Vol. 90, No. 2, 1979 September
BIOCHEMICAL
RESEARCH COMMUNICATIONS
AND BIOPHYSICAL
Pages 405-409
27, 1979
RESTRICTION
CLEAVAGE
MAP
FROM G.
RIOT
Laboratoire
de
Institut
Received
OF
KINETOPLAST
TRYPANCSOMA and
DNA
M.
BARROIS
Pharmacologic
Gustave
MINJCIRCLES
EQUIPERDUM
Molgculaire
Roussy,
94800
Villejuif
June 29,1979
Summary. The cleavage of the kDNA minicircles of Trypanosoma equiperdum by the restriction endonucleases Hinf I, w II, Mbo I, Taq I and -Mbo II revealed that this kDNA is homogeneous in base sequence. This is in contrast with the kDNA of minicircles of the other species of trypanoscmes so far studied. The 10 cleavage sites, obtained with these endonucleases, were ordered and a restriction cleavage map of the minicircles was thus drawn. We have
previously
(kDNA)
network
6 % of
the
circles of and
total
of
23
kb,
are
DNA and (I
kb)
the
cleaved
form
of
core Preliminary
homogeneous
In tion
T.
this
cruzi paper
cleavage
contribute ferent MATERIALS
to species AND
Strain. The Instmasteur. viously described
(8)
form
I
of
other
species
map
of
the
kDNA
the
analysis
of
results
the
represents
3ooO
that
the
contrast
T.
equiperdum.
to
draw
These
relationships
from
the
(Borst us
size
do
not
the
equiperdum
with
trypanosomes
in
minicircles
them
permitted T.
which the
fractionate
in
mini-
molecules
homogeneous
isolate
shown
of
about
supercoiled
are
to
(7)
of
phylogenetic
kDNA
DNA
supercoiled
to
and
which
minicircles
kinetoplast
endonucleases
have sequence
the
about
permits
base
present
of circular
restriction
property
experiments
and
The
the
maxicircles
in
we
of
several
network
(7).
of
by
kDNA
are
50
of
(7).
composed about These
This
minicircles
(5)
is of
(7).
maxicircles
circles
and
interlocked.
minicircles
components
equiperdum
cellular
1 kilobase topologically
the
the
Trypanosoma
selectively
cleave under
of
characterized
miniet
al.)(l).
a restricresults
between
also
the
dif-
trypanosomes.
METHODS kinetoplastic strain The trypanosomes (6).
of were
T. equiperdum fractionated
was frorr
Preparation of kDNA minicircles. Total DNA was extracted nosomes and kDNA form I fractionated in orooidium diiodide dients under conditions already describe; (9). The PDI was isopropanol (3). The core kDNA network form I, exclusively circles was obtained after of maxicircles, -Barn HI cleavage described (7).
obtained blood
rat
from as
the pre-
from the trypa(PDI)-CsCl araextracted with composed of minias previously
0006-291X/79/180405-05$01.00/0 405
Copyright All rights
@ 1979 by Academic Press. Inc. of reproduction in onyform reserved.
Vol. 90, No. 2, 1979
Digestion were obtained conditions
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by restriction endonucleases. from New England Biolabs recommended for each enzyme.
and
The restriction used under
the
endonucleases standard digestion
Gel electrophoresis. The endonuclease digests of kDNA minicircles were analysed after electrophoresis in 2 % agarose slab gels, as previously described (9). The sizes of the fragments were estimated by comparing their mobilities with those of the Hae III fragments of the bacteriophage @ X 174 RF, whose genome is composezf 5375 base pairs (Sanger et al. IO).
1
8
234567
9
10
11
12
13
1342 1078 872 606
118 72
Fig.
1.
Electrophoresis
in
2 % agarose
gel
of
the
fragments
generated
by
cleavage of kDNA minicircles of T. equiperdum by restriction endonucleases : (I) & II, (2) Taq I + & II, (3) Taq I, (4) Taq I + Mbo I, (5) Mbo I, (6) T. cruzi kDN4 fragments obtained by Hae III cleavage, (7)$ X 174 RF DNA fragments obtained by Hae IFcleavage and used as standard for size, (8) Hinf I, (9) Mboy+ Mbo II, (10) Hinf I + Mbo II, (11) l3& II + Mb=, (12) aI1 + Mbo I, (13) Hinf I+ Mbo 1.14) unreacted core Edna minicircles. The Gbers indicate themolecular weight in base pairs of the digest of -Hae III 0 x 174 DNA.
406
14
Vol. 90, No. 2, 1979
Table
I.
BIOCHEMICAL
Molecular dum kDNA triction
AND BIOPHYSICAL
weights (expressed minicircles generated endonucleases.
in
kb) of through
Restriction
Fragments Hinf
Blg
I
1.04
1
II
2
the fragments of T. cleavage by different
equiperres-
endonucleases
Mbo I
1.04
RESEARCH COMMUNICATIONS
Taq
I
Hinf I Elg II
Mbo II
Hinf Mbo
I I
0.71
0.85
0.47
0.69
0.69
0.34
0.20
0.31
0.36
0.35
1.05
1.04
3
0.15
4
0.12 -----
1.05
1.05
Hinf Mbo
Hinf Taq
I II
I
Bgl Taq
I
Bgl Mbo
II I
1.05
II I
Bgl Mbo
II II
Mbo Mbo
I II
Taq Mbo
I I
1
0.38
0.63
0.57
0.70
0.47
0.39
0.34
2
0.31
0.22
0.28
0.35
0.31
0.31
0.27
3
0.15
0.20
0.20
0.15
0.15
0.24
4
0.12
0.12
0.12
0.20
5
0.10 -------
0.09
1.06
1.05
1.05
1.05
1.05
1.06
1.05
RESULTS We have minicircles Hha
I,
Hind
Tat
I,
3
-Mbo
II,
the
other
sizes
different
I,
Ava
II, I
Hind and
the
shown
trarily secondary tion
per
listed
the
digests
double in
cannot
about
the
be 10
f3& Mbo
I
Kpn &l-
the
II,
Since
it
is
gel
-Hinf
enzyme
relatively
cleavage
the
DNA fragments
cleavage
by
pairs.
This I
and
gel
and
been
found
Hinf
407
1/B&
II,
;
the
the the
I
by
double
used
II
site
a arbias diges-
the
geno-
digest
estimated
results i3&
is
is
on
of
draw
II
double
is
results
number
ordered in and
by
I,
The the
Hinf
u
obtained
ccnfirmed
Sal
to of
of
electrophoresis is
I,
Pst
a limited
site
have
III,
significantly.
site
fragment
kDNA
Hae
-Taq I, -Mbo I and 2 and 4 fragments
2,
has
cleavage
sites
third
the II,
straightforward
unique
The
I,
1,
minicircles
The
The
cleave
electrophoresis
each
unique
to
-Eco RI, -Hae Mbo I, Mbo II, ----
1, kDNA
by
1.
of
II/Mb0site (2).
I,
the
observed
base
II,
2).
sizes
2.
B&
respectively
determined
1.
figure
II,
cleave
Table
and
Table in
I
not
molecule
The in
Hpa
I,
5 enzymes,
(figure origin,
presented
I/Mbo --Hinf consist of
included
DNA
endonucleases l3&
in
were 1 and
as
I,
Only
do
reference.
are
me as
I).
molecule
taken
HI,
minicircles
figure
this
@ Hinf
fragments
in sites
of
III,
Xba the
restriction
II,
endonucleases
cleavage map
21
(Alu --
cleave
of
are
used
to
observed
in
(4)
being
Vol. 90, No. 2, 1979
BIOCHEMICAL
2. Restriction
Fig.
Taq
I*
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
cleavage map of kDNA minicircle of T. equiperdum. is cleaved by incubation at 60°C but not at 37%
site
The (111.
DISCUSSION The to
be
kDNA
minicircles
cleavage
(7)
T.
cruzi
kDNA
molecules
(8).
in
base
in
(I).
kinetics
of
nucleotide
circles, unambiguously
having
and
found
problem
might
unique
position
_T.
to
be
quential
the
of
be
kDNA
also
they
of
of
a single
T.
equiperdum
Mbo
II
elucidated, (12).
M.
We do
not
and
A.
sequence
the
genetic
An
approach
of
minicircles.
cleavage
approach in and
for
408
ordered
sites
whether signals
to
this The
on
the
feasible.
useful
Lamarra
be
know
the thus
determination help
in
of the
their
technical
the
taxonomic
trypanosomes.
Gabillot
mini-
maxicircles
ACKNCWLECGMENTS We thank
and class
can
only
possess RN&.
DNA
minicircles
enzymes,
I and
RNA
endonucleases this
these
homogeneous
drawn.
messenger the
the of
are
presence
been
messenger
of
could
the
be yet
with
restriction
Taq
found enzyme
heterogeneity
cleavage
I,
whether
makes
of of
thus not
with
restriction
map
Mbo
can has
determination
restriction evolution
kDNA
synthesis
minicircles
classification
II,
map
of
the the
of
m
of
were
restriction
minicircles
the
sites
transcribed,nor
initiate
equiperdum The
I,
studied
obtained
with
indicate ten
hybridize
are to
Hinf
role
minicircles
necessary
The
kDNA
cleavage
also (7).
a physical
biological been
by
far
by
a sequence
equiperdum
demonstrated
with
so demonstrated kinetics
indicate
T.
reassociation
obtained
The
also
sequence
, as
reassociation
contrast, as
of
Trypanosomatidae
sequence
complex
minicircles In
the
base
The
sequence
the
the
of
heterogeneous
assistance.
se-
Vol.
90,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO.
11. 12.
Borst, P. and Hoeijmakers, J.H.J. (1979) Plasmid 2, 20-40. Gelinas, R.E., Myers, P.A. and Roberts, R.J. (1977) J. Mol. Biol. 114, 169-l 79. Ostrander, D.A. and Gray, H.B. (1973) Biopolymers 12, 1387-1419. Pirrotta, V. (1976) Nucleic Acids Res. 3, 1747-1760. Riou, G., La&me, A., Brack, Ch., Delain, E. and Pautrizel, R. (1971) C. R. Acad. Sci. Paris 273, 2150-2153. Riou, G. and Pautrizel, R. (1977) Biochem. Biophys. Res. Corrrn. 79, 1084-1091. Riou, G. and Saucier, J.M. (1979) J. Cell Biol. 81, July issue Riou, G. and Yot, P. (1975) C. R. Acad. Sci. Paris 280, 2701-2704. Riou, G. and Yot, P. (1977) Biochemistry 16, 2390-2396. Sanger, F., Air, G.M., Barrell, B.G., Brown, N.L., Coulson, A.R., Fiddes, J.C., Hutchison III, C.A., Slocombe, P.M. and Smith, M. (1977) Nature 265, 687-695. Sato, S., Hutchison III, C.A. and Harris, J.I. (1977) Proc. Nat. Acad. Sci. USA 74, 542-546. Simpson, L. and Simpson, A.M. (1978) Cell 14, 169-178.
409
BIOCHEMICAL
RESEARCH COMMUNICATIONS
AND BIOPHYSICAL
Pages 405-409
27, 1979
RESTRICTION
CLEAVAGE
MAP
FROM G.
RIOT
Laboratoire
de
Institut
Received
OF
KINETOPLAST
TRYPANCSOMA and
DNA
M.
BARROIS
Pharmacologic
Gustave
MINJCIRCLES
EQUIPERDUM
Molgculaire
Roussy,
94800
Villejuif
June 29,1979
Summary. The cleavage of the kDNA minicircles of Trypanosoma equiperdum by the restriction endonucleases Hinf I, w II, Mbo I, Taq I and -Mbo II revealed that this kDNA is homogeneous in base sequence. This is in contrast with the kDNA of minicircles of the other species of trypanoscmes so far studied. The 10 cleavage sites, obtained with these endonucleases, were ordered and a restriction cleavage map of the minicircles was thus drawn. We have
previously
(kDNA)
network
6 % of
the
circles of and
total
of
23
kb,
are
DNA and (I
kb)
the
cleaved
form
of
core Preliminary
homogeneous
In tion
T.
this
cruzi paper
cleavage
contribute ferent MATERIALS
to species AND
Strain. The Instmasteur. viously described
(8)
form
I
of
other
species
map
of
the
kDNA
the
analysis
of
results
the
represents
3ooO
that
the
contrast
T.
equiperdum.
to
draw
These
relationships
from
the
(Borst us
size
do
not
the
equiperdum
with
trypanosomes
in
minicircles
them
permitted T.
which the
fractionate
in
mini-
molecules
homogeneous
isolate
shown
of
about
supercoiled
are
to
(7)
of
phylogenetic
kDNA
DNA
supercoiled
to
and
which
minicircles
kinetoplast
endonucleases
have sequence
the
about
permits
base
present
of circular
restriction
property
experiments
and
The
the
maxicircles
in
we
of
several
network
(7).
of
by
kDNA
are
50
of
(7).
composed about These
This
minicircles
(5)
is of
(7).
maxicircles
circles
and
interlocked.
minicircles
components
equiperdum
cellular
1 kilobase topologically
the
the
Trypanosoma
selectively
cleave under
of
characterized
miniet
al.)(l).
a restricresults
between
also
the
dif-
trypanosomes.
METHODS kinetoplastic strain The trypanosomes (6).
of were
T. equiperdum fractionated
was frorr
Preparation of kDNA minicircles. Total DNA was extracted nosomes and kDNA form I fractionated in orooidium diiodide dients under conditions already describe; (9). The PDI was isopropanol (3). The core kDNA network form I, exclusively circles was obtained after of maxicircles, -Barn HI cleavage described (7).
obtained blood
rat
from as
the pre-
from the trypa(PDI)-CsCl araextracted with composed of minias previously
0006-291X/79/180405-05$01.00/0 405
Copyright All rights
@ 1979 by Academic Press. Inc. of reproduction in onyform reserved.
Vol. 90, No. 2, 1979
Digestion were obtained conditions
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by restriction endonucleases. from New England Biolabs recommended for each enzyme.
and
The restriction used under
the
endonucleases standard digestion
Gel electrophoresis. The endonuclease digests of kDNA minicircles were analysed after electrophoresis in 2 % agarose slab gels, as previously described (9). The sizes of the fragments were estimated by comparing their mobilities with those of the Hae III fragments of the bacteriophage @ X 174 RF, whose genome is composezf 5375 base pairs (Sanger et al. IO).
1
8
234567
9
10
11
12
13
1342 1078 872 606
118 72
Fig.
1.
Electrophoresis
in
2 % agarose
gel
of
the
fragments
generated
by
cleavage of kDNA minicircles of T. equiperdum by restriction endonucleases : (I) & II, (2) Taq I + & II, (3) Taq I, (4) Taq I + Mbo I, (5) Mbo I, (6) T. cruzi kDN4 fragments obtained by Hae III cleavage, (7)$ X 174 RF DNA fragments obtained by Hae IFcleavage and used as standard for size, (8) Hinf I, (9) Mboy+ Mbo II, (10) Hinf I + Mbo II, (11) l3& II + Mb=, (12) aI1 + Mbo I, (13) Hinf I+ Mbo 1.14) unreacted core Edna minicircles. The Gbers indicate themolecular weight in base pairs of the digest of -Hae III 0 x 174 DNA.
406
14
Vol. 90, No. 2, 1979
Table
I.
BIOCHEMICAL
Molecular dum kDNA triction
AND BIOPHYSICAL
weights (expressed minicircles generated endonucleases.
in
kb) of through
Restriction
Fragments Hinf
Blg
I
1.04
1
II
2
the fragments of T. cleavage by different
equiperres-
endonucleases
Mbo I
1.04
RESEARCH COMMUNICATIONS
Taq
I
Hinf I Elg II
Mbo II
Hinf Mbo
I I
0.71
0.85
0.47
0.69
0.69
0.34
0.20
0.31
0.36
0.35
1.05
1.04
3
0.15
4
0.12 -----
1.05
1.05
Hinf Mbo
Hinf Taq
I II
I
Bgl Taq
I
Bgl Mbo
II I
1.05
II I
Bgl Mbo
II II
Mbo Mbo
I II
Taq Mbo
I I
1
0.38
0.63
0.57
0.70
0.47
0.39
0.34
2
0.31
0.22
0.28
0.35
0.31
0.31
0.27
3
0.15
0.20
0.20
0.15
0.15
0.24
4
0.12
0.12
0.12
0.20
5
0.10 -------
0.09
1.06
1.05
1.05
1.05
1.05
1.06
1.05
RESULTS We have minicircles Hha
I,
Hind
Tat
I,
3
-Mbo
II,
the
other
sizes
different
I,
Ava
II, I
Hind and
the
shown
trarily secondary tion
per
listed
the
digests
double in
cannot
about
the
be 10
f3& Mbo
I
Kpn &l-
the
II,
Since
it
is
gel
-Hinf
enzyme
relatively
cleavage
the
DNA fragments
cleavage
by
pairs.
This I
and
gel
and
been
found
Hinf
407
1/B&
II,
;
the
the the
I
by
double
used
II
site
a arbias diges-
the
geno-
digest
estimated
results i3&
is
is
on
of
draw
II
double
is
results
number
ordered in and
by
I,
The the
Hinf
u
obtained
ccnfirmed
Sal
to of
of
electrophoresis is
I,
Pst
a limited
site
have
III,
significantly.
site
fragment
kDNA
Hae
-Taq I, -Mbo I and 2 and 4 fragments
2,
has
cleavage
sites
third
the II,
straightforward
unique
The
I,
1,
minicircles
The
The
cleave
electrophoresis
each
unique
to
-Eco RI, -Hae Mbo I, Mbo II, ----
1, kDNA
by
1.
of
II/Mb0site (2).
I,
the
observed
base
II,
2).
sizes
2.
B&
respectively
determined
1.
figure
II,
cleave
Table
and
Table in
I
not
molecule
The in
Hpa
I,
5 enzymes,
(figure origin,
presented
I/Mbo --Hinf consist of
included
DNA
endonucleases l3&
in
were 1 and
as
I,
Only
do
reference.
are
me as
I).
molecule
taken
HI,
minicircles
figure
this
@ Hinf
fragments
in sites
of
III,
Xba the
restriction
II,
endonucleases
cleavage map
21
(Alu --
cleave
of
are
used
to
observed
in
(4)
being
Vol. 90, No. 2, 1979
BIOCHEMICAL
2. Restriction
Fig.
Taq
I*
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
cleavage map of kDNA minicircle of T. equiperdum. is cleaved by incubation at 60°C but not at 37%
site
The (111.
DISCUSSION The to
be
kDNA
minicircles
cleavage
(7)
T.
cruzi
kDNA
molecules
(8).
in
base
in
(I).
kinetics
of
nucleotide
circles, unambiguously
having
and
found
problem
might
unique
position
_T.
to
be
quential
the
of
be
kDNA
also
they
of
of
a single
T.
equiperdum
Mbo
II
elucidated, (12).
M.
We do
not
and
A.
sequence
the
genetic
An
approach
of
minicircles.
cleavage
approach in and
for
408
ordered
sites
whether signals
to
this The
on
the
feasible.
useful
Lamarra
be
know
the thus
determination help
in
of the
their
technical
the
taxonomic
trypanosomes.
Gabillot
mini-
maxicircles
ACKNCWLECGMENTS We thank
and class
can
only
possess RN&.
DNA
minicircles
enzymes,
I and
RNA
endonucleases this
these
homogeneous
drawn.
messenger the
the of
are
presence
been
messenger
of
could
the
be yet
with
restriction
Taq
found enzyme
heterogeneity
cleavage
I,
whether
makes
of of
thus not
with
restriction
map
Mbo
can has
determination
restriction evolution
kDNA
synthesis
minicircles
classification
II,
map
of
the the
of
m
of
were
restriction
minicircles
the
sites
transcribed,nor
initiate
equiperdum The
I,
studied
obtained
with
indicate ten
hybridize
are to
Hinf
role
minicircles
necessary
The
kDNA
cleavage
also (7).
a physical
biological been
by
far
by
a sequence
equiperdum
demonstrated
with
so demonstrated kinetics
indicate
T.
reassociation
obtained
The
also
sequence
, as
reassociation
contrast, as
of
Trypanosomatidae
sequence
complex
minicircles In
the
base
The
sequence
the
the
of
heterogeneous
assistance.
se-
Vol.
90,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO.
11. 12.
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