BIOCHEMICAL
Vol.64,No.2,1975
HISTIN,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AN RNA POLYMERASE INHIBITOR ISOLATED FROM HISTOPLASMA CAPSULATUM
David
George Boguslawski*, Gerald Medoff, Schlessinger, and George S. Kobayashi
Departments of Medicine and Microbiology Washington University School of Medicine 660 South Euclid, St. Louis, Missouri 63110
Received
March
21,1975 Abstract
The dimorphic fungus Histoplasma capsulatum contains an extractable inhibitor of RNA polymerase in its mycelial phase. The inhibitor, called column chromatography cannot histin, binds to the inhibited enzymes: restore polymerase activity to extracts prepared from a mixture of mycelial cells and yeast phase cells. Histin is specific in that it preferentially affects those RNA polymerases which are resistant to the toxin from Amanita phalloides, a-amanitin. This relative specificity for some RNA polymerases is also found when histin is tested against the enzymes from Xenopus laevis and Escherichia coli as well as from H. capsulatum.
Introduction The pathogenic a mycelial that
phase
begun.
cell
(1);
We found
that
the yeast
(2).
by a-amanitin,
* Present
may involve
of the
RNA polymerases columns
Histoplasma
at 23O to a yeast
the conversion
machinery
Copyright AN rights
fungus
which
whereas
address:
phase
the other
Department Lawrence,
o 19 75 by Academic Press, 1~. of reproducrim in aq form reserved.
of the
of its
by chromatography species
two were
625
shown
RNA polymerase had at least
(PC I) was inhibited
University
was
three
on phosphocellulose
resistant.
of Microbiology, Kansas 66045
from
RNA-synthesizing
of H. capsulatum
be separated
converts
We have previously
an analysis
phase
RNA polymerase
reversibly
at 37'.
a modification
therefore,
could
The major
capsulatum
of Kansas,
strongly
Vol. 64, No. 2, 1975
The findings merases
were
fungal
in
from
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
phase
prompted
the study
of H. capsulatum.
mycelial,
The mycelial
inhibitor
specificity
phase
prepared
be detected.
potent
the
in the yeast
of the mycelial
extracts could
BIOCHEMICAL
only
cells
of RNA polymerase its
action
against
system.
This
suggests
Here we report
trace
activity.
RNA polymerases role
for
the
that
when
of RNA polymerase
to contain
The inhibitor
a possible
Materials
amounts
have proved
various
of the RNA poly-
a novel exhibits
which
a marked
extends
beyond
inhibitor.
and Methods
Organism. -.
iI yeast phase cells of H. capsulatum @own's strain, mating type (-) from our laboratory] were grown as before (2) using an initial inoculum density of lo7 cells/ml. The maintenance and growth of the mycelial phase of the same strain is described elsewhere (Boguslawski et al., submitted for publication). Cells were harvested by filtration, washed with distilled water and extracts prepared as described (2). the
RNA polymerases. Ribonucleic acid polymerases from H. ca sulatum were prepared by phosphocellulose chromatoqraphy and assayed as ---+described 2). The nomenclature of RNA polymerases from H.-capsulatum was discussed in the same paper and is followed throughout this report. Xenopus laevis RNA polymerases IA, IIB, and III were isolated and partially purified by chromatography on DEAE-Sephadex and CM-Sephadex as previously described (3). Each polymerase was subsequently concentrated and further purified by chromatography on phosphocellulose (R.G. Roeder, personal communication). Escherichia coli RNA polymerase was purified through the phosphocellulose step as described (4). The RNA polymerases from X. laevis and from E. coli were kindly donated by Dr. R. G. Roeder of the Washington University School of Medicine, St. Louis, Missouri. Inhibitor assay. Inhibition was tested by pipeting aliquots of mycelial extracts into the RNA polymerase re ct'on mixture prior to enzyme addition and comparing the incorporation of If3H UMP into RNA with the incorporation obtained in the absence of the inhibitor. Total reaction volume was 0.125 ml. When crude were made for the residual preparations of the inhibitor were used, corrections RNA polymerase activity. Purified inhibitor preparations contained no polymerase activity. Both crude and purified preparations of the inhibitor were stable for months when stored at -200. Results RNA polymerase
activities
When the extract phosphocellulose (Fig.
1).
polymerase
from yeast from
column,
In contrast, activity,
the yeast it
yielded
mycelial
and mycelium. phase three
extracts
and in a single
peak.
626
cells well
was chromatographed separated
contained
only
It is important
on a
peaks of activity very
little
to note
RNA that
the
BIOCHEMICAL
Vol. 64, No. 2,1975
AND f3lOPHYSlCAL RESEARCH COMMUNICATIONS
PC III
Figure
yeast
1.
Phosphocellulose chromatography of RNA polymerase from the yeast and mycelial phase of H. capsulatum. Slightly purified extracts of yeast (63 rng protein, panel A) and mycelium (83 mg protein, panel B) were prepared, chromatographed and assayed as described in reference 2. Column bed volumes were 20 ml and 16 ml, respectively, The RNA polymerase activity of yeast fractions was assayed using native calf-thymus DNA (u). The activity of mycelial fractions was assayed with denatured DNA (M). (NH4)2S04 concentration (------).
enzymes
merase
were
was assayed
assayed with
with
native
denatured
DNA was used as a template
for
DNA, whereas
DNA, a more active the mycelial
enzyme,
the mycelial
RNA poly-
template.
When native
activity
was barely
detectable. The two chromatographic separate
column
gradient, enzymes;
runs.
the mycelial it
elutes
patterns
On the
basis
polymerase
between
shown in Figure of the position
does not
the yeast
phase
627
correspond enzymes
1 represent of elution
two in the
salt
to any of the yeast PC II
and PC III.
Vol.64,No.2,
1975
BIOCHEMICAL
The chromatographic us that
mycelium
gestion
was supported
yeast
phase
could
cells
activities
profile
extracted
prepared
from
and the
residual
activity
(data
single with the
peak of activity the mycelial inhibitory
be removed
by column
showed
almost
of RNA The
no activity In contrast,
about
80% of the
from mixed
cells,
when mixed (not
exactly
cell
shown),
to the
observation
only
a
peak obtained
suggested
to RNA polymerase
total
that
and could
not
crude
and mixed
of RNA polymerase
extract.
From these
of RNA polymerase
cell
which
results
extracts did
not
probably undergo
we inferred
in mycelial
extracts
the and have
"histin". of inhibition
The three sensitivity
yeast
phase
to the crude
were
differences
levels,
enzymes inhibitor
PC I,
all
enzymes
PC II,
three
and PC III
were
tested
for
As can be seen in Fig.
preparation.
among the enzymes
Although
concentrations. high
activity.
extract
This
bound
levels
little
column
in mycelial
proportion
of an inhibitor
Specificity
very
high
In addition,
1).
was strongly
peak of activity
in the
it
activity.
(Fig.
and RNA polymerase
constitutes in the
and
chromatography.
inactivation
there
alone
substance
the minute
called
activity
to
sug-
mycelial
to a-amanitin.
was seen , corresponding
represented
presence
resistant
on phosphocellulose
extract
The single
also
in yeast
the mycelial
was chromatographed
had very
This
in which
contained
of cells
activity
The residual
resembles
extract
extract
suggested
of RNA polymerase.
phase extract
mixture
extract
or separately
was totally
sensitive
not shown).
therefore,
a 1:l
mycelial
of an experiment jointly
the mycelial
extract
the a-amanitin
result
The yeast
whereas
with
an inhibitor
by the
compared.
polymerase,
obtained
contain
were
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in response
polymerases
PC II
and PC III
were
in Figure
2 were
extended
were
to increasing totally
2,
histin
inhibited
much more sensitive
at than
was PC I. The findings systems,
using
shown
more purified
preparations
628
of the
to other inhibitor
RNA polymerase (Table
I).
RNA
BIOCHEMICAL
Vol. 64, No. 2, 1975
AND BIOPHYSICAL
0 0
60
40
Crude
Figure
2.
polymerase
from
X. laevis
also
includes
E. coli embryos
data
for
When a-amanitin
not
affected
of histin
(Table whereas,
was strongly
160
ug
inhibited
showed differential enzymes
from
was employed
PC I was inhibited
histin
I20
inhibitor,
Differential inhibition of yeast RNA polymerases by a crude histin preparation. Aliquots of yeast enzyme fractions were tested against increasing amounts of histin using native DNA as template. (O--0 ) PC II; (0-i; ) PC I; (a---A ) PC III. Maximal activity (100%) represents 561 cpm, 460 cpm, and 1,582 cpm, respectively.
from
were
mycelial
RESEARCH COMMUNICATIONS
by histin, sensitivity
the yeast
instead
even at 64 ug/ml.
These
in agreement
E. coli with
H. capsulatum
whereas
results
(6),
it
enzyme
with
was strongly
data
I
PC II and PC III
contrast
polymerase
published
Table
of H. capsulatum.
of histin,
of a-amanitin,
Moreover,
to histin.
phase
50% by 3 ug/ml
I).
and RNA polymerases
the action inhibited
was totally
by
resistant
to a-amanitin. Discussion Although merase
many naturally
have been described
has been extensively
occurring (5-lo),
used for
and synthetic
a-amanitin
the characterization
629
is
inhibitors
the most widely of transcription
of RNA polystudied, in cells
and
Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Effect
Organism
of of
purified* mycelial inhibitor RNA polymerase from various
RNA Polymerase
H. capsulatum ,ast phase
I
Amount Inhibitor
on the activity organisms.
of (ug)
Countsimin.
PC I
898 886 828 705 5197 1409 661 376 1227 117 67
0.45 1.80 18.00 PC II 0.45 1.80 18.00 PC III 3.20 16.00
--X. laevis embryo
IA
33184 16659 3553 3389 1012 1335 1439 7166 2498 1889 1748 21300 6045 4638 3025
O.i5 4.50 18.00 =B 1.80 4.50 III 1.80 4.50 18.00 E. coli -__
*The
COlX enzyme
inhibitor submitted
for
and extracts
(6,
a-amanitin,
sites,
E. coli
polymerase
I,
II,
and III
identical cytoma (the
is
suggest
is
inhibited
(R.G.
for that
inhibited
per ml,
and histin
I enzyme)
polymerase
IIB
complementary
probably
of RNA polymerase. by histin,
whereas
and only X. laevis
for
the
weakly showed
analogous
(the
major
630
H. capsulatum by histin.
Thus,
II
at
For example,
enzymes
insensitive Class
act
PC I
The Class
a-amanitin
communication). was totally
et al,
of activity
a-amanitin
from
personal
(Boguslawski
of transcription.
by a-amanitin
Roeder,
100.0 50.2 10.7 10.2 100.0 131.9 142.2 100.0 34.9 26.4 24.4 100.0 28.4 21.8 14.2
studies
only
(16)
treatment
a spectrum
subunits,
reported
Class
by heat
with
RNA polymerases
predominant
a-amanitin
Histin,
or different
to those cells
12-fold
be useful
results
different
polymerase
about
11-15).
may also
Several
0.90 1.80 4.50
was purified publication).
Percent Activity Remaining 100.0 98.7 92.5 78.5 100.0 27.1 12.7 7.2 100.0 9.5 5.5
sensitivities
from
mouse plasma-
polymerase
IA
to one mg of enzyme)
was 50% inhi-
to
BIOCHEMICAL
Vol. 64, No. 2, 1975
bited
by 0.004
inhibited
ug/ml
sensitivity
to w-amanitin
Whether
histin
histin
the cell
there
organisms
undergoing
fications
reported the
a small, the future
and histin
is
in whole
life
is
heat
stable,
should
cells
is
whether
no clear
et al., acidic
permit
phase,
during
at low
levels
between
the
not absolute.
or that
inhibitors
its
level
sporulation
there
in
phase
function
to another,
of the
It is possible
unknown.
to the yeast
can
in are modi-
of Bacilli
(17)
and will
report
evidence
that
and
19).
and characterize submitted
a clearer
was
RNA polymerases
a transition
(18,
protein.
though
at present
from one phase
to purify
correlation
against
case in which
of some fungi
(Boguslawski
strong,
effect
in RNA polymerases cycle
was most effective
The inverse thus
III
and polymerase
on the mycelial
transitions
We have continued elsewhere
I).
somehow determine
Although
during
(Table
may have no effect might
occur.
phases
toxin,
histin
has any inhibitory
or mycelial
that
of the Instead,
IA and III
polymerase
yeast
concentration
by 50% at 20 ug/ml.
against
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for
histin, publication)
The availability determination
of its
of purified cellular
it
is
material location
in and
function.
Acknowledgments We are grateful polymerases from 5. This work was training grants AI Hartford Foundation George Boguslawski AI 00284.
to Dr. R. G. Roeder for generous gifts of RNA laevis and g. coli and for helpful advice. supported by USPHS grants AI 10622 and AI 06213; 00459 and AM 05611 and grants from the John A. and the Research Corporation (Brown-Hazen Fund). was supported by Special N.I.H. Fellowship lF22-
References 1. 2. 3. 4. 5. 6. 7. 8.
Cheung, S. -S. C., Kobayashi, G. S., Schlessinger, D. and Medoff, G., (1974). J. of Gen. Microbial. 82, 301-307. Boguslawski, G., Schlessinger, D., Medoff, G., and Kobayashi, G. S., (1974). J. Bacterial. 118, 480-485. Roeder, R. G. (1974). J. Biol. Chem. 249, 241-248. Burgess, R. R. (1969). J. Biol. Chem. 244, 6160-6167. Lindell. T. J.. Weinberg. F.. Morris. P. W.. Roeder. I R. G. and Rutter, W. J., (1970): Science-i70,.447-4491 1 Wehrli, W. and Staehelin, M., (1970). Bact. Rev. 35, 290-309. Cano, F. R., KUO, S. -C. and Lampen, J. O., (1973). Antimicrob. Ag. Chemother. 3, 723-728. Arens, M. Q. and Stout, E. R., (1974). Biochim. Biophys. Acta 353, 12 l- 131
631
Vol. 64, No. 2,1975
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hasselbach, B. A., Yamada, T. and Nakada, D., (1974). Nature 252, 71-74. Wieland, T. and Wieland, O., (1972). In. Kadis, S., Ciegler, A., and Ajl, S. J. (eds.). Microbial Toxins. cl. VIII, pp. 249-280. Acad. Press, N.Y. and London. Weinmann, R. and Roeder, R. G., (1974). Proc. Natl. Acad. Sci. 71, 1790-1794. Price, R. and Penman, S., (1972). J. Mol. Biol. 70, 435-450. Reeder, R. H. and Roeder, R. G., (1972). J. Mol. Biol. 67, 433-441. Weinmann, R., Raskas, H. H. and Roeder, R. G., (1974). Proc. Natl. Acad. of Sci. 71, 3426-3430. Price, R. and Penman, S., (1972). J. Virol. 9, 621-626. Schwartz, L. B., Sklar, V. E. F., Jaehning, J. A., Weinmann, R. and Roeder, R. G., (1974). J. Biol. Chem. 249, 5889-5897. Leighton, T. J., Doi, R. H., Warren, R. A. J. and Kellin, R. A., (1973). J. Mol. Biol. 76, 102-122. Gong, C. -S. and Van Etten, Biochim. Biophys. Acta 272, J. L., (1972). 44-52. Sebastian, J., Takano, I. and Halvorson, H. O., (1974). Proc. Natl. Acad. Sci. 71, 769-773.
632
Vol.64,No.2,1975
HISTIN,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AN RNA POLYMERASE INHIBITOR ISOLATED FROM HISTOPLASMA CAPSULATUM
David
George Boguslawski*, Gerald Medoff, Schlessinger, and George S. Kobayashi
Departments of Medicine and Microbiology Washington University School of Medicine 660 South Euclid, St. Louis, Missouri 63110
Received
March
21,1975 Abstract
The dimorphic fungus Histoplasma capsulatum contains an extractable inhibitor of RNA polymerase in its mycelial phase. The inhibitor, called column chromatography cannot histin, binds to the inhibited enzymes: restore polymerase activity to extracts prepared from a mixture of mycelial cells and yeast phase cells. Histin is specific in that it preferentially affects those RNA polymerases which are resistant to the toxin from Amanita phalloides, a-amanitin. This relative specificity for some RNA polymerases is also found when histin is tested against the enzymes from Xenopus laevis and Escherichia coli as well as from H. capsulatum.
Introduction The pathogenic a mycelial that
phase
begun.
cell
(1);
We found
that
the yeast
(2).
by a-amanitin,
* Present
may involve
of the
RNA polymerases columns
Histoplasma
at 23O to a yeast
the conversion
machinery
Copyright AN rights
fungus
which
whereas
address:
phase
the other
Department Lawrence,
o 19 75 by Academic Press, 1~. of reproducrim in aq form reserved.
of the
of its
by chromatography species
two were
625
shown
RNA polymerase had at least
(PC I) was inhibited
University
was
three
on phosphocellulose
resistant.
of Microbiology, Kansas 66045
from
RNA-synthesizing
of H. capsulatum
be separated
converts
We have previously
an analysis
phase
RNA polymerase
reversibly
at 37'.
a modification
therefore,
could
The major
capsulatum
of Kansas,
strongly
Vol. 64, No. 2, 1975
The findings merases
were
fungal
in
from
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
phase
prompted
the study
of H. capsulatum.
mycelial,
The mycelial
inhibitor
specificity
phase
prepared
be detected.
potent
the
in the yeast
of the mycelial
extracts could
BIOCHEMICAL
only
cells
of RNA polymerase its
action
against
system.
This
suggests
Here we report
trace
activity.
RNA polymerases role
for
the
that
when
of RNA polymerase
to contain
The inhibitor
a possible
Materials
amounts
have proved
various
of the RNA poly-
a novel exhibits
which
a marked
extends
beyond
inhibitor.
and Methods
Organism. -.
iI yeast phase cells of H. capsulatum @own's strain, mating type (-) from our laboratory] were grown as before (2) using an initial inoculum density of lo7 cells/ml. The maintenance and growth of the mycelial phase of the same strain is described elsewhere (Boguslawski et al., submitted for publication). Cells were harvested by filtration, washed with distilled water and extracts prepared as described (2). the
RNA polymerases. Ribonucleic acid polymerases from H. ca sulatum were prepared by phosphocellulose chromatoqraphy and assayed as ---+described 2). The nomenclature of RNA polymerases from H.-capsulatum was discussed in the same paper and is followed throughout this report. Xenopus laevis RNA polymerases IA, IIB, and III were isolated and partially purified by chromatography on DEAE-Sephadex and CM-Sephadex as previously described (3). Each polymerase was subsequently concentrated and further purified by chromatography on phosphocellulose (R.G. Roeder, personal communication). Escherichia coli RNA polymerase was purified through the phosphocellulose step as described (4). The RNA polymerases from X. laevis and from E. coli were kindly donated by Dr. R. G. Roeder of the Washington University School of Medicine, St. Louis, Missouri. Inhibitor assay. Inhibition was tested by pipeting aliquots of mycelial extracts into the RNA polymerase re ct'on mixture prior to enzyme addition and comparing the incorporation of If3H UMP into RNA with the incorporation obtained in the absence of the inhibitor. Total reaction volume was 0.125 ml. When crude were made for the residual preparations of the inhibitor were used, corrections RNA polymerase activity. Purified inhibitor preparations contained no polymerase activity. Both crude and purified preparations of the inhibitor were stable for months when stored at -200. Results RNA polymerase
activities
When the extract phosphocellulose (Fig.
1).
polymerase
from yeast from
column,
In contrast, activity,
the yeast it
yielded
mycelial
and mycelium. phase three
extracts
and in a single
peak.
626
cells well
was chromatographed separated
contained
only
It is important
on a
peaks of activity very
little
to note
RNA that
the
BIOCHEMICAL
Vol. 64, No. 2,1975
AND f3lOPHYSlCAL RESEARCH COMMUNICATIONS
PC III
Figure
yeast
1.
Phosphocellulose chromatography of RNA polymerase from the yeast and mycelial phase of H. capsulatum. Slightly purified extracts of yeast (63 rng protein, panel A) and mycelium (83 mg protein, panel B) were prepared, chromatographed and assayed as described in reference 2. Column bed volumes were 20 ml and 16 ml, respectively, The RNA polymerase activity of yeast fractions was assayed using native calf-thymus DNA (u). The activity of mycelial fractions was assayed with denatured DNA (M). (NH4)2S04 concentration (------).
enzymes
merase
were
was assayed
assayed with
with
native
denatured
DNA was used as a template
for
DNA, whereas
DNA, a more active the mycelial
enzyme,
the mycelial
RNA poly-
template.
When native
activity
was barely
detectable. The two chromatographic separate
column
gradient, enzymes;
runs.
the mycelial it
elutes
patterns
On the
basis
polymerase
between
shown in Figure of the position
does not
the yeast
phase
627
correspond enzymes
1 represent of elution
two in the
salt
to any of the yeast PC II
and PC III.
Vol.64,No.2,
1975
BIOCHEMICAL
The chromatographic us that
mycelium
gestion
was supported
yeast
phase
could
cells
activities
profile
extracted
prepared
from
and the
residual
activity
(data
single with the
peak of activity the mycelial inhibitory
be removed
by column
showed
almost
of RNA The
no activity In contrast,
about
80% of the
from mixed
cells,
when mixed (not
exactly
cell
shown),
to the
observation
only
a
peak obtained
suggested
to RNA polymerase
total
that
and could
not
crude
and mixed
of RNA polymerase
extract.
From these
of RNA polymerase
cell
which
results
extracts did
not
probably undergo
we inferred
in mycelial
extracts
the and have
"histin". of inhibition
The three sensitivity
yeast
phase
to the crude
were
differences
levels,
enzymes inhibitor
PC I,
all
enzymes
PC II,
three
and PC III
were
tested
for
As can be seen in Fig.
preparation.
among the enzymes
Although
concentrations. high
activity.
extract
This
bound
levels
little
column
in mycelial
proportion
of an inhibitor
Specificity
very
high
In addition,
1).
was strongly
peak of activity
in the
it
activity.
(Fig.
and RNA polymerase
constitutes in the
and
chromatography.
inactivation
there
alone
substance
the minute
called
activity
to
sug-
mycelial
to a-amanitin.
was seen , corresponding
represented
presence
resistant
on phosphocellulose
extract
The single
also
in yeast
the mycelial
was chromatographed
had very
This
in which
contained
of cells
activity
The residual
resembles
extract
extract
suggested
of RNA polymerase.
phase extract
mixture
extract
or separately
was totally
sensitive
not shown).
therefore,
a 1:l
mycelial
of an experiment jointly
the mycelial
extract
the a-amanitin
result
The yeast
whereas
with
an inhibitor
by the
compared.
polymerase,
obtained
contain
were
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in response
polymerases
PC II
and PC III
were
in Figure
2 were
extended
were
to increasing totally
2,
histin
inhibited
much more sensitive
at than
was PC I. The findings systems,
using
shown
more purified
preparations
628
of the
to other inhibitor
RNA polymerase (Table
I).
RNA
BIOCHEMICAL
Vol. 64, No. 2, 1975
AND BIOPHYSICAL
0 0
60
40
Crude
Figure
2.
polymerase
from
X. laevis
also
includes
E. coli embryos
data
for
When a-amanitin
not
affected
of histin
(Table whereas,
was strongly
160
ug
inhibited
showed differential enzymes
from
was employed
PC I was inhibited
histin
I20
inhibitor,
Differential inhibition of yeast RNA polymerases by a crude histin preparation. Aliquots of yeast enzyme fractions were tested against increasing amounts of histin using native DNA as template. (O--0 ) PC II; (0-i; ) PC I; (a---A ) PC III. Maximal activity (100%) represents 561 cpm, 460 cpm, and 1,582 cpm, respectively.
from
were
mycelial
RESEARCH COMMUNICATIONS
by histin, sensitivity
the yeast
instead
even at 64 ug/ml.
These
in agreement
E. coli with
H. capsulatum
whereas
results
(6),
it
enzyme
with
was strongly
data
I
PC II and PC III
contrast
polymerase
published
Table
of H. capsulatum.
of histin,
of a-amanitin,
Moreover,
to histin.
phase
50% by 3 ug/ml
I).
and RNA polymerases
the action inhibited
was totally
by
resistant
to a-amanitin. Discussion Although merase
many naturally
have been described
has been extensively
occurring (5-lo),
used for
and synthetic
a-amanitin
the characterization
629
is
inhibitors
the most widely of transcription
of RNA polystudied, in cells
and
Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Effect
Organism
of of
purified* mycelial inhibitor RNA polymerase from various
RNA Polymerase
H. capsulatum ,ast phase
I
Amount Inhibitor
on the activity organisms.
of (ug)
Countsimin.
PC I
898 886 828 705 5197 1409 661 376 1227 117 67
0.45 1.80 18.00 PC II 0.45 1.80 18.00 PC III 3.20 16.00
--X. laevis embryo
IA
33184 16659 3553 3389 1012 1335 1439 7166 2498 1889 1748 21300 6045 4638 3025
O.i5 4.50 18.00 =B 1.80 4.50 III 1.80 4.50 18.00 E. coli -__
*The
COlX enzyme
inhibitor submitted
for
and extracts
(6,
a-amanitin,
sites,
E. coli
polymerase
I,
II,
and III
identical cytoma (the
is
suggest
is
inhibited
(R.G.
for that
inhibited
per ml,
and histin
I enzyme)
polymerase
IIB
complementary
probably
of RNA polymerase. by histin,
whereas
and only X. laevis
for
the
weakly showed
analogous
(the
major
630
H. capsulatum by histin.
Thus,
II
at
For example,
enzymes
insensitive Class
act
PC I
The Class
a-amanitin
communication). was totally
et al,
of activity
a-amanitin
from
personal
(Boguslawski
of transcription.
by a-amanitin
Roeder,
100.0 50.2 10.7 10.2 100.0 131.9 142.2 100.0 34.9 26.4 24.4 100.0 28.4 21.8 14.2
studies
only
(16)
treatment
a spectrum
subunits,
reported
Class
by heat
with
RNA polymerases
predominant
a-amanitin
Histin,
or different
to those cells
12-fold
be useful
results
different
polymerase
about
11-15).
may also
Several
0.90 1.80 4.50
was purified publication).
Percent Activity Remaining 100.0 98.7 92.5 78.5 100.0 27.1 12.7 7.2 100.0 9.5 5.5
sensitivities
from
mouse plasma-
polymerase
IA
to one mg of enzyme)
was 50% inhi-
to
BIOCHEMICAL
Vol. 64, No. 2, 1975
bited
by 0.004
inhibited
ug/ml
sensitivity
to w-amanitin
Whether
histin
histin
the cell
there
organisms
undergoing
fications
reported the
a small, the future
and histin
is
in whole
life
is
heat
stable,
should
cells
is
whether
no clear
et al., acidic
permit
phase,
during
at low
levels
between
the
not absolute.
or that
inhibitors
its
level
sporulation
there
in
phase
function
to another,
of the
It is possible
unknown.
to the yeast
can
in are modi-
of Bacilli
(17)
and will
report
evidence
that
and
19).
and characterize submitted
a clearer
was
RNA polymerases
a transition
(18,
protein.
though
at present
from one phase
to purify
correlation
against
case in which
of some fungi
(Boguslawski
strong,
effect
in RNA polymerases cycle
was most effective
The inverse thus
III
and polymerase
on the mycelial
transitions
We have continued elsewhere
I).
somehow determine
Although
during
(Table
may have no effect might
occur.
phases
toxin,
histin
has any inhibitory
or mycelial
that
of the Instead,
IA and III
polymerase
yeast
concentration
by 50% at 20 ug/ml.
against
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for
histin, publication)
The availability determination
of its
of purified cellular
it
is
material location
in and
function.
Acknowledgments We are grateful polymerases from 5. This work was training grants AI Hartford Foundation George Boguslawski AI 00284.
to Dr. R. G. Roeder for generous gifts of RNA laevis and g. coli and for helpful advice. supported by USPHS grants AI 10622 and AI 06213; 00459 and AM 05611 and grants from the John A. and the Research Corporation (Brown-Hazen Fund). was supported by Special N.I.H. Fellowship lF22-
References 1. 2. 3. 4. 5. 6. 7. 8.
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