Published February 1, 1961
A COMPARATIVE
STUDY
OF RNA
D.
DANON,
ELECTRON
FROM
M.D.,
MICROSCOPICAL
DIFFERENT
Y.
MARIKOVSKY,
SOURCES
and
U.
Z.
LITTAUER,
Ph.D.
From tile Weizmann Institute of Science, Rehovoth, Israel
ABSTRACT
In a previous communication electron micrographs
of E. coli r i b o s o m a l R N A w e r e p r e s e n t e d (1). T h e
R N A fibers were d e s c r i b e d as h a v i n g a d i a m e t e r
of a b o u t 10 A a n d a v a r i a b l e l e n g t h of 1500 to
4000 A. T h e s e fibers d e m o n s t r a t e d a t e n d e n c y to
coil u p o n t h e m s e l v e s a n d a g g r e g a t e in t h e prese n c e of salt. I n view of t h e fact t h a t R N A fibers
f r o m o t h e r s o u r c e s were d e s c r i b e d as h a v i n g a
l a r g e r d i a m e t e r (2, 3), a c o m p a r a t i v e s t u d y of
R N A f r o m five d i f f e r e n t s o u r c e s w a s u n d e r t a k e n .
MATERIALS
AND
METHODS
E. coli R N A was prepared as previously descrihed (4).
R N A from Bacillus cereus 5 6 9 / H was p r e p a r e d by a
slight modification of the p r o c e d u r e used for the isolation of E. coli R N A . Cells (10 gm.) were frozen with
liquid air a n d g r o u n d u n d e r liquid air in a m o r t a r ;
the crushed cells were vigorously stirred with 80 ml.
of p h e n o l - w a t e r mixture, (1 v o l u m e of Tris buffer
(0.01 M, p H 7.4) containing E D T A (10 -4 M) a n d
1 v o l u m e of 90 per cent phenol solution). T h e suspension was h o m o g e n i z e d in a n all glass h o m o g e n i z e r
a n d stirred at 20 ° for 60 minutes. T h e h o m o g e n a t e
was chilled a n d centrifuged for 3 m i n u t e s at 10,000 g
at 2 ° . T h e a q u e o u s phase was r e m o v e d a n d allowed
to stand. T h e phenol phase was m i x e d with 40 ml.
T r i s - E D T A buffer and, after 5 m i n u t e s in the cold,
the a q u e o u s phase was separated by centrifugation.
T h e a q u e o u s s u p e r n a t a n t solutions containing the
R N A were c o m b i n e d a n d centrifuged for 2(I m i n u t e s
at 10,000 g. T h e R N A solutions were precipitated
from the s u p e r n a t a n t by addition of 2 volumes of cold
96 per cent ethanol, containing 2 per cent K - a c e t a t e
(4), washed with 75 per cent ethanol, dialyzed against
10 3 M s o d i u m chloride solution a n d t h e n lyophilized.
Microsomal R N A from calf, rat, a n d chick liver
was extracted from the microsomal fraction with a
phenol-water m i x t u r e a n d precipitated with NaCI
using a modification of a previously described m e t h o d
(8).
Livers were h c m o g e n i z e d in 0.25 M sucrose solution containing 0.005 M MgCI2 a n d 0.131 M potassium
p h o s p h a t e buffer p H 6.8 a n d centrifuged for 10 m i n utes at 10,000 g. T h e s u p e r n a t a n t was r e m o v e d a n d
centrifuged for 45 m i n u t e s at 78,000 g. T h e microsomal pellet thus obtained was suspended in 10-4 M
cold E D T A solution a n d to this was a d d e d an equal
v o l u m e of 90 per cent freshly redistilled phenol. T h e
m i x t u r e was w a r m e d to 20°C., stirred at this temperature for 60 minutes, a n d then centrifuged at
10,000 g for 3 m i n u t e s at 0°C. T h e R N A was isolated
from the top aqueous phase by NaC1 (1 M) precipitation (details of the t e c h n i q u e to be published (5)).
Soluble R N A from Es~herichia coil was prepared as
described elsewhere (6). A suspension of "protoplasts"
was extracted with a phenol-water m i x t u r e a n d precipitated with ethanol. T h e dissolved precipitate contained b o t h the soluble a n d ribosomal R N A ; the
latter was separated from the soluble R N A by a m m o n i u m sulfate precipitation. For this purpose, the
R N A (300 mg.) was dissolved in 25 ml. of Tris buffer
(0.01 M, p H 7.4) a n d small a m o u n t s of insoluble
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E l e c t r o n m i c r o g r a p h s of r i b o s o m a l R N A f r o m Escherichia coli, m i c r o s o m a l R N A fl'om calf,
rat, a n d c h i c k liver, Bacillus cereus R N A a n d E. coli s o l u b l e R N A a r e p r e s e n t e d . F i l a m e n t s
of a b o u t 10 A in d i a m e t e r c o u l d be o b s e r v e d in p r e p a r a t i o n s o b t a i n e d f r o m a q u e o u s solutions of h i g h m o l e c u l a r w e i g h t R N A . W h e n a m m o n i u m a c e t a t e s o l u t i o n s were u s e d a
t e n d e n c y for coiling a n d a g g r e g a t i o n w a s o b s e r v e d . E. coli soluble R N A a p p e a r s as small,
s o m e t i m e s e l o n g a t e d p a r t i c l e s t h e s m a l l e s t d i a m e t e r b e i n g of a b o u t 10 A.
Published February 1, 1961
Abbreviations." E D T A ,
ethylenediaminetetraacetic
acid ; D N A , deoxyribonucleic acid ; R N A , ribonucleic
acid; T M V , tobacco mosaic virus; Tris, tris(hydroxymethyl) a m i n o m e t h a n e .
T h e u p p e r portion (1.5 ml.) was r e m o v e d a n d used
for spraying on the grids.
T h e air dried preparations were shadow-cast with
p l a t i n u m at shadow-to-height ratios of 8 to 1. T h e
films were backed with a thin s u p p o r t i n g layer of SiO
(7). An a q u e o u s suspension of polystyrene spheres of
0.34 # diameter was a d d e d to the solution, to aid in
the location of the microdroplets a n d in the exact
d e t e r m i n a t i o n of the shadow-casting angle a n d the
direction of shadow. M e a s u r e m e n t s of the length of
the s h a d o w were m a d e on filaments perpendicular
to the direction of the s h a d o w a n d in close vicinity
to one of the latex spheres. In rare cases w h e n the
film was stripped successfully following exactly the
m e t h o d of Hall (7), similar results were obtained.
R C A E M U - 2 A with an i m p r o v e d h o m e - m a d e
specimen stage, 25 # objective aperture, a n d a back
focal plane projector aperture, was used.
RESULTS
AND
DISCUSSION
Calf Liver Microsomal R N A : A p p e a r s r a r e l y as a
single l o n g fiber; it h a s a s t r o n g t e n d e n c y for
lateral a g g r e g a t i o n a n d is s o m e t i m e s coiled u p as
i r r e g u l a r g r a i n s (Fig. 1). T h e d i a m e t e r of t h e
single fiber is a b o u t 10 A w h i c h is s i m i l a r to t h a t
of E. coil ribosomal R N A (Fig. 7). H o w e v e r , in
regions of a g g r e g a t i o n larger d i a m e t e r s were o b served. I n rat liver microsomal R N A t h e single
fibers a r e m o r e f r e q u e n t , p r e s e n t i n g h e r e a n d
t h e r e a t h i c k e r d i a m e t e r as if t h e y were coiled u p
o n t h e m s e l v e s (Fig. 3). The chick liver microsomal
R N A s h o w s a p a r t i c u l a r f o r m of fibers f r e q u e n t l y
a t t a c h e d to a g r a n u l e at o n e e n d , as if t h e fiber
was d r a w n o u t of a g r a n u l a r a g g r e g a t i o n . T h e fiber
is t h i n n e r as it goes a w a y f r o m t h e g r a n u l a r b o d y
(Fig. 6). T h e B. cereus R N A r e v e a l e d n u m e r o u s
l o n g fibers of a b o u t 10 A in d i a m e t e r (Fig. 5).
Explanation of Figures
All the preparations were sprayed from a low pressure g u n a n d air dried at room temperature. Shadow-cast with Pt at a shadow-to-height ratio of 8 to 1. Electron microg r a p h s were taken at a n electronic magnification of 20,000 a n d photographically
magnified to 100,000.
FIGURE l
Calf liver microsomal R N A sprayed from a water solution.
FIGURE
Calf liver microsomal R N A sprayed fi'om 0.1 M a m m o n i u m acetate solution.
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m a t t e r were r e m o v e d by centrifugation for 20 m i n utes at 10,000 g a n d discarded. T o the clear solution
(NH4)2SO4 (0.365 g / m l . ) was added. After 30 m i n utes at 4 ° the solution was centrifuged for 10 m i n u t e s
at 10,000 g. T h e s u p e r n a t a n t , w h i c h contained the
soluble R N A , was dialyzed 36 hours against four
changes of NaCI (10 ~ M) a n d then lyophilized.
Sedimentation in the Ultracentr(/u~e: S e d i m e n t a t i o n
analyses were m a d e in a Spinco model E ultracentrifuge with phase-plate schlieren optics. Sedimentation
studies were performed on 0.3 to 0.5 per cent solutions
of R N A in 0.2 M NaC1, at r o o m t e m p e r a t u r e a n d
corrected to 20 ° .
Calf, rat, a n d chick liver microsomal R N A each
revealed two m a j o r boundaries in the ultracentrifuge.
T h e sedimentation constants (S) were: 17, 28; 18, 26;
a n d 19, 24 respectively. E. coli ribosomal R N A gave
two boundaries (S = 16.5 a n d 23.7) while soluble
R N A gave one b o u n d a r y (S = 4.1). B. cereus R N A
revealed four boundaries, S - 4, 8, 12, a n d 22.
Electron Microscopy: A modification of Hall's m e t h o d
for visualizing macromolecules (7) imposed by the
quality of the available mica was used as described
previously (1).
Freshly cleaved mica rectangles were dipped in a
0.5 per cent parlodion (Mallinckrodt C h e m i c a l
Works) solution in redistilled a m y l acetate. After
drying at room t e m p e r a t u r e in an erect position u n d e r
a cover, the film from the freshly cleaved m i c a surface
was floated onto glass doubly distilled water. T h e
grids were deposited on the floating film, a n d taken
up on a glass slide. Solutions were sprayed as fine
droplets onto the film covering the grids on the slide,
using a low pressure gun. All the samples of R N A
(2 ml.) were centrifuged for 20 m i n u t e s at 105,000 g.
Published February 1, 1961
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D. DANON, Y. MAItIKOVSKY, AND U. Z. ],ITTAUER Electron Microse,~py of RNA
255
Published February 1, 1961
individual threads became rare. O n the other
h a n d R N A deposited from aqueous solutions
showed this kind of aggregation to a m u c h lesser
degree, and single strands were observed. T h e
effect of salt on the appearance of rat a n d calf
liver microsomal R N A was therefore studied. W i t h
both R N A samples it was observed t h a t the addition of a m m o n i u m acetate resulted in a strong
tendency of the fibers to aggregate (Figs. 2 a n d 4),
usually laterally, causing the appearance of relatively thick threads, whereas in E. coli ribosomal
R N A g r a n u l a r forms of aggregation were more
frequent. These findings are in accordance with
the h y d r o d y n a m i c measurements and support
the idea t h a t both rat a n d calf liver R N A are
molecules capable of coiling. The fact that some
R N A threads a p p e a r to be coiled up upon themselves a n d aggregated even w h e n deposited from
aqueous solutions (containing less t h a n 10-4 M
salt), might be explained by the considerable
increase of the local sah concentration during the
drying of the micro droplets, occasionally reaching
sufficiently high values (greater t h a n 10-2 M) to
cause the observed effect. The present study with
calf liver R N A might explain the observations
made by Hall (3) who, using a m m o n i u m acetate
solutions, reported relatively short threads of an
a p p a r e n t thickness of a b o u t 30 A. T h e use of a
different method for the preparation of the R N A
might also contribute to the morphological
difference.
H a r t (9) observed t h a t R N A fibers which were
attached to the rod end of T M V , were extended
when sprayed from a salt solution. However, as
stated by h i m the R N A fibers " a r e resolvable in
the micrographs only because of a d h e r i n g cont a m i n a n t material (perhaps detergent or den a t u r e d protein)." It will be h a r d to evaluate the
effect of the presence or absence of salt on the
association of c o n t a m i n a n t s with R N A molecules.
FIGURE 3
Rat liver microsomal RNA sprayed from a water solution.
FIGURE 4
Rat liver microsomal RNA sprayed from 0.1 M ammonium acetate sohltion.
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Soluble R N A from E. coil appears as very small,
sometimes elongated particles or grains, longitudinally and laterally aggregated; the smallest
d i a m e t e r observed was a b o u t 10 A (Fig. 8). In
view of the low molecular weight of this material
(about 30,000) it is difficult to state if the elongated
particles are single molecules or a longitudinal
aggregation of smaller subunits.
T h e length of the fibers of all the high molecular
weight R N A preparations, is m u c h more variable
t h a n would have been expected from the sedim e n t a t i o n data. This variability in length of the
fibers was observed in R N A preparations of
various sources a n d to a lesser degree in different
samples of the same source. This variability seems
to be due to a certain a m o u n t of longitudinal
aggregation, which makes the measurements of
the length of the molecule a very approximate one.
T h e fact t h a t the sedimentation constants did not
vary to such an extent m i g h t suggest that, during
the preparation of the electron microscopical
specimens, the various R N A preparations were
differently affected by the spraying a n d the drying
of the micro droplets, thus showing different
degrees a n d forms of aggregation as well as coiling.
T h e effect of the spraying on the E. coli R N A was
d e m o n s t r a t e d in a previous study (1). Although
the sedimentation constants of the calf liver R N A
present the highest values, the length distribution
of the fibers is considerably lower as c o m p a r e d to
the other R N A preparations.
Effect of Salt." H y d r o d y n a m i c measurements of
E. coli ribosomal R N A (4), rat and calf liver microsomal R N A (5, 8) have indicated t h a t R N A
molecules fold u p into more compact structures
w h e n salt is added to the solution. These findings
were recently supported by an electron microscopical study of E. coli ribosomal RNA, where it
was shown t h a t when R N A was deposited from
a m m o n i u m acetate solutions, granules a n d various
forms of aggregates d o m i n a t e d the picture while
Published February 1, 1961
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O. DANON, Y. MARIKOVSKY, AND U. Z. LITTAUER Electron Micro~col~y of R N A
257
Published February 1, 1961
All the R N A fibers studied present a diameter of
about 10 A, which is similar to that found for
E. coli ribosomal R N A and is about half the diameter reported for D N A (20 A). The R N A fibers
are wavy and granular in appearance as c o m p a r e d
to the smooth and rigid D N A strands (1).
The authors are indebted to Dr. M. R. Pollock for
providing us with Bacillus cereus 569/H.
The authors wish to thank D. Givon for his technical
assistance.
This investigation was supported in part by a United
States Public Health Service grant RG-5217.
Received for publication, July 30, 1960.
REFERENCES
1. LITTAUER, U. Z., DANON, D., and MARIKOVSKY,
Y., Biochim. et Biopf~ysica Acta, 1960, 42, 435.
2. SHUSTER, H., SCHRAMM, G., and ZILHG, W., Z.
Natiirforsch., 1956, B l l , 339.
3. HALL, C. E., Proc IVth Intern. Cong. Biochem.,
1958, 9, 90.
4. LITTAUER, U. Z., and EISENBERG, H., Biochim. et
Biophysica Acta, 1959, 32, 320.
5. LITTAUER, U. Z., data to be published.
6. Cox, R. A., and LITTAUER,U. Z., J. Molec. Biol.,
1960, 2, 166.
7. HALL, C. E., .f. Biophysic. and Biochem. Cytol., 1956,
2,625.
8. LASKOV, R., MARGOLIASH, 1~., I,rrTAUER, U. Z.,
and EISENBERG, H., Biochim. et Biophrsica Acta,
1959, 33,247.
9. HART, G. R., Biochim. et Biophysica Acta, 1958, 28,
457.
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FIGURE 5
Bacillus cereus RNA sprayed from a water solution.
FIGURE 6
Chick liver microsomal RNA sprayed fi'om a water solution.
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Published February 1, 1961
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D. DANON, Y. MARIKOVSKY,AND U. Z. LITTAUER Electron Microscopy of RNA
2~59
Published February 1, 1961
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FIGVRE 7
E. coil r i b o s o m a l R N A
sprayed from a water solution.
FIGURE
E. coli s o ! u b l e R N A s p r a y e d f r o m a w a t e r s o l u t i o n .
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Published February 1, 1961
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I). I)ANON, Y. MARIKOVSKY, AND V. Z. LITTAUER Electron
Micro.~copy of R N A
26l