The base specificity of mutation induced by nitrous acid in phage T2

Vol. 2, No. 5

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 1960

THE BASE SPECIFICITY OF EUTATION INDUCEDBY NITROUSACID IN PHAGET2 V. Vielmetter Max-Planck-Institut

Received April

Nitrous

and 5. Schuster

f-lir Virusforschung,

14, 1960

acid has been shown to be an effective

mutagenic agent for

and Wieder,l%g),

fysed,

T4 (Freese,1959),

for

and mutation follow

changes are caused by single

U, respectively ster,

cases ana-

order kinetics lethal

or mutagenic

in the nucleic

acid.

is known to be the deaminconverted

into X, RX or

(Zamenhof, 1953; Schuster and Schramm, 1958; Schu-

for

deamination rate

different

ratios,

pH values (Schuster,

rA/sG and aC/sG, 1960). Therefore,

in

paper the deamination rates of G, A and C in phage T2 treated

with HN02 at different

pH values are compared with the corresponding

of mutation and. inactivation.

deaminations can lead to either

1

Therefore

all

1960).

are different

rates

first

0, 1 A or C, which is thereby

In DNA, the relative

this

Iin

chemical alterations

The chemical nature of these alterations of either

and

the phages T2 (Vielmetter

D&A (Litman and Ephrussi-Taylor,l959).

both inactivation

(Schuster

and

$ x 174 (Tessman,l959) and for

with respect to the time of treatment.

ation

inactivating

the RRA of tobacco mosaic virus

Schramm,l358; kiundry and Gierer,1958),

transforming

TGbingen,Germany

This reveals mutagenic or lethal

which types of base changes.

G = guanine, A = adenine, C = cytosine, or in phage T2 hydroxymethyl-cytosine (l&C), X = xanthine, HX = hypoxanthine, U = uracil, or in phage T2 hydroxy-methyl-uracil (RI&J). 324

Vol. 2, No. 5

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 1960

EXPERIL%bTAL T2 wild -2

mg/ml

phage,

experiment for

type

dialysis and Cohen graphed

each

the

increase

the

inactivation

rate

and Wieder,

chosen

so that

ly

mutants picked

at was

r-

least

plaques

the

number

The

50 r-

plaques

by assaying

on strain

E. coli

of

the

deamin-

same condi-

of r-

mutation

previously

for

each

observed.

the

chromato-

in G, A and E!kC and

of induction

were

of C'yatt

v!ere

extent

Under

plates

and after

methods

as described

of

at pti 5.00

bases

decrease

rate

measured

The

determined

the

One

g &Cl.

withdrawn

the

T to be constant.

were

1959).

using

as the

containing

and 6.00

(Schuster,l960).

pH values,

a mixture

were

hydrolysis

was measured

at various

metter

rII-

described

in

and another

samples

acid

in HW * 9 assuming

but

60 hrs

and purified

formic

sample

20°C buffer

for

intervals

DNA was extracted After

at

g acetate

pH 4.20

time

as previously for

at

regular

(1953).

ation

incubated C.25

pit NaN02,

1

At

the

tions,

were

was performed

550 hrs.

and

phage

(Viel-

sample

was

The fraction

purified

progeny

K (Benzer,

of

of random-

1957).

RESULTS AND COMCLUSIONS In vation the

the

Fig.1

rate

are

mutation

rate

was used

4.20

and pH 5.00. of

has been

the

it

the

three

The about

rate,

90 from

pR 4.20

by a factor

the

order the

kinetics

same applies and

the

inactilog

pH. Linear

with

the

at

pH

deamination

rate

of each

for

a long

period

also

for

inactivation

of regres-

constants

the have

A and C decreases

to pE 5.C0, only

the

base of

time.

beginning been

of

neasured.

conclusions.

of both

of

and that

rate

deamination

following

a,

found

increasing for

I).The

mutation

the

is

mutation

can be compared

first

to

with values

(Table

time

of r-

PH. It

linearly

that

which

deamination

G decreases

bases

lead

the

values

likely

reaction,at

induction

accurate

to follow

The results (1)

decreases

These

is

the

against

to obtain

found

Therefore,

of

plotted

sion

rates

rate

whereas 35.

325

The

the

by a factor

deamination

corresponding

of

rate factor

of for

Vol. 2, No. 5

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 1960

Figure

1.

Dependence

of

the

rate

r-

mutation

of

and

the

inactivation rate

on the

pH for

phage

treated

with

1 &I NaW2

at

20°C

PH-

Table

I

a (Deaminations/min Percent of Total DNA

pH 4.20

116

A

17 33

G

17

297

C

18.5

Average deamination per Total DNA j4 r-mutants

phage

lethal

76.3

survivors min hits/Din

326

x 106)

1.3

89

0.2

90

8.55

35

1.74

44

2.9

3.3x10-2

88

9

0.3

30

T2

Vol. 2, No. 5 the

BIOCHEMICAL

decrease

Therefore

(2)

but

This

result

#atson

the

is

concluded

not

of is

is

from

with

primarily

not

affect

like

G and T,

base

pairs

from

deamination

respectively.

(so

called

derived

have

found

tants

is

genone

that

proportion

the

65 f!, lead

of the

total

zer

(1957)

The

inactivation

tor

of only

base for

30.

This

deaminations

rise

region, related

1959))

should

CC (type

from that

of result

mutants more

mutation.

Since

induced

of about

376 base

roughly

l/2

as estimated

from

data

r-

If

in T2 the

rate

of

the

unspecific 327

we mu-

pairs to

I/:,

of Een-

to pH 5.00

contrast

to the

be caused

by the

remains also

by a facmutagenic

deamination

tentative,

contribute

beto

the

inactivation. (5)

as

T4.

pH 4.20

might

resul-

frequent

is

in

2)

of any one of about

This

however,

protein

that

mutants/total

phage

can also

the

of conversions

to a r

mutation.

conclusion in

pair

2.

of any one

indicates

This

(HX and U) which types

conversion

of rI1

decreases

inactivation I).

the

rI1

closely

rate

in

be j-times

type

gives

pairs/r11

the

should of

conversion

to a visible

inducibility, of G (Table

the

therefore However,

1) and AT ->

1

pH 5.CC

Z',

specificity.

Freese,

conversions or

specifici-

in G and T and the

can be calculated,

pairs/phage

should

cause

it

by

replication:

of type

from

pH 4.20

570 base

(4)

pH 4.2

by

specifically

in G is

two

C or

proposed

G. The pairing

to analowes

TA (type at

A,

of mutations.

scheme

keto-group

Therefore

to be 88.

induction

A pairs

pairing

subsequent

conversions

either

6'

May 1960

of mainly

pairing

with

"transitions",

and

T2 treated

At

the leads

CG ->

(3)

the

scheme

by the

found

deamination

base

this

been

in A and C. The amino-group

A and C deamination

those

has

for

the

In

determined

does

from

the

T and C pairs

both

ting

mutation

that / responsible

(1953).

bonding

removal

For

of r-

expected

61 amino-group its

rate

G, is

and Crick

hydrogen ty

of it

both,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inactivation

at a low

pH of

Vol. 2, No. 5

BIOCHEMICAL

treatment cated

does

from

not

p ersonal

the

account

Zeitschrift

Benzer,

S.,

in

"The

Baltimore

Brookhaven

R. and Ephrussi-Taylor,

Nundry,

K..Y.

and Gierer,

H. and Schramm, H.,

S.,

G.,

Z.Maturforsch.

the

as indi-

unpublished)

target

C and G can

size

for

be calculated total

results

of Heredity",

and

Crick,

Alexander,

in Biol.

l2,

inacto be

T2 DNA. will

be published

John

Hopkins

press,

63 (1959)

Compt.rend.Acad.Sci.,a,838

Z.f.Vererbl.,

3,

697

S.S., H.E.

Cold

Spring

Biochem.J., and Leidy,

328

(1958)

(1960)

375 (1959) C.I>., Z.I?aturforsch.,

T.H.C.,

(1959)

614 (1958)

Z.Naturforsch.,m, in press

Virology, 2, W. and Wieder,

2, 155 (1953) G.R. and Cohen,

Zamenhof,

the

of

[

(Vielmetter,

or 25;'0 of the

B.,

A.,

Schuster,

Xyatt,

of k,

Basis

Symp.

Schuster,

J.D.

FLT22

discussion

Chemical

Litman,

Watson,

inactivation

(1957)

E.,

I.,

total

May 1960

5aturforschung.

Freese,

Tessman, Vielmetter,

phage

pairs

and

fiir

of the

communication)]

5 x 104 nucleotide

A detailed

20$

with

due to dedmination

about

in

exceed

experiments

and T4 (Freese, tivation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I&, Harbor

312 (1959) Symp.Quant.Biol.,

2,

774 (1953)

L.,

J.exp.Ned.,

a,

373 (1953)