Vol. 70, No. 3, 1976

ESCHERICHIA

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

COLI

DEFICIENT

MUTANTS

IN

EXORIBONUCLEASES

Nikolai Nikolaev, Virginia Folsom and David Department of Microbiology and Immunology, Biology and Biomedical Sciences, Washington St. Louis, MO. 63110.

Received

March

Schlessinger, Division University,

of

18,1976

SUMMARY Strain s296, isolated by screening 2000 colonies after nitrosoguanidine mutagenesis, yields extracts with less than 1% of wild-type RNase activity Unlike other strains, S296 grows with a against (3H) poly(U). --E. coli doubling time 8f abouto2 hr., both in nutrient broth and in minimal medium, an 42 . The strain retains 10 to 20% of wild-type exonuclease and at 30°, 37 activity against ( $ H) rRNA or T4 phage-specific mRNA; but two further mutants, made by screening mutagenized colonies of strain S296, are reduced to 3% of wi Id-type activity against those substrates as we1 I. INTRODUCTION Many for

proteins

normal

growth.

lesions

in

cells

will

be

for In

up mutants whole

cells

in if

a limiting

in

cells

(reviewed

many

have

tRNA

at

large

can ref.

all,

level,

case,

excess

tolerate 1).

over

some

The

though

in

producing

1~

enough

useful

in

specific

the

level

severe

growth

rate

some

instances

a selective

of

inference to

required

mutational of

the

mutant

the

mutated

retardation

us

to

ask

-

and

in

particular,

For mutant screening, 10) and of colonies from in 1 ml of RNase II assay

10 ml mutagenized buffer

coli

DNA polymerase

(2.7.7.25;

ref.

from

of

a

levels that

whether

to this

activity

3);

and

the in

permit

critical

simple

method

analysis K+-activated E.

for

RNase

culture

might

co1 i cell

for would RNase extracts

III

turned studies

mutants

a similar for

1 (E.C.2.7.7.7;

a mutagenized

temperature-sensitive

led

exonuclease

AND

much

The

E.

colonies enzyme

the

MATERIALS

for

of

alternative (I)

for

transferase

extracts.

enzymes

isolated

screening

exoribonucleases

accounts

been

nucleotidyl

each

and

a generally

Copyright All r&hts

cells

result,

little

at

mutants

2);

(4-6).

for

in

process. Such

of

a

genes

be affected

particular

ref.

present

As

vital

may

element

are

in provide

the be

study possible

II

which (7-9).

METHODS

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

overnight cultures of E. coli strain C3 (ref. cultures (11) were centrifuged, resuspended (10 mM Tris-Cl, pH 7.5; 150 mbl NH4CIj I mM 920

Vol.

70, No. 3, 1976

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

MgCl2; ref. 7) and lysed in an ice-water bath by three with a Bios nik probe. 1 pl of C3 lysate was sufficient all added ( 3 H) poly(U) to alcohol-soluble nucleotides for 30 min (Fig. 1). Ten ul of each lysate was assayed with low activity, and confirmatory assays were carried crude extracts or soluble proteins cleared of ribosomes

COMMUNICATIONS

15-set burstsof cavitation to degrade essentially in a standard incubation to identify candidates out on alumina-ground by centrifugation (ref.

8). Ribonuclease activity was measured at with enzyme and 2 ug of rRNA (77,000 cpm), 0.8 ug of T4 mRNA (70,000 cpm). Twenty pl and 0.2 ml of chilled absolute ethanol were ice, the mixture was centrifuged at 8000 x and 150 ul of the supernatant was counted containing 10% protosol (New England Nuclear).

35 ’ 2 ug (200 then g for in 5

in 50 ul of assay buffer along of poly(U) (41,000 cpm), or ug) of carrier ribosomal RNA added. After 15 to 20 min on 10 min to remove the precipitate, ml of toluene scintillation fluid

E. coli B was labeled from 8 to 13 min To prepare T4 mRNA as a substrate, after infection at 37O (12), with 50 uCiofm) uridine/ml. RNA was phenolextracted and fractionated in a sucrose gradient (13). Fractions sedimenting faster than lOS, excluding the absorbance peaks of 16~ and 2 S rRNA, were pooled, brought to 0.1 M NaCl, and precipitated with ethanol. The ( 3 H) T4 mRNA, or rRNA similarly prepared from growing cells labeled for 90 min, was then stored frozen in water solution. RESULTS

AND

In

DISCUSS

each

of

tested

after

found,

extracts

first

set,

296th

colony

with

when

have

noted

that

that

unlike

proven, new (Gupta,

the

1% of

activity The and exonuclease R.S.,

which Kasai,

mutant

bacterial (Fig. strain

wild-type

the can

T.,

be and

more

that

it

was

truly

rate

compared

to

the

II

or in

the has

detected

Schlessinger,

D.,

921

mutant

(data

20%

been

of

to

extracts

submitted

of to

in shown).

reduced the

J.

to rjarental

(e.g.,

since

found

and

not

RNA

has

we

comparable

ribosomal

in ms.

is

were

10 to (3H)

but parental;

42’

poly(U)

retained

activity easily

and

against

mRNA RNase

37’,

rate

enzyme

deficient.

a slow

30°,

activity of

detail,

they

remaining

inhibition

in

at

(the

wild-type

no

growth

the

S296

analyzed

assayed

in

1% of

gave

the

of

strain

been

T&-specific

deficiency of

so

than

were

one

and

were

mutants

not

activity,

(3H)

less

extract

and

S256

Two

activity;

strains, 2),

colonies

requirement:

with

has at

C3.

phenotypic

extracts,

grows

of

specific bulk

The

5296

media

against

the

lot),

slow-growing

strain

enzyme

second

strain

reported

the

the

wild-type

strain

extracts

1000 of

wild-type

1).

with

fortified

Whereas

the

of

other

or

3).

loO/, of in

mixed

trials,

satisfied

(Figure

physiology

Fig.

screening mutagenesis

about

The

than

of

which

tested

activity

strain

sets

of

poly(U)

less

two

nitrosoguanidine

against

minimal

I ON

been

reside

in

the

mutant

Bioi.

a

Chem.).

BIOCHEMICAL

Vol. 70, No. 3, 1976

I

1

I

I

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

4 0

0

20

IO

Time

30

0

(min)

Time

(hr)

Fig.

1.

Degradation of (3H) poly(U) by crude extracts of strains C3 and S296. using samples from alumina-ground Assay as in MATERIALS AND METHODS, Note that from strain s296, 100 ug cells extracted in assay buffer. of protein is used, as compared with 5 ug from strain 13.

Fig.

2.

Growth of strain s296 in different media. The growth rate was followed by the optical density of the culture at 420 nm. Cultures were grown from single colonies in minimal salts containing glucose, tryptophan and methionine (ref. IO; -o-); in the same medium fortified with 0.4% technical grade casein hydrolysate (-A-) or L broth (1% tryptone - 0.5% yeast extract - 0.5% NaCl (-o-).

Strains by

repeating

From

two

with

even

the

screening

mutagenized

tively,

were

activity;

they mutant

S296

(Fig.

3).

activity One

induced result to

that

strains

(Fig. might by

of

with strain

colonies,

purified

as

against

(3H)

mRNA

515

lower

they

been

isolated

a substrate.

colonies,

culture, and

much

T4

have RNA as

and

each

S296/197

activity

S296

ribosomal 520

from

strains

than

(3H) S256,

one

showed

Tested

levels

procedure

Two

were

these

exonuclease

cultures

tested.

from

in

lower

respec-

showed

low

S256/680.

Cell

than

parental

showed

their

a comparable

levels extracts strain reduction

4). expect

that

the

nitrosoguanidine.

from

one

of

mutants

or

a very

few

scored

as

mutant

strains

However,

the

lesions,

because

lac-

or

RNase

922

would

show

mutants

I-

their in

complex

reported

control

lesions

here

frequency screenings.

in

very is

likely

comparable Also,

of

vol. 70, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

r-

I

poly(U

rRNA

2’

S296 S296/197 S296/660

Q

ug

500

t

protein

I

I

I

I

27

36

T4 mRNA S296i

400

300

200

100

0 18

4g

Fig.

3.

Fig.

4.

45

protein

Degradation of (3H) poly(U) (left) and (3H) rRNA (right) protein of strains C3, ~296, S296/197, and S296/680. MATERIALS AND METHODS, with portions of soluble protein ground cells. Degradation

of

(3H)

197, and S296/680. protein

from

T4

mRNA

Assay

alumina-ground

by

soluble

as in MATERIALS cells.

923

proteins AND

by Assay from

of strains METHODS, with

soluble as in alumina-

~296, soluble

~296/

BIOCHEMICAL

Vol. 70, No. 3, 1976

all

the

mutant

tryptophan

-

strain

strains i.e.,

grow with

the

at

30’

same

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on

minimal

medium

nutritional

containing

requirements

methionine

as

their

and

parental

C3. While

may

already

low

levels

the be of

genetic useful

analysis in

endogenous

many

of

these

studies

nuclease

mutants

of

RNA

activity

remains function

are

to and

frequently

be

pursued,

metabolism, an

they where

advantage.

ACKNOWLEDGEMENTS

the NSF

The screening for mutant S296 technical assistance of Mayumi Grant BMS - 74 - 11779.

was Ono.

carried The

out in work was

March-April, supported

1974, part

in

with by

REFERENCES 1.

2.

Schlessinger, Okazaki, Nature Deutscher, Kindler,

2: 65: i: 9. 10. II.

12. 13.

D. (1974) Ann. Rev. Genet. R., Sugimoto, K., Okazaki, T., 228, 223-32. M. and Hilderman, R. (1974) P., Keil, T.U. and Hofschneider,

126,.5340.

8, Imae, J.

135-151. Y.

and

Bacterial. P.H. (1973)

Sugino, 118, Mol.

A.

(1970)

621-627. Gen.

Genet.

.

Dunn, J.J. and Studier, F.W. (1973) Proc. Nat. Acad. Sci. USA 70,3298-3303. Nikolaev, N., Silengo, L. and Schlessinger, 0. (1973) Proc. Nat. Acad. Sci. USA. 70. 3361-3365. Spahr, P.F. and Schless inger, D. (1963) J. Biol. Chem. 238, P.C. 2251-2253. Spahr, P.F. (1964) J. Biol. Chem. 239, 3716-3726. Singer, M.F. and Tolbert, G. (1965niochemistry 5 1319-1330. Glazier, K. and Schlessinger, D. (1974) J. Bacterial. a 1195-1200. Adel berg, E.A., Mandel, M. and Chen, G.C. (1965) Biochem. Biophys. Res. Commun. l8, 788-795. Bolle, A., Epstein, R.H., Salser, W. and Geiduschek, E.P. (1968) J. Mol. Biol. 31, 325-333. Gupta, R.S. and Schlessinger, D. (1975) J. Mol. Biol. 2, 311-318.

924

Escherichia coli mutants deficient in exoribonucleases.

Vol. 70, No. 3, 1976 ESCHERICHIA BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS COLI DEFICIENT MUTANTS IN EXORIBONUCLEASES Nikolai Nikol...
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