On the regulatory mechanism of synthesis of early enzyme induced by bacteriophage T4

BIOCHEMICAL

Vol. 11, No. 3, 1963

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ONTHE REGULATORY MECBAtiISMOF SYi1THESISOF EARLYENZYMEINDUCEDBY BACTERIOPHAGE T4:/ Makiko Kozaka, Kenichi Matsubara and Yasuyuki Takagi Department of Biochemistry, Medical School, Kanazawa University, Kanazawa, Ishikawa (Japan)

Received

April

2, 1963

One of the interesting the regulation

of protein

problems for the biologist

is the mechanismof

synthesis (see review by Jacob and Monod, 1961).

The mode of synthesis of the so called early enzymes induced by T-even phage infection

and participating

in this respect.

in the production of viral

DNAdeserves attention

These enzymes are induced on introducing

some) into the host.

phage DNA (chromo-

Synthesis takes place only during the early stage of

phage growth, coming to halt at about 15 minutes (Flaks s &., Kornberg et al.,

1959).

Presumably there is a *regulatory

1959;

mechanism" which

brings about cessation of enzyme synthesis. The synthesis of enzyme induced by ultraviolet

light

(W) irradiated

phage does not cease at the normal time, but continues so that the final enzyme level

exceeds that produced in the normal phage replication

(Dirksen et al., information

1960; Delihas, 1961).

necessary to initiate

It

appears that the phage carries

susceptible to W-irradiation.

The present work was undertaken to determine if nism" is mediated by cytoplasmic materials.

11

the introduction

the

enzyme synthesis along with something that

regulates enzyme formation and is particularly

involved,

cycle

the "regulatory

mecha-

If cytoplasmic materials are

of both W-irradiated

and non-irradiated

phage

This investigation has been aided by grants from the Rockefeller Foundation and the Jane Coffin Childs Memorial Fund for Medical Research. 244

BIOCHEMICAL

Vol. 11, No. 3, 1963

must result

AND BIOPHYSICAL

in the dominance of the non-irradiated

increase of the enzyme should level

RESEARCH COMMUNICATIONS

chromosome; that is the

off at about 15 minutes after

infection.

The experiments were carried out by studying the formation of deoxycytidine triphosphatase (dCTPase) in Escherichia &

1(12(A).

MATKXIALS AJ!lDFXTHODS The wild type T4r+ phage and two deletion deletion

in A cistron,

LV-irradiation

r196: deletion

type mutants of T4 (h'88:

in I3 cistron;

was carried out by stirring

Benzer, 1955) were used.

the phage suspensions gently at a

distance of 00 cm from a 15 watt Mazda germicidal lamp. produce 5 lethal logarithmic

hits was used.

A dose sufficient

to

KlZ(h) organisms grown to the late

phase with aeration at 37°C in a glucose-salts

synthetic

medium

(Sekiguchi and Takagi, 1960) supplemented with lOOtig/ml of DL-tryptophan were infected

with phage at an input multiplicity

(zero time).

of 10 phage particles

Incubation was continued and aliquots

were taken out at intervals,

chilled

per cell

of the infected

to stop further

synthetic

culture

reactions and

centrifuged. The collected

cells were resuspended in 0.02 M Tris-acetate

buffer,

pH

7.5 and disrupted by sonication at O'C for 2 minutes (20 KC sonic oscillator, #easuring and Scientific

Equipment Ltd.).

12,000 xg for 10 minutes.

Almost all

The sonicate was centrifuged

the cell protein

at

appeared in the

supernatant which was used as the enzyme preparation. Deoqycytidine ically

triphosphate

labeled with P32 in D and y position

was chem-

synthesized according to the procedures described by Smith and Khorana

(1958).

The assay for dCTPaseconsisted of counting the charcoal-nonadsorb-

able P3' released after Kornberg, 1961).

incubation with enzyme for 20 minutes (Zimmermanand

Protein content was determined by a modified Folin reaction

(Lowry -et al. 1951). pyrophosphate liberated

Enzyme activity

was expressed as poles

of inorganic

from dCTP per minute per mg protein. RESULTSANDDISCUSSION

In the present paper %.mited synthesis " will of dCTPase that stops after

15 minutes of infection. 245

refer

to the synthesis

Synthesis of enzyme

BIOCHEMICAL

Vol. 11, No. 3, 1963

continuing

beyond 15 minutes

Mixed infection resulted

.

.02

-

/

:

synthesis"

of dCTPase.

I,/"VI+ , ,' #' :

.03

.02 -

02

30

.Ol

0

SO

30

60

0 L!!c 0

d. -

.03

.03

.02

D2

rl96

t

.Ol

60

30

1

1

UV-HI8

.Ol

r+

0

in Fig.

e.

.03 -

H88

.

tr0

with r+ alone

This is illustrated

C.

,.-+

r’ .Ol

synthesis".

lines.

a. .03

be termed "unlimited

with H88 and r196 as well as infection

in "limited

a and b solid

will

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0

30

60

4- r196

30

I

60

f.

0 L!!I UV-H88

0

+ r+

30

60

Figurel. Kinetics of dC!lPase synthesis in E. & K12(A) infected with bacteriophage T4. The bacteria at 7 tc 8 x 108/ml in synthetic medium were infected with various types of T4 phage as indicated in the figures at multiplicity = 10 of infection was in the case of a, c and d. In b, e and f the multiplicity 5 for each type of phage to make a total of 10. Ordinate: dC'IPase activity in poles PPij2 released / min / mg protein. Absissa: Time in minutes after infection.

"Unlimited

synthesisn

or on mixed infection

was observed on infection

with W-irradiated

with W-irradiated

H88 and W-irradiated

r+

rlg6 simulta-

neously (Fig.

confirm

of Dirksen

1 a and b dotted .lines). These observations et al. (1960) who studied the mode of synthesis

of the same enzyme

in & g&

J3 infected

with T2 phage. 246

the data

BIOCHEMICAL

Vol. 11, No. 3, 1963

The dotted

lines

AND BIOPHYSICAL

of Fig. 1 c and d show that infection

irradiated

H88 or with W-irradiated

Therefore,

there is some difference

formation

in organisms infected

phage mutants

occurred in the mixed infection.

ities

in E. coli

1961) may be responsible

rl96,

after

simultaneous

non-irradiated *Unlimited

complementary synthesisn,

Very different

of bacteria

phages: r',

results

H88,

r+),

were obtained

with one W-irradiated

and another

rI1 mutant (W-H88 + r196, or ~88 + ~496).

when bacteria

were infected

conditions,

only in those complexes mixedly

infected

production

of the

1 e) in such experiments.

that under these experimental

"unlimited

1 f).

with a W-irradiated It

should be noted

synthesis"

takes place

with the two kinds of phages.

phage behaved as if it were "dominant"

ated phage with respect

tc occur

r196 (but not W-irradiated

wild type phage (Fig.

infection

synthesis"

with any one of the following

rI1 mutant and non-irradiated

the W-irradiated

capac-

(Garen, 1961; Nomura,

that is extended and exaggerated

was obtained

of the rI1 func-

loss of synthetic

of QrU.mited

enzyme beyond 15 minutes was observed (Fig. The same result

of the rI1 bacterio-

to restoration

The general

synthesis".

infection

synthesis".

with wild type phage or mixedly

leading

H88 and W-irradiated

always leads tc "limited

W-

rI1 mutants.

of the host cell

W-irradiated

with either

infected

for the failure

with W-irradiated

Infection

in %.mited

caused by the rI1 character

KU(h)

with either

between the mechanism which stops enzyme

Complement&ion,

tion,

on infection

r196 results

separately

and that in organisms

with H88 and rl96.

RESEARCH COMMUNICATIONS

tc the mechanism for control

Thus

over the non-irradi-

of the level

of

induced dCTPase.

Wiberg --et al.

(1962) demonstrated

with the s

the occurrence of phage DNA synthesis the synthesis

of the early enzymes.

is closely It

mutants of T4 phage that related

to the cessation

seems that the accumulation

DNA is not the direct

cause for the cessation

"unlimited

of dCTPase took place in organisms mixedly

synthesis*

with both W-irradiated

and non-irradiated

phage growth ( and therefore

DNA synthesis). 247

of dCTPase synthesis,

of

of phage since infected

phages, and such organisms support Repression

due to the accumula-

BIOCHEMICAL

Vol. 11, No. 3, 1963

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

tion of enzymatic reaction products (feed back inhibition) mulation of a Qytoplasmic

repressor* (Jacob and Monod, 1961) may also be

ruled out by the samedata, since the W-irradiated as if it were dominant over the non-irradiated present together. non-irradiated

If

protein

it

is likely

that the

Therefore someother hypothesis

an operator (Jacob and Monod, 1961) on the phage chromosome

or somefunctional phage growth will

phage chromosomebehaved

chromosomewhen both were

such mechanismswere operating,

chromosomebehaves dominantly.

perhaps involving

or due to the accu-

differentiation

of the phage DNAmolecule in the course of

have to be sought to account for the regulation

synthesis in phage infected

of early

bacteria,

REFERENCES Benzer, S., Proc. Natl. Acad. S&., 4l, 344 (1955). Delihas, N., Virolopv, g, 242 (1961). Dirksen, M. L., tiiberg, J. S., Koerner, J. F., and Buchanan, J. M., proC. Natl. Acad. S&., 46, 1425 (1960). Flaks, J. G., Lichtenstein, J., and Cohen, S. S., J. Biol. Chem., 2& 1507 (1959). Garen, A., Virolorsv,

l4, 151 (1961).

Jacob, F., and Monod, J., 2. -Mol. u., 4, 318 (1961). Kornberg, A., Zimmerman,S. D., Kornberg, S. R., and Josse, J., Proc. Natl. m. a., 2, 772 (1959). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. u. e., m, 265 (1951). Nomura, M., Virology, l.4, 164 (1961). Sekiguchi, M. and Takagi, Y., Biochim, Biouhys. Acta., ,,41 434 (1960). Smith, M., and Khorana, H. G., J. &. w. %., 80, 1141 (1958). Wiberg, J. S., Dirksen, M. L., Epstein, R. H., Luria, S. E., and Buchanan, J. M., Proc. Natl. Acad. S&., g. 293 (1962). Zimmerman,S. B., and Kornberg, A., 2. a, 1480 (1961).

248