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Insect Mol Biol. Author manuscript; available in PMC Oct 6, 2008.
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PMCID: PMC2561890

Sex-, stage- and tissue-specific regulation by a mosquito hexamerin promoter


A portion of the 5′-flanking region of the female-specific hexamerin gene, Hex-1.2, from the mosquito Ochlerotatus atropalpus was used to drive expression of the luciferase reporter gene in Drosophila melanogaster. The proximal 0.7 kb of 5′-flanking DNA were sufficient to partially repress reporter gene activity in males and to drive tissue- and stage-specific expression comparable with that of the endogenous O. atropalpus Hex-1.2 gene. The Drosophila doublesex transcription factor (DSX), expressed in Escherichia coli, bound putative DSX sites of the Hex-1.2 gene differentially in vitro. Blocking expression of the female isoform of the Doublesex transcription factor in transgenic female flies resulted in reduction of luciferase expression to levels comparable with those in males, suggesting that Doublesex could contribute to regulation of female-specific expression of the O. atropalpus Hex-1.2 gene.

Keywords: hexamerin gene regulation, mosquito, fat body, gel-shift assays, sex specificity, doublesex


Combating the worldwide spread of many deadly infectious diseases requires the development of novel strategies for control of insect-borne pathogens. Such strategies will most likely involve the use of molecular tools, including promoters targeting gene activity to particular developmental stages, tissues and/or gender of the disease vector. One of the species-specific methods for control of diseases vectored by insects is the sterile insect technique (SIT), which involves the release of sterilized males to mate with wild females (Tan, 2000). However, the use of SIT is costly and labour-intensive. New approaches using transgenic insects have been proposed, such as the ‘release of insects carrying a dominant lethal’ (RIDL; reviewed in Alphey et al., 2002), in which a dominant sex-specific lethal gene can be expressed to self-destruct the offspring from the mating of a released transgenic male population with wild females. For RIDL and other transgenetic sexing strategies (including more conventional SIT) to be effective, sex- and tissue-specific promoters are needed to drive expression of a repressible lethal gene. Furthermore, any transgenic approach to modify natural disease vector populations will require the introduction of selective advantages for the transgenic insects such as increased or enhanced reproductive fitness.

The insect fat body is the major biosynthetic organ, which maintains homeostasis of haemolymph proteins, lipids and carbohydrates and plays an important part in metabolism, development and reproduction (Candy, 1985; Haunerland & Shirk, 1995). Owing to its critical roles in reproduction and immunity, the fat body is an important tissue to target for transgenic approaches. Molecular analysis of fat body-specific genes of the fruit fly and mosquito suggests that their tissue-specific expression is governed by specific transcription factors, including C/EBP, GATA, BBF-2, HNF-4, and a fat body-specific repressor, AEF-1, binding to their cognate cis-acting DNA sequences (Garabedian et al., 1986; Sofer & Martin, 1987; Abel et al., 1992; Falb & Maniatis, 1992a,b; An & Wensink, 1995a,b; Beneš et al., 1996; Kapitskaya et al., 1998; Kokoza et al., 2000; Martin et al., 2001).

Hexamerins are insect storage proteins that are synthesized in the fat body (Telfer & Kunkel, 1991) and belong to a large arthropod protein family that includes haemocyanins and prophenol oxidases (reviewed in Haunerland, 1996 and Burmester et al., 1998). Hexamerins or hexameric storage proteins of holometabolous insects are synthesized in the fat body, primarily at the end of larval development, secreted into the haemolymph and then taken up via receptor-mediated endocytosis into the larval fat body for storage in protein granules. These hexameric storage proteins, which are primarily rich in aromatic amino acids, are then utilized as an amino acid reserve during non-feeding periods of insect development or diapause. Diptera produce two immunologically distinct hexamerins typified by the larval serum proteins (LSPs), LSP-1 and LSP-2 of Drosophila melanogaster (reviewed in Burmester et al., 1998). Very similar hexamerins are also expressed by the fat body of different species of mosquitoes (Zakharkin et al., 1997; Korochkina et al., 1997).

Previously, in our laboratory we identified putative female-specific regulatory elements in the 5′-flanking region of the hexamerin gene, Hex-1.2, of the aedine mosquito, Ochleratatus atropalpus (Zakharkin et al., 2001). These elements contain putative overlapping C/EBP and DSX (doublesex) transcription factor binding sites and are similar in structure to the female- and fat body-specific element from the 5′-flanking region of the Yolk protein (Yp) genes of the fruit fly, Drosophila melanogaster (An & Wensink, 1995a). As one of the downstream regulators of the Drosophila sex determination pathway (Burtis et al., 1991), the doublesex locus encodes either male-specific DSXM or female-specific DSXF transcription factors that are produced through alternative RNA splicing (Burtis & Baker, 1989; Nagoshi & Baker, 1990). Action of the transformer (TRA) and transformer-2 (TRA-2) proteins is required to produce the female-specific DSXF isoform (Inoue et al., 1992). The two DSX proteins differ only in their C-termini and bind to the same DNA site in the o-r enhancer of the yolk protein (Yp) genes (An & Wensink, 1995a; Cho & Wensink, 1997). We hypothesized that female-specific activity of the mosquito Hex-1.2 gene could be regulated similarly to the Yp genes in D. melanogaster, with Doublesex playing an important role.

In this study, we show that the putative DSX sites in the 5′-flanking region of Hex-1.2 bind the Drosophila DSX protein and that in transgenic Drosophila a 738 bp promoter/enhancer sequence confers strong female-enhanced activity on a reporter gene. This female-enhanced activity is fat body- and stage-specific, recapitulating in a distant dipteran insect the regulation that is observed for the mosquito Hex-1.2 gene. In addition, using a null mutation (transformer1) for one of the critical components of the Drosophila sex determination pathway, we show that the Hex-1.2 DSX binding sequences are indeed functional in a heterologous dipteran insect.


Gel mobility shift assays for DSX binding

Previously, we identified three putative DSX binding sites in the 5′ flanking region of the O. atropalpus Hex-1.2 gene (Zakharkin et al., 2001; Fig. 1). To determine if these putative sites are able to bind a cognate DSX transcription factor from a related species, we performed electrophoretic mobility shift assays (EMSA) with bacterially expressed Drosophila DSX proteins. An initial experiment was designed to demonstrate the specificity of Escherichia coli-expressed DSXM and DSXF proteins, using as a probe the DSX binding site from the Drosophila Yp1 gene regulatory region (An & Wensink, 1995a). A shift in Yp1 probe was observed only with DSXM- and DSXF-containing E. coli extracts, but not with the control BL21 extracts (data not shown). DSXF-containing extract (also called ‘DSXF protein’) was used in all subsequent experiments, as DSXF and DSXM have identical DNA-binding domains and properties (Burtis et al., 1991; Erdman & Burtis, 1993; Cho & Wensink, 1996; Erdman et al., 1996).

Figure 1
Schematic diagram of the Hex-1.2 5′-flanking region in the 0.7Hex-luc fusion gene. The Hex-luc gene consists of 0.74 kb from the Hex-1.2 5′-flanking region, isolated using the indicated NsiI and XhoI sites and fused to the luciferase reporter ...

A comparison of the binding patterns of Yp1, DSX-1, DSX-2 and DSX-3 oligonucleotide probes in the presence of varying amounts of DSXF protein is shown in Fig. 2. The oligonucleotides containing putative DSX sites from the mosquito Hex-1.2 gene were able to bind the DSXF protein to varying degrees; retarded bands of the same mobility as ones obtained with the Yp1 probe were clearly recognizable in all reactions. This binding pattern was confirmed and quantified by competitive EMSA (Fig. 3A,B). DSXF protein was incubated with a fixed amount of radio-labelled Yp1 probe and competed by increasing amounts of unlabelled (cold) Yp1, DSX-1, DSX-2, DSX-3 or non-specific probes. For the Yp1 control reaction, binding was significantly competed away by inclusion of a 250-fold molar excess of unlabelled homologous Yp1 probe (lane 3). Unlabelled DSX-1 and DSX-2 probes also performed well as competitors in 250-fold molar excess, competing away 55% (Fig. 3A,B, lane 6) and 43% (lane 10) of binding to the Yp1 probe, respectively. One thousand-fold molar excess of unlabelled DSX-1 was needed to compete away 90% (lane 8) of Yp1 binding. No significant competition was observed with DSX-3 (lanes 13–16) and in particular, the non-specific (lanes 17–20) unlabelled DNA probes. Hence, the efficiency of competition indicated that the DNA binding affinities of the DSX sites for DSXF were in the following order: Yp1 [dbl greater-than sign] DSX1 > DSX2 [dbl greater-than sign] DSX3.

Figure 2
Putative sequences from Hex-1.2 bind Drosophila DSXF. E. coli extracts (0, 12.5, 25, 125, 250 and 500 ng) containing expressed DSXF protein were incubated with Yp1 (lanes 1–6), DSX-1 (lanes 7–12), DSX-2 (lanes 13–18) and DSX-3 ...
Figure 3
DSX-binding sites from the Hex-1.2 gene differ in affinity for Drosophila DSXF. (A) Different affinities of Hex-1.2 DSX-binding sites. E. coli-expressed DSXF protein (25 ng) was incubated with 2 fmol of 32P-labelled Yp1 probe in the absence (lane 1) and ...

The Hex-luc promoter induces female-enhanced activity in Drosophila

To examine the function of the Hex-1.2 DSX sites by a transgenic approach, we constructed a fusion gene, 0.7Hex-luc, containing positions −715 to +23 (relative to the transcription start site) of Hex-1.2 (Fig. 1) and subcloned it into the P-element-based transformation vector, pCaSpeR, as described in the Experimental procedures. Using the y1w67c23 strain (Beneš et al., 1996), four independent transgenic lines were established. Each of the transgenic lines contained a single-copy Hex-luc insert, as determined by Southern blot analysis (data not shown); the chromosome of transgene insertion was identified by genetic crosses. Homozygous flies of one line, TR-6, were viable and fertile. Homozygous flies of two other lines, TR-4 and TR-10, were not viable; while TR-1 homozygotes were viable but sterile. More recently in another context, Drosophila transgenics with the same Hex-1.2 mosquito DNA were obtained without an excessive number of homozygous lethals; hence, there is nothing unusual about the mosquito DNA used in the Hex-luc construct.

To analyse differences in Hex-luc activity between genders, three independent transgenic lines (TR-4, TR-6 and TR-10) were assayed for luciferase activity. Individual late third-instar female and male larvae were collected and whole-animal extracts were assayed as described in Experimental procedures. Both homozygous and heterozygous flies of the TR-6 line were tested. The mean luciferase expression levels and the resulting female/male ratios in expression were different for each line, with ratios ranging from 2 to 4, reflecting different effects of chromosomal position on the transgene in each line (Table 1 and Fig. 4A). We determined that luciferase activity was clearly higher in females than in males using a series of pair-wise t-tests (P < 0.001). In order to confirm that the sex-enhanced luciferase activity was based on transcriptional, and not translational regulation, real-time RT–PCR analysis was performed to assess the levels of luc mRNA levels in male and female L3 larvae of TR-6 line. Results of real-time PCR analysis (Fig. 4B) indicated that female luc mRNA was transcribed at about a 4.6-fold higher level in female larvae than in males of the same age.

Figure 4
Sex-specific regulation of Hex-luc in Drosophila larvae. (A) Female-enhanced expression in third-instar larvae. Normalized firefly luciferase activity (RLU/μg protein; mean ± SD) was measured for individual male or female larvae from three ...
Table 1
Sex-enhanced luciferase activity in transgenic Drosophila larvae

Expression of Hex-luc is tissue- and stage-specific

Hexamerins are insect storage proteins that are predominantly expressed in the fat body and occasionally in additional tissues, on a species-specific basis (Telfer & Kunkel, 1991). Hence, to determine if the Hex-luc transgene could elicit the expected fat body specificity in a heterologous dipteran insect (Drosophila), we assayed luciferase expression levels in fat bodies vs. the rest of the body (or carcass) in male and female late third-instar larvae. A typical, representative experiment is shown in Fig. 5(A); luciferase activity was almost exclusively confined to the fat body. Factorial ANOVA and a series of posthoc tests demonstrated that luciferase levels in male and female carcasses were not significantly different from each other (P = 0.695), while all others groups were significantly different (P < 0.001).

Figure 5
Conservation of mosquito promoter specificity in Drosophila. (A) Tissue-specific expression of the Hex-luc transgene. Extracts were prepared from individual L3 larvae of the TR-4 transgenic line and assayed for protein concentration and luciferase activity ...

To determine the developmental profile of Hex-luc expression, we assayed whole-animal extracts of both males and females at different stages; results for the TR-4 line are shown in Fig. 5(B). The developmental profile of transgene expression was similar to that of the Hex-1.2 gene in the mosquito (Zakharkin et al., 1997), with peak levels found at the last larval and early pupal stages and some expression in adult flies. Differences in luciferase activity levels between genders at different stages of the life cycle were analysed using a series of pairwise t-tests. Starting from the last larval stage, the ratio of mean luciferase levels in females to mean luciferase levels in males was approximately 2 throughout development.

Hex-luc expression in tra mutants

Activity of the transformer gene is required for female-specific splicing of dsx mRNA (Hoshijima et al., 1991); in a fly carrying a null transformer mutation (tra1) over a deficiency of the same locus, splicing produces only the default male dsx mRNA in both sexes. Similarly, in both male and female flies carrying dominant dsx mutations, only the male DSX isoform is produced in both sexes (Nagoshi & Baker, 1990). To confirm that Hex-luc transgene activity was under the control of the DSX transcription factor and dependent on sex-specific differential splicing of the dsx transcripts, we chose to use the tra1 rather than any dsx mutation. Hexamerins and yolk proteins are similar storage proteins produced by the fat body and are likely to use similar regulatory networks. As both tra and dsx mutations have the same effect on yolk protein expression (Artyom Kopp, personal communication) and dominant dsx mutants are difficult to maintain in the laboratory, we found little advantage to working with dsx mutations in our experimental system. Hence, we placed the Hex-luc transgene in the context of tra1 by genetic crossing of appropriate stocks (Fig. 6A). Among transgenic animals carrying the Hex-luc fusion gene, as judged by a red eye colour, we selected three groups: genetic males, genetic females, and pseudomales. Genetic males were identified by their bar-shaped eyes, due to a dominantly marked duplication chromosome, Dp(1;Y)BS, while pseudomales had a normal eye shape. Pseudomales were genetic females that carried the tra1 mutation uncovered by the Df(3L)st-j7,Ki1 deficiency, leading to a total absence of functional TRA protein and production of the male DSXM isoform instead of the female-specific DSXF. Individual adult animals at 1–2 days after eclosion were selected and whole-animal extracts assayed for firefly luciferase reporter gene activity as described in the Experimental procedures (Fig. 6B). Inactivation of the transformer protein, leading to production of the male DSXM factor in genetic females (or pseudomales), resulted in reduction of Hex-luc transgene expression to levels more similar to those in males. One-way ANOVA and a series of posthoc tests confirmed that mean luciferase levels in pseudomales were not different from males (P = 0.797), while both were significantly different from females (P < 0.001 in both cases).

Figure 6
Modification of female-enhanced Hex-luc activity by a tra1 mutation. (A) Genetic cross to test the effect of the tra1 mutation. Parental lines were constructed from the TR-6 line and appropriate stocks obtained from the Bloomington Drosophila Stock Center. ...


Molecular tools for transgenetic manipulation of numerous arthropod pests (of plants and animals) are needed to control the pests themselves as well as the diseases that they can spread. Such tools should include DNA sequences whose role in regulating gene activity can be reliably predicted in terms of sex, stage and tissue specificity. In particular, the insect fat body, as a central organ of intermediate metabolism, immune defence and reproduction, is an important tissue to target in such transgenic approaches. Accordingly, we have begun the characterization of a female, fat body-specific mosquito gene whose strongest activity is in the late larval stage but which continues at low levels uniquely in the adult female (Zakharkin et al., 2001). In this study, analysis of transgenic 0.7Hex-luc flies shows that 738 bp of Hex-1.2, including its transcription start site, constitute a very strong promoter and enhancer whose activities are not only significantly enhanced in females of an heterologous dipteran species but are also negatively affected by a tra mutation, which abrogates DSXF-regulated activity in female flies. Furthermore, we demonstrate that at least two DSX sites, previously proposed within the Hex-1.2 5′-flanking region (Zakharkin et al., 2001), are able to bind the Drosophila DSXF isoform with reasonably high affinity. These findings suggest that the mosquito Hex-1.2 enhancer/promoter is a target of Doublesex regulation and likely to function sex specifically in other transgenic insects, given the extensive conservation of the doublesex or DM domain, a cysteine-rich DNA-binding motif first identified in the Drosophila DSX protein, genes among eukaryotes as distantly related as worms and vertebrates (Cline & Meyer, 1996; Raymond et al., 1998; Kuhn et al., 2000; Hodgkin, 2002; Scali et al., 2005). Recently, characterization of sex-specific doublesex transcripts and the single dsx gene from the mosquito Anopheles gambiae showed that DSX isoforms of this mosquito possess the same domain structure as the Drosophila DSX proteins (Scali et al., 2005). The presence of specific genomic regulatory elements in the Agdsx gene suggests that in this mosquito the TRA/TRA2 regulatory pathway is used to produce the female-specific DSX domain by a mechanism more similar to the splicing of the Drosophila fruitless transcript.

Using an E. coli extract containing highly expressed Drosophila DSXF protein, we observed that the DSX-1 and DSX-2 sites (Fig. 1) from the Hex-1.2 gene were able to bind DSXF with a measurable (Fig. 3A,B) but somewhat weaker affinity than does the Drosophila Yp1 regulatory element. We estimate from the quantification of competitive EMSA that the Hex-1.2 DSX-1 site exhibits about 20-fold weaker affinity than the Yp1 site, and that the DSX-2 site is five times weaker than the DSX-1 site in binding DSXF. These relative DSXF binding strengths correspond in part to the extent of identity between Hex-1.2 DSX sites and the proposed consensus for the Drosophila DSX factor (Erdman et al., 1996). Six of the seven consensus positions are identical between both DSX-1 and DSX-2, and the Drosophila consensus with the divergent nucleotide being in the +1 or −1 position of the palindrome, respectively. Predictably, the DSX-3 site, which has significantly less homology with the Drosophila DSX consensus binding site, showed little if any binding to the Drosophila DSX protein. Given the high conservation of the DNA binding region of the different Doublesex proteins as well as of their DNA response elements (Zarkower, 2002; Hodgkin, 2002; Suzuki et al., 2003; Hediger et al., 2004; Scali et al., 2005), we maintain that there are at least two bona fide DSX sites in the Hex-1.2 enhancer/promoter region. As the DSX-3 site is placed very near the ‘TATA box’, in a regulatory region that is usually highly conserved among TATA-box-containing genes, we suggest that this site may also be functional in O. atropalpus. It is possible that the homologous mosquito DSX protein will be found to have higher affinity for the DSX sites of the O. atropalpus Hex-1.2 gene.

By testing the function of these mosquito DSX binding sites in vivo in transgenic Drosophila carrying the 0.7Hex-luc gene, we found that they could account for the significantly (two- to six-fold) higher reporter gene activity in female larvae, pupae and adult flies. The differences between the two sexes were entirely reproducible although influenced to some degree by transgene position effects. Despite considerable animal-to-animal variation in reporter gene activity (as reflected in the large SDs; Figs 4 and and5),5), the differences between male and female activities were statistically significant in all three lines examined. However, in none of the transgenic Drosophila lines did we obtain complete repression of the transgene in male larvae or adults, as occurs for the Hex-1.2 gene in male O. atropalpus. Other regulatory sequences, upstream, downstream or both, of the tested 738 bp enhancer/promoter may be necessary for full repression in males. Alternatively, one or more coactivators and/or corepressors are lacking in the heterologous fruit fly. In a previous analysis (Zakharkin et al., 2001), we found no potential regulatory factor binding sites downstream of the Hex-1.2 transcription start site. Hence, we are currently exploring the impact of DNA sequences located further upstream of the transcription start site.

Further confirmation that the DSX sites of the O. atropalpus Hex-1.2 gene are functional and at least partially determine sex-specific gene activity in a heterologous dipteran species came from our experiments with the transformer null mutant tra1. When only the default male isoform, DSXM, was produced in genetic females, there was no enhancement of reporter gene expression. This observation suggests that female larvae, pupae and adult flies need the DSXF isoform to express the transgene at a higher level. However, it is still possible that other TRA targets, regulators similar to Doublesex and Fruitless (Heinrichs et al., 1998; Dulac, 2005) but as yet undiscovered, may bring about sex-specific activity of the mosquito Hex-1.2 gene. Future experiments in which the Hex-1.2 DSX sites are mutated and tested in transgenic Drosophila and mosquitoes should directly demonstrate if these DSX-binding sites are indeed essential for female-specific activity of Hex-1.2 in O. atropalpus.

This study also demonstrates that the 738 bp of mosquito DNA tested are sufficient to confer the same tissue and similar stage specificity in Drosophila as in the mosquito but not complete sex specificity. The weak activity of the reporter gene detected in carcasses could be due to technical constraints: it is extremely difficult to remove all of the fat body cells; even a few cells remaining in the carcass could result in significant luciferase activity. Alternatively, the transgene could be expressed in tissues other than fat body, although we consider this possibility quite unlikely, given the results of our previous studies of hexamerin expression in mosquitoes (Zakharkin et al., 1997; Korochkina et al., 1997). Somewhat surprisingly, the relative level of reporter gene activity in 1–3-day and 7–10 day-old adult transgenic Drosophila was much higher than in adult female Ochlerotatus mosquitoes. The luciferase level in adult fruit flies was about half of the peak level in L3 larvae, while in adult female mosquitoes the Hex-1.2 mRNA level was only about 1/100 of the larval peak (Zakharkin et al., 2001). This may be due to differences between fruit flies and mosquitoes in gene regulation that may operate at the transcriptional or post-transcriptional levels. However, most studies of the use of heterologous DNA sequences in transgenic Drosophila have shown conservation of tissue-specific regulatory elements (from the first by Mitsialis & Kafatos, 1985 to a more recent one by Kokoza et al., 2001). Alternatively, some additional sequences, beyond what we have tested, might be necessary to achieve more restricted stage specificity in Drosophila.

Previous studies (Falb & Maniatis, 1992a,b; Attardo et al., 2005), including those in our laboratory (Beneš et al., 1996), have defined a limited number of transcription factors and their DNA response elements that are sufficient for targeting gene activity to the dipteran insect fat body. The fat body-specific regulatory unit was proposed to consist of at least one positive element binding a bZIP protein such as C/EBP and a negative element binding a repressor such as AEF-1, which restricts gene activity to the fat body (Falb & Maniatis, 1992a; An & Wensink, 1995b). Each of the Hex-1.2 DSX sites tested in this study is associated with one or more C/EBP sites whose specific roles will need to be tested in future experiments. Also critical to establishing tissue specificity may be binding sites for the GATA factor, which is highly conserved among vertebrates and invertebrates and harbours a very conserved DNA-binding domain associated with other more variable domains that allow interaction with numerous transcriptional activators and repressors (reviewed in Attardo et al., 2005). The roles of two putative GATA sites located in close proximity to the sex-specific DSX-C/EBP regulatory module will also be examined to fully understand the integration of sex- and fat body-specific activity of the Hex-1.2 gene.

In conclusion, we have demonstrated that 738 bp of Ochlerotatus mosquito DNA from the 5′-flanking region of the Hex-1.2 gene are capable of directing stage- and fat body-specific and sex-enhanced gene activity in a heterologous dipteran insect. To date, few target genes regulated by DSX have been identified in Drosophila, mosquitoes and other insects. The Ochlerotatus Hex-1.2 gene may be the first example for mosquito species. Further studies are now in progress to isolate a culicine mosquito homologue of the DSX factor and to test the Hex-1.2 regulatory sequences in transgenic mosquitoes.

Experimental procedures

Animals and genetic manipulations

Fruit flies, Drosophila melanogaster, were reared as described previously (Beneš et al., 1996). y1w67c23 flies were a kind gift of Jean-Antoine Lepesant. f1/Dp(1;Y)BS; Df(3L)st-j7,Ki1/TM6B, Tb1 (BL-5416) and wa/Y; tra1/TM2 (BL-675) flies were obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN, USA) and used to generate other lines as needed. Pseudomale, male and female flies carrying a Hex-luc transgene and the transformer mutation, tra1, were generated by crossing y1w67c23/Dp(1;Y)Bs; Sp/CyO; Df(3L)st-j7,Ki1/TM6B, Tb1 males with y1w67c23/y1w67c23; P{0.7Hex-luc}/P{0.7Hex-luc}; tra1/TM3, Sb females. Among the progeny, true males were selected by eye shape from expression of the Bar gene (BS) and pseudomales as non-Stubble, non-Tubby flies carrying the Ki dominant bristle marker; true females were identified as non-Kinked, non-Bar, Tubby flies.

Fusion gene constructs

Portions of the 5′-flanking regions of the Hex-1.2 gene were isolated and subcloned as described previously (Zakharkin et al., 2001; GenBank accession number AF430247). First, a 1.3Hex-luc (hexamerin/luciferase) fusion gene construct was created by insertion of a 1.3 kb MluI/XhoI DNA fragment of the Hex-1.2 5′-flanking region into the promoterless pGL2-Basic vector (Promega, Madison, WI, USA). Then, a 0.7Hex-luc fusion gene construct, containing positions −715 to +23 (relative to the transcription start site) of the Hex-1.2 gene, was derived from it by KpnI/NsiI digestion and ligation. All constructs were verified by DNA sequencing. To generate transgenic flies, a 3.9 kb XhoI/BamHI DNA fragment of 0.7Hex-luc was subcloned into the Drosophila P-element transformation vector, pCaSpeR (Pirrotta, 1988).

Generation of Drosophila transformants

P-element transformation was performed according to Rubin & Spradling (1982), using y1w67c23 host flies as previously described (Beneš et al., 1996). Surviving adults were individually backcrossed to the parental y1w67c23 line; stable transformants were selected by eye colour. Transgenes were localized to specific chromosomes by genetic crosses using dominantly marked balancer chromosomes according to standard protocols. Transgene copy number was determined by genomic Southern blot analysis for each transformed line; lines with multiple P-element insertions were discarded.

Luciferase activity assay

Individual animals were collected at different stages and either frozen or assayed immediately. TR-4, TR-6 and TR-10 lines were used to assay sex-enhanced activity in transgenic larvae. For analysis of tissue-specific gene expression, fat bodies of third-instar TR-4 larvae were separated from the carcasses by dissection (Beneš et al., 1996) and immediately frozen in liquid nitrogen. Samples were homogenized in lysis buffer from the Luciferase Reporter Gene Assay System (Promega) after adding a protease inhibitor cocktail at the manufacturer’s recommended concentration (Roche, Indianapolis, IA, USA) and aprotinin (Sigma, St Louis, MO, USA) at 1 μg/ml to reduce protein degradation. Luciferase activity was measured using the Victor Light 1420 Luminescence counter (Perkin Elmer, Shelton, CT, USA). For each sample, relative light units were normalized to protein concentration measured using the Protein Assay Reagent (Pierce, Rockford, IL, USA) detected with a Multiskan Ascent spectrophotometer (Labsystems, Madison, WI, USA).

Real-time PCR

Total RNA was isolated from five pooled, frozen male or female larvae using the TRIzol reagent according to the manufacturer’s specifications (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA from each sample was treated with 1 unit of RNase-free deoxyribonuclease I (Amplification Grade, Invitrogen) according to the manufacturer’s protocol. First-strand cDNA synthesis was carried out on 1 μg of DNase-treated RNA using a poly dT20 primer and SuperScript II reverse transcriptase (Invitrogen). Real-time quantitative PCR (q-PCR) amplification reactions were performed using the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen). Primers were as follows: Luciferase Forward: 5′-TACAACACCCCAACATCTTCG-3′; Luciferase Reverse: 5′-TCCACAAACACAACTCCTCC-3′; S3 ribosomal protein Forward: 5′-GCTGAATTGAACGAGTTCCT-3′; S3 ribosomal protein Reverse: 5′-ACCTTCTCGGCGTACAGCTC-3′. Samples were analysed using the DNA Engine Opticon 2 Detection System (MJ Research, Waltham, MA, USA) with Opticon 2 Monitor software. The ribosomal protein S3 transcript was used as an internal control to normalize RNA. Amplifications were performed as follows: initial incubation at 50 °C for 2 min, denaturation at 95 °C for 2 min, followed by 34 cycles of 94 °C, 15 s; 56 °C, 15 s; 72 °C, 15 s. Fluorescence was recorded after the 72 °C/15 s step of each cycle. The specificity of each reaction was determined after completion of PCR cycling by analysis of the melting-point dissociation curve generated for temperatures from 60 to 95 °C at 0.5 °C/s, and by visual inspection of DNA bands resolved by agarose gel electrophoresis. The optimal threshold cycle value (CT) was determined at the 0.05 fluorescence value, using the auto scale function provided by the analysis software, and was identified using regions of amplification curves that met the criterion of a two-fold increase in fluorescence per cycle. A validation experiment (data not shown) with eight-fold serial dilutions of cDNA, confirmed that the amplification efficiencies of luciferase and S3 ribosomal protein cDNAs were comparable and that data could be analysed using the comparative ΔΔCT method (Livak & Schmittgen, 2001) to calculate differences in transcript concentrations between samples (Fig. 4B).

Electrophoretic mobility shift assay

Expression of DSX proteins

To express the male- and female-specific Drosophila, DSXM and DSXF, proteins, we obtained the plasmids pT7-7 Dxfe and Dxme as a generous gift from Pieter Wensink (Brandeis University, Waltham, MA, USA) through Gyunghee Lee (University of Tennessee-Knoxville, Knoxville, TN, USA). Protein expression was performed according to Burtis et al. (1991) with some modifications. DSX protein expression was induced in 15 ml of E. coli BL-21 AI cells (Invitrogen) at mid-log phase with 0.002% arabinose. After 2.5 h incubation at 37 °C, cells were harvested by centrifugation, washed with phosphate-buffered saline, resuspended in 1.5 ml of Buffer L [20 mm Tris–HCl (pH 8.0), 1 mM EDTA and 10% sucrose] and frozen in liquid N2. Cells were thawed at 0 °C, and lysozyme added to 0.2 mg/ml. After 45 min at 0 °C, the cells were twice frozen in liquid N2 and thawed at 0 °C. The lysate was centrifuged at 19630×g at 4 °C for 20 min; the pellet was resuspended in 3 ml of buffer Z-50 (Burtis et al., 1991), aliquoted and stored at −80 °C. Control protein extracts from untransformed E. coli BL-21AI cells were prepared similarly. Protein concentrations were determined using the Bradford reagent (Sigma). Expression of the Drosophila DSX proteins within the bacterial lysates was confirmed by standard SDS–polyacrylamide gel electrophoresis and Western blotting (data not shown).

Double-stranded DNA probes were produced from oligonucleotides specifically designed for central placement of a putative or known DSX binding site, shown as underlined letters. The following were used as probes for the putative DSX sites of the Hex-1.2 gene, with each site numbered according to the female-specific element (or FSE) in which it is located (Fig. 1): DSX-1, 5′-TGCAACTTGCAACTTTGTGCTCTGTGTTAT-3′; DSX-2, 5′-CGGGTGAATATACTATGTTTCAATTTATAA-3′; and DSX-3, 5′-AGTGAGATCGAGAACATTTTTGCATATATA-3′. The dsxA binding site of the Yp1 gene and a non-specific DNA (Cho & Wensink, 1997) were used as positive and negative controls, respectively: 5′-TCGACACAACTACAATGTTGCAATCAGCTAGCC-3′ (sense strand of dsxA) and 5′-GTTACCCGATGGATACTTAATAACC-3′ (non-specific sense strand). These DNA probes were end-labelled using a DNA 5′-End-Labeling System (Promega) and [γ-32P]-ATP (Perkin). Unincorporated nucleotides were removed with Quick-Spin columns (Qiagen, Santa Clara, CA, USA). Protein-DNA binding reactions were performed with 0–500 ng of DSX protein extract mixed with 2 fmol of 32P-labelled, double-stranded probes in 40 μl Z-50 Buffer for 40 min at room temperature. For the competition assay (Fig. 3A), unlabelled 0–1000-fold molar excess of double-stranded oligonucleotide competitors were preincubated with DSX protein extract in Z-50 buffer for 20 min on ice, followed by the addition of labelled Yp1 probe and incubation for another 30 min at room temperature. The DNA–protein complexes were resolved on 6% non-denaturing polyacrylamide gels prerun for 1 h in Tris–glycine buffer (25 mM Tris–HCl; 190 mM glycine; 1 mM EDTA, pH 8.3) at 10 V/cm and 4 °C. The dried gels were exposed to a phosphor screen, scanned with a Personal Molecular Imager FX (Bio-Rad, Hercules, CA, USA). Quantification was performed with Quantity-One software from Bio-Rad: to determine the probe fraction bound, radioactivity associated with specific DNA-DSX complexes was divided by the sum of radioactivity present in the shifted and unshifted oligonucleotide probe.

Statistical analysis

Statistical analysis was performed using the SPSS-10 (SPSS Inc., Chicago, IL, USA) and SAS 9 (SAS Institute Inc., Cary, NC, USA) software packages. The sample distribution in each group was tested for normality using a one-sample Kolmogorov–Smirnov test. Pairwise comparisons of sample groups were done using independent samples Welch t-tests, which do not have an equal variance assumption. Luciferase levels were compared (in multiple groups) using either one-way ANOVA (for analysis of tra mutants) or factorial ANOVA (for analysis of tissue specificity). Individual groups were contrasted using Tamhane posthoc tests, which do not assume equal variance. P-values below 0.05 were considered significant. The validation experiment and calculation of the SD of real time PCR data were performed according to the Applied Biosystems (Applied Biosystems, Foster City, CA, USA) ‘Guide to performing relative quantification of gene expression using real-time quantitative PCR’ (available online at http://www.appliedbiosystems.com/support/apptech/).


This research was supported a grant (AI046738) from the National Institutes of Health, USA, to H.B and a grant from the Committee for Allocation of Graduate Student Resources at the University of Arkansas for Medical Sciences to SZ. Doublesex antisera and clones for bacterial expression of Drosophila Doublesex proteins were generous gifts from Pieter C. Wensink (emeritus of Brandeis University) and Gyunghee Lee (University of Tennessee-Knoxville, TN, USA. We are grateful to Artyom Kopp and Bruce S. Baker for their suggestions and critical comments.

Contributor Information

U. K. Jinwal, Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

S. O. Zakharkin, Section on Statistical Genetics, Department of Biostatistics, University of Alabama at Birmingham, Birmingham, AL, USA.

O. V. Litvinova, Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

S. Jain, Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA.


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