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First published online September 16, 2005
Journal of Experimental Biology 208, 3701-3709 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01819
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Identification of sex-specific transcripts of the Anopheles gambiae doublesex gene

Christina Scali*, Flaminia Catteruccia*, Qiuxiang Li and Andrea Crisanti{dagger}

Department of Biological Sciences, SAF Building, Imperial College London, Imperial College Road, London, SW7 2AZ, UK



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  		Fig. 1. Schematic representation of the putative female (F1, F2 and F3) and male (M1) Agdsx transcripts isolated from a mixed pupal A. gambiae cDNA library. The sequence of the putative female transcript was deduced by combining the information provided by clones F1, F2 and F3, while the sequence of the putative male transcript was based on clone M1. The diagonally hatched box represents the DBD/OD1 domain, and the stippled box represents the non-sex-specific region of the OD2 domain. The putative female- and male-specific regions are indicated by a horizontal and a vertical hatched box, respectively. Black and white triangles indicate the position of the initiation and stop codon, respectively. Vertical arrows in the male transcript indicate polyadenylation signal sequences. Numbers indicate the distance in bp from the start of clone F1, which was arbitrarily considered as the start for both female and male transcripts. The location of Probe 5' and Probe 3' is indicated by solid bars. Primers used in RT-PCR experiments are indicated as horizontal arrows.

 


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  		Fig. 2. RT-PCR and northern blot analyses of total A. gambiae RNA. (A-F) RT-PCR products were amplified from female and male adult A. gambiae total RNA using the following combinations of primers: (A) dsx1f and dsx1r; (B) dsx2f and dsx1r; (C) dsx1f and dsx2r; (D) dsx3f and dsx3r; (E) dsxef and dsxer; (F) S7for and S7rev (which amplify the housekeeping ribosomal S7 gene as a control). For primer locations, see Fig. 1. (G) Northern blot analysis was performed on total RNA (10 µg) extracted from male and female A. gambiae adults. Lane 1, female adult; lane 2, male adult. The upper panel shows hybridization performed with Probe 5', while the lower panel shows standardization with probe S7. The molecular size is shown in kb.

 


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  		Fig. 3. Exon/intron organization of AgdsxF and AgdsxM and comparison with the female and male transcripts of Dmdsx. Exons are shown as boxes, where black boxes represent untranslated regions and open boxes represent coding regions. The OD1 domain is indicated by diagonal hatched boxes, and the non-sex-specific region of the OD2 domain by stippled boxes. Male- and female-specific regions of the OD2 domains are indicated by vertical and horizontal hatched boxes, respectively. In Agdsx, a male-specific region of the OD2 could not be identified. Introns and exons are numbered according to the sequence of the female transcripts. The exon/intron boundary between the last common exon and the sex-specific regions is conserved between Dmdsx and Agdsx. Vertical arrows indicate the region where the dsxREs and PREs are found. Introns are not shown to scale.

 


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  		Fig. 4. Multiple sequence alignment of DSX homologues from D. melanogaster, B. tryoni, M. domestica, M. scalaris, B. mori and A. gambiae. The sequence is divided into (A) the region that is common to DSXF and DSXM, (B) the conserved female-specific parts and (C) the highly divergent male-specific regions. The DBD/OD1 and OD2 domains are indicated. Black boxes represent areas of amino acid identity; gray boxes represent areas of amino acid similarity; the asterisks indicate six amino acids whose replacement has been shown to abolish DNA-binding activity in D. melanogaster.

 


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  		Fig. 5. Determination of Agdsx gene copy number in male and female genomic DNA. Genomic DNA (5 µg) extracted from male and female A. gambiae was digested with XhoI, HindIII and BamHI and electrophoresed on a 1% agarose gel. Hybridization was performed with a radiolabeled Agdsx Probe 5' that has no restriction site recognized by the three restriction enzymes. The molecular size is shown in kb.

 

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© The Company of Biologists Ltd 2005