Volume 12 Number 16 1984
Nucleic Acids Research
Sequence analysis of Viciafaba repeated DNA, the Fokl repeat element
Atsushi Kato, Kimitaka Yakura1 and Shigeyuki Tanifuji
Department of Botany, Faculty of Science, Hokkaido University, Sapporo 060, Japan
Received 7 June 1984; Accepted 5 July 1984
ABSTRACT
A t y p e o f h i g h l y r e p e a t e d DNA sequences p r e s e n t i n t h e genome o f V i c i a
f a b a was d e t e c t e d by d i g e s t i o n i t s n u c l e a r DNA w i t h F o k l endonuclease and
f r a c t i o n a t i n g t h e d i g e s t s on p o l y a c r y l a m i d e g e l s . Four f r a g m e n t s o f 5 9 , 1 0 8 ,
177 and 246 bp o f t h e F o k l r e p e a t e d sequences were c o l l e c t e d f r o m t h e g e l s and
t h e i r p r i m a r y s t r u c t u r e s were d e t e r m i n e d by t h e method o f Maxam and G i l b e r t .
These r e p e a t e d DNA sequences were shown t o be a m u l t i p l e tandem a r r a y o f a
59 bp sequence e l e m e n t . And i t s n u c l e o t i d e sequence was almost c o m p l e t e l y
c o n s e r v e d among a l l t h e sequence members o f each t h e s i z e c l a s s and a l s o
among t h e s e c l a s s e s . T h i s sequence element c o n s i s t s o f a d u p l e t o f an about
20 bp s h o r t sequence and a n o t h e r u n r e l a t e d 20 bp sequence. Each r e p e a t u n i t o f
t h e d u p l e t has an imcomplete dyad s y m m e t r i c a l s t r u c t u r e .
INTRODUCTION
Highly
the
genomic
tions
with
high
consists
homogeneous
consisting
different
roles
zation
ported
of repeated
In Secale
of original
were
that
was l o c a l i z e d
i t s nucleotide
© IRL Press Limited, Oxford, England.
(1,2,3,
repeated
DNA s e q u e n c e
repeating
each
organized
of which
detected
tandemly
Several
have
using
determined
(7,8,9),
types
of
mouse
organi-
been r e p o r t e d
units,
and i n s e r t e d
various
t o knob
in a
sequences
had a c c u m u l a t e d
repeating
elements
exclusively
repeated
i n bovine
a 1 8 5 bp r e p e a t e d
sequence
functions
tandemly
by a n a l y s i s
( 1 6 ) . I n Zea m a y s ,
coupled
has l e d t o
sequence
DNA s e q u e n c e s
repetitive
found
of
func-
in eukaryotes,
cellular
(13,14,15).
cereale,
fraction
their
a simple
highly
were
a n d some p r i m a t e s
pattern
sequences,
enzymes
More
mutations,
a large
Although
occurrence
in several
o f some s u b r e p e a t s ,
plants.
consist
(5,6).
cells.
in heterochromatin,
o f a 20 bp u n i t
array
base
(10,11,12)
and
widespread
concentration
on t h e i r
comprise
eukaryotic
D r o s o p h i 1 a genome c o n t a i n s
which
in
DNA s e q u e n c e s
DNA i n m o s t
are not clear,
their
speculation
4).
repeated
also
which
unrelated
restriction
sequence
was r e -
heterochromatin
indicated
t h e absence
of
641 5
Nucleic Acids Research
a n y i n t e r n a l s u b r e p e a t s ( 1 7 ) . H o w e v e r , t h e r e is no c l e a r e v i d e n c e
of s e q u e n c e c o n s e r v a t i o n a m o n g m e m b e r s of t h e 185 bp s e q u e n c e
e l e m e n t ( 1 7 ) . S e v e r a l c l a s s e s of t h e r e p e a t e d s e q u e n c e s f o u n d in
t h e S c i l l a s i b e r i c a g e n o m e w e r e all s h o w n to be v e r y h i g h l y c o n s e r v e d in n u c l e o t i d e s e q u e n c e a m o n g m e m b e r s of e a c h c l a s s and
a l s o a m o n g t h e c l a s s e s , e x c e p t t h e r e g i o n of m u l t i p l e s u c c e s i v e
i n s e r t i o n of t h e t e t r a n u c l e o t i d e G - T - C - C . H o w e v e r , t h e r e is no
i n f o r m a t i o n on t h e r e l a t i o n s h i p in t h e a r r a n g e m e n t of t h e v a r i a n t s of t h e s e r e p e a t e d s e q u e n c e s w i t h i n t h e g e n o m e ( 1 8 ) . S o m e of
t h e v a r i o u s f a m i l i e s of r e p e a t e d D N A s e q u e n c e s f o u n d w i t h i n t h e
g e n o m e of an o r g a n i s m m a y b e r e l a t e d to o n e a n o t h e r and u s e f u l
to s t u d y t h e e v o l u t i o n of D N A s e q u e n c e s . T h e s u b j e c t of t h i s
c o m m u n i c a t i o n is t h e m o l e c u l a r o r g a n i z a t i o n of o n e t y p e of r e p e a t e d D N A s e q u e n c e s f o u n d in Vi ci a f a b a . A h y p o t h e t i c a l m o d e l of
t h e e v o l u t i o n of t h i s r e p e a t e d s e q u e n c e is p r e s e n t e d .
M A T E R I A L S AND
METHODS
E x t r a c t i o n of D N A
E m b r y o n i c a x e s u s e d f o r p r e p a r a t i o n of D N A w e r e o b t a i n e d
f r o m V i c i a f a b a s e e d s a f t e r s o a k i n g f o r 24h in t h e d a r k at 27 C.
A nuclear fraction was obtained ( 1 9 , 2 0 ) , from which DNA was prep a r e d by C s C l - e q u i 1 i b r i u r n c e n t r i f u g a t i o n a c c o r d i n g to t h e m e t h o d
of B e d b r o o k ( 1 6 ) .
R e s t r i c t i o n e n d o n u c l e a s e d i g e s t i o n , gel e l e c t r o p h o r e s i s and
sequence analysis
In m o s t c a s e s D N A was d i g e s t e d w i t h 2 u n i t s of a r e s t r i c t i o n
enzyme
p e r jug D N A in the b u f f e r s u g g e s t e d by t h e s u p p l i e r s . D N A
f r a g m e n t s w e r e f r a c t i o n a t e d by e l e c t r o p h o r e s i s on 1 - 2 % a g a r o s e or
5 % p o l y a c r y l a m i d e g e l s . DNA f r a g m e n t s s t a i n e d w i t h e t h i d i u m
b r o m i d e w e r e v i s u a l i z e d by a UV l a m p and e l u t e d e l e c t r o p h o r e t i c a l l y in d i a l y s i s b a g s . A f t e r l a b e l i n g 5 - e n d s of t h e D N A
f r a g m e n t s w i t h ( 7 " - 3 2 P ) - A T P , D N A w a s d e n a t u r a t e d in 0 . 3 % N a O H and
t h e s t r a n d s w e r e s e p a r a t e d by e l e c t r o p h o r e s i s on 5 or 8% p o l y a c r y l a m i d e g e l s . F a s t and s l o w s t r a n d s w e r e s e q u e n c e d a c c o r d i n g
t o t h e m e t h o d of M a x a m and G i l b e r t ( 2 1 ) .
P-end l a b e l i n g and D N A / D N A h y b r i d i z a t i o n
T h e t r e a t m e n t of DNA f r a g m e n t s w i t h a l k a l i p h o s p h a t a s e and
t h e i r 5 - e n d l a b e l i n g w e r e c a r r i e d o u t a c c o r d i n g to M a i z e l s ( 2 2 ) .
6416
Nucleic Acids Research
D N A f r a g m e n t s r e c o v e r e d f r o m a g a r o s e g e l s w e r e d e n a t u r e d by
a l k a l i ( 2 3 ) or g l y o x a l and d i m e t h y l s u l f o x i d e b e f o r e s u b s e q u e n t
e l e c t r o p h o r e s i s ( 2 4 ) . H y b r i d i z a t i o i of D N A b l o t t e d on n i t r o c e l l u l o s e f i l t e r s w a s p e r f o r m e d a f t e r p r e i n c u b a t i o n o f t h e f i l t e r s at
3 7 ° C f o r 4h in a s o l u t i o n c o n t a i n i n g 5 0 % f o r m a m i d e , 5 x SSC and
10 x D e n h a r d t s " s o l u t i o n ( 2 5 , 2 6 ) . T h e f i l t e r s w e r e h y b r i d i z e d
w i t h h e a t - d e n a t u r e d p r o b e s in t h e a b o v e - m e n t i o n e d s o l u t i o n c o n t a i n i n g a d d i t i o n a l l y 1 0 0 jug/ml h e a t - d e n a t u r e d s o n i c a t e d £ . coli
D N A f o r 2 4 h at 3 7 ° C . T h e n t h e f i l t e r s w e r e w a s h e d f o r 6 t i m e s
w i t h t h e p r e i n c u b a t i o n s o l u t i o n c o n t a i n i n g 0 . 1 % S D S at 37 C,
t w i c e w i t h 2 x S S C and t h e n e x p o s e d to F u j i - R X f i l m .
RESULTS
I s o l a t i o n and p u r i f i c a t i o n of F o k l r e p e a t e d s e q u e n c e
T h e p r e s e n c e of s a t e l l i t e D N A in t h e V. f a b a g e n o m e and its
u n u s u a l n o n - l o c a l i z e d d i s t r i b u t i o n on all c h r o m o s o m e s h a v e b e e n
r e p o r t e d ( 2 7 ) . T h e a m o u n t of V_. f a b a s a t e l l i t e D N A h a s b e e n s h o w n
to be v e r y s m a l l and t h e u s e of Ag + -Cs,,S0. g r a d i e n t s , i n s t e a d of
CsCl g r a d i e n t s , d i d not a l l o w to i n c r e a s e t h e d e t e c t a b l e a m o u n t
of s a t e l l i t e ( d a t a not s h o w n ) . H o w e v e r , w h e n n u c l e a r D N A w a s
d i g e s t e d w i t h F o k l and p r o d u c e d f r a g m e n t s w e r e s e p a r a t e d on 5%
polyacrylamide g e l s , several discrete bands were visualized,
w h i c h w e r e s u p p o s e d to be a b a s i c s e q u e n c e e l e m e n t of a c e r t a i n
r e p e a t e d s e q u e n c e and its m u l t i m e r s . T h e s e f r a g m e n t s w e r e r e s i s t ant to d i g e s t i o n w i t h E c o R I , H i n d H I , B a m H I , H i n f l , H p a l l , H a e l l l o r
S a u 3 A I . T h e r e f o r e , it s e e m e d t h a t t h e r e p e a t e d D N A s e q u e n c e s r e v e a l e d by F o k l c u t w e r e d i s t r i b u t e d w i t h h i g h f r e q u e n c i e s in s o m e
r e l i c D N A f r a c t i o n s " t h a t w e r e c h a r a c t e r i z e d by r e l a t i v e l a c k s
of t h e t a r g e t s e q u e n c e s f o r t h e s e e n z y m e s . In t h e p r e s e n t s t u d y ,
Haelll w a s u s e d to p r e p a r e a r e l i c D N A f r a c t i o n . A f t e r d i g e s t i o n
of t h e n u c l e a r D N A w i t h HaeIII, i t s p r o d u c t s w e r e l a y e r e d on a
1 0 - 4 0 % l i n e a r s u c r o s e g r a d i e n t (1M N a - a c e t a t e , 1 O m M T r i s - H C l ,
pH 8 . 0 , lmM E D T A ) in a H I T A C H I R P S - 2 7 c e n t r i f u g a t i o n t u b e , t h e n
f o l l o w e d by c e n t r i f u g a t i o n at 2 4 0 0 0 r p m f o r 15h at 2 0 ° C . A f t e r
f r a c t i o n a t i o n , s m a l l p o r t i o n s w e r e r e m o v e d f r o m e a c h f r a c t i o n and
e a c h w a s d i g e s t e d w i t h F o k l , t h e n s u b j e c t e d to e l e c t r o p h o r e s i s on
a g a r o s e or p o l u a c r y l a m i d e g e l s to s e a r c h t h e d i s t r i b u t i o n of the
r e p e a t e d s e q u e n c e s d e f i n e d by F o k l c u t a m o n g t h e s e f r a c t i o n s of
6417
Nucleic Acids Research
B
A
-IV
-III
-II
-I
1 2
3
4
F i g . l DNA fragments obtained by digestion of .V. faba nuclear DNA w i t h Fokl
were separated by e l e c t r o p h o r e s i s on 5% polyacrylamide gels and stained w i t h
ethidium bromide. A) The e f f e c t of enrichment of the Fokl repeated sequences
by preparing a H a e l H - r e l i c DNA f r a c t i o n . Twenty jjg of Fokl-digested ^/. faba
nuclear DNA (lane 1 ) . One ^ig of the H a e l l - r e l i c DNA digested w i t h Fokl ( I a n e 2 ) .
Repeated DNA sequences defined w i t h Fokl were enriched i n the r e l i c DNA f r a c t i o n more than 2 0 - f o l d . Molecular sizes of the major bands are as f o l l o w s :
1:60 bp, 11:120 bp, III : 180 bp, IV: 240 bp. B) Weak or extensive d i g e s t i o n of
the HaelH-relic DNA w i t h F o k l . Conditions of d i g e s t i o n were as f o l l o w s :
Lane 1:0.5 u n i t s / j j g DNA f o r 5 min. Lane 2:1.5 u n i t s / p g DNA f o r 5 min. Lane 3:
3.0 u n i t s / u g DNA f o r 60 min. Lane 4:20 u n i t s / u g DNA f o r 240 min.
various
sizes.
sequences
the Fokl
Next,
repeated
of
enrichment
of
DNA f r a c t i o n
and
DNA f r a c t i o n
with
gave
a typical
ples
in
6418
r e p e a t e d DNA
in the H a e i n - r e l i c
Fokl
of
the
degree
of
sizes.
length
containing
shows t h e
sequences
their
shows t h e
Fig.lA
Digestion
also
Fig.IB
fractions
were p o o l e d .
ladder
a basic
patterns
of
the H a e l l l - r e l i c
fragments
repeat
of
unit
of
w h i c h were e x a c t
approximately
electrophoresis
with
multi-
60 b p .
partial
di-
Nucleic Acids Research
F-3-2
GG
A
T
Fig.2 Autoradiographs of parts of the sequencing gels of the Fokl repeat
fragments. Target sequences recognized by Fokl are i n d i c a t e d by b r a c k e t s , the
cleavage points are i n d i c a t e d by arrow heads, and p o s i t i o n s of methylated
cytosines are i n d i c a t e d by M. F - 2 - 1 : One strand of a l k a l i - d e n a t u r e d 118 bp
fragment. F-2-2: Another strand of a l k a l i - d e n a t u r e d 118 bp fragment. F-3-2:
One strand of a l k a l i - d e n a t u r e d 177 bp fragment.
gestion
products
fragments
additional
with
decreased,
occurrence
Fokl.
whereas
of
Upon weak d i g e s t i o n s ,
larger
new s i z e s
fragments
of
smaller
increased
fragments.
without
Therefore,
it
6419
Nucleic Acids Research
I
TCATTATGGA AGGTAGTCTG TTGTCGAGAT TTTTGTTACT CCAATGGATG GTAGCCTTG"
n
TCATTATGGA AGGTAGTCTG TTGTCGAGAT TTTTGTTACT CCAATGGATG GTAGCCTTGT
CATTATGGAA GGTAGTCTGT TGT&AGATT TTTGTTACTC CAATGGATGG TAGCCTTG*
IH
TCATTATGGA AGGTAGTCTG TTGTCGAGAT TTTTGTTACT CCAATGGATG GTAGCCTTG'T
CATTATGGAA GGTAGTCTGT TGTCGAGATT TTTGTTACTC CAATGGATGG TAGCCTTGTC
ATTATGGAAG GTAGTCTGTT GTCGAGATTT TTGTTACTCC AATGGATGGT AGCCTTG
V
—
AATGGATG GTAGCCTTG'T
CATTATGGAA GGTAGTCTGT TGT&AGATT TTTGTTACTC CAATGGATGG TAGCCTTGTC
ATTATGGAAG GTAGTCTGTT GTC*GAGATTT TTGTTACTCC AATGGATGGT AGCCTTGTCA
TTATGGAAGG TAGTCTGTTG TCGAGATTTT TGTTACTCCA ATGGATGGTA GCCTTG*
Fig.3
N u c l e o t i d e sequences of t h e F o k l c l e a v e d f r a g m e n t s ,
I n d i c a t e d symboles a r e t h e same w i t h F i g . 2 .
was
concluded
that
sequence
elements.
Sequence
analysis
gels
The
60,
and
their
lysis.
Some
Although
rated
as
tides
it
the
shows
of
the
and
of
was
at
these
and
177
the
59
variation
in
nucleotide
constituting
again
tion.
exact
to
of
the
each
of
indicated
these
60,
118,
177
fragments
were
exact
element.
sequence
a very
sequence
determined,
among
fragment
but
high
of
the
degree
the
236
120,
236
of
bp
be
high-
sequence.
180
bp
and
of
240
nucleo-
and
and
also
trimer
there
sequence
a multiple
sequence
sepa-
ladders.
numbers
dimer
59
of
Fig.2.
two
should
Futhermore,
classes
probably
and
the
ana-
nucleotide
repeating
exact
59,
sequence
in
sequence
the
that
from
one
classes
basic
sequence
shown
the
of
bp
between
of
W.
was
no
eleseries,
conserva-
bp
fragment
class
this
fragment
should
rebe
a
tetramer.
Only
sequence
6420
be
are
position
were
these
indicating
A part
mained
bp
bp
to
contamination
fragment
60
eluted
identify
sequences
fragments
were
subjected
to
these
II, I I ,
only
possible
members
results
of
thus
of
most
118
fragments
were
autoradiography
each
nucleotide
bp
cross
quite
molecules
ments
the
sequence
The
240
DNA c o m p r i s e d
strands
a slight
among
fragments.
that
was
representive
homologous
Fig.3A
bp
examples
strands,
the
180
repeated
separated
there
Therefore,
ly
120,
this
I,
a band c o r r e s p o n d i n g
per
the
shortest
to
one c y t o s i n e
59 bp b a s i c
residue
in
s e q u e n c e was a b s e n t
C-G
in
the
Nucleic Acids Research
B
12
12 3 4
34
Fig.4
R e i t e r a t i o n o f t h e F o k l r e p e a t u n i t s i n t h e V.. f a b a genome.
A) A n e g a t i v e f i l m o f
2% a g a r o s e g e l s t a i n e d w i t h e t h i d i u m b r o m i d e .
B) An a u t o r a d i o g r a m o f S o u t h e r n b l o t h y b r i d i z a t i o n w i t h l a b e l e d 59 bp r e p e a t e d
sequence u n i t . Lane 1:20 j j g n u c l e a r DNA. Lane 2 : 1 0 ug n u c l e a r DNA. Lane 3 :
0 . 5 jug 59 bp F o k l r e p e a t e d s e q u e n c e , 0 . 0 5 pg 118 bp F o k l r e p e a t e d sequence
and 0 . 1 jug 177 bp F o k l r e p e a t e d s e q u e n c e . Lane 4 : t h e same F o k l r e p e a t e d
sequence w i t h t h a t i n l a n e 3 e x c e p t DNA amounts were one t e n t h . N u m e r i a l s
i n d i c a t e t h e s i z e (bp) o f t h e major bands.
sequence
were
was
ladders,
quite
concluded
were
To
within
trophoresed
the
ried
units
59
the
Fokl
repeated
the
the
V^.
Then,
the
as
shown
a faint
We d e t e r m i n e d
scanning
agarose
59,
118
the
gels
and
and
longer
the
than
the
number
of
Fokl
gels,
in
59
177
bp
whole
118
bp
residues
Fokl
in
both
bases
Then,
DNA
repeated
nuclear
parallel
with
bp
Fig.4,
blot
it
strands
of
repeat
units.
band
of
In
of
negative
The
were
amount
fragments
estimated
59
118
reiteration
fragments
of
known
DNA f r a g m e n t s
and
sequences
DNA w a s
was
the
bp
this
films
of
the
from
the
carcase
in
was
relative
estimated
from
bp
elec-
quantities
hybridization
a densitometer.
was
other
element.
the
radioactive
degree
and
radioactivity.
digested
Southern
in
of
sequences
autoradiograms
using
cytosines
sequence
P-labeled
and
of
cytosine
bp
faba,
2% a g a r o s e
using
filmes
quence
copy
of
units.
appear
by
stained
tive
two
this
experiment,
not
tected.
of
on
band
intensities
only
genome
repeat
out
this
did
of
the
high
in
estimate
the
whereas
with
that
methylated
Reiteration
of
clear
of
length
de-
repeat
of
the
amounts
the
related
negase-
autoradiograms.
6421
Nucleic Acids Research
As the r e s u l t of s u m m a t i o n
b a n d s , we estimated
of t h e r e l a t i v e v a l u e s w i t h all
t h e total c o p y n u m b e r of t h e r e p e a t e d
q u e n c e e l e m e n t s to be in t h e r a n g e o f 5 x 1 0
to 5 x 1 0
per
the
se-
diploid
genome.
DISCUSSION
We c o u l d c o n c e n t r a t e t h e F o k l r e p e a t e d s e q u e n c e s a b o u t
2 0 - f o l d in a c e r t a i n D N A f r a c t i o n o f h i g h m o l e c u l a r w e i g h t f r a g m e n t s t h a t w a s c h a r a c t e r i z e d by a r e l a t i v e l a c k of HaeIII s i t e s .
M o s t of c o n t a m i n a t i n g D N A s in t h e r e l i c O N A f r a c t i o n w e r e D N A
f r a g m e n t s w i t h m o l e c u l a r w e i g h t s h i g h e r t h a n t h o s e of t h e m u l t i p l e d r e p e a t e d s e q u e n c e s . T h e r e f o r e , as to t h e DNA f r a c t i o n s of
t h e b a n d s t h e m s e l v e s o f t h e F o k l - c l e a v e d f r a g m e n t s , t h i s t y p e of
r e p e a t e d s e q u e n c e s w a s e n r i c h e d m o r e t h a n 20 t i m e s . P r e p a r a t i o n
of r e l i c D N A f r a c t i o n s is g e n e r a l l y a b e n e f i c i a l m e t h o d , w h e n t h e
s i z e of a r e p e a t u n i t is r a t h e r s m a l l . T h i s t y p e of r e p e a t e d s e q u e n c e s r e v e a l e d by F o k l d i g e s t i o n c o n s i s t e d of o n l y o n e s e q u e n c e
e l e m e n t of 59 bp w i t h a p e r f e c t l y c o n s e r v e d n u c l e o t i d e s e q u e n c e .
S u c h t h e o r g a n i z a t i o n p a t t e r n is s i m i l a r to t h a t o b s e r v e d w i t h
s a t e l l i t e DNA I, II and IV o f D r o s o p h i 1 a mei a n o g a s t e r ( 5 , 6 ) . In
t h e c a s e s of r e p e a t e d s e q u e n c e s of b o v i n e ( 7 , 8 , 9 ) , m o u s e ( 1 0 , 1 1 ,
1 2 ) and s o m e p r i m a t e s ( 1 3 , 1 4 , 1 5 ) , t h e i r n u c l e o t i d e s e q u e n c e s e x h i b i t e d m o r e c o m p l e x v a r i a t i o n . In p l a n t s , a c l a s s of r e p e a t e d
s e q u e n c e s c o n s i s t i n g of o n l y b a s i c s e q u e n c e e l e m e n t w a s f o u n d in
S e c a l e c e r e a l e ( 1 8 ) , C u c u m i s sati v u s and C u c u m i s m e l o ( 2 8 ) , b u t
t h e s e e l e m e n t s h a v e n o t yet b e e n s e q u e n c e d . A h i g h l y r e p e a t e d s e q u e n c e l i m i t e d to k n o b h e t e r o c h r o m a t i n of Z e a m a y s ( 1 7 ) w a s s h o w n
to c o n s i s t of a r e p e a t i n g u n i t o f 1 8 5 b p . A l t h o u g h t h e n u c l e o t i d e
s e q u e n c e of o n e c l o n e d m e m b e r o f t h e r e p e a t i n g u n i t had b e e n d e t e r m i n e d , t h e r e w a s no e v i d e n c e f o r s e q u e n c e c o n s e r v a t i o n a m o n g
m e m b e r s of t h e e l e m e n t , nor e v i d e n c e f o r t h e i r o r g a n i z a t i o n
w i t h i n t h e g e n o m e . In the c a s e o f S c i 1 1 a si b e r i c a ( 1 6 ) , p r i m a r y
s t r u c t u r e s of s e v e r a l c l a s s e s o f r e p e a t e d s e q u e n c e s w e r e c o n s i d e r a b l y c o n s e r v e d a m o n g t h e i r m e m b e r s of e a c h c l a s s and a l s o a m o n g
t h e c l a s s e s , but c l e a r l y e x h i b i t e d s o m e e x t e n t s of v a r i a t i o n w i t h
several base s u b s t i t u t i o n s or i n s e r t i o n s of subsets of t e t r a n u c l e o t i d e . H o w e v e r , no i n f o r m a t i o n w a s g i v e n on t h e a r r a n g e m e n t
o f t h e s e c l a s s e s of S c i l l a s i b e r i c a r e p e a t e d s e q u e n c e s .
6422
Nucleic Acids Research
In \/. f a b a , t h e r e a r e o t h e r t y p e s o f r e p e a t e d s e q u e n c e s s h o w i n g
a b o u t 2 0 % s e q u e n c e v a r i a t i o n in e a c h s e q u e n c e f a m i l y b e s i d e s t h e
Fokl repeated s e q u e n c e s described here ( 2 9 , 3 0 ) . Then, the facts
that this r e p e a t e d DNA d e t e c t e d by Fokl cut c o n s i s t e d of only o n e
sequence element and its nucleotide sequence was extremely conserved are particularly important features among several types of
r e p e a t e d s e q u e n c e s f o u n d in t h e V_. f a b a g e n o m e .
When total nuclear DNA was digested with Fokl, several fragm e n t s as t h e p r o d u c t s of i m c o m p l e t e d i g e s t i o n a p p e a r e d r a t h e r
c o n s i s t e n t l y . T h e c a u s e of t h e i r o c c u r r e n c e is u n k n o w n . S o m e of
t h e F o k l s i t e s in t h e r e p e a t e d D N A w e r e n o t d i g e s t e d , e v e n w h e n
lug DNA w a s d i g e s t e d w i t h 20 u n i t s of Fokl e n d o n u c l e a s e of a
l a r g e a m o u n t f o r 6h of a l o n g e r p e r i o d ( F i g . I B ) . And t h e s e u n c l e a v e d F o k l s i t e s a m o u n t e d t o a b o u t 5 0 % o f t o t a l F o k l s i t e s in
the repeated DNA fraction, which value was calculated from the
r a t i o of t h e s u m of e s t i m a t e d n u m b e r s of t h e 59 bp b a s i c s e q u e n c e
u n i t p r e s e n t i n e a c h m u l t i m e r t o i t s t o t a l c o p y n u m b e r in t h e
whole g e n o m e . H o w e v e r , the bases of the r e c o g n i z a t i o n sequence of
t h e u n c l e a v e d F o k l s i t e s p r e s e n t in t h e s e m u l t i m e r f r a g m e n t s w e r e
s u p p o s e d to be not m e t h y l a t e d , b e c a u s e t h e b a n d s c o r r e s p o n d i n g to
t h e b a s e s o f t h e t a r g e t s e q u e n c e f o r F o k l w e r e v e r y c l e a r in t h e
a u t o r a d i o g r a p h s of s e q u e n c i n g g e l s . This i m p e r f e c t d i g e s t i o n
might be due to a c e r t a i n s e c o n d a r y s t r u c t u r e of the D N A .
G e n e r a l l y , plant DNAs have high c o n t e n t s of 5 - m e t h y l c y t o s i n e
a n d p a r t i c u l a r l y i t s m e t h y l a t i o n i s f o u n d in t h e c y t o s i n e r e s i d u e s in C - G d i n u c l e o t i d e s e q u e n c e . F o r e x a m p l e , in S c i 1 1 a
si b e r i c a a n d C u c u m i s s a t i v u s , a b o u t 5 5 o r 3 0 % c y t o s i n e s o f s a t e l l i t e D N A s a r e m e t h y l a t e d ( 1 8 ) a n d in w h e a t t o t a l D N A ( 3 1 )
c y t o s i n e s a r e m e t h y l a t e d in a b o u t 8 2 % C - G s e q u e n c e s . T h e f a c t
t h a t y_. f a b a D N A w a s n o t d i g e s t e d m o s t l y w i t h M l u l , H h a l o r Hpall
s u g g e s t s a h i g h l e v e l of m e t h y l a t i o n in m o s t C - G s e q u e n c e s . T h e r e
i s o n e C - G s e q u e n c e in t h e 5 9 b p r e p e a t e d s e q u e n c e e l e m e n t a n d i n
f a c t t h e c y t o s i n e r e s i d u e s at t h i s p o s i t i o n w e r e m o s t l y m e t h y lated. This result was similar to the case o b s e r v e d with the
r e p e a t e d D N A o f S c i 1 1 a si b e r i c a .
A s s h o w n in F i g . 5 A , t h e 5 9 b p s e q u e n c e e l e m e n t c o n s i s t s o f
a d u p l e t of an a b o u t 2 0 bp s h o r t s e q u e n c e a n d a n o t h e r u n r e l a t e d
20 bp s e q u e n c e . T h e s e q u e n c e h o m o l o g y b e t w e e n t w o s u b s e t s (19 and
6423
Nucleic Acids Research
22
41
TGTCGAGATTTTTGTTACTC
C
A
1
A.TGG.A T J G T A G D J T T IT
21
GT
CAI1ATGGAA
42 T A
CAA'GG TG
B
C2ATTA T G G A A
ii
ii
i
21GT- T r A
T C G
J.GTT CC r AT
|
Tr
G
42
C
CAATGG/TGGTAlgCCTTGT
CATTATGGAAGGTAGTCTffT
F i g . 5 O r g a n i z a t i o n o f t h e 59 bp repeated sequence u n i t . Bases a r e numbered
f r o m t h e f i r s t base o f t h e cleavage s i t e by F o k l . A) The sequences and
n u c l e o t i d e s showing homology between t h e two tandem subsets are boxed. Bar
u n d e r l i n e d i n d i c a t e s t h e t a r g e t sequence r e c o g n i z e d w i t h F o k l and an arrow
head i n d i c a t e s t h e c l e a v e d s i t e by F o k l . B) Dyad s y m m e t r i c a l s t r u c t u r e s o f
each o f t h e d u p l e t sequence. C) I n t r a s t r a n d symmetrical s t r u c t u r e s o f each
subsets o f t h e d u p l e t sequence.
20 b p ) o f t h e d u p l e t
that
this
cation
ation
o f an a n c e s t r a l
with
generation
repeating
Although
unit
formed
20 bp s e q u e n c e .
59 bp r e p e a t
o f about
unit
325 bp f o u n d
we c a n n o t i m a g e
i t s detail
common m e c h a n i s m
secondary
80%. T h e n ,
had been
1 9 - 2 0 bp s e q u e n c e
an u n r e l a t e d
of this
unit
a somewhat
shows
was m o r e t h a n
59 bp r e p e a t
structures
such
half
both
have
with
Sci1la s i b e r i c a
the other
hand,
metry,
which
bovine
satellite
mine which
6424
sequences,
resembles
model
imperfect
as s h o w n i n F i g . 5 C ,
19 a n d 20 bp s h o r t
that
have
assosi-
hypothesis
that
for
f o r a sub-
there
might
of the duplex.
dyad
symmetry,
satellite
each
found with
symmetry
dupli-
the structure.
half
imperfect
DNA ( 9 ) . A l t h o u g h ,
o f dyad
likely
i n t h e rDNA o f _V. f a b a ( 3 2 ) .
sembles
found
This
resembles
a n d 2 0 bp s e q u e n c e
that
seems
and s u b s e q u e n t
at present,
to yield
o f each
it
by a t a n d e m
we h a v e
T h e s e 19
which r e -
DNA ( 1 8 ) . On
of the duplex,
intra
sequence
no e v i d e n c e
strand
the
s t r a n d sym-
an a n c e s t r a l
or i n t r a
be
Fig.5B
to
of
deter-
symmetry r e -
Nucleic Acids Research
fleets i t s ancestral s t r u c t u r e , such t h e m o l e c u l a r s t r u c t u r e s ,
either or both, might be related with e s t a b l i s h m e n t and d e v e l o p ment of a certain repeated sequence.
We could n o t d e t e c t a n y s e q u e n c e s being h o m o l o g o u s t o t h i s
J/. f a b a F o k l r e p e a t s in t h e g e n o m e s o f o t h e r s o m e s p e c i e s o f
Legumi nosae by S o u t h e r n blot h y b r i d i z a t i o n (data n o t s h o w n ) .
F u t h e r m o r e , a n y D N A s e q u e n c e s c o n t a i n e d in t h e V . f a b a g e n o m e
except those Fokl repeated s e q u e n c e s were not hybridized with
t h i s 5 9 bp F o k l r e p e a t u n i t , w h e n d i g e s t s o f V^. f a b a D N A w i t h
v a r i o u s restriction e n z y m e s were analyzed (data not s h o w n ) . On
the basis of these r e s u l t s , w e speculate t h a t , although t a n d e m
duplication of t h e 1 9 - 2 0 bp short sequence had occurred at a
c e r t a i n v e r y o l d t i m e , r e i t e r a t i o n o f t h e 59 b p r e p e a t u n i t h a s
occurred rather recently during t h e course of e v o l u t i o n .
ACKNOWLEDGEMENTS
We thank P r o f e s s o r M . S u g i u r a and D r . K. S h i n o z a k i o f N a g o y a
U n i v e r s i t y a n d D r . J. K u s u d a a n d M r s . M . K u s u d a o f N a t i o n a l
Institute of G e n e t i c s , M i s h i m a f o r their kind help and d i s c u s sion. This study w a s supported by t h e grants from t h e M i n i s t r y
of E d u c a t i o n , S c i e n c e a n d C u l t u r e o f J a p a n ( N o . 5 8 1 1 9 0 0 2 ,
58212001, 58480009) and t h e Mishima Kaiun Science Foundation.
'Present address: Department of Biology, Faculty of Education, Kanazawa University, Kanazawa
920, Japan
REFERENCES
1. Appels,R. and Peacock,W.J. (1978) Int. Rev. Cytology, Suppl. 8, eds:
G.H. Bourne and J.F. Danielli, pp. 70-126.
2. Davidson,E.H. and Britten R.J. (1979) Science 204, 1052-1059.
3. Orgel.L.E. and Crick,F.H.C. (1980) Nature 284, 604-607.
4. Doolittle.W.F. and Sapienza,C. (1980) Nature 284, 601-603.
5. Endow,S.A. (1977) J.Mol.Biol. 114, 441-449.
6. Mullins.J.I. and Blumenfeld.M. (1979) Cell 17, 615-621.
7. Streeck.R.E. (1981) Science 213, 443-445.
8. P^ucienniczak.A., Skowronski,J. and Jaworski.J. (1982) J.Mol.Biol.
158, 293-304.
9. Taparowsky,E.J. and Gerbi.S.A. (1982) Nucl. Acids Res. 10, 1271-1281.
10. Hbrz.W. and Altenburger.W. (1981) 9, 683-696.
11. Manuelidis.L. (1981) FEBS Letters 129, 25-28.
12. Kalb.V.F., Glasser.S., King,D. and Lingrel.J.B. (1983) Nucl.Acds Res.
11, 2177-2184.
13. Rubin,CM., Deininger,P.L., Houck.C.M. and Schmid.C.W. (1980) J.Mol.
Biol. 136, 151-167.
6425
Nucleic Acids Research
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
6426
Frommer.M., P r o s s e r . J . , Tkachuk.D., Reisner.A.H. and Vincent,P.C. (1981)
Nucl.Acids Res. 10, 547-563.
M a n u e l i d i s , L . (1982) Nucl.Acids Res. 10, 3211-3219.
Bedbrook.J.R., J o n e s , J . , O ' D e l l . M . , Thompson,R.D. and F l a v e l l . R . B .
(1980) Cell 19, 545-560.
Peacock,W.J., Dennis,E.S., Rhoades.M.M. and P r y o r . A . J . (1981) P r o c . N a t l .
Acad.Sci.USA 78, 4490-4494.
Deumling.B. (1981) Proc.Natl .Acad.Sci.USA 78, 338-342.
Sakamaki,T., Fukuei.K., Takahashi.N. and T a n i f u j i . S . (1975) Biochim.
Biophys.Acta 395, 314-321.
Yakura.K., Fukuei.K. and T a n i f u j i . S . (1978) Plant & Cell P h y s i o l .
19, 1381-1390.
Maxam.A.M. and Gilbert,W. (1980) i n Methods i n Enzymology vol 65,
pp.499-560, Academic Press, New York.
Maizels,N. (1976) Cell 9, 431-438.
Southern,E.M. (1975) J . M o l . B i o l . 98, 503-517.
Thomas,P.S. (1980) Proc.Natl .Acad.Sci-USA 77, 5201-5205.
Denhardt.D.T. (1966) Biochem.Biophys.Res.Commun. 23, 641-646.
Rigby.P.W.J., Dieckmann.M., Rhodes,C. and Berg,P. (1977) J . M o l . B i o l .
113, 237-251.
Timmis.J.N., Deumling.B. and I n g l e , J . (1975) Nature 257, 152-155.
Hemleven.V., Leweke.B., Roth,A. and S t a d l e r . J . (1982) Nucl.Acids Res.
10, 631-644.
K a t o . A . , I i d a . Y . , Yakura,K. and T a n i f u j i . S . (1984) submitted.
K a t o . A . , I i d a . Y . , Yakura.K. and T a n i f u j i . S . (1984) submitted.
Gluenbaum.Y., Naveh-Many.T., Cedar,H. and Razin.A. (1981) Nature
292, 860-862.
Yakura.K., Kato.A. and T a n i f u j i . S . (1984) Molec.Gen.Gen. 193, 400-405.