Identification of sex-specific transcripts of the Anopheles gambiae doublesex gene
Department of Biological Sciences, SAF Building, Imperial College London, Imperial College Road, London, SW7 2AZ, UK
Author for correspondence (e-mail:
acrs{at}imperial.ac.uk)
Accepted 1 August 2005
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Summary |
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Key words: doublesex, Anopheles gambiae, sex determination, sex-specific transcript
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Introduction |
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In nature, sexual differentiation is achieved through a variety of
mechanisms, which determine morphological, physiological and behavioral traits
in most living organisms. Despite the striking differences in the machinery
determining sex and in the individual genes involved, a common pattern can be
recognized among distinct taxonomic groups: a primary signal, a key gene and a
regulatory control gene lead to a double-switch gene, which selects between
alternative sexual programs. In Drosophila melanogaster, one of the
best characterized organisms both genetically and molecularly, the primary
signal of somatic sex determination is the ratio of X chromosomes to autosomes
(X:A ratio). An X:A ratio of 1.0 (2X:2A) dictates female development while an
X:A ratio of 0.5 (1X:2A) determines male development
(Cline, 1993). The sole target
of the primary signal is the key gene sex-lethal (sxl),
which becomes active in females and is inactive in males. SXL then acts as a
splicing regulator of transformer (tra) pre-mRNA
(Boggs et al., 1987
). Although
tra is transcribed in both sexes, a splice form of its transcript
leading to a complete open reading frame (ORF) occurs only in females. The
final double-switch gene in the somatic sex determination cascade is
doublesex (dsx). Dsx codes for two sex-specific
transcription factors (DSXF in females and DSXM in
males) that activate or repress the final genes necessary for the
differentiation of sexually dimorphic traits. Sex-specific transcripts of
dsx result from differential splicing that depends on the function of
tra. In females, TRA, along with the product of the constitutively
active gene tra2, acts as a splicing regulator by binding to splice
enhancer sites (dsxREs) present on the pre-mRNA of dsx,
activating a weak female-specific 3' acceptor site preceding the
female-specific exon (Baker and Wolfner,
1988
; Burtis and Baker,
1989
; Hoshijima et al.,
1991
). In males, where TRA is inactive, the weak 3' acceptor
site is not recognized, causing the splicing of the female-specific exon and,
instead, the inclusion of two downstream male-specific exons.
Analysis of the cascade in other insects supports a bottom-up model of
evolution for sex determination (Wilkins,
1995). The last gene in the cascade is the most ancient and
conserved while upstream regulators have strongly diverged during evolution.
Homologues of the key gene sxl have been isolated from Chrysomya
rufifacies (Muller-Holtkamp,
1995
), Megaselia scalaris
(Sievert et al., 2000
),
Musca domestica (Meise et al.,
1998
) and Ceratitis capitata
(Saccone et al., 1998
), where
they do not play a role in sex determination. With the exception of C.
capitata (Pane et al.,
2002
), no homologues of tra have been found outside of
the genus Drosophila, and the sequence has diverged considerably even
within this genus (O'Neil and Belote,
1992
). Homologues of the gene tra2 have been found in
Drosophila virilis (Chandler et
al., 1997
), humans (Dauwalder
et al., 1996
; Nayler et al.,
1998
) and mice (Segade et al.,
1996
). On the other hand, homologues of Dmdsx in
Bactrocera tryoni (Shearman and
Frommer, 1998
), M. scalaris
(Kuhn et al., 2000
), C.
capitata (Saccone, 1996
)
and M. domestica (Hediger et al.,
2004
) are largely conserved in exon-intron structure and
sex-specific splicing regulation, suggesting functional conservation. These
dsx homologues share most of the features of Dmdsx, which
include cis-regulatory elements such as the TRA/TRA2 splice enhancer
elements dsxREs and a purine-rich enhancer (PRE), a weak 3'
splice acceptor site preceding the female-specific exon and the domains
necessary for functional properties related to DSX (DNA-binding domain DBD and
oligomerization domains OD1 and OD2). Some features are also conserved in the
more distantly related lepidopteran Bombyx mori, where, however, the
regulatory mechanisms of the sex-specific processing are different
(Ohbayashi et al., 2001
;
Suzuki et al., 2001
).
Dsx homologues have also been identified in Caenorhabditis
elegans (Raymond et al.,
1998
), humans (Raymond et al.,
1999b
), mice and chickens
(Raymond et al., 1999a
).
In the present study, we have characterized the sex-specific splicing forms of the A. gambiae dsx gene and elucidated its chromosomal exon-intron organization. The gene is distributed over an 85 kb region of chromosome 2R and is composed of multiple exons, which are alternatively spliced to produce female- and male-specific transcripts. Based on the sequence conservation, exon-intron organization and transcription pattern, our results strongly indicate a role for Agdsx in determining the sexual fate in A. gambiae.
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Materials and methods |
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RT-PCR and northern blot analyses
Total RNA was extracted from adult male and female A. gambiae
mosquitoes using Tri Reagent (Helena Biosciences, Sunderland, UK), according
to manufacturer's instructions. Pupae were sexed by comparing their terminalia
under a dissecting microscope. The Superscript First-Strand Synthesis System
(Gibco, Gaithersburg, MD, USA) was used for first-strand cDNA synthesis using
oligo d(T) primers and total RNA, according to manufacturer's instructions.
PCR was performed essentially as described above except that the annealing
temperatures were adjusted to individual primers. Standardization was
performed with primers S7for (5'-GGCGATCATCATCTACGTGC-3') and
S7rev (5'-GTAGCTGCTGCAAACTTCGG-3'), which amplify the housekeeping
S7 ribosomal gene. Other primers included: dsx1f
(5'-AAAGCACACCAGCGGATCG-3'), dsx2f
(5'-TCTACAATCAATCAATCCGTG-3'), dsx3f
(5'-ACCATCGTTCAACCAATACC-3'), dsx1r
(5'-CACCGAGATGTTCTCGTCC-3'), dsx2r
(5'-TCCACTCTGACGGGTGGTATTGCG), dsx3r
(5'-GATTGATTGATTGTAGAGTGG-3'), dsxef
(5'-TTTCGATCGTGCAACGAAGG-3') and dsxer
(5'-TTTGGTGGGAAATTGGGCG-3').
For northern blot analysis, 10 µg of total RNA from male and female
adults was isolated as described above and hybridized with a
32P-labeled Probe 5' (described above) and Probe 3', a
446 bp fragment amplified from genomic DNA with dsx3'f
(5'-GAAGTCATCGCTCGATCCG-3') and dsx3'r
(5'-CCAGGCTCTCGTACACG-3'), following well established protocols
(Sambrook, 1989).
Southern blot analysis
For Southern blot analysis, a total of 5 µg of genomic DNA from male and
female A. gambiae adults was digested with BamHI,
HindIII or XhoI and hybridized with Probe 5' as
previously described (Catteruccia et al.,
2000).
Sequencing of splice acceptor sites
In order to obtain the sequence of the 3' splice acceptors within
introns 2, 3, 4 and 5, PCR amplification from genomic DNA was performed as
described above. Primers intron2f (5'-CTCCGAGGTGAACAATCGG-3') and
intron2r (5'-AGGAGATTTACAGGTTCTGG-3') amplified a fragment of
intron 2; intron3f (5'-GCTGCCACGATTGACACC-3') and intron3r
(5'-TTCAGTATGACGTACATCAGG-3') amplified a fragment of intron 3;
intron4f (5'-TAAAGAGCGCCGATGGCG-3') and dsx2r (above) amplified
intron 4; and intron5f (5'-ACAATCAATCAATCCGTGCAG-3') and dsx1r
amplified intron 5. Amplification products were cloned into the pGEM T-easy
vector according to manufacturer's instructions. Sequencing reactions were
performed using the ABI Prism fluorescence sequencing kit (Applied Biosystems,
Foster City, CA, USA). Sequencing of the inserts was performed using primers
annealing on the vectors M13left (5'-GTAAAACGACGGCCAGT-3') or
M13rev (5'-GGAAACAGCTATGACCATG-3').
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Results |
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To determine whether sex-specific transcripts of dsx were present in A. gambiae, northern blot hybridization was performed. Probe 5' was hybridized to total RNA from male and female adults (Fig. 2G). In female adults, two transcripts of approximately 9 kb and 8.2 kb were detected, which possibly corresponded to identical transcripts resulting from the use of polyadenylation sites downstream of the 3' end of the isolated female cDNA clones. In male adults, a single transcript of approximately 6.5 kb was detected, consistent with the size of the cDNA clone M1. Hybridization experiments performed with Probe 3' (Fig. 1), which encompasses a 446 bp sequence downstream of the female-specific region, showed the same banding pattern observed with Probe 5', confirming the presence of this region in both female and male transcripts (data not shown).
Genomic organization of Agdsx
Transcript analysis and genomic sequence allowed us to draft the structural
organization of Agdsx, define exon-intron boundaries and characterize
alternative sex-specific splicing. In Drosophila, the Dmdsx
gene is spread over a 45 kb region on chromosome 3R and consists of three
common exons, followed by a female-specific and two male-specific exons
(Fig. 3). DmdsxF
translation initiates at the AUG within exon 2 and terminates within the
female-specific exon 4, while in the case of DmdsxM, translation
begins at the same AUG and terminates within the first male-specific exon 5.
Similarly, the Agdsx gene is spread over an 85 kb region of
chromosome 2R and consists of seven exons, the first four of which code for
the common region of the protein (705 bp)
(Fig. 3). Exon 5 (1.7 kb) is
female-specific, and exon 6 (1.2 kb) represents the male-specific coding
sequence and flanking UTR, which is transcribed completely as UTR in the
female. Finally, exon 7 (2.6 kb) is a non-coding exon in the UTR of both
female and male transcripts. The complete female-specific transcript contains
a 795 bp ORF, coding for a protein of 265 amino acids. By sequence comparison
with Dmdsx, it can be assumed that translation initiates at an AUG
within exon 2 and terminates in the female-specific exon 5 with two successive
stop codons, opal and ochre (UGAUAA), a feature conserved in other Diptera
(Kuhn et al., 2000). The
male-specific transcript contains a much longer (1866 bp) ORF, coding for a
622 aa protein. The ORF starts at the same position in exon 2 and terminates
within exon 6 (Fig. 3).
|
Multiple sequence alignment of A. gambiae DSXM and
DSXF with the DSX proteins from D. melanogaster and other
Diptera shows a high degree of sequence conservation in the N-termini up to
the unique zinc finger-like DBD/OD1 domain and the common part of the OD2
domain, whose end marks the beginning of the sex-specific regions
(Fig. 4). Furthermore, six
residues (Cys, His, His, Cys, Cys and Arg) within the DBD/OD1 that have been
shown to be essential for DNA-binding activity in D. melanogaster are
conserved in Agdsx (Erdman and
Burtis, 1993). The C-terminal regions of the DSXF
proteins show very high sequence conservation among the different insects. A
sequence corresponding to the OD2 domain in the female region is very highly
conserved, and the stop signal (UGAUAA) is identical among the different
species. On the other hand, multiple alignments show very little similarity in
the DSXM regions. One common feature that can be observed is the
greater length of the male-specific regions when compared with the
female-specific parts. Several of the DSXM orthologues contain
strings of repeated amino acids, and AgDSXM contains
regions rich in proline, serine, glutamic acid and threonine (PEST regions), a
characteristic observed in DmDSXM
(Fig. 4).
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Discussion |
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Agdsx is organized in an exon/intron structure spanning an 85 kb
region of chromosome 2R, with similarities to Dmdsx and homologues
from other insects. The female transcript consists of a 5' common
segment, a female-specific part and a 3' common region, while the male
transcript comprises only the 5' and 3' common segments. The
5' common region is organized into four exons (1-4), the female-specific
segment corresponds to exon 5, while the 3' common segment consists of
exons 6 (which encodes for the male-specific part) and 7. In both females and
males, translation initiates at the same start codon in exon 2, while it
terminates within the female-specific exon 5 in AgdsxF transcripts
and within exon 6 in AgdsxM transcripts. The male-specific region is
therefore transcribed as a UTR in females, a situation seen in M.
scalaris (Kuhn et al.,
2000) and B. mori
(Ohbayashi et al., 2001
;
Suzuki et al., 2001
)
dsx homologues.
Although this situation differs from D. melanogaster, where female transcription terminates before the appearance of male-specific exons, the composition of the proteins is the same in the two species. The AgDSXM and AgDSXF proteins consist of a common N-terminus and sex-specific C-termini. The N-terminus features a highly conserved zinc finger-like DBD/OD1 as well as the non-sex-specific part of the OD2. While the female-specific region is very highly conserved, the male-specific part shows little sequence conservation amongst all DSXM homologues. The only conserved features are the greater length with respect to the female regions and the presence of strings of repeated amino acids and certain `PEST' amino acids in some of the DSXM homologues.
Sex-specific transcripts were detected in both northern blots and RT-PCR
analyses. Two female-specific bands of different size were detected in the
northern blot experiments, probably representing transcripts containing
alternative polyadenylation sites located downstream of the 3' end of
the female F2 and F3 cDNA clones. The presence of alternative polyadenylation
sites has been reported in dsx homologues from other organisms
(Kuhn et al., 2000). The size
of the female clones isolated from the cDNA library is considerably smaller
than expected from the size of the female transcripts detected in the northern
blots. However, RT-PCR experiments showed that female and male transcripts
share similar 3' regions that extend beyond the 3' end of the
female cDNA clones, suggesting that these did not contain the entire 3'
UTR. Indeed, including the 3' UTR from clone M1 in the calculation for
the length of the female cDNA transcripts results in a good match with the
size of the bands detected in northern blot analyses in female adults
(Fig. 2G). On the other hand,
the cDNA clone M1 is likely to represent the full male transcript, as its size
is consistent with the band detected in the northern analysis.
Apart from the highly divergent sequence of the male-specific region, the
major difference between Agdsx and Dmdsx resides in the
splicing mechanism of the female-specific exon. In D. melanogaster,
the inclusion or excision of the female-specific exon depends on the presence
of a weak 3' acceptor site in the preceding intron. Activation of this
splice site in females is brought about by TRA and TRA2, which form a
multiprotein complex with RNA-binding protein 1 and bind to
cis-regulatory elements (dsxREs and PRE) present in the
3' UTR of the female-specific exon immediately downstream of the
3' splice acceptor site (Hoshijima
et al., 1991; Inoue et al.,
1992
; Lynch and Maniatis,
1995
). In males, the absence of TRA leads to a default
male-specific splicing in which the weak 3' splice site is not
recognized and the next downstream acceptor site is chosen by the splicing
machinery, resulting in the splicing of the female-specific exon. On the other
hand, in A. gambiae, the retention of exon 5 in females seems to
depend on the activation of the 5' donor site of the downstream intron
5. This scenario would resemble the splicing of fruitless
(fru) in D. melanogaster, where the TRA/TRA2 enhancer
complex activates a female-specific 5' splice site
(Lam et al., 2003
). Similarly
to Agdsx, fru contains repeat elements (fruREs) nearly
identical to the DmdsxREs but located immediately upstream of this
alternative 5' splice donor site, approximately 1.3 kb downstream of the
3' acceptor site of the preceding intron
(Table 2A). The remarkable
similarity between Agdsx and Dmfru suggests that splicing of
Agdsx follows the same mechanism, with the female mode of splicing
occurring though the activation of the 5' splice site of intron 5
following the binding of TRA and TRA2 to the AgdsxREs. On the other
hand, the female transcript may represent the default splicing mode, with the
5' donor site of intron 5 repressed in males. The region preceding
intron 5 shows the presence of putative silencer-binding sites such as
guanosine-rich motifs (GGGG and UAGG), which are involved in the regulation of
a cassette exon in the glutamate NMDA
(N-methyl-D-aspartate) R1 receptor (GRIN1)
(Grabowski, 2004
).
Alternatively, a decoy 3' acceptor site could engage the 5' donor
site of intron 5 in a non-productive interaction, conferring in turn a
competitive advantage to the skipping of exon 5, as found in the caspase-2
pre-mRNA (Cote et al.,
2001
).
Conservation of the exon/intron structure and of the functional regions (DBD/OD1, OD2) found in all known dsx homologues suggests a role for Agdsx in dimorphic differentiation in A. gambiae. The identification of female- and male-specific transcripts of Agdsx represents an important step towards the understanding of the sex differentiation process in A. gambiae and will facilitate the development of genetic tools to induce male sterility or manipulate sex ratios in mosquitoes, for instance by constitutively expressing the female-specific form of dsx in the male gonads or by inducing the sex-specific splicing of a dominant lethal.
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Acknowledgments |
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Footnotes |
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References |
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