In gynanders (XX/XO mosaics) feminised by tra^F.U2af50, tra^F protein in XO somatic cells of the gonad is sufficient (even in the absence of detectable Sxl^F protein) to elicit all nonautonomous feminising signals required by XX germ cells, as well as all other somatic-cell functions required for normal oogenesis, so that functional eggs are produced. However, some of these feminised gynanders fail to lay their eggs, indicating that there are diplo-X cells outside the gonad for which tra^F.U2af50-feminised haplo-X cells cannot substitute.

The Hrp59 protein binds preferentially to a subset of mRNAs, including tra mRNA.

tra is necessary in order to prevent the continued presence of male-specific somatic gonadal precursors in the female somatic gonad.

Ectopic somatic expression of the female product of tra is sufficient to feminise XY germ cells. This feminisation depends upon the tra2 gene, but does not seem to require a functional dsx gene. However, feminisation of XY germ cells by the female product of tra can be blocked by the male form of dsx protein.

The crystal structure has been determined at 2.6A resolution of the complex formed between two tandemly arranged RNA-binding domains of the Sxl protein and a 12 nucleotide, single stranded RNA derived from the tra polypyrimidine tract. The two RNA-binding domains have their β-sheet platforms facing each other to form a V-shaped cleft. The RNA is bound in this cleft, where the tra UGUUUUUUU sequence is specifically recognized by the Sxl protein.

tra is not required for the development of the internal organisation of the male terminal segment.

Mutants carrying a heat inducible female form of tra exhibit indiscriminate sexual behaviour. Studies suggest a disturbed nervous system, and not self-stimulation, is the most probable cause for this behaviour. Sexual behaviour is irreversibly programmed during a critical period as a result of the activity or inactivity or a single control gene.

The female nervous system is substantially responsible for controlling the process of sperm transport from the bursa copulatrix to the storage organs.

tra and tra2 regulate sex-specific splicing of fru, inducing female-specific splicing of fru by activating the female-specific fru 5' splice site.

Sex determining genes tra, ix and dsx have no role in regulating the template organisation of the X chromosome(s) for dosage compensation.

The Sxl RBD1+2/tra PPT interaction is analysed by chemical shift perturbation mapping of the protein backbone. Data suggests the two domains respond quite differently to the presence of tra PPT RNA.

The roX1 gene shows a male-specific expression pattern in adult flies. roX1 expression is dependent on Sxl, but is independent of tra activity, and is positively regulated by genes of the dosage compensation system such as mle.

The simultaneous influence of different male and female pheromones on male courtship behaviour is measured using tra mosaic flies.

fl(2)d function is necessary for the female-specific splicing of tra pre-mRNA, but not for the female-specific splicing of dsx pre-mRNA.

Both HeLa and Kc cell nuclear extracts have been used for UV cross-linking experiments to determine which proteins bind to dsxRE as part of the native tra- and tra2-dependent dsx enhancer complex (dsxEC). Rbp1 and SRp30 have been identified that bind the 13-nucleotide repeats and purine rich element (PRE), respectively, of the dsx repeat element (dsxRE).

Anatomical and behavioural studies of fru mutants are consistent with the function of fru being downstream of tra and tra2.

Both of the RNA-binding domains (RBDs) of Sxl are required for efficient and specific binding of Sxl protein to the tra cis-acting element.

Female specific form of the tra gene, expressed using the GAL4 system, has been used to give a pattern of feminization. Feminization of certain brain structures in the male is associated with different types of sexual behaviour. These intersexual flies produce a large variety of pheromonal bouquets which combine male and female pheromones in different amounts. There is a significant correlation between the production of different pheromones and the induction of different male behaviours.

Using the Scer\GAL4-Scer\UAS system to express tra males have been produced with regionally feminized brains. Flies feminized in a portion of the antennal lobes or in a subset of the mushroom bodies court both males and females.

Amino acid sequences required to direct the tra protein to nuclear speckles have been identified. The tra nuclear localisation can be uncoupled from localisation to the speckle domains. An amino sequence in tra is capable of directing a heterologous protein to nuclear speckles of mammalian cells, regions of the nucleus previously shown to contain high concentrations of spliceosomal small nuclear RNAs and splicing factors. tra2 and tra colocalise in the speckle domains.

Genetic and molecular analyses suggest that vir is required for female-specific splicing of Sxl and of tra pre-mRNA.

Female-specific expression of genes in the germline is dependent on a somatic signalling pathway which requires the sex-non-specific tra2 but not the sex-specific tra and dsx.

l(3)73Ah shares 3' untranslated sequence with tra.

Regulated alternative splicing of dsx pre-mRNA requires the dsxRE splicing enhancer, dsx repeat element. The activity of dsxRE requires tra and tra2 and one or more general splicing factors. A purine rich enhancer (PRE) sequence within the RE has been identified, this element functionally synergises with the dsxRE and is required for specific binding of tra2 to the dsxRE. Results demonstrate that positive control of dsx pre-mRNA splicing requires tra- and tra2- dependent assembly of a multiprotein complex on at least two distinct enhancer elements.

The expression of tra under the control of Scer\GAL4 in sub domains of the male mushroom bodies causes feminisation of the cells in which tra is expressed and a subsequent insertion-specific non-discriminatory courtship behaviour where males court other males as vigorously as females. This supports the notion that at least some sub-domains of the mushroom bodies develop in a sex-specific manner.

tra is expressed in genetically defined subregions of the male brain, in particular within different domains of the mushroom bodies. Expression in a Scer\GAL4 line that marks a component of the antennal lobe causes males to exhibit nondiscriminatory sexual behaviour (court males and females). Expression in other mushroom body domains has no such effect.

her^- intersexuality cannot be rescued by constitutive tra expression.

The Sxl splice site consensus is a highly specific sequence, explaining why it can regulate splicing of tra pre-mRNA and autoregulate splicing of its own mRNA.

The choice of female identity in the germ line is dependent upon a somatic signalling pathway that requires the sex-non-specific tra2 gene but not the sex specific genes tra and dsx.

The sex-specific requirement of sov in gonadal development is controlled by the somatic sex regulatory genes tra, tra2 and dsx.

The tra2 product interacts with itself, and with the tra and SF2 products in vitro and in the yeast two-hybrid system.

The tra gene is regulated by Sxl-dependent 3' splice site blockage. 40 nucleotides immediately upstream of the regulated splice site are sufficient to direct sex-specific regulated splicing.

tra and dsx control early inductive signals that determine the sex of XX germ cells. tra product present in somatic cells of XY animals, or in backgrounds lacking the sex-determining function of Sxl, is sufficient to support developing XX germ cells through oogenesis.

There is no cost to a female to receive sperm: lifespan, egg production, egg hatchability and remating rate of females intermittently exposed to males that could (tra mutants) or could not (tud mutants) transfer sperm are not significantly different.

The genetic hierarchy regulating female germ-line sex determination includes tra, tra2, dsx, fu, otu, ovo, snf and Sxl.

Female specific splicing of dsx is regulated by tra and tra2, which recruit general, serine/arginine-rich splicing factors to a regulatory element located downstream of a female-specific 3' splice site.

In vitro system that recapitulates the regulation by Sxl of tra sex-specific splicing developed. Sxl blocks splicing to the non-sex specific, default site in tra by specifically binding to its polypyrimidine tract, blocking the binding of the essential splicing factor U2AF: U2AF then acivates the lower-affinity female-specific site.

Mutants exhibit defective courtship song.

UV crosslinking/transfection of Kc cells showed female-specific tra protein binds to 13 nucleotide motif present in 6 copies in the female-specific fourth exon of dsx pre-mRNA.

The tra-homologous genes from D.simulans, D.erecta, D.hydei and D.virilis have been cloned, sequenced and compared to the D.melanogaster tra gene. This comparison reveals as unusually high degree of evolutionary divergence among the tra coding sequences. In addition there is a highly conserved region within the first intron that may define a cis-acting regulator of sex-specific alternative splicing.

tra function directs the development of sexually dimorphic skeletal muscles.

An in vitro splicing system to study the mechanism involved in positive control of dsx female specific splicing by tra and tra2 is used in HeLa cell nuclear extracts.

Wild type functions of tra and tra2 are necessary in females for the expression of the female specific dsx function. In the absence of tra or tra2 function the alternative pattern of processing produces the dsx mRNA that encodes the male specific dsx protein.

Cotransfection analyses in which dsx, tra and tra2 cDNAs are expressed in Kc cells revealed that female specific splicing of dsx transcript is positively regulated by tra and tra2 gene products.

The functions of the arginine/serine-rich RS domains of su(w^a) and tra have been studied.

Cotransfection assays to examine regulatory interactions between specific cis-acting sequence elements of dsx pre-mRNA, and tra and tra2 gene products establish that tra and tra2 function to activate the use of the female specific exon.

Co-transfection experiments in which Sxl cDNA and the tra gene are expressed in Kc cells demonstrate that the female Sxl-encoded protein binds specifically to the tra transcript at or near the non-sex-specific acceptor site. This implies that the female Sxl gene product is the trans-acting factor that regulates alternative splicing.

The mechanism of sex determination in the germ line has been analysed.

The information for proper 3' splice site choice in tra is contained within the regulated intron.

The nature and function of the 2 different tra RNAs is determined and the mechanisms of their sex-specific regulation is elucidated.

The tra locus has been molecularly isolated and characterized. P element mediated transformation has demonstrated that a fragment of 2kb is sufficient to supply tra⁺ function.

Mutant females are transformed into sterile males.

XX flies homozygous for tra transformed into sterile males with fully developed sex combs, male-colored abdomen, male abdominal tergites and plates, external and internal male genitalia. Mate readily with females. Testes rudimentary, without sperm and with ovarian nurse-cell-like cells <up>Brown and King, 1961</up>. Testes reduced in size, but of normal color and shape. Transformed female slightly larger than normal male, developmental rate about that of female. X/X/Y; tra/tra also sterile. tra not required in male since X/Y, tra/tra flies are normal males. X/X/X and X/X/Y, tra/tra/tra like diploid, i.e. male in phenotype, but with larger wing cells as expected of triploids. Normal testis anlagen transplanted into tra female becomes attached to duct apparatus and produces sperm. Not needed for female germ cell development since X/X, tra/tra pole cells transplanted into a wild-type female embryo give rise to progeny of both sexes <up>Marsh and Wieschaus, 1978</up>. Cell autonomous in mitotic clones <up>Baker and Ridge, 1980</up>.