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Restriction map of the hepatitis B virus DNA cloned in Escherichia coli
481
Gene, 20 (1982) 481-484 Elsevier Biomedical
Press
Restriction map of the hepatitis B virus DNA cloned in Escherichia coli (Endonuclease
mapping;
recombinant
DNA)
V.V. Bichko, T.M. Kozlovskaya, A. Dishler, P. Fumpen, A. Janulaitis and E.J. Gren Institute
of Organic Synthesis, Latvian
(Received
September
(Accepted
October
SSR Academy of Sciences, Airkraukles
21, Riga, 226006 (U.S.S.R.)
16th. 1982) 2nd, 1982)
SUMMARY
A plasmid carrying the complete genome of hepatitis B virus (HBV) inserted into the BamHI site of the pBR322 plasmid vector has been constructed. The physical map of HBV DNA established for 13 restriction endonucleases DNA of ayw subtype.
allows to conclude
that the cloned
DNA is similar,
but not identical
to the HBV
The problem of diagnosis and treatment of hepatitis B, a common infectious desease in man, as well as the mechanism of HBV replication, still await solution. This is primarily due to the fact that the blood plasma of affected persons and
and adw, (Valenzuela et al., 1980) and partial sequence of adyw subtype (Pasek et al., 1979) have been determined after cloning the entire HBV genome or its fragments in bacterial vectors. In this paper we present a detailed physical
chronic disease carriers remains the sole source of HBV and its antigens and no permissive cell culture is available for HBV replication studies.
mid pHB320
Up to date, physical
mapping
map of the cloned
of HBV DNA of
adw, subtype has been performed with eight restriction endonucleases without use of genetic engineering methods (Siddiqui et al., 1979). More
bp. base pairs;
0378-I 119/82/0000-0000/$02.75
HBV. hepatitis
site
of
from plas-
HBV genome
inserted
into
(Pumpen different
et al., 1981). For this purpose we used 22 restriction endonucleases, three of which,
pBR322
vector
previously
The cleavage products generated by single or double endonuclease digestion were analyzed electrophoretically in 1% agarose gels or, for the most part, in gradient (3.5-20%) polyacrylamide gels (150 x 300 X 0.75 mm) in TEA . NaCl buffer (0.05 M Tris . HCl, pH 8.0; 0.02 M CH,COONa; 0.002 M EDTA; 0.018 M NaCl) (Helling et al., 1974) for 16 h at 24 mA. The larger DNA fragments for consecutive restriction endonuclease cleavage were resolved electrophoretically in a 1% LGT-agarose
B virus.
0 1982 Elsevier Biomedical
BamHI
the complete
BcnI, CfrI and S&I, had not been applied in the study of HBV DNA.
detailed restriction endonuclease cleavage patterns are known for cloned DNA of ayw (Fritsch et al., 1978; Hirschman et al., 1980), adw, (Sninsky et al., 1979; Valenzuela et al., 1979), and adyw (Burrel et al., 1979) subtypes. On the other hand, the complete sequences of HBV DNA of subtypes ayw (Galibert et al., 1979)
Abbreviations:
HBV DNA derived
carrying
Press
482
gel according
to Wieslander
(1979).
The size of HBV DNA fragments
generated
by
several restriction endonucleases cleaving at multiple sites, is summarized in Table I and the resulting restriction map of the complete is presented in Fig. 1.
CfrI (Janulaitis
unpublished nuclease
results). attacks
MspI (HpaII) mapping.
The BcnI restriction
sequence
CC IEGG
EcoRV,
(our
subtype
a unique
generated
by several restriction
is in bp. The fragments
BamHI:
110-b
182
BgIII:
780+
197
B
420
BglII:
254+
170
C
400
BamHI:
316+
64
D
355
E
260
F
224
endonucleases eliminated
cleaving
G H
A* 2000
XhoI:
1600+330
Barn HI: 1600 + 350 B C
355
BglII:
220+ 130
BspI:
300-t.
44
250
D
190
BspI:
110+
76
160
BamHI:
87+
69
142
F
98
135
G
76
BglII:
60+
16
I
110
H
58
J
63
KI
59
K2
59
L
50
XhoI:
172+
85
Hind11
Msp I BglII:
1900+
XhoI:
1040+1000
100
BamHI:
1400+
610
B
750
BglII:
430+
360
C*
420
BamHI:
245+
175
A
1230
B
760
C*
690
D
490
1760 BamHI: 860
C
520
XhoI:
BumHI:
745 +520
BamHI:
410+283
Tag1
CfrI
B
SmaI, ClaI, KpnI, PvuII
of BcnI, C’rI and MuI
based on its primary
1520+ 350+
240 172
A
1800
B
950
C’
430
EarnHI:
at multiple
cleavage
structure
(Galibert
sites.
in the course of HBV DNA cloning
E
A*
sites for EcoRI,
et
The cloned DNA is identical to the viral DNA of ayw subtype relative to the location of BspI,
All41
Bsp I
2000
was also determined
al., 1979).
with an asterisk.
A
sites
I
The size of DNA fragments
960
PstI,
BspI
cleavage
sites determinated for the cloned HBV DNA coincides with that found for viral DNA of ayw
site which can be useful for physical
HBV DNA fragments
A*
HindIII,
The location
endo-
BcnI site and three CfrI cleavage sites. Since the CfrI cleavage site Py 1 GGCCPu comprises sequence GGCC attacked by BspI, restriction endo-
TABLE
A A GIGGCT C C
sequence
for mapping
of two S&I
and Sail.
containing
The cloned HBV DNA contains
sites. Location
on HBV DNA. Cloned HBV DNA lacks the cleavage the following restriction endonucleases:
HBV genome
et al., 1980) and MuI
CfrI can be applicable
cleavage attacking
The HBV DNA carries cleavage sites for several new enzymes: BcnI (Petrusyte and Janulaitis, 1982)
nuclease
1410+405
at the BamHI
site are indicated
483
the above restriction
Sdu I
Xhol
BarnHI
\
pression
endonucleases
of HBV
genes lead
ex-
in homologous
systems.
to expression
of so far
unidentified HBV genes, their existence cated by endogenous DNA-polymerase
being indi(Kaplan et
This
may possibly
to provide
al., 1973) and protein kinase (Albin 1980) activities of HBV particles.
and Robinson,
REFERENCES
Albin,
C. and
hepatitis Burrel,
I
Barn HI
Robinson,
C.J., Mackay,
and Murray,
SduI
W.S.:
P., Greenaway,
K.: Expression
B virus DNA sequences Fig. 1. Physical
map of cloned
structed
in kilobases.
HBcAg
(C) are boxed.
HBV
HBV DNA.
genes
encoding
The map is conHBsAg
(S) and
Protein
kinase
activity
in
B virus, J. Virol., 34 (1980) 297-302. P.J., Hofschneider,
P.H.
in Escherichia co/i of hepatitis
cloned in plasmid
pBR 322. Nature
279 (1979) 43-47. Fritsch,
A., Pourcell,
C., Charnay,
P. and Tiollais,
du gimome du virus de l’hepatite
P.: Clonage
B dans E. co/i, C.R. Acad.
Sci., Paris. Ser. D 287 (1978) 1453-1456. Galibert,
F., Mandart,
P.: Nucleotide (subtype
Hind11 and XhoI cleavage sites, but it lacks one site for each respective endonuclease BumHI, BglII and MspI and carries two new sites for A/u1 and TuqI and a single HpaI site. The position of a HpaI cleavage site is characteristic of HBV DNA of adw, subtype, whereas the alignment of MspI sites is similar to that in the DNA of both ayw and adw, subtype. Note that the cloned DNA lacks EcoRI cleavage site present in all the studied HBV DNAs. The
Helling,
features
of
restriction
endo-
nuclease cleavage map of the cloned HBV DNA used here and those of DNAs of other subtypes can be employed for cloning particular genes or the complete HBV genome to attain their expression in prokaryotes. By using 7’uqI restriction endonuclease the complete genes for HBsAg and HBcAg can be cleaved, the beginning of the HBsAg gene lying close to one of the ends of the DNA fragment. The location of TaqI and XhoI cleavage sites close to the beginning of HBsAg gene may be useful for its fusion to foreign regulatory signals. The unique HpaI and BcnI cleavage sites are located beyond the HBsAg and HBcAg genes and are probably detached from their regulatory regions which makes it possible to clone the complete genome of HBV and its tandem dimers using
F., Tiollais. P. and Charnay,
of the hepatitis
ayw) cloned in E. co/i. Nature
R.B., Goodman, R-EcoRI
bacteriophages
and
phoresis.
B virus genome
281 (1979) 646-650.
H.M. and Boyer, H.W.:
endonuclease
fragments
other
of DNA
viruses
Analysis
of
from lambdoid
by agarose-gel
electro-
J. Virol. 14 (1974) 1235-1244.
Hirschman,
S.Z.. Price,
cloned hepatitis
P. and Garfinkel,
E.: Expression
B virus DNA in human
cell cultures.
of Proc.
Natl. Acad. Sci. USA 77 (1980) 5507-5511. Janulaitis,
A.A., Stakenas,
E.N.
and
C/r1
from
Berlin,
P.S., JaskelevilSene,
Yu.A.:
Citrobacrer
B.P., Lebedenko,
A new restriction
freundii.
Bioorg.
endonuclease
Khim.
6 (1980)
1746- 1747. Kaplan,
inherent
E., Fitoussi,
sequence
P.M., Greenman,
merase associated
R.L. and Gerin,
with human
hepatitis
Y.L.:
DNA
B antigen.
poly-
J. Virol.
21 (1977) 96-104. Pasek,
M., Goto,
T.. Gilbert,
Kay, P., Leadbetter, genes
W., Zink, B., Schaller.
G. and Murray
and their expression
H., Mac-
K.: Hepatitis
in E. co/i. Nature
B virus
282 (1979)
575-579. Petrusyte, of
M. and Janulaitis,
the
restriction
cenrrosporus. Pumpen,
Eur. J. Biochem.
P.P.. Dischler,
Gren,
A.: Isolation
endonuclease
E.J.,
M.B.,
from
Bacillus
121 (1982) 377-381.
A.V., Kozlovskaya.
Rivkina,
R.A.: Cloning
and some properties BcnI
T.M.. Bichko, V.V.,
Grinberg,
of the hepatitis
A.P.
and
Kukain,
B virus DNA in Escherichia
co/i. Dokl. Akad. Nauk SSSR 260 (1981) lO22- 1024. Siddiqui,
A., Sattler,
nuclease
F. and Robinson,
cleavage
the DNA
map and location
of hepatitis
B virus.
W.S.: Restriction of unique
subtype
adw,.
endo-
features
of
Proc. Natl.
Acad. Sci. USA 76 (1979) 4664-4668. Sninsky,
J.J.. Siddiqui,
A., Robinson.
Cloning
and endonuclease
genome.
Nature
mapping
279 (1979) 346-348.
W.S. and Cohen, of the hepatitis
S.N.: B viral
484
Valenzuela, H.M. coding antigen. Valenzuela, W.J.: 1803/l.
P., Gray,
P., Quiroga,
and Rutter,
W.J.:
for the major Nature
protein
J., Goodman,
sequence
of hepatitis
of the gene
B virus surface
M., Zaldivar,
Scientific
F., Gray,
Memoranda,
Wieslander,
L.: A simple method
in Low gelling temperature
P. and Rutter,
1980,
Memo-H-
to recover intact high molecu-
lar weight RNA and DNA after electrophoretic (1979) 305-309.
280 ( 1979) 8 15-8 19.
P., Quiroga, Hepatitis
M., Zaldivar,
Nucleotide
Communicated
by Z. Hrade%
agarose
separation
gels. Anal. B&hem.
98
Gene, 20 (1982) 481-484 Elsevier Biomedical
Press
Restriction map of the hepatitis B virus DNA cloned in Escherichia coli (Endonuclease
mapping;
recombinant
DNA)
V.V. Bichko, T.M. Kozlovskaya, A. Dishler, P. Fumpen, A. Janulaitis and E.J. Gren Institute
of Organic Synthesis, Latvian
(Received
September
(Accepted
October
SSR Academy of Sciences, Airkraukles
21, Riga, 226006 (U.S.S.R.)
16th. 1982) 2nd, 1982)
SUMMARY
A plasmid carrying the complete genome of hepatitis B virus (HBV) inserted into the BamHI site of the pBR322 plasmid vector has been constructed. The physical map of HBV DNA established for 13 restriction endonucleases DNA of ayw subtype.
allows to conclude
that the cloned
DNA is similar,
but not identical
to the HBV
The problem of diagnosis and treatment of hepatitis B, a common infectious desease in man, as well as the mechanism of HBV replication, still await solution. This is primarily due to the fact that the blood plasma of affected persons and
and adw, (Valenzuela et al., 1980) and partial sequence of adyw subtype (Pasek et al., 1979) have been determined after cloning the entire HBV genome or its fragments in bacterial vectors. In this paper we present a detailed physical
chronic disease carriers remains the sole source of HBV and its antigens and no permissive cell culture is available for HBV replication studies.
mid pHB320
Up to date, physical
mapping
map of the cloned
of HBV DNA of
adw, subtype has been performed with eight restriction endonucleases without use of genetic engineering methods (Siddiqui et al., 1979). More
bp. base pairs;
0378-I 119/82/0000-0000/$02.75
HBV. hepatitis
site
of
from plas-
HBV genome
inserted
into
(Pumpen different
et al., 1981). For this purpose we used 22 restriction endonucleases, three of which,
pBR322
vector
previously
The cleavage products generated by single or double endonuclease digestion were analyzed electrophoretically in 1% agarose gels or, for the most part, in gradient (3.5-20%) polyacrylamide gels (150 x 300 X 0.75 mm) in TEA . NaCl buffer (0.05 M Tris . HCl, pH 8.0; 0.02 M CH,COONa; 0.002 M EDTA; 0.018 M NaCl) (Helling et al., 1974) for 16 h at 24 mA. The larger DNA fragments for consecutive restriction endonuclease cleavage were resolved electrophoretically in a 1% LGT-agarose
B virus.
0 1982 Elsevier Biomedical
BamHI
the complete
BcnI, CfrI and S&I, had not been applied in the study of HBV DNA.
detailed restriction endonuclease cleavage patterns are known for cloned DNA of ayw (Fritsch et al., 1978; Hirschman et al., 1980), adw, (Sninsky et al., 1979; Valenzuela et al., 1979), and adyw (Burrel et al., 1979) subtypes. On the other hand, the complete sequences of HBV DNA of subtypes ayw (Galibert et al., 1979)
Abbreviations:
HBV DNA derived
carrying
Press
482
gel according
to Wieslander
(1979).
The size of HBV DNA fragments
generated
by
several restriction endonucleases cleaving at multiple sites, is summarized in Table I and the resulting restriction map of the complete is presented in Fig. 1.
CfrI (Janulaitis
unpublished nuclease
results). attacks
MspI (HpaII) mapping.
The BcnI restriction
sequence
CC IEGG
EcoRV,
(our
subtype
a unique
generated
by several restriction
is in bp. The fragments
BamHI:
110-b
182
BgIII:
780+
197
B
420
BglII:
254+
170
C
400
BamHI:
316+
64
D
355
E
260
F
224
endonucleases eliminated
cleaving
G H
A* 2000
XhoI:
1600+330
Barn HI: 1600 + 350 B C
355
BglII:
220+ 130
BspI:
300-t.
44
250
D
190
BspI:
110+
76
160
BamHI:
87+
69
142
F
98
135
G
76
BglII:
60+
16
I
110
H
58
J
63
KI
59
K2
59
L
50
XhoI:
172+
85
Hind11
Msp I BglII:
1900+
XhoI:
1040+1000
100
BamHI:
1400+
610
B
750
BglII:
430+
360
C*
420
BamHI:
245+
175
A
1230
B
760
C*
690
D
490
1760 BamHI: 860
C
520
XhoI:
BumHI:
745 +520
BamHI:
410+283
Tag1
CfrI
B
SmaI, ClaI, KpnI, PvuII
of BcnI, C’rI and MuI
based on its primary
1520+ 350+
240 172
A
1800
B
950
C’
430
EarnHI:
at multiple
cleavage
structure
(Galibert
sites.
in the course of HBV DNA cloning
E
A*
sites for EcoRI,
et
The cloned DNA is identical to the viral DNA of ayw subtype relative to the location of BspI,
All41
Bsp I
2000
was also determined
al., 1979).
with an asterisk.
A
sites
I
The size of DNA fragments
960
PstI,
BspI
cleavage
sites determinated for the cloned HBV DNA coincides with that found for viral DNA of ayw
site which can be useful for physical
HBV DNA fragments
A*
HindIII,
The location
endo-
BcnI site and three CfrI cleavage sites. Since the CfrI cleavage site Py 1 GGCCPu comprises sequence GGCC attacked by BspI, restriction endo-
TABLE
A A GIGGCT C C
sequence
for mapping
of two S&I
and Sail.
containing
The cloned HBV DNA contains
sites. Location
on HBV DNA. Cloned HBV DNA lacks the cleavage the following restriction endonucleases:
HBV genome
et al., 1980) and MuI
CfrI can be applicable
cleavage attacking
The HBV DNA carries cleavage sites for several new enzymes: BcnI (Petrusyte and Janulaitis, 1982)
nuclease
1410+405
at the BamHI
site are indicated
483
the above restriction
Sdu I
Xhol
BarnHI
\
pression
endonucleases
of HBV
genes lead
ex-
in homologous
systems.
to expression
of so far
unidentified HBV genes, their existence cated by endogenous DNA-polymerase
being indi(Kaplan et
This
may possibly
to provide
al., 1973) and protein kinase (Albin 1980) activities of HBV particles.
and Robinson,
REFERENCES
Albin,
C. and
hepatitis Burrel,
I
Barn HI
Robinson,
C.J., Mackay,
and Murray,
SduI
W.S.:
P., Greenaway,
K.: Expression
B virus DNA sequences Fig. 1. Physical
map of cloned
structed
in kilobases.
HBcAg
(C) are boxed.
HBV
HBV DNA.
genes
encoding
The map is conHBsAg
(S) and
Protein
kinase
activity
in
B virus, J. Virol., 34 (1980) 297-302. P.J., Hofschneider,
P.H.
in Escherichia co/i of hepatitis
cloned in plasmid
pBR 322. Nature
279 (1979) 43-47. Fritsch,
A., Pourcell,
C., Charnay,
P. and Tiollais,
du gimome du virus de l’hepatite
P.: Clonage
B dans E. co/i, C.R. Acad.
Sci., Paris. Ser. D 287 (1978) 1453-1456. Galibert,
F., Mandart,
P.: Nucleotide (subtype
Hind11 and XhoI cleavage sites, but it lacks one site for each respective endonuclease BumHI, BglII and MspI and carries two new sites for A/u1 and TuqI and a single HpaI site. The position of a HpaI cleavage site is characteristic of HBV DNA of adw, subtype, whereas the alignment of MspI sites is similar to that in the DNA of both ayw and adw, subtype. Note that the cloned DNA lacks EcoRI cleavage site present in all the studied HBV DNAs. The
Helling,
features
of
restriction
endo-
nuclease cleavage map of the cloned HBV DNA used here and those of DNAs of other subtypes can be employed for cloning particular genes or the complete HBV genome to attain their expression in prokaryotes. By using 7’uqI restriction endonuclease the complete genes for HBsAg and HBcAg can be cleaved, the beginning of the HBsAg gene lying close to one of the ends of the DNA fragment. The location of TaqI and XhoI cleavage sites close to the beginning of HBsAg gene may be useful for its fusion to foreign regulatory signals. The unique HpaI and BcnI cleavage sites are located beyond the HBsAg and HBcAg genes and are probably detached from their regulatory regions which makes it possible to clone the complete genome of HBV and its tandem dimers using
F., Tiollais. P. and Charnay,
of the hepatitis
ayw) cloned in E. co/i. Nature
R.B., Goodman, R-EcoRI
bacteriophages
and
phoresis.
B virus genome
281 (1979) 646-650.
H.M. and Boyer, H.W.:
endonuclease
fragments
other
of DNA
viruses
Analysis
of
from lambdoid
by agarose-gel
electro-
J. Virol. 14 (1974) 1235-1244.
Hirschman,
S.Z.. Price,
cloned hepatitis
P. and Garfinkel,
E.: Expression
B virus DNA in human
cell cultures.
of Proc.
Natl. Acad. Sci. USA 77 (1980) 5507-5511. Janulaitis,
A.A., Stakenas,
E.N.
and
C/r1
from
Berlin,
P.S., JaskelevilSene,
Yu.A.:
Citrobacrer
B.P., Lebedenko,
A new restriction
freundii.
Bioorg.
endonuclease
Khim.
6 (1980)
1746- 1747. Kaplan,
inherent
E., Fitoussi,
sequence
P.M., Greenman,
merase associated
R.L. and Gerin,
with human
hepatitis
Y.L.:
DNA
B antigen.
poly-
J. Virol.
21 (1977) 96-104. Pasek,
M., Goto,
T.. Gilbert,
Kay, P., Leadbetter, genes
W., Zink, B., Schaller.
G. and Murray
and their expression
H., Mac-
K.: Hepatitis
in E. co/i. Nature
B virus
282 (1979)
575-579. Petrusyte, of
M. and Janulaitis,
the
restriction
cenrrosporus. Pumpen,
Eur. J. Biochem.
P.P.. Dischler,
Gren,
A.: Isolation
endonuclease
E.J.,
M.B.,
from
Bacillus
121 (1982) 377-381.
A.V., Kozlovskaya.
Rivkina,
R.A.: Cloning
and some properties BcnI
T.M.. Bichko, V.V.,
Grinberg,
of the hepatitis
A.P.
and
Kukain,
B virus DNA in Escherichia
co/i. Dokl. Akad. Nauk SSSR 260 (1981) lO22- 1024. Siddiqui,
A., Sattler,
nuclease
F. and Robinson,
cleavage
the DNA
map and location
of hepatitis
B virus.
W.S.: Restriction of unique
subtype
adw,.
endo-
features
of
Proc. Natl.
Acad. Sci. USA 76 (1979) 4664-4668. Sninsky,
J.J.. Siddiqui,
A., Robinson.
Cloning
and endonuclease
genome.
Nature
mapping
279 (1979) 346-348.
W.S. and Cohen, of the hepatitis
S.N.: B viral
484
Valenzuela, H.M. coding antigen. Valenzuela, W.J.: 1803/l.
P., Gray,
P., Quiroga,
and Rutter,
W.J.:
for the major Nature
protein
J., Goodman,
sequence
of hepatitis
of the gene
B virus surface
M., Zaldivar,
Scientific
F., Gray,
Memoranda,
Wieslander,
L.: A simple method
in Low gelling temperature
P. and Rutter,
1980,
Memo-H-
to recover intact high molecu-
lar weight RNA and DNA after electrophoretic (1979) 305-309.
280 ( 1979) 8 15-8 19.
P., Quiroga, Hepatitis
M., Zaldivar,
Nucleotide
Communicated
by Z. Hrade%
agarose
separation
gels. Anal. B&hem.
98