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