Reprinted from

Proc. Nat. Acad. Sci. USA
Vol. 70, No. 12, Part IJ, pp. 3810-3814, December 1973

Unique Nuclear DNA Sequences in the Involved Tissues of Hodgkin’s

and Burkitt’s Lymphomas

(eancer/virus)

D. W. KUFE, W. P. PETERS*, AND 8. SPIEGELMAN

Institute of Cancer Research, College of Physicians and Surgeons, Columbia University, Department of Human Genetics and

Development, 99 Fort Washington Ave., New York, N.Y. 10032

Contributed by S. Spiegelman, August 28, 1973

ABSTRACT Previous studies have demonstrated that
Hodgkin’s, Burkitt’s, and other human lymphomas
contain particulate elements encapsulating 70S RNA and
RNA-dependent DNA polymerase. PH]DNA probes endo-
genously synthesized by these particles were used to
demonstrate that the nuclear DNA of the lymphomas
contain particle-related sequences that cannot be detected
(ess than 1/20th of a copy per genome) in the DNA of
normal cells. This result agrees with our earlier findings in
human leukemias. The data are inconsistent with any
etiologic concept that invokes germ-line transmission
of at least one complete copy of the particulate infor-
mation associated with the malignancy. The unique se-
quences found in the nuclear DNA of Burkitt’s and
Hodgkin’s tissues are related to each other but not to the
DNA of the Epstein-Barr virus.

 

We have been searching for evidence in human neoplasias of
possible viral agents. First we used appropriate murine RNA
tumor viruses (i.e., mouse mammary tumor virus and Rau-
scher leukemia virus) to generate highly radioactive DNAs
complementary to the viral RNAs. Molecular hybridizations
with these radioactive DNAs revealed that human adeno-
carcinomas of breast (1, 2), leukemias (3), sarcomas (4), and
lymphomas (5-7) contain RNA molecules possessing a small
but significant homology to the RNAs of the tumor viruses
that cause the corresponding malignancies in mice. Using
the simultaneous detection test (8, 9) we demonstrated that
the RNA identified in these human cancers was 70 S in size
and encapsulated with an RNA-directed DNA polymerase in
a particle of density between 1.16 and 1.19 g/ml (10-13).
The DNAs synthesized endogenously by these particles
possessed complementarity to the RNAs of the analogous
murine tumor viruses.

Collectively, these experiments documented the existence in
various human neoplastic tissues of particulate elements
possessing four features diagnostic of animal RNA tumor
viruses. Here we focus on the question of whether normal cells
contain in their genome at least one complete copy of the in-
formation necessary and sufficient for particle production.
We have previously considered this problem in human
leukemias in which the methodology had already been de-
veloped (10) to separate the particles. These were then used
to generate the [7H]DNA-labeled DNA probes required to
search for the corresponding sequences in nuclear DNA. The

 

Abbreviation: Cot, product of concentration of nucleotide se-
quences of DNA and time of incubation.

* This author is a member of the Medical Scientist Training
Program.

data obtained (14) with eight leukemic patients demonstrated
that the DNA of leukemic cells contained particle-related se-
quences that could not be detected in the leukocyte DNA of
normal individuals. This conclusion was further strengthened
and made biologically more precise by showing (15) that the
leukemic members of two sets of identical twins contained
particle-related sequences in their leukemic cell DNAs that
could not be detected in the leukocytes of their healthy
siblings. These findings imply that the additional specific in-
formation found in the DNA of the leukemic individuals must
have been inserted subsequent to fertilization. Thus, the out-
comes argue against any etiologic concept that invokes vertical
transmission through the germ line of the particle-related in-
formation found uniquely in the DNA of leukemic cells.

We wanted to see whether these findings hold also for some
other human neoplasias in which similar particles have been
identified (12, 13). In this paper we describe experiments with
Hodgkin’s and Burkitt’s lymphomas. The data show that the

‘ nuclear DNA of both types of lymphomas contain unique se-

3810

quences that are not detectable in normal cellular DNA.

METHODS

Preparation of [(}H|DNA. Ten grams of lymphoma tissues
were disrupted with a Silverson homogenizer at 4° in 2
volumes of TNE buffer (0.01 M Tris-HCl, pH 8.3-0.15 M
NaCl-0.01 M EDTA). The suspension was centrifuged at
4000 X g for 10 min at 4°, and the supernatant was recentri-
fuged at 10,000 x g for 10 min at 4°. The resulting super-
natant fluid was then layered on a 138-ml column of 20%
glycerol in TNE and centrifuged at 98,000 x g for 1 hr
at 4° ina SW-27 rotor (Spinco). The pellet was resuspended
in TNE and layered on a linear gradient of 15-55% sucrose
in TNE and spun in a SW-27 (Spinco) rotor at 4° for
210 min. The gradient was dripped from below, and the
density region between 1.15 and 1.19 g/ml was collected and
pooled. After dilution with TNE, this region was pelleted at
100,000 X g for 1 hr. The resulting pellet was resuspended in
1.0 ml of 0.01 M Tris: HCl, pH 8.3, brought to 0.15% Nonidet
P-40 (Shell Chemical Co.), and incubated at 0° for 15 min.
DNA was synthesized in an RNA-instructed DNA polymer-
ase reaction mixture (final volume 2.0 ml) containing: 200
umole of Tris-HCl, pH 8.3, 80 wmol of NaCl, 24 wmol of
MegCh, 400 pmol each of dATP, dGTP, dCTP, and 200 pmol
of PH]JdTTP (50 Ci/mmol). Actinomycin D (100 ng/ml) was
added to inhibit DNA-instructed DNA synthesis. After in-
cubation at 37° for 15 min, the reaction was adjusted to 0.2 M
Proc. Nat. Acad. Sci. USA 70 (1978)

NaCl and 1% sodium dodecy] sulfate. After phenol extrac-
tion, the aqueous phase was subjected to Sephadex G-50
chromatography and the DNA region of the column was
pooled and collected. The [7H]DNA product was then ad-
justed to 0.4 M NaOH, incubated at 37° for 6 hr to destroy
RNA, and then neutralized.

Preparation of Nuclear DN A. The nuclear pellet (1000 X g
for 10 min) was resuspended in TNE and lysed by addition of
sodium dodecyl sulfate to a concentration of 1% in the pres-
ence of 1 M NaClOs. After repeated extraction with phenol-
cresol and chloroform—isoamy! alcohol, DNA was spooled out
of solution. The DNA was redissolved in 83 mM EDTA and
sheared to 6-8 S as measured in a 10-30% glycerol gradient.
Shearing was accomplished with a Bronwill Biosonic IV soni-
fier using the microtip at maximum power for two 30-sec
intervals. The sample was then incubated with 0.4 M NaOH
for 16 hr at 37°. After neutralization, DNA was precipitated
with alcohol and redissolved in 3mM EDTA. The DNA prep-
arations had an A20/Aos9 ratio greater than 1.9 and renatured
more than 80%, as measured by hydroxyapatite chroma-
tography. :

Annealing Conditions. Annealing reactions contained >60
Asgp units of nuclear DNA, 0.1-1.0 pmol of [7H]DNA, and
G.1% sodium dodecyl! sulfate at a final DNA concentration of
>15 mg/ml. The reaction was brought to 100° for 10 min and
0.04 mmol of NaCl was added. The reaction mixture was then
incubated at 65°, and stopped by addition of 4 ml of 0.01 M
NaH,PQu., pH 6.8. The sample was then passed over a hy-
droxyapatite (16, 17) column of 2-ml bed volume/mg DNA
at 60°. The column was washed with 16 ml of 0.01 M phos-
phate buffer at 60° and then 20 ml of 0.15 M NaH PO,, pH
6.8, at 60°, 80°, 88°, and 95°. Fractions of 4 ml were collected,
and the [H]DNA counts in each fraction were assayed by
scintillation counting in 10 ml of Aquasol (NEN). The method
identifies unpaired strands that elute at 60° and poorly paired
duplexes that disassociate at 80°. Only the duplexes disassociat-
ing and eluting at 88°-95° are counted here as hybridized.

Recycling of Probe on Normal DNA to Remove Normal
Sequences. The reactions were done with normal DNA as
above and the eluates were collected in 1-ml fractions at 60°.
The peak fractions were combined and the resulting pools
were passed over a Sephadex G-50 column of 10-ml bed
volume. The DNA regions were collected and the DNA was
precipitated with 2 volumes of 100% ethanol. The material
eluted at 60° was used as the recycled product unable to
hybridize to normal DNA.

RESULTS

RNA of the human leukemic particles shared sequences with
the DNA of normal cells (14), a feature also exhibited (18-25)
by some animal RNA tumor viruses and the normal DNA of
their putative indigenous hosts. Sequences common to both
normal DNA and the [7H ]DNA synthesized endogenously by
the particle system can be removed by exhaustive hydridiza-
tion to a vast excess of normal DNA followed by hydroxy-
apatite chromatography to separate duplexes from unpaired
[SH}DNA. The residue of unpaired [7H]DNA can then be
used to obtain an answer to the question: ‘Does the DN A of the
neoplastic cells contain sequences not found in normal cells?”
These considerations dictate the following strategy for the
experiments to be described: (1) Isolate the particles encap-

Unique DNA Sequences in Lymphomas 3811

sulating 70S RNA and the RNA-directed DNA polymerase
from human lymphoma specimens; (2) use the particle fraction
to endogenously synthesize [7H]DNA in the presence of a
high concentration (100 ug/m!) of actinomycin D to inhibit
host or viral DNA-directed DNA synthesis; (3) purify the
[7H ]DNA by Sephadex chromatography and hydroxyapatite;
(4) remove the [3H ]DNA sequences shared with normal DNA
by exhaustive hydridization in the presence of vast excess of
normal DNA followed by hydroxyapatite chromatography
to separate paired from unpaired [HJDNA; and (5) test the
unpaired residue for specific hybridizability to lymphoma
DNA.

(7H|DNA probes were synthesized from four Burkitt’s
tumors, three Hodgkin’s disease specimens, and one lympho-
sarcoma. In all instances the lymphoma (*H]DNA probe
hybridized 35-40% to normal spleen nuclear DNA, in agree-
ment with our experience with human leukemias (14).

The sequences shared with normal nuclear DNA were then
removed by exhaustive annealing of the [7H]DNA with nor-
mal spleen DNA in vast excess. In this step, annealing reac-
tions were set up to contain 60 Ao units of normal cellular
DNA per 0.1 pmol of [7H]DNA (1000 cpm) corresponding to
a 100- to 1000-fold excess of the relevant sequences in normal
DNA. The annealing was continued for Cot values (26) in
excess of 10,000, and the unpaired [7H ]DNA was recovered by
hydroxyapatite chromatography. These should no longer con-
tain sequences complementary to those found in normal DNA,
and exclusive hybridizability of such recycled [7H]DNA to
lymphoma DNA would then establish that the genome of
lymphoma cells contains specific sequences not present in
normal DNA.

We illustrate a representative elution result with recycled
{3H JDNA made with the particles derived from Hodgkin’s and

14,

>
_
°o

 

 

 

 

 

 

 

   

 

13} [2 B
oF 71 ab 6 Cc 6p
8 8
Tr 7
% 6F 6 ~ 4 4b
- oO
x sy 5 i L
E x
Oo 4+ 4 E
| 3
3 3 2 2b
2r 2
1 1 LQ
a
60 80 88 95 60 80 88 95 60 80 88 95 60°80 68 95
Elution Temperature, °C Elution Temperature, °C /
Fig. 1. Hydroxyapatite elution profiles of hybridization

reactions of recycled Hodgkin’s disease [7H]DNA to nuclear
DNA from (A) normal human spleen, (B) a spleen involved with
Hodgkin’s disease; and recycled Burkitt’s lymphoma [7H] DNA
to nuclear DNA from (C) normal human spleen and (D) Burkitt’s
tumor. Hybridization reactions contained 2000 cpm of recycled
(3H}]DNA and 2 mg of nuclear DNA (concentration >15 mg/
ml). Hybrid formation was analyzed by hydroxyapatite chro-
matography at a phosphate buffer (pH 7.2) elutent concentration
of 0.15 M. Five fractions of 4 ml were collected at each of the
four temperatures, and the [3H] DNA counts in each fraction were
assayed by scintillation counting in 10 ml of Aquasol (NEN).
All radioactive samples were counted for 10 min to assure the
necessary accuracy.
3812 Medieal Sciences: Kufe ef al.

157 © Normal Spleen

& Hodgkins Disease
no. 302

® Burkitt(Na)

Percent Duplex [SH]ONA

 

 

 

oO 5000 10000
; Cyt

Fic. 2. Hybridization of recycled Hodgkin’s disease no. 302
[7H] DNA to nuclear DNA isolated from normal spleen (O—O),
Hodgkin’s disease no. 302 (a——a), and Burkitt’s lymphoma
(Na) (g@——-). Hybridization reactions in which duplex forma-
tion was analyzed at multiple C.t values contained propor-
tionately increased amounts of recyeled [8H]DNA probe and
nuclear DNA. Equal aliquots were removed from the hybridiza-
tion vessel at each Cot value, and hybrid formation was analyzed
by hydroxyapatite chromatography (see legend to Fig. 1).

Burkitt’s lymphoma. In each case the recycled [7H]DNA is
rechallenged against normal DNA and against the nuclear
DNA of the neoplastic tissue from which the particles were
derived. In neither case do the recycled probes enter into com-
plexes with normal DNA that are stable above 80° (Fig. 1).
However, each probe forms well-paired duplexes with the
DNA from the tumor of origin.

Fig. 2 shows similar outcomes at two different Cot values.
Recycled Hodgkin’s disease (7HJDNA is challenged with nu-
clear DNA from normal spleen, Hodgkin’s disease spleen, and
from Burkitt’s lymphoma. The input counts for each Cot point
were 1500-2000 cpm. Only those duplexes disassociating and
eluting above 88° are counted here as stably hybridized. Few,
if any, stable complexes are formed with normal DNA; over
10% of the input [7H]DNA pairs with both Hodgkin’s and
Burkitt’s disease nuclear DNA. Although the probe was made
with Hodgkin’s disease particles, clear evidence is seen of
homology with sequences in the nuclear DNA of Burkitt’s
lymphoma. That the converse also obtains is shown in Fig. 3,
in which recycled Burkitt’s [7H ]DNA is challenged with vari-
ous nuclear DNAs, including one from Hodgkin’s disease to
which it does hybridize. The Burkitt’s [SH]DNA complexes
to nuclear DNA from two Burkitt’s tumors and to nuclear
DNA from a pool of Burkitt’s lymphoma cell lines (EB-2 and
EB-3). In contrast, no significant hybridization was detect-
able when the same probe was challenged with normal spleen
DNA, infectious mononucleosis spleen DNA, or against NC-
37 cell DNA, a “normal” leukocyte cell line containing multi-
ple copies of the Epstein-Barr virus genome (27).

Table 1 summarizes all of our findings with the recycled
lymphoma [H]DNA probes annealed with nuclear DNA
from normal and malignant tissues. Normal DNA is unable to
form significant amounts of stable complexes with the [?H]-
DNA. In all instances, the lymphoma [*H]DN As hybridized
to the nuclear DNA of the original lymphoma types from

Proc. Nat. Acad. Sct. USA 70 (1973)

which the particles were obtained and used to generate the
DNA probes.

With one exception, all of the Burkitt’s and Hodgkin’s
disease [7H]DNA cross-hybridized with each other’s nuclear
DNA. This outcome suggests that particle-related informa-
tion found in these two lymphomas share sequences in common
in agreement with our earlier finding that all types of lym-
phomas, including Burkitt’s and Hodgkin’s contain particulate
RNA homologous to Rauscher leukemia virus RNA. One
would predict then that [?H]JDNAs made from Burkitt’s and
Hodgkin’s particles would hybridize with the RNA from both
neoplastic tissues (Fig. 4). The recycled [7H ]DNA synthesized
by Burkitt’s particles does hybridize to the RNA from a
Hodgkin’s spleen (Fig. 4A) and not to the RNA from a nor-
mal spleen (Fig. 4B). The converse of hybridizing recycled
[]H]DNA generated by Hodgkin’s particles to the RNA of
Burkitt’s tumors has been done, but is not detailed here.
Recycled [7H ]DNA synthesized by particles from lymphoma
hybridized to Rauscher leukemia virus RNA (Fig. 4C) but
not (Fig. 4D) to the RNA of the unrelated mouse mammary
tumor virus.

DISCUSSION

Whether our inability to detect the lymphoma-specific
sequences in normal DNA means that the latter contains less
than one copy per genome equivalent depends on the sensi-
tivity of the detection and the multiplicity of the specific
sequences in the malignant DNA. The specific activity of the
[7H ]TTP used, when corrected for counting efficiency and

20--
@|Normai Spleen

4\|NC-37

O\IM

@ Nya (Burkitt)

@ Nalw (Burkitt)

4'EB2, EB3 (Burkitt Cell Lines)
O:HD no. 302

   

% High Meeting
3

 

 

T q
0 5000 10000
Ct

Fic. 3. Hybridization of recycled Burkitt’s lymphoma (Nya-
T) (HJDNA to nuclear DNA isolated from normal human
spleen (O——O), a “normal” lymphoblastoid cell line containing
Epstein-Barr virus (NC-37) (4——), Burkitt’s lymphoma
(Nya) tumor (@ @), Burkitt’s lymphoma (Na) tumor
(®——-6), a pool of Burkitt’s lymphoma cell lines, (EB-2 and EB-3)
(A--—-A), and a Hodgkin’s disease no. 302 spleen (G——Q).
Hybridization reaction conditions and duplex formation analysis
are as described in the legend to Fig. 2. Only duplexes dis-
sociating and eluting at 88° and 95° are counted as high melting.

 
Proc. Nat. Acad. Sci. USA 70 (1978)

composition of the product, results in a specific activity of 3 x
10’ cpm/ug or 10,000 epm/pmol. Since actinomycin D was in-
cluded in all syntheses, the single-stranded DNA transcripts
would be expected to represent a substantial portion of the
particle RNA sequence (28). If the normal DNA contained
one copy of the probe per genome equivalent, the 300 ug of DNA
used in our hybridizations would contain about 3 X 107? ug
of the relevant sequences, and the specificity of our probe
would permit us to detect at saturation 1/1000 of a single
copy per genome. The question of multiplicity of the lym-
phoma-specific sequences in the tumor DNA must be con-
sidered in comparing the results obtained with normal DNA.
Thus, if the lymphoma-specific sequences were represented 100
times per malignant genome, and if the annealings were done
at saturation so that all the multiple copies were being counted,
then one would have to demonstrate that normal DNA
contains less than 1/100 that found in malignant DNA.

TABLE 1. Hybridization of recycled [7H]DNA probes
synthesized by human particles with nuclear DN A obtained
from normal and tumor tissues

 

High melting

 

Recycled
[3H] DNA probe nDNA hybridized Cpm G*
Burkitt lymphoma
(Ny) Burkitt (Ny) 209 9.17
(Ny) Normal spleen 24 1.83
(Na) Burkitt (Ny) 52 11.12
(Na) Normal spleen 0 0.00
(NaT) Burkitt (NaT) 197 15.43
(NaT) Burkitt (Nya) 30 11.09
(NaT) H.D. no. 302 40 11.64
(NaT) Normal spleen 3 0.56
(NyaT) Burkitt (Nya) 28 16.05
Burkitt (Na) 21 12.33
H.D. no. 302 29 9.80
EB-2 and EB-3 (Burkitt
cell lines) 26 16.60
NC-37 (“normal”
leukocyte cell line) 0 0.00
Infectious mononucleosis
spleen 2 1.65
Normal spleen 1 0.39
Hodgkin’s disease
no. 302 H.D. no. 302 63 12.12
no. 302 Burkitt (Na) 28 11.72
no. 302 Normal spleen 7 0.47
no. 304 H.D. no. 304 106 4.87
no. 304 Burkitt (Ny) 56 5.97
no. 304 Normal spleen 4 0.66
no. 306 H.D. no. 306 119 9.607
no. 306 Burkitt (Ny) 5 0.82
no. 306 Normal spleen 7 0.83+
Lymphosarcoma
no. 300 H.D. no. 302 154 14.2
no. 300 Normal spleen 16 1.8

 

nDNA, nuclear DNA; H.D., Hodgkin’s disease.

* Values represent percentage of counts dissociating and
eluting at 88°-95° at a Cot of 10,000 unless otherwise noted.
Scintillation counting was performed for 10 min in all cases to
achieve the necessary accuracy and are expressed as corrected
for background.

+ Cot = 5000.

Unique DNA Sequences in Lymphomas 3813

  
  

 

100-
A DNA B ONA
18 18
100} p | p g
16 80 16 @
146
asl 14
42 60F 12=
E RNA
S 50h
40+
25-
20+
1 1 Nee
5 10 15 20 25 30 5 10 15 20 25 30
DNA
c 180-P
160+ ' 18 18 9
] 460L p DNA 8
a
140+ 116 163
140
120+ {14
4, , 120
100+ 12
E 100
@ BOF 180
607 160
407 40
20+ 20)

 

 

 

--—1___1_ .
5 10 15 20 25 30 35 5 10 15 20 25 30
. Fraction Number
Fre. 4. Cs.SO, equilibrium density gradient centrifugation of
recycled Burkitt’s lymphoma (Na) [3H]DNA hybridized to

(A) Hodgkin’s disease no. 304 polysomal RNA (pRNA) and

(B) normal spleen IR.NA, and of recycled lymphosarcoma [3H]-
DNA hybridized to (C) Rauscher leukemia virus 70S RNA and
(D) mouse mammary tumor virus 70S RNA. pRNA was iso-
lated from the Hodgkin’s and normal spleens by disruption with
a Silverson homogenizer at 4° in 2 volumes of 5% sucrose in
TNM buffer (0.01 M Tris-HCl, pH 7.4-0.15 M NaCl-2 mM
MgCl.). The suspension was centrifuged at 15,000 < g for 40
min at 0°. The supernatant fluid was then layered on 20 ml of
25% sucrose in TNM buffer and centrifuged for 180 min at
180,000 x g in a Spinco 60 Ti rotor. The pellet (P-180) was
resuspended in TNM and 1% sodium dodecyl sulfate, and the
RNA was extracted twice with an equal volume of cresol-phenol
(pH 8.4). Nucleic acid was precipitated from the aqueous phase
by addition of two volumes of ethanol and !/1 volume of 4 M
LiCl. Recycled [7H] DNA (2000 cpm reaction) was first incubated
at 80° for 10 min in 50% formamide to denature the DNA.
After quick chilling to 0°, 400 ug of pRNA or 1 ug of purified
70S viral RNA was added. The annealing mixtures were brought
to 0.4 M NaCl-50% formamide in a total volume of 100 yl and
incubated at 18 hr at 37°. After incubation the reaction mixtures
were added to 5.5 ml of 5mM EDTA, mixed with an equal volume
of saturated Cs,SO, to yield a starting density of 1.52, and
centrifuged at 44,000 rpm in a 50 Ti rotor (Spinco) for 60 hr at
15°. 0.4-ml fractions were collected and assayed for trichloro-
acetic acid-precipitable radioactivity.

However, complications due to multiplicity are minimized by
the quantitative aspects and design of the experiments. First,
the Cot values required to detect the lymphoma-specific
sequences by the recycled [H]DNA probes insure that we are
dealing not with highly reiterated sequences, but rather with
sequences possessing a multiplicity close to unity.
Examination of the results with normal spleen DNAs (Figs.
2 and 3) show that they contain less than 1/20th of an
equivalent found in the DNAs from Burkitt’s and Hodgkin’s
tissue at a Cot value of 10,000. Indeed, one of the normal spleen
3814 Medical Sciences: Kufe et al.

DNAs was carried out to a Cot value of 50,000 with no
evidence of sequences complementary to a Hodgkin’s [8H]-
DNA probe.

One is led to conclude that a normal genome contains less
than 1/20th of an equivalent of the DNA sequences found in
lymphoma DNA and homologous to the DNA synthesized by
the particles found in these neoplastic diseases. Any value
clearly less than unity for normal DNA argues against any
concept (29) that postulates germ-line transmission of a com-
plete set of the information contained in the particulate ele-
ments found in the tumor tissue.

Several other features emerged from this study. The par-
ticle-related sequences found in Burkitt’s and Hodgkin’s
lymphomas possess sequences in common, suggesting a rela-
tion between their respective particles, and in accord with our
earlier findings (5, 6, 12, 13) that Hodgkin’s and Burkitt’s
particles both share sequences with the murine Rauscher
leukemia agent. Further, in view of the previous association of
the Epstein—Barr virus with Burkitt’s disease (30-35) and the
non-neoplastic infectious mononucleosis (36, 37), it is of
interest that the leukocyte DNA of patients with infectious
mononucleosis were devoid of Burkitt’s particle-related
sequences, indicating that these latter sequences are specific for
neoplastic tissues. The fact that the particle-related sequences
in Hodgkin’s and Burkitt’s tumors are homologous adds fur-
ther weight to this conclusion. Finally, the fact that cells
(e.g., NC-37, infectious mononucleosis leukocytes) carrying
multiple copies of the DNA of Epstein-Barr virus do not ecom-
plex with the (7H]DNA synthesized by either Hodgkin’s or
Burkitt’s particles proves that these sequences have no rela-
tion to DNA of Epstein-Barr virus.

We thank Dr. I. T. Magrath, Uganda Cancer Institute,
Uganda, Africa, and Dr. J. Ziegler, National Cancer Institute,
Bethesda, Md., for supplying the Burkitt’s tumors. The excellent
technical assistance of E. Gordon is appreciated. This research
was supported by the National Institutes of Health, National
Cancer Institute, Virus Cancer Program Contract NO1-CP-3-
3258 and by the National Cancer Institute Grant CA-02332
and the Medical Scientist Training Program GM-02042.

1. Axel, R., Schlom, J. & Spiegelman, 8. (1972) Nature 235,
32-36.

2. Schlom, J. & Spiegelman, 8. (1973) Amer. J. Clin. Pathol.
60, 44-56.

3. Hehlmann, R., Kufe, D. & Spiegelman, 5. (1972) Proc.
Nat. Acad. Sci. USA 69, 435-439,

4. Kufe, D., Hehlmann, R. & Spiegelman, 8. (1972) Science
175, 182-185.

5. Hehlmann, R., Kufe, D. & Spiegelman, 8. (1972) Proce.
Nat. Acad. Sci. USA 69, 1727-1731.

6. Kufe, D., Hehlmann, R. & Spiegelman, 8S. (1973) Prec.
Nat. Acad. Sci. USA 70, 5-9.

7. Hehlmann, R., Baxt, W., Kufe, D. & Spiegelman, S. (1973)
Amer. J. Clin. Pathol. 60, 65-79.

10.
11.
12.
13.
14.

15.

16.
17.
18,

19.

20.

21.
22.

23.
24.
25.

26.
27.

28.
29.

30.
3l.

32,

33.

34.
35.
36.

37.

Proc. Nat. Acad. Sct. USA 70 (1978)

Schlom, J. & Spiegelman, S. (1971) Science 174, 840-843.
Gulati, 8. C., Axel, R. & Spiegelman, S. (1972) Proc. Nat.
Acad. Sct. USA 69, 2020-2024.

Baxt, W., Hehlmann, R. & Spiegelman, S. (1972) Nature
New Biol. 240, 72-75,

Axel, R., Gulati, 8. C. & Spiegelman, 8. (1972) Proc. Nat.
Acad. Sci. USA 69, 3133-3137.

Spiegelman, S., Kufe, D., Hehlmann, R. & Peters, W. P.
(1973) Cancer Res. 33, 1515-1526.

Kufe, D., Magrath, 1. T., Ziegler, J. H. & Spiegelman, S.
(1973) Proc. Nat. Acad. Set. USA 70, 737-741.

Baxt, W. G. & Spiegelman, S. (1972) Proc. Nat. Acad. Sci.
USA 69, 3737-3741.

Baxt, W., Yates, J. W., Wallace, Jr., H. J., Holland, J. R. &
Spiegelman, 8. (1973) Proc. Nat. Acad. Sci. USA, 70, 2629—
2632.

Manly, K. F., Smoler, D. F., Bromfield, E. & Baltimore, D.
(1971) J. Virol. 7, 106-111.

Varmus, H. E., Levinson, W. E. & Bishop, J. M. (1971)
Nature New Biol. 223, 19-21.

Baluda, M. A. & Nayak, D. P. (1970) Proc. Nat. Acad. Sct.
USA 66, 329-336.

Rosenthal, P. N., Robinson, H. L., Robinson, W. S§.,
Hanafusa, T. & Hanafusa, H. (1971) Proc. Nat. Acad. Sci.
USA 68, 2336-2340.

Baluda, M. A. (1972) Proc. Nat. Acad. Sci. USA 69, 576-
580.

Wilson, D. E. & Bauer, H. (1967) Virology 33, 754-757.
Yoshikawa-Fukada, M. & Ebert, J. P. (1969) Proc. Nat.
Acad. Sci. USA 64, 870-877.

Gelb, L. D., Aaronson, 8. A. & Martin, M. (1971) Seience
172, 1353-1355,

Varmus, H. E., Weiss, R. A., Friis, R. R., Levinson, W. &
Bishop, J. M. (1972) Proce. Nat. Acad. Sci. USA 69, 20-24.
Varmus, H. E. & Bishop, J. M. (1972) Nature New Biol.
238, 189-191.

Britten, R. J. & Kohne, D. E. (1968) Science 161, 529-540.
Zur Hausen, H., Diehl, V., Wolf, H., Schulte-Holthausen,
H. & Schneider, U. (1972) Nature New Biol. 237, 189-190.
Garapin, A. C., Varmus, H. E., Faras, A., Levinson, W. E. &
Bishop, J. M. (1973) Virology, in press.

Huebner, R. J. & Todaro, G. J. (1969) Proc. Nat. Acad. Sct.
USA 64, 1087-1094.

Wright, D. H. (1967) Cancer Res. 27, 2424-2438.

Henle, G., Henle, W., Clifford, P., Diehl, V., Kafuko, G. W.,
Kirya, B. G., Klein, G., Morrow, R. H., Munube, G. M. R.,
Pike, M., Tukel, P. M. & Ziegler, J. L. (1969) J. Nat.
Cancer Inst. 43, 1147-1157.

Gunven, P., Klein, G., Henle, G., Henle, W. & Clifford, P.
(1970) Nature 228, 1053-1055.

Zur Hausen, H., Schulte-Holthausen, H., Klein, G., Henle,
W., Henle, G., Clifford, P. & Santesson, L. (1970) Nature
228, 1056-1058.

Epstein, M. A., Achong, B. G. & Barr, Y. M. (1964) Lancet
i, 702-703.

Epstein, M. A., Henle, G., Achong, B. G. & Barr, Y. M.
(1965) J. Hap. Med. 121, 761-770.

Henle, G., Henle, W. & Diehl, V. (1967) Proc. Nat. Acad.
Sct. USA 54, 94-101.

Niederman, J. C., Evans, A. 8., Subrahmanyan, L. &
McCollum, R. W. (1970) N. Engl. J. Med. 282, 361-365.