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Tra Tra _TOP_

RS domain directs localization of proteins to the speckled subnuclear compartment and the purpose of this localization is to allow colocalization and co-concentration of components of the splicing and splicing regulatory machinery to permit relatively high rates and/or efficiencies of reaction and interaction.

Tra Tra

For each fully sequenced cDNA the DGRC maintains various forms of the cDNA (e.g tagged or untagged) in several different host vectors for subsequent cloning and expression in Drosophila and Drosophila cell lines.

In gynanders (XX/XO mosaics) feminised by traF.U2af50, traF protein in XO somatic cells of the gonad is sufficient (even in the absence of detectable SxlF 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 traF.U2af50-feminised haplo-X cells cannot substitute.

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.

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 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.

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).

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.

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.

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.

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.

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.

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 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.

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 Brown and King, 1961. 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 Marsh and Wieschaus, 1978. Cell autonomous in mitotic clones Baker and Ridge, 1980.

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