Cell, Vol. 11, 845-857, August 1977, Copyright 0 1977 by MIT

The Nucleotide Sequence of Repetitive Monkey DNA
Found in Defective Simian Virus 40

Martin Rosenberg
Laboratory of Molecular Biology

Shoshana Segal, Edward L. Kuff and
Maxine F. Singer
Laboratory of Biochemistry
National Cancer Institute
Bethesda, Maryland 20014

Summary

DNA fragments Containing monkey DNA se-
quences have been isolated from defective SV40
genomes that carry host sequences in place of
portions of the SV40 genome. The fragments were
isolated by restriction endonuclease cleavage
and contain segments homologous to sequences
in both the highly repetitive and unique (or less
repetitive) classes of monkey DNA. The complete
nucleotide sequence of one such fragment [151
base pairs (bp)] predominantly homologous to the
highly reiterated class of monkey DNA was deter-
mined using both RNA and DNA sequencing
methods. The nucleotide sequence of this homo-
geneous DNA segment does not contain dlscernl-
ble multiple internal repeating units but only a few
short oligonucleotide repeats. The reiteration fre-
quency of the sequence in the monkey genome is
>lo6. Digestion of total monkey DNA (from unin-
fected cells) with endonuclease R-Hind Ill pro-
duces relatively large amounts of discrete DNA
fragments that contain extensive regions homolo-
gous to the fragment isolated from the defective
SV40 DNA.
A second fragment, also containing monkey se-
quences, was isolated from the same defective
substituted SV40 genome. The nucleotide se-
quence of the 33 bp of this second fragment that
are contiguous to the 151 bp fragment has also
been determined.
The sequences in both fragments are also pres-
ent in other, independently derived, defective
substituted SV40 genomes.

Introduction

Several defective variants of simian virus 40 (SV40)
with genomes that contain DNA sequences de-
rived from the host (monkey) genome have been
reported in recent years (Lavi and Winocour, 1972;
Martin et al.. 1973; Lavi et al., 1973; Rozenblatt et
al., 1973; Brockman and Nathans, 1974; Frenkel,
Lavi and Winocour, 1974; Lee, Brockman and Na-
thans, 1975; Oren, Kuff and Winocour, 1976; Davoli
et al., 1977; Rao and Singer, 1977b). The genomes
of the defective variants are closed circular duplex

molecules (DNA I), and typically consist of several
tandem repeats of a DNA segment containing the
monkey sequences and a portion of the wild-type
SV40 DNA sequences including the origin of repli-
cation. The monkey sequences in a given defective
may be derived from either the highly repetitive
portion of the monkey genome or from the less
repetitive or unique portion or from both. Further-
more, there is evidence indicating that the monkey
sequences present in independently derived defec-
tive variants are not a random selection from the
monkey genome. Rather, certain sequences, both
of the repetitive and unique classes, are more likely
to occur than are others (Frenkel et al., 1974; Oren
et al., 1976). It is presumed that recombinational
events between monkey DNA and infecting or repli-
cating viral DNA give rise to these "substituted"
variants, but the mechanism of the recombination
is unknown. It may involve chromosomal DNA or
extrachromosomal DNA known to exist in monkey
kidney cells in tissue culture (Smith and Vinograd,
1972). The recombination may reflect site-specific
integration into the cellular DNA and subsequent
excision, but it is also possible that nonspecific
recombination is involved.
Segments containing the monkey sequences and
few, if any, SV40 sequences can be isolated from
purified, substituted defective genomes by cleav-
age with restriction endonucleases (Rozenblatt et
al., 1973; Lee et al., 1975; Segal et al., 1976; Rao
and Singer, 1977b). The substituted defective DNA
called CVP8/11P2 (Eco RI res) DNA I (Figure 1)
yields two fragments of interest (Rao and Singer,
1977b). [Restriction endonucleases and fragments
of DNA derived from cleavage with restriction en-
donucleases are abbreviated according to the rec-
ommendations of Smith and Nathans (1973).] The
(Hind II/Hind Ill)-E fragment is approximately 3.1%
of a wild-type SV40 genome in length and contains
monkey DNA of high reiteration frequency: the
(Hind II/Hind Ill)-C fragment is approximately 4.3%
of a wild-type genome in length and does not hy-
bridize to filters containing either SV40 or monkey
DNA, and may therefore consist predominantly of
monkey DNA from the low repetitive or single-copy
class (Segal et al., 1976; Rao and Singer, 1977b).
Since the CVPB/l/P2 (Eco RI res) DNA I consists of
a 4 fold tandeom repeat, each fragment occurs 4
times within the defective genome. Fragments
(Hind II/Hind Ill)-C and (Hind II/Hind Ill)-E are con-
tiguous within each tandem repeat, and each has
one end generated by endonuclease ReHind II and
one generated by endonuclease R.Hind 111; the
Hind 111 cleavage site separates fragments C and E.
No SV40 sequences are detectable in either frag-
ment by filter hybridization. We have undertaken
experiments designed to characterize the se-

Cell
846

rplpl monkey 'unique' sequences
w monkey 'repetitive' sequences

SV40 sequences

Figure 1. A Schematic Drawing of the Structure of the Substi-
tuted Defective Genome CVP8/1/P2 (Eco RI res) DNA I
The figures shows the sites sensitive to endonucleases R. Hind II.

R.Hind Ill, R.Bam I and R.Hap 11, as well as (inner circle) the
fragments generated by combined cleavage with endonuclease
R.Hind II and R'Hind 111 (Rao and Singer, 1977b). Fragments
(Hind IllHind ill)-C (WOO) and (Hind IVHind Ill)- (NW) are indi-
cated.

-

quences in (Hind II/Hind Ill)-C and -E in a variety of
ways.
Radioactively labeled cRNA copies of (Hind II/
Hind Ill)-E have been prepared and used to study
the location of the sequences in the fragment in
African green monkey chromosomes by in situ hy-
bridization (Segal et al., 1976). The results indi-
cated many possible chromosomal origins for the
(Hind IVHind Ill)-E sequences, both centromeric
and noncentromeric. The sequence was detected
in the arms of between 9 and 11 chromosomes; this
location is consistent with an interspersion of re-
petitive and unique sequences in the monkey ge-
nome as has been observed in a variety of other
eucaryotic DNAs (Davidson and Britten, 1973).
In the present paper, we report the complete
nucleotide sequence of (Hind II/Hind Ill)-E frag-
ment (151 residues), as well as the sequence of the
first 33 nucleotide residues of (Hind II/Hind Ill)-C
from the endonuclease R.Hind 111 site that sepa-
rates it from (Hind II/Hind Ill)-E. The monkey se-
quences in fragment E are shown to have a reitera-
tion frequency of approximately 1.6 x lo6 in the
monkey genome. It was recently reported that
cleavage of total monkey DNA with endonuclease
R.Hind 111 results in a series of discrete DNA frag-

ments resolvable on polyacrylamide or agarose
gels (Gruss and Sauer, 1975). Our results indicate
that the monkey sequence in the (Hind WHind Ill)-E
fragment hybridizes to each of the fragments ob-
tained directly from monkey DNA. Finally, we pres-
ent hybridization data indicating that sequences in
both (Hind II/Hind Ill)-E and (Hind IVHind Ill)-C are
found within the genomes of other independently
derived, substituted SV40 defectives.

Results

The Complete Nucleotide Sequence of (Hind Ill
Hind Ill)-E Fragment
Both RNA and DNA sequencing techniques were
used to determine an unambiguous sequence for
the 151 residues of the (Hind II/Hind Ill)-E fragment
(Figure 2). The RNA sequencing methods involved
preparing and characterizing a cRNA transcript of
the DNA fragment, and analyzing the oligonucleo-
tide products generated from both complete and
partial digests of the cRNA with T1 and pancreatic
RNAase. As previously described (Segal et al.,
1976). the major transcription product was an es-
sentially full-length copy of one strand of (Hind II/
Hind Ill)-E fragment. A second (minor) transcript,
readily separable from the major product on poly-
acrylamide gels, yielded oligonucleotides consist-
ent with its being a covalently linked RNA copy of
both strands of (Hind IVHind III)-E. Analysis and
comparison of the T1 and pancreatic oligonucleo-
tide products obtained from these two transcripts
designated two separate sets of oligonucleotides
that derived from each of the two complementary
strands of the DNA fragment (Figure 3). As shown
in Figure 2, the complementary products were used
to help specify the relative order of certain of the
oligonucleotides from the major transcript. These
data, in conjunction with the data obtained from
the partial T1 ribonuclease digestions (see Figure
2, RNA), allowed deduction of a unique sequence
for most of the fragment, although some ambigui-
ties remained.

Direct DNA sequence analysis was also carried
out on (Hind IVHind Ill)-E, using both the tech-
niques of partial snake venom analysis (Sanger et
al., 1973) and the new chemical procedures intro-
duced by Maxam and Gilbert (1977). For both pro-
cedures, (Hind II/Hind Ill)-E was labeled with 32P at
one or the other of its 5'-hydroxy termini, as de-
scribed in Experimental Procedures.

The dimethyl sulfate-hydrazine (DMS-Hz)
(Maxam and Gilbert, 1977) technique for direct
DNA sequencing was applied to (Hind II/Hind Ill)-E
labeled separately at either its Hind II or Hind 111
end, and the nucleotide sequences were deduced
from a comparison of the various polyacrylamide
gels obtained. Representative examples of these

Monkey DNA in Defective SV40
847

t Fragment C ""I 'I' Fragment E -

-- Fragment E mm I"

Hlnd 111

Fragment C            with  -30 -20 -10 ->*I(
DNA        pah Hind 111 end        ... GTT"C.CCTEICTTCW 5'

SVD

OMS- Hind 111 end ... ~TCE~~~~O~.O~FYTI~~.~~,~~,,. 5'

HZ

Figure 2. The Derivation of the Nucleotide Sequence of (Hind il/Hind Ill)- Fragment and Part of (Hind IllHind Ill)-C Fragment
Ail nucleotide positions are numbered from the R. Hind 111 site separating the C and E fragments (-1/+1). RNA (fragment only): numerical
designation of TI (RNAase (1) and pancreatic RNAase (P) oligonucleotides associated with either the major strand or minor strand
transcript are those indicated in Figure 3. Only oligonucleotides which were actually used to deduce the relative order of the T1 RNAase
products from the major strand transcript are depicted. Partial T1 RNAase digestion products (see Experimental Procedures) obtained from
the major strand transcript are designated by brackets (m). Nearest-neighbor nucleotides to each oligonucleotide product are underlined
(for example, T23, AAACUGC). The nucleotide sequence of each oligonucleotide product was determined by standard RNA sequencing
techniques (see Experimental Procedures). These sequences and their relative order within the overall sequence were confirmed by direct
DNA sequencing methods. DNA: the sequence of nucleotides depicted for each of the DNA sequencing methods (that is, DMS-Hz; partial
SVD) denote only those residues which could be unambiguously determined from that technique at the level of single-nucleotide
resolution.

gel patterns are shown in Figure 4, and the derived
sequences are indicated in Figure 2 (DNA:DMS-
Hz). Partial snake venom analysis of the same end-
labeled fragments allowed correlation of the first
identifiable nucleotide residue in the DMS-Hz gel
patterns with a particular nucleotide position
within the fragment (Figure 2, DNA:SVD). Analysis
of the products of the partial snake venom diges-
tions by both two-dimensional "homochromatog-
raphy" (Brownlee and Sanger, 1969) (Figure 5) and
one- and two-dimensional paper electrophoresis
(Rf values given in Table 1) gave nucleotide se-
quences for 20-25 nucleotide residues at each end
of the fragment. These sequences overlapped the
sequences obtained by both the RNA and DNA
sequencing techniques, and also confirmed the se-
quences predicted from the known specificity of
the restriction endonuclease cleavage sites at the
termini of the fragment (Old, Murray and Roizes,
1975; Smith, 1974).
The results summarized in Figure 2 indicate that
the RNA and DNA sequencing methods gave com-
pletely consistent data. Although each approach

generated certain sequence ambiguities, in con-
junction these methods allowed deduction of an
unambiguous sequence of 151 nucleotide residues
for (Hind II/Hind lll)-E. It is important to point out,
for assessing the accuracy of the sequencing data,
that in addition to using the RNA and DNA se-
quence information separately, as confirmatory of
one another, the techniques were also used to
complement one another. Given a relatively easily
obtainable catalogue of all the T1 and pancreatic
RNAase products obtained from the RNA analysis,
they may be ordered within the sequence by refer-
ence to the gels obtained from the DNA sequencing
method. Knowing the sequence and the nearest-
neighbor nucleotide of a particular T1 or pan-
creatic RNAase product predicts a very specific
pattern on the DNA sequencing gels. These pat-
terns can readily be picked out and the oligonucle-
otides ordered far beyond the single residue reso-
lution of the gel. Thus when used in conjunction,
the RNA and DNA sequencing methods allow for
greater flexibility and accuracy than does either
one alone.

Cel I
e48

B

:-I 102

\,

pH 3.5 -

3

00'01

D

,-,

! ., l111

pH 3.5 -

Figure 3. T1 RNAase Fingerprint (A, Autoradiograph; 6, Schematic Sketch) and Pancreatic RNAase Fingerprint (C, Autoradiograph; 0,
Schematic Sketch) of Total cRNA Transcription Product Labeled with &*P-GTP
First dimension separation: electrophoresis on Cellogel in 8.0 M urea at pH 3.5 (Sanger et al., 1965). Second dimension: ascending thin-

layer homochromatography on DEAE-cellulose (9/1, celluloselDEAE-cellulose, 40 x 20 cm) (Brownlee and Sanger, 1969). Closed circles
(0) depict oligonucleotides derived from the major strand transcript; stippled circles (.:.) depict oligonucleotides derived from the minor
strand transcript (see Figure 2).

Monkey DNA in Defective SV40
849

I

GATC

iGCY

T. -
,T -
c, -

.c -

A-
'G -
A-
A-
A-
A-
A-

A-
G-
T-

T-
T-

T-

T-

A-

G-

T-


G-



G-

2

GATC
AGC?

v II

3

AGTC

GX

4

AGTC

Kii

- T,
- - Gs:

- T:
- TIG

-c
-T

-

-

- c_T


- T/G

-

-

-c
--G
-T

-c

-A

-A

-A

-G

-A


-G

-T


-c


-T

Figure 4. Representative Autoradiographs of DNA Sequencing Gels Obtained from the DMS-Hz Chemical Sequencing Methods (Maxam
and Gilbert, 1977) as Applied to (Hind IllHind Ill)-E Fragment
(1) a2P-labeled at the Hind II end; first discernible residue (at the bottom) isthe G 6 nucleotides from the end at position 146 in the sequence
shown in Figure 2: (2) identical to (1). except for longer electrophoresis such that first discernible residue is 22 nucleotides from the end, at
sequence posltion 130; (3) "P-labeled at the Hind 111 end; first discernible residue (at the bottom) is at position 6 (Figure 2); (4) identical to
(3), except that first discernible residue is at position 17.

Partial Nucleotide Sequence of (Hind IVHind Ill)-C
Fragment
A sequence of 33 nucleotide residues at the Hind Ill
end of (Hind WHind Ill)-C fragment [contiguous

with (Hind IVHind Ill)-E) was determined using only
the direct DNA sequencing techniques (Figures 2
and 58). The fragment was labeled with 32P at the
Hind 111 terminus (see Experimental Procedures)

Cell
850

Figure 5. Autoradiographs of Two-Dimensional Fractionation (as Described In Experimental Procedures and Identical to That Used in
Figure 3) of the Products Resulting from Partial Digestion with Snake Venom Exonuclease of (Hind IllHind Ill)-E Fragment (A) and (Hind 111
Hind Ill)-C Fragment (6) Labeled with V-at Their Hind 111 5' Termini

and analyzed by partial snake venom digestion
(Figure 2, SVD, and Figure 56) and the DMS-Hz
sequencing technique (Figure 2, DMS-Hz).

The Occurrence of Sequences in (Hind II/Hind 111)-
E in the Monkey Genome-Cot Analysis
3H-labeled (Hind IVHind Ill)-E fragment was allowed
to reanneal in the presence of heterologous DNA
(E. coli) or DNA prepared from the BSC-1  line of
monkey kidney cells, and the percentage of (Hind
II/Hind)-E in double-stranded form was determined
by chromatography on hydroxylapatite (Figure 6).
(Hind IVHind Ill)-E alone reannealed with a CotllZ of
1.7 x    mole-sec/l; the reaction was accelerated
about 7 times in the presence of a 126 fold weight
excess about 7 times in the presence of a 126 fold
weight excess of BSC-1 DNA. Reactants harvested
in the plateau regions of the two reannealing
curves were almost equally resistant to digestion
with single-strand-specific nuclease S1 (82% in the
case of fragment E alone, and 72% for the mixture

with BSC-1 DNA). In addition, the melting of rean-
nealed (Hind II/Hind Ill)-E itself was compared with
the melting of duplexes between (Hind IVHind Ill)-E
and BSC-1 DNA (Figure 6, insert). Melting was
sharp in both cases. The Tm value for the duplex
between (Hind II/Hind Ill)-E and BSC-1 DNA was
about 1C lower than was that of the reanneled
fragment itself. Both the S1 nuclease data and the
melting curves indicate extensive matching be-
tween (Hind IVHind Ill)-E and the homologous se-
quences in the cellular DNA, but suggest the
possibility of a small percentage of mismatching.
The observed Tm values, 83-84"C, are consistent
with the fact that (Hind IVHind Ill)-E contains 59.6%
A.T bp (Figure 2).
From the acceleration in reannealing of (Hind II/
Hind Ill)-E by a known quantity of BSC-1 DNA (see
above), it can be calculated that fragment se-
quences may represent as much as 4.8% of the cell
genome. Since monkey cells contain about 5  x
lo8 bp of DNA, (Hind II/Hind Ill)-E sequences may

Monkey DNA in Defective SV40
851

Table 1. Rf Values of Oligonucleotide Products of Partial Snake
Venom Phosphodiesterase Digestion of (Hind II/Hind Ill)-E

Rf Values

Oligonucleotide
Product pH 3.5 pH 1.7

'pdA 1.90 2.25

'pdApdG                   0.78 2.00
'pd ApdGpdC                 0.42 1 .85


'pdApdGpdCpTpTpT            0 0.08

'pdApdApdC                 0.72 2.00
'pdApdApdCpdC              0.47 1.95
'pdApdApdCpdCpdA            0.15 1 .80
' pdApdApdCpdCpdApdG          0.05 1.22

'pdApdApdCpdCpdApdG pdGpT 0 0.21

'pdApdG pdCpT               0.20 0.72
'pdApdGpdCpTpT              0.09 0.25

'pdApdA                   1.06 2.05

'pdApdApdCpdCpdApdG pdG 0 0.56

e Denotes Rf values = mobility relative to blue marker dye, xylene
cyano1 FF, determined by electrophoresis on DEAE paper at either
pH 3.5 (2.5 hr at 250 ma) or pH 1.7 (4.5 hr at 250 ma) (Brownlee.
1972); appropriate markers were obtained from other DNA frag-
ments of known sequence or purchased from Collaborative Re-
search.
The first six oligonucleotides listed were obtained from the diges-
tion of (Hind WHind Ill)-E labeled with 31P at the 5'-hydroxyl of the
Hind 111 terminus. The last seven oligonucleotides listed were
obtained from fragment labeled at the 5'-hydroxyl of the Hind II
terminus. * Denotes a radioactive phosphate; dA, dC, dG and T
are the four deoxyribonucleosides.

comprise as much as 2.4 x IO8 bp per cell. This
value corresponds to a reiteration frequency of
about 1.6 x lo6 in the monkey genome.

The Occurrence of Sequences in (Hind II/Hind 111)-
E in the Monkey Genome-Hybridization to
Fragments of Monkey DNA on Filters
As previously observed (Gruss and Sauer, 1975; F.
L. Brown, P. R. Musich and J. J. Maio, personal
communication; M. Israel and M. Martin, personal
communication), cleavage of total DNA from Afri-
can green monkey kidney cells with endonuclease
I?. Hind 111 and analysis of the products on agarose
or polyacrylamide gels yielded several prominent
fragments of defined size, as well as a mass of
unseparable fragments of relatively high molecular
weight (Figure 7). Additional cleavage of the mon-
key DNA fragments with Hind II [which is necessary
to generate (Hind II/Hind Ill)-E from the defective
SV401 did not alter the Hind 111 digestion pattern
seen in Figure 7 in a detectable manner (not
shown).

At least five discrete bands were usually visible.
The smallest discrete class of fragments [called
AGMr (Hind 111)-1] is also the predominant class and
is slightly larger than the 151 bp (Hind IIIHind Ill)-E
marker (Figure 7). Other experiments using the
endonuclease R. Hind 111 fragments of wild-type
SV40 as markers indicated that AGMr (Hind 111)-1 is

Cot [mole x recllilerl

Figure 6. Reassociation of (Hind II/Hind Ill)-E
JH-labeled (Hind II/Hind Ill)-E fragment (349,600 cpmlpg, 4.8 ngl
ml) was allowed to reanneal at 68% in 0.12 M sodium phosphate
(pH 6.8) in the presence of 605 nglml of denatured sonicated
BSC-1 DNA (0) or E. coli DNA (0). The extent of hybridization at
each time was analyzed by hydroxyapatite chromatography. The
abscissa is the molar concentration of nucleotide residues of
(Hind IllHind ill)-E fragment times seconds. The insert shows a
thermal denaturation of samples first reannealed under the same
conditions as above to a Cot value of lo-*.

approximately 170 bp in length (preliminary se-
quence analysis indicates a chain length of 172 bp;
unpublished experiments). The next largest class
AGMr (Hind 111)-2 is about 350 bp in length, and the
remaining fragments appear to be consecutively
larger multiples of the size of AGMr (Hind 111)-1.

The products of the digestion of BSC-1 DNA with
endonuclease R. Hind 111 were transferred from the
agarose slab gel (see Figure 7) directly to nitrocel-
lulose strips and hybridized against 32P-labeled
cRNA from (Hind II/Hind Ill)-E fragment as de-
scribed in Experimental Procedures. As shown in
Figure 7, each of the five discrete bands detected
by staining with ethidium bromide hybridized with
the cRNA; no other hybrid was detected. Thus all or
part of the sequences contained in the (Hind Ill
Hind Ill)-E fragment of the defective SV40 genome
also occur in each size class of discrete monkey
Hind 111 fragments. Although all the classes of dis-
crete monkey fragments contain some sequences
homologous to at least a portion of (Hind IVHind
III)-E, the nature of the additional sequences in the
fragments longer than AGMr (Hind 111)-1 is not
known.

Sequences Present in (Hind IVHind Ill)-E and -C
Fragments Are Present in Other Substituted
Defective SV40 Variants
Previous work (Rozenblatt et al., 1973; Frenkel et
al., 1974; Oren et al., 1976) indicated that the DNA
associated with the defective SV40 variant CVBIII
P4 contains segments homologous to both highly
reiterated and infrequently reiterated or unique
monkey sequences. Furthermore, using cRNA

Cell
852

AB C

-Frg.E

Figure 7. DNA Fragments Obtained from the Digestion of BSC-1
DNA with Endonuclease R.Hind 111 and Hybridization of the Frag-
ments with (Hind ll/Hind Ill)-E
BSC-1 DNA (3 pg, total volume 50 rl) was digested with endonu-
clease R.Hind 111, and the material was electrophoresed on a 1.4%
agarose gel (slot 6). A mixture of defective SV40 DNAs (CVP8/1/
P2) was digested with endonuclease R.(Hind II/Hind Ill) to provide
a marker of (hind II/Hind Ill)-E fragment which is the shortest
fragment in the digest (slot C) (Rao and Singer, 1977b). The gel
was stained with ethidium bromide and photographed (slots B
and C). The BSG1 digest was transferred to nitrocellulose sheets
and hybridized with a2P-labeled cRNA to fragment E (spec. ra-
dioact. about 1 X 10' dpmlpg), and an autoradiogram of the
nitrocellulose strip was made (slot A).

probes copied from the monkey sequences present
in CVB/l/P4 and other independently derived de-
fective viral DNAs, Oren and his co-workers (1976)
demonstrated that the same sequences occur in
several of the independently isolated populations
of substituted SV40 variants. It seemed probable
that the same sequences might be present in
CVP8/1/P2 (Eco RI res) DNA 1, since it shares a
common ancestry with CVB/l/P4 (Rao and Singer,
1977a). The data in Table 2 indicate that sequences
in (Hind IVHind Ill)-E fragment hybridize signifi-
cantly to several of the independent SV40 variants
previously described. We consider that hybridiza-
tion of >5% of the radioactive probe represents a
clear indication of positive homology. It is espe-
cially noteworthy that the two defectives 776-P11
and 1103 contain sequences homologous to those
of (Hind II/Hind Ill)-E, since these were derived
from passage of wild-type strain 776 and not wild-
type strain 777, as was CVPB/I/P2 (Eco RI res). No
hybridization of the cRNA to (Hind II/Hind Ill)-E was
detected with the defective variants 1101 or DAR-
d5, which only contain monkey sequences that are
infrequently reiterated (Lee et al., 1975; Davoli et

Table 2. Hybridization of (Hind II/Hind Ill)-E Fragment Sequences
to Independently Derived, Substituted Variants of SV40

DNA on Filter

% Input

Experiment Type Pg Hybridized

1 CVP8/1/P2 0.5 16

         CVGIllP10 1.25 29

         777-T0 1.25 13

         CVB14lP8 0.5 12

         CVB/2/P5 1.25 4

         RH91 1 0.5 3

         CV112/P7 1.25 1

         CVl/I/P5 1.25 0.4

2

BSC-I 18 65

777(CVB) 1.8 0.8

776-P11 1.2 25

3 777(CVB) 6 2

         CVP8/11P2     1.25 15

         DAR-d5       1.5 0.8

4 BSC-1       12.5 24

         1101        1.25 23

         1103        1.25 10

The filter hybridization procedure is described in Experimental
Procedures. In experiments 1, 2 and 3, the input was 12P-cRNA
(7.5 x lo*, 4.5 x lo4 and 3.0 x 10` cpm. respectively). In experi-
ment 4, the input was (Hind IllHind Ill)-E fragment (DNA) labeled
with snP at the Hind II 5'-hydroxyl terminus (9.5 x IO'cpm). When
first prepared, the cRNA had a specific radioactivity of about 1 .I X
10ncpm/pg. The inputs of cRNA correspond, in each instance, to
approximately 0.7 ng of RNA. The defective CVP8lllP2 is the
source of (Hind II/Hind Ill)-E fragment. The other defectives have
been described previously by Oren et al. (1976), except for DAR-
d5, whichwasdescribed byDavolietal. (1977), and1011 and1103
which were described by Lee et al. (1975). The viral stocks 777-T8,
RH 911 and 776-PI1 were derived from the indicated strains of
SV40, and have been carried at high multiplicity of infection over a
period of years in the laboratory of E. Winocour (Oren et al.,
1976). 777(CVB), a plaque-purified strain 777 SV40, and DNA
isolated from BSC-1 cells SeNed, respectively, as negative and
positive controls for hybridization. The percentage of the input
radioactivity bound to blank filters (no DNA) was subtracted from
the values shown. In experiments 1,2 and 3, the blank values were
<0.4% of the input; in experiment 4, the blank values were <1 5%.
It should be noted that the cRNA-DNA hybridization procedure
(experiments 1, 2 and 3) affords lower blank values and greater
efficiency of hybridization than the DNA-DNA procedure (experi-
ment 4).

al., 1977). We conclude that the (Hind II/Hind 111)-E
sequences are frequently incorporated into substi-
tuted SV40 defectives.

Discussion

The mechanisms responsible for the formation of
defective SV40 genomes that are substituted with
host (monkey) DNA are not known. Furthermore,
those properties of the substituted genomes that

Monkey DNA in Defective SV40
853

account for their perpetuation during multiple se-
rial passages of defective viral stocks are also not
known. It is clear from the available data (reviewed
in the Introduction) that such molecules are gener-
ated during permissive infection of monkey kidney
cells in tissue culture by SV40 and, after high multi-
plicity passaging, may come to represent a sub-
stantial portion of the viral genomes synthesized
during infection. The formation of these genomes
and their subsequent evolution during serial high
multiplicity passage (Brockman, Lee and Nathans,
1973; Martin et al., 1973; Rozenblatt et al., 1973)
must involve recombinational events within the
SV40 genome, as well as between SV40 and mon-
key genome DNA. These events may include inte-
gration of infecting wild-type SV40 genomes into
the chromosomal DNA of the cells and subsequent
faulty excision, or exchanges of DNA segments
between the DNA of the two genomes in the ab-
sence of true integration. It is also possible that
they arise by recombination between the wild-type
SV40 genomes and nonchromosomal host DNA
molecules such as the small polydisperse circular
DNA molecules known to occur in uninfected cells
of the BSC-1 monkey kidney line (Smith and Vino-
grad, 1972).
Both low and high reiteration frequency classes
of monkey DNA occur in the substituted SV40
defective genomes. Winocour and his co-workers
(Frenkel et al., 1974; Oren et al., 1976) have
demonstrated that in a variety of independently
isolated defectives, a nonrandom selection from
the total monkey genome is present. A set of
highly reiterated monkey sequences and a set of
sequences of low reiteration frequency were de-
tected in several independently isolated defective
variants. Other substituted variants did not appear
to contain the same monkey sequences. With re-
gard to the reiterated sequences, the data in this
paper confirm the earlier observation. Sequences
contained in fragment (Hind IVHind Ill)-E of the
defective SV40 genome CVP8/1/P2 (Eco RI res)
DNA I are present in several of those defectives
previously characterized as sharing common mon-
key reiterated sequences. In addition, a completely
separate isolate, variant 1103 (Leeet al., 1975), also
has (Hind II/Hind Ill)-E sequences. While it might
appear that even random recombination would fre-
quently result in the incorporation of a sequence
reiterated 1.6 x lo6 times in the monkey genome, in
fact, the (Hind II/Hind Ill)-E sequences represent
10% of the genome and yet are found in about half
the substituted variants tested. Thus particular
monkey sequences containing at least portions of
(Hind II/Hind Ill)-E fragment are commonly found in
substituted variants. Selection of these sequences
may occur during the initial recombination events
or during the subsequent replication of DNA.

The availability of well characterized, substituted
SV40 variants offers an opportunity to elucidate the
structures involved in recombination and, perhaps,
thereby to increase understanding of the corre-
sponding mechanisms. The incorporation of the
monkey sequences present in (Hind II/Hind Ill)-E
fragment into the defective SV40 variant has also
provided a convenient tool for the isolation and
characterization of a portion of the monkey ge-
nome. We describe here the entire nucleotide se-
quence of the (Hind II/Hind Ill)-E fragment isolated
from the defective CVP8/1/P2 (Eco RI res) DNA I, as
well as that of 33 residues in (Hind II/Hind Ill)-C that
are contiguous with (Hind IVHind Ill)-E in the defec-
tive genome. The direct DNA sequencing data indi-
cate that (Hind IVHind Ill)-E and (Hind IVHind Ill)-C
are each single homogeneous sequences, and not
mixtures of sequences as might have been ex-
pected if the molecules of CVP8/P2 (Eco RI res)
DNA I contained a variety of host sequences.
(Hind II/Hind Ill)-E fragment is 151 residues long,
has an A-T content of 59.6%, and is bounded by an
endonuclease R. Hind 111 restriction site at one end
and an endonuclease R.Hind II restriction site at
the other. Although the (Hind II/Hind Ill)-E se-
quence has a high reiteration frequency in the
monkey genome, the nucleotide sequence is mark-
edly different from the sequences reported previ-
ously for reiterated satellite and spacer regions of
eucaryote genomes (Brownlee, Cartwright and
Brown, 1974; Biro et al., 1975; Endow, Polan and
Gall, 1975; Saker et al., 1976). The latter are char-
acterized by extensive internal repetitions of rela-
tively short segments (6-15 residues). On the other
hand, inspection of the sequence of (Hind II/Hind
Ill)-E (Figure 2) does not indicate extensive internal
repetition, although there are a few short repeats.
Recent data (Roizes, 1976) suggest that the highly
repetitive bovine satellite DNA I (Botchan, 1974)
may also contain rather long basic internal repeat
units.

There are several short regions of 2 fold rota-
tional symmetry within the fragment (Hind II/Hind
Ill)-E sequence. The longest contain a total of 10
and 12 symmetrically positioned residues, and oc-
cur at positions 4-14 and 68-84, respectively (Fig-
ure 2). The longer region of symmetry (positions
68-84) contains an unsymmetrical core sequence
of five residues (positions 74-78), and potentially
gives rise to a stem and loop structure in the single-
stranded cRNA copy of this region. In fact, under
the conditions used for partial endonuclease diges-
tion of the cRNA transcript, this region was found
to be most resistant to ribonuclease attack (Figure
2).
Most strikingly, the sequence contains relatively
long stretches of asymmetrically distributed, alter-
nating arrays of purine- and pyrimidine-rich seg-

Cell
854

ments. Examination of either strand indicates that
most of these stretches vary from between six and
twelve consecutive purine or pyrimidine residues,
and have little, if any, exact sequence homology
among them.
There is also an interesting array of sequences at
the R.Hind II end of (Hind IVHind Ill)-E. The G at
position 149 is in the recognition site GTTPuAC for
endonuclease R.Hind II (Smith, 1974). The same G
residue is contained within a recognition site for
endonuclease R. Eco RII-that is, CCTGG, residues
145-149 (Bigger, Murray and Murray, 1973; Boyer
et al., 1973). Similarly, the two C residues at the 5'
end of the endonuclease R.Eco RII recognition site
are the last two residues of a recognition site for
the endonuclease R.Hph, TCACC (residues 142-
146) (Kleid et al., 1976). There is no apparent rea-
son to expect that sequences for three different
bacterial restriction endonucleases should occur in
a row in this fragment. (Hind IVHind Ill)-E also
contains two sites susceptible to cleavage by endo-
nuclease R.Eco RI under the relatively nonspecific
conditions termed RI" (Polisky et al., 1975). These
sites are the sequences AATT at positions 32-35
and 95-98.
Digestion of total monkey DNA with endonucle-
ase R.Hind 111 yields a series of well defined DNA
fragments of various molecular weights (Gruss and
Sauer, 1975). As shown here, all size classes of
these fragments contain sequences that hybridize
with (Hind II/Hind Ill)-E. The monkey fragments are
of various molecular weights, the smallest and
most abundant [AGMr (Hind 111)-1] being about 172
bp in length. The other fragments are of increasing
size and appearto be multimers of AGMr (Hind 111)-1
in length. It is not known whether these longer
fragments contain multiple copies of all or of only a
part of the (Hind II/Hind Ill)-E sequence. However,
studies on the nucleotide sequence of AGMr (Hind
111)-1 (M. Rosenberg, H. Rosenberg and M. F.
Singer, manuscript in preparation) indicate that a
sequence of 124 residues from one end of AGMr
(Hind 111)-1 is identical to the 124 residue sequence
from the endonuclease Re Hind 111 terminus of (Hind
II/Hind Ill)-E. Similarly, the 33 determined residues
of (Hind II/Hind Ill)-C fragment are identical, in 32
positions, to the 33 residues at the end of AGMr
(Hind 111)-1 distal to the (Hind II/Hind Ill)-E se-
quences. These data provide direct sequence con-
firmation of the origin of portions of both (Hind II/
Hind Ill)-C and -E in the monkey genome, and allow
us to define the exact positions at which the highly
reiterated monkey DNA contained in the SV40-de-
fective genome diverges from the AGMr (Hind 111)-1
sequence as it is found in the monkey genome.
Subsequent to residue 124, (Hind IVHind Ill)-E is
different from AGMr (Hind 111)-1. This sequence

might be an SV40 sequence or, alternatively, a
monkey sequence not present in AGMr (Hind 111)-1.
As reported earlier by Gruss and Sauer (1975), no
Hind II sites are detectable in the series of frag-
ments generated from the BSC-1 DNA by cleavage
with endonuclease R. Hind 111. Thus if residues 125-
151 of (Hind IVHind Ill)-E were derived from the
monkey genome, they must occur in an infrequent
segment containing a portion of the highly reiter-
ated AGMr (Hind 111)-1 sequence. Furthermore,
while it is improbable that the sequence is derived
from wild-type SV40 DNA, since nowhere within the
wild-type SV40 genome are three contiguous sites
specific for endonucleases ReEco RII and R.Hind
II, as there are in (Hind IVHind Ill)-E, it could repre-
sent an altered wild-type sequence. Sequence de-
termination at the Hind ll end of the (Hind WHind
III)-B fragment known to contain SV40 sequences
and to be contiguous to (Hind II/Hind Ill)-E in the
substituted defective CVP8/1/P2 (Eco RI res) DNA I
(Rao and Singer, 1977b) should allow a clearer
identification of the origin of the sequences at the
Hind II end of (Hind WHind Ill)-E. It is possible, of
course, that recombinational events themselves
generated the nucleotide sequence of residues 125
through 151, and that the observed sequence can-
not be characterized either as monkey or SV40 in
origin.
About 20-25% of the chromosomal DNA of Afri-
can green monkey cells is contained in the highly
repetitive fraction designated as component CY
(Maio, 1971; Kurnit, Shafit and Maio, 1973). Gruss
and Sauer (1975) reported that component CY DNA
gives the same series of well defined low molecular
weight DNA fragments as does total monkey DNA
upon digestion with endonuclease R.Hind 111. The
reiteration frequency of component CY has been
estimated to be about 7 x lo6, but it has not yet
been shown that a is a homogeneous sequence
element. It appears probable that the sequence
described in this report is a sequence that is in-
cluded in component CY.

Experimental Procedures

Materlals
The following materials were prepared as previously described:
defective substituted SV40 variant CVP8/1/P2 (Eco RI res) DNA I
(Rao and Singer, 1977a); (Hind ll/Hind Ill)- fragment from CVP8/
1/P2 (Eco RI res) DNA I (both unlabeled and lH-labeled), purified
by electrophoresis on polyacrylamide gels after digestion with the
endonuclease R.(Hind Ii/Hind 111) (Segal et al., 1976); Rao and
Singer. 1977b; "P-labeled cRNA to fragment E (Segal et al.,
1976). Monkey DNA was isolated from BSC-1. an established line
of monkey kidney cells, by the procedure of Aloni et al. (1969). E.
coli DNA (type VIII) was obtained from Sigma. Endonucleases
R.Hind 111 (Smith, 1974) and R.Hind 11, an isochizomerof R.Hind
II (Landy et al., 1974), were obtained from New England Bioiabs.
NE-260 scintillatlon fluid was obtained from Nuclear Enterprlses,
Inc. (San Carlos, California).

Monkey DNA in Defective SV40
855

RNA Sequencing Methods
"P-labeled cRNA to fragment E was prepared by labeling the RNA
separately with each of the four a-"P-ribonucleoside triphos-
phates (New England Nuclear). The transcription products were
analyzed either directly or subsequent to electrophoresis on 5%
polyacrylamide gels in 8 M urea (Segal et al., 1976). The RNA was
digested with either T1 or pancreatic RNAase, and the resulting
oligonucleotides were fractionated by two-dimensional homo-
chromatography (Figure 3) (Brownlee and Sanger, 1969). These
products were further characterized by a variety of standard se-
quencing techniques (Erownlee, 1972) identical to those used
previously (Rosenberg, Weissman and de Crombrugghe, 1975;
Kramer and Rosenberg. 1976). These methods include subse-
quent digestion of oligonucleotides with the appropriate enzymes
(Tl, pancreatic and U2 RNAases) and fractionation of the prod-
ucts in one dimension on DEAE paper (Whatman DE 81) at pH 1.7
and 3.5 (Brownlee, 1972); determination of base composition and
nearest-neighbor analyses by alkaline hydrolysis; and analysis of
certain oligonucleotide products subsequent to their modification
with a carbodiimide reagent (Barrell, 1971).

In addition, partial digestion of the cRNA with T1 RNAase was
carried out as previously described (Rosenberg and Kramer,
1977). The products were fractionated two-dimensionally as de-
scribed above using homochromatography solutions which effec-
tively separated oligomers up to 45 residues in chain length. All
partial products were further characterized by complete,digestion
with the appropriate enzymes and fractionation in either one or
two dimensions by standard electrophoretic and/or chromato-
graphic techniques (Sanger. Brownlee and Barrell, 1965; Brown-
lee and Sanger, 1969; Brownlee, 1972). The results of these partial
enzymatic digestions are summarized in Figure 2.

Preparatlon of Fragments Labeled wlth 32P at a Slngle S'-Hy-
droxy Terminus
The 5' ends of the DNA fragments were labeled, after dephospho-
rylation with bacterial alkaline phosphatase (Sigma), by phospho-
rylation with y-12P-ATP (ICN; spec. act. >1 Ci/pmoie) using T4
polynucleotide kinase as described by Maniatis, Jeffrey and Kleid
(1975). The strategy for obtaining the appropriate fragment [(Hind
IllHind Ill)-C or -E] labeled selectively at only one end will be
understood by reference to Figure 1 and as follows: CVP8/1/P2
(Eco RI res) DNA I was cleaved with restriction endonuclease
R.Hind 111, and the 5' ends of the mixed DNA fragments were
labeled with 12P as described above. These fragments were then
redigested with endonuclease R. Hind 11, and the products were
resolved by electrophoresis on polyacrylamide gels and eluted
from the gels as previously described (Rosenberg et al., 1975).
Hind IVHind Ill-C and -E fragments, labeled uniquely at their Hind
111 termini, were well resolved (Rao and Singer, 1977a) and can be
obtained in pure form directly from these gels.

(Hind IVHind Ill)- fragment, labeled selectively at its Hlnd II
end, was obtained by simply reversing the order of the restriction
endonuclease digestions. CVP8/1/P2 (Eco AI res) DNA I was first
cleaved with endonuclease R.Hinc II, end-labeled and then
cleaved with endonuclease R.HInd 111. Again, gel electrophoresis
yielded the purified fragments.

DNA Sequenclng by Partial Venom Phosphodletterase Digeo-
tlon
DNA fragments labeled selectively at one end were partially di-
gested with snake venom exonuclease (Sanger et al., 1973; Man-
iatis et al., 1975). The resulting products were fractionated by
electrophoresis on Cellogel (Kalex) at pH 3.5 followed by either
homochromatography on DEAE-cellulose thin-layer plates (see
Figure 5) or electrophoresis on DEAE paper at pH 1.7 and/or 3.5
(Sanger et al.. 1965; Brownlee. 1972).

DNA Sequencing by Dimethyl Sulfate and Hydrazlne Analysis
DNA fragments labeled at one 5'-hydroxy terminus were sub-

jected to direct DNA sequencing according to the methods de-
scribed by Maxam and Gilbert (1977).
Only representative autoradiograms of the many gel electro-
pherograms obtained in the course of this work are presented
here. The autoradiograms of all additional gels used to determine
the reported sequences are available to interested investigators
upon request to the authors.

Determination of Reatroclation Kinetics
BSC-1 and E. coli DNA were sonicated for 30 min at full strength in
a 10 kc Raytheon sonifier: the average length of the resulting
fragments was about 150 bp as determined on alkaline sucrose
gradients. The unlabeled BSC-1 or E. coli DNA was mixed with %HH-
labeled (Hind II/Hind Ill)- fragment in 8 mM NaOH and denatured
by heating at 100C for 5 min. Thereafter thesamples were cooled
to 68C and neutralized with 8 mM HCI. The salt concentration
was adjusted to 0.12 M sodium phosphate (pH 6.8). and incuba-
tion was carried out at 68'C. At various time intervals, samples
were removed and diluted with cold distilled HzO to give a final
concentration of 0.04 M sodium phosphate. The extent of hybridi-
zation was determined by hydroxyapatite chromatography at 60C
as described (Britten, Graham and Neufeld. 1974). In brief, unhy-
bridized (Hind II/Hind Ill)- was eluted from the columns at 0.12 M
sodium phosphate, and hybridized molecules were removed at
0.4 M sodium phosphate. Radioactivity was determined in NE-260
scintillation fluid.

Thermal Denaturation of Reannealed "-Labeled (Hlnd Ii/Hlnd

3H-labeled (Hind IllHind Ill)- fragment was allowed to reassociate
as described above in the presence of sonicated denatured BSC-1
or E. coli DNA to a Cot value of   (based on the concentration
of the fragment as in Figure 6). The samples were diluted to a final
concentration 0.04 M sodium phosphate and applied to hydroxy-
apatite columns at 60C. The columns were washed at 60C with
0.12 M sodium phosphate until no additional radioactivity
emerged. The temperature was then increased at 5'C intervals (up
to 98"C), and the total radioactivity eluted by 0.12 M sodium
phosphate at each temperature was determined in NE-260 scintil-
lation fluid.

Digestion of BSC-1 DNA wlth Endonuclease R.Hlnd 111 (and
Hybridization of Resulting Fragments wlth cRNA lo (Hlnd Ii/Hlnd
ill)-E
BSC-1 DNA was digested with 2 units of endonuclease R.Hind 111
per pg for 44 hr at 37C in 0.06 M NaCI, 7 mM MgClr, 7 mM Tris-
HCI (pH 7.4). The reaction mixture was analyzed by electrophore-
sis through 1.4% agarose slab gels for 5 hrat 40 V: the electropho-
resis buffer was 0.04 M Tris-HCI, 0.02 M sodium acetate, 2 mM
EDTA (pH 7.8). The gel was then stained with a 0.5 pglml solution
of ethidium bromide (Sharp, Sugden and Sambrook. 1973). and
the DNA was visualized with ultraviolet light. Photographs of gels
were taken with a Polaroid camera and high speed type 57 Kodak
film. The fragments were then transferred to nitrocellulose sheets
by the method of Southern (1975) as modified by Botchan. Topp
and Sambrook (1976). and hybrldlzatlon was carried out with s2P-
labeled cRNA to (Hind IllHind III)-E. Hybridization was in the
presence of 50% formamide (Fluka) and 0.75 M NaCI, 0.5% 50-
dium dodecylsulfate, 0.05 M Tris-HCI (pH 7.8) for 24 hr at 37C.
After appropriate washing, the nitrocellulose sheet was placed in
contact with Kodak No-Screen film for 48 hr.

Filter Hybrldlzatlon
Hybridization of aaP-labeled cRNA transcribed from (Hind IIIHind
Ill)-E fragment or of lPP-labeled (Hind WHInd Ill)-E itself to DNA
immobilized on nitrocellulose filters was carried out by the proce-
dures described previously (Lavi and Winocour. 1972: Segal et el.,
1976), except that the samples were treated at 68C for 30 min in
99% formamide prior to dilution to 50% formamide and addition to

Ill)-E

Cell
856

the filter suspended in hybridization buffer. DNA from a variety of
independently derived, substituted SV40 variants (Oren et al.,
1976) was prepared in the laboratory of Dr. Ernest Winocour. The
DNA of the substituted variant DAR-d5 (Davoli et al.. 1977) was
supplied by Dr. George Fareed. The DNA of variants 1103 and
1101 (Lee et al., 1975) was prepared in this laboratory using
stocks provided by Dr. Daniel Nathans.

Acknowledgments

We are grateful to Edward Bieber and Cathy Brady for excellent
technical assistance. We thank Drs. Ernest Winocour, Daniel Na-
thans and George Fareed for generously supplying substituted
SV40 defectives obtained in their laboratories. We also thank A.
Maxam and W. Gilbert for providing us with their DNA sequencing
protocols prior to publication.

Received April 8, 1977; revised May 16, 1977

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