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Studies of nucleic acid metabolism in embryonic tissue culture with the aid of C14-labeled purines
Experimental
Cell Research,
6, 34635%
(1954)
345
STUDIES OF NUCLEIC ACID METABOLISM IN EMBRYONIC TISSUE CULTURE WITH THE AID OF C14-LABELED PURINESl K. Radiation
Research
H.
Laboratory and University
LU
and
T.
WINNICK
the Department of Iowa, Iowa
Received
October
of Biochemistry, City, U.S.A.
College
of Medicine,
6, 1953
from this laboratory have emphasized the use of tissue culture methods for biochemical investigations of cells under controlled conditions (5, 9). Up to the present time isotopic studies concerned with the biosynthesis and intermediate metabolism of nucleic acids have employed only radiophosphorus, P32 (7). The important advances resulting from the use of purines and pyrimidines labeled with radiocarbon, C14, in animal experiments suggest the value of these tools for in vitro systems. Thus, it has been shown that, following the administration of isotopic adenine to rats, both adenine and guanine of the tissue nucleic acids become labeled; but isotopic guanine is not effectively utilized for nucleic acid synthesis in several species (2). The indications are that guanine is most effective at the nucleotide level, i.e., as guanylic acid, derived from adenine via adenylic acid. In many studies emphasis is placed on the relative rates of renewal of ribose- and desoxyribose-nucleic acids (RNA and DNA) in various tissues. In this connection, the possible interconvertability of these two substances is receiving attention. The present paper is concerned with the relative rates of incorporation of C14-labeled adenine and guanine into DNA and RNA of chick heart tlbroblasts. In studying the pathway of nucleic acid synthesis, nutrient media have been employed with both free- and polynucleotide-bound labeled purines. For the latter type of experiment, isotopic adenine was injected into fertile eggs, and radioactive DNA and RNA preparations subsequently isolated. Each was shown to be labeled in both its purine constituents. By employing these nucleic acids in the embryonic extract (EE) medium, it has been possible to study the transfer of bound adenine and guanine from the nutrient solution to the explanted tissue. PUBLICATIONS
1 Aided
by a grant
from
the National
Institutes
of Health,
U.S.
Public
Health
Experimental
Service. Cell Research
6
K. H. Lu and T. Winnick MATERIALS
AND
METHODS
Adenine-8-W and guanine-8-C’” with specific activities of 1.0 and 2.2 mC per mM, respectively, were purchased from the Isotopes Specialties Co., Glendale, California, on allocation from the US. Atomic Energy Commission. Their radiochemical purity was at least 95 per cent, as determined by chromatography on starch columns. Labeled nucleic acids were prepared as follows: 3.9 pc of adenine-g-Cl4 (0.6 mg) were injected into each of a number of g-day fertile eggs. Four days later the embryos were dissected, and used for the isolation of DNA and RNA by the method of Hurlbert and Potter (8). The final preparations were free of protein, as judged by negative ninhydrin tests, and each‘displayed its characteristic ultraviolet absorption spectrum. The DNA gave a negative orcinol test (for RNA), and the RNA a negative diphenylamine reaction (for DNA). Neutral solutions of the adenine-8-C”, guanine-8-C14, labeled DNAl, and labeled RNA were sterilized by autoclaving, prior to addition to the EE medium. The latter represented a centrifuged 25 per cent extract of whole 13-day chick embryos (5). Embryonic chick heart fragments were cultivated in this nutrient according to the quantitative roller tube method previously described (5), with changes of medium at a-day intervals. A cultivation period of 6 days was employed, since this interval represented the time of maximum cellular proliferation (7). Approximately 20 tubes were used in each tissue culture experiment, in order to provide sufficient nucleoprotein for isolation purposes. After removal of the tubes from the roller, the medium was aspirated off, and the cultures washed with saline. The tissues were pooled, and a radioactive nucleoprotein fraction isolated (8). A portion of the latter was then converted to sodium nucleates. The specific radioactivities of both fractions were determined by counting weighed solid layers (5). The observed counts per minute per mg were converted to corresponding FM of labeled adenine or guanine, with the aid of the known specific activities of these purines. A carrier method was then employed for the isolation of pure nucleic acids from the main nucleoprotein fraction. To the latter was added 4 to 8 parts of a stock non-labeled nucleoprotein preparation (from 13-day embryonic thick tissues). The resulting isotopically-diluted material (about 0.5 to I gm in quantity) was used to isolate DNA and RNA. The radioactivities of these nucleic acids were determined by counting aliquots of solutions dried on glass cover slips. The appropriate numerical factor was used to convert these values into the original activities of the DNA and RNA of the heart cultures. As before, the results were expressed in PM of labeled purine incorporated, per mg nucleic acid. In order to determine the Cl4 distribution in the isolated nucleic acids, 3 to 4 mg of the latter were hydrolyzed in 1 N HCl for 1 hour at 100”. The hydrolysates were subjected to starch column chromatography, according to the Daly-Mirsky modification (3) of the Moore-Stein procedure. One ml fractions of effluent were collected 1 The DNA solution could not be filtered, evidence of decomposition upon autoclaving, DNA upon addition of acid. Experimenial
Cell Research
6
because as judged
of its collodial character. There by the complete reprecipitability
was no of the
Nucleic
acids in tissue culture
347
in the purine regions, and were individually transferred to glass cover slips, dried, and counted. Subsequently each cover slip was rinsed with 0.1 N HCl, and the dissolved purine measured in the Beckman spectrophotometer at appropriate wave length. In measuring radioactivity, the stronger samples were determined in the mica window Geiger counter, while with samples of relatively weak radioactivity, the windowless gas-flow counter was used, and sufficiently long counting times were employed to reduce the probable error to 2 to 5 per cent. RESULTS
AND
DISCUSSION
The addition of labeled adenine to EE medium resulted in a rather extensive incorporation of C l4 into the nucleoprotein of heart cultures (Table I). The value, 15.4 PM represents a utilization by the tissue of about 15 per cent of the total radioactivity employed in the three changes of nutrient. By contrast, the 0.7 PM value in the labeled guanine experiment corresponds to an uptake of less than one per cent of the total Cl*. This marked difference between the degrees of utilization of free adenine and guanine for nucleic acid synthesis in tissue culture is in agreement with in uiuo findings for several animal species (2). Following removal of the protein component, the mixed nucleic acid fraction from the adenine-S-Cl* experiment exhibited enhanced activity. Upon resolution of this fraction to its components, the specific activity of the DNA was 2.2 times that of the RNA. However, chemical analysis indicated 65 pgrn of RNA, but only 30 pgrn of DNA, per mg nucleoprotein. approximately equally into the two Accordingly the Cl* was incorporated types of nucleic acids. From the 93 PM value it may be calculated that approximately 7 per cent of the total purines in the DNA were labeled, while the corresponding RNA value (42 PM) reflects a 3 per cent labeling. TABLE
I
Relative rates of incorporation of free labeled purines into nucleic acids of heart cultures. Adenine and guanine were added to the 25 per cent EE at 0.100 and 0.109 mM concentrations, respectively. PM incorporated
I Purine added to medium
Adenine-8-W Guanine-8-W
Nucleoprotein
(
Protein
per gm isolated material I”~~~l:s~~~m/
DNA
/
RNA
...... ...... Experimental
Cell Research
6
K. H. Lu and T. Winnick HYDROLYZED RNA
10.6
HYDROLYZED
3
GUANINE ADENINE
r
40
n
50
60
VOLUME
OF
HYDROLYZED
RNA n
40
50
EFFLUENT
60
tz
I
(ML.)
HYDROLYZED
,
DNA
ADENINE GUANINE
94
,0.6
t0.3
t z ii5 tiY z 2
@” n 50 VOLUME
60
40
OF
EFFLUENT
50
6
c
60 (ML.)
Fig. 1. Chromatograms of nucleic acids isolated from embryonic chick heart cultivated in the presence of adenine-8G4. O-spectrophotometric determinations; Xradioactivity.
%
Fig. 2. Chromatograms of nucleic acids from heart cultivated in the presence of guanine-8-C14. O-spectrophotometric determinations; X-radioactivity.
In the guanine-S-Cl4 experiment, both DNA and RNA had very low radioactivity. However, RNA was higher in both specific activity and total Cl4 content (per mg nucleoprotein) than DNA. Figs. 1 and 2 indicate the total purine concentrations and the distribution of Cl4 between adenine and guanine in the RNA and DNA preparations of Table I. In no case was there significant radioactivity in the pyrimidine regions (not shown) of the chromatograms. The results are interpreted in Table II. Considering first the adenine-S-Cl4 experiment, it may be seen that both the adenine and guanine had higher Cl4 concentration in DNA than in RNA. The values 0.053 and 0.0325 indicate that approximately 5 per cent of the adenine and 3 per cent of the guanine residues in the RNA were labeled. The corresponding percentages were Experimental
Cell Research
6
Nucleic
acids in tissue culture
about 9 and 4 for DNA. These figures agree reasonably well with the average labeled purine values calculated for RNA and DNA from Table I. It will be indicated later that the polynucleotides of the EE were actually the major source of purines for tissue nucleic acid synthesis in the tibroblasts, particularly DNA. The higher activities in DNA appear to be characteristic of growth processes. Thus regenerating liver and tumor tissue incorporate adenine rapidly into DNA4 in uiuo, while in non-growing organs, such as liver, RNA has a higher turnover rate (1). In the last column of the upper half of Table II the adenine to guanine Cl4 ratios differed markedly for RNA and DNA. This difference may be due to the existence of discrete metabolic pools of nucleotides for RNA and DNA synthesis. The presence of guanine-8-C l4 in the nutrient medium (lower half of Table II) led to low Cl4 concentrations in the guanine of both RNA and DNA, and almost negligible radioactivities in adenine. Experiments such as those of Friedkin (6) with isotopic thymidine have indicated the usefulness of the embryonated egg for the biosynthetic preparation of labeled nucleic acids in relatively large quantities. Such preparations were needed in the present study for determinations of the utilization of polynucleotide purines in tissue culture media. Following the injection of adenine-g-Cl4 into fertile eggs (Table III), approximately 14 per cent of the radioactivity was recovered in 2 gm of nucleoprotein, isolated from the embryos. By way of comparison, thymidine-Cl4 (but not thymine) was incorporated into DNA of chick embryos to the extent of 23 per cent, between the fifth and eighth days of incubation (6). As in the cultivation experiments of Table I, the DNA in Table III had TABLE Summary Purine added medium
Guanine-S-Cl4
to
. . . . .
of chromatographic
Nucleic acid isolated
RNA DNA
Ratio
II data
of Figs.
of labeled Adenine
0.0004 0.0001
1 and
to total
2. purine
Guanine
0.0032 0.0040
Experimental
Ratio of adenine-Cl4 to guanine-Cl*
0.08 0.07
Cell Research
6
350
K. H. Lu and T. Winnick III
TABLE Incorporation
of labeled
adenine
Six g-day embryonated embryos were harvested.
eggs were
each
PM
CrP-purines
Nucleoprotein
Mixed
1.5
per
gm isolated
sodium 5.4
I
nucleic
acid
of labeled
of nucleic purine
acid
RNA DNA
I
0.0050 0.0070
Guanine 0.0042 0.0060
4 days
later
the
RNA
I
7.8
to total
embryos. and
fractions’
DNA
analysis
Adenine
of chick
adenine-8-P,
nucleic
I
Ratio
acids
with
nucleate
Chromatographic
Nucleic
into injected
3.2
I acids
Ratio to
of adenine-Cl4 guanine-Cl4
0.75 1.17
a higher specific activity than RNA. The chromatographic analyses indicated that from 0.4 to 0.7 per cent of each purine was labeled. The adenine to guanine C l4 ratios differed somewhat from those in the tissue culture experiments, as might be expected. Because of extensive dilution by nucleic acids in the eggs, the radioactivities of the isolated DNA and RNA in Table III were relatively low. However, the Cl4 concentrations were adequate for cultivation experiments, as is indicated in Table IV. In the second column of this Table, the value, 16, represents an uptake into tissue nucleoprotein of 11 per cent of the total RNA-Cl4 employed in the medium, while 40 corresponds to a 13 per cent utilization of the DNAC14. These figures are comparable in magnitude to the 15 per cent uptake of free adenine-S-C14, mentioned in connection with Table I. It is significant that both DNA and RNA of the cultures became radioactive when either type of labeled nucleic acid was present in the EE. The most probable assumption is that at least partial hydrolysis occurred, either in the medium, or following the absorption of the original labeled nucleic acid into the tissue cells. With either labeled RNA or DNA in the nutrient, the resulting DNA to RNA Cl4 ratio of the cultures was greater than unity, hut in view of the 1 No Experimental
carrier
was required Cell Research
in these 6
isolations.
Nucleic
acids in tissue culture TABLE
Relative
rates
of incorporation
of Cl*-labeled
DNA
IV and RNA
into
The radioactive DNA and RNA preparations of Table III specific activities of adenine and guanine were 915 and 785 and 655 and 550 cm/y&f for the RNA (calculated from the III). The averages of these pairs of values, 850 and 600 were in each fraction. Labeled nucleic acid added per 100 ml of 25 per cent EE
20 mg RNA.
. . . .
FM of labeled purines rated per gm isolated Nucleoprotein , DNA
16
81
351
incorpomaterial , RNA
50
nucleic
acids
were employed. counts/min./pM chromatographic used to calculate
of heart
cultures.
The theoretical for the DNA, data of Table FM of pmines
. 1 D~~%~~o
r!$o~~~$!k
1.6
0.63 RNA) 3.25 (for DNA) (for
25 mg DNA
. . . . .
40
412
176
2.3
greater abundance of RNA in the cultures (previously mentioned), it may be concluded that the RNA utilized as much of the labeled nucleic acid of the medium as did DNA, in tissue synthesis. Calculated in terms of percentage labeling, the nucleic acid values indicate that, with radioactive RNA in the medium, 6 and 3.7 per cent of the purines in DNA and RNA, respectively, became labeled. When radioactive DNA was employed, the corresponding percentages were 30 and 13. These relatively high values cannot safely be interpreted as indicating corresponding degrees of net nucleic acid synthesis, since probably turnover processes were also operative. It is of interest that the adenine-to-guanine Cl4 ratio of the tissue RNA (0.63) differed only slightly from that of the original labeled RNA of the medium, 0.75 (Table III), while the corresponding ratios for the DNA were 3.25 and 1.17 (Table III). In considering these changes it is likely that hydrolysis of the labeled nucleic acids released isotopic adenine and guanine nucleotides. The isotopic adenylic acid could then form additional guanylic acid, and this process would favor a decrease in the adenine-to-guanine Cl4 ratio of the tissue nucleic acids. However, the lowering observed for RNA was insignificant, while the ratio increased with DNA. The interpretation of the present results is rendered complex by several factors: 1) The exact degrees of hydrolysis of the labeled nucleic acids are not known. Probably free purines were not released. If this had occurred, guanine could not be re-utilized effectively. Experimental
Cell
Research
6
352
K. H. Lu and T. Winnick
2) The bound labeled purines in the medium were utilized for both DNA and RNA synthesis. Probably adenine was incorporated in preference to guanine into tissues, as in Table III. 3) The nucleic acids normally present in the EE are known to play an important role in the growth of the cultures. As the studies of Davidson and co-workers (4) indicate, the DNA and (particularly) the RNA content increases in heart tibroblasts cultivated in embryonic extract medium. 4) Recent chemical studies suggest that both DNA and RNA are heterogeneous substances of exceedingly complex structure. Studies now in progress in this laboratory are concerned with the further elucidation of the pathway of nucleic acid synthesis in tissue culture, with emphasis on the exact nutritional requirements of embryonic cells, and the effect of experimental variations in the environment on synthetic processes. SUMMARY
C14-labeled adenine was readily utilized for nucleic acid synthesis in embryonic chick heart cultures. DNA and RNA, isolated from these cultures, contained radioactivity in both adenine and guanine constituents. C14labeled guanine was relatively poorly utilized, and was not significantly converted to polynucleotide adenine. Labeled DNA and RNA, prepared by injecting Cr4-adenine into fertile eggs, were used to study the transfer of bound purines from the nutrient medium to the cultivated tissue. The DNA, containing isotopic adenine and guanine, gave rise to double labeling in both the DNA and RNA of the cultures. Similar results were obtained with radioactive RNA in the medium. These findings were interpreted as indicating hydrolysis of the nucleic acids, with a subsequent utilization of resulting nucleotides for tissue synthesis. REFERENCES 1. 2. 3. 4. 5. 6.
RROWN, G. B., J. Cellular ~ Ann. Reu. Biochem., DALY, M. M.. and MIRSKY.
Comp. Physiol., 38, Supplement 1, 121 (1951). 22, 141 (1953). A. E., J. Biol. Chem.. 179, 981 (1949). DAVIDSON, J: N., LESLIE, i., and’ WAYMOUTH, C:, B&hem. ‘.I., 44, 5 (1949). FRANCIS, M. D., and WINNICK, T., J. Biol. Chem., 202, 273 (1953). FRIEDKI&, M., Paper presented at the September 1953 meeting of the American Society, Chicago. I. GERARDE. H. W.. JONES, M., and WINNICK, T., .I. Biol. Chem., 196, 69 (1952). R. B:, and POTTER, V. R., ibid.., 165, 257 (1952). 8. HURLBE&, 9. WINNICK, R. E., and WINNICK, T., J. Nat!., Cancer Inst., 14, 519 (1953).
Experimental
Cell Research
6
Chemical
Cell Research,
6, 34635%
(1954)
345
STUDIES OF NUCLEIC ACID METABOLISM IN EMBRYONIC TISSUE CULTURE WITH THE AID OF C14-LABELED PURINESl K. Radiation
Research
H.
Laboratory and University
LU
and
T.
WINNICK
the Department of Iowa, Iowa
Received
October
of Biochemistry, City, U.S.A.
College
of Medicine,
6, 1953
from this laboratory have emphasized the use of tissue culture methods for biochemical investigations of cells under controlled conditions (5, 9). Up to the present time isotopic studies concerned with the biosynthesis and intermediate metabolism of nucleic acids have employed only radiophosphorus, P32 (7). The important advances resulting from the use of purines and pyrimidines labeled with radiocarbon, C14, in animal experiments suggest the value of these tools for in vitro systems. Thus, it has been shown that, following the administration of isotopic adenine to rats, both adenine and guanine of the tissue nucleic acids become labeled; but isotopic guanine is not effectively utilized for nucleic acid synthesis in several species (2). The indications are that guanine is most effective at the nucleotide level, i.e., as guanylic acid, derived from adenine via adenylic acid. In many studies emphasis is placed on the relative rates of renewal of ribose- and desoxyribose-nucleic acids (RNA and DNA) in various tissues. In this connection, the possible interconvertability of these two substances is receiving attention. The present paper is concerned with the relative rates of incorporation of C14-labeled adenine and guanine into DNA and RNA of chick heart tlbroblasts. In studying the pathway of nucleic acid synthesis, nutrient media have been employed with both free- and polynucleotide-bound labeled purines. For the latter type of experiment, isotopic adenine was injected into fertile eggs, and radioactive DNA and RNA preparations subsequently isolated. Each was shown to be labeled in both its purine constituents. By employing these nucleic acids in the embryonic extract (EE) medium, it has been possible to study the transfer of bound adenine and guanine from the nutrient solution to the explanted tissue. PUBLICATIONS
1 Aided
by a grant
from
the National
Institutes
of Health,
U.S.
Public
Health
Experimental
Service. Cell Research
6
K. H. Lu and T. Winnick MATERIALS
AND
METHODS
Adenine-8-W and guanine-8-C’” with specific activities of 1.0 and 2.2 mC per mM, respectively, were purchased from the Isotopes Specialties Co., Glendale, California, on allocation from the US. Atomic Energy Commission. Their radiochemical purity was at least 95 per cent, as determined by chromatography on starch columns. Labeled nucleic acids were prepared as follows: 3.9 pc of adenine-g-Cl4 (0.6 mg) were injected into each of a number of g-day fertile eggs. Four days later the embryos were dissected, and used for the isolation of DNA and RNA by the method of Hurlbert and Potter (8). The final preparations were free of protein, as judged by negative ninhydrin tests, and each‘displayed its characteristic ultraviolet absorption spectrum. The DNA gave a negative orcinol test (for RNA), and the RNA a negative diphenylamine reaction (for DNA). Neutral solutions of the adenine-8-C”, guanine-8-C14, labeled DNAl, and labeled RNA were sterilized by autoclaving, prior to addition to the EE medium. The latter represented a centrifuged 25 per cent extract of whole 13-day chick embryos (5). Embryonic chick heart fragments were cultivated in this nutrient according to the quantitative roller tube method previously described (5), with changes of medium at a-day intervals. A cultivation period of 6 days was employed, since this interval represented the time of maximum cellular proliferation (7). Approximately 20 tubes were used in each tissue culture experiment, in order to provide sufficient nucleoprotein for isolation purposes. After removal of the tubes from the roller, the medium was aspirated off, and the cultures washed with saline. The tissues were pooled, and a radioactive nucleoprotein fraction isolated (8). A portion of the latter was then converted to sodium nucleates. The specific radioactivities of both fractions were determined by counting weighed solid layers (5). The observed counts per minute per mg were converted to corresponding FM of labeled adenine or guanine, with the aid of the known specific activities of these purines. A carrier method was then employed for the isolation of pure nucleic acids from the main nucleoprotein fraction. To the latter was added 4 to 8 parts of a stock non-labeled nucleoprotein preparation (from 13-day embryonic thick tissues). The resulting isotopically-diluted material (about 0.5 to I gm in quantity) was used to isolate DNA and RNA. The radioactivities of these nucleic acids were determined by counting aliquots of solutions dried on glass cover slips. The appropriate numerical factor was used to convert these values into the original activities of the DNA and RNA of the heart cultures. As before, the results were expressed in PM of labeled purine incorporated, per mg nucleic acid. In order to determine the Cl4 distribution in the isolated nucleic acids, 3 to 4 mg of the latter were hydrolyzed in 1 N HCl for 1 hour at 100”. The hydrolysates were subjected to starch column chromatography, according to the Daly-Mirsky modification (3) of the Moore-Stein procedure. One ml fractions of effluent were collected 1 The DNA solution could not be filtered, evidence of decomposition upon autoclaving, DNA upon addition of acid. Experimenial
Cell Research
6
because as judged
of its collodial character. There by the complete reprecipitability
was no of the
Nucleic
acids in tissue culture
347
in the purine regions, and were individually transferred to glass cover slips, dried, and counted. Subsequently each cover slip was rinsed with 0.1 N HCl, and the dissolved purine measured in the Beckman spectrophotometer at appropriate wave length. In measuring radioactivity, the stronger samples were determined in the mica window Geiger counter, while with samples of relatively weak radioactivity, the windowless gas-flow counter was used, and sufficiently long counting times were employed to reduce the probable error to 2 to 5 per cent. RESULTS
AND
DISCUSSION
The addition of labeled adenine to EE medium resulted in a rather extensive incorporation of C l4 into the nucleoprotein of heart cultures (Table I). The value, 15.4 PM represents a utilization by the tissue of about 15 per cent of the total radioactivity employed in the three changes of nutrient. By contrast, the 0.7 PM value in the labeled guanine experiment corresponds to an uptake of less than one per cent of the total Cl*. This marked difference between the degrees of utilization of free adenine and guanine for nucleic acid synthesis in tissue culture is in agreement with in uiuo findings for several animal species (2). Following removal of the protein component, the mixed nucleic acid fraction from the adenine-S-Cl* experiment exhibited enhanced activity. Upon resolution of this fraction to its components, the specific activity of the DNA was 2.2 times that of the RNA. However, chemical analysis indicated 65 pgrn of RNA, but only 30 pgrn of DNA, per mg nucleoprotein. approximately equally into the two Accordingly the Cl* was incorporated types of nucleic acids. From the 93 PM value it may be calculated that approximately 7 per cent of the total purines in the DNA were labeled, while the corresponding RNA value (42 PM) reflects a 3 per cent labeling. TABLE
I
Relative rates of incorporation of free labeled purines into nucleic acids of heart cultures. Adenine and guanine were added to the 25 per cent EE at 0.100 and 0.109 mM concentrations, respectively. PM incorporated
I Purine added to medium
Adenine-8-W Guanine-8-W
Nucleoprotein
(
Protein
per gm isolated material I”~~~l:s~~~m/
DNA
/
RNA
...... ...... Experimental
Cell Research
6
K. H. Lu and T. Winnick HYDROLYZED RNA
10.6
HYDROLYZED
3
GUANINE ADENINE
r
40
n
50
60
VOLUME
OF
HYDROLYZED
RNA n
40
50
EFFLUENT
60
tz
I
(ML.)
HYDROLYZED
,
DNA
ADENINE GUANINE
94
,0.6
t0.3
t z ii5 tiY z 2
@” n 50 VOLUME
60
40
OF
EFFLUENT
50
6
c
60 (ML.)
Fig. 1. Chromatograms of nucleic acids isolated from embryonic chick heart cultivated in the presence of adenine-8G4. O-spectrophotometric determinations; Xradioactivity.
%
Fig. 2. Chromatograms of nucleic acids from heart cultivated in the presence of guanine-8-C14. O-spectrophotometric determinations; X-radioactivity.
In the guanine-S-Cl4 experiment, both DNA and RNA had very low radioactivity. However, RNA was higher in both specific activity and total Cl4 content (per mg nucleoprotein) than DNA. Figs. 1 and 2 indicate the total purine concentrations and the distribution of Cl4 between adenine and guanine in the RNA and DNA preparations of Table I. In no case was there significant radioactivity in the pyrimidine regions (not shown) of the chromatograms. The results are interpreted in Table II. Considering first the adenine-S-Cl4 experiment, it may be seen that both the adenine and guanine had higher Cl4 concentration in DNA than in RNA. The values 0.053 and 0.0325 indicate that approximately 5 per cent of the adenine and 3 per cent of the guanine residues in the RNA were labeled. The corresponding percentages were Experimental
Cell Research
6
Nucleic
acids in tissue culture
about 9 and 4 for DNA. These figures agree reasonably well with the average labeled purine values calculated for RNA and DNA from Table I. It will be indicated later that the polynucleotides of the EE were actually the major source of purines for tissue nucleic acid synthesis in the tibroblasts, particularly DNA. The higher activities in DNA appear to be characteristic of growth processes. Thus regenerating liver and tumor tissue incorporate adenine rapidly into DNA4 in uiuo, while in non-growing organs, such as liver, RNA has a higher turnover rate (1). In the last column of the upper half of Table II the adenine to guanine Cl4 ratios differed markedly for RNA and DNA. This difference may be due to the existence of discrete metabolic pools of nucleotides for RNA and DNA synthesis. The presence of guanine-8-C l4 in the nutrient medium (lower half of Table II) led to low Cl4 concentrations in the guanine of both RNA and DNA, and almost negligible radioactivities in adenine. Experiments such as those of Friedkin (6) with isotopic thymidine have indicated the usefulness of the embryonated egg for the biosynthetic preparation of labeled nucleic acids in relatively large quantities. Such preparations were needed in the present study for determinations of the utilization of polynucleotide purines in tissue culture media. Following the injection of adenine-g-Cl4 into fertile eggs (Table III), approximately 14 per cent of the radioactivity was recovered in 2 gm of nucleoprotein, isolated from the embryos. By way of comparison, thymidine-Cl4 (but not thymine) was incorporated into DNA of chick embryos to the extent of 23 per cent, between the fifth and eighth days of incubation (6). As in the cultivation experiments of Table I, the DNA in Table III had TABLE Summary Purine added medium
Guanine-S-Cl4
to
. . . . .
of chromatographic
Nucleic acid isolated
RNA DNA
Ratio
II data
of Figs.
of labeled Adenine
0.0004 0.0001
1 and
to total
2. purine
Guanine
0.0032 0.0040
Experimental
Ratio of adenine-Cl4 to guanine-Cl*
0.08 0.07
Cell Research
6
350
K. H. Lu and T. Winnick III
TABLE Incorporation
of labeled
adenine
Six g-day embryonated embryos were harvested.
eggs were
each
PM
CrP-purines
Nucleoprotein
Mixed
1.5
per
gm isolated
sodium 5.4
I
nucleic
acid
of labeled
of nucleic purine
acid
RNA DNA
I
0.0050 0.0070
Guanine 0.0042 0.0060
4 days
later
the
RNA
I
7.8
to total
embryos. and
fractions’
DNA
analysis
Adenine
of chick
adenine-8-P,
nucleic
I
Ratio
acids
with
nucleate
Chromatographic
Nucleic
into injected
3.2
I acids
Ratio to
of adenine-Cl4 guanine-Cl4
0.75 1.17
a higher specific activity than RNA. The chromatographic analyses indicated that from 0.4 to 0.7 per cent of each purine was labeled. The adenine to guanine C l4 ratios differed somewhat from those in the tissue culture experiments, as might be expected. Because of extensive dilution by nucleic acids in the eggs, the radioactivities of the isolated DNA and RNA in Table III were relatively low. However, the Cl4 concentrations were adequate for cultivation experiments, as is indicated in Table IV. In the second column of this Table, the value, 16, represents an uptake into tissue nucleoprotein of 11 per cent of the total RNA-Cl4 employed in the medium, while 40 corresponds to a 13 per cent utilization of the DNAC14. These figures are comparable in magnitude to the 15 per cent uptake of free adenine-S-C14, mentioned in connection with Table I. It is significant that both DNA and RNA of the cultures became radioactive when either type of labeled nucleic acid was present in the EE. The most probable assumption is that at least partial hydrolysis occurred, either in the medium, or following the absorption of the original labeled nucleic acid into the tissue cells. With either labeled RNA or DNA in the nutrient, the resulting DNA to RNA Cl4 ratio of the cultures was greater than unity, hut in view of the 1 No Experimental
carrier
was required Cell Research
in these 6
isolations.
Nucleic
acids in tissue culture TABLE
Relative
rates
of incorporation
of Cl*-labeled
DNA
IV and RNA
into
The radioactive DNA and RNA preparations of Table III specific activities of adenine and guanine were 915 and 785 and 655 and 550 cm/y&f for the RNA (calculated from the III). The averages of these pairs of values, 850 and 600 were in each fraction. Labeled nucleic acid added per 100 ml of 25 per cent EE
20 mg RNA.
. . . .
FM of labeled purines rated per gm isolated Nucleoprotein , DNA
16
81
351
incorpomaterial , RNA
50
nucleic
acids
were employed. counts/min./pM chromatographic used to calculate
of heart
cultures.
The theoretical for the DNA, data of Table FM of pmines
. 1 D~~%~~o
r!$o~~~$!k
1.6
0.63 RNA) 3.25 (for DNA) (for
25 mg DNA
. . . . .
40
412
176
2.3
greater abundance of RNA in the cultures (previously mentioned), it may be concluded that the RNA utilized as much of the labeled nucleic acid of the medium as did DNA, in tissue synthesis. Calculated in terms of percentage labeling, the nucleic acid values indicate that, with radioactive RNA in the medium, 6 and 3.7 per cent of the purines in DNA and RNA, respectively, became labeled. When radioactive DNA was employed, the corresponding percentages were 30 and 13. These relatively high values cannot safely be interpreted as indicating corresponding degrees of net nucleic acid synthesis, since probably turnover processes were also operative. It is of interest that the adenine-to-guanine Cl4 ratio of the tissue RNA (0.63) differed only slightly from that of the original labeled RNA of the medium, 0.75 (Table III), while the corresponding ratios for the DNA were 3.25 and 1.17 (Table III). In considering these changes it is likely that hydrolysis of the labeled nucleic acids released isotopic adenine and guanine nucleotides. The isotopic adenylic acid could then form additional guanylic acid, and this process would favor a decrease in the adenine-to-guanine Cl4 ratio of the tissue nucleic acids. However, the lowering observed for RNA was insignificant, while the ratio increased with DNA. The interpretation of the present results is rendered complex by several factors: 1) The exact degrees of hydrolysis of the labeled nucleic acids are not known. Probably free purines were not released. If this had occurred, guanine could not be re-utilized effectively. Experimental
Cell
Research
6
352
K. H. Lu and T. Winnick
2) The bound labeled purines in the medium were utilized for both DNA and RNA synthesis. Probably adenine was incorporated in preference to guanine into tissues, as in Table III. 3) The nucleic acids normally present in the EE are known to play an important role in the growth of the cultures. As the studies of Davidson and co-workers (4) indicate, the DNA and (particularly) the RNA content increases in heart tibroblasts cultivated in embryonic extract medium. 4) Recent chemical studies suggest that both DNA and RNA are heterogeneous substances of exceedingly complex structure. Studies now in progress in this laboratory are concerned with the further elucidation of the pathway of nucleic acid synthesis in tissue culture, with emphasis on the exact nutritional requirements of embryonic cells, and the effect of experimental variations in the environment on synthetic processes. SUMMARY
C14-labeled adenine was readily utilized for nucleic acid synthesis in embryonic chick heart cultures. DNA and RNA, isolated from these cultures, contained radioactivity in both adenine and guanine constituents. C14labeled guanine was relatively poorly utilized, and was not significantly converted to polynucleotide adenine. Labeled DNA and RNA, prepared by injecting Cr4-adenine into fertile eggs, were used to study the transfer of bound purines from the nutrient medium to the cultivated tissue. The DNA, containing isotopic adenine and guanine, gave rise to double labeling in both the DNA and RNA of the cultures. Similar results were obtained with radioactive RNA in the medium. These findings were interpreted as indicating hydrolysis of the nucleic acids, with a subsequent utilization of resulting nucleotides for tissue synthesis. REFERENCES 1. 2. 3. 4. 5. 6.
RROWN, G. B., J. Cellular ~ Ann. Reu. Biochem., DALY, M. M.. and MIRSKY.
Comp. Physiol., 38, Supplement 1, 121 (1951). 22, 141 (1953). A. E., J. Biol. Chem.. 179, 981 (1949). DAVIDSON, J: N., LESLIE, i., and’ WAYMOUTH, C:, B&hem. ‘.I., 44, 5 (1949). FRANCIS, M. D., and WINNICK, T., J. Biol. Chem., 202, 273 (1953). FRIEDKI&, M., Paper presented at the September 1953 meeting of the American Society, Chicago. I. GERARDE. H. W.. JONES, M., and WINNICK, T., .I. Biol. Chem., 196, 69 (1952). R. B:, and POTTER, V. R., ibid.., 165, 257 (1952). 8. HURLBE&, 9. WINNICK, R. E., and WINNICK, T., J. Nat!., Cancer Inst., 14, 519 (1953).
Experimental
Cell Research
6
Chemical