Published May 1, 1961
Chromatographic Separation, and
Characteristics of Nucleic Acids
from HeLa Cells
LENNART PHILIPSON
From The RockefellerInstitute
INTRODUCTION
C h r o m a t o g r a p h y of D N A from different tissues indicates heterogeneity of
the nucleic acid (1-6). Separation on an anion exchanger, ecteola cellulose,
gave a n u m b e r of fractions in a pattern characteristic of the tissue (1). Later
studies, however, indicate that the extraction procedure has a definite influence on the elution pattern (2--4). Chromatography of transforming D N A
on a basic histone column showed a concentration of the transforming principle in some fractions (5). Heterogeneity of D N A has also been reported o n
the basis of ultracentrifugation studies (6). T h e chromatographic separation
of R N A (7, 8) has also been interpreted to indicate heterogeneity.
W h e t h e r the heterogeneity of the nucleic acids is an experimental artifact
caused by procedures used in extraction and the subsequent chromatography
or whether it reflects the properties of native D N A is unsettled.
Recently new methods have been described, which appear to cause very
little d a m a g e to the nucleic acid molecules during extraction (9-1 l) since
biological properties, such as infectivity of virus RNA, are partially retained
by the extracted material (9). T h e report of chromatographic separation on
an esterified bovine serum albumin column (12) of D N A extracted by the
899
The Journal of General Physiology
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ABSTRACT The application of the phenol-duponol method to extraction
of nucleic acids from HeLa cells is described. Chromatography of the phenol
extract on an esterified bovine serum albumin column with a salt gradient of
sodium chloride gives separation of soluble RNA, DNA, and two different
high molecular RNA fractions. Ultracentrifugation of the DNA eluted from
the column gives a sedimentation coefficient (s~0.~,) of 38, which agrees with
ultracentrifugation data on the phenol extract. The eluted RNA appears
polydisperse at low ionic strength, but at high ionic strength and after alcohol
precipitation two fractions with the sedimentation coefficients of 16 and 25
to 29, respectively, were obtained.
Published May 1, 1961
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p h e n o l m e t h o d f r o m b a c t e r i o p h a g e led to a n investigation of the a p p l i c a b i l i t y
o f this c o l u m n t e c h n i q u e to nucleic acids f r o m m a m m a l i a n cells, a n d of the
c o n d i t i o n s for successful e x t r a c t i o n of nucleic acids f r o m cells in culture. T h e
c o m m u n i c a t i o n also presents a p r e l i m i n a r y c h a r a c t e r i z a t i o n of the nucleic
acids isolated.
MATERIALS
AND
METHODS
Cultured Cells A strain of H e L a cells adapted to horse serum was used. In some
experiments a cloned line of H e L a cells ($3) was used (13). The cells were grown in
suspension in Eagle's m i n i m u m essential medium (14) without Ca ++, but with tenfold higher concentration of phosphate and 5 per cent undialyzed horse serum. T h e
ceils were fed every day by replacing half of the medium with fresh medium. Under
these conditions the doubling time was 17 to 20 hours, and the cells could be maintained in logarithmic growth for periods of 1 to 2 weeks.
Extraction of Nucleic Acids with Phenol-Duponol The ceils were centrifuged (600
R.P.M. for 10 minutes) immediately after removal from the suspension cultures, and
washed once in 0.1 M NaCI buffered at p H 7.2 with 0.1 M phosphate. T h e y were then
resuspended in the same solution in concentrations of 2 to 4 X l0 T ceils per ml.,
distributed in 2 ml. amounts in tubes, and frozen at --20°C. until extracted. Control
cells extracted without freezing showed results similar to those with the frozen cells.
Duponol was added to the thawed cell suspension to a final concentration of 2 per
eent (w/v) at room temperature and the mixture was shaken briefly. In some experiments the duponol concentration was varied as described below. T o the duponoltreated cells was added an equal volume of recently distilled water-saturated phenol,
and the suspension was shaken in a mechanical shaker for 4 minutes at 4°C. (240
excursions/minute). In some experiments the extraction temperature was varied
as described below. The water and phenol phases were separated by centrifugation
at 2,000 R.P.M. for 5 minutes, and the bottom phase of phenol was pipetted offleaving
the water phase and the interphase. Equal amounts of phenol were added to the
water phase and the interphase again and the same extraction procedure was repeated. After separation of the phases by centrifugation the water phase was carefully removed and transferred to a new tube. Ether treatment to remove the phenol
was performed 5 to 6 times using ethyl ether recently distilled over ferrous sulfate.
The residual ether was removed by vacuum. The water phase was then analyzed
for nucleic acids and chromatographed.
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P32-Labeled Cells In some experiments P3~-labeled cells were used. The cells
were grown in suspension in Eagle's m i n i m u m essential medium without Ca ++, but
containing 5 per cent dialyzed horse serum. To this medium was added radioactive
carrier-free orthophosphate to a specific activity of 0.01 mc./mg. P. T h e cells multiplied normally in this medium if the concentration of ceils did not exceed 4 X
10~/ml. The cells were grown for four divisions in the radioactive medium, which
was changed daily by removing half the cell suspension, and adding half the amount
of fresh medium.
Published May 1, 1961
LENNART PmLIPSON NucleicAcids from HeLa Cells
9oi
Chemical Methods. DNA DNA content of unlabeled material was determined
according to the Burton modification of the diphenylamine reaction (15), except that
optical density readings were made as O.D.~0 subtracted from O.D.595 to reduce
non-specific effects. Hydrolyzed DNA was used as a standard. Labeled material
was analyzed according to a modified Sehmidt-Thannhauser method (16). DNA
eluted from the column was precipitated, before hydrolysis, with 0.3 M cold trichloracetic acid (final concentration) with serum albumin as carrier in order to separate
DNA from a material which eluted from the column, and gave a green color in the
diphenylamine reaction.
RNA Unlabeled material was analyzed for RNA by the orcinol reaction according to Kerr and Seraidarian (17), except that optical density readings were
made as O.D.sen subtracted from O.D.se5 to reduce non-specific effects. Hydrolyzed
RNA was used as a standard. Labeled material was analyzed according to SchmidtThannhauser (16).
Determination of RNA and DNA in Cell Suspension T o evaluate the amount of
DNA and RNA extracted with phenol-duponol, the cell suspensions were defatted
by extraction with alcohol and twice with alcohol: ether (3: 1) at 70 °C. for 15 minutes.
The sediment was slurried in ether at 4°C. and the ether removed. The acid-soluble
fraction was then obtained by treating the sediment at 0°C. with 4 per cent perchloric acid. The diphenylamine and the orcinol reactions were carried out on the
supernate. The RNA and DNA were then extracted by 6 per cent perchloric acid
at 90°C. for 15 minutes, and, after cooling, the DNA and RNA were determined on
the supernate by the diphenylamine and orcinol reactions, respectively.
Ultracentrifugation A Spinco analytical ultracentrifuge model E was used. The
eluted fractions were analyzed by ultraviolet optics using Eastman commercial safety
film and the plates read in a microdensitometer. The phenol extracts were investigated both by schlieren optics and ultraviolet optics at different dilutions of the
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Chromatographic Technique The preparation of the column and the principles
of operation have recently been described in detail (12). In essence the column is
composed of diatomaceous earth and bovine serum albumin, the latter made basic
by esterification with methyl alcohol. The column is built up in three layers to prevent chanelling, the bottom layer containing approximately 2.5 rag. methylated
bovine albumin/gm, hyflo supercel, the middle layer, 0.7 reg./gin., and the top
layer, none. New columns were prepared for each experiment except when samples
were rechromatographed. The phenol extract to be adsorbed to the columns was
diluted to the desired salt concentration. The volume of diluted samples was 50 ml.
Ten to fifteen O.D.260 units (Beckman D. U.) of phenol-extracted material was
absorbed to each column. Elution was carried out with a concentration gradient of
sodium chloride. All solutions were buffered at p H 6.7 with 0.05 w phosphate. T h e
optical density of eluates was automatically recorded in arbitrary optical density
units using a Type UV-254 absorption meter (Gilson Medical Electronics, Middleton, Wisconsin). When P3~-labeled nucleic acids were investigated, the radioactivity
of the eluates was automatically determined with a ratemeter (Nuclear Chicago
model 1620BS) and recorded. The eluates were then collected in a fraction collector.
Published May 1, 1961
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THE
JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 44 " t 9 6 I
phenol extract. DNA fractions were centrifuged in 0.7 M NaC1 buffered at pH 6.7
with 0.05 ~ phosphate at a concentration of 1 mg./dl, at a speed of 35,600 mP.M.
with an AN-E rotor. RNA fractions were centrifuged in 0.8 M NaC1 buffered at pH
6.7 with 0.05 M phosphate at a concentration of 4 mg./dl, with an AN-D rotor at
59,780 mP.M. The phenol extract was centrifuged after dialysis against 0.1 M NaC1
buffered at pH 6.7 with 0.05 M phosphate at different dilutions corresponding to
20 to 135 mg./dl, of DNA in the samples. The AN-D rotor was used and the speed
was 50,740 R.P.M. Sedimentation coefficients were corrected for temperature and
viscosity of the solvent using 0.57 for the partial specific volume of RNA (18) and
0.58 for the partial specific volume of DNA (19).
TABLE
I
A COMPARISON OF THE AMOUNTS OF NUCLEIC ACIDS
E X T R A C T A B L E BY T H E P H E N O L - D U P O N O L A N D T H E P E R C H L O R I C
ACID EXTRACTION METHODS
Method of extraction
pgm.~:
Phenol-duponol
31.2; 21.2;
30.4
26.4; 22.0;
26.0
Perehloric acid
cell*
DNA
Mean
pgm.
27.6
24.8
pgm.
17.4; 16.3;
18.2
17.7; 16.6;
15.6
Acid-soluble ribose
Mean
pgm.
pgm. X 102
Mean
pgm.X 10
17.3
--
--
16.6
31.0; 31.0;
16.2
22.7
* T h r e e different b a t c h e s of cells were analyzed.
:~ p g m . , p i e o g r a m = 10-12 gm.
RESULTS
The Phenol-Duponol Method T h e efficiency of the phenol-duponol
method of extraction of nucleic acids from H e L a cells was compared to that
of the perchloric acid technique. An extraction temperature of 4°C. and a
duponol concentration of 2 per cent (w/v) were used. T h e conditions of the
perchloric acid extraction procedure were described under Methods. Results
obtained with three different batches of cells are shown in Table I. T h e
average values for R N A insoluble in cold acid were 27.6 and 24.8 pgm./cell
for the phenol-duponol and the perchloric acid methods, respectively. This
difference is not significant. T h e corresponding values for DNA were 17.3
and 16.6 pgm./cell for the two methods. R e m a r k a b l y little acid-soluble
ribose, i.e. about 1 per cent of the total a m o u n t of RNA, was found in the
H e L a cells in the logarithmic phase as seen in Table I.
T h e effect of variations in the phenol-duponol method on the amounts of
DNA and R N A extracted was also investigated. The concentration of duponol
added to the cell suspension was varied first. In these experiments the phenol
extraction was carried out at 4°C. The temperature of the phenol extraction
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A m o u n t per
RNA
Published May 1, 1961
LENNART PHILIPSON Nucleic Acids from HeLa Cells
9o3
was varied second. This was done b y carrying out extraction in a water bath
at the different temperatures. In these experiments the concentration of
d u p o n o l was constant at 2 per cent. T h e samples were stirred at 600 R.r.M.
with a dull edged stirrer. T a b l e II presents the results of typical experiments
in which the duponol concentration and the extraction temperature were
varied. It is evident that extraction of R N A was maximal at a 1 per cent
concentration of duponol, but to obtain a m a x i m u m yield of DNA, a 2 per
cent concentration was necessary. W h e n the extraction temperature was
varied it was found that extraction of R N A was slightly more efficient at
higher temperatures, whereas that of D N A appeared to be less efficient at
such temperatures.
Amount per cell
Experiment
Final concentration
of duponol
Extraction temperature
RNA
DNA
°C.
pgm.
pgm.
fi
+4
+4
+4
20.0
30.4
31.2
9.75
9.90
17.4
i
+4
+25
+37
+50
21.2
21.6
22.0
23.0
16.3
12.0
12.8
7.8
Perce~
Chromatography of Nucleic Acids The phenol extract was adsorbed to the
esterified bovine serum albumin column in 50 ml. amounts diluted in appropriate buffers. Elution was carried out b y a salt gradient in sodium chloride
buffered at p H 6.7 with 0.05 M phosphate. Fig. 1A shows a typical elution
diagram for the phenol extract at a gradient from 0.62 to 1.5 M sodium
chloride. T h e material running right through the column at 0.62 M contains
free bases and other ultraviolet-adsorbing materials that did not adsorb to
the column even at 0. 1 M sodium chloride, and also soluble RNA. W h e n a
different gradient was used soluble R N A eluted at 0.5 u sodium chloride.
After this non-adsorbing fraction, the first peak was eluted at a salt concentration from 0.70 to 0.77 M sodium chloride with the peak at 0.71 U. This
material contained only DNA, and the amount of D N A in the peak as determined by the diphenylamine reaction corresponded with the optical density
of 260 m o considering 1 #g. of D N A -- 0.0231 O.D. units. Further the
orcinol reaction gave a color yield corresponding to the amount of D N A
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TABLE II
EFFECT OF VARIATIONS IN THE PHENOL-DUPONOL
EXTRACTION TECHNIQUE ON THE AMOUNT OF H I G H MOLECULAR
WEIGHT RNA AND DNA EXTRACTED
Published May 1, 1961
904
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"
I96I
A
0.5,
0.3
OI
"1
I
I
20
,o
30
4'0
~o
6'o
~o
Fraction number
0.7
B
0.3
0.1
,b
~o
3'o
~o
4'o
~o
7'o
Fraction number
o.~
C
0.5
Jb
2'o
.~o
4'o
50
6'0
i
70
Fraction number
FIGtrRE l. Elution pattern of DNA and high molecular weight RNA from a bovine
serum albumin column. (A) Elution pattern of phenol-extracted material from HeLa
cells at 0.62 to 1.5 M NaC1 gradient. The DNA eluted in fractions 22 to 30. High molecular weight RNA cluted in fractions 38 to 59. (B) Kechromatography of the DNA from
A at a gradient of 0.62 to 0.84 M NAG1. (G) Rechromatography of high molecular RNA
from A at a gradient of 0.84 to 1.2 M NaCl.
(20). T h e a v e r a g e c o n c e n t r a t i o n o f s o d i u m c h l o r i d e c a l c u l a t e d f o r t h e e l u t i o n
o f t h e p e a k o f D N A f r o m t e n e x p e r i m e n t s w a s 0.705 M. W h e n all t h e D N A
f r o m t h e p e a k in Fig. 1A w a s r e c h r o m a t o g r a p h e d in a n a r r o w e r g r a d i e n t ,
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0.5~
o
Published May 1, 1961
LENNART PHILIPSON Nucleic Acids from HeLa Cells
9o5
0.62 to 0,84 ~ sodium chloride, as seen in Fig. 1B, only one peak was obtained
which eluted at 0.70 to 0.72 M sodium chloride with the m a x i m u m of the
peak at the calculated concentration of 0.71 M.
In the original run (Fig. 1A) high molecular weight R N A eluted after the
DNA in a pattern suggesting two peaks. This elution pattern was reproducible.
The sodium chloride concentrations for the R N A peaks ranged from 0.86 to
0.96 M. The first peak eluted at 0.86 M and the second at 0.90 M. W h e n a third
of the pooled fractions 40 to 55 from the experiment shown in Fig. 1A was
0.6
-6
0.5
r~
>, 0.5
<
0.2
II
It
I
I
I
i
-2
n
'
0.1
i
P
I
.
,.
~
il~.
l\
J \
I
I
I
I
[
I
I0
20
50
40
50
60
" ~ -
70
Fraction number
FIOURE 2. Elution pattern of soluble RNA, DNA, and high molecular weight RNA
from the column at a gradient of 0.1 to 2.1 M NaC1. Soluble RNA eluted in fractions
36 to 41; DNA and high molecular weight RNA eluted in fractions 44 to 51 and 54 to
66, respectively. Solid line, arbitrary O.D. units. Broken line, pa~ counts.
rechromatographed in a narrower gradient, 0.84 to 1.2 M, the two peaks
separated clearly as seen in Fig. 1C. The eluting concentrations were 0.86
and 0.90 M for the two peaks. Both R N A peaks in Fig. 1A and 1C gave a
positive orcinol test and negative diphenylamine reaction. The results described were obtained with an uncloned strain of H e L a cells. Similar results
were also obtained with the $3 cloned line of H e L a cells. However, the
DNA of the cloned cell line eluted at 0.68 M sodium chloride (average of
two experiments).
To obtain additional information concerning the apparent homogeneity
of the DNA and the two peaks of RNA, a more sensitive method than the
recording of optical density was used. P3Mabeled cells were extracted according to the same methods used with unlabeled cells. Automatic recording of
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g
%
0.4
Published May 1, 1961
P
9ot~
THE
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PHYSIOLOGY
• VOLUME
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• ~9 o I
III
RECOVERY FROM THE COLUMN OF HIGH MOLECULAR WEIGHT RNA AND
DNA PREPARED BY DIFFERENT EXTRACTION PROCEDURES
Recovery in eluate as per cent of input
RNA
Duponol
concentration
in per cent
Extraction
temperature
w/v
oc.
2
2
2
2
+4
+25
-/-37
+50
Orcinol
reaction
74.5
92.5
104.0
100.3
DNA
Over-all recovery
P~
Diphenylamine
reaction
p32
O . D . units
Pa~
81.0
81.4
---
102.0
73.5
62.4
41.3
92.0
75.5
---
99
102
100
98
88.3
90.1
---
FIeURE 3. Schlieren pattern from dialyzed phenol extract in 0.1 M NaC1 buffered at
pH 7.2 with 0.05 M phosphate centrifuged at 50,740 mV.M. with a bar angle of 30 °. Pictures, from left to right were taken at 70, 78, and 86 minutes, respectively, after speed
was reached. Phenol extract diluted seven parts extract and three parts buffer.
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p3~ w a s m a d e o n t h e e l u a t e s . A s s e e n in F i g . 2 t h e e l u t i o n o f r a d i o a c t i v i t y
c o r r e s p o n d e d t o t h e o p t i c a l d e n s i t y c o m p l e t e l y . T h e g r a d i e n t i n this e x p e r i m e n t w a s 0. 1 to 2.1 M s o d i u m c h l o r i d e i n o r d e r to s h o w a l l t h e f o u r d i f f e r e n t
fractions separable from the phenol extract by the column. Non-adsorbing
m a t e r i a l a n d u n i n c o r p o r a t e d p32 e l u t e d e a r l y . A t a p p r o x i m a t e l y 0.5 M s o d i u m
c h l o r i d e s o l u b l e R N A e l u t e d f o l l o w e d , a t t h e s a m e c o n c e n t r a t i o n s as r e p o r t e d
above, by DNA and the two high molecular RNA fractions. The soluble
R N A e l u t i n g a t 0.5 M N a C I w a s s o l u b l e in 1 M N a C 1 a n d it w a s a l k a l i - s o l u b l e
in t h e S c h m i d t - T h a n n h a u s e r
r e a c t i o n (16).
Recovery of the different types of nucleic acid from the column was determ i n e d e i t h e r b y o r d i n a r y c h e m i c a l m e t h o d s o r w i t h p32 for p r e p a r a t i o n s
extracted by different methods. In these calculations the input was always
c o n s i d e r e d 100 p e r c e n t . A s s e e n in T a b l e I I I t h e r e c o v e r y o f R N A s e e m e d
to i n c r e a s e w i t h h i g h e r e x t r a c t i o n t e m p e r a t u r e s a l t h o u g h t h i s w a s n o t as
Published May 1, 1961
LENNART PmLIPBON NucleicAcids from HeLa Cells
907
apparent with the p32 tests made. On the other hand the recovery of DNA
was less at higher temperatures of extraction. The over-all recovery of optical
density and p3~ was between 88 to 100 per cent.
sssO
12
soj sU
s~
ss
, OS
I0
%
js S
6 sSSSSj•
I
I
I
,/4o lo'
I
I
I
I
I
I
I
1/4
I
I
i
I
I
I
I
I
l
1/2
L
I
I
!
I
I
l
I
I
I
Dilution factor of phenol extract
i f
I
2'o
I
4'o
I
to
i
8'o
T
,6o
I
I
,2o
I
I
|
I I I
i4o
Corresponding cone.of DNA mg./dl.
FlOURE 4. Reciprocal of s~0,, in Svedberg units (10-18 see.) plotted against dilution
factor of the phenol extract. Corresponding amount of DNA in the preparation is also
given on the abscissa.
Solid llne, values from present experiments. Broken line, drawn from values obtained
with purified DNA as published by Rice and Dory (21). X, point obtained with DNA
fraction from the column (1 mg./dl.).
Ultracentrifugation Characteristics The eluted fractions of high molecular
DNA and RNA were investigated in the ultracentrifuge by ultraviolet optics.
The DNA peaks showed a sharp boundary in two of three preparations
giving a s~0,w of 37.9. The third preparation gave a diffuse boundary with
an average sedimentation coefficient of 32. The RNA material showed
diffuse boundaries in many preparations but in 0.8 ~ sodium chloride buffered
at pH 6.7 with 0.05 Mphosphate a sharpening of the boundaries was observed.
In materials from the original chromatography (Fig. 1A), where the two
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8
Published May 1, 1961
908
THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 44 " I 9 6 I
peaks did not clearly separate, a material with an average S2o,,o value of 16. 1
was found in material from the front of the peak and from the top of the
peak. The tail of the peak did, however, contain increasing amounts of a
component moving faster than the 16S component. In a single preparation
two different fractions of RNA were separable in the microdensitometer
tracings of the photographs from the centrifugation. A value of 29S was
calculated for the faster moving component.
To compare the S values of the nucleic acid components after chromatography with those of the high molecular DNA and RNA in the original
phenol extract, different dilutions of the phenol extract were centrifuged
using schlieren or ultraviolet optics depending on the concentration of nucleic
acids. As seen in Fig. 3 the DNA appeared as a sharp band because of selfTABLE
IV
s~0,w X 101a
Nucleic acid
Eluate
Phenol extract
DNA
R N A peak 1
peak 2
37.9 4- 0.5
16.1 4-. 0.6
29
38.0
15.6
25
sharpening (21), but the RNA showed a broad, barely visible peak. The
sedimentation constants of DNA could be easily calculated, and, as seen in
Fig. 4, when plotting the reciprocal of S2o,~, against the dilutions used, a
linear relationship seems to exist, giving a S~o,~, value at infinite dilution of
about 38. The concentration dependence of the S2o,,o paralleled values found
by Rice and Doty for purified DNA (21). However, the s~0,w of their preparation at infinite dilution was 21. The RNA in the phenol extract was found
to move slower than expected from the S values obtained from the eluates
of the column. However, when the nucleic acids in the phenol extracts were
precipitated by alcohol and resuspended in 0. 1 u sodium chloride buffered
at pH 6.7 with 0.05 M phosphate, RNA showed two distinct peaks in the
schlieren optics which, when corrected for the concentration dependence of
s20,~ according to Kurland (18), gave estimated s~0,~ values of 15.6 and 25,
respectively.
Table IV gives a comparison of the s~0,w values obtained with the eluates
from the column with those obtained with the original phenol extract. With
the concentration of cells used no soluble RNA was observed in the ultracentrifugation studies.
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S E D I M E N T A T I O N C O E F F I C I E N T S A T I N F I N I T E D I L U T I O N (s~o,,,)
OF THE ELUTED FRACTIONS OF HIGH MOLECULAR DNA AND RNA COMPARED
TO THE VALUES OBTAINED FROM THE PHENOL-DUPONOL EXTRACT
Published May 1, 1961
LENNARTPHILIP$ON NucleicAcidsfrom HeLa Cells
909
DISCUSSION
s20,~
° = 0.063 X M °'37
this would give a figure of about 32 )< 106, which is larger than most values
reported for DNA except the phage DNA from the T series (25).
In many centrifugations RNA showed diffuse boundaries indicating polydispersity of the RNA. However, by increasing the salt concentration or by
alcohol precipitation of the RNA, sharper boundaries were obtained indicating two fractions in both the phenol extract and the eluates from the
column. The s~0,~ values were 16 and 25 to 29, respectively, which agree
with values obtained with RNA from purified ribosomes (18). No determinations of the amount of protein in the phenol extracts were made, but in
previous studies with a similar extraction method less than 1 per cent protein
was found (10). Furthermore, the column used has been reported to reduce
the protein content of phage DNA preparations about tenfold (12).
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The extraction of nucleic acids from HeLa cells with phenol-duponol followed
by chromatography on an esterified bovine serum albumin column gives a
separation of soluble RNA, DNA, and probably two different types of high
molecular weight RNA.
The amounts of DNA and RNA per HeLa cell found in the present study
seem to agree with previous results obtained with HeLa (22) and KB cells
(23). The phenol-duponol method extracts DNA quantitatively when the
extraction is carried out at 4°C. At higher temperatures of extraction less
DNA is obtained in the water phase. Possible explanations for this are that
phenol may interact with the DNA or that the DNA-protein complex may
not dissociate at higher temperatures. In contrast, RNA recovery is not
affected at the higher temperatures.
The chromatographic separation is similar to the one obtained with a
phenol extract from E. coli and the same type of column (12). The recovery
of DNA is, however, much higher with HeLa cells than with E. coli.
The recovery of DNA from the column is influenced by the temperature of
extraction. A decrease in recovery from 100 to 40 per cent was observed when
the extraction temperature was increased from +4°C. to 50°C. With regard
to similar findings for this column with T2 and T4 bacteriophage DNA (12),
this indicates that DNA is denatured by phenol extraction at high temperatures.
The ultracentrifugation characteristics of the DNA showed a sedimentation
coefficient of 38 (s~0,~) for the material eluting from the column. The same
value was obtained when the sedimentation of DNA was studied in the
phenol extract. Applying the formula reported by Doty et al. (24) for the
correlation between molecular weight and sedimentation coefficient of DNA
Published May 1, 1961
9Io
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
44
"
I96I
The author wishesto express his sincere thanks to Dr. A. D. Hershey,in whoselaboratorythe main
part ofthisinvestigationwas carried out, for facilitiesand advice.The assistanceof Dr. D. A. Yphanfis
in the ultracentrifugationis acknowledged.
This research was aided by a grant from The National Foundation. Dr. Philipsonis a Sophie Fricke
Fellow of the Royal SwedishAcademyof Sciencein The RockefellerInstitute.
Receivedfor publication, November8, 1980.
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