

The

protein

from

densities

--de novo.

increase from

buoyant

AND

A.

These sized

BIOCHEMICAL

point

derived

have of 50%

indicated

appeared of by

Vol.

42,

arrow

No.

No.

results

(Fig.

experiments DNA

are

adequately

1, 1971

2.

BIOCHEMICAL

Again, 1F).

The

is of the shown

lower order

in Fig.

labeled.

frozen-thawed

if progeny

cell

limit

is mixed

The

with

light

marker

that

peak

in these

density

DNA in the

particles

a single

transfer

isolated

infectious

demonstrate virus

phage,

of the

labeled

new

COMMUNlCATlONS

to progeny

densities

heavy

again

completely

RESEARCH

of parental

relative

that

experiments system

BIOPHYSICAL

of detection

indicating

These assay

phage

of 15%.

2,

AND

was indeed

infectious

including

labeled

DNA

DNA are

synthesized. I

/

L Jc +

Fig.

2

Relative I, unlabeled unlabeled

buoyant DNA; DNA.

densities II,

DENSITY

of infectious D-labeled

DNA;

DNA. III,

equal

mixture

of D-labeled

and

ACKNOWLEDGMENTS This work was supported by a research grant from the Inc., and the Dr. Wallace C. and Clara A. Abbott Memorial Chicago.

John

A. Hartford Foundation, Fund of the University of

REFERENCES 1.

Mackal, U.S.,

R.P ., Werninghaus, 51, 1172 (1964). -

B.,

and

Evans,

95

E.A.,

Jr.

Proc.

Natl.

Acad.

Sci .,

Vol.

42,

No.

1, 1971

2.

Weinstein, Acad.

3.

Weinstein, in press.

4.

Villarejo,

M.,

5.

Fangnan,

W.

6.

Gierer,

BIOCHEMICAL

B.I., Mackal, Sci., U.S., -62,

A.,

B.I.,

L., and

and

R.P.,

S.,

and

Feiss,

Schramm,

BIOPHYSICAL

R.P., Werninghaus, 420(1969).

Mackal,

Hua,

AND

Werninghaus,

Evans, M.

G.

J. Z.

E.A., Mol.

Jr.

96

COMMUNICATIONS

B.,

and

Evans,

E .A.,

Jr.

Proc.

B.,

and

Evans,

E.A.,

Jr.

Virology,

J.

Virol

Biol . , 4&

Naturforsch.,

RESEARCH

5,

.,

1,

928

103 (1969). 138

(1956).

(1967).

Natl.