volume 14 Number 12 1986
Nucleic Acids Research
Structural variations In the Drosophila retrotransposon, 17.6
Satoshi Inouye, Kazue Hattori, Shunji Yuki and Kaoru Saigo*
Department of Biochemistry, Kyushu University 60 School of Medicine, Fukuoka 812, Japan
Received 23 April 1986; Revised and Accepted 23 May 1986
ABSTRACT
More than 21 members of 17.6, a Drosophila retrotransposon,
were isolated and t h e i r possible s t r u c t u r a l changes were
examined by r e s t r i c t i o n
mapping,
blot
hybridization,
heteroduplex analysis and nucleotide sequence determination of
long terminal repeats (LTRs). At least 7 members were found to
suffer with terminal or internal long deletions. No pair of LTRs
having an identical nucleotide sequence was found either within
an element or between elements. Although an i n i t i a t i o n s i t e
for the presumable genome-sized transcript of 17.6, a potential
substrate for reverse transcription on trans location, was
identified within the left-hand LTR, our r e s u l t s as a whole
support the notion that the majority of 17.6s have continued to
reside for a long period of time at their present chromosomal
loci and hence the rate of translocation of 17.6 is very low.
INTRODUCTION
17.6 i s a member of c££_ia-like t r a n s p o s a b l e g e n e t i c
elements (retrotransposons) in Drosophila melanogaster and has
been suggested to have about 40 copies per haploid chromosome
(1). In a previous experiment (2), we determined the complete
nucleotide sequence of a 17.6 element inserted in a histone gene
and found the 17.6 genome to include three long open reading
frames (ORFs) comparable in size and location with r e t r o v i r a l
gag, pol and env. We also found the second ORF of 17.6 not only
to contain the n u c l e o t i d e sequence coding for r e v e r s e
transcriptase-like enzyme but al3o to be very similar in overall
structure to the pol gene of Moloney murine leukaemia virus
(2,3). Since subsequent nucleotide sequence analyses (4-10) have
revealed the presence of similar pol-like
genes in
several
other copia-like elements in Drosophila and yeast, i t is now
quite reasonable to presume that the translocation of copia-like
© IRL Pren Limited, Oxford, England.
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elements i s generally carried out via RNA intermediates by
reverse t r a n s c r i p t i o n . In fact, Boeke e_t a_l. (11) have recently
presented more direct evidence for t h i s notion at least in the
case of a yeast copia-like element, Ty.
Here, we examined possible structural changes among more
than 21 members of a 17.6 family, representing about half the
t o t a l per haploid chromosome, to see how fast these elements
change their chromosomal positions. Although an initiation site
for the presumable, genome-sized t r a n s c r i p t of 17.6 , a
potential substrate for reverse transcription on translocation,
was identified within the left-hand LTR, our results as a whole
support the notion that the majority of 17.6s have continued to
reside for a long period of time at t h e i r present chromosomal
loci and hence the rate of translocation of 17.6, if i t actually
occurs, is very low.
MATERIAL AND METHODS
Hateria"is.
The r e s t r i c t i o n enzymes, DNase, DNA ligase and
reverse transcriptase were obtained from commercial sources.
P - l a b e l l e d nucleotides were purchased from New England
Nuclear.An oligonucleotide primer(3'GGAACTACGCCATTGGTCTAAACGG5')
for p r i m e r e x t e n t i o n was s y n t h e s i z e d using a Model 380A
synthesizer (Applied Biosystems).
General Methods.
Restriction enzyme digestions, agarose gel
e l e c t r o p h o r e s i s , subcloning of r e s t r i c t i o n segments and b l o t
h y b r i d i z a t i o n were carried out e s s e n t i a l l y as described by
Maniatis et a_l_. (12). As a cloning vehicle for sub-cloning., only
pOC9 (13) was used. The DNA probe was labelled with 32 P by
k i n a t i o n . DNA sequencing was conducted by the Maxam-Gilbert
method (14). Heteroduplex analysis was carried out according to
Modolell et al.(15).
Ph_£g_es a.£? pTa£raid£.
The o r i g i n of X h i s t l 7 . 6 has been
d~escrTbed previously (16). Other 17.6 containing phages were
screened from an EcoRI p a r t i a l l i b r a r y of D. melanogaster
(Oregon R) embryonic DNA e s s e n t i a l l y as descrTbed elsewhere
(16) .
Preparation of RNA and a primer extension experiment.
GM2
c e lTT1
l s were c u l t u r e d inlOOml D20 medium to l a t e log phase as
described
previously
(17) and c o l l e c t e d by low-speed
centrifugation. RNA was prepared from GM2 c e l l s essentially as
described by Deeley et aj_-(18). Using lmg/ml of GM2 total RNA, a
primer e x t e n t i o n experiment was c a r r i e d out as described
elsewhere (4).
RESULTS AND DISCUSSION
V a r i a t i o n j.n o v e r a l l s t r u c t u r e of 17.6 e l e m e n t s
Using a 2.1 kb (kb=»1000 b a s e p a i r s ) H i n d l l l
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1
504
L±_
t
t_
2
2J
t-LJ
SOB
t T t
511
514
516
518*
519* -
525
T
t
T
T7
536
T
t T t
T
tTt
530
5J4B
d
f
T y T
t T t O
Q t
0 0 Ttt
1
Fig. 1.
Restriction maps of cloned Drosophila
fragments
containing 17.6-related sequences along with tne locations and
deduced structures of 17.6s. The top line labelled 17.6 shows
the restriction map of the Drosophila insert in Xhistl7.6. The
fragments used for probes for hybridization are marked by three
underlines a, b and c.
T , Hindlll; • , EcoRI; + , BamHI; O ,
Clal;V SinI. In the case of Clal and SinI, only the sites within
some LTRs are shown.
, homology with probe a for detection
of LTR sequences;
, homology with probe b;
, homology
with probe c. Asterisks show the presence of plural clones
exhibiting identical restriction maps. The locations and the
deduced structures of 17.6s are schematically illustrated just
beneath the restriction map of each clone. The locations of
deletions are shown by triangles, while those of LTRs, by boxes.
The estimated lengths of the deletions are as follows; X510, 0.6
kb; X525, 0.5kb;X530, 3.3kb;X540, 1.0kb;X602, 2.2kb;X605, 1.2kb;
X534B,0.4kb.
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17.6 in Xhistl7.6 (see an underline b in Fig.l) as a probe, 50
recombinant clones were isolated from about 5x 10 phages in a
cloned l i b r a r y of Drosophila me lanogaster DNA. Mapping of
r e s t r i c t i o n enzyme s i t e s followed by Southern
hybridization
showed that these recombinant clones as a whole represent at
least 21 chromosomal loci corresponding to half the total 17.6i n s e r t i o n s i t e s per haploid chromosome (Fig.l). Since 17.6
genomes in s e v e r a l clones were found to be a r t i f i c i a l l y
truncated at i n t e r n a l EcoRI s i t e s , t h e i r structures were not
analyzed furthermore except for that in X526. The precise
locations of the ends of 17.6s together with those of possible
i n t e r n a l deletions were determined in the majority of the
remaining clones by mapping the cutting s i t e s of r e s t r i c t i o n
enzymes, heteroduplex a n a l y s e s and/or p a r t i a l nucleotide
sequencing. The structures of t h i r t e e n 17.6 elements thus
determined are schematically shown in Fig.l.
About half these t h i r t e e n elements
( those in X510,
X525,X530, X534B,X540, X602 and X605) were each found to have a
large deletion, internal or terminal, with respect to the
authentic 17.6 in Xhistl7.6. The distribution of these deletions
appears random. Some examples of electron micrographs of
heteroduplex molecules between
Xhistl7.6 and other clones are
shown in Fig.2.
In the case of 17.6 in X605, which contains
a 1.2kb-long i n t e r n a l deletion, the duplex region with the
right-hand LTR (see Fig.l) was occasionally observed to be
single-stranded (see the regions with large arrowheads in
Figs.2D and E). Though the reason for t h i s is unclear, i t i s
unlikely that t h i s structural change a r i s e s from possible
variation in nucleotide sequence
in that particular region of
17.6 in
X605. In fact, except for a 50bp(base pair)-long,
highly reshuffled region located immediately next to the
deletion s i t e (Fig.3), only one nucleotide change could be found
in a t o t a l of 1100 nucleotides flanking the deletion s i t e in
17.6 in X605 (data not shown). 17.6 in X534B contained a very
short (about 200bp-long), internal deletion which could not be
clearly visualized by electrontnicroscope (Fig.2B): the location
of t h i s short deletion was determined by fine r e s t r i c t i o n
mapping not shown here.
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Fig. 2.
Electron micrographs of heteroduplex molecules between
the DNA from a cloned phage having 17.6-related sequence and
Ahistl7.6 DNA.
A, X530; B, X534B; C, X540; D and E,
two
different examples of X605; F,X634. M, marker DNA (pBR322 with
a molecular weight of 4.4kb); D, double-stranded region; S,
single-stranded region. Large arrows in D and E show two
variations in the heteroduplex molecules of Xhistl7.6 and
X
605. Arrows labelled X indicate stem and loop structures found
in the DNA from
X605. The lengths of the duplex regions
expected to include 17.6 are estimated as follows. A, (4.1 ±
0.2)kb. B, (7.0 ± 0.3)kb. C; (5.3 ± 0.3 and 1.1 ± l.OJkb; D,
(3.9 ± 0.2)kb; E, (3.8 ±0.2 and 2.5 ± 0.2)kb; F, (7.1 ± 0.3)kb.
(A slightly reduced value in F with respect to the authentic
17.6 is at least partly due to a high degree of variation in
nucleotide sequence at the 3'terminal regions of LTRs (see
Fig.3) .)
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605
17.6
AAUCTTTTCTTGGACTUAI AGGATATTATCGTAAATTTATTXCAAACTTTGCAGACAT
AAGCTTTTCTTGGACTGACAGGATATTATCGTAAATTTATTCCAAACTTTGCAGACAT
AGCCAAACCCATGACTAACTGTTTAAAAAAGAACATGAAAATTGACACTACCAACCCAGAATAT
AGCCAAACCCATGACTAAGTGTTTAAAAAAGAACATGAAAATTGACACTACCAACCCAGAArAT
CCCGA]
CCCGACTTTI
rr>
12|l lit
IGAGATGCOT'GGAGACTGAGGAAGTCGAAT rGCAGC
TTAACA(;AACAAAAACTGGTCT(K3CGGACATAGAAAGACTACATAAAACAATTAATGAAAAGAT
TTAACACAACAAAAACTGGTGTGGCGGACATAGAAAGACTACATAAAACAAT1AATGAAAAGAT
Fig. 3.
Nucleotide sequence variation near the deletion site
in X605. Horizontal lines with arrowheads and numbers show the
locations and sizes of direct repeats. Note that, although
repeats 1 and 3 are located at identical loci in the 17.6s of
X605
and
Xhistl7.6, respectively, their sequences differ
considerably from each other.
One additional finding that should be mentioned is that the
restriction map of 17.6 in X602 extensively differs from those
of other 17.6s including the authentic 17.6 in Xhistl7.6. That
is, 17.6 in X602 lacks all the conserved cutting sites of
EcoRI, BamHI and Sail, although no deletion/insertion was
observed in the corresponding regions by electronmicroscopy (see
Fig.l). Because of the deletion of its left-hand LTR-containing
region, 17.6 in X602 must be a kind of dead element at least
with respect to translocation. There is the possibility that
this 17.6 became a dead element a very long time ago, followed
by introduction of the observed variations in the restriction
sites.
Variations j^n nucleotide sequences of LTRs.
To ascertain the degree of variation at the level of
nucleotide sequence, we determined the complete nucleotide
sequences of the right-hand LTRs of nine 17.6 elements. As shown
in Fig.4, these nucleotide sequences are similar but not
identical to one another. Since we determined the LTR sequences
of about one quarter of 17.6s in a haploid chromosome, the above
finding that there is no pair of 17.6s with LTRs having
identical sequences may suggest that the last duplication and
insertion of the majority of these 17.6s occurred long ago so
that even newly inserted 17.6a have become the targets for
additional mutations with respect to parental ones.
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In addition to many 1-3 bp long nucleotide changes, several
long (6-34bp) insertions and deletions were detected, most
explainable by duplication of neighbouring sequences or deletion
of already duplicated sequences, as indicated by the horizontal
lines with arrowheads in Fig.4. In particular, the 3'terminal
one third of the LTR of 17.6 in
X634 was found to have an
exceptionally varied nucleotide sequence with only 45 % homology
with the counterpart of the authentic 17.6 in
Xhistl7.6. As
described below, this highly varied region corresponds to R + U5
in the LTR of the authentic 17.6. To our knowledge, no other
copia-like elements have been shown to include a member having
LTRs with such a high degree of variation (19-23). However, this
unusual variation in LTR- of
17.6 in
X6J4 appears not to
influence the sequence-specific insertion of 17.6 (24), since,
as shown previously (24), the insertion of this 17.6 resulted in
a duplication of ATAT as also in the case of all other 17.6
elements.
In addition to these, 9 changes in nucleotide sequences
were found to be shared by plural elements (Fig.4) , indicating
17.6 LTR to possibly contain many polymorphic nucleotide
sequences having equal biological activity. Since combinations
of polymorphic changes among 17.6s appear to be rather random,
the present distribution of combinations of polymorphic changes
in 17.6s may possibly result in part from either cumulative
recombinations between LTRs of different 17.6s or plural
independent mutational events or both.
The nucleotide sequences of both LTRs of a copia-like
element must be perfectly corrected at the time of insertion, if
its translocation is carried out via an RNA intermediate by a
mechanism similar to that in retrovirus (25). In fact, in the
case of copia, such a phenomenon has been reported (19). To
examine this possibility in 17.6, all or some of the nucleotide
sequences of the left-hand LTRs of five 17.6 elements were
determined and compared with those of their right-hand
counterparts.The results shown in Fig.4 are consistent to some
degree with our expectation, since all the 17.6 elments examined
here as well as the authentic 17.6 in Xhistl7.6 have at least a
few minor base differences in both LTRs. In particular, the LTRs
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17.1
ACTGACATATTCACATACAAAACCACATAACATAGAGTAAACATATTGAAAAaXC--
M5
-CAT-ACGTAAACAATAAGTGACCACCATGCTAATGTGGATCAAA
-C-
134
534B
T
T
T.
T.
:«.:::•::•?:.
..fl.». . .
. . ..
]
C-
jf.
/
A.A
•
A . A .
.A.A.
17 a TAACAAAAATATCCACTCTOCATTTTC- •03
T.CA
cTTT-S34
T
5J4B
CT
Q
53U
540
S2fl
314
SU
G
t.*...A.A
m"- : • g :
-ACAaXCC-ATACTCTATGCC-ATCTGCCCAGTATGCATTCTAATAAACAAATTCTTTC .T
CAG. G . .
CAG..G.
•:.::
.. .c
.CA.. . . .c
. . .c
T .CA.. . . . c
334S
341
17.8
•OB
T .CA..
-B-CA
...c
C
CAG..G..
.CAG - G . . .
:*.?
-
TTACCO>TTCnOTAAA<^UUTCTTAAAcicTlgCTCCTCTCTCTgAaarrrcTCCTCC
-
-
ACTTAAC-AATCCAAGA
R+U5
420
b
GAACGTTAATCAATAAAC
TCATTTGA
. . .GT
TTTGAI 11 IlL
CATTTCATTTm
TCATTTGATTTTlT
AC...AATC...A.C
AC. . .AATC.. & C
AC...AATC..X.C
TCATTTGA1111C
P- ^ T l
A C . . AATC . . A . . C
AC...AATC...A..C
AC...AATC . . A . . C
T-
S- - - - -H5341
529
514
533
HI
53«a
S40
A..A.AA..GA'
A..A.AA..SA'
..A.AA..SA'
..A.AA..SA'
..A.AA..SA'
..A.AA..CA'
..A.AA..GATT.C
-.A...CT
-
T-
S^l
-.A...ST
-.A...ST
T- . . . . I
T-.
-.A...CT
-.A...CT
- . A . . .GT
-.A...CT
TTTT-
A
AAAACTCAATT
AAAACTCAATT.. . .
AAAAC1QUTT.. . .
G.C.
G.C.
C.C.
G.C.
G.C.
G.C.
G.C.
.TTCOSA...G.C
.TTCCOA...C.C
.TTCCSA...G.C
.TTCGGA...G.C
.TTCSCA ..G.C
.TTCGOA...G.C
.TTCCSA ..G.C
US
at
514B
523
514
in
5U
33(0
...C..TTTATT.C...C.TTTATTT.C
...C..TTTATT.C...C.IlBllll.C
.. .c..TTTATT.C. . . c . r W c
. . .C. .TTTATT.C. . .C.mATTT.C
...C..TTTATT.C...C.TTTATTT.C
...C..TTTATT.C...C.TTTATTT.C
Fig.
4.
The nucleotide sequences of LTRs of 17.6s from
various clones. Capital letters on the line labelleH 5y 17.6
show the nucleotide sequence of the left-hand LTR of the
authentic 17.6 in
Xhistl7.6, whereas those on other lines
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exhibit the sequences of the right-hand LTRs of the 17.6s from
various clones examined in the present research. Small letters
appearing above these sequences indicate nucleotide sequence
changes observed in the opposite LTRs of the same elements. Note
that the complete nucleotide sequences of the left-hand LTRs
were determined in 17.6s in X605, X634 and X508, while only
the 5'terminal one third and two thirds of the left-hand LTRs
were sequenced for 17.6s in X535 and X538B, respectively. Bars
indicate deletions, and dotted lines the absence of nucleotide
change with respect to the left-hand LTR of the authentic 17.6.
Boxes indicate variation in the nucleotide sequences from the
left-hand LTR of the authentic 17.6. Direct repeats and
locations of RNA starts are indicated by horizontal and L-shaped
arrowheads, respectively. Dots in some horizontal arrowed lines
denote mismatches and X identifies a particular repeat discussed
in the text. A large lozenge and triangle, located respectively
at 450 and 536 nucleotides from the 51 end, indicate the
locations of Clal and SinI characteristic of 17.6 LTR.
of 17.6 in X538B was found to have many right-left differences.
However, at the same time, that the degree of difference in LTR
sequence within an element is much less than that among
different elements in most cases should be mentioned. Note the
right-left difference in LTR sequence in 17.6 in X634 having an
unusually high degree of variation, is also very small. We
interpret these results as indicating that 17.6 possibly has,
b a s i c a l l y , the a b i l i t y to c o r r e c t LTR s e q u e n c e s on
translocation, but since they may have resided for a long period
of time at the present chromosomal loci, the LTRs of the
majority of 17.6s have incurred mutations, the number and extent
of which depending on the duration of this period.
Variation in RNA start sites from within left-hand LTR.
Our previous nucleotide sequence analysis of the entire
17.6 element (2) showed the direction of transcription of all
three major open reading frames of 17.6 to be from left to right
in the case of orientation of 17.6s as in Fig.l. The genomesized RNA of copia was also shown to be transcribed from within
LTR (4,27). To see if transcription of the 17.6 genome starts
from within left-hand LTR, all the RNA extracted from Drosophila
tissue cultured cells (GM2 cells) was examined by primer
extension, using a 25 nucleotide-long synthetic oligonucleotide
complementary to the region P in Fig.5 as a primer. In Fig.6,
seven intensive bands along with many faint ones and smear in
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297 5'_
17.6
i
297
GTTCAAATACT i A G G C T T c T C C T C A l M T C A A T T T G C A T T T i
17 6
AAGCAAGCAf AliAGGCTTCTCCTCA r T T T C A C T T T C A T T T
297
CTTAAIJCTGAGATCCAAAO
17.6
CTTAAGCTGAACGTTAATC AATAAA CAACACAATCGATACCGAAATTT
JTCGTGAAACTATTTCTCCTAAA ( 3 3 2 )
I 401))
Fig. 5.
Locations of RNA s t a r t s and n u c l e o t i d e sequence
conservation between 17.6 and 297. The locations of RNA s t a r t s
a-g of 17.6 (see Fig.6 ) a n d T h a t of the major RNA of 222
(unpublished observation) are shown by vertical arrowheads. Wavy
l i n e s i n d i c a t e host DNA, while the boxes show the s i z e and
l o c a t i o n s of LTRs. A bar l a b e l l e d P i n d i c a t e s the l o c a t i o n of
the primer used for primer extention (region P; nucleotides 542547 from the 5'end of the a u t h e n t i c 17.6 (see Fig.l of ref.
2).).
Triangles
show t h e l o c a t i o n s of t h e
putative
polyadenylation s i g n a l s . The nucleotide sequences of 17.6 and
297, d e l i m i t e d by broken l i n e s , are shown in the lower h a l f .
Note t h a t the l o c a t i o n of RNA s t a r t b of 17.6 and t h a t of the
major 29 7 RNA are i d e n t i c a l to each other
within a l i m i t of
e r r o r . Boxes A and B i n d i c a t e the nucleotide sequences assumed
in a previous experiment to correspond to the Hogness box and
polyadenylation signal of avian leukaemia-saroma v i r u s (1),
while box X shows the sequence conserved between 17.6 and 2 9 7.
As shown by arrowheads X in Fig.4, t h i s conserved sequence Fs
repeated within the 17.6
LTR. A s t e r i s k s show the homology
between 17.6 and 297 sequences.
the high molecular-weight region can be seen. Since t h i s banding
p a t t e r n did not change with the further a d d i t i o n of r e v e r s e
t r a n s c r i p t a s e (data not shown), 17.6s may p o s s i b l y have
heterogeneous RNA s t a r t s i t e s in or near left-hand LTRs.
As summarized in Fig.7, RNA s t a r t s , c-g, are located near
or w i t h i n a 9 n u c l e o t i d e - l o n g canonical sequence, CCATTTATT,
whereas RNA s t a r t b i s localized in the very v i c i n i t y of TCAGT,
a sequence proposed as being near the major RNA s t a r t s of
v a r i o u s Drosophila c o p i a - l i k e elements including mdgl
(26).
Since a p u t a t i v e polyadenylation signal AATAAA i s located
upstream of RNA s t a r t s c-g for a l l LTR sequences presented in
F i g . 4 , t h e s e s i t e s do not appear t o p a r t i c i p a t e in t h e
transcription of the genome-sized RNA of 17.6 expected to serve
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160
147
122
110
J
76 - « •
67 J
• e
• g
Fig. 6.
A primer extention pattern of 17.6 RNA.
Horizontal
arrowheads labelled by alphabetical letters show the major RNA
starts of 17.6 in or near the left-hand LTR. Minor RNA starts
are indicated by dots. The range of left-hand LTR is shown by an
L-shaped arrowhead. The markers used were HpalI digests of
pBR322 (4).
as a substrate for reverse transcription on translocation. RNA
start a .located upstream of the 5'end of the left-hand LTR of
the authentic 17.6, must be the strongest initiation site for
the transcription of 17.6 from an outside promoter.
Similar
transcription from outside of LTR has been found in the case of
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17.6 c
] 7.6 d
17.6
AATTATTTfTTI
efg
17.6 b
297
TTtpSUDCTTA
»dg 1
Fig.7. Consensus sequences near RNA starts. Deduced consensus
sequences are enclosed by boxes. Thick bars with L-shaped
arrowheads indicate the uncertainty in determining RNA starts.
The RNA start of mdgl is as shown by Arkhipova et. al.(26).
copia (27). RNA start b is located within a region previously
proposed as corresponding to Hogness box of avian leukaemiasarcoma virus (1). In Fig.5, the location of RNA start b is
within the region corresponding to that with the major RNA start
of 29 7, a sibling of 17.6, within a limit of error. Since it is
the only major RNA start located within 17.6 LTR and upstream of
a putative polyadenylation signal (see box B in Fig.5), RNA
start b is tentatively concluded to be the initiation site of
transcription of the 17.6 genome RNA expected to be reverse
transcribed. It is also suggested from these results that, as
with copia (4), LTR of 17.6 can be divided into three regions
U3, R, and U5 and the location of the boundary between U3 and R
+ U5 is as indicated in Fig.4. (Note that this location differs
somewhat from that proposed on the basis of the homology with
the avian retrovirus (1).) However, the above findings do not
necessarily mean that the genome RNA of 17.6 is always reverse
transcribable for its
translocation. In fact, no change was
found in the blotting hybridization pattern of 17.6 among DNAs
extracted from GM2 cells cultured separately for more than two
years (unpublished observation).
In this connection, it should perhaps be mentioned that a
14-nucleotide-long sequence, GAGGCTTCTCCTCA,
is present 20-30
nucleotides upstream of both 17.6 RNA b and the major RNA of 297
(see box labelled by arrowed underline X in Fig.5). Because of
its location, this sequence is likely related to the
transcriptional regulation of these RNAs. Interestingly, the
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same sequence is repeated in the 5'-upstream region in the case
of 17.6 and is the longest in the repeated sequences found in
the LTR of the authentic 17.6 (see arrowed underlines X in
Fig.4) .
ACKNOWLEDGEMENTS
The authors express their appreciation to Prof. M. Sekiguchi for.
his critical comments. This work was supported in part by grants
from the Ministry of Education, Science and Culture, Japan to
K.S.
T o whom correspondence should be addressed
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