Volume 14 Number 20 1986
Nucleic Acids Research
Molecular cloning and characterization of small polydisperse circular DNA from mouse 3T6 cells
Per Sunnerhagen, Rose-Marie Sjoberg, Anne-Li Karlsson, Leif Lundh and Gunnar Bjursell
Department of Medical Biochemistry, University of Goteborg, PO Box 33031, £400 33 Goteborg,
Sweden
Received 18 September 1986; Accepted 25 September 1986
ABSTRACT
We h a v e i s o l a t e d ,
cloned
and a n a l y z e d s m a l l
polydisperse
c i r c u l a r ( s p c ) DNA f r o m m o u s e 3 T 6 c e l l s . T h e r e p r e s e n t a t i o n o f
h i g h l y r e p e a t e d m o u s e g e n o m e s e q u e n c e f a m i l i e s i n spcDNA h a s
b e e n e x a m i n e d , a n d t h e B1 r e p e a t a p p e a r s o v e r r e p r e s e n t e d i n
spcDNA by t w o c r i t e r i a . The m a j o r i t y o f spcDNA c l o n e s ,
however,
i s made o u t by a s y e t u n c h a r a c t e r i z e d
middle
repetitive
s e q u e n c e s . We h a v e i n v e s t i g a t e d
t h e i n c r e a s e i n t h e spcDNA
p o p u l a t i o n upon c y c l o h e x i m i d e t r e a t m e n t of i n d i v i d u a l s e q u e n c e s ,
which a r e found t o a m p l i f y
differentially.
IHTRODUCTIOH
In
addition
c e l l s
t o chromosomal
contain
small
extrachromosomal
identified
are present
avian
and insect
a
origin
per cell
such as t r e a t m e n t
cultivation
in
DNA,
cDNA
from
stationary
excluded.
include
excision
reverse
s o u r c e s of spcDNA,
been
that
i s no fixed
chromosomal
of
(8,9).
genomic r e a r r a n g e m e n t s
by p r o d u c t
c i r c u l a r
been
lines
examined
of sequences,
copy
number.
increases
with
from
Since
limited
on
and
and
of
or
circular
homologous
to
circularization
the
replication
formation
as
to
inhibitors
considered
developmental
is
of a v a r i e t y
spcDNA i s
the
spcDNA
t h e number of
synthesis
are
mammalian,
and in contrast
episomal
(3,6,7)
may l e a v e
been
of
Rather,
chromosomes
hypotheses
certain
These
have
(1-5).
as a result
protein
phase.
DNA.
sizes
0 . 0 0 1 t o 0 . 1 % o f t o t a l DNA.
and c e l l
have
misfiring"
In
( m t ) DNA, m a m m a l i a n
varying
transcription
although
Current
"repllcon
transposition
from
population
there
spcDNA m o l e c u l e s
stimuli,
ranging
of
in a l l tissues
heterogeneous
plasmids,
polydisperse
DNA c i r c l e s
i n amounts
They
and mitochondrial
main
has not
of
spcDNA
intermediates
stages,
in
specific
DNA m o l e c u l e s
as a
(5,10).
The main p a r t of work on mammalian spcDNA on t h e m o l e c u l a r
© IRL Press Limited, Oxford, England.
level
7823
Nucleic Acids Research
has
been
BSC-1
carried
individual
that
Schlndler
and
distinct
size
showed
that
discrete
cell
in
primate
There
sequences
indicating
Our
out
(2,9,12).
is
represented
spcDNA
Rush
(12)
middle
molecular
BSC-1
not
lengths
in
species,
1^ r e p e a t s
Paulson
an
or
are
event.
THE 1
from
(3,11)
there
discrete
Kpn
and
element
spcDNA
HeLa
that
a random
that
spcDNA
repetitive
as
spcDNA a s
is
demonstrated
in
such
evidence
in
formation
classes
the
cells
growing
Thus,
show
as
e_t j i ] ^ ( 8 )
hybridizes
established
to
human
line.
previous
electron
possibility
provokes
effect
an
was
work
was
issue,
such
that
increased
demonstrated
done
and
as
with
also
their
study
and
cloned
of
of
cell
several
3T6
more
of
spcDNA.
but
most
We w a n t
to
of
settle
spcDNA from
our
this
spcDNA,
involvement
DNA. As a f i r s t
characterized
This
of
regarding
and p o s s i b l e
the
cells
lines,
questions
Induction
indicated
mammalian
(cellular)
cells.
general
(13)
DNA t o
in
amplification
and
studies
foreign
production
mouse
mechanism
recombination
Isolated,
microscopic
transfer
step
we
m o u s e 3T6
in
have
cells.
MATERIALS AHD METHODS
Cel 1 c u l t u r e .
Eagle's
penicillin
p e r ml
Purification
time
of
h
of
3
purified
by
cccDNA
bromide
50
alkaline
was
calf
density
added
to
on
M o u s e 3T6 c e l l s ,
to
a 5% a l i q u o t
all
was
10^ a t
Fractions
DNA w e r e
dissolved
in
the
to
the
as
final
DNA
was
described
(4),
CsC1-ethidium
from
pooled,
TE.
cells.
a
Circular
successive
gradients.
of
added
cells.
three
circular
and
modified
100 U of
t o n e a r c o n f l u e n c e . A t 24 h b e f o r e
denaturation essentially
purified
serum,
streptomycin per ml.
cyc1oheximide
ug/ml
precipitated
The
the
third
destained,
purity
of
the
p r e p a r a t i o n w a s e x a m i n e d by e l e c t r o n m i c r o s c o p y .
Electron
droplet
microscopy.
Visualization
t e c h n i q u e and m e a s u r e m e n t of
(16295 base
described
7824
was
containing
ethanol
mtDNA
of
10% f e t a l
cloning.
were grown
harvest,
(EtBr)
gradient
final
were grown i n D u l b e c c o ' s
100 ug of
H-thymidine
before
concentration
but
and
cccDNA f o r
harvest,
harvest
16
M o u s e 3T6 c e l l s
medium s u p p l e m e n t e d w i t h
earlier
pairs
(15).
<14>)
as
a
of
DNA
with
an
aqueous
contour
l e n g t h s u s i n g mouse
length
standard
have
been
Nucleic Acids Research
Construction
of
spcDNA
Aliquots,
50 ng e a c h ,
separately
with
DNA f r a g m e n t s
llgated
and
separation
on
of
Hanahan
MtDNA
clones
1 through
s i x - c u t t e r
preparation
digested
with
with
Xho ^ .
were
screening
into
A 100
with
ng
Kpn
no. 2 with
by
ligation
colonies
pooled,
methylation.
were
mtDNA
pUC 9 ,
as
were
Bam H_I
probes.
of
the
I_ a n d
plus
the
hybridization
The f i r s t
Hae I I
by
containinlng
cccDNA
Sjil^
I_ a n d
aliquot
P s t I., n o . 3 w i t h
were
to
Xma
was
clones
14) a s
portion
aliquots.
ng)
resistance
mouse
I_,
linkers
pUC 9 ( 1 6 ) .
by c o l o n y
unpubl.,
of
(10
cleaved
cloned
S a c I_, n o . 5 w i t h
protected
of
of
six
The a l i q u o t s
I-linkers,
portion
on p l a t e s
eliminated
digested
£ _ l £ I,
Hae 11 p l u s
cleavage
recombinant
4 (G. B J u r s e l l ,
divided
with
and
inserts
l i b r a r y .
was
thereafter
Ps t
were
the gel-purified
fragments
The
and
to a m p i c i l l l n
(17),
of
digested
TaqI).
a
Lac Z phenotype
library.
were
T 4 DNA p o l y m e r a s e
After
CL-6B,
transformed
by s c o r i n g
sites
end with
Sepharose
identified
The
blunt
Bam H I - , P s t _ I - d o u b l e
procedure
Xgal.
The f o u r - c u t t e r
preparation
o r T t h HJ3 JU ( a n i s o s c h l z o m e r
made
t o 25 ng of
C J D ] ^ RR1 w e r e
using
1lbrarles.
t h e cccDNA
t o Bam H I - a n d P s t l - l I n k e r s .
ligated
E.
Mspl
were
clone
of
was
Bj5l_ _II^, n o . 4
Sph I,
and no. 6
a n d Bam H I - a n d P s t _IAddition
of
Bam H I - a n d
transformation
described
for
of
the
RR1 a n d
four-cutter
library.
Radlolabellng
of
t r a n s l a t i o n
electrophoresis
total
cccDNA
Southern
by
labeled
Total
of
in Tris-borate
transferred
S . l k k —
saturation
density.
acid
treatment
(22)
After
fractions
were
,w e r e
of
DNA
containing
3
gel
3 T 6 DNA o r
essentially
out in
or
g r o w n
with
described
was c e n t r i f u g e d
fractionation,
nick-
by
(19).
1%
treatment,
control
8
1 0
as
by
as
agarose
t h e DNA w a s
filters.
o f DNA f r o m
Labeling
supernatants
gradients.
was c a r r i e d
After
c e l l s ,
cycloheximide
total
"oligo-1abeling"
(21) to n i t r o c e l l u l o s e
3Tei
labeled
purified
3 T 6 DNA w a s p r e p a r e d
buffer.
^^c^t b l o t t i n g
were
clones,
electro-elution,
using
(20). Electrophoresis
Gradlent
and
plasmids
I n s e r t s
followed
were
blotting.
described
gels
DNA. W h o l e
(18).
cycloheximidet o a b o u t
3
H-thymidlne
above.
DNA
to e q u i l i b r i u m
H-radloactivity
circular
DNA
In
was
were
from
h a l f
and
Hirt
CsCl-EtBr
determined
pooled
and
7825
Nucleic Acids Research
33
30
25
za
13
Innn n n
nn
n n
B
!ii!
! i
I'
i
i
n
?0
ES
GO
55
30
43
«0
35
30
25
20
13
10
5
n
iiii
c
II
iiK
|
h1
1n]
kb
7826
Nucleic Acids Research
recentrifuged.
The second
fractions
were
stored
treated,
denatured
gradient
i n t h e dark
was a l s o
until
and a p p l i e d
f r a c t i o n a t e d , and
use.Aliquots
to n i t r o c e l l u l o s e
were
acid-
filters
as
described (4).
Hybridization.
DNA, s l o t
from
colonies,
bacterial
filters,
DNA
was h y b r i d i z e d
probes
of
representing
provided
conserved"
of
(EC) s e q u e n c e
R1/R2,
Rotival
the
was g i v e n
mouse
I n s e r t s
of
electrophoresis.
was
provided
triple
recovered
from
above
A mouse
together
fragment
satellite
were
this
satellite
(23) was
288,
gift
sequences
D r . M. M e u n i e r -
c o m p r i s i n g most of
(28).
purified
containing
After
Mm 3 1
a kind
of
(MIF)r e p e a t
The
by g e l
plasmid,
C l o n e 16
T a g _ I - , H h a 1^- a n d E c o R1 -
and g e l e l e c t r o p h o r e s i s ,
17 m o u s e
were
of t h e R group
clones
b y D r . W. H d r z .
digestion
encompassing
Plasmld
and t h e " e v o l u t i o n a r i l y
respectively,
pMRB1-1 a n d pMRB5,
t h e
DjJA^
t o u s b y D r H. Z a c h a u .
interspersed
or
on n i t r o c e l l u l o s e
pMR 1 4 2 a n d pMR
(24,25)
(24,26),
gels
o f t h e mouse genome
Arepresentative
furnished
long
mous£
Plasmids
t h e B2 r e p e t i t i o n
agarose
as described (13).
family
Fort.
from
immobilization
repeated
t h e B1 r e p e a t
D r . N. H a s t i e .
(27),
after
blotted
and washed
h^ghl^y
by D r . P.
representing
blotted,
a T a g _I
monomer r e p e a t s
fragment
in tandem was
plasmid.
RESULTS
Preparation
than
o_f cccDNA.
99% o f
corresponding
after
3
the
treatment
In
(29), t h i s
CsCl-EtBr
DNA. S i n c e
labeling
o r spcDNA,
cccDNA. E x t e n s i v e
the t h i r d
H-rad 1 o a c t i v i t y
to c i r c u l a r
H-thymidine
mitochondrial
3
figure
electron
before
is a valid
in
the s p e c i f i c
is identical
both
gradient,
was f o u n d
estimate
better
peak
radioactivity
in e i t h e r
and a f t e r
the
chromosomal,
eye 1 o h e x l m l d e
of t h e p u r i t y of
microscopic examination
r e v e a l s mtDNA
F i g . 1.
Length d i s t r i b u t i o n s ,
o b t a i n e d by e l e c t r o n microscopy,
of
c i r c u l a r and l i n e a r DNA in the cccDNA p r e p a r a t i o n used for
c l o n i n g before and a f t e r c l e a v a g e with r e s t r i c t i o n enzymes.
Solid l i n e , c i r c u l a r DNA; broken l i n e , l i n e a r DNA.
A, n a t i v e preparation; n = 268
B, f o u r - c u t t e r cleaved; n = 254
C, s i x - c u t t e r cleaved; n = 498
7827
Nucleic Acids Research
m o l e c u l e s , spcDNA m o l e c u l e s of v a r i o u s s i z e s , and a few l i n e a r
m o l e c u l e s . F i g . 1 A d i s p l a y s t h e s i z e d i s t r i b u t i o n of c i r c u l a r
DNA i n t h e cccDNA p r e p a r a t i o n . The n u m b e r r a t i o of spcDNA t o
mtDNA m o l e c u l e s i s 7 to 1, which e q u a l s a 7 0 - f o l d a m p l i f i c a t i o n
of spcDNA compared t o 3T6 c e l l s n o t t r e a t e d w i t h c y c l o h e x i m l d e
( 1 3 ) . The l o w a m o u n t of l i n e a r DNA, 1.5 %, i s i n a g r e e m e n t w i t h
t h e 3 H p r o f i l e of t h e g r a d i e n t . I n c l o s e a g r e e m e n t w i t h o u r
e a r l i e r r e s u l t s f o r 3T6 c e l l s n o t t r e a t e d w i t h c y c 1 o h e x i m i d e
( 1 3 ) , a mean s i z e of 2.04 k i l o b a s e p a i r s (kb) i s f o u n d f o r
spcDNA. T h u s , i n c o n t r a s t t o t h e s i t u a t i o n i n D r o s o p h l 1 a ( 3 0 ) ,
t h e s i z e of spcDNA d o e s n o t d e c r e a s e u p o n c y c 1 o h e x i m i d e
amplification.
spcDNA c l o n e 1 i b r a r l e s . As t h e c l o n i n g p r o t o c o l p e r m i t s c l o n i n g
of a l l c o n t a m i n a t i n g l i n e a r DNA m o l e c u l e s , we found i t n e c e s s a r y
t o e s t i m a t e t h e p r o p o r t i o n of c i r c u l a r DNA r e p r e s e n t e d i n t h e
c l o n e b a n k s . T h i s c a n be d o n e by c o m p a r i n g t h e p r o p o r t i o n s of
c i r c u l a r v s . l i n e a r DNA in t h e n a t i v e and f o u r - c u t t e r and s i x c u t t e r d i g e s t e d cccDNA p r e p a r a t i o n ( F i g . 1 ) . I t I s n o t e w o r t h y
t h a t s h o r t l i n e a r m o l e c u l e s ( l e s s t h a n a p p r o x . 200 b a s e p a i r s
<bp>) a r e i n d i s t i n g u i s h a b l e from t h e c y t o c h r o m e c b a c k g r o u n d .
Hence, no c o n c l u s i o n s can be drawn a b o u t t h e s i z e d i s t r i b u t i o n
of t h e r e s u l t i n g
clone I n s e r t s ,
which in f a c t
have a
c o n s i d e r a b l y s h o r t e r mean l e n g t h t h a n s u g g e s t e d from F i g . 1 B
and C ( s e e T a b l e 1 ) . I t i s s e e n , h o w e v e r , t h a t i n d e e d t h e
m a j o r i t y of spcDNA m o l e c u l e s h a v e been l i n e a r i z e d , a t l e a s t 83%
a f t e r f o u r - c u t t e r d i g e s t i o n and a t l e a s t 18% a f t e r s i x - c u t t e r
digestion.
A f t e r e l i m i n a t i o n of mtDNA c l o n e s ,
2 3 0 a n d 4 7 0 spcDNA
r e c o m b i n a n t c l o n e s r e m a i n i n t h e f o u r - c u t t e r and s i x - c u t t e r
library,
r e s p e c t i v e l y . The p r o p e r t i e s of c h a r a c t e r i z e d c l o n e s
a r e summarized In T a b l e 1. The i n s e r t s of t h e f o u r - c u t t e r c l o n e s
h a v e a mean s i z e o f a p p r o x i m a t e l y 250 b p , and i n s e r t s up t o 2 kb
h a v e been f o u n d . In the s i x - c u t t e r l i b r a r y , i n s e r t s i z e s r a n g e
from 200 bp t o 3 k b , w i t h a mean a r o u n d 750 b p .
R e p r e s e n t a t i o n of h i g h 1y r e pea t e d s e q u e n c e f a m i l i e s i n spcDNA
c l o n e l i b r a r i e s . T a b l e 2 3 hows t h e n u m b e r o f c l o n e s i n t h e two
spcDNA l i b r a r i e s t h a t a r e p o s i t i v e i n c o l o n y h y b r i d i z a t i o n w i t h
h i g h s t r i n g e n c y w a s h i n g ( s e e M a t e r i a l s and Methods) f o r h i g h l y
7828
Nucleic Acids Research
Table 1 . Properties of individual spcDNA clones.
Hybridization Stronger hybHomology
Clone (a) Insert
size (kb ) with highly
pattern in
ridization
repeated DNA 3T6 DNA (b)
with cccDNA
Four-cutter library
0.3
pSPC-5
IS
0.4
-8
B1
IS
2.0
-20
B
+
0.2
B1
-29
0. 1
-31
MIF
0.5
IS
+
-47
1 .0
B1
-49
0.2
-50
IS
0.6
-52
IS
+
0.2
-53
B1
0.4
-83
R
0.4
_
-86
IS
+
0.3
_
-100
IS
-102
0.5
IS + B
0.3
IS
-127
2.0
IS+B
+
-145
0.2
IS
-165
0.2
-170
IS
-174
0.3
IS
-201
0.5
IS
Six-cutter library
IS
+
-238
1 .0
0.8
-243
IS
0.4
-249
R
0.2
_
-291
IS
-292
1 .3
B2
-371
0.7
IS
-372
0.3
B1
1 .0
-389
IS
+
IS + B
-420
0.4
-492
0.3
IS
-518
0.5
IS
0.2
MIF
-537
0.2
IS
-547
-580
_
1 .5
IS
+
0.4
R
-585
IS
-612
R
0.5
-621
0.8
B1
0.6
-627
IS
+
IS + B
-631
1 .2
-641
0.2
IS
+
-644
1 .0
IS
-648
MIF
1 .5
0.4
-669
IS
MIF
1 .7
-679
0.6
-688
IS
+
(a) C l o n e s of which only insert size and lack of homology to
highly repeated DNA or only homology to highly repeated DNA are
known are not included in this table.
(b) B = one or several discrete bands
IS rhybridizati-on smear c h a r a c t e r i s t i c of interspersed
sequences
7829
Nucleic Acids Research
Table 2.
Representation of highly r e p e t i t i v e mouse DNA sequences in the
spcDNA clone l i b r a r i e s .
Fraction of
Repeat
No. of posi tive clones in spcDNA libraries
chromosomal
element
Four-cutter Six-cutter
Total
%
DNA (24,31)
library
library
B1
10
25
35
5.0
0.9-1.3%
B2
-
2
2
0.3
0.5-0.8%
EC
1
5
6
0.8
0.5-0.8%
R
2
8
10
1. 4
1.0-1.5%
MIF
4
1 0
14
2.0
4.0-5.0%
Satellite
-
-
repetitive
DNA. When s t r i n g e n c y
hybridization
were
and w a s h i n g
obtained
When
(data
calculating
the f o l l o w i n g
(1)
When
the expected
the cloned
elements,
5-1 0%
by l o w e r i n g
t o 48°C,
number
identical
identical
fragments
frequency
the c l o n e d
fragments
elements,
the f r a c t i o n
of p o s i t i v e
to that
into
the
results
colonies,
of c h r o m o s o m a l DNA,
consideration:
are considerably
the proportion
the r e p e a t
equal
was r e d u c e d
temperature
h a s t o be t a k e n
the r e p e t i t i o n
-
n o t shown).
assuming a d i s t r i b u t i o n
repeated
-
of p o s i t i v e
longer
clones
than the
d e p e n d s on
of t h e r e p e a t e l e m e n t s . By c o n t r a s t ,
are shorter
t h a n o r n o t much l o n g e r
the proportion
of p o s i t i v e
clones
if
than
should
o f t h e genome c o n s t i t u t e d by a c e r t a i n
repeat
element.
(il)
The d i s t r i b u t i o n
elements.
arranged
than
<24>)
dispersed
to being
total
dispersed
used
sites
i s stronger
flanking
elements.
short
sequences
t h e enzymes
The f i v e
7830
since
and dispersed,
prototype
in
dependence
elements,
for short
addition
for
This
of restriction
sites
in the repeated
f o r long
will
or
tandemly
be f a r t h e r
( 2 0 0 b p f o r B1 a n d B 2 , 4 0 0 - 5 0 0
a l l contain
contained
apart
T h e B1 , B2 a n d R e l e m e n t s ,
bp f o r R
In t h e i r
published
i n t h e EMBL d a t a b a s e
(23,25,27)
for cloning
sites
in
a t a density
comparable
to that
m o u s e DNA.
sequence
sequences
families
repeated
B 1 , B 2 , R, M I F a n d EC r e p r e s e n t a l l
more
than
30 000 t i m e s
per haplold
Nucleic Acids Research
3.7 -
II
**••
0.7 -
w
Fig. 2.
S o u t h e r n b l o t s of c h r o m o s o m a l 3T6 DNA probed w i t h i n d i v i d u a l
spcDNA clones. In each lane is shown to the left Bam H1- cleaved
and to the right Eco RJ_-cl e a v e d 3T6 DNA. Lanes 1 - 5~j~ random ly
chosen spcDNA c l o n e s . 1, p S P C - 6 3 1 ; 2, p S P C - 5 0 ; 3, p S P C - 4 2 0 ; 4,
pSPC-243; 5, pSPC-100. Lanes 6 - 10, spcDNA clones identified by
d i f f e r e n t i a l h y b r i d i z a t i o n . 6, p S P C - 5 8 0 ; 7, p S P C - 3 8 9 ; 8, pSPC20; 9, p S P C - 4 7 ; 10, p S P C - 8 6 .
genome
(24). The o n l y s i g n i f i c a n t d e v i a t i o n
number
of
colonies
overrepresented
is
for
B1,
element
MIF
sites
near
repeat
was
sufficient
the expected
has
not
been
to permit
The long
in
the average density of sites. A slight
<24>)
although
(31).
sequence
a minor
The
precludes
be drawn
sequences
tandem
cloning
from
in
Chromosomal
(31) contains
no sites
underrepresentation
clones. The
for
fraction of satellite DNA
arrangement
in flanking
these data about
of
most
sites, and
but
deviation
absence
of s a t e l l i t e DNA from the c l o n e banks was e x p e c t e d , since
prototype
in
this
length,
parts reveals no drastic
is found in both libraries for MIF-positive
of
(up to 7 kb
its e n t i r e
(26)
cloning
representation
frequency.
is
sequence
used. A p p a r e n t l y
sequenced
examination of the sequenced
from
apparently
about 5-fold. The EC p r o t o t y p e
c o n t a i n no s i t e s for the e n z y m e s
flanking
from the expected
which
the enzymes
is cleaved
satellite
by Tag 1^
sequences
so no conclusions
the representation
of
the
used,
can
satellite
spcDNA.
organization of spcDNA clones. Fig. 2 shows Southern
7831
Nucleic Acids Research
1
i iilMllilli 1
11
2
tI I I I I I lilt I
Iff
3
4
I
III
5
Fig.
3.
H y b r i d i z a t i o n o f c l o n e s o f h i g h l y r e p e a t e d DNA a n d o f mtDNA t o
CsCl-EtBr
gradient
slot
blots
of
DNA f r o m
normal
and
cycloheximide-treated
3T6 c e l l s ,
prepared
as d e s c r i b e d
in
M a t e r i a l s and M e t h o d s . The f r a c t i o n c o r r e s p o n d i n g t o t h e l o w e r
p a r t of t h e t u b e i s o r i e n t e d t o t h e l e f t . A f t e r
immobilization
on n i t r o c e l l u l o s e ,
t h e DNA w a s h y b r i d i z e d w i t h t h e
purified
i n s e r t s o f t h e f o l l o w i n g p r o b e s : Rows 1 a n d 6 , mtDNA; 2 a n d 7 ,
Mm31 ( B 1 ) ; 3 a n d 8 , p M R B 1 - 1 ( M I F ) ; 4 a n d 9 , R 1 / R 2 ( R ) ; 5 a n d 1 0 ,
c l o n e 16 ( s a t e l l i t e ) . R o w s 1 - 5 , s l o t b l o t s o f o n e g r a d i e n t
from
untreated
c e l l s ;
6 - 10,
of
one
gradient
from
cycloheximide-treated cells.
A, n o r m a l c e l l s . T h e a u t o r a d i o g r a m s w e r e s c a n n e d w i t h an o p t i c a l
densitometer.
Hybridization
intensities
were c o n v e r t e d
to
a b s o l u t e a m o u n t s o f DNA u s i n g c a l i b r a t i o n c u r v e s
p r e p a r e d by
a p p l y i n g t o t a l 3 T 6 DNA, s o n i c a t e d , d i l u t e d i n 4 7 . 8 % ( w / w ) o f
C s C l a n d 0.4 m g / m l o f E t B r , a n d t h e r e a f t e r t r e a t e d a s a b o v e , i n
a m o u n t s r a n g i n g from 3 pg t o 1 ug and
h y b r i d i z e d w i t h t h e same
p r o b e . The c u r v e s shown w e r e n o r m a l i z e d s u c h t h a t t h e sums of
t h e DNA a m o u n t s o f t h e f r a c t i o n s f r o m t h e p e a k o f l i n e a r DNA
( f r a c t i o n s 1 4 - 1 6 f r o m t h e l e f t ) w e r e s e t e q u a l . R e l a t i v e DNA
a m o u n t s a r e g i v e n o n t h e Y - a x i s . F o r mtDNA, n o r m a l i z a t i o n w a s
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Nucleic Acids Research
i n s t e a d d o n e s u c h t h a t t h e sums of t h e DNA a m o u n t s from t h e
c i r c u l a r peak ( f r a c t i o n s 6-8 from t h e l e f t ) were e q u a l i n A and
B. B, a s i n A b u t c e l l s t r e a t e d w i t h c y c l o h e x i m i d e .
b l o t s where i n d i v i d u a l spcDNA c l o n e s were h y b r i d i z e d t o t o t a l
3T6 DNA, d i g e s t e d w i t h Bam HI o r Eco R1. Out o f 20 r a n d o m l y
c h o s e n spcDNA c l o n e s , of w h i c h o n l y two h y b r i d i z e d w i t h any of
t h e h i g h l y r e p e a t e d p r o b e s (one f o r B1, one f o r R), a l l p r o d u c e d
the h y b r i d i z a t i o n
smear c h a r a c t e r i s t i c
of
interspersed
r e p e t i t i v e s e q u e n c e s . I n some c a s e s , b a n d s of d i s c r e t e s i z e s
were v i s i b l e o v e r t h e smear. T h i s
p r e d o m i n a n c e of d i s p e r s e d
r e p e t i t i v e s e q u e n c e s was u n e x p e c t e d s i n c e r e p e t i t i v e ,
nons a t e l l i t e DNA makes o u t o n l y 16 - 24 % of t h e mouse genome ( 2 4 ) .
Of t h i s , t h e f i v e p r e v i o u s l y i d e n t i f i e d h i g h l y r e p e a t e d s e q u e n c e
f a m i l i e s c o n s t i t u t e 8 % of t o t a l DNA ( 2 4 ) , l e a v i n g 8 - 16 % f o r
m i d d l e r e p e t i t i v e s e q u e n c e s . S i n c e a s i m i l a r p r o p o r t i o n (9 %) of
h i g h l y r e p e a t e d DNA i s f o u n d i n o u r spcDNA l i b r a r i e s , on t h e
o r d e r of 90 % of o u r spcDNA c l o n e s r e p r e s e n t d i s p e r s e d , m i d d l e
r e p e t i t i v e s e q u e n c e s . By c o m p a r i n g h y b r i d i z a t i o n s w h e r e t h e
i n s e r t s a r e of s i m i l a r l e n g t h , and where l a b e l i n g of p r o b e s has
been e q u a l l y e f f i c i e n t
( b a s e d on t h e h y b r i d i z a t i o n t o a
s t a n d a r d i z e d a m o u n t of p l a s m l d DNA) we c o n c l u d e t h a t
the
m a j o r i t y of o u r i n t e r s p e r s e d spcDNA c l o n e s h y b r i d i z e
less
i n t e n s e l y w i t h 3T6 DNA t h a n do t h e c l o n e s of h i g h l y r e p e t i t i v e
DNA.
R e p r e s e n t a t i o n of i n d i v i d u a l s e q u e n c e s j^n spcDNA from u n t r e a t e d
and c y c l o h e x l m l d e - t r e a t e d e e l I s .
By p r o b i n g d u p l i c a t e
filters
w i t h c o l o n i e s from t h e c l o n e banks w i t h e i t h e r t o t a l chromosomal
3T6 DNA or t o t a l 3T6 cccDNA, c l o n e s were i d e n t i f i e d t h a t r e a c t e d
somewhat s t r o n g e r w i t h cccDNA t h a n w i t h chromosomal DNA, which
would s u g g e s t an o v e r r e p r e s e n t a t i o n i n spcDNA. In t h i s g r o u p , no
c l o n e s h y b r i d i z e d w i t h any of t h e h i g h l y r e p e a t e d p r o b e s . T w e l v e
s u c h c l o n e s w e r e u s e d a s p r o b e s a g a i n s t S o u t h e r n b l o t s of
c h r o m o s o m a l DNA ( F i g . 2 , l a n e s 6 - 1 0 ) . J u s t a s w i t h t h e
r a n d o m l y c h o s e n c l o n e s , a l l showed a d i s p e r s e d p a t t e r n of
h y b r i d i z a t i o n w i t h one e x c e p t i o n , pSPC-20, which l a b e l e d a s e t
of d i s t i n c t b a n d s , r e p e a t e d on t h e o r d e r of 10 - 100 t i m e s i n
t h e genome ( F i g . 2 , l a n e 8 ) .
In F i g . 3 , i n d i v i d u a l c l o n e s of h i g h l y r e p e a t e d DNA h a v e b e e n
7833
Nucleic Acids Research
h y b r i d i z e d t o C s C l / E t B r g r a d i e n t s of p a r t i a l l y p u r i f i e d cccDNA
from
u n t r e a t e d or
cycloheximide-treated c e l l s .
Approximately
90 % of t h e
H - a c t i v i t y was f o u n d i n t h e l i n e a r p e a k i n t h e s e
g r a d i e n t s . The p o s i t i o n of .the c i r c u l a r peak can be v e r i f i e d by
h y b r i d i z i n g t o mtDNA ( F i g . 3 , rows 1 and 6 ) . H y b r i d i z a t i o n by
p r o b e s o t h e r t h a n mtDNA t o t h e c i r c u l a r p e a k
consistently
occured a t a s l i g h t l y h i g h e r p o s i t i o n in the g r a d i e n t . This i s
in a g r e e m e n t w i t h t h e o r i g i n a l o b s e r v a t i o n of Smith and V i n o g r a d
( 2 9 ) , who a t t r i b u t e d i t t o a h i g h e r s u p e r h e l i c a l d e n s i t y i n
.spcDNA t h a n i n mtDNA. Of c o u r s e , i t i s e s s e n t i a l t o t h i s t y p e of
a s s a y t h a t no h o m o l o g i e s e x i s t b e t w e e n mtDNA and t h e p r o b e s
u s e d . T h i s was v e r i f i e d by c o m p a r i n g t h e s e q u e n c e s of mtDNA and
of t h e h i g h l y r e p e a t e d p r o b e s ( t h e s e q u e n c e d p a r t s i n t h e c a s e
of MIF) u s i n g t h e GENEUS (32) Dot M a t r i x p r o g r a m .
The p r o b e c o n s i s t e n t l y y i e l d i n g t h e most d i s t i n c t c i r c u l a r peak
i s B1 ( F i g . 3 A and B, r o w s 2 and 7 ) . T h i s p e a k i s f o u n d b o t h i n
u n t r e a t e d and c y c l o h e x i m i d e - t r e a t e d c e l l s , and t h e i n c r e a s e upon
c y c l o h e x i m i d e t r e a t m e n t i s o n l y moderate (about t w o - f o l d in t h i s
a s s a y ) . A l l o t h e r h i g h l y r e p e a t e d p r o b e s (B2, EC, R, MIF and
s a t e l l i t e ) give comparable r e s u l t s with very l i t t l e m a t e r i a l
h y b r i d i z i n g b e l o w t h e l i n e a r peak i n u n t r e a t e d c e l l s ( F i g . 3 A,
rows 3 - 5 ,
and d a t a n o t shown). However, a f t e r c y c l o h e x i m i d e
t r e a t m e n t a m a s s i v e i n c r e a s e of h y b r i d i z i n g c i r c u l a r DNA i s s e e n
w i t h t h e s e p r o b e s ( F i g . 3 B, rows 8 - 10). T h i s a m p l i f i c a t i o n i s
p a r t i c u l a r l y prominent
( f i v e - to t e n f o l d in t h i s assay) with
MIF and R, w h i c h i n m o s t c a s e s a r e f o u n d a d j a c e n t i n t h e
chromosome (33).
DISCUSSIOM
When c o n s t r u c t i n g c l o n e l i b r a r i e s of spcDNA,
between two major i n t e r e s t s :
there is a
conflict
i ) t o c l o n e i n t a c t c i r c l e s , and
11) t o o b t a i n a l i b r a r y t h a t i s u n b i a s e d w i t h r e s p e c t
to
s e q u e n c e . The s t r a t e g y i n t h i s work h a s been t o u s e r e s t r i c t i o n
enzymes
that
cleave
c o m b i n a t i o n s of s e v e r a l
DNA
frequently
(four-cutters )
enzymes ( s i x - c u t t e r s )
in order to
w i t h i n an a s w i d e a s p o s s i b l e v a r i e t y of s e q u e n c e s . T h i s
h a s b e e n a c h i e v e d s i n c e t h e m a j o r i t y o f spcDNA h a s
7834
or
cut
goal
been
Nucleic Acids Research
l i n e a r i z e d . T h e r e i s t h u s r e a s o n t o b e l i e v e t h a t o u r spcDNA
l i b r a r i e s r e p r e s e n t a l a r g e f r a c t i o n of t h e t o t a l
sequence
c o m p l e x i t y i n t h e spcDNA p o p u l a t i o n .
A c o m p a r a t i v e l y l a r g e number of spcDNA c l o n e s h a v e been examined
f o r t h e p r e s e n c e of t h e s i x m o s t h i g h l y r e p e a t e d
sequence
e l e m e n t s , p e r m i t t i n g an e v a l u a t i o n of t h e d i s t r i b u t i o n
between
h i g h l y r e p e a t e d e l e m e n t s . I t a p p e a r s t h a t t h e B1 r e p e a t i s
o v e r r e p r e s e n t e d i n spcDNA i n 3T6 c e l l s r e l a t i v e t o t h e r e s t of
h i g h l y r e p e a t e d f a m i l i e s , b o t h from t h e n u m b e r of p o s i t i v e
c l o n e s i n t h e l i b r a r i e s and from h y b r i d i z a t i o n s t o C s C l - E t B r
g r a d i e n t s . In t h i s c o n t e x t i t i s i n t e r e s t i n g to n o t e t h a t t h e r e
e x i s t s e q u e n c e h o m o l o g l e s b e t w e e n B1 and t h e p a p o v a v i r u s o r i g i n
of r e p l i c a t i o n ( 3 4 ) . A r l g a (35) d e m o n s t r a t e d
autonomous
r e p l i c a t i o n of a c l o n e d Alu e l e m e n t , c o n s i d e r e d t o c o r r e s p o n d t o
B1 i n p r i m a t e s , i n e x t r a c t s from C o s I c e l l s . T h i s d o e s n o t
n e c e s s a r i l y i m p l y t h a t A l u - or B 1 - c o n t a i n i n g spcDNA m o l e c u l e s
r e p l i c a t e as e p i s o m e s iji v i v o , a l t h o u g h s t r u c t u r e s r e s e m b l i n g
r e p l i c a t i v e i n t e r m e d i a t e s of c i r c u l a r m o l e c u l e s h a v e b e e n
i d e n t i f i e d i n t h e e l e c t r o n m i c r o s c o p e by u s and o t h e r s ( 3 6 ) .
R a t h e r , B1- c o n t a i n i n g c i r c u l a r m o l e c u l e s can h a v e a r i s e n as a
r e s u l t of " m i s f i r i n g " from
chromosomal o r i g i n s of r e p l i c a t i o n
( 6 ) , s i n c e l a b e l i n g d a t a p o i n t a t e x c i s i o n from c h r o m o s o m e s a s
t h e main mechanism of spcDNA f o r m a t i o n ( 2 9 ) .
I n t h e o n l y p r e v i o u s l y p u b l i s h e d c l o n i n g s t u d y o f spcDNA from
mouse c e l l s , F u j i m o t o £_t a l . (5) a r g u e f o r t h e R r e p e a t t o be
p r e f e r e n t i a l l y i n v o l v e d i n spcDNA f o r m a t i o n i n mouse t h y m o c y t e s .
T h i s may be a c o n s e q u e n c e of t h e s p e c i f i c genomic r e a r r a n g e m e n t s
in t h y m o c y t e s s i n c e t h e i m m u n o g l o b u l i n g e n e s a r e a s s o c i a t e d w i t h
R r e p e a t s . We f i n d no a p p a r e n t d e v i a t i o n from t h e e x p e c t e d
a m o u n t of R r e p e a t s i n m o u s e 3T6 spcDNA. J o n e s a n d P o t t e r ( 1 1 )
screened for sequences o v e r r e p r e s e n t e d in c y c 1 o h e x i m i d e a m p l i f i e d HeLa spcDNA and found s e q u e n c e s r e p r e s e n t i n g a m i n o r ,
tandemly repeated,
c o m p o n e n t o f s a t e l l i t e DNA. S e v e r a l
e x p l a n a t i o n s c a n be g i v e n f o r t h i s r e s u l t ,
apparently
in
c o n f l i c t w i t h t h o s e of us and o t h e r s ( 3 7 ) . F i r s t , t h e r e a s o n may
r e s i d e in the d i f f e r e n c e s in c e l l type or s u b c e l l u l a r l o c a t i o n
of spcDNA ( J o n e s and P o t t e r i s o l a t e d spcDNA from t h e c y t o p l a s m
of HeLa c e l l s ) . S e c o n d , t h e i r u s e of c o l d c o m p e t i t o r Alu DNA in
7835
Nucleic Acids Research
differential
hybridization
precluded
representation
o f A l u i n spcDNA.
Overabundance
of r e p e t i t i v e ,
sequences
paper).
be
The c i r c u l a r
form
an i n t e r m e d i a t e
genome.
of
a
mainly
i n spcDNA h a s b e e n
When
highly
repeated
found
interspersed,
in the process
clones
with
repeated
sequence
repetitive
of t h e i r
long
sequences.
(2-4,
dispersal
in the
the presence
overshadows
By v i r t u e
this
e l e m e n t s may
inserts,
element
on t h e
v s . unique
by u s a n d o t h e r s
of d i s p e r s e d
analyzing
and unique
any conclusions
middle
of t h e s h o r t
mean
l e n g t h o f o u r c l o n e d i n s e r t s , we h a v e b e e n a b l e t o s o r t o u t t h a t
highly
repeated
spcDNA.
I t seems
population
sequences
Our
form
indicate
that
directly
exist
strongly
main
adressed
i t s high
repetitive
group
upon
group,
repetition
unknown
whether
cycloheximide
increase
unique
i n spcDNA.
certain
sequence
such
in the amplified
This
spcDNA
question
Some s e q u e n c e s ,
such
in
form,
circular
in
cells
has not
a s B1 ,
seem
whereas
a s MIF a n d R, a p p e a r s
t o b e more
work
clones
degree
for this
clones
task
that
would
a r e B 1 , by v i r t u e
and o v e r r e p r e s e n t a t i o n
by v i r t u e
of i t s apparent
eye 1 o h e x i m i d e
treatment.
entails
t h e same
treatment
a lower
or other
as after
stimuli
i n spcDNA,
degree of
although
sensitivity.
sequences
other
high
Of t h e
s o m e c l o n e s may b e s u i t a b l e ,
degree
i n spcDNA
was t o i d e n t i f y
p r o b e s f o r spcDNA t o s t u d y t h e p r o c e s s o f i t s
Candidate
amplification
lower
earlier.
repetition
t h e MIF/R
while
by c y c l o h e x i m l d e .
aim of t h i s
amplification.
and
counterpart.
of o t h e r s ,
a
in
i n t h e spcDNA
and c y c 1 o h e x i m i d e - t r e a t e d
"cons11tu11ve 1y"
induced
extent
of
composition
t h e normal
serve as efficient
of
t h e amount
t h e sequence
from
enriched
DNA, a s a g r o u p ,
t o any g r e a t
of
is not overrepresented
is really
repetitive
in untreated
circularization
The
a s i f what
a r e n o t found
may d i f f e r
been
as a whole,
i s middle
investigations
circular
to
DNA,
It
middle
their
remains
a r e a m p l i f i e d on
that
p r o v o k e an
content.
ACKHOWLEDGEHEHTS
This work was supported
Council
7836
by the Swedish Natural
and the Swedish Cancer
Society.
Science
Research
Nucleic Acids Research
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