University of Southern California Dissertations and Theses

Metabolism of mammalian deoxyribonucleic acid and nuclear ribonucleic acid

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Metabolism of mammalian deoxyribonucleic acid and nuclear ribonucleic acid

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Content METABOLISM OF MAMMALIAN
DEOXYRIBONUCLEIC ACID
AND
NUCLEAR RIBONUCLEIC ACID
by
Nancy Joy Stone
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Biology)
January 1964
UNIVERSITY O F SOUTHERN CALIFORNIA
GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES 7, C ALIFORNIA
This dissertation, written by
Nancy Joy Stone
under the direction of hQXt...Dissertation C o m
mittee, and approved by all its members, has
been presented to and accepted by the Graduate
School, in partial fulfillment of requirements
for the degree of
D O C T O R O F P H I L O S O P H Y
Dean
1.963......
DISSERTATIO N C O M M IT T E E *
.
n Chairman
..
ACKNOWLEDGEMENTS
I would like to express my gratitude to the following
people who have helped me during the course of my graduate
studies: Dr. Walter Martin, who served as chairman of
both my dissertation and guidance committees, and provided
the facilities of his laboratory for the research reported
in this dissertation; Dr. Leslie Chambers, who served on
both my dissertation and guidance committees, and kindly
assumed the responsibility of serving as chairman of my
dissertation committee in Dr. Martin's absence; Dr. Donald
Visser, who served on both my dissertation and guidance
committees, and spent many hours imbuing me with the prin
ciples of biochemical research; Dr. Myles Maxfield, who
served on my dissertation committee in Dr. Martin's ab
sence; Dr. T. T. Chen and Dr. Lyell Thomas Jr., who served
as members of my guidance committee.
I would like to thank Dr. Louis Stearns, Dr. Matthew
Meselson, Dr. Hans Bichsel, and Dr. Marcelo Nimni for
many helpful discussions. I am grateful to Mr. Prank
Ciofalo, Mr. Robert Gee, and Mr. Stephen Zam for help with
the radioactivity measurements.
This work would not have been possible without the
support and encouragement of my parents to whom I give my
sincerest thanks.
- iii -
TABLE OF CONTENTS
I. METABOLIC CHARACTERISTICS OF MAMMALIAN
DEOXYRIBONUCLEIC ACID............................... 1
Purpose of the Project
History of DNA Heterogeneity
Theoretical Background for Studying Formate
Incorporation into DNA Fractions
Experimental Methods
Animal Preparation and Nucleic Acid Isolation
Fractionation of DNA and Its Bases
Radioactivity Measurements
Results and Discussion
Derivation of the Mathematical Model
Summary
II. FORMATE METABOLISM IN NUCLEAR NUCLEIC ACIDS......... 22
Purposes of the Project
History of Precursor Incorporation into
Nuclear Nucleic Acids
Cytological Studies
Incorporation of Nucleic Acid Precursors
into Nucleic Acids of Different
Mammalian Organs
Kinetics of Isotope Incorporation into
Synchronously Dividing Cells Including
Regenerating Liver
Synthesis of Nuclear Nucleic Acids in
Isolated Nuclei
Theoretical Background for Studying Isotope
Incorporation into Nucleic Acid Fractions
Experimental Methods
Results
Discussion of Results
Formate Metabolism in Mammalian Organs
A Paradigm of Normality
Summary
BIBLIOGRAPHY
60
LIST OF FIGURES
FIGURE PAGE
1 The Folic Acid Interconversion System........ 77
2 Flow Diagram of Isolation Procedures of
Nucleic Acids....................... 78
v -
LIST OF TABLES
TABLE PAGE
I Specific Radioactivity of High A-T DNA.......80, 81
II Specific Radioactivity of Low A-T DNA........82, 83
III Specific Radioactivities of Adenine,
Guanine, and Thymine in High A-T DNA.......84-
IV Specific Radioactivities of Adenine,
Guanine, and Thymine in Low A-T DNA........83
V Mean Values of the Specific Radioactivities
of the Bases, Adenine, Guanine, and
Thymine in Extractable-DNA (High A-T
DNA and Low A-T DNA)....................... 86
VI Ratios of the Specific Radioactivities of
High A-T DNA and Low A-T DNA in Ex-
tractable-DNA. Ratios of the Mean
Values for the Specific Radioactivities
of Adenine, Guanine, and Thymine in
Extractable-DNA............................ 87
VII Specific Radioactivities of Adenine, Guanine
and Thymine in Nonextractable-DNA.......... 88
VIII Specific Radioactivities of Adenine and
Guanine in Chromatin-RNA................... 89
IX Specific Radioactivities of Adenine and
Guanine in Ethanol-RNA..................... 90
- vi -
i
LIST OP TABLES
(Continued)
TABLE PAGE
X Ratios of the Specific Radioactivities of
the Bases in Nonextractable-DNA,
Chromatin-RNA, and Ethanol-RNA and
the Specific Radioactivities of the
Bases in Extractable-DNA............. 91, 92
XI Ratios of the Values for the Specific
Radioactivities of Adenine, Guanine,
and Thymine in Nonextractable-DNA......... 93
- vii -
CHAPTER I
METABOLIC CHARACTERISTIC OF
MAMMALIAN DEOXYRIBONUCLEIC ACID
Purpose of the Project
The purpose for which this project was performed was
to derive a mathematical model which would explain appar
ent and real metabolic heterogeneity in fractions of
mammalian deoxyribonucleic acid (DNA).
History of DNA Heterogeneity
Goldthwait and Bendich (1952) and Bendich, Russell,
and Brown (1953) reported demonstrating heterogeneity in
14
the metabolism of C -labeled formate by studying its in
corporation into the purines of DNA in different organs of
rats. Their experiments involved daily administration of
labeled formate for a period of three days and extraction
of DNA from dried tissue residues with ten percent sodium
chloride (NaCl) at 85° Centigrade (C.)* The DNA which
they isolated could be separated into fractions with dif
ferent solubility properties in the various organs, and
there was concommitant heterogeneity in the formate in
corporation into guanine. Holbrook, Irvin, Irvin, and
Rotherham (I960), studying the incorporation of glycine-
14
1-C in liver and hepatoma nuclear nucleic acids twenty
- 1 -
2
hours after the administration of the labeled glycine,
found heterogeneity in labeling of adenine and guanine in
different fractions of the two types of tissues.
Meselson, Stahl, and Vinograd (1957) and Meselson and
Stahl (1958) developed a technique whereby the heterogene
ity of DNA with respect to adenine + thymine: guanine +
cytosine (A + T: G + C) ratios and weight average molecu
lar weight could be demonstrated from the buoyant density
of DNA, which forms narrow bands in density gradients of
cesium chloride (CsCl) solutions during equilibrium centri
fugation. Using this method Rolfe and Meselson (1959),
Sueoka, Marmur, and Doty (1959), and Schildkraut, Marmur,
and Doty (1962) found that A + T: G + C ratios are distri
buted over a fairly narrow range in any species. Kit
(1961) demonstrated that the DNA from different mouse or
gans and tumors has the same distribution of A + T: G + C
ratios using the CsCl method. Doty, Marmur, and Sueoka
(1959) correlated ultraviolet absorbance characteristics
of DNA solutions as a function of temperature with the
A + T: G + C ratios. The rise in absorbance of DNA solu
tions was interpreted to be the result of disruption of
the hydrogen bonds stabilizing the DNA double helix and
subsequent denaburation or melting of the helix. At this
time DNA was transformed from a stiff double helical
structure to a random coil, and its molecular weight was
halved. The results they obtained using this technique
3
were similar to those obtained using the CsCl method. The
midpoints (Tm) of the melting curves were found to depend
on the mole percentage of guanine plus cytosine in the DNA
and the salt concentration of the incubation medium. The
T of calf thymus DNA was found to be 86° c. in 0.15 Molar
m
(M.) NaCl, 0.001 M. sodium citrate. Kit (I960), using
melting curve data, found a Tf f i of 84-° C. for mammalian
DNA. Kleinschmidt (1959) found no significant differences
in base composition of normal and neoplastic mouse and
chicken DNA using quantitative biochemical techniques.
Agrell and Bergqvist (1962), using cytochemical tech
niques, found a bimodal peak in a heat-stability versus
hydrolysis^time plot of DNA in synchronously dividing
embryonic cells. However, in cells which are not dividing
synchonously, biochemical demonstration of this manner of
heterogeneity would be impossible.
One can infer from the foregoing discussion that com
positional heterogeneity which Goldthwait and Bendich
(1952) and Bendich et al. (1953) found in different mam
malian organs was an artifact produced by the isolation
procedure. The differences in solubility characteristics
of the DNA reflected differences in physical state and
molecular weight and not genetic differences in these or
gans. Holbrook et al. (I960), who found differences in
glycine incorporation into adenine and guanine of ribonu
cleic acid (RNA) and DNA fractions in normal and tumor
4
tissue which were probably significant, did not provide
more general information about the metabolism of glycine
in several types of normal tissue.
Gerber, Gerber, and Altman (1960a) administered tri-
tiated thymidine to rats and calculated the replacement
times of DNA in various organs fit>m the rates of incorpora
tion and loss of label. They found both fast and slow
components in intestine, spleen, and thymus. However,
this can be explained by loss of cells from these tissues
under normal conditions.
Kit (1961) and Sueoka (1962), using mammalian DNA
isolated by a phenol extraction method, found satellite
bands during equilibrium centrifucation of DNA in CsCl
density gradients. The metabolic characteristics of
these satellite bands were not investigated. Rolfe (1963),
who found a less buoyant satellite band containing ten
percent of the cellular DNA within the main band of Es
cherichia coli DNA, postulated that the satellite band
contained DNA which was combined with protein and in the
process of replication.
Theoretical Background for Studying Formate Incor
poration into DNA Fractions
When formate is administered to animals, the carbon
atom becomes incorporated into the two' and eight' posi
tions of the purines (Buchanan and Hartman, 1959) and the
methyl groups of thymine and 5-methylcytosine (Reichard,
1959) with a high degree of efficiency via pathways in
volving folic acid derivatives (Figure 1). Since the
purines, adenine and guanine, and the pyrimidine, thymine,
are major constituents of animal DNA (Chargaff, 1955),
much information regarding the biosynthesis of DNA can be
14-
obtained by administration of C -labeled formate and
studying its incorporation into adenine, guanine, and thy
mine of various types of nucleic acids.
The specific radioactivity of DNA and its constituent
in the various organs may be a reflection of the rate at
which cell division is proceeding in these tissues. Men
delsohn, Dohan, and Moore (I960) using mouse breast cancer,
and Sisken and Kinosita (1961), using kitten lung and hu
man amnion cells in tissue culture, showed that, "While
the progress of cells through the early part of the mito
tic cycle varies in different populations, once DNA syn
thesis commences the cells proceed to division at constant
rate."
Living cells have populations of DNA molecules in
which the A + T: G + C ratios are distributed over a cir
cumscribed and measurable range (Sueoka et al., 1959)•
Sueoka (1961a) has correlated these ratios with the con
tents of most of the amino acids found in bacteria cells
and found significant dependence of the content of some
amino acids on the A + T: G + C ratios. Because of this
one might infer that DNA molecules with differing A + T:
G + C ratios would he metabolically distinct.
The possible existence of metabolic differences in
two mammalian DNA fractions, which contained equimolar
populations of DNA molecules differing in their A + T: G +
C ratios, was investigated in the following normal rat
organs: brain, kidney, pancreas, liver, salivary gland,
testis, thymus, spleen. Since much of the data on isotope
incorporation into DNA has been reported in terms of in
corporation into purines and pyrimidines of DNA, the DNA
of the two fractions was subsequently hydrolyzed and also
analyzed.
Experimental Methods
Animal Preparation and Nucleic Acid
Isolation
Seven normal, sixty-gram male rats were fasted over
night. They were then given intraperitoneal injections of
1*l *
C -labeled sodium formate, containing a total of 500 mi-
14
crocuries of C in 0.87 percent NaCl buffered to pH 7.4-
with 0.001 M. Tris(hydoxyethyl)aminomethane-Hydrochloride
(Tris-HCl). After twenty-four hours the animals were
killed by exsanguination, and their organs were quickly
*Purchased from California Corporation for Biochemical Re
search, Lot # 108653, 9.5 millicuries per millimole.
7
excised and frozen on dry ice. All organs which are alike
were pooled. Organs taken were brain, kidney, pancreas,
liver, salivary gland, testis, thymus, and spleen.
DNA was isolated by the method of Perry and Walker
(1958) This DNA has been called Extractable-DNA. Other
fractions of the cell were saved for later analysis by
storage at - 15° C. Figure 2 shows the flow diagram used
for preparation of nucleic acid fractions.
Fractionation of DNA and Its Bases
Extractable-DNA was separated into two fractions dif
fering in their A + T: G + C ratios by utilization of the
fact that DNA with a larger proportion of adenine plus
thymine base pairs is more susceptible to heat denatura-
tion and degradation than DNA with lesser amounts of
adenine plus thymine (Doty et al., 1959; Sueoka et al.;
Kit, 1961). Solutions containing 10 optical density
units of DNA were incubated in 0,15 M. NaCl, 0.001 M.
Tris-HCl, pH 9*0 at 86.2° C. for four hours. The solu
tions were maintained at this high pH level to prevent de-
purination of the polynucleotides (Thomas and Doty, 1956;
Rice and Doty, 1957; Rice, Wada, and Geiduschek, 1958;
Ginoza and Guild, 1961; Ginoza and Zimm, 1961). At 86.2°
C. part of the population of DNA molecules waB selective
ly denatured and degraded (Marmur and Doty, 1959; Eigner,
Boedtker, and Michaels, 1961). These molecules had
8
A + T: G + C ratios which were higher than the mean values
for the total solution.
After the incubation period the solutions were quick
ly chilled in an ice-water bath to prevent renaturation of
the denatured molecules (Marmur and Doty, 1959). They
were acidified by dropwise addition of concentrated acetic
acid and precipitated with three volumes of 95 percent
ethanol. The precipitates were dried with absolute ethan
ol and ether and then air dried.
Three milliliters of 0.15 M. NaCl, 0.001 M. Tris-HCl
pH: 8.Q were added to each of the precipitates and the mix
tures were stirred with magnetic stirrers for twenty-four
hours at 5° C. The suspensions were then transferred to
12 milliliter volumetric test tubes, and the volumes were
adjusted to 4.5 milliliters by addition of distilled
water. The suspensions were then centrifuged in an Inter
national refrigerated centrifuge for 50 minutes at 1,500
revolutions per minute. The supernatant was decanted, and
5.0 milliliters of 0.15 M. NaCl was added to the undis-
*
solved DNA, which had formed a transparent precipitate.
Both the supernatant fraction, containing DNA high in
adenine plus thymine (High A-T DNA), and the precipitate
fraction, containing DNA low in adenine plus thymine (Low
A-T DNA), were heated for two minutes at 100° c. an^ then
quickly cooled. The volume of the precipitate fraction
was adjusted to 4.5 milliliters by addition of distilled
9
water. The solutions were stored in the 5° 0, cold room
overnight. The optical density of the solutions was de
termined by measuring the ultraviolet absorbance of the
solutions at 260 millimicrons in a Beckman DU spectropho
tometer, equipped with thermospacsrs through which filtered
tapwater, 20° C. or less, was circulated. These determi
nations were made in quartz cuvettes with a light path of
0.5 centimeters. Amounts of DNA were calculated from the
optical density measurements assuming that the molar ex
tinction coefficient per nucleotide unit of denatured DNA
is 10,000 (Chargaff, 1955; Marmur and Doty, 1959). The
DNA solutions were then pipetted onto stainless steel
planchets in 2.0 milliliter aliquots and evaporated to
dryness in a hot air oven.
After the radioactivity measurements were made the
DNA was recovered from the planchets by dissolving it in
warm 1.0 M. sodium hydroxide. The solutions were then
acidified by dropwise addition of concentrated acetic acid,
and the DNA was precipitated with three volumes of 95 per
cent ethanol. The precipitates were dried with absolute
ethanol and ether and then air dried. Enough 12 Normal
perchloric acid was added to each of the precipitates to
cover them, and they were heated for one hour at 100^ C.
(Marshak and Vogel, 1951)• After the solutions had cooled,
they were diluted with approximately 0.5 milliliter of
distilled water. Concentrated potassium hydroxide solu
10
tions were added dropwise until potassium perchlorate pre
cipitates ceased to form. The precipitates were succes
sively washed with distilled water and decanted. The
aspirated solutions, containing purines and pyrimidines,
were tranferred to small test tubes an stored at - 15° C.
until they were used.
Nucleic acid bases were separated by chromatography
on Whatman Number 1 Eilterpaper, using an isopropanol-
hydrochloric acid solvent system (Wyatt, 1951)* After
development of the chromatograms, positions of the purines
and pyrimidines were determined by observing their visible
fluorescence upon ultraviolet irradiation. The adenine,
guanine, and thymine spots were marked and cut out. Each
was eluted with 3.0 ipillilters of 5 x lO-^ M. Tris-HCl,
pH 7.2 in distilled water. The optical densities of the
resultant solutions were determined at 250, 260, and 280
millimicrons for each base. Data on the ratios of absor
bance at the wave lenghs 250:260 and 280:260 were used in
addition to the values for positive identification of
the bases. Two milliliter aliquots of each solution were
pipetted onto aluminum planchets and dried in a hot air
oven. Amounts of each base per planchet were calculated
from the optical density readings at 260 millimicrons
using the following molar extinction coefficients: ade
nine = 13,500; guanine = 7,200; thymine = 7,500. These
values were calculated from graphs of ultraviolet spectra
11
of nucleic acid components at various pH values (Beaven,
Holiday, and Johnson, 1955)*
Radioactivity Measurements
The radioactivity was measured with a gas flow Gei-
ger-Mueller counter and a Baird Atomic Multiscalar II,
Model 132. The radioactivity (counts per minute) was di
vided by the number of micromoles on each planchet to give
the specific radioactivity (counts per minute/ micromole).
14
A stock solution of denaturedC -labeled thymus DNA was
successively diluted with 0.10 M. NaCl solutions, and
planchets were prepared as described above. Measurements
of the radioactivity of these planchets show that there
was essentially no absorption of the beta radiation farom
14
the C by the NaCl. The lack of apparent self-absorption
by the NaCl may have been due to the tendency for counter
ions to become adsorbed to the DNA during the dehydration
process, coating all the DNA molecules with a NaCl layer
of constant thickness.
Results and Discussion
The data obtained by the experiments involving the
unhydrolyzed DNA are presented in Tables I and II. The
specific radioactivities of the less heat-stable fractions
are higher than the specific radioactivities of the more
heat-stable fractions, and the ratio between the specific
radioactivities of these two fractions varies between 1.05
and 1,41 with a mean of 1.15 and a standard deviation of
+ 0.12 (Table VI). This means that the ratio between the
specific activities of the two fractions is fairly con
stant. Since mammalian DNA has 4-2 mole percent guanine
plus cytosine and 58 mole percent adenine plus thymine
(Sueoka, 1961b), it can be concluded from the data in
Tables I and II that the less heat-stable fraction of DNA
is richer in adenine plus thymine base pairs, and High A-T
DNA and Low A-T DNA have been correctly identified.
The specific radioactivities of adenine, guanine, and
thymine in High A-T DNA are presented in Table III. The
specific radioactivities of the bases in Low A-T DNA are
presented in Table IV. No significant difference between
the specific radioactivities of the two groups of bases is
evident. The specific radioactivity of adenine in High
A-T DNA is the same as the specific radioactivity of ade
nine in Low A-T DNA. The specific radioactivity of gua
nine in High A-T DNA is the same as the specific radio
activity of guanine in Low A-T DNA. The specific radio
activity of thymine in High A-T DNA is the same as the
specific radioactivity of thymine in Low A-T DNA. The
mean values for the specific radioactivities of adenine,
guanine, and thymine in Extractable-DNA (High A-T DNA and
Low A-T DNA) are presented in Table V. The relative dis
tribution of specific radioactivities of the bases is
similar in all organs and is in the following order from
13
highest to lowest: adenine, guanine, thymine. This is
consistent with the data of Langen and Lies (1961a and
1961b), who found a constant relative distribution of
P -labeling in DNA-nucleotides.
When comparing the specific radioactivities of ade
nine to thymine and adenine to guanine, adenine has a
higher specific radioactivity than either guanine or thy
mine. Also there is a slight trend towards higher speci
fic radioactivities in the thymine moieties relative to
adenine in the organs which have incorporated the most
isotopic label (Table VI). This observation was not made
by Zbarsky, Hori, and Findley (1958), who compared the
14
incorporation of C -labeled formate into the tissues of
normal rats to its incorporation into the nucleic acids of
tissues of rats bearing Novikoff hepatoma. This may pos
sibly be explained by the results of Totter (1954), who
reported very high specific radioactivity in the 5-methyl
group of thymine within several hours after incubating
14
rabbit bone marrow cells with C -labeled formate. Since
DNA does not break down the excess of label would remain
in the thymine moieties of DNA until enough radioactively
labeled DNA was synthesized to mask the initial deviation
14
from equilibrium values. After several hours the C may
become more evenly distributed among the compounds in
Figure 1 than it is shortly after administration to the
animalo
None of the data reported in this phase of the pre
sent experiments explains the results of Holbrook et al.
(i960). These authors found significant differences in
the specific radioactivities of adenine and guanine in DNA
fractions differing in their extractability from isolated
nuclei. The fraction, which they designated Fraction II,
would be analogous to Extractable-DNA, which was investi
gated here. Since no further fractionation of Fraction II
of Holbrook et al. (i960) was reported, a more thorough
comparison of their results with the present results will
be deferred to the next chapter.
Derivation of the Mathematical Model
The ratio of the specific radioactivity of High A-T
DNA and the specific radioactivity of Low A-T DNA was
shown to be a constant, the High A-T DNA having a signifi
cantly higher specific radioactivity than the Low A-T DNA.
It can be showa mathematically that the specific radio
activities of the labeled compounds comprising any DNA
fraction are a function of the mole fractions of the
constituent compounds in the DNA fraction being studied,
and the specific radioactivities of the labeled constitu
ent compounds in the precursor pool from which the DNA
was synthesized. The specific radioactivities of the
compounds in the deoxyribonucleotide pool are proportional
to their corresponding members in macromolecular DNA be-
cause DNA does not turnover (Kihara and Sibitani, 1955?
Nygaard and Rusch, 1955s Takagi, Hecht, and Potter, 1956;
Bennett, Simpson, and Skipper, I960; Fresco and Bendich,
I960; Ives and Barnum, 1962). Thus, for every new mole
cule of DNA that is synthesized there is an old molecule
with exactly the same base composition.
Assumptions made in deriving a mathematical model to
explain the results are as follows.
1. A fractionation method was used to separate
different species of DNA, and it distinguished
among the molecules on the basis of their
A + T: G + C ratios (see page 7)*
2. The average A + T: G + C ratio in DNA mole
cules and the distribution of this ratio are
the same in all organs of the same species, and
not all molecules have the same A + T: G + C
ratios (Kit, 1961; Sueoka et al., 1959).
5. The data of Chargaff (1955), saying that the
molar proportions of adenine equal thymine and
of guanine equal cytosine, are correct.
4. The DNA had been labeled with an isotopic
precursor so that the labeled compounds, which
had been incorporated into its constituent bases,
had the following properties: the specific
radioactivity of adenine was greater than the
specific radioactivity of guanine, and the spe
16
cific radioactivity of thymine was greater than
the specific radioactivity of cytosine.
Now the specific radioactivities of the bases in an
isotopically labeled deoxyribonucleotide precursor pool,
and hence in DNA can be designated as follows.
Specific activity of adenine = SA
Specific activity of guanine = SG
Specific activity of thymine = SO?
Specific activity of cytosine = SC
In a similar manner the mole fractions of these bases
in DNA can be designated as follows.
Mole fraction of adenine = f^
Mole fraction of guanine = f^
Mole fraction of thymine = fg.
Mole fraction of cytosine = f^
The specific activity and mole fraction of 5-methyl-
cytosine will be disregarded for this discussion, because
5-methylcytosine constitutes less than two percent of the
total base components in DNA of animal cells (Chargaff,
1955).
The fractionation method used separated the DNA mole
cules into two equally sized groups differing in their
A + T: G + C ratios such that the average A + T: G + C
ratio of one fraction (I) was higher than the unfractiona
ted DNA sample, and the average A + T: G + C ratio
17
of the other fraction (II) was lower than in the unfrac
tionated DNA sample. Therefore, new designations can he
used for the mole fractions of the bases in each of these
two fractions. The change in mole fraction of any base
will be called "x." The new designations for the mole
fractions of the bases in DNA fractions are as follows.
Mole fraction of adenine in I = f^ + x
Mole fraction of adenine in II = f^ - x
Mole fraction of guanine in I = f^ - x
Mole fraction of guanine in II » fg + x
Mole fraction of thymine in I = f^ + x
Mole fraction of thymine in II = f^, - x
Mole fraction of cytosine in I = f^ - x
Mole fraction of cytosine in II = f^ + x
The specific activities of the DNAs. in the two frac
tions can be represented by S-DNA-I and S-DNA-II, and the
ratio between them is S-DNA-I/S-DNA-II « R.
The specific activity of S-DNA-I is a function of the
specific activities of its components (Equation 1).
S-DNA-I . (fA + x)(SA) + (fT + x)(ST) +
(fG - x)(SG) + (fc - x)(SC) (1)
This can be simplified because the mole fractions of
adenine equal thymine and of guanine equal cytosine (Equa
tion 2).
18
S-DNA-I « (fA + x)(SA + ST) + (fQ - x)(SG + SC) (2)
In a similar manner the specific activity of DNA in
fraction II can be represented (Equation 3)«
S-JDKA-II = (fA -x)(SA) + (fT - x)(ST) +
(fQ + x)(SG) + (fc + x)(SC) (3)
Equation 3 can also be simplified (Equation 4-).
S-DNA-II = (fA - x)(SA + ST) + (fQ + x)(SG + SC) (4)
The ratio of the specific activities of these two DNA
fraction would then be as depicted in Equation 5*
S—DNA—T " * * ^0(SA + ST) + (fp< — x)(SG + SC)
E ’ s-dWa-IT “ Cf~ - x)(SA V ftt) + (fG + xT(fia + SS) (5)
If both the numerator and the denominator of Equation
5 are divided by a factor of (SG + SC), the result will
be Equation 6,
(SA + ST)
(fA + x)(SS' V T O + (fr - x)
R ■ - - - -r g"J«i 2- - - - ( 6 >
(fA - x)(£6 + SC) + (fG + x)
Now SS can be define (Equation 7)•
SA + ST
ss “ SS + g<2 (7>
The ratio of the specific activities of the two DNA
fractions is finally simplified to Equation 8.
19
(fA + x)(SS) + (ffi - x)
E - CfA - x)(SS) + (fG + x}
It can be seen that the only term in Equation 8
which is not a constant is SS. The term becomes smaller
when there is conversion of adenine to guanine (Brown,
Roll, Plentl, and Cavalieri, 194-8) and a significant
14-
amount of C has been incorporated into cytosine. The
ratio, R, was found experimentally to be a constant. The
significance of this observation is that it is very tin-
likely that metabolic differences, which are indicative of
organ specificity and/or metabolic heterogeneity, can be
detected by studies of isotope incorporation into DNA
molecules differing in their A + T: G + C ratios. The
fact that no metabolic differences could be detected
between the adenines, guanines, and thymines of the two
fractions studied further confirmed this conclusion.
Summary
Formate labeled with radioactive carbon was adminis
tered to seven male rats weighing approximately sixty
grams each. After twenty-four hours the animals were
sacrificed by exsanguination, and their internal organs
were excised and frozen on dry ice. DNA was isolated from
the organs by the method of Perry and Walker (1958). The
DNA from each organ was separated into two fractions dif
fering in adenine + thymine: guanine + cytosine ratios by
20
a method utilizing the heat-lability dependence on these
ratios. Determinations of specific radioactivity of mac-
romolecular DNA in each fraction were made. The ratio,
R, between the specific radioactivities of the two DNA
fractions was found to be a constant (1.15 + 0.12) and
independent of the rate of cell growth. When the DNA
fractions were hydrolyzed and the specific radioactivties
of adenine, guanine, and thymine were measured, the spe
cific radioactivities of the bases in the two DNA frac
tions were found to be identical. The results were com
bined. The ratios of the mean specific radioactivities
of adenine: thymine were found to be related to the total
amount of isotope incorporated into the particular organ
being studied. The specific radioactivities of the bases
were found to be in the following order from highest to
lowest: adenine, guanine, thymine.
On the basis of the data presented and several as
sumptions the following mathematical model describing the
ratio, R, of the specific radioactivities of the two types
of DNA was derived,
(fA + x)(SS) + (fQ - x)
R * (fA - x)(SS) + (fG + x)
where fA and fG are the mole fractions of adenine and
guanine in DNA and x is a constant. SS represents the
specific radioactivities of the bases, adenine, thymine,
21
guanine, and cytosine (SA, ST, SG, and SC) such that
SS « (SA + ST)/(SG + SC).
It was concluded that organ specificity and true
metabolic heterogeneity in DNA species differing in their
adenine + thymine: guanine + cytosine ratios could not be
detected by isotope incorporation studies in mammalian
organs.
CHAPTER II
FORMATE METABOLISM IN NUCLEAR NUCLEIC ACIDS
Purposes of the Project
The experiments discussed in this chapter were per
formed for the following objects.
1. To investigate the formate metabolism in
nuclear ribonucleic acid (RNA) and DNA in the
residual chromosomes of mammalian organs.
2. To establish a paradigm of normality for
the pattern of isotope incorporation in nuclear
nucleic acids.
History of Precursor Incorporation into
Nuclear Nucleic Acids
Cytological Studies
Rudkin and Corlette (1957), using ultraviolet light
absorption data, found disproportionate synthesis of DNA
in Balbiani rings, bulbs, and puffs in the tissues of the
dipteran, Rhychsciara angelae. Pelling (1959) demonstra
ted metabolic activity of specific chromosomal regions by
autoradiographically observing the incorporation of tri-
tiated uridine into the puffs and Balbiani rings of the
salivary gland chromosomes in Chironomus tentans. He
found that the nucleolar RNA was synthesized at only the
- 22 -
23
nucleolar organizers. In a similar experiment Rudkin and
Woods (1959) observed the incorporation of tritiated
cytidine and tritiated thymidine into salivary gland
chromosomes of Drosophila larvae during puff formation.
They found RNA synthesis without concommitant DNA synthe
sis in the puff regions.
Gall and Callan (1962) studied the incorporation of
tritiated uridine into the loops of the lampbrush chromo
somes of the newt. When they observed that the label
moved from one end of the loops to the other, they postu
lated that the loops are continuously uncoiling at one end
and coiling at the other. The incorporation pattern of
phenylalanine was not asymmetrical, but the phenylalanine
was incorporated evenly throughout the loops.
Hyden and Egyhazi (1962) induced RNA and protein pro
duction in isolated Deiters nerve cells of rabbits with
tricyanoaminopropene, and then made microbiochemical anal
yses of the content and composition of nuclear RNA. They
found the newly synthesized RNA was richer in adenine and
uracil than cytoplasmic RNA, but the guanine to cytosine
ratio was 0.86 in the nuclear RNA as compared to 1.16 in
the cytoplasmic RNA. They explained their data on the
basis of the extraordinary development of the nucleolus
during the process of RNA and protein induction.
Bloch and Hew (1960a and 1960b) provided evidence
that histones may be involved in the process of cellular
24
differentiation. During spermatogenesis in the pulmonate
snail, Helix aspersa. there is a progressive loss of
lysine-rich histone from the nuclear material. The ly
sine-rich histone is regained in the process of cellular
differentiation during embryogenesis. They discussed the
acquisition of this type of histone with the loss of toti-
potency of embryonic cells during gastrulation.
Although they did not propose structures for the com
plexes involved, Agrell and Bergqvist (1962) demonstrated
that two types of DNA exist, differing in their stability
to heat hydrolysis during different stages of embryogene
sis. This was attributed to differences in replication
times of heterochromatin and euchromatin. Taylor (I960)
also showed that asynchronous duplication of DNA occurs in
animal cells by autoradiographic studies of tritiated
thymidine incorporation in cultured cells of the Chinese
hamster.
Huang and Bonner (1962) related the histone-DNA com
plex to a lack of ability of DNA to function in DNA-de-
pendent RNA synthesis, thus providing a reasonable explana^
tion for the role of histones in cellular differentiation.
Allfrey, Littau, and Mirsky (1963) found that ar
ginine-rich histones are more effective than lysine-rich
histones in preventing DNA-dependent RNA synthesis in thy
mus nuclei. When histones were removed from DNA by di
gestion with trypsin, the type of Messenger-RNA synthe-
25
sized was richer in guanine and uracil than the usual
Messenger-RNA.
From the data summarized above the following infer
ences can be made.
1. It might be possible to isolate DNA-protein
complexes from animal cells which exist in two
forms differing in their physical properties.
2. The process of DNA replication involves
demonstrable alteration of the physical state
of DNA.
3. Some of the DNA which is not complexed with
histone may have RNA associated with it.
4. Metabolic activity alters the composition
of nuclear RNA.
Incorporation of Nucleic Acid Precursors into
Nucleic Acids of Different Mammalian Organs
Tyner, Heidelberger, and LePage (1955) administered
32 14
P -labeled phosphate and C -labeled glycine simultane
ously to rats with Flexner-Jobling carcinoma and to nor
mal rats. The animals were killed at various subsequent
intervals. Although the pool sizes for phosphate and gly
cine were found to be the same, phosphate was incorporated
into RNA precursors more rapidly than glycine. The radio
activity of the RNA in different cell fractions was found
to be be proportional to the total quantity of the
26
nucleotides present. Edmonds and LePage (1955) studied
14
the incorporation of C -labeled glycine into the acid
solubles and RNA in the livers of normal rats and those
with Flexner-Jobling carcinoma. They found that hypo-
xanthine was labeled first. At early times after adminis
tration of glycine there was considerable heterogeneity in
the labeling of the different purines in the various
nucleic acid fractions. However, at equilibrium the la
beling was the same in all of the purines.
Balis and Samarth (1962) studied the incorporation of
14
C -labeled glycine and adenine into RNA in various organs
of young and old hamsters. They found that the glycine
pools were larger in young animals, and that there is a
difference in the sizes of the adenine pools, which de
pends on the organ involved. Holbrook et al. (I960) in-
14
vestigated the incorporation of C -labeled glycine in
nucleic acid fractions of normal rat liver and hepatoma.
They found that there was a higher specific activity in
DNA-adenine than in DNA-guanine in hepatoma. In the RNA
from the r63idual chromosomes of hepatoma the specific
activity of the guanine was found to be significantly
higher than the specific activity of the adenine.
Hotta and Osawa (1958) studied the incorporation of
2^-labeled phosphate into the nucleic acids of rabbit
appendix, thymus, and liver. They found that the specific
activity of the guanine in RNA extracted with 1.0 M. NaCl
27
was much higher in the appendix than in other tissues.
Stein, Murakami, and Visser (1959), who studied the
incorporation of C -labeled glycine and formate into
purine bases of the hydrolyzed acid solubles of different
tissues of mice, found data consistent with the concept
that hypoxanthine is a precursor to both adenine and gua
nine. However, since they found that the specific activi
ty of guanine was greater than the specific activity of
hypoxanthine in the acid-soluble nucleotides of the in-
14
testine after administration of C -labeled formate, they
suggested that the pathway from formate to guanine should
be studied in greater detail.
Kier and Davidson (1958) studied the incorporation of
14
C -labeled formate into the appendix, intestinal mucosa,
and liver of rabbits. At fifteen minutes after the ad
ministration of the isotope there was a significant dif
ference in the specific activities of the components of
the acid-soluble ribonucleotide pool. After two hours
the specific activities of these components were equal,
showing that there is rapid equilibration in the acid-
soluble pool.
Smellie, Humphrey, Kay, and Davidson (1955) studied
the incorporation of P^-labeled phosphate into DNA,
nuclear RNA, cytoplasmic RNA, and inorganic phosphate of
various rabbit tissues. The specific activities of the
DNA-nucleotides from highest to lowest were found to be in
28
the following order: thymidylic acid, guanylic acid,
adenylic acid, cytidylic acid. The time of maximum iso
tope incorporation appeared to be a function of the amount
of labeling in the fraction involved. Langen and Liss
(1961a and 1961b) studied the incorporation of P^-labeled
phosphate in the DNA of various rat and mouse organs at
one, four, and sixteen hours after administration of the
isotope. They found that the specific activities of the
DNA-nucleotides were in the following order from highest
to lowest: thymidylic acid, cytidylic acid, guanylic
acid, adenylic acid. Since they used different species of
animals at different times after the administration of
isotope, the reason for the discrepancy between the re
sults of Langen and Liss (1961a and 1961b) and Smellie et
al. (1955) cannot be readily ascertained.
Gerber, Gerber, and Altman (1960a and 1960b), study
ing the replacement times and turnover of DNA, RNA, and
free nucleotides in various rat tissues, found both slow
and fast components in these substances. They also found
vast differences in turnover times within subcellular
fractions of the liver.
Other than studies of isotope incorporation into the
various organs or into various subcellular fractions, no
systematic investigation of isotope incorporation into
various subcellular fractions of different mammalian or
gans has hertofore been conducted. Because of this, no
29
no unifying principles have been proposed, which would
give insight into the interrelationships involved in the
biosynthesis of nucleic acids in mammalian organs.
Kinetics of Isotope Incorporation into
S.ynchonously Dividing Cells Including
Regenerating Liver
Anderson and Aqvist (1956) studied the incorporation
15
of N -'-labeled glycine into regenerating rat liver at
various time intervals after partial hepatectomy. The
peaks of isotope incorporation were as follows: cytoplas
mic RNA— 18 hours, nuclear RNA — 26 hours, DNA— 26 to 37
hours, protein— 26 and 37 hours. Hammarsten, Aqvist,
Anderson, and Eliasson (1956) in a similar study found
peaks in the specific activity of RNA at 13 and 56 hours
after hepatectomy. The peaks in specific activity of DNA
were at 30 and 56 hours. They also found that the ratio
of the specific activities of guanine to adenine increase
with time.
Takagi, Hecht, and Potter (1956) studied the kinetics
of orotic acid-C^ incorporation into cell fractions of
regenerating liver. They found the peak specific activity
of DNA was obtained 14 hours after the injection of orotic
acid, and that the specific activity of DNA remained at a
high level for days. During this time the specific acti
vities of all the other nucleic acid components decreased,
30
with the exception of cytoplasmic RNA.
Looney (I960) followed the incorporation of tritiated
thymidine in regenerating livers both spectrophometrically
and autoradiographically. He found that the onset of DNA
synthesis is at approximately 15 hours after hepatectomy.
The upper limit of the doubling time is about eight hours
for DNA. He also observed that DNA synthesis and mitosis
occur in cycles.
Kelly, Hirsh, Beach, and Palmer (1937) injured the
livers of intact mice by injection of massive doses of
carbon-tetrachloride. They then made a time study of the
32
incorporation of P-' -labeled phosphate into liver DNA and
of mitotic activity of the liver cells. They found an in-
32
crease in P incorporation began 30 hours after carbon-
tetrachloride administration, and the maximum was at ap
proximately 40 hours. Mitotic activity was not apparent
until after DNA synthesis had reached its maximum.
Morin, Zajdela, and Costerousse (1937) studied the
32
time course of P-' -labeled phosphate incorporation into
different subcellular fractions of mouse liver. They
found that the chromatin-rich fraction associated with the
nucleolus had the highest specific activity at one and
one-quarter and five hours after administration of the
isotope.
Lieberman and Ove (1962) cultured rabbit kidney tis
sue . There was doubling of nuclear RNA between 20 and 40
31
hours after explantation of the tissue. After a lag of 36
to 40 hours DNA synthesis and cell division commenced.
Ethylenediaminetetraacetate (EDTA) inhibited nucleic acid
synthesis. The only cation capable of reversing EDTA in
hibition was found to be zinc ion.
Harbers (1961) investigated the incorporation of oro
tic acid into nuclear RNA fractions of ascites tumor cell^
and normal and regenerating rat liver. In the regenera
ting livers there was a higher specific activity in the
nucleolar fraction than in nuclear ribosomes, but the re
verse was true for normal livers.
Zbarskii and Georgiev (1959) devised a method for
fractionation of cell nuclei from rat livers after using
a sucrose-glycerolphosphate solution to isolate the nu
clei. DNA was extracted with 1.5 M. NaCl and the nucleoli
were in the residual chromosomes. Georgiev, Samarina,
Mantieva, and Zbarskii (1961) injected P^-labeled phos
phate into intact rats, sacrificed them after thirty
minutes, and isolated the nuclei from their livers. They
found the highest incorporation of phosphate to be in the
chromatin fraction associated with the nucleolus, and the
second highest in the acid soluble fraction.
Logan and Davidson (1957) injected P^-labeled phos
phate into intact rabbits, sacrificed the animals two
hours later, isolated the thymus nuclei in a sucrose-cal-
cium chloride (sucrose-CaCl^) solution, and fractionated
52
the nuclei. They found a RNA fraction, which could he ex
tracted from the isolated nuclei with a buffer solution,
which had a much lower specific activity than the remain
ing fraction. Both nuclear RNA fractions had higher
specific activities than the cytoplasmic RNA.
Guschlbaur and Williamson (1963) studied the regenerar
xp
tion of wound tissue in the rat. They found that the P
pool is constant and the change in rate of DNA synthesis
during the regeneration process is due to different cell
types being synthesized.
Christensson (1962) studied the quantitative changes
in different RNA fractions during cell growth and syn
chronous division in cultures of Tetrahymena p.yriformis.
A fraction which stayed in the phenol phase during RNA
extraction was shown to be poorer in guanine than the
fraction which was concentrated in the aqueous phase. The
phenol-RNA showed a rapid increase prior to cell division.
The RNA in the aqueous phase was proportional to cell size
up to division.
Swick, Koch, and Handa (1956) studied nucleic acid
turnover in the livers of young rats during the continuous
14-
administration of C Mathematical parameters describ
ing the movement of isotopic precursors were derived using
assumptions, such as all RNA is metabolically unstable,
which have been shown to be invalid since their publica
tion (Davern and Meselson, I960).
33
Levinthal, Keynan, and Higa (1962) studied Messenger-
RNA turnover and protein synthesis in B. subtilus during
inhibition by Actinomycin D and calculated flow rates and
pool sizes of the substances involved. They found Messen
ger RNA to be a single molecule which is utilized several
times in protein synthesis before it breaks down with a
time constant of two minutes into acid-soluble, low mole
cular weight material. They found the relative sizes of
the various pools and flow rates remain constant in the
steady state, and their absolute values increase at the
same rate as the cell population.
Jardezky and Barnum (1958) provided a mathematical
explanation of why metabolic reactions involving isotopi-
cally labeled precursors and products can be considered
"unidirectional" in the absence of continuous administra
tion of isotope.
It can be concluded from the experiments summarized
above that after administration of a single dose of
isotopically labeled precursor to synchronously dividing
cells, the peaks of labeling occur at different times for
different nucleic acid components. Although the time
schedules are not identical for all cell systems used, in
most cases investigated the nuclear RNA appears more
highly labeled at earlier times than does the cytoplasmic
RNA or the DNA. DNA synthesis always precedes mitosis.
34
Synthesis of Nuclear Nucleic
Acids in Isolated Nuclei
Allfrey, Mirsky, and Osawa (1937) isolated calf thy
mus nuclei in sucrose-CaC^ solutions, and studied the
14
incorporation of C -labeled amino acids into nuclear
proteins in vitro. The nuclei were fractionated by a
technique which is similar in principle to the techniques
used for the experiments reported in this chapter. Part
of the DNA was associated with non-histone protein in a
linkage which was stable to salt and alkali. Using simi
lar methods Allfrey and Mirsky (1959) studied the uptake
14
of C -labeled adenosine, orotic acid, and uridine into
nuclear RNA in nuclei which had been isolated in sucrose-
CaC^ solutions. They found more rapid synthesis and de
gradation of nucleolar RNA than any other RNA fraction.
Adenine and cytosine moieties were found to be more stable
than uracil. This is interesting in light of the more re
cent experiments of Allfrey, Littau, and Mirsky (1963)
discussed on page 24 and of Hyden and Egyhazi (1962) dis
cussed on page 23. Wang (1962) found that nuclear ribo
somes of calf thymus have a high content of guanine and
cytosine like cytoplasmic ribosomes, but both nuclear and
cytoplasmic ribosomes of thymus have a higher cytosine to
guanine ratio than the ribosomes from other types of cells
Davern (I960), studying 5-bromouracil incorporation into
KB cell DNA in cesium chloride density gradients, found
35
that one polynucleotide chain had more adenine than thy
mine, while the other had more thymine than adenine.
Osawa, Takata, and Hotta (1958) fractionated calf
thymus nuclear RNA and found that the RNA in the fraction,
which could be extracted from the nuclei with 1.0 M. NaCl,
was similar in composition to the RNA which was in the
residual chromatin.
Frenster, Allfrey, and Mirsky (I960) studied the in-
14
corporation of C -labeled orotic acid and adenosine into
different fractions of RNA in isolated lymphocyte nuclei.
They extracted nuclear ribosomes with dilute buffer solu-
14
tions and found that most of the C was in the RNA of
the residual nuclei.
Georgiev, Mantieva, and Zbarskii (i960) studied the
ratio of "acid protein" and RNA to DNA in various types
of nuclei isolated by the sucrose-glycerolphosphate and
phenol methods. The nuclei with highly developed nucleo
lar apparatuses had higher "acid protein" to DNA and RNA
to DNA ratios. "Acid protein" is called non-histone pro
tein by most American and European workers.
Bonner, Huang, and Maheshwari (1961) isolated chroma
tin from pea seedlings, which was able to synthesize RNA
from the four ribonucleoside triphosphates. The chromatin
contained DNA, RNA, and protein in a complex which dis
sociated at 60° C. There was one RNA to two DNAs on a wet
weight basis.
Osawa, Allfrey, and Mirsky (1957) found that inhibi
tors of mitochondrial oxidative phosphorylation did not
prevent mononucleotides from being converted to dinucleo
tides in isolated thymus nuclei. McEwen (1965) found
that adenosine triphosphate (ATP) was synthesized in iso
lated thymus nuclei in the presence of inhibitors of oxi
dative phosphorylation. Ord and Stocken (1961) studied
52
the uptake of P^ -labeled phosphate in rat thymus nuclei
in vivo and in vitro. Prior synthesis of ATP was not
needed for incorporation of labeled phosphate into nuclear
52
nucleic acids. P uptake was not inhibited by incubation
of the isolated nuclei at 0° C. or with iodoacetate.
Rees, Rowland, and Varcoe (1963) isolated nuclei from
rat livers in sucrose-CaC^ solutions and incubated them
14-
with C -labeled amino acids and orotic acid. By dif
ferential centrifugation they separated five fractions
plus a nucleolar fraction. All fractions incorporated the
labeled compounds into proteins and nucleic acids without
an external energy source. The nucleoli and the lipid-
rich fractions had the highest specific activities.
The following conclusions can be inferred from the
data summarized above.
1. Isolated nuclei are capable of synthesizing
proteins and nucleic acids as well as intact
cells.
2. At least two types of nuclear RNA exist; a
more labile RNA which is rich in guanine and
uracil and a more stable RNA which is rich in
adenine and cytosine.
3. The most active site of RNA synthesis and
degradation or diffusion is the chromatin as
sociated with the nucleolus.
4-. The energetic requirements for intranuclear
phosphorylation, nucleic acid synthesis, and
protein synthesis differ from that of the cyto
plasmic system. The intranuclear system does
not derive energy from oxidative phosphoryla
tion.
Theoretical Background for Studying Isotope
Incorporation into Nucleic Acid Fractions
14-
In the present experiments incorporation of C -la
beled formate was studied in several types of nuclear RNA
and DNA of various mammalian organs. In order to ascer
tain the significance of the results several assumptions,
based on the results of other workers, were made. These
assumptions and justifications for them are as follows.
1. DNA is metabolically stable. Once a poly-
deoxyribonucleotide chain is polymerized, it re
mains intact until the cell in which it resides
is destroyed. When isotopic precursors are in
corporated into DNA, the resultant specific
38
radioactivity is decreased only by subsequent
synthesis of more DNA from non-radioactive pre
cursors (Kihara and Sibitani, 1953; Nygaard and
Rusch, 1955; Takagi, Hecht, and Potter, 1956;
Bennett, Simpson, and Skipper, I960; Fresco and
Bendich, I960; Ives and Barnum, 1962).
2. The bulk of cellular RNA is metabolically
unstable (Levinthal et al., 1962). Only ribo-
somal RNA is stable for a period of time greater
than the time required for one cell division
(Davern and Meselson, I960).
3. A greater amount of radioactive label in
corporated into DNA is indicative of more cell
growth (Franzen and Binkley, 1961). It is
probable that in mammalian organs this means
that either there are more cells in the process
of DNA replication or the period of time before
the onset of DNA synthesis differs according to
cell type (Mendelsohn et al., I960; Sisken and
Kinosita, 1961). Heterogeneity in isotopic
labeling of DNA in various organs is not to be
interpreted as indicative of differences in the
rate of DNA synthesis in these organs.
4. At the time the DNA and RNA samples were
taken for analysis, twenty-four hours after ad-
14
ministration of the C -labeled formate, equi
librium had been attained in the ribonucleotide
precursor pools (Edmonds and LePage, 1955; Kier
and Davidson, 1958; Ives and Barnum, 1962).
The possibility exists that the sizes of the
various ribonucleotide pools differ in different
organs. Pranzen and Binkley (1961) studied the
ribonucleotide pool sizes in Escherichia coli
which were cultured in various media which al
tered the growth rate. They found that the
molar proportions of adenosine monophosphate:
adenosine diphosphate: adenosine triphosphate
were 1: 6: 50 regardless of the rate of
growth. They also found that guanosine triphos
phate (GTP) and uridine triphosphate (UTP) in
crease at a faster rate, the larger cells having
more GTP and UTP. Levinthal et al. (1962) found
constant relative pool sizes in B. subtilis.
If such phenomena exist in mammalian cells, it
is possible that the pool sizes of the nucleo
tides in nuclei of mammalian cells are either in
constant molar relationship to one another or
dependent on the growth rate of the cells in
volved (Guschlbaur and Williamson, 1965). The
fact that higher proportions of guanine and ura
cil were found in nascent Messenger-RNA after
removal of histones from isolated nuclei with
40
trypsin (Allfrey et al., 1963) would be con
sistent with this analogy. The amount of la
beling of the various poolsi, which would deter
mine the labeling in the nucleic acids, would
depend on the size of the pools diluting the
isotopic precursor. Balis and Samarth (1962)
studied the size of the glycine pool in the
livers and kidneys of young and old hamsters
and found that the differences in the pool siz
es were proportional to the relative specific
radioactivities in the cellular RNA. There was
no significant difference in the specific radio
activity of glycine in kidney and liver. Since
glycine as well as formate is a precursor in the
de novo synthesis of purines, results of studies
on glycine may be considered applicable to
formate.
5. Both adenine and guanine are derived from
hypoxanthine, so in de novo synthesis of purines
14
utilizing C -labeled formate as a precursor
the initial specific radioactivities of adenine
and guanine are approximately equal (Stein et
al., 1959).
6. There is a net conversion of adenine to
guanine (Brown et al., 1948; Hammarsten et al..
1956; Swick et al., 1956).
41
The types of RNA being considered here are RNA spe
cies which turnover rapidly. Newly synthesized polymer
chains are composed of nucleotides taken from a large
isotopically labeled precursor pool. Therefore, the
specific radioactivities of adenine and guanine in each
RNA species tend to become representative of the mole
fractions of these purines in the RNA species being con
sidered soon after introduction of the isotope in ribo
nucleotide precursor pool (Volkin, Astrachan, and Country
man, 1958)* Therefore, at early times the values for the
specific radioactivities of the bases are neither the same
as nor proportional to the values for the specific activi
ties of the same bases in macromolecular RNA, as was seen
to be true for DNA in Chapter I. At later times, however,
the specific activities of the purines in RNA equilibrate
with those of the ribonucleotide precursor pool. The
specific radioactivities of RNA and its bases would then
depend on how much of the unlabeled material was depleted
and replaced by labeled molecules at the time the RNA
sample was taken for assay.
The nucleic acid fractions investigated in the pres
ent experiments are as follows.
1. Extractable-DNA — This is the DNA which was
extracted from the nuclear fragments with 1.0 M.
NaCl at 5° C. and represents the bulk of cellu
lar DNA. The specific radioactivity data was
42
discussed in Chapter I, and the data from the
nucleic acid fractions described below were
compared with these results. This is analogous
to the DNA of fraction II of Holbrook et al.
(I960).
2. Nonextractable-DNA — This is the DNA which
remained in the nuclear fragments after ex
haustive extraction with 1.0 M. NaCl. It is
the DNA of the residual chromosomes (Allfrey,
Mirsky, and Osawa, 1957) or possibly satellite
band DNA (Rolfe, 1963). It is analogous to the
DNA of fraction III of Holbrook et al. (I960).
Chromatin-RNA — This is the RNA which re
mained in the nuclear fragments after exhaus
tive extraction with 1.0 M. NaCl. It is
Nucleolar-RNA (Allfrey et al., 1957; Pogo, Pogo,
Littau, Allfrey, and Mirsky, 1962).
4. Ethanol-RNA — This is the RNA which re
mained in the organic phase after DNA had been
precipitated from a saturated NaCl solution
with three volumes of 95 percent ethanol. It
is probable that this RNA is part of the Messen-
ger-RNA species for the following reasons.
Bonner et al. (1961) and Huang and Bonner (1962)
isolated chromatin from pea seedlings by ex
tracting cellular homogenates with dilute buffer
4 - 3
solutions. This chromatin fraction was able to
synthesize RNA from the four nucleoside tri
phosphates, and the nascent RNA was firmly
bound to the DNA. When this complex was sub
sequently centrifuged in 4.0 M. CsCl solutions,
the DNA formed a pellet, while a portion of the
nascent RNA remained in solution or in the pro
tein skin. In the present experiments most of
the DNA-protein complex was first dispersed in
1.0 M. NaCl. This ionic strength is not suf
ficiently high to dissociate the DNA-RNA com
plex. However, this solution was subsequently
saturated with NaCl, and both the DNA-histone
and DNA-RNA complexes were dissociated. It is
assumed that since Huang and Bonner (1962)
found the DNA-RNA complex to be labile in solu
tions of high ionic strength in pea seedlings,
the same would be true in mammalian tissue.
DNA was precipitated from the saturated NaCl
solution with three volumes of 93 percent ethan
ol. Sibitani, deKloet, Allfrey, and Mirsky
(1962) found a RNA fraction associated with DNA
in a 1.0 M. NaCl extract of calf thymus nuclei
which incorporated isotopic precursors more
rapidly than the nucleolar fraction. Although
they had identified a Messenger-RNA fraction,
44
which they had obtained by phenol fractionation
of nucleolar RNA, it is probable that the nu
cleolar fraction did not contain the Messenger-
RNA fraction in its entirety. It is more prob
able that a large portion of the Messenger-RNA
was associated with the DNA which had been sep
arated from the nucleolar fraction by extrac
tion with 1.0 M. NaCl. Since the Messenger-RNA
fraction could be isolated by phenol extraction
of aqueous solutions containing it, it is like
ly that it could also be isolated by utilizing
ethanol as the organic solvent. Although
Ethanol-RNA will be considered to be part of
the Messenger-RNA fraction, it is likely that
much of the Messenger-RNA can be found in other
subcellular fractions.
Experimental Methods
Formate labeled with radioactive carbon was adminis
tered to seven male rats weighing approximately sixty
grams each. After twenty-four hours the animals were sac
rificed, and their internal organs were excised and fro
zen. Nuclear fragments were obtained by the method of
Perry and Walker (1958). The bulk of the tissue DNA, Ex-
tractable-DNA, was separated from the nuclear fragments
with 1.0 M. NaCl for the experiments described in Chapter
45
I, leaving chromosomal residues which were stored at - 15°
C. Chromatin-RNA and Nonextractable-DNA were extracted
from these residues in the following manner. The residues
were transferred to 12 milliliter test tubes, and five
milliliters of 1,5 M. NaCl and approximately 50 milligrams
of sodium bicarbonate were added to each tube. The sus
pensions were refluxed for three hours at 100° C. and then
centrifuged at 1,500 revolutions per minute for 20 min
utes. The supernatant solutions were decanted and trans
ferred to 4-0 milliliter test tubes. The precipitates were
extracted again by the method just described, and the
supernatants were combined with those from the first ex
traction. The sodium nucleates were precipitated with
three volumes of 95 percent ethanol. They were dtfied with
absolute ethanol and ether, and air dried. DNA and RNA
were separated by the Schmidt-Thanhauser procedure
(Schmidt and Thanhauser, 1945). The basic solution of DNA
and ribonucleotides was acidified with acetic acid, and
the DNA was precipitated with three volumes of 95 percent
ethanol. The procedure for isolating the bases of DNA was
the same as the one described in Chapter I.
The supernatant solutions were made basic and ribo
nucleotides were precipitated by dropwise addition of
saturated barium acetate. The barium nucleotides were
dried with ethanol and ether, and air dried. Purines
were hydrolyzed by refluxing for one hour in 1.0 M.
46
hydrochloric acid. Purine solutions were made "basic by
dropwise addition of concentrated solutions of potassium
hydroxide and then chilled in an ice-water bath. Barium
phosphate and barium hydroxide precipitated, and the super
natant solutions were decanted an transferred to small
test tubes. The volumes were reduced with heat. Purines
were separated by paper chromatography using an isopropan-
ol-hydrochloric acid solvent system (Wyatt, 1951)• De
terminations of the specific radioactivity were made
using the methods described in Chapter I.
Ethanol-RNA was obtained in the following manner.
After the DNA-protein complex was extracted from the
nuclear fragments with cold 1.0 M. NaCl the protein was
dissociated from the DNA by saturating the solution with
NaCl and stirring it for three days at 5° C. The solu
tion was clarified by centrifugation and the DNA was pre
cipitated with three volumes of 95 percent ethanol. This
ethanolic solution was stored in the freezer at - 15° C.,
and a precipitate formed. The precipitate was dried with
ethanol, ether, and air dried. It was then subjected to
the Schmidt-Thanhauser procedure. The purines from the
RNA were isolated and analyzed by the methods described
above.
I
Results
The results of the specific radioactivity measure-
47
ments of the bases in Nonextractable-DNA, Chromatin-RNA,
and Ethanol-RNA are reported in Tables VII, VIII, and IX,
respectively. Several conclusions can be drawn from
these data.
1. Both adenine and guanine in Chromatin-RNA
have higher specific radioactivities than the
adenine and guanine in Ethanol-RNA.
2. The specific activity of guanine in Chroma
tin-RNA is significantly higher than the speci
fic activity of adenine. This is not the case
in any other nucleic acid fraction.
3. The order of magnitude of specific radio
activities in the various organs is the same in
the two DNA fractions, but it differs in the
two RNA fractions.
The ratios of the specific radioactivities of adenine
in Nonextractable-DNA, Chromatin-RNA, and Ethanol-RNA to
the specific radioactivity of adenine in Extractable-DNA,
and the ratios of the specific radioactivities of guanine
in Nonextractable-DNA, Chromatin-RNA, and Ethanol-RNA to
the specific radioactivity of guanine in Extractable-DNA,
show the following (Table X).
1. There is a significant difference between
Extractable-DNA and Nonextractable-DNA in the
adenine and thymine labeling in the organs
which have incorporated the most label.
48
2. The incorporation of precursors labeled with
radioactive carbon into the adenine and guanine
of the two types of RNA parallels the amount of
labeled precursors incorporated into adenine and
guanine in Extractable-DNA in most organs. How
ever, in the organs which have incorporated the
most label into Extractable-DNA this is not the
case.
5. The guanine in Chromatin-RNA appears to have
a high specific radioactivity relative to the
specific radioactivty of guanine in Extractable-
DNA from most organs.
Discussion of Results
The results will be discussed in terms of their
achievement of the purposes set forth at the beginning of
this chapter.
Formate Metabolism in Mammalian Organs
The present results represent a more complete study
of incorporation of formate into subnuclear fractions of
nucleic acids in mammalian organs than has heretofore been
attempted by any other worker in this field. Since all
possible permutations of metabolic studies involving the
14
utilization of C -labeled formate would not have been
feasible for a project of the scope intended here, the
metabolism of formate in nuclear nucleic acids of mammali-
4 - 9
an organs, twenty-four hours after the administration of
a single dose of the isotop^ was studied.
The fractionation method was based on the following
rationale. It is quite probable that mammalian DNA ex
ists in at least two forms; one which is complexed with
histone and is not being used as a template for the syn
thesis of RNA, and one which is complexed with non-histone
protein and/or RNA and is in the process of transmitting
genetic information. In addition, DNA may enter some
stable complex when it is in the process of replication.
A fractionation scheme, which is similar to that of
Allfrey, Mirsky, and Osawa (1957), was used. The DNA
was fractionated further, however, in an attempt to dem
onstrate metabolic differences in fractions differing in
their solubility in 1.0 M. NaCl (Holbrook et al., I960).
Only two fractions of RNA were studied here, Ethanol-RNA
and Chromatin-RNA.
Since the data on the specific radioactivities of the
bases in the subnuclear fractions were compared to the
specific radioactivity data of the bases in Extractable-
DNA, the metabolism of formate in the two types of DNA,
Extractable-DNA and Nonextractable-DNA, can be examined in
greater detail (Tables YI and XI). The findings are
summarized as follows.
1. The specific radioactivities of the bases in
the two types of DNA are in the same range of
magnitude in all organs studied.
2. The ratios of the specific radioactivities
of adenine to guanine were fairly constant in
Extractable-DNA, but they varied in Nonextracta
ble-DNA .
5. The ratios of the specific radioactivities
of adenine to thymine in Extractable-DNA were
lower in the tissues which had incorporated the
most isotope, but in Nonextractable-DNA these
ratios were approximately the same in all or
gans studied.
The most plausible explanation for these findings is
the Nonextractable-DNA fraction contains molecules which
are in the process of replication. The form of the pro
cess of replication would be detectable only if the time
required for preparation of the DNA molecules for this
process and for return of the molecules to the non-repli
cating state is large when compared with the twenty-four
hour incubation period. The most recently synthesized
molecules would then have to be composed of molecules
from a deoxyribonucleotide precursor pool which would
have the following characteristics.
1. The specific radioactivity of adenine rela
tive to guanine in the deoxyribonucleotide pre
cursor pool has decreased since the initiation
of isotope incorporation into DNA (Brown et al.,
51
1948; Hammarsten et al., 1956; Swick et al.,
1956).
2. The specific radioactivity of adenine rela
tive to thymine in the deoxyribonucleotide pre
cursor pool has increased since the initiation
of isotope incorporation into DNA (Totter,
1954).
The present findings would not affect the validity
of the mathematical model derived in Chapter I, because
in the absence of synchronous cell division it is reason
able to assume that the synthesis of molecules with the
complete range of mammalian A + T: G + C ratios took place
at random during the twenty-four hour incubation period.
Therefore, the specific radioactivities of the deoxyribo-
nucleotides in the DNA would be a function solely of the
specific radioactivities of the bases in the deoxyribo
nucleotide precursor pool at the time DNA was synthesized.
Although Langen and Liss (1961a and 1961b) found the same
order of magnitude in the specific radioactivities of DNA
nucleotides in different rodent organs at various time
intervals after the administration of P^-labeled phos
phate, the conditions of their experiments were such that
they studied only one DNA fraction. Therefore, DNA which
is in the process of replication would not have been de
tected by the methods that they used.
Now a comparison of the specific radioactivities of
52
adenine and guanine in Ethanol-RNA and Chromatin-RNA with
those in Extractable-DNA may be considered (Table X).
The increase in specific radioactivities of the purines in
the two RNA fractions was found to parallel the increase
in specific radioactivities of the purines of Extractable-
DNA in most of the organs studied. However, in the organs
which had incorporated the most isotope into Extractable-
DNA this was not found to be the case. Possible explana
tions for the parallel synthesis of RNA and DNA are as
follows.
1. DNA synthesis is necessary for RNA synthesis
to take place. Cohen and Barner (1955) studied
Escherichia coli, 15T_, & mutant which requires
an exogenous supply of thymine in the medium to
survive. When this mutant was grown in a thy-
mineless medium, RNA and protein synthesis took
place but DNA synthesis was blocked. The cells
enlarged in size but failed to divide and died.
These authors concluded that, "A selective in
hibition of DNA synthesis appears to be capable
of inducing death by unbalanced growth.”
Izawa, Allfrey, and Mirsky (1962) found that
Actinomycin-D not only blocked RNA synthesis in
loops of lampbrush chromosomes, but it led to
the disappearance of the chromosomal loops.
They concluded that the morphology of an active
55
chromosomal site is not only closely related to
its capacity to synthesize RNA hut is also de
pendent on it.
2. The combined effects of an inherent limita
tion on cell size, the stability of ribosomal
RNA (Davern and Meselson, I960), and the paral
lel synthesis of all types of cellular RNA
(Levinthal et al., 1962) in populations of mam
malian cells, provided conditions for which
parallel synthesis of RNA and DNA can be demon
strated by metabolic studies using isotopic
tracers.
The fact that the specific radioactivities of the
purines in the two types of DNA were greater than the
specific radioactivities of the purines in the RNA of
spleen and thymus may be a function of the greater growth
rate of these tissues. Also, it can be attributed to the
14
phenomenon whereby C -labeled molecules, which are in
corporated into DNA, are retained in the cell nuclei.
The net effect is an increase in the amount of labeled
DNA as cell division proceeds. The net increase in label
ed DNA would cease at a time when there is considerable
depletion of isotope from the deoxyribonucleotide precur
sor pool. In unstable RNA, however, maximum isotopic
labeling is reached shortly after maximum labeling in the
ribonucleotide precursor pool, and the labeling remains
54
at a level which depends on the labeling in the ribonucle
otide precursor pool. The maximum isotopic labeling of
purines in RNA would then be at a level at which the num
ber of labeled molecules entering a RNA fraction by RNA
synthesis equals the number of molecules leaving the same
fraction by degradation or diffusion.
The ratio of the specific radioactivity of guanine in
Chromatin-RNA to the specific radioactivity of guanine in
Extractable-DNA was not the same in all organs studied.
The following possibilities may explain the guanine data
in Chromatin-RNA.
1. The measurements are in error. This is un
likely because the trend for decreasing specific
radioactivity in the guanine of Chromatin-RNA
after a peak in specific radioactivity is mani
fested in the data from four different organs
(Table VIII), and all of the determinations of
specific radioactivity were made independently.
2. The metabolism of guanine in Chromatin-RNA
is a manifestation of organ specificity. This
is unlikely because if organ specificity were
involved, one would expect the ratios of the
specific activity of guanine in Chromatin-RNA to
the specific activity of guanine in Extractable-
DNA to be much higher in liver than in testis.
This would be expected because liver cells
produce greater amounts of RNA and protein than
testis cells but are mitotically less active.
It would be expected that the nuclear RNA of
liver cells would be much richer in guanine than
the nuclear RNA of testis cells (Franzen and
Binkley, 1961; Wang, 1962; Allfrey et al., 1963;
Guschlbaur and Williamson, 1963). The higher
molar proportion of guanine would be reflected
in its specific radioactivity after administra
tion of labeled precursors (Volkin et al..1958)♦
5* Chromatin-RNA is so structured that there
is a bias (Davern, I960) in the orientation of
the oases. This would affect the values of the
specific radioactivities of macromolecular RNA
species at a time when the specific radioacti
vities of the bases in these species are a
function of their mole fractions (Volkin et al.,
1958)* This factor alone would be insufficient
to explain the guanine data in Chromatin-RNA.
When the specific radioactivities of the bases
in the RNA species are in equilibrium with the
specific radioactivities of the bases in the
ribonucleotide precursor pool, no bias in base
orientation would be reflected by the specific
radioactivities.
4-. Chromatin-RNA is not one but several species
of RNA with different base compositions, turn
over times, and rates of diffusion away from
the site of synthesis. This is the most likely
of all possibilities. Scherrer, Lathan, and
Darnell (1963) demonstrated an unstable RNA in
HeLa cells which they interpreted to be a pre
cursor to ribosomal RNA. They found 55 to 60
percent of this rapidly synthesized RNA was
similar in base composition to ribosomal RNA,
and 35 to 40 percent was similar in base compo
sition to DNA. Midgely and McCarthy (1962)
found similar results in five species of bac
teria in which they studied the incorporation
14 32
of C -labeled uracil and -labeled phosphate.
Sibitani et al. (1962) found that following the
administration of isotopically labeled precur
sors, phenol treatment of a thymus nucleolar
RNA fraction separates the RNA into two phases;
1) an "aqueous phase" RNA fraction of relative
ly low specific radioactivity, and 2) an "inter
phase" fraction with a very high rate of syn
thesis and a base composition resembling DNA.
Heterogeneity in the composition of Chromatin-RNA
compounded with the possibility that a fraction of Chroma
tin-RNA is not yet in equilibrium with the ribonucleotide
precursor pool would account for the data that was ob-
57
tained.
A Paradigm of Normality
The most significant fact, that can he inferred from
the results of the present experiments, is that there is
parallel incorporation of isotopically labeled precursors
into the components of nucleic acids in some organs of
young rats. It follows that tissue specificity would not
be discernible by isotope incorporation studies such as
the present ones.
The present results also indicate that it might be
possible to measure turnover times of rapidly synthesized
RNA species by measuring the time it takes for the speci
fic activities of isotopically labeled compounds incor
porated into RNA to deviate from the representations of
their mole fractions in these species. However, it is
probable that specific activities of bases in RNA species,
such as ribosomal RNA would always be somewhat representa
tive of their mole fractions in these species, because
the intranuclear ribosomal RNA is not degraded in situ
but diffuses to the cytoplasm.
A corollary of conclusions based on the present re
sults is that the specific radioactivity data on nucleic
acids and nucleic acid components of any tissue, normal or
cancerous, would be a function of the rate of cell divi
sion within that tissue. In order to illustrate this, the
work of authors cited previously may be reviewed. Hol
brook et al. (I960) found significant differences in the
metabolism of glycine in subnuclear fractions of normal
rat liver and hepatoma. In a fraction which would be
analogous to Chromatin-RNA of the present experiments,
the guanine of hepatoma had a significantly higher speci
fic radioactivity than it did in normal rat liver. Also,
the ratio of the specific activity of guanine to adenine
was higher in hepatoma. Without adequate investigation
of isotope incorporation into normal tissues, many ex
periments of this sort have been erroneously interpreted
to indicate that tumorous tissue is metabolically dis
tinct from normal tissue. If such qualitative distinc
tions exist, they are probably demonstrable with great
difficulty (Vogt and Dulbecco, 1963).
Summary
Formate labeled with radioactive carbon was adminis
tered to seven male rats weighing approximately sixty
grams each. After twenty-four hours the animals were
sacrificed, and their internal organs were excised and fro
zen. DNA was extracted from the nuclear fragments by the
method of Perry and Walker (1958), leaving a chromosomal
residue. Extraction of the residue with 1.5 M. NaCl at
100° C. yielded DNA and RNA fractions which were called
Nonextractable-DNA and Chromatin-RNA. Another RNA frac
59
tion, Ethanol-RNA, was obtained from material soluble in
ethanol after precipitation of the sodium salt of Ex
tractable-DNA from a saturated NaCl solution. The bases
were separated by paper chromatography, and their specific
radioactivities were determined.
Specific radioactivity data, obtained on the purines
of Nonextractable-DNA, Chromatin-RNA, and Ethanol-RNA,
were compared with the data on the specific radioactivi
ties of the purines in Extractable-DNA. The data on the
specific radioactivities in thymine moieties were also
compared. The following conclusions were drawn on the
basis of these data.
1. There is parallel incorporation of isotopi
cally labeled precursors in RNA and DNA in some
organs of young rats. An explanation was of
fered for the cases in which parallel incorpora
tion was not observed.
2. There is no evidence for organ specificity
in the metabolism of nuclear nucleic acids in
young rats.
3. Nonextractable-DNA probably consists of a
form of DNA which is in the process of replica
tion.
4. Chromatin-RNA probably consists of several
molecular species which differ in base composi
tion and turnover time.
BIBLIOGRAPHY
61
BIBLIOGRAPHY
Agrell, I. and Bergqvist, H-l. 1962 Cytochemical Evi
dence for Varied DNA Complexes in the Nucleic Acids
of Undifferentiated Cells. J. Cell Biol.. 15: 604-
606
Allfrey, V. G., Littau, V. C., and Mirsky, A. E. 1965
On the Role of Histones in Regulating Ribonucleic
Acid Synthesis in the Cell Nucleus. Proc. Nat. Acad.
Sci.,4$: 414-421
Allfrey, V. G. and Mirsky, A. E. 1957 Some Aspects of
Ribonucleic Acid Synthesis in Isolated Cell Nuclei.
Proc. Nat. Acad. Sci.. 43; 821-826
Allfrey, V. G. and Mirsky, A. E. 1959 Contrasts in the
Metabolic Stability of Different Nucleotides in the
Ribonucleic Acids of Isolated Nuclei. Proc. Nat.
Acad. Sci.. 4£: 1325-1344
Allfrey, V. G., Mirsky, A. E., and Osawa, S. 1957 Pro
tein Synthesis in Isolated Cell Nuclei. J, Gen.
Physiol.. 40: 451-490
Anderson, E. P. and iqvist, S. 1956 Further Studies on
the Turnover of Polynucleotides in Regenerating Rat
Liver. Acta Chem. Scand.. 10: 1576-1582
Balls, M. E. and Samarth, K. 1962 Influence of Age on
Glycine and Purine Metabolism. Arch. Biochem.
Biophys.. 22: 517-521
Beaven, G. H., Holiday, E. R., and Johnson, E. A, 1955
Optical Properties of Nucleic Acids and Their Com
ponents. The Nucleic Acids. Volume I, E. Chargaff
and J. N. Davidson, (eds.), Academic Press, New York,
PP. 4-93-553
Bendich, A., Russell, P. J. Jr., and Brown, G. B. 1953
On the Heterogeneity of Desoxyribonucleic Acids.
J. Biol. Chem.. 205: 305-318
Bennett, L. L. Jr., Simpson, L., and Skipper, H. E. I960
On the Metabolic Stability of Nucleic Acids in Mito-
tically Inactive Adult Tissues Labeled During Embry
onic Development. Biochim. Biophys. Acta. 42: 239-
243
Bloch, D. P. and Hew, H. Y. C. 1960a Changes in Nuclear
Histone During Fertilization and Early Embryonic
Development in the Pulmonate Snail Helix aspersa.
J. Biophys. Biochem. Cytol.. 8: 69-81
Bloch, D. P. and Hew, H. Y. C. 1960b Schedule of Sperma
togenesis in the Pulmonate Snail Helix aspersa with
Special Reference to Histone Transition. J. Biophys.
Biochem. Cytol.. 515-532
Bonner, J., Huang, R-C. C., and Maheshwari, N. 1961 The
Physical State of Newly Synthesized RNA. Proc. Nat.
Acad. Sci.. 42*. 1548-1554
Brown, G. B., Roll, P. M. , Plentl, A. A., and Cavalieri,
L. F. 194-8 The Utilization of Adenine for Nucleic
Acid Synthesis and as a Precursor of Guanine.
£• Biol. Chem.. 172; 4-69-4-84-
Buchanan, J. M. and Hartman, S. C. 1959 Enzymic Re
actions in the Synthesis of the Purines. Advances
in Enzymology and Related Subjects of Biochemistry.
Volume 21, F. F. Nord, (ed.), Interscience Publishers,
New York, pp 199-261
Chargaff, E. 1955 Isolation and Composition of Deoxypen-
tose Nucleic Acids and of the Corresponding Nucleo-
proteins. The Nucleic Acids. Volume I, E. Chargaff
and J. N. Davidson, (eds.), Academic Press, New York,
pp. 507-371
Christensson, E. 1962 Different RNA Fractions During
Cell Growth and Synchronous Division in Mass Cultures
of Tetrahymena pyrlformis. Acta Physiol. Scand., £4:
1-8
Cohen, S. S. and Barner, H. D. 1955 The Death of Bacter
ia as a Function of Unbalanced Growth. Pediatrics.
16: 704—708
Davern, C. I. I960 Bias in Base Pair Orientation in DNA.
Nature. 183: 208-209
Davern, C. I. and Meselson, M. I960 The Molecular Con
servation of RNA During Bacterial Growth. J. Molec.
Biol.. 2: 153-160
Doty, P., Marmur, J. and Sueoka, N. 1959 The Hetero
geneity in Properties and Functioning of Deoxyribo
nucleic Acids. Brookhaven Symposium on Structure
and Function of Genetic Elements. Upton, New York,
pp. 1-16
Edmonds, M. P. and LePage, G. A. 1955 The Incorporation
14
of Glycine-2-C into Acid Soluble Nucleotide Purinea
Can. Res., 1£: 95-99
Ehrlich, P. and Doty, P. 1958 The Alkaline Denaturation
of Deoxyribonucleic Acid. J. Am. Chem. Soc., 80;
4251-4255
Eigner, J., Boedtker, H., and Michaels, G. 1961 Thermal
Degradation of Nucleic Acids. Biochim. Biophys.
Acta. £1: 165-168
Franzen, J. S. and Binkley, S. J. 1961 Comparison of
Acid Soluble Nucleotides in Escherichia coli at
Different Growth Rates. J. Biol. Chem.. 256; 515-519
Frenster, J. H., Allfrey, V. G., and Mirsky, A. E. I960
Metabolism and Morphology of Ribonucleoprotein Parti
cles from the Cell Nucleus of Lymphocytes. Proc.
Nat. Acad. Sci.. *6: 452-444
Fresco, J. R. and Bendich, A. I960 The Metabolic Stabil
ity of Rat Liver DNA; A Turnover Study. J. Biol.
Chem., 255: 1124-1128
65
Gall, J. G. and Callan, H. G. 1962 Uridine Incorpora
tion in Lampbrush Chromosomes. Proc. Nat. Acad. Sci. .
48: 562-570
Georgiev, G. P., Mantieva, V. L., and Zbarskii, I. B.
I960 ENA Fractions in Cell Nuclei Isolated by Phenol
and Sucrose-Glycerolphosphate. Biochim. Biophys.
Acta. J2: 375-374
Georgiev, G. P., Samarina, 0. P., Mantieva, V. L., and
Zbarskii, I. B. 1961 Further Studies on the Nuclear
Ribonucleoproteins. Biochim. Biophys. Acta. 46:
399-400
Gerber, G., Gerber, G., and Altman, K. I. 1960a The
Catabolism of Tissue Nucleic Acid in the Rat, I. Re
placement Time of DNA. J. Biol. Chem., 235: 1433-
1436
Gerber, G., Gerber, G., and Altman, K. I. 1960b The
Catabolism of Tissue Nucleic Acid in the Rat, II.
Turnover Time of Ribonucleic Acid and Free Nucleo
tides. J. Biol. Chem.. 255: 2682-2687
Ginoza, W. and Guild, W. R. 1961 On Inactivation of
Transforming DNA by Temperatures below the Melting
Point. Proc. Nat. Acad. Sci.. 47: 633-639
Ginoza, W. and Zimm, B. H. 1961 Mechanism of Inactiva
tion of Deoxyribonucleic Acids by Heat. Proc. Nat.
Acad. Sci.. 42: 639-652
Goldthwait, D. A. and Bendich, A. 1952 Effects of a
Folic Acid Antagonist on Nucleic Acid Metabolism.
J. Biol. Chem.. 196: 841-852
Guschlbaur, W. and Williamson, M. B. 1963 Metabolism of
Nucleic Acids during Regeneration of Wound Tissue,
III. The Rate of Formation of DNA. Arch. Biochem.
Biophys.. 100:251-254
Hammarsten, E., Iqvist, S., Anderson, E. B., and Eliasson,
N. A. 1956 The Turnover of Polynucleotides and Pro
teins in Regenerating Rat Liver Studied with Glycine-
N15. Acta Chem. Scand.. 10: 1568-1575
Harbers, E. 1961 Untersuchungen der Ribonucleinsaure aus
Zellkernen, II. Stoffwechselverhalten verschiedener
Ribonucleinsaure Fraktioner aus den Kernen „ruhender"
und proliferiender Zellen. Hoppe-Seyler's Z. fur
Physiol. Chem., 327: 3-12
Herbert, V. and Zalusky, R. 1962 Interrelation of Vita
min B^g Folic Acid Metabolism. Folic Acid Clear
ance Studies. J. Clin. Invest., 41: 1263-1276
Holbrook, D. J. Jr., Irvin, J. L., Irvin, E. M., and
Rotherham, J. I960 Incorporation of Glycine into
Protein and Nucleic Acid Fractions of Nuclei of Liver
and Hepatoma. Onn. Res^ 2Q: 1329-135?
Hbtta, Y. and Osawa, S. 1958 Further Studies on Nuclear
and Cytoplasmic Ribonucleic Acids. Biochim. Biophys.
Acta. 28: 642-643
Huang, R-C. C. and Bonner, J. 1962 Histone, A Suppressor
of Chromosomal RNA Synthesis. Proc. Nat. Acad. Sci..
48: 1216-1222
Hyden, H. and Egyhazi, E. 1962 Changes in the Base Com
position of Nuclear Ribonucleic Acid of Neurons Dur
ing a Short Period of Enhanced Protein Production.
J. Cell Biol.. 1£: 37-44
Ives, D. H. and Barnum, C. P. 1962 Radiochemical Equili
bration of Phosphorous-containing Precursors of
Deoxyribonucleic Acid in Regenerating Rat Liver. J.
Biol. Chem.. 237: 2599-2610
Izawa, M., Allfrey, V. G., and Mirsky, A. E. 1963 Rela
tionship between RNA Synthesis and Loop Structure in
Lampbrush Chromosomes. Science. 140: 382
Jardetzky, C. D. and Barnum, C. P. 1958 Estimation of
Metabolic Reaction Rates from a Mathematical Analysis
of Tracer Experiments. Arch. Biochem. Biophys.. 73'
435-450
Kelly, L. S., Hirsh, J. D., Beach, G., and Palmer, W.
1957 The Time Function of P ^ Incorporation into
Deoxyribonucleic Acid of Regenerating Liver, the
Effect of Irradiation. Can. Res.. 17: 117-121
Kier, H. M. and Davidson, J. N. 1958 Purine and Pyrimi
dine Derivatives in the Acid-Soluble Fraction of
Animal Tissues and Cell Nuclei. Arch. Biochem. Bio
phys. . 77: 68-80
Kihara, H. K. and Sibitani, A. 1955 Stability of Desoxy-
pentosenucleic Acid Phosphorous (DNA-P) in Growing
Tissue. Biochim. Biophys. Acta. 17:579-580
Kit, S. I960 Compositional Heterogeneity of Normal and
Malignant Tissue Deoxyribonucleic Acids. Biophys.
Biochem. Res. Comm.. 301-307
Kit, S. 1961 Equilibrium Sedimentation in Density Gradi
ents of DNA Preparations from Animal Tissues. J.
Mol. Biori.. *>: 711-716
Kleinschmidt, W. J. 1959 The Composition of Ribonucleic
Acid and Deoxyribonucleic Acid of Normal and Neoplas
tic Tissues. Can. Res.. 19:966-969
Langen, P. and Liss, E. 1961a Uber die Etnhau von Radio-
activem Phosphat in die Nukleotide der Desoxyribo-
nucleinsaure. Biochem. Z., 335: 187-198
Langen, P. and Liss, E. 1961b Unterschiede im Einbau von
radioactivem Phosphat in die Nukleotide der Desoxy-
ribonucleinsaure. Acta Biol. Med. Germ., 6: 186-188
Levinthal, C., Keynan, A., and Higa, A. 1962 Messenger
RNA Turnover and Protein Synthesis in B. subtilis
Inhibited by Actinomycin D. Proc. Nat. Acad. Sci.,
1631-1638
Lieberman, I. and Ove, P. 1962 Deoxyribonucleic Acid
Synthesis and Its Inhibition in Mammalian Cells Cul
tured from the Animal. J. Biol. Chem.. 237: 1634-
1642
Logan, R. and Davidson, J. N. 1957 Heterogeneity in
Nuclear Ribonucleic Acid. Biochim. Biophys. Acta.
24: 196-203
Looney, W. B. I960 The Replication of Deoxyribonucleic
Acid in Hepatocytes. Froc. Nat. Acad. Sci.. 46:
690-698
McEwen, B. S. 1963 Studies of Energy Yielding Reactions
in Thymus Nuclei, I. Comparison of Nuclear and Mito
chondrial Phosphorylation. J. Biol. Chem. . 238: 758-
765
Marmur, J. and Doty, P. 1959 Heterogeneity in Deoxyribo
nucleic Acids, I. Dependence on Composition of the
Configurational Stability of Deoxyribonucleic Acids.
Nature, 18^: 1427-1429
Marmur, J. and Doty, P. 1961 Thermal Renaturation of
Desoxyribonucleic Acids. J. Mol. Biol.. 585-595
Marshak, A. and Vogel, H. J. 1951 Microdetermination of
Purines and Pyrimidines in Biological Material. J.
Biol. Chem., 18%: 597-605
Mendelsohn, M. L., Dohan, P. C. Jr., and Moore, H. A. Jr.
I960 Autoradiographic Analysis of Cell Prolifera
tion in Spontaneous Breast Cancer of C^H Mouse, I.
Typical Cell Cycle and Timdng of DNA Synthesis.
J. Nat. Cancer Inst. . 25: 477-486
Meselson, M. S. and Stahl, F. W. 1958 The Replication of
Deoxyribonucleic Acid in Escherichia coli. Proc.
Nat. Acad. Sci., 44: 671-682
Meselson, M. S., Stahl, F. W., and Vinograd, J. 1957
Equilibrium Sedimentation of Macromolecules in Den
sity Gradients. Proc. Nat. Acad. Sci.. 43: 581-588
Midgley, J. E. M. and McCarthy, B. T. 1962 The Synthesis
and Kinetic Behavior of Deoxyribonucleic Acid-Like
Ribonucleic Acid in Bacteria. Biochim. Biophys. Acta
61: 696-717
Morin, G. A., Zajdela, F., and Costerousse, 0. 1957
Metabolic Heterogeneity of Mouse Liver Deoxyribonu
cleic Acid. Eago. Cell Res.. 1^: 204-207
Nygaard, 0. F. and Rusch, H. P. 1955 Incorporation of
Radioactive Phosphate into Nucleic Acids of Regenera
ting Rat Liver. Can. Res.. 15: 240-245
Ord, M. G. and Stocken, L. A. 1961 Acid Soluble Phos
phate Metabolism in Nuclei from Rat Thymus Gland.
Biochem. J., 81: 1-8
Osawa, S., Allfrey, V. G., and Mirsky, A. E. 1957 Mono
nucleotides of the Cell Nucleus. J. Gen. Physiol..
40:491-513
Osawa, S., Takata, K., and Hotta, Y. 1958 Nuclear and
Cytoplasmic Ribonucleic Acids of Calf Thymus.
Biochim. Biophys. Acta. 28: 271-277
Felling, G. 1959 Chromosomal Synthesis of Ribonucleic
Acid as Shown by Incorporation of Uridine Labeled
with Tritium. Nature. 184: 655-656
Perry, T. L. and Walker, D. 1958 Failure of Deoxyribo
nucleic Acid to Effect Somatic Transformation in
Rats. Soc. Exp. Biol. Med. Proc. . 99: 717-720
Pogo, A. 0., Pogo, E. G. T., Littau, V. C. , Allfrey, V. G.,
and Mirsky, A. E. 1962 The Purification and Proper
ties of Ribosomes from the Thymus Nucleus. Biochim.
Biophys. Acta. 55:849-864
Prescott, D. M. and Kimball, R. F. 1961 Relation be
tween RNA, DNA, and Protein Synthesis in the Replica
ting Nucleus of Euplotes. Proc. Nat. Acad. Sci.,
42: 686-695
Rees, K. R., Rowland, G. F., and Varcoe, J. S. 1963 The
Metabolism of Rat Liver Nucleoli and Other Subnuclear
Fractions. Biochem. J., 86: 130-156
Reichard, P. 1959 The Enzymic Synthesis of Pyrimidines.
Advances in Enzymology and Related Sub.jects of Bio
chemistry. Volume 21, F. F. Nord, (ed.), Interscience
Publishers, New York, pp. 263-294
Rice, S. A. and Doty, P. 1957 The Thermal Denaturation
of Deoxyribonucleic Acid. J. Am. Chem. Soc.. 79:
3937-3947
Rice, S. A., Wada, A., and Geiduschek, E. P. 1958 Some
Comments on ttie Theory of Denaturation. Disc. Farad.
Soc.. 2£: 150-137
Rolfe, R. 1963 Changes in the Physical State of DNA
During the Replication Cycle. Proc. Nat. Acad. Sci.,
386-392
Rolfe, R. and Meselson, M. S. 1959 The Relative Homo
geneity of Microbial Deoxyribonucleic Acid. Proc.
Nat. Acad. Sci.. 4^: 1039-1043
Rudkin, G. T. and Corlette, S. L. 1957 Disproportionate
Synthesis of Deoxyribonucleic Acid in a Polytene
Chromosome Region. Proc. Nat. Acad. Sci., 4^: 964-
968
Rudkin, G. T. and Woods, P. S, 1959 Incorporation of
x
Cytidine and Yr Thymidine into Giant Chromosomes of
Drosophila during Puff Formation. Proc. Nat. Acad.
Sci., ££: 997-1003
Schemer, K., Lathan, H., and Darnell, J. E. 1963 Demon
stration of an Unstable RNA and a Precursor to Ribo-
somal RNA in HeLa Cells. Proc. Nat. Acad. Sci.. 49:
240-248
Schildkraut, C. L., Marmur, J., and Doty, P. 1962 Deter
mination of the Base Composition of Deoxyribonucleic
Acid from Its Buoyant Density in CsCL J. Mol. Biol.,
Schmidt, G. and Thanhauser, S. J. 194-5 A Method for the
Determination of Desoxyribonucleic Acid, Ribonucleic
Acid, and Phosphoprotein in Animal Cells. J. Biol.
Chem., 161: 85-89
Sibitani, A., deKloet, S. R., Allfrey, V. G., and Mirsky,
A. E. 1962 Isolation of a Nuclear RNA Fraction
Resembling DNA in Its Base Composition. Proc. Nat.
Acad. Sci.. 46: 4-71-4-77
Sisken, J. E, and Kinosita, R. 1961 Time of DNA Synthe
sis in the Mitotic Cycle in vitro. J. Biophys.
Biochem. Cytol.. 2: 509-518
Smellie, R. M. S., Humphrey, G. F., Kay, E. R. M., and
Davidson, J. N. 1955 The Incorporation of Radio
active Phosphorous into Nucleic Acids of Different
Rabbit Tissues. Biochem. J., 60: 177-185
Stein, A. M., Murakami, V. T., and Visser, D. W. 1959
Purine Synthesis in Mouse Tissues. Can. Res.. 19:
84-88
Sueoka, N. 1961a Correlation between Base Composition of
Deoxyribonucleic Acid and Amino Acid Composition of
Protein. Proc. Nat. Acad. Sci.. 4-7: 114-1-114-9
Sueoka, N. 1961b Variation and Heterogeneity of Base
Composition of Deoxyribonucleic Acids: A Compilation
of Old and New Data. J. Mol. Biol.. 51-4-0
Sueoka, N., Marmur, J., and Doty, P. 1959 Heterogeneity
of Deoxyribonucleic Acids, II. Dependence of the
Density of Deoxyribonucleic Acids on Guanine-Cytosine
Content. Nature. 185: 1429-14-31
Swick, R. W., Koch, A. L., and Handa, D. T. 1956 The
Measurement of Nucleic Acid Turnover in Rat Liver.
Arch. Biochem. Biophys., 6%: 226-242
Takagi, Y., Hecht, L. I., and Potter, V. R. 1956 The
Nucleic Acid Metabolism in Regenerating Rat Liver,
II. Studies on Growing Rats. Can. Res.. 16: 994-998
Taylor, J. H. I960 Asynchronous Duplication of Chromo
somes in Cultured Cells of the Chinese Hamster.
J. Biophys. Biochem. Cytol., 2: 455-464
Thomas, C. A. Jr. 1956 The Mild Acidic Degradation of
Deoxyribonucleic Acid. J. Am. Chem. Soc., 2§: 1854-
1860
Totter, J. R. 1954 Incorporation of Isotopic Formate
into the Thymine of Bone Marrow Deoxyribonucleic
Acid in vitro. J. Am. Chem. Soc.. 76: 2196-2197
Tyner, E. P., Heidelberger, C., LePage, G. A. 1953
Intracellular Distribution of Radioactivity in Nu
cleic Acids, Nucleotides, and Proteins Following
Simultaneous Administration of P ^ and Glycine-2-C1^.
Vogt, M. and Dulbecco, R. 1963 Steps in the Neoplastic
Transformation of Hamster Embryo Cells by Polyoma
Virus. Proc. Nat. Acad. Sci.. 4-9: 171-179
Volkin, E., Astrachan, L„, and Countryman, J. L. 1958
Metabolism of RNA Phosphorous in Escherichia coli
Infected with Bacteriophage T 7. Virology. 6: 545-
555
Wang, T-Y. 1962 The Chemical Composition of Nuclear
Ribosomes of Calf Thymus. Arch. Biochem. Biophys..
2Z:387-392
Wyatt, G. R. 1951 The Purine and Pyrimidine Composition
of Deoxypentose Nucleic Acids. Biochem. J., 4-8:
584-390
Zbarskii, I. B. and Georgiev, G. P. 1959 New Data on the
Fractionation of Cell Nuclei of Rat Livers and the
Chemical Composition of Nuclear Structures. Biokhimi-
ya. 24: 177-184 (Translated from Russian)
Zbarsky, S. H., Hor.i, A. and Findley, B. S. 1958 The
Incorporation of Labeled Formate into the Tissue
Nucleic Acids of Rats Bearing Novikoff Hepatoma.
Can. J. Biochem., £6: 1185-1192
FIGURES
FIGURE 1
77
The Folic Acid Interconversion System (after Herbert
and Zalusky, 1962). Abbreviations are as follows:
FA— Folic Acid (pteroylglutamic acid); DHFA— Dihydro-
folic Acid; THFA— Tetrahydrofolic Acid; AICAR—
Aminoimidazolecarboxamide Ribotide.
^hymidylic Acid
Uridylic Acid
I Methionine
A
Vitamin B
Dependent
joxyuridylic
Acid
Homocysteine
N7 Methyl-THFA
FA Conjugates
in Food
FA(pga)
Inosmic
Acid
ICAR
Formyl-THFA
Histadine
Urocanic Acid
k Formiminoglu-
tamic Acid
Glutamic Acid
»Serine
'Glycine
Formimino-THFA
Hr» Methenyl-THFA
(Anhydroleucovorin)
Formaldehyde j
Methylene-THFA
Formic Acid
Formyl-THFA
(Folinic Acid,
Leucovorin)
FIGURE 2
78
Flow Diagram of Isolation Procedures of Nucleic Acids
Frozen Tissues
Homogenization in EDTA-Fluo
ride; Centrifugation
Supernatant Nuclear Fragments
Extraction with 1.0
M. NaCl at 5 C.;
Centrifugation
Supernatant
Dissociation with
Saturated NaCl; Pre
cipitation with Ethanol
1
Nuclear Residue
Extraction with 1.5
M. NaCl at 100u C.;
Centrifugation
(-----
Supernatant
Ethanol-RNA
r
Extractable- Supernatant
DNA
__ L
High A-T DNA Low A-T DNA
I -----------
Supernatant
Precipitate
Precipitation with
Ethanol; Centrifugation
1
Precipitate
I -------
Supernatant
Chromatin-RNA
Hydrolysis with 1.0
M. KOH; Acidifica
tion; Precipitation
with Ethanol;
Centrifugation
,
Precipitate
Nonextractable-
WK-----
TABLES
80
TABLE I
Specific Radioactivity of High A-T DNA
Sample
Number Brain Kidney Pancreas Liver
1 3.8
63
75
81
2 8.2
57 72 86
3 8.3
62
77 75
4 8.4 50
73
78
5 67 77 85
6 61 70 81
7 53 79
8 60
75
9 70
71
10 54
85
11
71 73
12
71 79
13 75
14
77
Mean
7
61 74
79
Standard
Deviation 3
7 5
81
TABLE I
(Continued)
Specific Radioactivity of High A-T DNA
Sample
Number
Salivary
Gland Testis Thymus Spleen
1
134 131
460 706
2
125
138
459
695
3
138
139 468
699
4
125 127
444
763
5 131
138 486 776
6 130
143
434
711
7 127 489 749
8 124 702
9 138
733
10
139
Mean 131 136 465
723
Standard
Deviation 6 6
15 30
82
TABLE II
Specific Radioactivity of Low A-T DNA
Sample
Number Brain Kidney Pancreas Liver
1 5.4
57 75 77
2 7.2 60 64 62
3
3.0 56 66 56
4 5.0 54 66
55
5 53 56
53
6 62
71
76
7 50 82
8
45 49
9 47 54
10 52 66
11 60 78
12
77
13 64
14
83
Mean
5
$8
66
63
Standard
Deviation 2 6 6 12
83
TABLE II
(Continued)
Specific Radioactivity of Low A-T DNA
Sample
Number
Salivary
Gland Testis Thymus Spleen
1
113
130 473 635
2 114
129
448 654
3
126 132 454 696
4 121
133
464
685
5 105
126 446 641
6
119
130
455 645
7 123
456 670
8
107
650
9
130
679
10 120 693
Mean 118 130 454
669
Standard
Deviation 10 6
7
10
84 -
TABLE III
Specific Radioactivities of Adenine, Guanine, and
Thymine in High A-T DNA. Each number is aniaverage
of three determinations.
Organ Adenine Guanine Thymine
Brain 10 11 —
Kidney 98
91
24-
Pancreas 130 120 32
Liver
125
112 28
Salivary
Gland
209
186
67
Testis 229 203 70
Thymus 800
731
238
Spleen 1,220 1,007
373
85
TABLE IV
Specific Radioactivities of Adenine, Guanine,* and
Thymine in Low A-T DNA. Each number is an average
of three determinations.
Organ Adenine Guanine Thymine
Brain 10
13
—
Kidney 96
87
21
Pancreas
153
116 34
Liver 121
115 33
Salivary
Gland
213
178 65
Testis
231 191 73
Thymus
793
732 236
Spleen 1,204
1,013 382
86
TABLE V
Mean Values of the Specific Radioactivities of
the Bases, Adenine, Guanine, and Thymine in
Extractable-DNA (High A-T DNA and Low A-T DNA).
Organ Mean of Mean of Mean of
Adenines Guanines Thymines
Brain 10 12
Kidney
97 89
22
Pancreas 132 118
33
Liver
123
114 30
Salivary
Gland
211 182 66
Testis 230
199 71
Thymus
797
732 237
Spleen 1,212 1,010 388
TABLE VI
Ratios of the Specific Radioactivities of High
A-T DNA and Low A-T DNA in Extractable-DNA„
Ratios of the Mean Values for the Specific Radio
activities of Adenine, Guanine, and Thymine in
Extractable-DNA
Organ
High A-T DNA
TowTTTSHI
Adenine
Thymine
Adenine
Guanine
Brain 1.41 — 0.82
Kidney
1.07
4.4
1.09
Pancreas 1.11 4.0 1.12
Liver
1.25
4.1 1.08
Salivary
Gland
1.11 3.2 1.16
Testis
1.05
3.2
1.35
Thymus
1.13 3.4 1.09
Spleen 1.09 3.2 1.20
88
TABLE VII
Specific Radioactivities of Adenine, Guanine and
Thymine in Nonextractable-DNA. Each number is
an average of three determinations.
Organ Adenine Guanine Thymine
Brain
83
81
25
Liver 136 116 42
Salivary
Gland
342
219 83
Testis 364 236 100
Thymus 750
775
210
Spleen 1,042
1,153
308
89
TABLE VIII
Specific Radioactivities of Adenine and Guanine
in Chromatin-RNA. Each number is an average of
at least two determinations.
Organ Adenine Guanine
Brain
69
72
Liver 184
557
Salivary
Gland
325 1,309
Testis
377
904
Thymus
400 656
Spleen
410 647
TABLE IX
90
Specific Radioactivities of Adenine and Guanine
in Ethanol-RNA
Organ Adenine Guanine
Kidney
Pancreas
Liver
Salivary
Gland
Testis
Thymus
Spleen
89
138
177
394
274
366
305
76
125
162
339
261
345
293
91
TABLE X
Ratios of the Specific Radioactivities of the
Bases in Nonextractable-DNA, ChromatineRNA, and
Ethanol-RNA and the Specific Radioactivities of
the Bases in Extractable-DNA.
Base Nucleic Acid Brain Kidney Pancreas Liver
Nonextracta-
ble-DNA
8.3
1.10
Adenine
Chromatin-
RNA
6.9
—
1.50
Ethanol-RNA
—
0.92 0.96 1.44
Nonextracta-
ble-DNA
6.7
1.02
Guanine
Chromatin-
RNA 6.0
— —
4.89
Ethanol-RNA —
0.85
1.06 1.42
Thymine
Nonextracta-
ble-DNA
—
— — 1.40
92
TABLE X
(Continued)
Ratios of the Specific Radioactivities of the
Bases in Nonextractable-DNA, Chromatin-RNA, and
Ethanol-RNA and the Specific Radioactivities of
the Bases in Extractable-DNA.
Base Nucleic Acid Salivary
Gland
Testis Thymus Spleen
Adenine
Nonextracta-
ble-DNA
Chromatin-
RNA
1.62
1.54
1.58
1.60
0.94
0.50
0.86
0.34
Ethanol-RNA
1.87 1.19
0.46
0.25
Guanine
Nonextracta
ble-DNA
Chromatin-
RNA
1.20
7.20
1.18
4-.52
1.06
0.90
1.14
0.64
Ethanol-RNA 1.86
1.51 0.47 0.29
Thymine
Nonextracta-
ble-DNA
1.25 1.41 0.89 0.79
9 3 5
TABLE XI
Ratios of the Values for the Specific Radioactivi
ties of Adenine, Guanine, and Thymine in
Nonextractable-DNA.
Adenine Adenine
Organ Thymine’ Guanine
Brain 3.3 1*0
Liver 3.2 1.17
Salivary
Gland
4.1 1.56
Testis 3.6 1.54
Thymus 3*6 0.97
Spleen 3.4 0.90

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Asset Metadata

Creator Stone, Nancy Joy (author)

Core Title Metabolism of mammalian deoxyribonucleic acid and nuclear ribonucleic acid

School Graduate School

Degree Doctor of Philosophy

Degree Program Biology

Degree Conferral Date 1964-01

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Chambers, Leslie A. (committee chair), Maxfield, Myles (committee member), Visser, Donald W. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-324043

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Document Type Dissertation

Rights Stone, Nancy Joy

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