THE JOURNAL OP BIOLOGICAL CHEMISTRS
    Vol. 237, No. 6, June 1962
      Printed in U.S.A.

Enzymatic Synthesis of Deoxyribonucleic Acid*

XI. FURTHER STUDIES ON NEAREST NEIGHBOR BASE SEQUENCES IN DEOXYRIBONUCLEIC ACIDS

       b1. K. SWARTZt T. A. TRAUTNER,$ AND ARTHUR KORNBERG

From the Department of Biochemistry, Stanford University School of Jfedicine, Palo Alto, California

           (Received for publication, January 26, 1962)

The examination of nearest neighbor base sequences in deoxy-
ribonucleic acid (DNA) with the technique described by Josse,
Kaiser, and Kornberg (1) established (a) that DNA from a given
source directs the synthesis of a product in which the four bases
occur next to one another in the 16 possible arrangements, not at
random but in a pattern of frequencies unique for that DNA; and
(b) that enzymatically synthesized DNA, and by inference the
primer DNA, shows complementary pairing of adenine to thy-
mine and of guanine to cytosine between two strands of opposite
polarity, as proposed in the Watson and Crick model.
This paper describes esperiments with the same technique
undertaken to explore the following questions.  (a) Does the
replication of a single stranded DNA (with noncomplementary
base composition) proceed by base pairing as in double stranded
DN.%?  (b) Are the nearest neighbor sequence patterns in the
DNA's from different tissues and tumors of a given species the
same? (c) Do sequence patterns in the DNA's of various bio-
logical forms reveal any relationships among or within groups of
organisms?

-

EXPERIMEXTAL PROCEDURE

-1 faterials

Substrates and Enzymes-Labeled and unlabeled deoxynucleo-
side triphosphates, micrococcal DNase, and calf spleen diesterase
were prepared as described previously (2-5).  The DNA-synthe-
sizing enzymes, Escherichia coli polymerase and the T2 phage-
induced polymerase, used in most experiments were prepared
from Fraction VII, refractionated on diethylaminoethyl cellulose
(DEAE-cellulose) (2) or on phosphocellulose resin (6) ; the specific
activities of these enzymes were approximately 1,500.  E. coli
DN.4 endonuclease (7) (carboxymethyl cellulose fraction, 2,500
units per ml) and E. coli DIU phosphodiesterase (8) (DEAE
fraction, 20,000 units per ml) were kindly supplied by Dr. I. R.
Lehman.  Crystalline pancreatic DNase was purchased from
the Worthington Biochemical Corporation.

DNA Preparations-The DNA's used in these experiments
generally had eM values (based on deoxypentose) ranging be-
tween 6.3 and 7.5; their protein contents were in most cases less
than 6% as determined by the method of Lowry et al. (9).  Un-

* This investigation was supported by research grants from the
National Institutes of Health, United States Public Health Serv-
ice.
t Present address, Massachusetts General Hospital, Bosto4
Massachusetts.
3 Fellow of Deutsche Forschungsgemeinschaft ; present address,
Institut fur Genetik, Universitat Koln, Koln-Lindenthal, Ger-
many.

less otherwise noted, DNA's were isolated by homogenizing tis-
sues or cells in a Waring Blendor in 0.15 M NaCl + 0.01 M so-
dium citrate and centrifuging according to the procedure of Kay,
Simmons, and Dounce (10).  The crude tissue fractions thus
obtained were again stirred in the Waring Blendor and then sub-
jected either to repeated shaking with chloroform-octanol (1 1)
and precipitation with 95% ethanol, or to treatment with sodium
lauryl sulfate according to the method of Kay, Simmons, and
Dounce (IO); in some cases both procedures were used.  When
necessary, purified DNA's were treated with pancreatic RNase
to remove RNA.

DNA's of bacteriophages T1 (T1 phage was kindly provided by
H. Modersohn) and T5 were prepared by phenol treatment of
virus stocks initially purified by differential centrifugation and
freed from bacterial DNA by treatment with pancreatic DKase.
Paracentrotus lividus (sea urchin) DNA was obtained by extrac-
tion with 3 M NaCl of a sperm homogenate, kindly supplied by
Dr. R. Hinegardner.  Release of DNA from bull sperm, kindly
provided by Dr. S. W. Mead, required treatment with 1 N
NaOH for 5 hours at 37"; after neutralization, the DNA was pre-
cipitated with 95% ethanol and further purified.

We are indebted to Dr. R. L. Sinsheimer for DNA of bacterio-
phage ChX 174 (ax); to Dr. N. Sueoka for Tetrahymena pyri-
formis DNA, prepared essentially by the Marmur method (12),
and for Cancer borealis (crab) testis DNA; to Dr. R. Sager for
Chlamydomonas DNA prepared by a sodium lauryl sulfate pro-
cedure; and to Dr. I<. Burton for Echinus esculenfu (sea urchin)
DNA.

Aiethods

Nearest Neighbor Frequency AnalysisThis method (1) in
volves enzymatic replication of a given DNA primer and employs
as substrates, deoxyribonucleoside triphosphates in which the
sugar-esterified 5`-phosphate is labeled with Pa.  Subsequent
cleavage between the phosphate and carbon 5` of the synthesized
polynucleotide chains yields Ps*-labeled 3'-mononucleotides; P3*
introduced into the DNA by the substrate nucleotide conse-
quently labels the adjacent nucleotide, ita "nearest neighbor."
By determining the Pa content of each of the four 3`-mononu-
cleotides isolated from the digested product of a reaction with a
given labeled substrate, one can calculate the frequency with
which this nucleotide is linked to each of the four nucleotides
found in the DNA.

The methods of enzymatic synthesis of DNA, digestion to 3`-
mononucleotides, separation of 3`-mononucleotides, and calcula-
tion of nearest neighbor frequencies were similar to those reported

1961

1962 Enzymatic Synthesis of Deoxyribonucleic Acid. XI Vol. 237, No. 6

Cornpsition
detemuned by
chemical analysis'

0.246
0.328
0.242
0.185

earlier (1). As before, the extent of DNA synthesis generally
represented a 20% increment over the amount of primer added.
Determination of Experimental Error-An estimate of error in
determination of nearest neighbor frequencies was obtained by
duplicate analyses of mouse thymus, mouse lymphoma, starfish
testis, and salmon sperm DNA samples.  Standard deviations,
expressed as percentage of the mean ("coefficients of variation"),
were calculated for each of the 16 dinucleotide sequences in each
of the four duplicate runs; the average of the coefficients of varia-
tion obtained for each sequence was found to vary between 2.33
and 10.0%.  The over-all average coefficient of variation was

5.8%.

Deviations in Total Isolated Ap and Tp Nucleotides-The total
amount of Tp (TpA +- TpT + TpG + TpC) has consistently
exceeded the total Ap (ApA + ApT + ApG + ApC) by 2 to
20%.  This deviation was not observed in our earlier studies
(l), which, although largely confined to bacterial DNA's, also
included calf thymus DNA.  Since the enzymatic digestions
and chromatographic procedures were complete and quantita-
tive, it was possible that the character of the polymerase prepara-
tion used or the state of the DNA primer might be responsible
for this deviation.  These factors were explored by (a) the use
of different polymerase preparations and (b) pretreatment of
the DNA primer with heating or nuclease cleavage.

Mouse ascites tumor cell DNA was analyzed with three dif-
ferent E. coli polymerase preparations, and heated salmon sperm

20% synthesis

Predicted
c/::ccal Observed
analysis
--

0.328  0.310
0.246  0.242
0.185  0.202
0.242  0.246

4

TTCAOTG

Pre-
dictedt
from

analysis

0.287
0.287
0.214
0.214

A/! a33        SO 1.15 (1.00)
G/cU         0.5 0.91 (1.00)
E 1.33         1.33 1.33 (1.33)

FIG. 1. Scheme for replication of single stranded DNA.  An
arbitrarily selected sequence of bases in a hypothetical single
stranded DNA primer is designated in bold print.  The base ratios
for limited and extensive replication refer to the values for the
newly synthesized DNA molecules, designated by standard print.

         TABLE I
Composition of products after limited and
  extensive replication of ipx DNA

Pre-
dictedt
from Observed
synt zoip esis .

---

0.276  0.271
0.276  0.293
0.224  0.213
0.224  0.224

Base

A
T
G
C

I

Composition determined by nearest
    neighbor analysis

Mx)% synthesis

* See (13).
t Based on unlimited replication (see Fig. 1).

DNA, with the distinctive polynierases from normal and T2
phage-infected cells. The coefficients of variation in these
analyses did not exceed the experimental error (see above).
A comparison of heated with native calf thymus DNA showed
no significant change in sequence frequencies in the earlier study
(1) or in the present one.  \17ith heated salmon sperm DNA com-
pared with native, the coefficients of variation for three of the
sequences (GpA, TpC, and CpT) differed by more than 10%.
Pretreatment of calf thymus DNA with E. coli endonuclease, a
variable contaminant of polymerase preparations, had a pro-
found effect upon the sequence frequencies; after endonuclease
treatment until 53 % of the nucleotides were released, coefficients
of variations between treated and untreated DNA were greater
than 10% for six of the sequences.  However, prior action by
pancreatic DNase, E. coli phosphodiesterase, or a polymerase
preparation on calf thymus DNA did not seem to alter the se-
quence frequencies significantly.

It must be concluded that, although distortions of sequence
frequencies can be introduced by alteration of the primer, the
basis for the systematic deviations between the Tp and Ap analy-
ses encountered in these studies is not yet clear.  These devia-
tions, however, do not alter the principal conclusions which may
be drawn from the experiments to be reported.

RESULTS

Replication of Single Stranded Primer-The DNA of phage
OX has been demonstrated by Sinsheimer (13) to be single
stranded and to be further distinguished by the absence of equiva-
lence between A and T and between G and C.  The DNA is a
primer for polymerase and can lead to 10-fold or greater net
synthesis of a product which has the characteristics of double
stranded DNA (14).  Two nearest neighbor analyses were car-
ried out, one under conditions of limited replication (20% increase
over the amount of primer), and the other with extensiue replica-
tion (600% increase).

In limited replication, DNA synthesis will be directed mainly
by original primer molecules.  In order to minimize participation
of newly synthesized, double stranded molecules as primers, syn-
thesis was restricted to a 20% increase over the initial primer
added.  If such replication follows the base composition of the
primer, the nucleotide composition of the newly synthesized
product, identified by its P32 label, should be the complement of
the base composition of the primer; Le. the A content of the
product should be equal to the T content of the primer, and the
T content of the product to the A content of the primer (Fig. 1).
The A:T ratio of the product should therefore be the reciprocal
of the A:T ratio of the primer.  Similarly, the G:C ratio of the
product should be the reciprocal of the G:C ratio of the primer.
As a consequence, the ratio, (A + T)/(G + C), of the product
should be identical with that of the primer.  The results in Table
I show that these predictions are fulfilled.

In extensive replicatiora, the priming molecules after the initial
period are double stranded, and accordingly their replication
should yield a product with identical nucleotide composition.
Specifically, equivalence of purine to pyrimidine nucleotides in
the product (A = T, G = C) should obtain, and the A (or T)
value, for example, should be equal to one-hdf the sum of the .4
and T values of the primer (or limited replication product).
Thus, the mole fraction of A in the original strand was 0.246,
and that of T, 0.328; consequently, the mole fractions in the
complementary strand will be 0.328 for A and 0.246 for T.  The

June 1962

M. N. Swartz, T. 8. Trautner, and A. Kornberg

1963

ApA, TpT
CpA, TpG
GpA,TpC
CpT, ApG
GpT, ApC
GpG, CpC

APT
CPG
GPC

TPA

over-all mole fractions should be, A = (0.246 + 0.328)/2 and
T = (0.328 + 0.246)/2.  These predictions are borne out by
the results obtained (Table I).

Individually, the nearest neighbor frequencies obtained under
both conditions of synthesis support the replication mechanism
discussed here.  Matching of complementary base pairs (in the
Pn-labeled product) is observed under conditions of ezlensive
replication but not in limited replication (Table 11).  The fre-
quencies of matching nearest neighbor pairs are close to values
predicted from the frequencies obtained in limited replication by
the same reasoning which had been applied to predict over-all
base composition in 600% synthesis.  Since each of the four se-
quencies, TpA, APT, CpG and GpC, is its own match (1), the
frequencies of each of these sequences should remain unaltered,
whether replication is limited or extensive; the results fit this
prediction closely.

Sequence Frequencies in T1 and T5 Bacteriophage DNA-The
base composition and the nearest neighbor pattern of the DNA
from temperature coliphage X are similar to those of its host, E.
coli (1).  The DNA's of the virulent T-even phages, T2, T4, and
T6, have identical base compositions; their nearest neighbor pat-
terns differ from those of E. coli (1).  The sequence frequencies
in the DNA of T1, a phage intermediate between the typically

  ate and virulent phages, and that of T5, a virulent phage
  in many respects to the T-even phages, can be distin-
  d from one another (Table 111) but do not deviate strik-
  from values for random association of the nucleotides.
  ost tenable conclusion to be drawn from these compariSons
f sequence patterns is to regard differences as significant but
entity as no more revealing than identity of the base composi-

e Frequencies in DNA of Several Tissues of a Species-
ues for DNA's of three different bovine organs and of
use tissues, including two tumors, are compared in Ta-

V and V.  The coefficients of variation for the bovine
  for the mouse DNA's were not significantly different
  obtained for repeated analyses of identical DNA's.

   e, any differences that may exist among the several bo-
ine DNA's or among any of the mouse DNA samples are within

   Freqzrencies in Crab Testis DNA-Examination
     of crab testes has revealed discrete and distinctive
       density gradient centrifugation of DNA iso-
d from the testes of five specimens of Cancer borealis, Sueoka
   two components, the lighter one representing as much
        a1 DNA.  The buoyant density of the main
       to a G-C content of 42%, which is character-
         .  However, the buoyant density of the minor
   indicated a low G-C content (20% or less) and was
   the density of dAT polymer, an alternating copolymer
nd T synthesized de novo by polymerase (16).  In order to
ate the possibility that adventitious materials, such as pro-
, might be responsible for the low buoyant density of the
A band, and with the thought that this band might even be
natural" dAT polymer, Dr. Sueoka asked us to examine
t neighbor frequency patterns of the crab DNA prepara-

  light component of C. borealis primed DNA synthesis at a
  mparable to dAT, but unlike the latter, all four deoxy-
ucleoside triphosphates were required. When dGTP and
CTP were omitted, the rate of synthesis was only 19% of that

or of the analysis.

0.071, 0.076 0.072, 0.082 0.079, 0.093 0.105, 0.100
0.071, 0.071 0.072, 0.070 0.065, 0.069 0.058, 0.057
0.055, 0.0560.056, 0.0620.062, 0.0650.054, 0.060
0.055, 0.0550.051, 0.0470.060, 0.0530.064, 0.056
0.055, 0.0540.054, 0.0550.056, 0.0540.048, 0.052
0.056, 0.0560.051, 0.0590.046, 0.0440.035, 0.038

0.051      0.050     0.057     0.098
0.068     0.076     0.076     0.103
0.067     0.068      0.058     0.032
0.083     0.074     0.063     0.043

          TABLE I1
Nearest neighbor frequencies* of ax DNA in
  limited and extensive replication

(1.29)' (1.24)
           Liver
(1.28) (1.29)

Thymus

Nearest neighbor
  sequence

ApA, TpT    0.080, 0.085   0.079, 0.090
CpA, TpG    0.078, 0.076   0.072, 0.077
GpA, TpC    0.066, 0.072   0.063, 0.071
CpT, ApG    0.074, 0.069   0.079, 0.071
GpT, ApC    0.049, 0.053   0.052, 0.051
GpG, CpC    0.051, 0.060   0.053, 0.057

TPA 0.052       0.055
APT 0.074        0.071
CPG 0.016       0.015
GPC 0.045       0.046

Nearest neighbor
  sequence

(1.39)
Srm

K35)

0.084,0.094
0.077, 0.069
0.059, 0.069
0.073, 0.069
0.049, 0.052
0.050, 0.060

0.062
0.076
0.014
0.043

Limited re lication
(20%): Otserved

0.101, 0.069
0.096, 0.048
0.054,0.064
0.052, 0.069
0.047, 0.068
0.040,0.053

0.061
0.072
0.045
0.061

Extensive replication (600%)

Predicted from
limited replica- Observed
  tiont

0.085, 0.085
0.072, 0.072
0.059, 0.059
0.061,0.061
0.057, 0.057
0.046, 0.046

0.061
0.072
0.045
0.061

0.085, 0.099
0.070, 0.070
0.058, 0.065
0.064, 0.058
0.053, 0.053
0.041, 0.045

0.059
0.075
0.045
0.061

* Expressed, in this and subsequent tables, as decimal propor-

t Based on unlimited replication (see Fig. 1).

tions of 1.OOO.

    TABLE I11

bacteriophages and E. coli

Nearest neighbor frequencies in DNA's of TI and T6

1964 Enzymatic Synthesis of Deoxym'bonucleic Acid. XI Vol. 237, No. 6

ApA, TpT
CpA, TpG
GpA, TpC
CpT, ApG
GpT, ApC
GpG, CpC

TPA
APT
CPG
GPC

observed with the four triphosphates; when dTTP was also
omitted from the incubation mixture, the rate was reduced to
less than 0.1%.  These results suggested at once that at least
a few G and C residues were interspersed in the chains of the
light crab DNA.

0.091, 0.1010.088, 0.0930.091, 0.0940.084, 0.100
0.076, 0.0830.072, 0.0780.077, 0.0790.074, 0.074
0.060, 0.0620.061, 0.0630.059, 0.0650.059, 0.068
0.076, 0.070 0.075, 0.070 0.075, 0.071 0.072, 0.072
0.057, 0.053 0.057, 0.054 0.051, 0.053 0.056, 0.050
0.051, 0.046 0.051, 0.050 0.052, 0.051 0.048, 0.052

0.060      0.067     0.063     0.062
0.072     0.075     0.075     0.078
0.011      0.009     0.011     0.011
0.038     0.039      0.037     0.039

                TABLE V
Nearest neighbor frequencies in DNA's of mouse tissues and tumors

  (0.56)'
Chlamydomonas
  (0.87)

Nearest 1 (1.38) (1.38) (1.38)
neighbor 1 Tf$rs+

Liver 1 Ly$ph$na I Asci;;!4;;lmor

sequence (1.43) i (1.42)

(1.15)
Wheat germ
(1.21)

* See Table 111.
t Each value is the average of two analyses.

             TABLE VI
Nearest neighbor frequencies of two DNA components of
      Cancer borealis (crab) testis

Nearest neighbor
  sequence

Main component
  (1.80).

0.085, 0.092
0.067, 0.066
0.053, 0.054
0.061, 0.055
0.057, 0.063
0.032, 0.038

0.113
0.116
0.019
0.030

Light componentt
  (36.6)'

0.0127, 0.0126
0.0100, 0.0089
0.0042, 0.0015
O.OOO4, 0.0018
0.0081, 0.0069
o.m, 0.0009

0.504
0.429
O.OOO7
0.0015

* The number in parentheses is the (A + T)/(G + C) ratio

t Each value is the average of two analyses.

determined by nearest neighbor analysis.

Nearest neighbor sequence

Nucleotide incorporation into DNA in the first stage of the
nearest neighbor analysis was in the ratio, A:T:G:C - 0.84:
1.07 :0.030:0.028 mpmoles, indicating a G-C content of approxi-
mately 3%.  A similar value was obtained by determining the
actual nearest neighbor frequencies (Table VI).

The most remarkable result of the nearest neighbor analysis is
that the light crab DNA appears very similar to the dAT co-
polymer, with alternating A and T residues comprising 93% of
the sequences.  However, all 16 possible sequences are observed,
and in strikingly nonrandom distribution.  The matching of se-
quences follows the predictions of Watson-Crick base pairing,
except in those instances in which the very low frequencies are
difficult to measure accurately.

Also included in Table VI are the nearest neighbor frequencies
of the main, heavy component of Cancer borealis DNA.  The
ratio, (A + T)/(G + C), was 1.8, compared with a value of 1.6
calculated by Sueoka from the buoyant density.  Although the
heavy crab DN.4 showed no gross contamination with the light
component, trace amounts might have escaped detection.  Since
the light component is a better primer, the reaction product of a
nearest neighbor analysis of the heavy component would appear
to have a relatively higher A + T content (and higher APT
and TpA sequences) than that of the heavy primer.

The possibility might also be considered that the light com-
ponent is pure dAT and that its contamination by the heavy
component is responsible for the presence of G and C residues.
This is unlikely, however, since replication of the light com-
ponent, as mentioned, is markedly reduced when G and C are
omitted from the reaction mixture.  Furthermore, the nearest
neighbor sequences involving G and C are distinctly different in
the two DNA components.

Sequence Frequencies in DNA of Various Animal and Plant
Species and Bacteria Compared-Analyses of DNA's from several
animal and plant species are shown in Tables VI1 and VIII.
In the previous study (l), the frequency patterns of DNA's
from six bacteria, five phages, and calf thymus were analyzed
for their fit to frequencies predicted for a random arrangement
of bases in DNA molecules.  In random ordering of the nucleo-
tides, the frequency of any nearest neighbor pair should be pre-
dictable as the product of the frequencies of its constituent mono-
nucleotides (e.g. fApT = ffpA = fAp x ffp).  Although most
of the observed sequence frequencies fell within these predictions,
several differed sharply.  This is true also in the frequency pat-

             TABLE VI1
Nearest neighbor frequencies of animal and plant DNA's

0.060, 0.059
0.077, 0.073
0.044, 0.046
0.057, 0.060
0.055, 0.060
0.071, 0.074

0.053
0.054
0.063
0.092

0.072, 0.089
0.068, 0.070
0.062, 0.071
0.067, 0.060
0.056, 0.053
0.051, 0.059

0.058
0.075
0.039
0.050

0.100, 0.116
0.076, 0.069
0.049, 0.064
0.064, 0.047
0.053, 0.062
0.031, 0.046

0.075
0.091
0.022
0.035

0.110,0.102

0.067, 0.067
0.057, 0.059
0.059, 0.053
0.053, 0.058
0.033, 0.038

0.090
0.104
0.020
0.031

Tctralrymcna (3.00) pyriformis

(3.25)

0.153, 0.176
0.045, 0.044
0.045, 0.048
0.052, 0.049
0.034, 0.036
0.016, 0.017

0.127
0.133
0.007
0.020

June 1962

Nearest neighbor sequence

APA, TPT
CPA, TPG
GPAi TPC
CPT, APG
GPT, APC
GPG, CPC

TPA
APT

GPC

CPG

M. N. Swartz, T. A. Trautner, and A. Kornberg

  (1.42).
Human s leen
(1.477

0.097, 0.097
0.074, 0.074
0.061, 0.057
0.071, 0.070
0.049, 0.054
0.050, 0.047

0.067
0.081
0.010
0.043

1965

           TABLE VI11
Nearest neighbor frequencies of animal tissue DNA's

  (1.34)
Chicken red cell
  (1.36)

(1.43)
Salmon liver
(1.33)

Rabbit liver
(1,35)

Starfish testis
  (1.45)

0.091, 0.096
0.073, 0.077
0.060, 0.060
0.071, 0.069
0.051, 0.049
0.062, 0.047

0.059
0.074
0.013
0.048

0.074, 0.083
0.077, 0.075
0.057, 0.067
0.075, 0.069
0.062, 0.062
0.049, 0.052

0.068
0.073
0.017
0.040

0.087, 0.097
0.078, 0.077
0.053, 0.060
0.077, 0.068
0.050, 0.054
0.048, 0.054

0.062
0.072
0.011
0.052

0.102, 0.104
0.072, 0.067
0.058, 0.056
0.058, 0.058
0.064, 0.060
0.043, 0.050

0.066
0.078
0.025
0.038

terns presented in this paper; variances from predicted (random)
frequencies for the 16 nearest neighbor pairs were found to be
considerably greater than experimental error in the cases shown
in Table IX.  A comparison of variances of isomeric nearest
neighbor pairs such as APT versus TpA, for example, reveals sharp
differences.

Fig. 2 is an attempt to determine whether variances from ran-

1

             TABLE IX
Variances from random nearest neighbor frequencies in
    animal, plant, and bacterial DNA's

12 animal and plant DNA's

6 bacterial DNA's

Deviation
from random
frequency'

Variance X 10`

Variance X 104

Deviation
:rom randon
frequency*

-

calcu-
lated
from
error:

-

0.04
0.04
0.07
0.07
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.04
0.04
0.07
0.07

-

-

-

2
2
6
6

4

4
3
6
3
6
6

-

-

-

1
2

10
1
10
8
4
6
3
12
9
12
6

-

+

-

11
10
11
12
12
1
11
2
3
8
3
7

1


5

-

0

-

1

1

1


3

2


2


1

-

Ob-
served

-

0.74
1.13
0.27
0.62
2.62
0.49
2.12
0.77
1.05
0.97
0.38
0.67
4.30
0.80
6.20
0.59

-

t

-

4
4

5
2
6
2
3

3

6
6
6

Ob-
served

-

1.35
2.04
1.87
1.83
0.52
1.03
0.56
1.01
0.77
0.58
0.55
0.61
3.69
0.13
1.88
3.26

hlculsted
from
error$

0.20
0.21
0.14
0.16
0.01
0.09
0.25
0.09
0.01
0.16
0.09
0.09
0.21
0.19
0.32
0.14

FIG. 2. Sequence frequencies in animal and plant cell DNA's
compared with values predicted from random association.  The
product of frequencies of constituent nucleotides of a nearest
neighbor pair is described by the line passing through the origin.
The values represented by the lower cuse letters are the observed
nearest neighbor frequencies.  a, Chlamydomonas; b, wheat germ;
c, calf thymus; d, salmon liver; e, rabbit liver; f, chicken red cells;
g, mouse lymphoma; h, starfish testis; i, human spleen; I, Echinus
esculenta; m, Paracentrotus lividus; 0, Tetrahymena pyriformis.

dom distribution have phylogenetic significance.  The observed
nearest neighbor frequencies are plotted against the values pre-
dicted from random association, the product of frequencies of
constituent nucleotides of a nearest neighbor pair.'  If there
were agreement between observed and predicted values, the
points for the sequences whose constitutent nucleotides occur
with equal frequency would fall on a straight lime through the
origin with a slope of 1.  As seen in Fig. 2, the sequence, CpG,
occurs in animal and plant cells with a frequency which is in-

1 This method of plotting the data was suggested by the obser-
vation of A. D. Kaiser and R. L. Baldwin that the nearest neigh-
bor frequencies are related to the product of the base frequencies
(submitted for publication to the Journal of Molecular Biology).

* Key: +, observed frequency > random expectancy; 0, ob-
served frequency = random expectancy (rt0.005); -, observed
frequency < random expectancy.

z(fob. - fpred)'

n-1 *

t
t."(Sfp.ed)P; n = number of entries; s = coefficient of variation

(for animal and plant DNA's, determined as in "Methods"; for
bacteria, an averagecoefficient of variation of 3% was assumed for
each nearest neighbor frequency).

n-1

1966 Enzymatic Synthesis of Deoxyribonucleic Acid. XI Vol. 237, No. 6

FIG. 3. Sequence frequencies in bacterial DNA's compared
with values predicted from random association, as in Fig. 2.  a,
~4ycrococcus lysodeikticus; b, Mycobacterium phlei; c, Aerobacter
aerogenes; d, Escherichia coli; e, Bacillus subtilis; f, Haemophilua
influenzae.

variably less than random and in some cases only one-third the
random value; by contrast, the frequencies of the isomeric se-
quence, GpC, come very close to the expectation for random-
ness. In bacteria, the reverse of this pattern appears to be the
case (Fig. 3).

In the case of the TpA and ApT sequence frequencies, bacterial
DNA's as well as animal and plant cell DNA's show a TpA fre-
quency that is decidedly below the prediction for random associa-
tion; the APT frequencies, however, are close to the predicted
values (Figs. 2 and 3).

DISCUSSION

Mechanism of Enzymatic Replication of Single Stranded DNA-
Replication of the single stranded DNA from phage (PX was
studied by analysis of a product after 20% increase in DNA over
the primer added (limited replication) and after a 600% increase
(extensive replication).  The base composition of the DNA syn-
thesized under conditions of limited replication was complemen-
tary to that of the primer.  A comparison of nearest neighbor
frequencies after extensive replication with those from limited
replication showed that the nearest neighbor sequences also con-
formed with complementary base pairing with the primer.  The
fact that a copy of only 20% of the primer chains is representa-
tive of the total can be interpreted in two ways: (a) only 1 out
of 5 molecules primes and is completely replicated or (a) replica-
tion of an average of # of the 5000 nucleotide sequences of each
molecule is representative of the entire molecule.  Density gra-
dient centrifugation experiments indicating that virtually all
(>85%) of the (PX DNA is combined with new DNA when a
20% increase is reached2 make the first interpretation unlikely
and the second preferred.

3 I. R. Lehman, R. L. Sinsheimer, and A. Kornberg, unpublished

observations.

Earlier viscometric and optical measurements had shown that
a double helical molecule is produced from the single stranded
primer (14). The chemical determinations presented here indi-
cate that the enzymatically synthesized strand (of the size of the
"20% synthesis product") already primes as effectively as the
original strand of phage DNA and that both strands of a double
helix may prime in the ensymatic replication.

Although these findings are restricted to the product of enzy-
matic action in Vitro, they are consistent with Sinsheimer's (17)
recent isolation from infected cells of a "replicative form" of (PX
DNA which has many features of a double helii.  It would follow
from these observations that at least the initial step in replication
of @X DNA is like that of other DNAs' in the formation of a com-
plementary strand and that some unique process is responsible
for including only one type of strand into mature phage.

Nearest neighbor sequence analyses of DNA have now been
performed with an RNA polymerase directed specifically by DNA
(18-20). The composition and the sequence frequencies of en-
zymatically synthesized RNA's primed by various DNA's (dAT,
dGdC, DNA's of phage QX, phage T2, calf thymus) are identi-
cal with those obtained with DNA polymerase.  Synthesis of
this type of RNA seems clearly to involve pairing of uracil to
adenine and cytosine to guanine in the manner of DNA synthesis
by E. coli polymerase.

A bothersome, frequent deviation in the current studies was
the absence of matching between certain of the complementary
sequences, resulting in a lack of correspondence between the
over-all frequencies of Tp and Ap nucleotides.  Although the
basis for this deviation has not been pinpointed, it is clear that
exposure of DNA to one of the E. coli nucleases known to be a
variable contaminant of the polymerase preparation leads to
some distortion in the sequence pattern when this DNA is sub-
sequently used as primer.

Comparison of Sequence Patterns of DNA's Within a Specie-
Although DNA's isolated from different tissues of the same spe-
cies have the same base composition, it is conceivable that dif-
ferences might exist in the arrangement of the bases.  A com-
parison by nearest neighbor analysis of bovine sperm, thymus,
and liver revealed no variations beyond the error of the method;
the DNA's of two normal tissues and two tumors of the mouse
also failed to reveal significant differences.

A surprising development has provided two separable and dis-
tinguishable DNA entities within a species.  Sueoka identified
and isolated two DNA components from crab testis on the basis
of distinctive buoyant densities (15). Sequence analysis of the
"light" crab DNA uncovered the fact that it contains G and C
residues as 3% of the total bases interspersed among A and T
residues, which are almost invariably in alternating sequence.
This DNA is therefore similar to the simple and well ordered
dAT, the copolymer synthesized de mu0 by polymerase.  The
main crab DNA component, by contrast, has a sequence pattern
resembling other animal DNA's of similar base composition.
The biological significance of the "light" crab DNA is an intrigu-
ing subject and one which immediately suggests studying the dis-
tribution of this DNA in other somatic cells and the sperm of
this species.

Comparison of Sequence Patterns Among Bacterial, Bacterio-
phage, Animal, and Plant DNA's-In the previous report,
confined largely to the sequence patterns of bacterial and bac-
teriophage DNA's, it was established in many cases that the
nucleotide arrangements did not conform to predictions of ran-

June 1962

;M. N. Swartz, T. A. Trautner, and A. Kornberg

1967

dom distributions.  In the present work, concerned principally
with animal DNA's, it is clear that deviations from random ar-
rangements also obtain.  The most striking example is the fre-
quencies of the CpG sequence, which are only approximately one-
third of the values calculated from the base composition, whereas
those of the isomeric GpC sequence are close to the calculated
values.  The consistency with which certain nearest neighbor
sequences, determined from a wide variety of DNA's, deviate
from the random expectancy is a surprising finding if one con-
siders that in each nearest neighbor experiment, on the order of
10'5 dinucleotides, representative of the dinucleotides of many
different primer molecules of a given DNA, are analyzed.  This
result suggests that determinants other than random arrange-
ment must have governed the development of the sequence pat-
tern of a species.

When all of the sequence frequencies are surveyed, the data
appear to distinguish the animal and plant cells from the bacteria
and bacteriophages. Other distinctions between and within
these groups are suggestive but less striking than the CpG se-
quence.  Were it possible to refine the accuracy of the sequence
frequency analyses, these examples might increase and permit
more meaningful phylogenetic correlations.  The connection be-
tween the relative abundance of certain sequences and their
phenotypic expression as amino acid sequence remains an ulti-
mate objective.

SUMMARY

1. Replication of the single stranded deoxyribonucleic acid
(DNA) from phage (PX 174 was studied under conditions of lim-
ited synthesis (20% increase over the primer) and extensive syn-
thesis (600% increase).  The base composition of the products
and the nearest neighbor frequencies conform to the model based
on pairing of adenine to thymine and of guanine to cytosine be-
tween strands of opposite polarity.  This experiment further
indicates that the enzymatically synthesized DNA strands prime
as effectively as the natural strand of phage DNA and that both
strands of a double helix may prime in the enzymatic replication.

2. A comparison of the nearest neighbor sequence analyses of
the DNA's of several bovine tissues (sperm, thymus, liver)
revealed no variations beyond the error of the method; similar re-
sults were obtained in a comparison of the DNA's of mouse tis-

sues, including two tumors.  However, the two DNA compo-
nents isolated by Sueoka from crab testis were shown to have
distinctive sequence patterns, one remarkably similar to the dAT
copolymer synthesized de novo by polymerase.

3. A survey of the sequence patterns of a variety of animal and
plant DNA's revealed that certain sequence frequencies were
strikingly different from predictions based on random arrange-
ment of the bases.  The data appear to distinguish animal and
plant cells from bacteria by the very low frequency of the cytidyl-
(3'-5')-guanosine (CpG) sequence in the former group and the
high frequency of the isomeric guanyl- (3'-5`) -cytosine (GpC)
sequence in the latter group.

1.

2.

3.

4.

5.

6.

7.


8.

9.

10.

11.

12.

13.
14.
15.
16.

17.
18.

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