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Separation of rapidly labeled RNA of animal cells into DNA-type and ribosomal RNA-type components
Vol. 15, No. 1, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SFPARATIONOF RAPIDLYLABLEJI FUiAOF ANIMAL CELLSINTO DNA-TYPEANDRIw)SOMALRNA-TYPECOMPONENTS M. Yoshikawa, T. Fukada and Y. Kawade Imtitute
Received
December
30,
for Virus Research, Kyoto University,
1963
The validity
of the messengerRNAhypothesis (1) has been well
supported by evidence from a variety (2-5).
Kyoto, Japan
of experiments in bacterial.
The messengerRNAis in general distinguished
from other cellular
RNA'S by its high turnover rate and a base composition similar animal cells, so clearly
however, the characteristics
defined,
systems
to DNA. In
of messenger RNAhave not been
although metabolically
active RNAwhich is rapidly
labeled by radioactive
precursors has been detected and studied in various
animal cells (6-12).
The results of Scherrer et al. (11, 12) appear to be
especislly
significant
groving HeLa cells
in that they obtained the rapidly
labeled RNAof
in the form of two molecular species with sedimentation
constants of 45 S and 35 S as estimated by sucrose density fugation.
gradient centri-
Roth of these were found to have a base composition similar
ribosomsl RNA(rRNA), and were shown to be essentially
to
composedof rRNA
precursor by "chase" experiments, although a small amount of metabolically unstable fraction rapidly
was also detected in them.
labeled RNAof growing FL cells
F'romour studies on the
(an established cell line derived
from humansmnion), we can now demonstrate its
separation into two compo-
nents by using the methylated albumin column (13), one having a base composition
similar
to DNA and the other similar
techniques provide a meansof isolating RNAof animal cells.
to rRNA. Thus, the present
what is likely
Such a DNA-like RNAfraction
to be the messenger
has been found in some
animal cells (14, 15), but it should be noted that in Our study the RNA appeared to be of a very large size and might represent the undegraded state* 22
BIOCHEMICAL
Vol. 15, No. 1, 1964
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FL cells uere grown in monolayers in 500 ml Roux bottles
uith 50 ml
of a phosphate-free Eagle's medium (16) containing double strength amino acids and vitamins and 10% calf serum. Labeling of nucleic acids was accomplished by adding P32 of varying period,
the labeling
After
labeling,
activities,
depending on the length of
together with 0.25 p/ml. each of the four nucleosides.
the cells were suspended in 0.14 M NaCl-0.01 M tris
buffer
at pH 8.1 containing 5 x 10B4 M EDTA. Nucleic acids were extracted dodecyl sulfate
sodium
(0.5$)-phenol method at 0-4'C.
95% of RNA and 9096of DNAcould be extracted, tion of labeled
by the
Under these conditions,
and no significant
fractiona-
RNAsuch as described by Sibatani et al. (17, 14) seemed
to take place. The nucleic acid thus obtained was fractionated albumin column according to Mandell and Hershey (13).
on a methylated The resolution
of the
two components of animal cell rRNA is poorer than in the case of bacterial rRNA (18-20). but could be much improved by keeping the column at 30-3pC (20).
A typical
chromatogram is shown in Ng. 1.
Soluble RNA, DNAand
rRNA were eluted at about 0.5, 0.75 and 0.95 M NaCl. respectively, rapidly
labeled RNAappeared later
material,
two radioactive
than rRNA. With the 30 min. pulse-labeled
peaks were observed at about 1.05 and 1.15 M NaCl,
which we call ql and q2, respectively
(Fig. 1).
peak soon becamesmall compared to ql, toward rRNA. After the radioactivity
and the
On longer labeling,
and the ql peak shifted progressively
24 hours (the generation time of the cell) almost paralleled
the q2
the absorbance profile
of labeling,
of rRNA.
Since the methylated albumin column separates RNAmolecules mainly according to their
size. ql and q2 may be supposed to have larger sizes than
rRNA. This was shown to be the case by sucrose density gradient gation of RNArecovered from the chromatographic fractions. 2, two radioactive
centrifu-
As seen in Fig.
peaks were observed ahead of rRNA, which are most likely
to correspond to the ql and q2 RNA. heir
sedimentation constants were
estimated to be about 40 and 50 S, respectively. 23
BIOCHEMICA!.
Vol. 15, No. 1, 1964
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cm .--. DNA
Tube Fig.
1.
Number
Chromatographic analysis of nucleic acids of FL cells labeled by P” (20 &/ml medium) for 30 min. Chromatography was performed according to Mandell and Hershey (13) but at 30°C. Elation was done by a NaCl gradient containing 0.01 M phosphate buffer at pH 6.7 and 5 x IO-’ M EDTA. with a flow rate of 3 ml/tube/IO min.
A258
:pm I--. 300
Fraotlon Fig.
2.
Number
Sucrose density gradient cennifugation of RNA labeled for 30 min. Ckomatographic fractions shown in Fig. 1 was collected by ethanol with rRNA added as carrier, and, after phenol treatment (to remove albumin released from the column), centrifuged on a linear sucrose ing 0.1 M NaCl, 0.1 mM MgC1, aod 0.01 M acetate buffer at pH 5.2, min. in a Hitachi SW40 rotor.
The labeled RNA in the precipitation together contaminating metbylated gradient (5-20s) containat 36,000 ‘pm for 210
The base compositions of various RNAuere determined by P32 counts incorporated It
into the four nucleotides.
The results
are given in Table 1.
can be seen that the rRNA has Fore guanine and cytosine than adenine and
uracil, trast
as is also found with other mammalianrRNA's
The DNA in con-
The composition of ql uas found to
is rich in adenine and thymine.
agree fairly
(21).
well with that of rRNA. q2 has a very different 24
base composi-
BIOCHEMICAL
Vol. 15, No. 1, 1964
Table 1.
AND BIOPHYSICAL
The base composition of FL cell Adenine
Uracil (Thymine)
RESEARCH COMMUNICATIONS
RNAand DNA (mole fk). Guanine
Cytosine
r-RNA(18 S)
19.4
19.4
34.1
27.5
rFtNA (31 S)
17.9
la.8
37.9
28.1
17.5
22.9 22.7
31.4 31.7
28.4
17.8
30.0 29.7
30.6 30.3
20.3 19.9
19.4
27.7
30.5
20.5
21.3
q1 RNA, Wt. ”
2
1
q2 RNA, Wt. n
1 2
DNA
28.1 19.6
rHNA: P32 was given to FL cells for 24 hours and the RNA extracted and fractionated by the methylated albumin column. The two i-RNA components were then separated by zone centrifugation on a sucrose gradient, collected, and hydrolyzed with 0.3 N KOHat 37oC for 18 hours. The resulting nucleotides uere separated on a Dcwex 1 (formate) column (Osaua, S., Takata, K. and Hotta, Y., Biochim. Biophys. Acta 28, 271 (1958)), and the radioactivity in the four nucleotide fractions was determined. The average of three determinations on a single sample is given. q and q2 RNA: RNAfrom 30 min. labeled cells was fractionated as in fig. 1. &everal fractions centering around the ql and q2 peaks were pooled separately, and the RNAwas collected by ethanol precipitation. To reduce cross contamination, each was further subjected to sucrose density gradient centrifugation, and the materisl in the region corresponding to ql and (12 of Pig. 2 was recovered (l/Z-Z/3 of the total radioactivity recovered). The RNAwas then analyzed as above with cold carrier RNAadded. The results in two independent experiments are given. DNA: DNAobtained from the column chromatography was purified by RNase treatment, then hydrolyced with 9546 formic acid at 17pC (sealed tube) for 30 min. The resulting bases mere then separated by paper chromatography and estimated by ultraviolet absorption. The average of three detenninations on one sample is given.
tion which is strikingly
similar
be of entirely
nature.
different
The rapidly
to DNA. The ql and q2 RNA's must therefore
labeled RNAwas thus shown to be separated into two com-
ponents, q1 and q2, with the size and base composition distinct other. indicated
from each
The "chase" experiments done in the presence of Actinomycin D also marked difference
more unstable.
between the two, q2 being metabolically
much
An obvious working hypothesis is that q1 is the precursor
of rRNA and q2 the messenger RNAof these cells. 25
Our results are in harmony
Vol. 15, No. 1, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with those of Schemer
et al.
(IZ),
pond to our q2 grossly
contaminated
these RNA's now in progress will
if their by ql.
be reported
45 S RNA is assumed to corresMetabolic in detail
We thank Miss Yoshiko Ogawa for her excellent aided in part by grants from the Waksmn Foundation Ministry
and other studies
of
elsewhere. help.
This work was
of Japan, Inc.,
and the
of Education.
REFERENCES
1. ;:
Jacob, F. and Monod, J., J. Mol. Biol. 2, 318 (1961). Nomura, M., Hall, B. D. and Spiegelman, S., J. Mol. Biol. 2, 306 (1960). Gras, F., Biatt, H., Gilbert, W.. Kurland, 0. C., Risebrough, R. W. and Watson, J. D., Nature m, 581 (1961). 4. Brenner, S., Jacob, F. and Meselson, M., Nature lj& 576 (1961). 5. Ishihama, A., Mizuno, N., Takai, Il.. Otaka, E. and Osawa, S., J. Mol. mol. 1. 2~. (1962). 6. Cheng, P., Biochim. Biophys. Acta 235 (1961). Hiatt, H. If., J. Mol. Biol. 2, 217 4 i962). 87:Marks, P. A., Willson, C., Kruh, J. and Gras, F., Biochem. Biophys. Res. Cam. f3, 9 (1962). H.. Nature s, 184 (1963). 9. Harris, 10. Kubinski, H. and Koch, G., J. Mol. Biol., 6, 102 (1963). Il. Schemer, K. and Darnell, J. E.. Biochem. Biophys. Res. Comm. 2, 486 (1962)
l
12. Scherrer, K., Latham, A. and Darnell, J. E., Proc. Natl. Acad. Sci. 3, 240 (1963). 13. Mandell, J. D. and Hershey, A. D., Anal. Biochem. 1, 66 (1960). 14. Sibatahi, A., de Kloet, S. R., Allfrey, V. G. and Mirs~y, A. E., Proc. Natl. Acad. Sci. 48, 471 (1962). 15. Georgiev, G. P. and Mantieva, V. E., Biochim. Biophys. Acta 61. 153 (1962). 16. Eagle, H., Science a, 432 (1959). A., Yamana, K., Kimura, K. and Takahashi, T., Nature lS6, 17. Sibatani, 215 (1960). 18. Pwrada, T. and Kawade. Y., Virology 2, 409 (1963). A., Exptl. Cell Research, Suppl. 2, 289 (1963). 19. Sibatani, 20. Kubinski, H., Koch, G. aud Drees. 0.. Biochim. glophys. Acta 6l, 332 (1962). 21. Magasahik, B., in "The Nucleic Acids" (E. Chargaff and 0. N. Davidson, eds.), Vol. 1, p. 373. Academic Press, New York 1955..
26
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SFPARATIONOF RAPIDLYLABLEJI FUiAOF ANIMAL CELLSINTO DNA-TYPEANDRIw)SOMALRNA-TYPECOMPONENTS M. Yoshikawa, T. Fukada and Y. Kawade Imtitute
Received
December
30,
for Virus Research, Kyoto University,
1963
The validity
of the messengerRNAhypothesis (1) has been well
supported by evidence from a variety (2-5).
Kyoto, Japan
of experiments in bacterial.
The messengerRNAis in general distinguished
from other cellular
RNA'S by its high turnover rate and a base composition similar animal cells, so clearly
however, the characteristics
defined,
systems
to DNA. In
of messenger RNAhave not been
although metabolically
active RNAwhich is rapidly
labeled by radioactive
precursors has been detected and studied in various
animal cells (6-12).
The results of Scherrer et al. (11, 12) appear to be
especislly
significant
groving HeLa cells
in that they obtained the rapidly
labeled RNAof
in the form of two molecular species with sedimentation
constants of 45 S and 35 S as estimated by sucrose density fugation.
gradient centri-
Roth of these were found to have a base composition similar
ribosomsl RNA(rRNA), and were shown to be essentially
to
composedof rRNA
precursor by "chase" experiments, although a small amount of metabolically unstable fraction rapidly
was also detected in them.
labeled RNAof growing FL cells
F'romour studies on the
(an established cell line derived
from humansmnion), we can now demonstrate its
separation into two compo-
nents by using the methylated albumin column (13), one having a base composition
similar
to DNA and the other similar
techniques provide a meansof isolating RNAof animal cells.
to rRNA. Thus, the present
what is likely
Such a DNA-like RNAfraction
to be the messenger
has been found in some
animal cells (14, 15), but it should be noted that in Our study the RNA appeared to be of a very large size and might represent the undegraded state* 22
BIOCHEMICAL
Vol. 15, No. 1, 1964
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FL cells uere grown in monolayers in 500 ml Roux bottles
uith 50 ml
of a phosphate-free Eagle's medium (16) containing double strength amino acids and vitamins and 10% calf serum. Labeling of nucleic acids was accomplished by adding P32 of varying period,
the labeling
After
labeling,
activities,
depending on the length of
together with 0.25 p/ml. each of the four nucleosides.
the cells were suspended in 0.14 M NaCl-0.01 M tris
buffer
at pH 8.1 containing 5 x 10B4 M EDTA. Nucleic acids were extracted dodecyl sulfate
sodium
(0.5$)-phenol method at 0-4'C.
95% of RNA and 9096of DNAcould be extracted, tion of labeled
by the
Under these conditions,
and no significant
fractiona-
RNAsuch as described by Sibatani et al. (17, 14) seemed
to take place. The nucleic acid thus obtained was fractionated albumin column according to Mandell and Hershey (13).
on a methylated The resolution
of the
two components of animal cell rRNA is poorer than in the case of bacterial rRNA (18-20). but could be much improved by keeping the column at 30-3pC (20).
A typical
chromatogram is shown in Ng. 1.
Soluble RNA, DNAand
rRNA were eluted at about 0.5, 0.75 and 0.95 M NaCl. respectively, rapidly
labeled RNAappeared later
material,
two radioactive
than rRNA. With the 30 min. pulse-labeled
peaks were observed at about 1.05 and 1.15 M NaCl,
which we call ql and q2, respectively
(Fig. 1).
peak soon becamesmall compared to ql, toward rRNA. After the radioactivity
and the
On longer labeling,
and the ql peak shifted progressively
24 hours (the generation time of the cell) almost paralleled
the q2
the absorbance profile
of labeling,
of rRNA.
Since the methylated albumin column separates RNAmolecules mainly according to their
size. ql and q2 may be supposed to have larger sizes than
rRNA. This was shown to be the case by sucrose density gradient gation of RNArecovered from the chromatographic fractions. 2, two radioactive
centrifu-
As seen in Fig.
peaks were observed ahead of rRNA, which are most likely
to correspond to the ql and q2 RNA. heir
sedimentation constants were
estimated to be about 40 and 50 S, respectively. 23
BIOCHEMICA!.
Vol. 15, No. 1, 1964
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cm .--. DNA
Tube Fig.
1.
Number
Chromatographic analysis of nucleic acids of FL cells labeled by P” (20 &/ml medium) for 30 min. Chromatography was performed according to Mandell and Hershey (13) but at 30°C. Elation was done by a NaCl gradient containing 0.01 M phosphate buffer at pH 6.7 and 5 x IO-’ M EDTA. with a flow rate of 3 ml/tube/IO min.
A258
:pm I--. 300
Fraotlon Fig.
2.
Number
Sucrose density gradient cennifugation of RNA labeled for 30 min. Ckomatographic fractions shown in Fig. 1 was collected by ethanol with rRNA added as carrier, and, after phenol treatment (to remove albumin released from the column), centrifuged on a linear sucrose ing 0.1 M NaCl, 0.1 mM MgC1, aod 0.01 M acetate buffer at pH 5.2, min. in a Hitachi SW40 rotor.
The labeled RNA in the precipitation together contaminating metbylated gradient (5-20s) containat 36,000 ‘pm for 210
The base compositions of various RNAuere determined by P32 counts incorporated It
into the four nucleotides.
The results
are given in Table 1.
can be seen that the rRNA has Fore guanine and cytosine than adenine and
uracil, trast
as is also found with other mammalianrRNA's
The DNA in con-
The composition of ql uas found to
is rich in adenine and thymine.
agree fairly
(21).
well with that of rRNA. q2 has a very different 24
base composi-
BIOCHEMICAL
Vol. 15, No. 1, 1964
Table 1.
AND BIOPHYSICAL
The base composition of FL cell Adenine
Uracil (Thymine)
RESEARCH COMMUNICATIONS
RNAand DNA (mole fk). Guanine
Cytosine
r-RNA(18 S)
19.4
19.4
34.1
27.5
rFtNA (31 S)
17.9
la.8
37.9
28.1
17.5
22.9 22.7
31.4 31.7
28.4
17.8
30.0 29.7
30.6 30.3
20.3 19.9
19.4
27.7
30.5
20.5
21.3
q1 RNA, Wt. ”
2
1
q2 RNA, Wt. n
1 2
DNA
28.1 19.6
rHNA: P32 was given to FL cells for 24 hours and the RNA extracted and fractionated by the methylated albumin column. The two i-RNA components were then separated by zone centrifugation on a sucrose gradient, collected, and hydrolyzed with 0.3 N KOHat 37oC for 18 hours. The resulting nucleotides uere separated on a Dcwex 1 (formate) column (Osaua, S., Takata, K. and Hotta, Y., Biochim. Biophys. Acta 28, 271 (1958)), and the radioactivity in the four nucleotide fractions was determined. The average of three determinations on a single sample is given. q and q2 RNA: RNAfrom 30 min. labeled cells was fractionated as in fig. 1. &everal fractions centering around the ql and q2 peaks were pooled separately, and the RNAwas collected by ethanol precipitation. To reduce cross contamination, each was further subjected to sucrose density gradient centrifugation, and the materisl in the region corresponding to ql and (12 of Pig. 2 was recovered (l/Z-Z/3 of the total radioactivity recovered). The RNAwas then analyzed as above with cold carrier RNAadded. The results in two independent experiments are given. DNA: DNAobtained from the column chromatography was purified by RNase treatment, then hydrolyced with 9546 formic acid at 17pC (sealed tube) for 30 min. The resulting bases mere then separated by paper chromatography and estimated by ultraviolet absorption. The average of three detenninations on one sample is given.
tion which is strikingly
similar
be of entirely
nature.
different
The rapidly
to DNA. The ql and q2 RNA's must therefore
labeled RNAwas thus shown to be separated into two com-
ponents, q1 and q2, with the size and base composition distinct other. indicated
from each
The "chase" experiments done in the presence of Actinomycin D also marked difference
more unstable.
between the two, q2 being metabolically
much
An obvious working hypothesis is that q1 is the precursor
of rRNA and q2 the messenger RNAof these cells. 25
Our results are in harmony
Vol. 15, No. 1, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with those of Schemer
et al.
(IZ),
pond to our q2 grossly
contaminated
these RNA's now in progress will
if their by ql.
be reported
45 S RNA is assumed to corresMetabolic in detail
We thank Miss Yoshiko Ogawa for her excellent aided in part by grants from the Waksmn Foundation Ministry
and other studies
of
elsewhere. help.
This work was
of Japan, Inc.,
and the
of Education.
REFERENCES
1. ;:
Jacob, F. and Monod, J., J. Mol. Biol. 2, 318 (1961). Nomura, M., Hall, B. D. and Spiegelman, S., J. Mol. Biol. 2, 306 (1960). Gras, F., Biatt, H., Gilbert, W.. Kurland, 0. C., Risebrough, R. W. and Watson, J. D., Nature m, 581 (1961). 4. Brenner, S., Jacob, F. and Meselson, M., Nature lj& 576 (1961). 5. Ishihama, A., Mizuno, N., Takai, Il.. Otaka, E. and Osawa, S., J. Mol. mol. 1. 2~. (1962). 6. Cheng, P., Biochim. Biophys. Acta 235 (1961). Hiatt, H. If., J. Mol. Biol. 2, 217 4 i962). 87:Marks, P. A., Willson, C., Kruh, J. and Gras, F., Biochem. Biophys. Res. Cam. f3, 9 (1962). H.. Nature s, 184 (1963). 9. Harris, 10. Kubinski, H. and Koch, G., J. Mol. Biol., 6, 102 (1963). Il. Schemer, K. and Darnell, J. E.. Biochem. Biophys. Res. Comm. 2, 486 (1962)
l
12. Scherrer, K., Latham, A. and Darnell, J. E., Proc. Natl. Acad. Sci. 3, 240 (1963). 13. Mandell, J. D. and Hershey, A. D., Anal. Biochem. 1, 66 (1960). 14. Sibatahi, A., de Kloet, S. R., Allfrey, V. G. and Mirs~y, A. E., Proc. Natl. Acad. Sci. 48, 471 (1962). 15. Georgiev, G. P. and Mantieva, V. E., Biochim. Biophys. Acta 61. 153 (1962). 16. Eagle, H., Science a, 432 (1959). A., Yamana, K., Kimura, K. and Takahashi, T., Nature lS6, 17. Sibatani, 215 (1960). 18. Pwrada, T. and Kawade. Y., Virology 2, 409 (1963). A., Exptl. Cell Research, Suppl. 2, 289 (1963). 19. Sibatani, 20. Kubinski, H., Koch, G. aud Drees. 0.. Biochim. glophys. Acta 6l, 332 (1962). 21. Magasahik, B., in "The Nucleic Acids" (E. Chargaff and 0. N. Davidson, eds.), Vol. 1, p. 373. Academic Press, New York 1955..
26