branes in lysates of FMDV-infected
baby hamster kidney cells (3, 4). This
report concerns the dissociation of the
active polymerase complex from these
structures, and its activity in a cellfree system.
Direct analyses of the reaction mixtures which contain the soluble polymerase have shown a_ heterogeneous
RNA-containing component (140 to
3005) and a 50S component containing
RNA, in addition to virus-specific
RNA (see 3). The 140 to 300S RNA
component is found before the appearance of 37S virus RNA.
The FMDV RNA polymerase was
prepared according to Polatnick and
Arlinghaus (3), It is known to be active in 0.5-percent deoxycholate (3).
However, the virus-specific RNA synthesized in the presence of deoxycho-
late was largely degraded by contami-
nating nucleases, yielding 20S RNA re-
sistant to ribonuclease, and 4 to 125
RNA fragments unless bentonite was
present. By use of this deoxycholatebentonite polymerase mixture, antibody
to an antigen associated with FMDV
infection
(5)
was
found
to
inhibit
FMDV-RNAsynthesis by about 90 per-
cent (6).
We used dextran sulfate-500 (7) to
inhibit rrbonuclease (8). Preliminary ex-
time at which cell-free synthesis has
stopped), showed that 90 percent of the
radioactive RNA insoluble in trichloroacetic acid was in the pellet; no significant peak was seen in the gradient.
The results were similar when the sample was centrifuged for only 2 hours.
It was also determined that active polymerase forms pellets under the same
conditions, These results indicate that
the FMDV polymerase, as well as its
attached RNA template, and its RNA
products, are membrane-bound in cell
lysates.
Direct sucrose-gradient analysis of reaction mixtures, containing polymerase
and both 0.25-percent deoxycholate and
dextran sulfate (140 ,g/ml) gave the
following results after 60 minutes at
37°C: The amount of *H-uridine
phosphate incorporated into RNA
soluble in trichloroacetic acid was
to 25 percent greater than in the
sence of deoxycholate and dextran sulfate. The RNA products were released
from the membrane, since 60 to 70
percent of the radioactive RNA insoluble in trichloroacetic acid was found
in the gradient. The optical-density profile showed peaks of 185 and 285
ribosomal RNA that originated from
ribosomal subparticles present in the
periments showed that dextran sulfate at 10 to 20 ug/ml caused some
30%
stimulation of incorporation of ?H-uri-
corporation. Addition of 0.25 percent
of deoxycholate to an intermediate
concentration of dextran sulfate (140
pg/ml) gave maximum incorporation.
The
sodium
dodecylsulfate-extracted
RNA products of the polymerase treated with deoxycholate-dextran sulfate
in the cell-free synthesizing system contained alf three virus-specific RNA’s
(3, 9): 37S virus RNA, 20S ribonu-
clease-resistant RNA,
geneous RNA.
and
a hetero-
Cell-free reaction mixtures were examined directly, without prior RNA extraction, by centrifugation on linear
sucrose gradients of from 5 to 25 percent in 0.01M tris(hydroxymethyl) aminomethane HCI (pH 7.5) and 0.001M
MgCl., (tris-MgCl.) for 17 hours at
25,000 rev/min in the SW-25.1 rotor.
The sucrose-gradient profile of the
polymerase reaction mixture, containing
neither deoxycholate nor dextran sulfate, after 60 minutes at 37°C (the
8 DECEMBER 1967
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component near the bottom of the tube,
which was not found after treatment
with sodium dodecylsulfate or pretreat-
ment with either 0.01M_ ethylenediaminetetraacetate or 0.04M pyrophos-
phate; a 37S zone containing infectious
virus RNA; and 20S RNA resistant
to ribonuclease. No attempt has been
made to demonstrate net synthesis of
37S infectious RNA in the cell-free
system.
A minor peak of 26 to 305
heterogeneous RNA was always present in the reaction mixture (peak D:
3, 9), but the amount varied with the
activity of the polymerase (that is, ac-
tivity presumably lost by denaturation
of the enzyme and not by ribonuclease).
The 50S and 375 zones were made
soluble in trichloroacetic acid by treatment with ribonuclease (10 »g/ml) for
30 minutes at 37°C in 0.15M KCl
and 0.1M tris-HCl, pH 7.0. Also, the
infectivity of the 37S zone was lost on
treatment with trace levels of ribonuclease. The 20S zone was almost completely resistant to ribonuclease, and
vitro
Sucrose
1600
dient (for example, Fig. 1B): a 50S
Fig, 1. Sucrose-gradient profiles of the iu
10%
sucrose
dine triphosphate in the cell-free
FMDV-polymerase system (Fig. 1 legend), whereas high concentrations (1
to 2 mg/ml) strongly inhibited in-
triin15
ab-
polymerase preparation. This deproteinizing of ribosomes is attributed to
the action of dextran sulfate (4). Three
major zones were detected in the gra-
chase
of whole-cell,
pulse-labeled,
virus-specific RNA. Baby hamster kidney
cells (6 x 10°) were infected as in text.
The soluble polymerase complex was isolated; it contained protein at 2.8 mg/ml
and 125,000 count/min mg” protein of
4C-uridine, as RNA insoluble in trichloroacetic acid, at 48-percent counting efficiency. The complete cell-free reaction mixture contained: 10 wmole of tris-HCI, pH
8.1 (23°C); 5 umole of phospho(enol)pyruvate; 20 y~g of pyruvate kinase; 25
mumole of each of adenosine triphosphate, cytidine triphosphate, uridine triphosphate, and guanosine triphosphate;
12.5 nmole of MgCl; 0.1 ml of polymerase; and water to a final volume of 0.7
ml. The mixture was incubated (see text).
Casein (300 ug} and 10 ml of 5-percent
trichloroacetic acid were added to each
gradient fraction. After 20 minutes at O0°C, the precipitate was collected on type-B6
membrane filters (25 mm in diameter; Schleicher and Schuell), and the filter was
washed five times with 5-percent trichloroacetic acid. Samples were counted in a liquid
scintillation spectrometer (9). (A) The cell-free reaction mixture was held at 0°C
for 60 minutes with 10 uc of *H-uridine triphosphate. The reaction mixture contained
100 wg of dextran sulfate, 0.05 percent deoxycholate, 0.1 ml of soluble polymerase
complex, and all components of the cell-free system in a volume of 0.7 ml. The
mixture was diluted to 2.2 mi with 0.01M tris-HC], pH 7.5, prior to layering on
the gradient. Two milliliters were applied, and the tube was centrifuged for 17 hours
at 20,000 rev/min on a 10- to 30-percent linear sucrose gradient in tris-MgCl. in the
SW-25.1 rotor. Carbon-14 at 35,344 count/min was applied to the gradient: 21,147
count/min was in the pellet; 12,336 count/min, in the gradient. (B) The reaction
mixture was the same as for (A), and the tube was incubated for 60 minutes at
37°C. chilled and treated as for (A). Carbon-14 at 32.634 count/min was applied
to the gradient: 5,862 count/min was in the pellet; 24,350 count/min, in the gradient.
1321