It was of interest to examine the
RNA associated with the polymerase
preparation; one could then discriminate between free. unbound, virus-specific RNA and active protein-RNA
complexes (polymerase-template). The
latter is the structural complex that
replicates virus RNA. The polymerase
was harvested, 3.5 hours after infection, from FMDV-infected baby ham-
ster
kidney
cells
pulse-labeled
with
14C-uridine under such conditions that
only virus-specific RNA was labeled.
Actinomycin D (5 g/ml) was added
30 minutes before harvest, and 20 yc
polymerase complex was
nent is not found after extraction with
sodium dodecylsulfate; after treatment
The soluble polymerase complex la-
minutes at 37°C; or in reaction mixture incubated for 60 minutes. In all
the
soluble
stable for long periods at —60°C, even
after repeated freezing and thawing.
beled with '4C-uridine was incubated
50%
sucrose
1405
+
805
tube was kept at O°C; another was
and FMDV polymerase) appears to be
incubated for 1 hour at 37°C. The
reaction mixture was analyzed directly
+
700
>
6000
3
&
=
5
soo
400
Us
>
«>
2
2
5
oo
35
E
2
=
200
°o
=
a
a
foo
o
5
Fig. 2. Sucrose-gradient profile of the 140
to 3005S RNA containing component of
the soluble polymerase complex. The soluble polymerase complex (0.9 mg) was
incubated in 2.1 mi of the complete cell-
free reaction mixture (Fig. 1) for 5 minutes
at 37°C, with 30 uc of *H-uridine triphos-
phate. The solution was rapidly chilled
and adjusted to 0.1M tris-HCl, pH 8.1
(0°C). After addition of ammonium sul-
fate to 50 percent saturation and standing
for 20 minutes at 0°C, the suspension was
centrifuged
for
15
minutes
at
10,000.
The precipitate was suspended in 2.2 ml
of tris-MgCl,, and 2 ml was layered on
a 10- to 50-percent linear sucrose gradient
in tris-MgCl». The gradient was centrifuged
for
17
hours at
15,000 rev/min in the
SW-25.1 rotor. Tritium at 18,375 count/
min was applied to the gradient; 4032
count/min was in the pellet; 12,662 count/
min. in the gradient. Samples were pre-
cipitated
Fig. 1,
with
trichloroacetic
acid
as
in
identical with the heterogeneous RNA
(peak D; 3, 9). Difficulties in demon-
stration of the 140 to 3005S component
have been encountered because of ag-
min,
gregation in solutions containing magnesium. It should be noted that intact
ribosomesare not detected in the soluble
The
whole-cell
labeled with
material,
pulse-
1!4C-uridine, in soluble
polymerase complex, contained both
375 and 20S virus-specific RNA, and a
small amount of the 50S component
in the pellet (S-
Tate exceeding 100).
After incubation in the cell-free system at 37°C, nearly all thé whole-cell
RNA labeled with ™“C-uridine was
found in the gradient in the three ma-
jor zones (Fig. 1B). This test consti-
tuted a true chase experiment, since total counts per minute of “C in both
the zero-time and the incubated sample
were identical; only the distribution of
radioactivity varied. This fact suggests
that the pellet material (exceeding
1005) is a precursor to the 50S, 37S,
Tube Number
300S complex (presumably of RNA
(no RNA extraction) on a 10- to 30-
percent linear gradient at 20,000 rev/
activity was found
800
three instances all the RNA product
occurred only at the top of the gradient.
The RNA contained in the 140 to
%H-uridine triphosphate—specific activity, 400 »c/ymole (Fig. 1 legend). One
ever, 65 percent of the 44C_ radio-
10%
sucrose
with ribonuclease at 1 pg/ml for 5
in the complete cell-free system with
appeared to be present (Fig. 1A). How-
0.4
3005, and another is at the top of the
tube. The 140 to 3005 RNA compo-
and 20S components, Moreover, nearly
all the polymerase complexes active
in the whole cell (labeled with MCuridine) are active im the cell-free
system. The product of the cell-free
synthesis of RNA @H-RNA) gave an
identical profile, and 85 percent of the
*7H-RNA was found in the gradient
(Fig. 1B).
Since the polymerase complex incorporates *H—uridine triphosphate into
RNA for only 60 minutes at 37°C
in the cell-free system, analysis of products formed early (after 5 to 10 minutes) should identify precursors to 375
virus RNA. Therefore a soluble polymerase complex was incubated for 5
minutes in the cell-free system containing dextran sulfate at 140 pg/ml. The
reaction mixture was rapidly chilled to
polymerase complex (Fig. 2). The
methods of both isolation and storage
of the soluble polymerase complex in
dextran sulfate at 1 mg/ml and 0.5percent deoxycholate destroy ribosomes
and polyribosomes.
These results suggest that the 140 to
3005S RNA containing component is a
precursor to FMDV 375 RNA.Studies
with poliovirus in intact HeLa cells
indicated that synthesis of poliovirus
RNA takes place in complex structures possessing an average S-rate value
of 250 (2).
RALPH B. ARLINGHAUS
JEROME POLATNICK
Plum Island Animal Disease
Laboratory,
U.S. Department of Agriculture,
Greenport, New York 11944
References and Notes
1. L. Daigarno, E. M. Martin, 5S. L. Liv, T.
S. Work, J. Mol, Biol. 15, 77 (1966); D.
Baltimore and R. M. Franklin, J. Biol.
Chem, 238, 3395 (1963).
- M, Gerard, D. Baltimore, J. F. Darnell, J.
PN
(4).
Analysis of the precipitate material
‘by zonal centrifugation gave the profile
shown in Fig. 2. A peak of radioactivity is found ranging from 140 to
Mol. Biol. 24,
59
(1967).
. J, Polatnick and R. B. Arlinghaus, Virology
31, 601 (1967).
R. B. Arlinghaus and J. Polatnick, in preparation.
K. M. Cowan and J. H. Graves, Virology
30, 528 (1966).
. J. Polatnick, R. B. Arlinghaus, J. H. Graves,
K. M. Cowan, ibid. 31, 609 (1967).
From Pharmacia Fine Chemicals, Inc,
L. Katz and S. Penman, Biochem. Biophys.
Res, Commun, 23, 557 (1966).
. R. B. Arlinghaus, J. Polatnick, G. F. Vande
Woude, Virelogy 30, 541 (1966).
10, J. Polatnick and R. B. Arlinghaus, J, Virol.,
in press.
11, We thank Barbara Montgomery for technical
assistance.
wR we
overlaps the 20S double-stranded RNA
PH 8.1.
A
partially sensitive to ribonuclease. and
reduction in production of polymerase
(10). The enzyme was prepared as
usual (3) and made 1 mg/mlin dextran
sulfate and 0.5 percent in deoxycholate
before storage at —60°C; this preparation will be referred to as the “soluble polymerase complex.” Activity of
0°C and adjusted to 50 percent of
saturation with ammonium sulfate at
oa
tained larger amounts of the above-
mentioned heterogeneous RNA. This
heterogeneous RNA (peak D; 3, 9) is
of 4C-uridine was added 15 minutes
before harvest (9). This schedule of
addition of actinomycin D caused no
@
most likely is double-stranded RNA.
It has been reported that the 205 zone
is only partially resistant to ribonuclease
(3, 9); however. such results were obtained from RNA, extracted with sodium dodecylsulfate and phenol, that con-
20 October 1967