Volume 16 Number 19 1988
Nucleic Acids Research
Structure and genomlc organization of I elements involved in I—R hybrid dysgenesis in
Drosophila melanogaster
Michele Crozatier*, Chantal Vaury, Isabelle Busseau, Alain Pelisson and Alain Bucheton"1"
Laboratoire de Ge'netique, Unit^ Associee au CNRS no. 360, University Blaise Pascal,
63177 Aubiere Cedex, France
Received June 15, 1988; Revised and Accepted August 30, 1988
ABSTRACT
I-R hybrid dysgenesis in D. melanoqaster is controlled by
transposable elements known as I factors which terminate at their
31 ends by an A-rich sequence. Inducer strains contain active I
factors. Both reactive and inducer stocks possess defective I
elements. We have cloned various I elements from both categories
of strains. The I elements having recently transposed in inducer
strains have a structure closely related to that of active I
factors. However we have isolated one such I element that is
truncated at its 5' end. The I elements common to reactive and
inducer strains are affected by various rearrangements and many
point mutations. They do not appear to be simple derivatives of
complete I factors.
INTRODUCTION
Hybrid dysgenesis is the production of abnormal characteristics in the progeny of crosses between particular strains of
Droaophila melanoqaster (1). Three independent systems of hybrid
dysgenesis have been identified so far and are controlled by
different
transposable
elements. They
are
the
IR
system
controlled by the I factor (2), the PM system controlled by the P
factor (3) and a third system named HE which appears to be
controlled by the element hobo(4. 5 ) .
IR hybrid dysgenesis is produced in the germ-line of F1
females resulting from crosses between males from a class of
strains known as inducer and females from the other class of
strains known as reactive (6). The progeny of reciprocal crosses
are apparently normal. The abnormalities characteristic of this
system include sterility and high frequencies of mutations and
chromosome rearrangements (7).
Unlike reactive strains, inducer strains contain active I
factors. These are stable when maintained in inducer strains, but
© IRL Press Limited, Oxford, England.
9 1
"
Nucleic Acids Research
are activated when introduced into the cellular environment of
reactive strains. In these conditions, they transpose with
unusually high frequencies (8).
Active I factors have been cloned from white gene mutations
produced during IR hybrid dysgenesis (9, 10). They are all 5.4 kb
long and appear to be very similar in structure. The ends of
these elements are highly conserved and are flanked by target
site duplications varying in length from 10 to 14 bp. They have
no terminal repeats and their 3' ends have 4 to 7 tandem copies
of the sequence TAA (11). This organization is similar to that of
F and G elements of Drosophila (12, 13), LINEs or LI elements
which are repeated several thousand times in mammalian genomes
(14-17), ingi elements of Trypanosoma (18) and cin4 elements of
Maize
(19). These
elements
are
bounded
by
target
site
duplications varying in length. They are terminated at their 3'
ends by an A-rich sequence but are very often truncated at their
5 ' ends.
The complete sequence of an active I factor has been
determined (11). It contains two long open reading frames, ORF1
and ORF2, of 1278 and 3258 bp. The amino-acid sequence of ORF2
shows
similarities
with
viral
reverse
transcriptases
and
polypeptides encoded by various elements such as LI, F, G, ingi
and cin4. Extensive homologies have been found between the
product
of
this
ORF
and
the domains
of
viral
reverse
tranacriptases corresponding to RNA dependent DNA polymerase and
RNAase H activities (11, 20). All these data suggest that the I
factor could transpose by reverse transcription of an RNA
intermediate. The detailed genetic analysis that has been carried
on about the determination of IR hybrid dysgenesis allows more
advanced investigations to elucidate the mechanism of I factor
transposition that could be thereafter extended to other elements
structurally related to it.
Sequences homologous to the I factor are not restricted to the
inducer strains. Indeed, Southern transfer experiments
have
shown that defective and probably incomplete I elements occur in
both reactive and inducer strains (9). Most of them appear to be
at constant locations in various stocks suggesting that they are
old
components
of
the
genome
of
the
species. In
situ
9200
Nucleic Acids Research
hybridization
are
to
restricted
strains
sites
also
on
salivary
to
the
contain
the
gland
chromosomes
peri-centromeric
about
chromosome
15
indicates
I elements
arras, most
that
heterochromatin.
of
located
which
at
are
they
Inducer
different
thought
to
be
complete and active.
We have
reactive
cloned
and analysed
strains,
in
order
I elements
to
study
the
from
both
relationships
active and defective elements. Our data indicate
specific to inducer
strains have very well
One of them has an intact
I
elements
found
in
that
conserved
3' end but is truncated
The relationships between active
both
I
factors
inducer
and
inducer
and
between
I elements
structures.
at its 5' end.
and
the
reactive
defective
strains
are
complex, and suggest that these defective elements are not recent
derivatives
of
the
I factors
of Drosophila
melanoqaster.
MATERIALS AND
METHODS
Bacterial
Plasmids were propagated
the
inducer
populations
in E. coli strains HB101
(21), NM522
(23). Bacteriophages lambda were grown in strain C600
(24) or Q359
Enzymes and
Enzymes
(25), and
M13
in
strain
NM522.
isotopes
were
purchased
Laboratories,
and Pharmacia
[
in
strains
(22) or DH1
Research
present
~ 3 2 P ] dATP
from
Amersham
Boehringer
International,
Mannheim,
and were used as recommended
(3000 Ci/mMol) and [
New
Bethesda
England
Biolabs
by the manufacturers.
- 3 5 S ] dATP were from
Amersham
International.
DNA
preparation,
aqarose
of DNA. hybridization and
gel
electrophoresls,
in
vitro
labeling
autoradioqraphy.
Phage, plaamid and Drosophila DNAs were prepared as previously
described
(26, 23, 9) and
the other procedures
were
carried
out
as reported ( 9 ) .
Construction of libraries
The
libraries
of wliLL
and
Cha
DNAs
cloned
in
1059
were
constructed as previously described ( 2 7 ) .
The
library
digesting
of
10 Jig of
wIR1
DNA
Drosophila
in
DNA
the
and
vector
pAT153
10 |)g of
was
plasmid
made
DNA
by
with
9201
Nucleic Acids Research
BamHI and EcoRI. The resulting fragments were mixed and ligated,
and then used to tranform the strain DH1.
DNA sequencing
DNA fragments to be sequenced were subcloned into M13 mp18 and
mp1 9 vectors
(28). Templates
were
made
and
sequenced
by
the
dideoxynucleotide chain termination method (29) using [ 35 S] dATP
and were analyzed on buffer gradient polyacrylamide gels (30).
RESULTS
Cloning of I elements from inducer and reactive strains
We have cloned I elements from three libraries. The first two
were made from DNA of the inducer strain wULL
which carries the
IR
a
induced white
mutant wliLl (31). One was
random
library
produced by cloning 15 to 25 kb fragments of wULL DNA generated
by partial digestion with Sau3A in the lambda vector
1059 (25).
The other was a plasmid library obtained by cloning BamHI EcoRI
fragments of wiJLL DNA in the plasmid vector pAT153
(32). These
two enzymes do not cut within the I factor. The third library
established from DNA of the reactive strain
sane way as the random library of w I R 1
in
cha was made in the
1059.
The probes used to screen these libraries were two fragments
called 1770 and 1771 containing respectively, the left-hand
(5'end) and right-hand (3'end) extremities of the I factor (see
figure 1 ) . Among a large number of positives, we have chosen
four clones from the wULL
library in
1059 and three clones from
the w I R 1 library in pAT153 on the basis of a strong hydridization
with both
library
probes. Two
of wiELl DNA in
other
clones
were
selected from the
pAT153
because they hybridized strongly
with the 1771 fragment but did not hydridize with 1770. We have
also isolated at random 14 clones from the cha DNA library. These
hydridized either with one or both probes.
We have determined the regions of the clones showing homology
with 1770 and 1771. The results are presented in Figure 1. Some
clones have related restiction maps. This is the case for
and
9202
R508,
R502 and
R510, and
R507 and
R501
1170. We have
Nucleic Acids Research
1770
IFACTOfl |
A
BEEHEH
P*"*
' '
)
« . aw '
HH
Ic
1771
H H
H
HS EH
HH
C
"
SH
'—'
'"""
SH
H HEE
"»0» • • • • • •
H•
H E H H H
E
E
EB
>
1—>—&••••••••••••••••••••••
H
1
11171 •
E
)
E
E
S
H
'
^
H E
—' '
HE H
S
B
1M70 aaf
1Kb
H
'
FM
E BE 3 E 8
*—*-* —'
SB
EH
8 E E
E
E
B
m{ "
WO)
BH
BH
E
H
S
U B 0 4
E E H
*—^kmmi
8 9 8 H
S
EH
H H H
H
BEE
1H50S
B
B
pliMI
H
1
•
E HH
^ ^
B
l_ -9m •
H
E E
S
Mm
Ha
f
P
pi,«7
HBH|HBBBBB|BB
E H HH
S E E
f\}
MEW
H
E
H HH H
SH
E H •
E
E
f
V<
fH
HS
H
H
B^EEE; HH E H
B
I—I^HB^BBBBBJ
H
."
mf
lflBIO
B
B
E B S SH B HK^
H
H
pliM
H
H
MB07
i.i
H
BHE H
EH H B
u»»
H
E
E
J'I i i i T 4 M ^
p
H
E-SEB
E
H
MS11
B ' SH
)
B
H
UB11
HHBE E
B
HSE E
a
HSE E
H
EH
S
US13
UB14
B
H HE H
HE
Fig. 1. Structure of clones containing I elements from inducer
and reactive strains. The figure represents the restriction maps
of the clones from the libraries of the w I M stock in 1059 (A)
and pAT153 (B), and of the Cha strain (C). To the top of the
figure is given a map of the I factor indicating the positions
of ORF1 and ORF2, and of the sequences corresponding to the 1770
and 1771 probes (see text). The back boxes represent repeated
sequences other than I, the hatched boxes unique sequences and
the thin lines sequences homologous to I factor DNA. When it has
been possible to determine the orientation of I elements, this
is indicated by arrow heads corresponding to the 3' ends. B :
BamHI ; H : Hindlll ; E : EcoRI ; S : Sail.
verified that the sequences
flanking the I elements contained in
similar clones cross-hybridized.
similar
and are probably
genome. As
R507 and
and w-IiLl this confirms
Therefore, these I elements are
located
1170 come
in the same
from
region
the unrelated
of
the
strains cha
that reactive and inducer strains contain
common I elements.
I elements of reactive strains are flanked by repeated
He
have
determined
whether
the I elements
sequences
we have
isolated
were inserted in unique or repeated sequences. Various digests of
the 23 clones were hydridiied to total genomic DNA of the strain
from
which
they
were
obtained
(data
not shown).
The results.
9203
Nucleic Acids Research
h
Ss
h
3 * 1 2 3 4
3 * 1 2 3 4
H
16.9 K b — - •
11.5Kb-
1234
^ ;
«•«-
-
•
11.1Kb—
8Kb-
2.6Kb-
- « - • -
3.5Kb-»
(Pl—M)
Fig. 2. Hybridization of unique sequences flanging I elements to
digests of DNAB from various Drosophlla strains. The 2 kb
EcoRI, 1.5 kb EcoRI-Sal and 1.9 kb EcoRI-Hindlll fragments from
1158, 1175 and pI166 were eubcloned in the plasmid pAT153 and
called pAi84, pA187 and pA186 respectively (see figure 1). They
were
used
as probes to hybridize
in Southern
transfer
experiments to digests of DNAs from clones
1158, 1175, pI166
from the stock, wiill and from four other unrelated Drosophila
strains. Two of these strains were the Canton-S (1) and I. (2)
inducer stocks, and the other two were the Paris (3) and Cha (4)
reactive stocks. (A) BamHI digests hybridized with pA184. (B)
Sail digests probed with pA187. (C) BamHI-EcoRI digests probed
with pA186. The 3.5 kb fragment observed in the track of pI166
corresponds to plasmid DNA.
given in Figure 1 , indicate that six clones from the inducer
strain and all 14 clones from the reactive strain contain mostly
or entirely repetitive sequences. This is in agreement with the
fact that both inducer and reactive strains
have I elements
located in peri-centromeric regions and these would be expected
to be associated with other repeated sequences.
Only three clones, 1158, 1175 and pI166, that come from the
inducer stock contain single copy DNA and therefore are probably
located
on the chromosome arms. We have confirmed this by
hybridizing unique sequences flanking each of these elements to
salivary gland chromosomes. The results indicate that they map on
the arms of the chromosomes in regions 82, 98 and 45 respectively
(data not shown).
He have tested DNAs from several strains for the presence of I
9204
Nucleic Acids Research
elements at these sites. The DNAs of the three clones, and of
various
inducer
appropriate
and
restriction
transfer experiments
elements.
reactive
The
enzymes
stocks,
and
with the unique
results
arc
shown
were
digested
hydridized
sequences
in
Figure
in
Southern
flanking
2.
In
with
each
the I
case
fragments of the same size are observed in the clone and in w I R 1
DNA. They are replaced by shorter fragments in all other strains.
The differences in size are 5.4 kb for
for
pI166.
This
suggests
that
these
1158 and
elements
1175 and 3.1 kb
have
recently
transposed in the genome of the stock wULl are the correct size
to be full-length
1175 and pI166, w
elements, except
IR1
for pI166. In the case of
DNA produces fragments with and without the
I elements (Figure 2B and C ) . This is not very surprising since
in situ hybridization experiments to salivary gland chromosomes
have shown that numerous sites in
this
stock are polymorphic
for the presence of I elements (unpublished observations).
I elements specific to the Inducer strain have a structure
closely related to that of the complete I factors
He have analysed in more detail the structure of the I elements
contained in clones 1158, 1175 and pI166. The elements present
in 1158 and 1175 appear to possess the characteristic internal
restriction fragments of the I factor included between the Aval
and PstI sites (see Figure 3 ) . We have sequenced the first 160 bp
from the 5' end and the last 210 bp from the 3' end of the I
element contained in 1158. The results, summarized in figure 5,
confirm that it is a full-length element terminated at its 3' end
by five tandem repeats of the triplet TAA, and flanked by a 13 bp
direct repeat. All our data indicate that it is similar to
functional I factors. The same is probably true for the I element
cloned
in
1175 which
hybridizes
with
I factor
probes
1
corresponding to the most extreme 190 bp from the 5 end, and 130
bp from the 31 end.
The I element cloned in pI166 contains all the internal
fragments of I factor that lie to the right of the Hindlll site
at position 2539 (figure 3 ) . It does not show any homology with
the left-hand end of I present in 1770 (black boxes in Figure 3 ) .
Therefore, it could have an intact 3' end and a deletion starting
9205
Nucleic Acids Research
5'
AC CH
I factor °
IT \
H
C
P y
i
>
,_g
I
Xn501/J08
. J ^ H .
X«OJ«
l s t r a i n
I....
*?<^H
I?
'
v
X
'"°
p)i39
P1104
P1187
i
H I
'' " f "
t
I-
-
t?
'
H
I I 11 11 •
t*f
•
_..._
* t
loci
™""^^^
L
Ansoa
"' ^
AC CH
L
P
*
tY^r
^jk.j
Jim
AR MI b, 5 ,o
H
— ^ ^ ^
I
Rstra]n
<
'
-.
AC
_-_^l__z>
'
An50
^B309
AH311
ARSIJ
->
'9 ^
'
^ •
B J W ^ l
—Hsbb
*?
J
C
H
'
AR5V4
Fig. 3. Structure of I elements from the w I R 1 and Cha strains.
The figure represents simplified restriction maps of the I
elements contained in clones of Figure 1. They have been
established
on the criterion
of comigration
and crosshybridization with characteristic internal restriction fragments
of the I factor. In some cases the data obtained from sequencing
have been used to determine the structure of the elements. The
black boxes represent sequences homologous to the part of the I
factor lying to the left of the Hindlll site at position 1516,
the white boxes sequences homologous to the part situated to the
right of the Hindlll site at position 2539, and the hatched
boxes sequences homologous to the part of I between the two
Hindlll sites. The dotted lines correspond to deletions (the
dotted
line to the right of
R501/508 represents a DNA
substitution instead of a deletion). The positions of the
restriction
sites of the I factor
refer to the sequence
previously reported (11). A: Aval ; C: H i n d u ; E : EcoRI ; H :
Hindlll ; P : Pstl.
within the two internal Hindlll sites and removing all the lefthand part.
370 bp of the 3' end have been sequenced
identical
TAA
to those of active
repeats. We have
element
from
its 5
1
I factors
determined
(Figure 5 ) . They are
and terminate with
the base
end to the Hindlll
sequence
restriction
of
four
the
site at
position 2539. It is flanked by a 14 bp direct repeat and starts
at position 2316. It has lost all of ORF1 and part of ORF2. The
first 220 bp of its sequence are indentical to those reported by
Fawcett et al.
9206
(11) except for three base substitutions.
Nucleic Acids Research
A(682) H(1516)
H(2539)
P(4842)
Fig.
4. Heteroduplex between phaqes
R508 and
II5B.
1158
contains a complete I factor. The two arrows show the ends of
the
R508 I element. Arrow heads indicate the ends of the two
loops generated by the R508 internal deletion and substitution.
Their position and length are indicated with an asterisk on the
map of the
R508 I element when they were obtained from
sequence data instead of measurements on the heteroduplex. The
deletions and substitution are represented by white and hatched
boxes respectively. A : Aval ; H : Hindlll ; P : Pstl.
I elements in repetitive DMA show sequence divergences compared
to complete I factors
Five of the 19 clones containing only repetitive DNA have more
than one I element (see Figure 1 ) . In each case, elements on the
same clone have similar structures and are bounded by sequences
showing related restriction maps. This suggests that these
tandem
repeats
result from rearrangements involving the
sequences in which the I elements were inserted rather than from
multiple insertions of I elements within tanderaly repeated
sequences.
Most of the I elements inserted in repetitive DNA are more
different from active I factors than are I elements inserted in
unique
sequences.
They
all
contain
large
deletions
or
rearrangements located inside and/or at one or both ends (Figure
3). The presence of deletions has been confirmed by making
heteroduplexes between some of these elements and a complete I
factor. In some cases, I factor DNA from within the element is
substituted by DNA of unknown origin. A typical experiment shown
in Figure 4 indicates that the element in
R508 has a 1.2 kb
internal detetion, and that a 1.4 kb internal sequence of I is
9207,
Nucleic Acids Research
I
5
138
TAACAACAAAAA
I
1 T
'
1
[ m n i m i i l CATTAC
A 1158
217
AAAATCACTTCA
2.316
—CCTCTAATCAAT
-
|0T7
CAACAAACACTC—-
}30«
CTAGCTAATGCT
?359
-CTATCA(TAA).tumm«nt
—
)316
tattactmctn ATCAAT
pl\66
5359
CT ATCAtTAAhtaltmctHlctn
I
!3«
ARJ07
tuttgatftcuttCAGTAC
ARS12
taftamcitctctaCAGTAC
GTAGCTttac|ttffCt
CAACAAtllacaltllcgcutmactc
138
u g i g a t u t c f t f ccaccCAAAAA
1 RM1
?JS9
CTATCATAATAAATAAATAAATAAA tctttcaactmcgcgt
J.17
utuuttglutlggACTTCA-
pIlM
5)59
I'
CTATCA(TAA)
?3J9
-CTATCATAATAATAAATAAACAAATAAATAAAAAGCAAAAtlcat'tctll
-
1 R510
53S9
CTATCATAATAAATAAATAAATAAATAAAc«tlctlt«mct»CIC
pllU
?3J9
-CTATCATAATAAATAAAAAAAAAllgctmgtcimccc
Fig. 5. Sequences of the ends of some I elements. The sequences
of the ends of the I elements are shown in upper case, and the
A-rich sequences at their 3' ends are underlined. The sequences
of adjacent DNA are shown in lower case, and the target site
duplications are underlined. The positions refer to the sequence
of the I factor previously reported (11).
replaced by unrelated DNA of 0.5 Kb. This has been confirmed with
R501 , and independent
clone
containing
the same
I element as
R508.
We have
strain
sequenced
( R501 ,
both
R507
stock (pI164), and
end from
and
three
elements
R512) and one element
the 3' ends of
from
from
two other elements,
the Cha
the wiiLL
R510 and
pI163. The results, given in Figure 5, indicate that none of them
is flanked by a target site duplication.
The four elements having an intact 3' end ( R501,
R510, pI163
and pI164) have unusual A-rich sequences in this region which are
not
exactly
the
similar
I elements
to the TAA repeats
typical
seem
TAA(TAAA) n
(see Figure 5 ) .
In
these
to
of
sequences
elements cloned in
of those
Their
cloned
base
functional
I
9208
we have
R501 and
in
R510,
sequences
substitutions
the inducer
be characterized
experiments,
have
factors.
compared
found
by
at the 3' ends of
strains.
These
A-rich
the pattern
sequenced
1800 bp of the I
R507 and 440, 1050, 300 and 400 bp
R512, pI163
diverged
They
all
to active
and pI164
considerably
contain
I factors,
respectively.
from
many
that of
base
as well
pair
as small
Nucleic Acids Research
deletions and substitutions. The average percentage of homology
is 935C, additions and deletions of more than one contiguous base
being counted as one change.
By contrast, only five base pair substitutions have been
scored among 370 and 590 bp in elements
1158 and pI166 located
on the chromosome arms. Only one of them would change an aminoacid in the polypeptides encoded by the I factor. The others
either are located outside the large open reading frames of the I
factor, or do not change the sense of the codons in which they
occur. These data confirm
that
the
I elements specific to
inducer strains have a very well conserved sequence even when
truncated at the 5' end.
DISCUSSION
We have cloned and analysed I elements from inducer and
reactive strains. It appears that the structure and genomic
organization of I elements that are confined to inducer strains
are very different from those common to inducer and reactive
strains. The I elements characteristic of inducer stocks are
dispersed at various sites on the chromosome arms. Most of them
appear to be complete and probably functional I factors (9,10).
However, we have cloned an I element (pI166) which has an intact
31 end and is deleted at its 5' end. It is 3055 bp long. 5'
truncated I elements are not exceptional since five others that
were associated with yellow gene mutations induced by IR hybrid
dysgenesis have been isolated (unpublished results). This is an
additional similarity between I elements and other non-viral
retrotransposons such as F, G, LI, ingi and cin4 elements which
are heterogeneous in size and often truncated at their 5' ends.
However a large proportion of I elements characteristic of
inducer strains seem to be complete, whereas most of the members
of
the
other
families
of
non-viral
retrotransposons
are
incomplete.
The truncated I element described in the present paper, though
having a terminal deletion, is flanked by a
target
site
duplication, suggesting that it inserted as a truncated element
rather than having been deleted after insertion. This indicates
that the sequences near the left-hand end of complete I factors
are not required absolutely for integration.
9209
Nucleic Acids Research
Truncated
elements
elements,
or
truncation
being
think, that
the
could
could
be
exact
originate
from
produced
second
during
copies
the
hypothesis
of
deleted
complete
transposition
is
most
donor
elements,
the
process. We
likely.
Indeed,
if
transposition requires an RNA intermediate as it is reasonable to
assume,
it
is
probably
synthesised
from
an
internal
pol
II
promoter located near the left-hand end of the I factor in Figure
1 (11). Therefore truncated
could
be unable
elements that have lost
to produce a full-length
RNA
and
this region
could
elements. The I elements
that come from the reactive
and
common
which
are probably
have a sequence
elements
organization
that are confined
to all
heterochromatin
cases,
than one
related
some
I element,
and
restriction
maps
from
the
the
(9) and
clones
we
sequences
suggesting
got
flanking
that
are
flanked
have
strains,
that of
to inducer strains. They
in peri-centromeric
In
D. melanogaster
very different
sequences.
be dead
strain Cha
by
the I
located
repeated
contain
them
often
duplications
more
have
occured
after their insertion in these regions.
All I elements from heterochromatin contain deletions and other
rearrangements
situated
at
different
places.
Deletions
can
be internal and/or terminal. There are no direct repeats flanking
the four elements
for which we have sequence
informations
both ends. As all
of these elements have at
least
one
from
terminal
deletion, duplications of the target could have been
removed in
the
additional
same
time
as
difference between
inducer
strains
adjacent
these
which
regions.
This
is
an
I elements and those characteristic of
are
always
flanked
by
target
site
duplications.
He have sequenced the ends of four elements having complete 3'
ends. Two were
from
the Cha
reactive
strain
and
the other
two
from the wiJLl inducer stock. The latter were inserted in repeated
sequences
and
for
chromocenter. All
this
reason
are
thought
of them have an A-rich
that of the I elements that
to
3' end
come
from
the
reminiscent of
have recently transposed
in inducer
strains.
It seems therefore that there are two types of I elements. The
first
type are restricted
They are complete
9210
to the genome of the inducer strains.
I factors or have structures derived
directly
Nucleic Acids Research
from
them.
They
are
the
products
of
recent
insertions,
are
preferentially in unique sequences located on chromosome arms and
are flanked by target site duplications. They have intact
with
TAA
repeats.
exhibiting
The
various
second
type
are
3'ends
defective
I
elements
rearrangements. They are bounded
by
repeated
sequences and are located at the same places in peri-centromeric
heterochromatin
not
flanked
apparently
are
not
of both
by
reactive
target
31
complete
necessarily
site
ends,
and
inducer
strains. They are
duplications.
they
When
are A-rich
they
have
sequences
that
exactly similar to the characteristic TAA
repeats of I. At least part of these A-rich sequences is of the
type TAA(TAAA) n .
Finally, the degree of divergence between active I factors and
the
ubiquitous
strikingly
much
peri-centromeric
defective
higher
between
the D. melanoqaster
than
that
and D. slmulans
I
elements
mobile
sibling
appears
I factors
from
species. Indeed all
the internal fragments characteristic of the I factors have been
found in all strains
studied
from D. simulans
(33, 3 4 ) , whereas
none of the I elements from the Cha reactive strain exhibits this
typical restriction map. For example, the nucleotide sequences of
the
I elements
of clones
R501,
R507,
R510
and
R512
differ from that of the complete I factor by as much as 7% that
is
the order
of magnitude
alcohol
dehydrogenase
the
simulans
determined
genes
(35). We conclude
centromeric
heterochromatin
inactivation
of
They
be
could
transposable
complete
this
not
I factors
vestiges
element
from
from
are
to
D.
sequences
melanoqaster
that
the
I elements
results
of
of present-day
resulting
related
for intronic
from
I
before
and
of
D.
of perimutational
populations.
inactivation
speciation
of
of
a
D.
melanoqaster.
It is also interesting
we
have
isolated
corresponding
from
to notice
the
that none of the I elements
reactive
strain
contains
to a significant fraction of ORF2.
these elements could have resulted from
sequences
Inactivation of
the loss of function of
this ORF which is though to play a crucial role in transposition.
Six elements bounded by repeated sequences have almost all the
sequence
corresponding
characteristic
to
restriction
ORF1
(see
sites
Figure
3 ) . They
which
delimit
contain
fragments
9211
Nucleic Acids Research
conigrating
and
hybridizing
with
the corresponding
fragments of
active I factors that cover a large part of ORF1. Southern blot
experiments have shown that the 0.8 kb Aval-Hindlll fragment of I
is
also
strains
present
at
(unpublished
several
copies
in
the
data). This indicates
genome
that
of
some
reactive
I elements
in reactive strains could possess an intact 0RF1. Fawcett et al.
(11)
have
reported
that
the
polypeptlde
encoded
by
this
open
reading frame contains a nucleic acid binding domain that is also
found
in
elements
gag
proteins
of reactive
of
retroviruses.
strains produces
If
ORF1
present
such a molecule,
in
I
it could
play an important role in the regulation of IR hybrid dysgenesis.
Therefore,
elements
it
are
should
be particularly
transcribed
in
reactive
interesting
strains
to
and
study
if
if I
eventual
transcripts can encode polypeptides.
ACKNOWLEDGEMENTS
M.C. and C.V. should be considered as equal first authors. We
are
grateful
manuscript,
excellent
to
J.C.
D.
Finnegan
Bregliano
technical
for
for
helpful
discussions
assistance. This work
comments
and
has
A.
been
on
the
Lenoir
for
financed
grants from the Centre National de la Recherche Scientifique
by
(UA
360 and ATP 8304), University Blaise Pascal, Association pour la
Recherche sur le Cancer, and Fondation pour la Recherche Medical
Francaise.
•Present address: Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121,
1066 CX Amsterdam, The Netherlands
+
To whom requests for reprints should be sent
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