Proc. Natl. Acad. Sci.

USA
Vol. 76, No. 5, pp. 2222-2226, May 1979

Biochemistry

Cloning in Escherichia coli and physical structure of hepatitis B

virion DNAt
(Dane particle/bacteriophage X vector/restriction mapping/SI endonuclease/exonuclease III)

PATRICK CHARNAY, CHRISTINE POURCEL, ANNE LouISE, ALEXANDRE FRITSCH, AND PIERRE TIOLLAIS
Recombinaison et expression genetique (Institut National de la Sante et de la Recherche MWdicale U. 163), Unite de Genie G6n4tique, Institut Pasteur,
28 rue du Docteur Roux, 75015 Paris, France

Communicated by Andre Lwoff, February 23, 1979

ABSTRACT
A restriction map of hepatitis B virion DNA
was established after cloning of the whole viral genome in
Escherichia coli. By use of EcoRI, Xho I, Bgl II, Xba I, BamHI,
HincII, and Hae III endonucleases, a total of 28 restriction sites
were mapped. The single-stranded region was localized on the
restriction map and the 5' end of the short strand was mapped
at a fixed position.
Hepatitis B is a frequent and sometimes severe disease. In some
areas of Asia and tropical Africa, 10% of the population carry
the viral surface antigen (1). The Dane particle (2), found in
the sera of patients, is considered to be the etiological agent (3).
It contains a circular DNA molecule, hepatitis B virion (HBV)
DNA, with a nick (or a short gap) and a single-stranded region
(4-6). HBV DNA is the smallest known mammalian viral genome. These unique characteristics of the genome, the frequency of hepatitis B, and the likely relationship between this
virus and primary liver carcinoma (7, 8) justify an accurate
investigation of the structure of the HBV genome.
In this article we present a physical map of the HBV genome.
Twenty eight restriction sites are located, and new information
concerning the physical structure of HBV DNA is also established. In particular, the single-stranded region is localized on
the restriction map. The position of the nick is discussed.

Enzymatic Digestions and Chemicals. Digestions by the

following restriction nucleases (New England BioLabs), EcoRI,
BamHI, Sma I,Xho I, Pst I, Xba I, Bgl II, Sst I, Sal I, Kpn I,
HincII, and Hae III, were performed in the buffers recommended by New England BioLabs. Alkaline phosphatase (P-L
Biochemicals) was used in 10 mM Tris-HCI, pH 8.5/10 mM
MgCl2. The 5' ends were labeled by means of [y-32P]ATP (New
England Nuclear, 3000 Ci/mmol) and polynucleotide kinase
(P-L Biochemicals) in 50 mM Tris-HCI, pH 9.5/10 mM
MgCl2/5 mM dithiothreitol. SI endonuclease (generous gift
from F. Rougeon) was used at 25C in 25 mM NaCOOCH3,
pH 4.5/1 mM ZnSO4/125 mM NaCl. DNA was labeled by nick
translation with [a-32P]dATP (New England Nuclear, 300
Ci/mmol) and the three other unlabeled dNTPs at a concentration of 200,uM and with Escherichia coli DNA polymerase
I (Boehringer Mannheim) in 50 mM Tris-HCl, pH 7.8/5 mM
MgCl2/10 mM 2-mercaptoethanol; DNA concentration was
10 ,g/ml; DNA was purified from free dNTPs by gel filtration
on Sephadex G100 (10). E. coli exonuclease III (New England
BioLabs) digestion was done in 67 mM Tris-HCI, pH 7.3/90
mM NaCl/4 mM MgCl2/4 mM dithiothreitol at 25C for 30
min; the DNA concentration was 1 ug/ml.
Other Methods. Polyacrylamide gel electrophoresis of restriction DNA fragments was carried out by the method of
Adams et al. (11). DNA fragments from polyacrylamide gels
were purified as described by Maxam and Gilbert (12). Phage
plate stocks, extraction of DNA, electrophoretic analysis of
restriction fragments on agarose gels, and electron microscopic
analysis of DNA heteroduplexes were done as described
(13).
Biohazards. Biohazards associated with the experiments
described in this article have been examined previously by the
French National Control Committee. Growth of recombinant
bacteriophages was done under L3B11* conditions. L3 is
equivalent to P3 physical containment and B1* is intermediate
between the EK1 and EK2 biological safety levels (14).

MATERIALS AND METHODS

DNA Preparations and Cloning. HBV were purified as
described by Summers et al. (4) and their DNA was labeled
with the endogenous DNA polymerase (6), with [32P]dTTP (22
Ci/mmol, 1 Ci = 3.7 X 1010 becquerels) as the radioactive
precursor. After purification of the HBV DNA (4), it appeared
from electron microscope observation that, under the action
of the endogenous DNA polymerase, the single-stranded region
of about 10% of the DNA molecules was totally repaired.
The purified HBV DNA was used for cloning. The circular
HBV genome was cleaved by EcoRI endonuclease at its unique
restriction site (J. Summers, personal communication) and inserted in vitro into the DNA of bacteriophage Xgt WES. XB
as described (9). After transfection of C600 recBC bacteria with
the hybrid DNA, recombinant phage clones were purified three
times. For each recombinant clone a first phage stock was made
and, in order to avoid any genetic drift, all subsequent stocks
were made from this initial sample. The cloned EcoRI DNA
fragment will be referred to as Eco HBV DNA.
After digestion of the DNA of a recombinant clone with
EcoRI endonuclease, Eco HBV DNA was purified from the two
vector arms by zone centrifugation in a 5-30% sucrose gradient.

RESULTS
Analysis of Various X-HBV Recombinant Clones. The
DNAs from five different clones, in which an EcoRI DNA
fragment of about 3200 base pairs had been identified, were
analyzed. These DNAs were digested by EcoRI plus Xho I,
EcoRI plus BamHI, EcoRI plus Bgl II, and EcoRI plus Hpa I
and submitted to electrophoresis on 2% agarose gels. The restriction patterns were identical for the five clones (results not
shown). Heteroduplexes were formed between the DNA from
one of these clones (clone X-HBV1) and HBV DNA. The nu-

The publication costs of this article were defrayed in part by page

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this fact.

Abbreviation: HBV, hepatitis B virion.

t In order to conform to the usual terminology, the Dane particle is
designated as hepatitis B virion.
2222

Biochemistry: Charnay et al.

Proc. Natl. Acad. Sci. USA 76(1979)

:<~~~~~~~~M
0:B

I..; ..;.i.*-

f,
, ,

....

7.. 05 ;

j,

,t

2223

. ;

;7b

..

...

., ..... .. .'.b

FIG. 1. Heteroduplexes between DNA from the X-HBV1 recombinant and DNA from Dane particles. (A) One DNA strand from X-HBV1
recombinant is hybridized with the long strand from Dane particle DNA. The loop (a) is entirely double stranded. A HBV DNA homoduplex
is also observed (b). (B) The other DNA strand from the X-HBV1 recombinant is hybridized with the short strand from Dane particle DNA.
The heteroduplex loop (a) contains a single-stranded region. A HBV DNA homoduplex is also seen (b). The bar represents 0.5 ,gm.

Restriction Map of HBV Genome. The restriction map was

established on the Eco HBV fragment from clone X-HBV1.
The fragment was labeled by nick translation, except for the
experiments in which 5'-end labeling was necessary. No restriction site was observed for Sma I, Pst I, Sst I, Kpn I, and
Hpa I endonucleases.

merous heteroduplexes observed by electron microscopy have

the expected structure (Fig. 1). No apparent base-pairing defects were observed. Both experiments suggest that there was
no major heterogeneity in the original HBV DNA preparation.
Clone X-HBV1 was arbitrarily chosen for all further experiments.

6 B

2 3 4 5 6

A s
1 7 70

1170

1100

A
B

E-

'Affi.bb

BC
C
520

--

450

C-.

xc

xC -wow:

E_220

G
H

C---xc

E _

.
D
B

J
K

bb -bb
FIG. 2. Electrophoretic analysis of restriction DNA fragments obtained from Eco HBV DNA. (A) Autoradiogram of a 4% polyacrylamide
gel. Arrows indicate the positions of simian virus 40 DNA fragments obtained from digestion with HindIII endonuclease. Fragment sizes are
given in base pairs. Eco HBV DNA was digested with the following endonucleases: lane 1, Hae III; lane 2, HincII; lane 3, Bgl II; lane 4, BamHI;
lane 5, Xba I; lane 6, Xho I. (B) Autoradiogram of an 8% polyacrylamide gel. Eco HBV DNA was digested with the following endonucleases:
lane 1, Hae III; lane 2, Hae III + HincII; lane 3, Hae III + BamHI; lane 4, Hae III + Bgl II; lane 5, Hae III + Xba I; lane 6, Hae III + Xho I.
xc and bb indicate the respective positions of the xylene cyanol and bromophenol blue dyes.

2224

Proc. Natl. Acad. Sci. USA 76 (1979)

Biochemistry: Charnay et al.

Table 1. Electrophoretic analysis of restriction DNA fragments

obtained from Eco HBV DNA*
Enzyme
Xho I
BamHI

Bgl II

Fragment

Size, base pairs

A
B
A
B
C
D
A
B
C
D

2950
135

Xba I

HincII

B
C
D
A
B
C
D
E

Hae III

B
C
D
E
F
G
H
I
J

K
L
M
N

1450
900
475
280
1880
430
420
350
1675
1030
245
155
1000
740
650
465
220
920
400
370
340
250
205
150
140
105
60
55
45
42
15

Pst I, Hpa I, Kpn I, Sst I, Sal I, and Sma I do not cleave Eco HBV
DNA.
* See Fig. 2.

The size of the restriction fragments obtained from digestion

by Xho I, BamHI, Bgl II, HincII, Xba I, and Hae III endonucleases was determined by the method of Maniatis et al. (15).
The results (Fig. 2) are presented in Table 1.
The position of the Xho I, BamHI, Bgl II, Xba I, and HincIl
restriction sites was determined as follows. Restriction fragments
corresponding to the extremities of Eco HBV DNA were
identified through 5'-end labeling with 32P and size measurement of restriction fragments from mixed digestions with the
preceding enzymes taken in pairs. The end closest to the Xho
I restriction site is arbitrarily defined as the right end of the Eco
HBV DNA (Fig. 3A).
The Hae III restriction sites have been positioned by a
combination of several methods. (i) Identification of the end
fragments by 32p labeling of the 5' ends; (ii) mixed hydrolysis
of Eco HBV DNA with Hae III and each of the above-mentioned endonucleases (Fig. 2B); (iii) hydrolysis by Hae III of
the purified HincII A, B, C, D, and E restriction fragments; and
(iv) partial hydrolysis of Eco HBV DNA with Hae III and purification of the corresponding restriction fragments on a 4%
polyacrylamide gel, followed by complete hydrolysis of these
fragments with Hae III and their analysis on an 8% polyacrylamide gel. The evidence for the location at the left end of Eco
HBV DNA of the 15-base-pair Hae III N fragment (observed

on an 8% gel) is the following: after 5'-end labeling of Eco HBV

DNA, only one fragment was observed, Hae III M, which
corresponds to the right end of Eco HBV DNA. On the other
hand, partial hydrolysis of the Eco HBV DNA by Hae III gives
a fragment of about 150 base pairs which, upon complete hydrolysis with Hae III, leads to the Hae III H fragment of 140
base pairs. Finally, from other partial hydrolysis, this Hae III
H fragment has been placed in the vicinity of the left end of the
Eco HBV fragment. The Hae III restriction map is presented
in Fig. 3A.
The analysis of restriction fragments from mixed digestions
allowed not only the positioning on the physical map of the
fragments obtained with a given enzyme, but also the accurate
location of restriction sites for different enzymes relative to each
other (e.g., the Bgl II and HincHI sites at the extreme right).
These results are given in Fig. SB.
Structure of HBV DNA. The model for the structure of HBV
DNA as initially proposed by Summers et al. (4) has been tested
and some additional properties have been determined. In
particular, this model implies that the 5' ends of the two DNA
strands overlap over a short stretch.
To determine how the circular structure of the HBV DNA
is maintained, we denatured the DNA, renatured it, and observed it by electron microscopy. (This experiment was the
same as the one described in Fig. 1. Because there was an excess
of Dane particle DNA, many HBV DNA homoduplexes were
obtained.) DNA molecules having the initial circular and partially single-stranded structure were observed, but also a few
"dimer" molecules with double the initial length and with two
single-stranded gaps (results not shown). These "dimers" were
not observed in the initial HBV DNA preparation, and their
formation is easily explained by end-to-end polymerization
through cohesive ends.
HBV DNA was also treated by exonuclease III in order to
digest the 3' ends on each strand. From observation by electron
microscopy it follows that a great majority of the molecules
remained circular whereas their single-stranded region was
lengthened by about 50%. It was also observed that about
one-third of the circular molecules had a second single-stranded
region apparently located at random relative to the first one and
probably due to an accidental single-stranded nick. Both these
observations prove that digestion by exonuclease III had actually occurred. Since the majority of the molecules remain circ'ular, this experiment and the preceding one prove that the
circular structure is due to base pairing of the 5' ends of each

strand.
To determine the position of the single-stranded region, three
samples of HBV DNA were treated with S1 endonuclease, labeled by nick translation, and then digested with EcoRI, Xho
I, and BamHI endonucleases. The resulting restriction fragments were analyzed (Fig. 4). Some major bands were observed:
one with EcoRI (corresponding to a 1580-base-pair fragment),
one with Xho I (corresponding to 1700 base pairs), and two with
BamHI (corresponding to 1310 and 750 base pairs). With this
last enzyme, an additional minor band, corresponding to 900
base pairs, was also observed. These results are easily explained
by the model given in Fig. 5: the 5' end of the short strand is at
a fixed position located at 1580 base pairs from the EcoRI site,
1700 base pairs from the Xho I site, and 1310 base pairs from
the BamHI A site. This location is strengthened by measurements made on heteroduplexes identical to the one of Fig. 1B:
the long double-stranded stretch which necessarily has the
EcoRI site at one end has a constant length of 1500 150 base
pairs. The 750-base-pair fragment obtained after BamHI digestion corresponds to the sum of the BamHI C and D fragments from the Eco HBV DNA. Finally, the broad band is

Biochemistry: Charnay et al.

Proc. Natl. Acad. Sci. USA 76 (1979)

2225

1000

Nucleotide ISlI
K,

I..

1pp

2000

1lii

3000

PPIi
li
Ip

Il

Xho
D1

BglI I

CI
B

Xba

B|

aI

Bam H

NH J

.~

C
B

KLG F

III

ii

B
D

Hinc I
Hae III

I,

D |

IDI

lil
Ai

IIl lI

E
D

l l

B
M

E3M

E3=-DB
-=3
y

cr
I

ZI

1 1

15 140 606565 240

- oE

-Ea,

CD

m
E

cD 8

II)()A

240 35

155

300

E3
c

PIT7
I

)IJ

180

EaI E3B

265

II

185554512525 205

oD xIxxI

II

105

165

190 50808542

FIG. 3. Restriction map of Eco HBV DNA. Eco HBV DNA is about 3100 base pairs long. The end closest to the Xho I restriction site is
defined as the right end. (A) Position of Xho I, Bgl II, Xba I, BamHI, HincII, and Hae III restriction fragments. (B) Relative positions of restriction
sites corresponding to the preceding enzymes. Lengths of DNA fragments were estimated from their electrophoretic mobility and are expressed
in number of base pairs.
generated by

fragment located

in the variable

single-stranded

region of HBV DNA and corresponds to the Eco HBV BamHI

B fragment. Hence, the single-stranded stretch of HBV DNA
A

94

t ff

-,-5200
_-w 3835

I
I

that is of variable length has one fixed end at 1580 nucleotides

from the EcoRI site.
Several minor bands have also been observed. From EcoRI
digestion, at least one band 1320 base pairs long is obtained. Xho
I digestion gives two minor bands, 1630 and 1460 base pairs
long. Similarly, from BamHI digestion one obtains two minor
bands 1220 and 1060 base pairs long. From these results it follows that SI endonuclease partially cuts the HBV DNA at positions 1500 and 1330.

_-1 770
_ 11170 +
1100

700 --

+1170
1100

--6-

"' I

i
--1390

520
--450

FIG. 4. Electrophoretic analysis of DNA fragments obtained

after digestion of HBV DNA by S1 endonuclease and by EcoRI, Xho
I, and BamHI restriction enzymes. (A) Autoradiogram of a 1% agarose
gel. HBV DNA was mixed with a 10-fold excess of XWES AB DNA,
treated with S1 endonuclease, and nick-translated (lane 4). Two
samples were treated in addition with Xho I (lane 1) and EcoRI (lane
2) endonucleases. Lane 3 represents Eco HBV DNA. (B) Autoradiogram of a 2% agarose gel. HBV DNA was mixed with a 10-fold excess
of AWES AB DNA, treated with S1 endonuclease, and then digested
with BamHI endonuclease (lane 1). Eco HBV DNA was digested with
BamHI (lane 2). Arrows indicate the positions of linear simian virus
40 DNA and simian virus 40 DNA fragments obtained from digestion
with HindIII or Pst I endonucleases. Fragment sizes are given in
number of base pairs.

FIG. 5. Physical structure of the genome of hepatitis B Dane

particles. The circular structure is maintained through base pairing
of the 5' extremities of the two DNA strands. These two ends and the
single-stranded region (dashed line) are located on the restriction
map. The EcoRI restriction site is chosen as the origin of the physical
map, and distances are given in number of base pairs. The 5' end of
the short strand is located at a position estimated to be at 1580 base
pairs.

2226

Proc. Natl. Acad. Sci. USA 76 (1979)

Biochemistry: Charnay et al.

DISCUSSION
The size of the cloned HBV genome was estimated to be 3200
base pairs, in agreement with the results of Landers et al. (6)
and J. Summers (personal communication). The cloned DNA
was digested by various restriction enzymes, and the sum of the
fragment sizes from each digest was 3100 base pairs. The
number of fragments and their sizes obtained upon Hae III
endonuclease digestion (Fig. 2) are similar to those obtained
from identical digestions of HBV DNA by Summers et al. (4)
and Landers et al. (6). In agreement with Summers (personal
communication), we locate the cohesive ends of the two Eco
HBV DNA strands in the Hae III A fragment and find the
EcoRI restriction site just opposite the cohesive region. In
contrast, the sizes of certain HincIl restriction fragments (Fig.
2A) are different from HincII fragments obtained by Landers
et al. (6).
Because digestion of HBV DNA by BamHI endonuclease
gives a fragment of 750 base pairs, equal to the sum of the
lengths of BamHI C and D fragments of the cloned DNA, we
conclude that the original HBV DNA does not contain two
EcoRI restriction sites close to each other and, therefore, that
no HBV DNA was lost upon cloning of the EcoRI fragment.
The digestion of HBV DNA by exonuclease III and the formation of HBV DNA "dimers" prove that the circular structure
is maintained through base pairing of the 5' ends of each DNA
strand, which confirms the structural model of Dane particle
DNA proposed by Summers et al. (4). We have now located the
single-stranded region relative to the restriction map (Fig. 5).
Taking the EcoRI restriction site as the origin of the map, the
5' end of the short strand is at position 1580 and the singlestranded region is almost completely contained in the BamHI
B fragment of HBV DNA.
From the analysis of the restriction fragments of HBV DNA
and, particularly, from the existence of the Hae III AA' doublet,
Robinson and coworkers (6, 16) suggest that the HBV DNA
population is heterogeneous. On heteroduplexes and HBV DNA
homoduplexes, as exemplified in Fig. 1, we did not detect any
apparent base-pairing defect, which could mean that the
eventual heterogeneity is not localized in a particular region
of HBV DNA. Digestion patterns of this DNA by Si plus Xho
I, Si plus EcoRI, and Si plus BamHI nucleases (Fig. 4) prove
that, in the great majority of the molecules, the Xho I, EcoRI,
and BamHI restriction sites are at fixed positions relative to the
5' end of the short HBV DNA strand and that no major amount
of heterogeneity exists in the double-stranded region.
After digestion of HBV DNA with SI plus Xho I, Si plus
EcoRI, and Si plus BamHI nucleases, minor DNA bands are
also observed (Fig. 4); we propose three possible explanations
for their origin. (i) Si endonuclease does not cut HBV DNA at
the nick of the long strand; the DNA preparation then contains

two additional minor populations in which the 5' end of the

short strand is shifted to positions 1500 and 1320. (ii) S1 endonuclease cuts part of the DNA molecules at the nick and two
DNA populations are present with a different location of the
nick. (iii) Each HBV DNA molecule contains two nicks and S1
endonuclease cuts only part of them. The first two hypotheses
imply that the HBV DNA population is heterogeneous. Upon
increase of the S1 endonuclease concentration, the minor bands
corresponding to position 1500 become weaker relative to the
other minor bands. We have no explanation for this observation.
A comparison of restriction patterns from different clones
of Eco HBV DNA should give some more insight into this
heterogeneity problem.
We thank Dr. P. Maupas for the supply of virion-rich serum, Dr. J.
Summers for his help in the purification of HBV DNA, and Dr. F.
Rougeon for helpful discussion. This work was supported by grants
from the Institut National de la Sante et de la Recherche Medicale
(INSERM), the Centre National de la Recherche Scientifique (CNRS),
the Delegation Generale a la Recherche Scientifique et Technique
(DGRST), the Universite Paris VII, the North Atlantic Treaty Organization (NATO), and the Fondation pour la Recherche Medicale.
1. Blumberg, B. S. (1977) Science 197, 17-25.
2. Dane, D. S., Cameron, C. H. & Briggs, M. (1970) Lancet i,
695-698.
3. World Health Organization (1977) Progress in Viral Hepatitis,
WHO Technical Report Series no. 602 (WHO, Geneva, Swit-

zerland).

4. Summers, J., O'Connel, A. & Millman, I. (1975) Proc. Nat!. Acad.

Sci. USA 72, 4597-4601.
5. Hruska, J. F., Clayton, D. A., Rubenstein, J. L. R. & Robinson,
W. S. (1977) J. Virol. 21, 666-682.
6. Landers, T. A., Greenberg, H. B. & Robinson, W. S. (1977) J.
Virol. 23, 368-376.
7. Maupas, P., Coursaget, P., Goudeau, A., Drucker, J., Sankale, M.,
Linhard, J. & Diebolt, G. (1977) Ann. Microbiol. (Paris) 128,
245-253.
8. Summers, J., O'Connel, A., Maupas, P., Goudeau, A., Coursaget,
P. & Drucker, J. (1978) J. Med. Virol. 2, 207-214.
9. Fritsch, A., Pourcel, C., Charnay, P. & Tiollais, P. (1978) C. R.
Hebd. Seances Acad. Sci., Ser. D 287, 1453-1456.
10. Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448.
11. Adams, J. M., Jeppesen, P. G. N., Sanger, F. & Barrel, B. G. (1969)
Nature (London) 223, 1009-1014.
12. Maxam, A. M. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA

74,560-564.
13. Charnay, P., Fritsch, A., Louise, A., Perrin, D. & Tiollais, P. (1979)
Mol. Gen. Genet. 170, 171-178.
14. Commission franoaise de recombinaison genetique in vitro:
rapport d'activite (1977) Prog. Sci. 191, 86-94.
15. Maniatis, T., Jeffray, A. & Van de Sande, H. (1975) Biochemistry

14,3787-3794.
16. Robinson, W. S. (1977) Annu. Rev. Microbiol. 31,357-377.