Interspecific Comparison of the Transformer Gene of Drosophila Reveals an Unusually High Degree of Evolutionary Divergence

The transformer (tra) gene of Drosophila melanogaster occupies an intermediate position in the regulatory pathway controlling all aspects of somatic sexual differentiation. The female-specific expression of this gene's function is regulated by the Sex lethal (Sxl) gene, through a mechanism involving sex-specific alternative splicing of tra pre-mRNA. The tra gene encodes a protein that is thought to act in conjunction with the transformer-2 (tra-2) gene product to control the sex-specific processing of doublesex (dsx) pre-mRNA. The bifunctional dsx gene carries out opposite functions in the two sexes, repressing female differentiation in males and repressing male differentiation in females. Here we report the results from an evolutionary approach to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interspecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event.     

Sex determination in the Drosophila germline is dictated by the sexual identity of the surrounding soma

It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway and an autonomous X chromosome counting system. In the studies reported here, we have examined the role of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway. We asked whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. We find that Tra(F) is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2) gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by Tra(F) can be blocked by the male form of the Dsx protein (Dsx(M)). Expression of the female form of dsx, Dsx(F), in XY animals also induced germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX germ cells in XX animals, our findings indicate that the somatic signaling pathway is redundant at the level tra and dsx. Finally, our studies call into question the idea that a cell-autonomous X chromosome counting system plays a central role in germline sex determination.     

The sex-determining gene tra of Drosophila: molecular cloning and transformation studies.

In Drosophila, the primary signal for sex determination is given by the ratio of X chromosomes to sets of autosomes (X:A). The primary signal is read by a key gene (Sxl) and transmitted down to the differentiation genes by the subordinate control genes tra, tra-2, ix and dsx. Mutations in tra transform chromosomal females (X/X; tra/tra) into sterile males (pseudomales). We have cloned the tra region by microdissection and chromosomal walking. We identified the gene using deficiency breakpoints, DNA aberrations in three different alleles of tra and by P-mediated transformation. A 3.8-kb fragment perfectly rescued the mutant phenotype of X/X; tra/tra flies, showing that it contained all the necessary information to restore female-specific functions in the mutant flies. We present evidence that most of the function of tra can be provided by a subsegment of 2 kb that is differentially transcribed or processed in males and females.     

Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro.

Somatic sex determination in Drosophila involves a hierarchy of regulated alternative pre-mRNA processing. Female-specific splicing and/or polyadenylation of doublesex (dsx) pre-mRNA, the final gene in this pathway, requires transformer (tra) and transformer-2 (tra-2) proteins. The mechanisms by which these proteins regulate RNA processing has not been characterized. In this paper we show that tra-2 produced in Escherichia coli binds specifically to a site within the female-specific exon of dsx pre-mRNA. This site, which contains six copies of a 13 nucleotide repeat, is required not only for female-specific splicing, but also for female-specific polyadenylation. These observations suggest that tra-2 is a positive regulator of dsx pre-mRNA processing.     

Interactions between sex-transformation mutants of Drosophila melanogaster. I. Hemolymph vitellogenins and gonad morphology

In Drosophila, vitellogenins (yolk protein precursors) are synthesized by the female fat body, secreted into the hemolymph and subsequently taken up by the developing oocytes. The male fat body, on the other hand, does not do this even when immature ovaries are transplanted into the body cavity and grow. Thus, the hemolymph vitellogenins serve as an easily detectable sexually dimorphic biochemical marker.--We have examined hemolymph vitellogenins by SDS polyacrylamide gel electrophoresis in flies carrying various sex-transformation mutants (dsx, tra, tra-2 and tra-2OTF) singly and in all possible combinations. Chromosomal females homozygous for tra or tra-2 have no detectable hemolymph vitellogenins, while those homozygous for tra-2OTF exhibit appreciable levels of these proteins. Flies homozygous for dsx, both X/X and X/Y, have hemolymph vitellogenins, although the amount is consistently smaller in the latter. Indeed, X/Y; dsx/dsx is the only genotype in which hemolymph vitellogenins are detected in the X/Y flies. A clear hierarchy of epistasis exists among these sex-transformation mutants when they are examined in various combinations: dsx greater than tra, tra-2 greater than tra-2OTF. Moreover, an interaction between tra-2OTF and tra was seen in these experiments: X/X; tra-2OTF/tra-2OTF flies show the presence of only a trace of hemolymph vitellogenins when they are made heterozygous for tra. These results, combined with observations on gonad morphology, are discussed with respect to the Baker and Ridge (1980) hypothesis of sex determination.     

Ectopic expression of the female transformer gene product leads to female differentiation of chromosomally male Drosophila

The transformer (tra) gene of Drosophila is necessary for all aspects of female somatic sexual differentiation. tra uses a single set of precursor RNAs to produce female- and non-sex-specific RNAs by alternative splicing. Ectopic expression of the female-specific RNA causes chromosomal males to develop as females, indicative of a linear pathway of regulated genes controlling sex. Genetic and molecular tests with this ectopically expressed gene are consistent with the following order of gene action: X chromosome to autosome ratio----Sex lethal----transformer----transformer-2----doublesex----intersex--- - terminal differentiation. Expression of the female-specific tra RNA in tra mutants is sufficient to lead to female differentiation. Expression of the non-sex-specific tra RNA in tra mutants is not sufficient to lead to female differentiation. The tra female-specific activity is not required for female-specific splicing of the tra precursor RNAs.     

The Doublesex Locus of Drosophila Melanogaster and Its Flanking Regions: A Cytogenetic Analysis

The region of the third chromosome (84D-F) of Drosophila melanogaster that contains the doublesex (dsx) locus has been cytogenetically analyzed. Twenty nine newly induced, and 42 preexisting rearrangements broken in dsx and the regions flanking dsx have been cytologically and genetically characterized. These studies established that the dsx locus is in salivary chromosome band 84E1-2. In addition, these observations provide strong evidence that the dsx locus functions only to regulate sexual differentiation and does not encode a vital function. To obtain new alleles at the dsx locus and to begin to analyze the genes flanking dsx, 59 lethal and visible mutations in a region encompassing dsx were induced. These mutations together with preexisting mutations in the region were deficiency mapped and placed into complementation groups. Among the mutations we isolated, four new mutations affecting sexual differentiation were identified. All proved to be alleles of dsx, suggesting that dsx is the only gene in this region involved in regulating sexual differentiation. All but one of the new dsx alleles have equivalent effects in males and females. The exception, dsxEFH55, strongly affects female sexual differentiation, but only weakly affects male sexual differentiation. The interactions of dsxEFH55 with mutations in other genes affecting sexual differentiation are described. These results are discussed in terms of the recent molecular findings that the dsx locus encodes sex-specific proteins that share in common their amino termini but have different carboxyl termini. The 72 mutations in this region that do not affect sexual differentiation identify 25 complementation groups. A translocation, T(2;3)Es that is associated with a lethal allele in one of these complementation groups is also broken at the engrailed (en) locus on the second chromosome and has a dominant phenotype that may be due to the expression of en in the anterior portion of the abdominal tergites where en is not normally expressed. The essential genes found in the 84D-F region are not evenly distributed throughout this region; most strikingly the 84D1-11 region appears to be devoid of essential genes. It is suggested that the lack of essential genes in this region is due to the region (1) containing genes with nonessential functions and (2) being duplicated, possibly both internally and elsewhere in the genome.     

Genes expressed in the Drosophila head reveal a role for fat cells in sex-specific physiology

The downstream effectors of the Drosophila sex determination cascade are mostly unknown and thought to mediate all aspects of sexual differentiation, physiology and behavior. Here, we employed serial analysis of gene expression (SAGE) to identify male and female effectors expressed in the head, and report 46 sex-biased genes (>4-fold/P < 0.01). We characterized four novel, male- or female-specific genes and found that all are expressed mainly in the fat cells in the head. Tsx (turn on sex-specificity), sxe1 and sxe2 (sex-specific enzyme 1/2) are expressed in males, but not females, and are dependent on the known sex determination pathway, specifically transformer (tra) and its downstream target doublesex (dsx). Female-specific expression of the fourth gene, fit (female-specific independent of transformer), is not controlled by tra and dsx, suggesting an alternative pathway for the regulation of some effector genes. Our results indicate that fat cells in the head express sex-specific effectors, thereby generating distinct physiological conditions in the male and female head. We suggest that these differences have consequences on the male and female brain by modulating sex-specific neuronal processes.     

Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene

The transformer (tra) gene regulates female somatic sexual differentiation and has no known function in males. It gives rise to two sizes of RNA, one non-sex-specific and one female-specific. These two RNAs are shown to be present throughout the life cycle, and related by the use of alternative first intron splice acceptor sites. The non-sex-specific RNA has a 73 base first intron, while that in the female-specific RNA is 248 bases. The non-sex-specific RNA has no long open reading frame, while the female-specific RNA has a single long open reading frame beginning at the first AUG. Substitution of a heat shock promoter for the tra promoter still leads to female-specific differentiation of otherwise tra-females. We suggest a mechanism by which Sex-lethal controls itself and tra.