Download Cloning and analysis of the mobile element gypsy from D. virilis

Nucleic Acids Research, Vol. 19, No. 4 913
Cloning and analysis of the mobile element gypsy from
D. virilis
Lev J.Mizrokhi* and Alexander M.Mazo1
Laboratory of Biochemistry, National Cancer Institute and laboratory of Molecular Genetics, National
Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892,
USA
Received September 26, 1990; Revised and Accepted December 20, 1990
GenBank accession no. M38438
ABSTRACT
The homologue of the Drosophila melanogaster mobile
element gypsy was cloned from the distantly related
species D. virilis. It has three ORFs highly homologous
to those of the element from D. melanogaster. gypsy
from D. virilis appears to be actively transcribed and
is capable of transposition. Comparison of the
untranslated regions of both elements revealed
conserved sequences including those which had
previously been demonstrated to be important in
transcription regulation. Distribution of gypsy among
the different strains of D. virilis and different species
within the D. virilis group was analyzed. Possible
involvement of horizontal transmission in the process
of spreading and evolution of gypsy is discussed.
INTRODUCTION
The mobile element gypsy was originally cloned from D.
melanogaster (1). It belongs to the LTR-containing class of the
retroelements (2) and is responsible for a number of mutations
described in D. melanogaster (3). gypsy appears to be the best
studied LTR-containing mobile element in Drosophila. Its
structure (4) and regulation of transcription (5) have been
investigated in great detail as well as the phenomenon of
suppression of its mutations (5, 6) and transposition memory (7).
The distribution of gypsy within the Drosophila genus has also
been investigated previously (8). It was found by Southern
hybridizations with the entire gypsy DNA as a probe that genomes
of some species—D. virilis, D. busckii, D. pinicola and few
others—have some homologies with the D. melanogaster element.
However, the extent of the homologies and the relatedness of
these sequences to gypsy were unclear.
In this paper we report cloning and sequencing of gypsy
homologues from D. virilis. The analysis of the nucleotide and
putative amino acid sequences has shown that D. virilis indeed
possesses highly homologous mobile element which is capable
of transposition and is actively transcribed. This element is widely
distributed within different strains of D. virilis and other species
• To whom correspondence should be addressed
of the same group. We speculate that the existence of the highly
homologous gypsy in distant species might be a result of the
horizontal transmissions of this element.
MATERIALS AND METHODS
Drosophila strains
Drosophila strains were obtained from Bowling Green (Ohio)
stock center except for strain '9', which was isolated by Dr.
M.Evgen'ev in a vineyard in Batumi, USSR. The cell culture
of D. virilis was a gift from Dr. I.Schneider.
DNA preparation, electrophoresis and hybridization
DNA was prepared as described previously (7), digested with
HindHI, electrophoresed in 1 % agarose and transferred to a nylon
filter. Hybridization was performed in 6x SSPE, 10 X Denhardt,
100 /t/ml sheared salmon sperm DNA, 0.5% SDS for 24 h at
55°C. The filter was washed three times for 15 min in 2 x SSC,
0.5% SDS at 37°C.
Library construction and screening
Genomic DNA from adult D. virilis (strain '9') flies was partially
digested with Sau3A, ligated and packaged into phage EMBL4.
Hybridization conditions for screening were the same as described
above.
DNA sequencing
Nucleotide sequence was determined for both strands by using
the dideoxynucleotide chain-termination method (9) for double
stranded DNA and Sequenase™ (United States Biochemical).
For sequence comparisons, the programs from the University
of Wisconsin, Genetics Computer Group (UWGCG) were used
(10).
RNA preparation and hybridization
Poly(A) RNA from adult flies (strain '9') were purified as
described (11). Northern hybridization was conducted under the
same conditions as for the Southerns (see above). Washing
conditions were O.lx SSC, 0.5% SDS at 50°C.
914 Nucleic Acids Research, Vol. 19, No. 4
RESULTS
Structure of gypsy from D. virilis and its similarity to gypsy
from D. melanogaster
The D. virilis genomic library was screened with the probes
derived from regions corresponding to the first and the third
ORFs and LTR of gypsy from D. melanogaster. Two clones
containing presumably full-length copies were picked for
subcloning and sequencing. The nucleotide sequence of one of
the clones was determined completely. Only the untranslated
regions of the other clone were sequenced. No differences were
found between the two copies in the sequenced regions. The
nucleotide and putative amino acid sequences of gypsy from
D. virilis are presented in Fig. 1A. This element consists of 7156
bp and contains two 411 bp long LTRs. In the central part of
the element we have found three ORFs whose sizes, sequences
and locations correspond to those of the D. melanogaster element
(Fig. IB). The very high homology between amino acid
sequences of the corresponding ORFs from both species (see Fig.
IB) did not permit to define any conservative motifs in addition
to those found earlier (4). In contrast, comparison of the
untranslated regions revealed distinct conservative sequences.
Comparison of the sequences of the gypsy LTRs (Fig. 2A)
disclosed two long conservative domains in the 5' part of the
LTR and also a short conserved sequence 18 nucleotides upstream
of the site of the transcription initiation. Sites of transcription
initiation and termination were determined previously for the
D. melanogaster element (12). It was also noted that the sites
of initiation of the several LTR-containing mobile elements are
located within or immediately after the sequence TCAGTPy
{gypsy RNA begins at A and G in this sequence). We did not
find the same sequence in the appropriate region of D. virilis
gypsy. At the same time sequences surrounding the
polyadenylation signal and the site of termination are well
conserved in D. virilis gypsy. There are no other conserved
sequences in the LTR downstream of the site of initiation;
moreover, this region in the D. virilis element is significantly
( — 70 nucleotides) shorter (not shown). This results in the
difference in the lengths of the LTRs (see Fig. IB). Two
sequences important for the cycle of replication of retroelements,
the tRNA binding site adjacent to the 5' LTR (Fig. 2B) and the
oligopurine stretch adjacent to the 3' LTR (Fig. 2 Q are conserved
in D. virilis gypsy. Conservation of these sequences indicates that
the new element is capable of replication and that the same
tRNA, Lvs is involved in the priming of the synthesis of the first
strand.
MELANOGASTER
UOM0LOG?
m
—
VIRILIS
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1
451**
IOIOH
451**
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470a*
I
I
I
Bi
.•:•:•«.•:•:•.•:•
ORT2^ 1
479**
BB
OR73
A.
AGTTAAOACTAACCATAAAIATATTGC. . CCTACCAATTAJLCT.
kCOCTCCACGCOAJU
I I I I I I I _,
47 gTATCHCHTGCTOi
49
97 UTGCCI
I I I I
99
CTaAaciTccT.
147 GCTGCMCAUTGCTGAO'
I I I I I I
TJCCGAC
OCATT..CCTCGACgCTA
11 I I I
TTGTGGCGGCGCCA'
I I I
123
222
I 1111111 I
CCTJUTTTTTTAa 221
I
223 TJkTTOTCTTCTACTCAOTTCJUUU
I
II I I
II
Mil I I
CTTO 272
II
II II
IAM
222
• TATACTO 26S
273 CTCCOCCTC*TTOCCOTTAAJU^TC«TOTTCITATTIAC»»TC»»»
I I II II
I I I I I I I I I I II
I I III II I
I I I
269 CTCCOa. .CAnOCCaATAAAACATATCTTTaSTAATAmCTOQU
319
313
B.
469
I I
I I I I I I I I I I I I I I I I I
39S
c.
6971
6727
Figure 2. Comparison of the nucleotide sequences of the LTRs (bold letters)
and adjacent regions of gypsy from D. melanogaster (top line) and D. virilis (bottom
line). A. Nucleotide sequences of the 5' parts of the LTRs. Tworegionsof
extensive homology are shadowed; sites of initiation and termination of transcription
of D. melanogaster gypsy are indicated by horizontal arrow and vertical
arrowheads, respectively (12); polyadenylation signal is underlined. B. Nucleotide
sequences of gypsy adjacent to the 5' LTRs; tRNA(Lyl binding site is underlined.
C. Nucleotide sequences of gypsy adjacent to the 3' LTR; polypurine stretch
is underlined.
7kb
482bp
41 lbp
/
H
Figure 1. General comparison of the elements from D. \irilis and D. melanogaster.
The amino acid sequence of the putative proteins encoded by the three ORFs
is shown below the nuclcotide sequence, the percentages of homology are indicated
for identical amino acids. The region homologous to the region containing the
enhancer in gypsy from D. melanogaster is underlined; the LTRs are double
underlined. For the homology between LTRs see text and Figure 2. Abbreviations:
Pr, protease; RT, reverse transcriptase; End, endonuclease.
Figure 3. The gypsy element is transcribed in D. virilis. Northern Hot containing
5 mg of poly(A)-containing RNA from D. melanogaster and D. virilis adult flics
was hybridized with a randomly labeled internal fragment of gypsy from D. virilis
(EcoRI-Hindm fragment, nucleotides 4197-5975 on the Fig. 1A).
Nucleic Acids Research, Vol. 19, No. 4 915
Analysis of the region located between 5' LTR and ORF1
revealed conservation of the sequence that was shown earlier to
act as an enhancer of D. melanogaster gypsy (5) and to bind the
product of the suppressor gene su(Hw) (5, 6). In gypsy from both
species, this domain is comprised of the motif
5'PyPuT/cTGCATAc/TPyPy which is repeated 12 times (8 in
some copies of D. melanogaster gypsy, see ref. 5). It seems very
likely that this region acts also as an enhancer in D. virilis and
binds a product of the same gene, su(Hw). A poly(A) block is
also present in both species upstream of the enhancer. The
conservation suggests that it is functional; perhaps it imparts some
flexibility to the DNA necessary for the functioning of the
enhancer. The sequence between the poly(A) block and the
enhancer, which provides negative regulation of transcription in
gypsy from D. melanogaster and probably interacts with the
product of the su(f) gene (5), is substituted in D. virilis gypsy
DC
HI
LU
H
5kb
-4kb
-3kb
2kb
1.6kb
AM TX
NO
VIRILIS PHYLAD
LU VI
LT MO
T
MONTANA PHYLAD
VIRILIS GROUP
Figure 4. Hybridization of the D. virilis gypsy DNA to genomic DNAs from
different strains and cultured cells of D. virilis and from other species of D. virilis
group (top) and the genealogical tree of the D. virilis species group (bottom).
The 74Obp Hindlll-Aval fragment (nucleotides 5975-6712 on the Fig. 1A) was
used as a probe. Because this fragment is not confined between internal Hindlll
sites, it gives different bands for different copies of the element when hybridized
to Hindm digests of genomic DNA. The following strains from Bowling Green
collection were used (stock number and origin are in parentheses): VIR1-VIR4,
D. virilis C9\ Batumi, USSR; 1051.0, California; 1051.9, Japan; 1051.51, Chile);
VIR C, D. virilis cell culture (gift from Dr. I.Schneider); LUM, D. lummei
(1011.1, Moscow, USSR); LIT, D. littoralis (1001.0, Switzerland), MEL, D.
melanogaster (Oregon R). The tree was drown in accordance with (13);
abbreviations are: AM, D. americana; TX, D. texana; NO, D. novamexicana;
LU, D. lummei; VI, D. virilis; LT, D. littoralis; MO, D. momana.
by the motif GTAAA repeated three times. Whether this domain
binds some protein(s) and regulates gypsy transcription in
D. virilis is unknown.
Transcription of gypsy in D. virilis
Northern blots with poly(A) RNA from D. virilis and D.
melanogaster were probed with DNA of the D. virilis element
(Fig. 3). The results of this experiment show that gypsy is actively
transcribed in at least adult D. virilis flies. This implies that gypsy
from D. virilis possesses all sequences essential for its
transcription.
Distribution of gypsy in the D. virilis strains and in other
species of the D. virilis species group
We analyzed the distribution of gypsy within different strains of
D. virilis from various geographical regions. Southern
hybridization of genomic DNAs from these strains with a DNA
probe from D. virilis gypsy showed that this element is present
in approximately the same numbers of copies in all the strains
and the cultured cells tested. (Fig. 4). The experiment was
designed such that elements from different chromosomal locations
should give different bands on the autoradiograph (see legend
for Fig. 4). Thus, one can conclude from the results presented
in Fig. 4 that there are variations in the location of gypsy in
different strains. The same observation has also been made from
in situ hybridization experiments (M. Evgen'ev, personal
communication).
The presence of gypsy in other species of the D. virilis species
group was also analyzed. For this experiment we chose
D. lummei, which is, so far as is known, the species closest to
D. virilis, and D. littoralis, also from D. virilis species group,
but from a different philad (see bottom of Fig. 4 and ref. 13).
We found that elements highly homologous to D. virilis gypsy
are present in D. lummei but with an apparently lower copy
number. D. littoralis probably also possesses a few copies of
gypsy.
DISCUSSION
We have cloned and sequenced homologues of D. melanogaster
(subgenus Sophophora) gypsy from D. virilis, the species which
belongs to the other subgenus, namely Drosophila. Comparison
of the nucleotide sequences of the elements from both species
shows that they are highly homologous and that certain domains
(both upstream and downstream of the initiation site) some of
which were proven earlier to be involved in the transcriptional
regulation of gypsy in D. melanogaster are present in the new
element.The gypsy family of mobile elements is broadly
distributed in D. virilis and its relatives where it appears to be
active in terms of transcription and transpositional capability (Fig.
3,4). This is so far the first example of the active homologue
of the D. melanogaster mobile element in the different subgenus
of the genus Drosophila.
The finding of the intermediates of the reverse transcription
of gypsy and other LTR-containing mobile elements has shown
that the step of the reverse transcription is included in its cycle
of replication (12). The reverse transcription by the enzymes from
different sources is highly inaccurate (see ref. 14 and refs.
within). Thus, it is assumed that an active mobile element evolves
much more rapidly than the 'usual' gene. This point of view is
supported by sequence comparisons of the different groups of
mobile elements (15). Although the elements of the 'gypsy group'
916 Nucleic Acids Research, Vol. 19, No. 4
are among the slowly diverging elements, it was found that the
rate of divergency of their most conserved enzyme reverse
transcriptase is significantly higher than that of the 'usual' genes.
The percent of the identity of the amino acid sequences of
different genes cloned from D. melanogaster and D. virilis vary
from 60% to more than 80% (16-23). Our finding of the active
gypsy element in D. virilis with the degree of amino acid identity
comparable to that of the 'usual' genes suggests that this element
may have appeared in this species by horizontal transmission and
has not yet changed significantly according to the predicted rate
of divergence for an active element.
Recently we obtained evidence for the horizontal transmission
of another Drosophila retroposon jockey (24) between distant
Drosophila species (25). This is also shown to be the case for
the P-element (26) which is apparently not a retrotransposon (for
rev. see ref. 27). In both cases direct evidence was obtained due
to the absence of these mobile elements in related species along
with its presence in more distant species. Our search for the
homologues of gypsy in the other species of D. virilis group
revealed its presence in these species (Fig.4); this does not allow
to prove directly the fact of the transmission of gypsy into D.
virilis. At the same time this does not exclude the possibility of
the horizontal transmission but rather shows that it might have
occurred before the irradiation of the D. virilis species group.
Previous studies (8) indicate that homologous to gypsy sequences
(as determined by Southern hybridizations) are present in higher
number of species than P-element (26) and jockey (25) within
the genus Drosophila. Authors argue that the observed patchy
distribution of gypsy might be the result of the multiple
transmissions of this element between different species. If so,
it should not be surprising that LTR-containing mobile elements
which are remarkably similar in structure to retroviruses would
have the ability to transmit between different species more
frequently than other mobile elements for which this ability has
already been proven.
ACKNOWLEDGMENTS
Authors thank Maxine Singer for advice and help throughout and
Julie McMillan for critical reading of the manuscript. L.J.M.
thanks V.Corces for support while working in his lab.
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