Japan. J. Microbiol. Vol. 19(6), 433-439, 1975
Ribonucleic
Acid-Dependent
Polymerase
in the
Kazuko
SAITO and
Ribonucleic
Immune SUSUMU
Acid
Response
MITSUHASHI
Departmentof Microbiology,Schoolof Medicine, Gunma University, Maebashi (Received for publication, April 17, 1975)
ABSTRACT
Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 m column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA preparation (iRNA) as template made from the spleens of immunized mice but very low activity was found with an nRNA preparation made from the spleens of normal mice. Incorporation of 3H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12-13 S was most active as a template. It was followed by a fraction corresponding to 6-7 S. Sucrose gradient analysis of the 3H-UTP-labeled product was attempted. Some properties of this enzyme are described.
In
previous
immune
papers,
extracted cells
acid
from or
induced cells
or
memory
the
lymph
[10,
antigenic
of
of
Salmonella
enteritidis
antibodies
against
These
results
replicated In
in the
incorporation
iRNA decreased
in
evoking [11].
the
iRNA
Antigen. animals,
capable
of
3H-UTP a
MATERIALSAND METHODS Animals. Seven-week-old C3H strain. mice (raised by the Central Animal Laboratory, Gunma University) were used.
cells.
immunized
enzyme
in
in with
fla•¬ella that
of
fraction
serial
successful
and
recipient of
an
of
21]
a
25].
infection
suggested
incorporation
presence
was
Salmonella
the
14, that
against [18,
spleens
demonstrated
insoluble
iRNA
animals
[12] to
[9-11,
disclosed
transfer
immunizing
cells responding
stimulus
experiments
passive
animals
antibody-carry-
pyroninophilic capable
RNase but not of DNase. This activity was also inhibited by two derivatives of rifamycin, i.e., 2, 6-dimethy1-4-benzy1-1-4-demethyl rifamycin and 3-formyl rifamycin SV Ooctyloxime, while it was not inhibited by actinomycin D, mitomycin C, and bleomycin A2 [19, 20]. This paper deals with partial purification of the enzyme and some of its properties.
the
exudate
immunized of
14-16], cells
that preparation
peritoneal of
proliferation
secondary Further
reported (iRNA)
spleens,
nodes
the
ing
the
we
ribonucleic
into
cell-free as
a in
inducing an
system template. the
presence
acidin
Flagellae
of
Salmonella
tennessee
(O-6, 7; H-Z29) were used as antigen. They were prepared by the method described previously [14]. Twenty micrograms of flagella antigen were suspended in 0.1 ml of 0.85% saline. Immunization. Mice were singly immunized with 0.1 ml of flagella suspension by intravenous injection. Preparation of immune RNA. Mice were sacrificed 3 days after immunization and the
we
the The of
Requests for reprints should be addressed to Dr. Kazuko Saito, Department of Microbiology, School of Medicine, Gunma University, 3-39-22 Showa-machi, Maebashi 371, Japan. 433
434
K. SAITO
AND S. MITSUHASHI
immune(i) RNA preparation was extracted from the spleens of immunized mice by the modified method of Kidson et al [8]. Spleens of 20 immunized mice were weighed and disrupted into small pieces. After addition of nine volumes of 0.01 Macetate buffer (pH 5.0) containing 0.5% di-sodium naphthalene1, 5-disulfonate, the tissue was homogenized with a teflon homogenizer in an ice bath. The homogenate was mixed with an equal volume of 90% (v/v) phenol containing 0.1°/o (w/v) 8-hydroxy quinoline and centrifuged at 10 000 rpm for 10 min. To the water phase, 0.1 volume of 20% sodium dodecyl sulfate was added and the mixture was extracted again by the addition of 90% phenol. Phenol treatment of the aqueous phase was repeated four to six times until no denatured product was seen in the interphase. Two volumes of cold ethanol were added to the final aqueous phase. The precipitate formed was dissolved in a small amount of 0.15 MNaC1 containing 2 ,ug/ml of potassium polyvinyl sulfate (PVS) and precipitated again with ethanol. All procedures were carried out at 4 C. The ethanol suspension of the precipitate was stored at -20 C and ethanol was removed by centrifugation and evaporation before use. The amount of RNA was determined by measurement of absorption at 260 nm. Sucrose density gradient centrifugation. Two milligrams 0.01 M
of
iRNA
preparation
buffer
(pH
M acetate NaC1,
were
10-4
layered
linear
sucrose
buffer.
Ltd.,
was
RPS-25
(1
ml
bottom.
16
hr
each)
by
tubes
containing
in
24
000
2 ƒÊg/ml
0.15 of
PVS,
the
concen-
dialysis
through solution
and
precipitated
ethanol. Preparation
sacrificed immune(i) from
of 3
the
DNA spleens
method
described
Twenty
spleens
standard
immune
days
saline
DNA.
after preparation of by
were citrate
was
immunized Mach cut
Mice
were
immunization
and extracted
mice and
by
Vassalli
into
pieces
(SSC:
0.15
in
in
25
C,
60 for
the
60
C and 10 min.
at
washing
with
suspended
for
30
al
DNA
After
precipitation
with
Co.,
was
glass
U.S.A.),
out
C
and
then
(Nutritionat
37
was
C
for
30
extracted alcohol
Two volumes aqueous phase
spooled
37 (Wor-
chloroform-isoamyl
: 1 by volume). added to the
DNA
at
pronase
solution
with
and
RNase
U.S.A.)
DNA
times
of
of
pre-
preparation
incubated
Co.,
100 ƒÊg/m1
The
(24 were
and
with
was
DNA
100 ƒÊg/ml
Biochemicals
min. three
SSC
The
mixed
ethanol;
Biochemicals
treated
phenol. and
C.
with
and
performed
of
in
min
thington
separated
fresh
the
The
warmed
were
pooled
ethanol,
phenol C.
vigorously in a slow cooling to
was
with
0
0
then
rotated After
was
volumes
cipitated
was
was
phase
each
phase
three
at
extractions
C
of
blender
aqueous
aqueous
suspension
volume
phenol
additional
at
The
equal
a Waring in
to bath
four
citrate). an
from
of ethanol and the
solution
with
a
rod.
Preparation of RNA polymerase. Spleens of 30
immunized
and
mice
were
homogenized
buffer The
(pH
in
8.0)
buffer
for
3
of
cell
suspension
by
differential
000 •~
with and
g for
The the
10
same
mm
min
was
9000 •~ and
sediments
sedimented
25
buffer again
ice
cold bath.
Tris-HC1 and 1 The
mm ho-
sequentially
centrifugation
min, 60
of
an
acid.
sedimented for
in
dithiothreitol
mogenized
g
weighed
volumes
min
consisted
(pH 8.0), 4 mm ethylenediaminetetraacetic
excised,
two
in
168
g
for 000 •~
at 20 g for
were
homogenized
a teflon
homogenizer
by
min, 120
centrifugation.
Biogel column chromatography of RNA poly-
Frac-
M NaC1
with
quickly water
106
from
sodium
homogenate
min.
a
M mixed 5 min
20%
(Hitachi
were
for
900 •~
same
rpm.
was
PVS
in
collected
pressure against
the
rotor
at
fractions
negative
collodion
of a 5 to
centrifuged
were
'Collected
trated
ml
swinging
for
of 0.1
2 ƒÊg/m1
of 30 prepared
tube
1 ml
containing
and
top
gradient
Tokyo)
tions
by
the
The
Hitachi
5.0)
M MgC12 on
in
0.015
the [13].
10
M NaC1-
ml
merase. Five milligrams of protein of crude enzyme extract solubilized by Triton X-100 from microsomal and ribosomal fraction were applied to a 2 x 80 cm column of Biogel A 1.5 m (BIO-RAD Laboratories, Richmond, Calif.) which had been equilibrated with 0.05 M Tris-20% glycerol-0.01 M 2-mercaptoethanol buffer (pH 8.0). The column was eluted with the same buffer and 5-ml fractions were collected. Assay
of enzyme activity.
The
reaction
mixture contained the following in a final volume of 0.2 ml : 4 mm Tris-HC1(pH 8.3), 2 mm MgC12, 0.4 mm dithiothreitol, 0.2 mm
RNA-DEPENDENT Table
1.
Distribution
RNA POLYMERASE
of RNA-dependent
RNA
IN IMMUNE
polymerase
RESPONSE
activities
435
in subcellular
fractions
Reaction mixture contained 25 ƒÊgof iRNA preparation and 0.16-ml portions (400 ƒÊgprotein) of each fraction. A background incorporation of radioactivity from unincubated controls was subtracted to obtain the values listed above. Number in parentheses shows percentage of total activity. See Materials and Methods for details.
each of CTP, ATP and GTP, and 4 ÊCi of 3H-UTP (15.9 Ci/mmole, the Radiochemical Center, England), 25 ƒÊg of i(or n) RNA, 0.16-ml portions of the column eluate and 1 μg
of
actinomycin
D(Merck,
Sharp
and
Dome Lab., U.S.A.). After 3 hr of incubation at 37 C, 400 ƒÊgof bovine serum albumin and 3 ml of 10% trichloroacetic acid(TCA) containing 2 mm sodium pyrophosphate were added to the reaction mixture. After standing for 1 hr in an ice bath, the resulting precipitate was filtered through a glass filter (GF/F, Whatman), washed with 30 ml of cold TCA, dried and dissolved in 10 ml of omuniflour-toluene solution. A sample from the glass filter was counted in a liquid scintillation counter. RESULTS
Distributionof RNA-DependentRNA Polymerase Activity in the Subcellular Fractions from Immune Spleen Cell Homogenate
Each fraction obtained by differential centrifugation was dispersed in buffer and its RNA polymerase activity was assayed. As shown in Table 1, fraction No. 4 (microsomal and ribosomal fraction) was most active, and followed by fractions No. 3 and No. 5 in decreasing order of activity when the iRNA preparation was used as a template.
Fig. 1. Biogel A 1.5 m column chromatography of RNA-dependent RNA polymerase. A crude enzyme preparation was extracted with Triton X-100 from a fraction No. 4 (Table 1) and applied to the Biogel A column. A 0.16-m1 portion of each enzyme fraction (14 ƒÊg/mlof protein) was added to the reaction mixture.
against
1
liter
of
glycerol-0.01 (pH 1.5
M
8.0) for m column
in
Fig.
about of
a
single
60 •~ the
104
4
Specific
purified compared
solubilized
was
(molecular
daltons).
fortyfold
No.
Biogel A As shown
activity peak
enzyme
increased
fraction
buffer to
polymerase
in
activity
of
Tris-HC1-20%
2-mercaptoethanol
RNA
out
weight:
Biogel
M
3 hr and subjected chromatography.
1,
eluted
0.05
through with
with
that
Triton
X-
RNA
and
100.
Column Chromatography of a Solubilized Fraction No.
4
Fraction No. 4 was suspended in 3 ml of buffer containing 1% Triton X-100 and incubated at 37 C. After 10 min of incubation, the suspension was centrifolged at 52 000 rpm for 120 min. The supernatant thus obtained was dialyzed in cellulose tubing
To DNA spleens
examine
of in
Table
plate, from
while normal
made
from
or 2,
the mice
extracted
iRNA
or as
from
immunized
RNA and the
normal
inactive
specificity, were
normal
shown
almost
template
preparations
was
the
best
As tem-
preparation made DNA preparations
immunized
templates.
the
mice.
mice The
were
incorpo-
436
K.
Table
2.
Template
specificity
Twenty-five ƒÊg for
of each
details. a) Fraction b)
separated 0
template
. 4 shown in No . 4 was treated by
AND S. MITSUHASHI
for
was
Table
the
incorporation
added
to
0.2
ml
of 3H-UTP
of reaction
into
acid-insoluble
mixture.
See Materials
min
the
fraction
and
Methods
1.
with
Triton
X-100
at
with
Triton
X-100
was
37
C for
10
and
solubilized
fraction
was
centrifugation.
Fraction
graphy d)
of iRNA
No
Fraction
SAITO
No.
4 solubilized
as shown Twenty-five ƒÊg/0
in
Fig. .02
DNase(100 ƒÊg/ml)
at
purified
by
Biogel
A
1.5
m
column
chromato-
1.
37
ml
of iRNA
C for
10
was
min
and
pretreated was
with
added
to
the
each
same
volume
reaction
mixture
of
RNase(100 ƒÊg/m1) without
removal
or of the
nucleases.
by a fraction
corresponding
to 6-7 S.
Sucrose Gradient Analysis of the 3H-Labeled Product Synthesized by Purified Enzyme
The 3H-labeled product was sedimented in a sucrose gradient to estimate roughly the molecular size of newly synthesized RNA. After the reaction was completed, the mixture was concentrated about tenfold by negative pressure dialysis in a collodion bag (Sartorius Membran Filter, Gottingen) against 0.15 M NaCl solution containing 2 μg/ml Fig.
2.
Template
sucrose
density
portion tein)
of shown
added
to
ration the
activity gradient
the 0.2
ml
fractionated
centrifugation.
purified in Fig.
reaction
was
greatly
of RNase
In active tion
order
Gradient
but
decreased
not
was
density
profile RNA
shown with
corresponding most
the
of iRNA,
fractionated
sucrose
active
by
gradient. in Fig. template to
in
of DNase.
Fractionated
by
Sedimentation
to estimate
fractions
0.16-ml
mixture.
Template Activity of iRNA Sucrose
A
by
enzyme (14 ƒÊg/ml of pro1 and 20 ƒÊg of RNA were
of the
of 31-1-UTP presence
of iRNA
molecular
the
iRNA
centrifugation The
2 revealed activity.
12-13
as template.
size
S was This
of
preparain
a
sedimentation three A found was
peaks of fraction to followed
be
of
PVS.
A
0.2-ml
portion
of
the
concentrated sample was layered on the top of 5 ml of a 5 to 20% linear sucrose gradient in 0.01 M acetate buffer (pH 5) containing 0.15 MNaCl and 2 ,ug/m1of PVS. The tube was centrifuged in a Hitachi RPS-40 swinging rotor at 40 000 rpm for 7 hr at 4 C. Fivedrop fractions were collected from the bottom and the radioactivity of a 0.1 ml aliquot spotted on a Whatman GF/F glass filter was counted in a liquid scintillation counter after drying. The molecular size of the synthesized product was compared with that of the iRNA fraction used as template. As shown in Fig. 3, a single radioactive peak was seen in the sucrose gradient and its sedimentation property was indistinguishable from that of the iRNA fraction (6-7 S) used as template.
RNA-DEPENDENT
RNA
POLYMERASE
IN
IMMUNE
Fig. 3. Sedimentation velocity pattern of the 3Hlabeled product. Reaction mixture is shown in Materials and Methods. Twenty-five yg of iRNA (6-7 S) prepared in the experiment shown in Fig. 2 was used as a template.
Fig. 5. Effect of pH on enzyme. The series of pH 5.0 to 8.5 were prepared
Fig.
Fig.
4.
UTP
Time into
course
RNA
of
the
by purified
incorporation enzyme.
of
3H-
Two-tenth-ml
portions were withdrawn from the reaction mixture at various time intervals after incubation and the radioactivity in the acid-insoluble fraction was counted. The shown in Fig.
iRNA 3.
preparation
was
the
same
437
RESPONSE
the activity of purified values ranging from pH by 2 M of Tris—malate-
NaOH buffer instead of Tris-HC1 buffer. See Materials and Methods for assay conditions. The iRNA preparation was the same as shown in Fig. 3.
6.
Effect
of
temperature
on
the
activity
purified enzyme. See Materials and Methods assay conditions. The iRNA preparation was same as shown in Fig. 3.
of for the
as
Time Course of the Reaction
The incorporation of 3H-UTP continued linearly for at least 3 hr with iRNA as template (Fig. 4). Optimum pH and Temperature
Optimum pH of the reaction was determined in the pH range of 5 to 8.5 in 0.2 Tris—malate—NaOH buffer (Fig. 5). The highest activity was observed at pH 7.6. The optimum temperature of this reaction was 37 C and its activity decreased to less than 50% at 30 or 45 C (Fig. 6).
Fig. 7. Effect of divalent activity. See Materials condition preparation
except was
cations and
for ion the same
on purified enzyme Methods for assay
concentration. as shown
The in Fig.
iRNA 3.
438
The Divalent
K.
Cation
SAITO
AND S. MITSUHASHI
Requirement
The effect of divalent cations on the rate of RNA synthesis is shown in Fig. 7. MgC12 at its optimum concentration (5-10 mM) best satisfied the requirement for divalent cations, while less stimulation was seen at the optimum concentration (1.25 mM) of MnC12.
In
the
present
characterized lows:
1)
is
in
fraction
of
homogenate;
enzyme
2)
increased
There have been many reports concerning the RNA-dependent RNA polymerases on RNA viruses and RNA bacteriophages [1, 23] but only a few reports have been presented on mammalian cells : e.g., virusinduced leukemia cells [6, 24] and immunized lymphoid tissues [7, 17, 19, 20]. A cytoplasmic microsome-bound RNA-dependent RNA polymerase was also demonstrated in a rabbit reticulocyte lysate. The synthesis of RNA was absolutely dependent on the addition of RNA template and the best template was hemoglobin messenger RNA [3].
Previous studies in our laboratory have disclosed that passive transfers of iRNA were successful in establishing immunity in mice against infection with S. enteritidis [16] or immunity to Salmonellaflagella [11]. It was also found that iRNA was able to confer ability for secondary response of antibody formation on recipient cells [10-12, 14, 25]. This ability of iRNA was also serially and passively transmissible [11, 18, 21]. In addition, we reported the following results [20] ; 1) a crude extract(iEXT) obtained from the homogenate of immune spleen cells catalyzed the incorporation of 3H-UTP into acidinsoluble fraction when the iRNA preparation was used as a template; 2) the incorporation was abolished by treatment of iRNA with RNase but not with DNase; 3) two derivatives of rifamycin, which are known to be inhibitors of reverse transcriptase of oncogenic RNA viruses, inhibited the incorporation of 3H-UTP, while mitomycin C, bleomycin A2 and actinomycin D did not show any inhibitory effect; 4) four ribonucleoside triphosphates were required for this reaction; and 5) the product of the reaction resisted RNase treatment but was rendered sensitive after heat treatment. Thus the product RNA appeared to be in a hybrid form with the template RNA [20].
crude of
this
eluted the
was with
at
1.42
most to
iRNA
be
divalent and behavior
from of pH,
cation 6)
the of
microsomal
specific by
3)
the
of
Biogel
column
void
for
S RNA
this
be
volume; enzyme from followed
source;
5)
requirements
are gradient
3H-UTP-labeled
4) was
it was
this enzyme, i.e., optimum temperature
density
around activity
prepared
same
of
weight
to
the
the
that
molecular
polymerase
of
the
and spleen
with
the
fol-
activity
compared
preparation;
S RNA
as
immunized
template
12-13
further
polymerase
estimated
times
active
crude
properties optimum
4;
daltons,
found
7-6
No.
enzyme
6 •~ 105
the the
fortyfold as
fraction
have
polymerase RNA
localized
ribosomal
chromatography DISCUSSION
we
RNA
RNA-dependent
activity
cell
article
this
the by
various kinetics, and
described; centrifugal product
is
presented.
The results reported previously and those described in this article suggest that this enzyme is an RNA-dependent RNA polymerase and that the enzyme is induced or its activity is enhanced by antigenic stimulation in the spleen cells. This implies that the enzyme acts as an amplifier of antigenic stimulation in the processes of the antibody formation. The iRNA preparations of two different S values can serve as template for this enzyme. A larger iRNA fraction (12-13 S) corresponds in S value to the mRNA of immunoglobulin (light chain) which shows an S value of 9 to 13 [22]. A smaller iRNA fraction (6-7 S) may contain a portion of the immunoglobulin mRNA for the variable part of the immunoglobulin. Although we have no evidence at the moment, possible involvement of the iRNA (6-7 S) in the production of immunoglobulin should be the subject of future experiments. Gene amplification or the production of multiple gene copies was reported to occur in the nucleoli of amphibian oocytes of genes for ribosomal RNA [2, 4, 5], suggesting that these cells can synthesize large quantities of rRNA in a relatively short time. The RNA-dependent RNA polymerase in mammalian cells may offer an additional site for control of gene expression as well as providing
RNA-DEPENDENT
a mechanism
for
amplification
RNA
POLYMERASE
of the
ex-
pression of specific genes. Especially, the possible production of iRNA by means of RNA-directed RNA synthesis may offer a new clue in the study of antibody formation and may account for the transmission and amplification of antigenic information. REFERENCES
[1]
Baltimore, D. 1971. Expression of animal virus genomes. Bacteriol. Rev. 35: 235-241. [ 2 ] Brown, D., and David, I. B. 1968. Specific gene amplification in oocytes. Science 160: 272-280. [3]
Downey, K. M., Byrnes, J. J., Jurmark, B. S., and So, A. G. 1973. Reticulocyte RNA-dependent RNA polymerase. Nature 70: 3400-3403. [ 4 ] Evans, D., and Birnsteil, M. L. 1968. Localization of amplified ribosomal DNA in the oocyte of Xenopus laevis. Biochim. Biophys. Acta 166: 274276. [ 5 ] Gall, J. F. 1968. Differential synthesis of the genes for ribosomal RNA during amphibian oogenesis. Proc. Nat. Acad. Sci. U.S. 60: 553-560. [ 6 ] Haruna, I., Ohno, T., and Watanabe, I. 1970. Isolation of an RNA-dependent RNA polymerase from friend murine leukemia cells. Proc. Japan Acad. 46: 1016-1021. [ 7 ] Jacherts, D., Opits, U., and Opits, H. G. 1972. Gene amplification in cell-free systems. Zschr. Immunitatsforsch. 144: 260-272. [ 8 ] Kidson, C., Kirby, K.S., and Ralph, R.K. 1963. Isolation characteristics of rapidly labeled RNA from normal rat liver. J. Mol. Biol. 7: 313-315. 9 ] Kitamura, K., Kurashige, S., and Mitsuhashi, S. 1973. Dose effect of immune ribonucleic acid fraction on antibody formation. Japan. J. Microbiol. 17: 29-33. [10] Kurashige, S., Kitamura, K., Akama, K., and Mitsuhashi, S. 1970. Transfer agent of immunity. IV. Antibody formation against diphtheria toxin by an immune ribonucleic acid fraction. Japan. J. Microbiol. 14: 41-47. [11] Kurashige, S., and Mitsuhashi, S. 1972. Serial passive transfers of immune response by an immune ribonucleic acid preparation. J. Immunol. 108: 1034-1038. [12] Kurashige, S., Saito, K., Fukai, K., Kitamura, K., and Mitsuhashi, S. 1973. Proliferation of pyroninophilic cells in lymphoid tissues by stimula-
IN IMMUNE
RESPONSE
439
tion with immune ribonucleic acid. Japan. J. Microbiol. 17: 217-219. [13] Mach, B., and Vassalli, P. 1965. Biosynthesis of RNA in antibody-producing tissues. Proc. Nat. Acad. Sci. U.S. 54: 975-982. [14] Mitsuhashi, S., Kurashige, S., Kawakami, M., and Nojima, T. 1968. Transfer agent of immunity. I. Immune ribonucleic acid which induces antibody formation of Salmonella flagella. Japan. J. Microbiol. 12: 261-268. [15] Mitsuhashi, S., Saito, K., Kurashige, S., Osawa, N., and Kitamura, K. 1973. The role of RNA in cell-mediated immunity. Ann. New York Acad. Sci. 207: 380-388. [16] Mitsuhashi, S., Saito, K., Osawa, N., and Kurashige, S. 1967. Experimental slmonellosis. XI. Induction of cellular immunity and formation of antibody by transfer agent of mouse mononuclear phagocytes. J. Bacteriol. 94: 907-913. [17] Neuhoff, V., Schill, W. B., and Jacherts, D. 1970. Nachweiss einer RNA abhangigen RNAReplicase aus immunologisch kompeteten Zellen durch Micro-Disk-Electrophorese. Hoppe-Seyler's Zschr. Physiol. Chem. 351: 157-162. [18] Saito, K., Kurashige, S., and Mitsuhashi, S. 1969. Serial transfers of immunity through immune RNA. Japan. J. Microbiol. 13: 122-124. [19] Saito, K., and Mitsuhashi, S. 1972. Inhibitory effect of rifamycin derivatives on immunogenic RNA. J. Antibiotics 25: 477-479. [20] Saito, K., and Mitsuhashi, S. 1973. Ribonucleic acid-dependent ribonucleic acid replicase in the immune response. Japan. J. Microbiol. 17: 117121. [21] Saito, K., Osawa, N., and Mitsuhashi, S. 1971. Transfer agent of immunity. VI. Serial passive transfers of cellular immunity to Salmonella infection by immune ribonucleic acid. Japan. J. Microbiol. 15: 159-168. [22] Stewart, P. R., and Letham, L, D. (eds) 1973. The ribonucleic acids. Springer-Verlag, Berlin, Heidelberg, New York, p. 76. [23] Sugiyama, T., Korant, B. D., and LonbergHolm, K. K. 1972. RNA virus gene expression and its control. Ann. Microbiol. 26: 467-495. [24] Watanabe, I., and Haruna, I. 1969. Selfreplicating RNA in leukemic cells. Acta Haematol. Japon. 32: 593-602. [25] Yamaguchi, N., Kurashige, N., and Mitsuhashi, S. 1971. Antibody formation against Salmonella flagella by an immune ribonucleic acid fraction. J. Immunol. 107: 99-103.
Ribonucleic
Acid-Dependent
Polymerase
in the
Kazuko
SAITO and
Ribonucleic
Immune SUSUMU
Acid
Response
MITSUHASHI
Departmentof Microbiology,Schoolof Medicine, Gunma University, Maebashi (Received for publication, April 17, 1975)
ABSTRACT
Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 m column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA preparation (iRNA) as template made from the spleens of immunized mice but very low activity was found with an nRNA preparation made from the spleens of normal mice. Incorporation of 3H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12-13 S was most active as a template. It was followed by a fraction corresponding to 6-7 S. Sucrose gradient analysis of the 3H-UTP-labeled product was attempted. Some properties of this enzyme are described.
In
previous
immune
papers,
extracted cells
acid
from or
induced cells
or
memory
the
lymph
[10,
antigenic
of
of
Salmonella
enteritidis
antibodies
against
These
results
replicated In
in the
incorporation
iRNA decreased
in
evoking [11].
the
iRNA
Antigen. animals,
capable
of
3H-UTP a
MATERIALSAND METHODS Animals. Seven-week-old C3H strain. mice (raised by the Central Animal Laboratory, Gunma University) were used.
cells.
immunized
enzyme
in
in with
fla•¬ella that
of
fraction
serial
successful
and
recipient of
an
of
21]
a
25].
infection
suggested
incorporation
presence
was
Salmonella
the
14, that
against [18,
spleens
demonstrated
insoluble
iRNA
animals
[12] to
[9-11,
disclosed
transfer
immunizing
cells responding
stimulus
experiments
passive
animals
antibody-carry-
pyroninophilic capable
RNase but not of DNase. This activity was also inhibited by two derivatives of rifamycin, i.e., 2, 6-dimethy1-4-benzy1-1-4-demethyl rifamycin and 3-formyl rifamycin SV Ooctyloxime, while it was not inhibited by actinomycin D, mitomycin C, and bleomycin A2 [19, 20]. This paper deals with partial purification of the enzyme and some of its properties.
the
exudate
immunized of
14-16], cells
that preparation
peritoneal of
proliferation
secondary Further
reported (iRNA)
spleens,
nodes
the
ing
the
we
ribonucleic
into
cell-free as
a in
inducing an
system template. the
presence
acidin
Flagellae
of
Salmonella
tennessee
(O-6, 7; H-Z29) were used as antigen. They were prepared by the method described previously [14]. Twenty micrograms of flagella antigen were suspended in 0.1 ml of 0.85% saline. Immunization. Mice were singly immunized with 0.1 ml of flagella suspension by intravenous injection. Preparation of immune RNA. Mice were sacrificed 3 days after immunization and the
we
the The of
Requests for reprints should be addressed to Dr. Kazuko Saito, Department of Microbiology, School of Medicine, Gunma University, 3-39-22 Showa-machi, Maebashi 371, Japan. 433
434
K. SAITO
AND S. MITSUHASHI
immune(i) RNA preparation was extracted from the spleens of immunized mice by the modified method of Kidson et al [8]. Spleens of 20 immunized mice were weighed and disrupted into small pieces. After addition of nine volumes of 0.01 Macetate buffer (pH 5.0) containing 0.5% di-sodium naphthalene1, 5-disulfonate, the tissue was homogenized with a teflon homogenizer in an ice bath. The homogenate was mixed with an equal volume of 90% (v/v) phenol containing 0.1°/o (w/v) 8-hydroxy quinoline and centrifuged at 10 000 rpm for 10 min. To the water phase, 0.1 volume of 20% sodium dodecyl sulfate was added and the mixture was extracted again by the addition of 90% phenol. Phenol treatment of the aqueous phase was repeated four to six times until no denatured product was seen in the interphase. Two volumes of cold ethanol were added to the final aqueous phase. The precipitate formed was dissolved in a small amount of 0.15 MNaC1 containing 2 ,ug/ml of potassium polyvinyl sulfate (PVS) and precipitated again with ethanol. All procedures were carried out at 4 C. The ethanol suspension of the precipitate was stored at -20 C and ethanol was removed by centrifugation and evaporation before use. The amount of RNA was determined by measurement of absorption at 260 nm. Sucrose density gradient centrifugation. Two milligrams 0.01 M
of
iRNA
preparation
buffer
(pH
M acetate NaC1,
were
10-4
layered
linear
sucrose
buffer.
Ltd.,
was
RPS-25
(1
ml
bottom.
16
hr
each)
by
tubes
containing
in
24
000
2 ƒÊg/ml
0.15 of
PVS,
the
concen-
dialysis
through solution
and
precipitated
ethanol. Preparation
sacrificed immune(i) from
of 3
the
DNA spleens
method
described
Twenty
spleens
standard
immune
days
saline
DNA.
after preparation of by
were citrate
was
immunized Mach cut
Mice
were
immunization
and extracted
mice and
by
Vassalli
into
pieces
(SSC:
0.15
in
in
25
C,
60 for
the
60
C and 10 min.
at
washing
with
suspended
for
30
al
DNA
After
precipitation
with
Co.,
was
glass
U.S.A.),
out
C
and
then
(Nutritionat
37
was
C
for
30
extracted alcohol
Two volumes aqueous phase
spooled
37 (Wor-
chloroform-isoamyl
: 1 by volume). added to the
DNA
at
pronase
solution
with
and
RNase
U.S.A.)
DNA
times
of
of
pre-
preparation
incubated
Co.,
100 ƒÊg/m1
The
(24 were
and
with
was
DNA
100 ƒÊg/ml
Biochemicals
min. three
SSC
The
mixed
ethanol;
Biochemicals
treated
phenol. and
C.
with
and
performed
of
in
min
thington
separated
fresh
the
The
warmed
were
pooled
ethanol,
phenol C.
vigorously in a slow cooling to
was
with
0
0
then
rotated After
was
volumes
cipitated
was
was
phase
each
phase
three
at
extractions
C
of
blender
aqueous
aqueous
suspension
volume
phenol
additional
at
The
equal
a Waring in
to bath
four
citrate). an
from
of ethanol and the
solution
with
a
rod.
Preparation of RNA polymerase. Spleens of 30
immunized
and
mice
were
homogenized
buffer The
(pH
in
8.0)
buffer
for
3
of
cell
suspension
by
differential
000 •~
with and
g for
The the
10
same
mm
min
was
9000 •~ and
sediments
sedimented
25
buffer again
ice
cold bath.
Tris-HC1 and 1 The
mm ho-
sequentially
centrifugation
min, 60
of
an
acid.
sedimented for
in
dithiothreitol
mogenized
g
weighed
volumes
min
consisted
(pH 8.0), 4 mm ethylenediaminetetraacetic
excised,
two
in
168
g
for 000 •~
at 20 g for
were
homogenized
a teflon
homogenizer
by
min, 120
centrifugation.
Biogel column chromatography of RNA poly-
Frac-
M NaC1
with
quickly water
106
from
sodium
homogenate
min.
a
M mixed 5 min
20%
(Hitachi
were
for
900 •~
same
rpm.
was
PVS
in
collected
pressure against
the
rotor
at
fractions
negative
collodion
of a 5 to
centrifuged
were
'Collected
trated
ml
swinging
for
of 0.1
2 ƒÊg/m1
of 30 prepared
tube
1 ml
containing
and
top
gradient
Tokyo)
tions
by
the
The
Hitachi
5.0)
M MgC12 on
in
0.015
the [13].
10
M NaC1-
ml
merase. Five milligrams of protein of crude enzyme extract solubilized by Triton X-100 from microsomal and ribosomal fraction were applied to a 2 x 80 cm column of Biogel A 1.5 m (BIO-RAD Laboratories, Richmond, Calif.) which had been equilibrated with 0.05 M Tris-20% glycerol-0.01 M 2-mercaptoethanol buffer (pH 8.0). The column was eluted with the same buffer and 5-ml fractions were collected. Assay
of enzyme activity.
The
reaction
mixture contained the following in a final volume of 0.2 ml : 4 mm Tris-HC1(pH 8.3), 2 mm MgC12, 0.4 mm dithiothreitol, 0.2 mm
RNA-DEPENDENT Table
1.
Distribution
RNA POLYMERASE
of RNA-dependent
RNA
IN IMMUNE
polymerase
RESPONSE
activities
435
in subcellular
fractions
Reaction mixture contained 25 ƒÊgof iRNA preparation and 0.16-ml portions (400 ƒÊgprotein) of each fraction. A background incorporation of radioactivity from unincubated controls was subtracted to obtain the values listed above. Number in parentheses shows percentage of total activity. See Materials and Methods for details.
each of CTP, ATP and GTP, and 4 ÊCi of 3H-UTP (15.9 Ci/mmole, the Radiochemical Center, England), 25 ƒÊg of i(or n) RNA, 0.16-ml portions of the column eluate and 1 μg
of
actinomycin
D(Merck,
Sharp
and
Dome Lab., U.S.A.). After 3 hr of incubation at 37 C, 400 ƒÊgof bovine serum albumin and 3 ml of 10% trichloroacetic acid(TCA) containing 2 mm sodium pyrophosphate were added to the reaction mixture. After standing for 1 hr in an ice bath, the resulting precipitate was filtered through a glass filter (GF/F, Whatman), washed with 30 ml of cold TCA, dried and dissolved in 10 ml of omuniflour-toluene solution. A sample from the glass filter was counted in a liquid scintillation counter. RESULTS
Distributionof RNA-DependentRNA Polymerase Activity in the Subcellular Fractions from Immune Spleen Cell Homogenate
Each fraction obtained by differential centrifugation was dispersed in buffer and its RNA polymerase activity was assayed. As shown in Table 1, fraction No. 4 (microsomal and ribosomal fraction) was most active, and followed by fractions No. 3 and No. 5 in decreasing order of activity when the iRNA preparation was used as a template.
Fig. 1. Biogel A 1.5 m column chromatography of RNA-dependent RNA polymerase. A crude enzyme preparation was extracted with Triton X-100 from a fraction No. 4 (Table 1) and applied to the Biogel A column. A 0.16-m1 portion of each enzyme fraction (14 ƒÊg/mlof protein) was added to the reaction mixture.
against
1
liter
of
glycerol-0.01 (pH 1.5
M
8.0) for m column
in
Fig.
about of
a
single
60 •~ the
104
4
Specific
purified compared
solubilized
was
(molecular
daltons).
fortyfold
No.
Biogel A As shown
activity peak
enzyme
increased
fraction
buffer to
polymerase
in
activity
of
Tris-HC1-20%
2-mercaptoethanol
RNA
out
weight:
Biogel
M
3 hr and subjected chromatography.
1,
eluted
0.05
through with
with
that
Triton
X-
RNA
and
100.
Column Chromatography of a Solubilized Fraction No.
4
Fraction No. 4 was suspended in 3 ml of buffer containing 1% Triton X-100 and incubated at 37 C. After 10 min of incubation, the suspension was centrifolged at 52 000 rpm for 120 min. The supernatant thus obtained was dialyzed in cellulose tubing
To DNA spleens
examine
of in
Table
plate, from
while normal
made
from
or 2,
the mice
extracted
iRNA
or as
from
immunized
RNA and the
normal
inactive
specificity, were
normal
shown
almost
template
preparations
was
the
best
As tem-
preparation made DNA preparations
immunized
templates.
the
mice.
mice The
were
incorpo-
436
K.
Table
2.
Template
specificity
Twenty-five ƒÊg for
of each
details. a) Fraction b)
separated 0
template
. 4 shown in No . 4 was treated by
AND S. MITSUHASHI
for
was
Table
the
incorporation
added
to
0.2
ml
of 3H-UTP
of reaction
into
acid-insoluble
mixture.
See Materials
min
the
fraction
and
Methods
1.
with
Triton
X-100
at
with
Triton
X-100
was
37
C for
10
and
solubilized
fraction
was
centrifugation.
Fraction
graphy d)
of iRNA
No
Fraction
SAITO
No.
4 solubilized
as shown Twenty-five ƒÊg/0
in
Fig. .02
DNase(100 ƒÊg/ml)
at
purified
by
Biogel
A
1.5
m
column
chromato-
1.
37
ml
of iRNA
C for
10
was
min
and
pretreated was
with
added
to
the
each
same
volume
reaction
mixture
of
RNase(100 ƒÊg/m1) without
removal
or of the
nucleases.
by a fraction
corresponding
to 6-7 S.
Sucrose Gradient Analysis of the 3H-Labeled Product Synthesized by Purified Enzyme
The 3H-labeled product was sedimented in a sucrose gradient to estimate roughly the molecular size of newly synthesized RNA. After the reaction was completed, the mixture was concentrated about tenfold by negative pressure dialysis in a collodion bag (Sartorius Membran Filter, Gottingen) against 0.15 M NaCl solution containing 2 μg/ml Fig.
2.
Template
sucrose
density
portion tein)
of shown
added
to
ration the
activity gradient
the 0.2
ml
fractionated
centrifugation.
purified in Fig.
reaction
was
greatly
of RNase
In active tion
order
Gradient
but
decreased
not
was
density
profile RNA
shown with
corresponding most
the
of iRNA,
fractionated
sucrose
active
by
gradient. in Fig. template to
in
of DNase.
Fractionated
by
Sedimentation
to estimate
fractions
0.16-ml
mixture.
Template Activity of iRNA Sucrose
A
by
enzyme (14 ƒÊg/ml of pro1 and 20 ƒÊg of RNA were
of the
of 31-1-UTP presence
of iRNA
molecular
the
iRNA
centrifugation The
2 revealed activity.
12-13
as template.
size
S was This
of
preparain
a
sedimentation three A found was
peaks of fraction to followed
be
of
PVS.
A
0.2-ml
portion
of
the
concentrated sample was layered on the top of 5 ml of a 5 to 20% linear sucrose gradient in 0.01 M acetate buffer (pH 5) containing 0.15 MNaCl and 2 ,ug/m1of PVS. The tube was centrifuged in a Hitachi RPS-40 swinging rotor at 40 000 rpm for 7 hr at 4 C. Fivedrop fractions were collected from the bottom and the radioactivity of a 0.1 ml aliquot spotted on a Whatman GF/F glass filter was counted in a liquid scintillation counter after drying. The molecular size of the synthesized product was compared with that of the iRNA fraction used as template. As shown in Fig. 3, a single radioactive peak was seen in the sucrose gradient and its sedimentation property was indistinguishable from that of the iRNA fraction (6-7 S) used as template.
RNA-DEPENDENT
RNA
POLYMERASE
IN
IMMUNE
Fig. 3. Sedimentation velocity pattern of the 3Hlabeled product. Reaction mixture is shown in Materials and Methods. Twenty-five yg of iRNA (6-7 S) prepared in the experiment shown in Fig. 2 was used as a template.
Fig. 5. Effect of pH on enzyme. The series of pH 5.0 to 8.5 were prepared
Fig.
Fig.
4.
UTP
Time into
course
RNA
of
the
by purified
incorporation enzyme.
of
3H-
Two-tenth-ml
portions were withdrawn from the reaction mixture at various time intervals after incubation and the radioactivity in the acid-insoluble fraction was counted. The shown in Fig.
iRNA 3.
preparation
was
the
same
437
RESPONSE
the activity of purified values ranging from pH by 2 M of Tris—malate-
NaOH buffer instead of Tris-HC1 buffer. See Materials and Methods for assay conditions. The iRNA preparation was the same as shown in Fig. 3.
6.
Effect
of
temperature
on
the
activity
purified enzyme. See Materials and Methods assay conditions. The iRNA preparation was same as shown in Fig. 3.
of for the
as
Time Course of the Reaction
The incorporation of 3H-UTP continued linearly for at least 3 hr with iRNA as template (Fig. 4). Optimum pH and Temperature
Optimum pH of the reaction was determined in the pH range of 5 to 8.5 in 0.2 Tris—malate—NaOH buffer (Fig. 5). The highest activity was observed at pH 7.6. The optimum temperature of this reaction was 37 C and its activity decreased to less than 50% at 30 or 45 C (Fig. 6).
Fig. 7. Effect of divalent activity. See Materials condition preparation
except was
cations and
for ion the same
on purified enzyme Methods for assay
concentration. as shown
The in Fig.
iRNA 3.
438
The Divalent
K.
Cation
SAITO
AND S. MITSUHASHI
Requirement
The effect of divalent cations on the rate of RNA synthesis is shown in Fig. 7. MgC12 at its optimum concentration (5-10 mM) best satisfied the requirement for divalent cations, while less stimulation was seen at the optimum concentration (1.25 mM) of MnC12.
In
the
present
characterized lows:
1)
is
in
fraction
of
homogenate;
enzyme
2)
increased
There have been many reports concerning the RNA-dependent RNA polymerases on RNA viruses and RNA bacteriophages [1, 23] but only a few reports have been presented on mammalian cells : e.g., virusinduced leukemia cells [6, 24] and immunized lymphoid tissues [7, 17, 19, 20]. A cytoplasmic microsome-bound RNA-dependent RNA polymerase was also demonstrated in a rabbit reticulocyte lysate. The synthesis of RNA was absolutely dependent on the addition of RNA template and the best template was hemoglobin messenger RNA [3].
Previous studies in our laboratory have disclosed that passive transfers of iRNA were successful in establishing immunity in mice against infection with S. enteritidis [16] or immunity to Salmonellaflagella [11]. It was also found that iRNA was able to confer ability for secondary response of antibody formation on recipient cells [10-12, 14, 25]. This ability of iRNA was also serially and passively transmissible [11, 18, 21]. In addition, we reported the following results [20] ; 1) a crude extract(iEXT) obtained from the homogenate of immune spleen cells catalyzed the incorporation of 3H-UTP into acidinsoluble fraction when the iRNA preparation was used as a template; 2) the incorporation was abolished by treatment of iRNA with RNase but not with DNase; 3) two derivatives of rifamycin, which are known to be inhibitors of reverse transcriptase of oncogenic RNA viruses, inhibited the incorporation of 3H-UTP, while mitomycin C, bleomycin A2 and actinomycin D did not show any inhibitory effect; 4) four ribonucleoside triphosphates were required for this reaction; and 5) the product of the reaction resisted RNase treatment but was rendered sensitive after heat treatment. Thus the product RNA appeared to be in a hybrid form with the template RNA [20].
crude of
this
eluted the
was with
at
1.42
most to
iRNA
be
divalent and behavior
from of pH,
cation 6)
the of
microsomal
specific by
3)
the
of
Biogel
column
void
for
S RNA
this
be
volume; enzyme from followed
source;
5)
requirements
are gradient
3H-UTP-labeled
4) was
it was
this enzyme, i.e., optimum temperature
density
around activity
prepared
same
of
weight
to
the
the
that
molecular
polymerase
of
the
and spleen
with
the
fol-
activity
compared
preparation;
S RNA
as
immunized
template
12-13
further
polymerase
estimated
times
active
crude
properties optimum
4;
daltons,
found
7-6
No.
enzyme
6 •~ 105
the the
fortyfold as
fraction
have
polymerase RNA
localized
ribosomal
chromatography DISCUSSION
we
RNA
RNA-dependent
activity
cell
article
this
the by
various kinetics, and
described; centrifugal product
is
presented.
The results reported previously and those described in this article suggest that this enzyme is an RNA-dependent RNA polymerase and that the enzyme is induced or its activity is enhanced by antigenic stimulation in the spleen cells. This implies that the enzyme acts as an amplifier of antigenic stimulation in the processes of the antibody formation. The iRNA preparations of two different S values can serve as template for this enzyme. A larger iRNA fraction (12-13 S) corresponds in S value to the mRNA of immunoglobulin (light chain) which shows an S value of 9 to 13 [22]. A smaller iRNA fraction (6-7 S) may contain a portion of the immunoglobulin mRNA for the variable part of the immunoglobulin. Although we have no evidence at the moment, possible involvement of the iRNA (6-7 S) in the production of immunoglobulin should be the subject of future experiments. Gene amplification or the production of multiple gene copies was reported to occur in the nucleoli of amphibian oocytes of genes for ribosomal RNA [2, 4, 5], suggesting that these cells can synthesize large quantities of rRNA in a relatively short time. The RNA-dependent RNA polymerase in mammalian cells may offer an additional site for control of gene expression as well as providing
RNA-DEPENDENT
a mechanism
for
amplification
RNA
POLYMERASE
of the
ex-
pression of specific genes. Especially, the possible production of iRNA by means of RNA-directed RNA synthesis may offer a new clue in the study of antibody formation and may account for the transmission and amplification of antigenic information. REFERENCES
[1]
Baltimore, D. 1971. Expression of animal virus genomes. Bacteriol. Rev. 35: 235-241. [ 2 ] Brown, D., and David, I. B. 1968. Specific gene amplification in oocytes. Science 160: 272-280. [3]
Downey, K. M., Byrnes, J. J., Jurmark, B. S., and So, A. G. 1973. Reticulocyte RNA-dependent RNA polymerase. Nature 70: 3400-3403. [ 4 ] Evans, D., and Birnsteil, M. L. 1968. Localization of amplified ribosomal DNA in the oocyte of Xenopus laevis. Biochim. Biophys. Acta 166: 274276. [ 5 ] Gall, J. F. 1968. Differential synthesis of the genes for ribosomal RNA during amphibian oogenesis. Proc. Nat. Acad. Sci. U.S. 60: 553-560. [ 6 ] Haruna, I., Ohno, T., and Watanabe, I. 1970. Isolation of an RNA-dependent RNA polymerase from friend murine leukemia cells. Proc. Japan Acad. 46: 1016-1021. [ 7 ] Jacherts, D., Opits, U., and Opits, H. G. 1972. Gene amplification in cell-free systems. Zschr. Immunitatsforsch. 144: 260-272. [ 8 ] Kidson, C., Kirby, K.S., and Ralph, R.K. 1963. Isolation characteristics of rapidly labeled RNA from normal rat liver. J. Mol. Biol. 7: 313-315. 9 ] Kitamura, K., Kurashige, S., and Mitsuhashi, S. 1973. Dose effect of immune ribonucleic acid fraction on antibody formation. Japan. J. Microbiol. 17: 29-33. [10] Kurashige, S., Kitamura, K., Akama, K., and Mitsuhashi, S. 1970. Transfer agent of immunity. IV. Antibody formation against diphtheria toxin by an immune ribonucleic acid fraction. Japan. J. Microbiol. 14: 41-47. [11] Kurashige, S., and Mitsuhashi, S. 1972. Serial passive transfers of immune response by an immune ribonucleic acid preparation. J. Immunol. 108: 1034-1038. [12] Kurashige, S., Saito, K., Fukai, K., Kitamura, K., and Mitsuhashi, S. 1973. Proliferation of pyroninophilic cells in lymphoid tissues by stimula-
IN IMMUNE
RESPONSE
439
tion with immune ribonucleic acid. Japan. J. Microbiol. 17: 217-219. [13] Mach, B., and Vassalli, P. 1965. Biosynthesis of RNA in antibody-producing tissues. Proc. Nat. Acad. Sci. U.S. 54: 975-982. [14] Mitsuhashi, S., Kurashige, S., Kawakami, M., and Nojima, T. 1968. Transfer agent of immunity. I. Immune ribonucleic acid which induces antibody formation of Salmonella flagella. Japan. J. Microbiol. 12: 261-268. [15] Mitsuhashi, S., Saito, K., Kurashige, S., Osawa, N., and Kitamura, K. 1973. The role of RNA in cell-mediated immunity. Ann. New York Acad. Sci. 207: 380-388. [16] Mitsuhashi, S., Saito, K., Osawa, N., and Kurashige, S. 1967. Experimental slmonellosis. XI. Induction of cellular immunity and formation of antibody by transfer agent of mouse mononuclear phagocytes. J. Bacteriol. 94: 907-913. [17] Neuhoff, V., Schill, W. B., and Jacherts, D. 1970. Nachweiss einer RNA abhangigen RNAReplicase aus immunologisch kompeteten Zellen durch Micro-Disk-Electrophorese. Hoppe-Seyler's Zschr. Physiol. Chem. 351: 157-162. [18] Saito, K., Kurashige, S., and Mitsuhashi, S. 1969. Serial transfers of immunity through immune RNA. Japan. J. Microbiol. 13: 122-124. [19] Saito, K., and Mitsuhashi, S. 1972. Inhibitory effect of rifamycin derivatives on immunogenic RNA. J. Antibiotics 25: 477-479. [20] Saito, K., and Mitsuhashi, S. 1973. Ribonucleic acid-dependent ribonucleic acid replicase in the immune response. Japan. J. Microbiol. 17: 117121. [21] Saito, K., Osawa, N., and Mitsuhashi, S. 1971. Transfer agent of immunity. VI. Serial passive transfers of cellular immunity to Salmonella infection by immune ribonucleic acid. Japan. J. Microbiol. 15: 159-168. [22] Stewart, P. R., and Letham, L, D. (eds) 1973. The ribonucleic acids. Springer-Verlag, Berlin, Heidelberg, New York, p. 76. [23] Sugiyama, T., Korant, B. D., and LonbergHolm, K. K. 1972. RNA virus gene expression and its control. Ann. Microbiol. 26: 467-495. [24] Watanabe, I., and Haruna, I. 1969. Selfreplicating RNA in leukemic cells. Acta Haematol. Japon. 32: 593-602. [25] Yamaguchi, N., Kurashige, N., and Mitsuhashi, S. 1971. Antibody formation against Salmonella flagella by an immune ribonucleic acid fraction. J. Immunol. 107: 99-103.
